Effect of modulator sorption on gradient shape in ion-exchange chromatography

NASA Technical Reports Server (NTRS)

Velayudhan, A.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

1995-01-01

Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

PubMed

Tsonev, Latchezar I; Hirsh, Allen G

2008-07-25

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

PubMed

Ishihara, Takashi; Kadoya, Toshihiko; Endo, Naomi; Yamamoto, Shuichi

2006-05-05

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

Two-column sequential injection chromatography--new approach for fast and effective analysis and its comparison with gradient elution chromatography.

PubMed

Chocholous, Petr; Satínský, Dalibor; Sklenárová, Hana; Solich, Petr

2010-05-23

This work presents novel approach in low-pressure chromatography flow systems--two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab, USA) with two commercially available monolithic columns the "first column" Chromolith Flash RP-18e (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.) and the "second column" Chromolith RP-18e (10 mm x 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min(-1) (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min(-1) (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 microL of filtered supernatant was injected on each column (2 x 10 microL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 microg mL(-1) for paracetamol, at 0.5 microg mL(-1) for caffeine and at 0.7 microg mL(-1) for propyphenazone. The new two-column chromatographic set-up developed as an alternative approach to gradient elution chromatography shows evident advantages (time and solvent reduction more than one-third) as compared with single-column gradient SIC method with Chromolith Flash RP-18 (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.). Copyright 2010 Elsevier B.V. All rights reserved.

Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography.

PubMed

Joshi, Varsha; Kumar, Vijesh; Rathore, Anurag S

2015-08-07

A method is proposed for rapid development of a short, analytical cation exchange high performance liquid chromatography method for analysis of charge heterogeneity in monoclonal antibody products. The parameters investigated and optimized include pH, shape of elution gradient and length of the column. It is found that the most important parameter for development of a shorter method is the choice of the shape of elution gradient. In this paper, we propose a step by step approach to develop a non-linear sigmoidal shape gradient for analysis of charge heterogeneity for two different monoclonal antibody products. The use of this gradient not only decreases the run time of the method to 4min against the conventional method that takes more than 40min but also the resolution is retained. Superiority of the phosphate gradient over sodium chloride gradient for elution of mAbs is also observed. The method has been successfully evaluated for specificity, sensitivity, linearity, limit of detection, and limit of quantification. Application of this method as a potential at-line process analytical technology tool has been suggested. Copyright © 2015 Elsevier B.V. All rights reserved.

Size exclusion chromatography-gradients, an alternative approach to polymer gradient chromatography: 2. Separation of poly(meth)acrylates using a size exclusion chromatography-solvent/non-solvent gradient.

PubMed

Schollenberger, Martin; Radke, Wolfgang

2011-10-28

A gradient ranging from methanol to tetrahydrofuran (THF) was applied to a series of poly(methyl methacrylate) (PMMA) standards, using the recently developed concept of SEC-gradients. Contrasting to conventional gradients the samples eluted before the solvent, i.e. within the elution range typical for separations by SEC, however, the high molar mass PMMAs were retarded as compared to experiments on the same column using pure THF as the eluent. The molar mass dependence on retention volume showed a complex behaviour with a nearly molar mass independent elution for high molar masses. This molar mass dependence was explained in terms of solubility and size exclusion effects. The solubility based SEC-gradient was proven to be useful to separate PMMA and poly(n-butyl crylate) (PnBuA) from a poly(t-butyl crylate) (PtBuA) sample. These samples could be separated neither by SEC in THF, due to their very similar hydrodynamic volumes, nor by an SEC-gradient at adsorbing conditions, due to a too low selectivity. The example shows that SEC-gradients can be applied not only in adsorption/desorption mode, but also in precipitation/dissolution mode without risking blocking capillaries or breakthrough peaks. Thus, the new approach is a valuable alternative to conventional gradient chromatography. Copyright © 2011 Elsevier B.V. All rights reserved.

Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros.

PubMed

Fasoula, S; Zisi, Ch; Gika, H; Pappa-Louisi, A; Nikitas, P

2015-05-22

A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances. Copyright © 2015 Elsevier B.V. All rights reserved.

Fast, comprehensive two-dimensional liquid chromatography

PubMed Central

Stoll, Dwight R.; Li, Xiaoping; Wang, Xiaoli; Carr, Peter W.; Porter, Sarah E. G.; Rutan, Sarah C.

2011-01-01

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two dimension liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20 seconds for a full gradient elution run. Thus fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 minutes. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed. PMID:17888443

Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

PubMed

Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

2015-04-21

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

Refractive index-based detection of gradient elution liquid chromatography using chip-integrated microring resonator arrays.

PubMed

Wade, James H; Bailey, Ryan C

2014-01-07

Refractive index-based sensors offer attractive characteristics as nondestructive and universal detectors for liquid chromatographic separations, but a small dynamic range and sensitivity to minor thermal perturbations limit the utility of commercial RI detectors for many potential applications, especially those requiring the use of gradient elutions. As such, RI detectors find use almost exclusively in sample abundant, isocratic separations when interfaced with high-performance liquid chromatography. Silicon photonic microring resonators are refractive index-sensitive optical devices that feature good sensitivity and tremendous dynamic range. The large dynamic range of microring resonators allows the sensors to function across a wide spectrum of refractive indices, such as that encountered when moving from an aqueous to organic mobile phase during a gradient elution, a key analytical advantage not supported in commercial RI detectors. Microrings are easily configured into sensor arrays, and chip-integrated control microrings enable real-time corrections of thermal drift. Thermal controls allow for analyses at any temperature and, in the absence of rigorous temperature control, obviates extended detector equilibration wait times. Herein, proof of concept isocratic and gradient elution separations were performed using well-characterized model analytes (e.g., caffeine, ibuprofen) in both neat buffer and more complex sample matrices. These experiments demonstrate the ability of microring arrays to perform isocratic and gradient elutions under ambient conditions, avoiding two major limitations of commercial RI-based detectors and maintaining comparable bulk RI sensitivity. Further benefit may be realized in the future through selective surface functionalization to impart degrees of postcolumn (bio)molecular specificity at the detection phase of a separation. The chip-based and microscale nature of microring resonators also make it an attractive potential detection technology that could be integrated within lab-on-a-chip and microfluidic separation devices.

Incorporating high-pressure electroosmotic pump and a nano-flow gradient generator into a miniaturized liquid chromatographic system for peptide analysis.

PubMed

Chen, Apeng; Lynch, Kyle B; Wang, Xiaochun; Lu, Joann J; Gu, Congying; Liu, Shaorong

2014-09-24

We integrate a high-pressure electroosmotic pump (EOP), a nanoflow gradient generator, and a capillary column into a miniaturized liquid chromatographic system that can be directly coupled with a mass spectrometer for proteomic analysis. We have recently developed a low-cost high-pressure EOP capable of generating pressure of tens of thousands psi, ideal for uses in miniaturized HPLC. The pump worked smoothly when it was used for isocratic elutions. When it was used for gradient elutions, generating reproducible gradient profiles was challenging; because the pump rate fluctuated when the pump was used to pump high-content organic solvents. This presents an issue for separating proteins/peptides since high-content organic solvents are often utilized. In this work, we solve this problem by incorporating our high-pressure EOP with a nano-flow gradient generator so that the EOP needs only to pump an aqueous solution. With this combination, we develop a capillary-based nano-HPLC system capable of performing nano-flow gradient elution; the pump rate is stable, and the gradient profiles are reproducible and can be conveniently tuned. To demonstrate its utility, we couple it with either a UV absorbance detector or a mass spectrometer for peptide separations. Copyright © 2014. Published by Elsevier B.V.

Fast methodology of analysing major steviol glycosides from Stevia rebaudiana leaves.

PubMed

Lorenzo, Cándida; Serrano-Díaz, Jéssica; Plaza, Miguel; Quintanilla, Carmen; Alonso, Gonzalo L

2014-08-15

The aim of this work is to propose an HPLC method for analysing major steviol glycosides as well as to optimise the extraction and clarification conditions for obtaining these compounds. Toward this aim, standards of stevioside and rebaudioside A with purities ⩾99.0%, commercial samples from different companies and Stevia rebaudiana Bertoni leaves from Paraguay supplied by Insobol, S.L., were used. The analytical method proposed is adequate in terms of selectivity, sensitivity and accuracy. Optimum extraction conditions and adequate clarification conditions have been set. Moreover, this methodology is safe and eco-friendly, as we use only water for extraction and do not use solid-phase extraction, which requires solvents that are banned in the food industry to condition the cartridge and elute the steviol glycosides. In addition, this methodology consumes little time as leaves are not ground and the filtration is faster, and the peak resolution is better as we used an HPLC method with gradient elution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Secretory immunoglobulin purification from whey by chromatographic techniques.

PubMed

Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

2017-08-15

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000bar.

PubMed

De Pauw, Ruben; Swier, Tim; Degreef, Bart; Desmet, Gert; Broeckhoven, Ken

2016-11-18

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental pressure-induced limitations. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

PubMed

Pinto, Nuno D S; Uplekar, Shaunak D; Moreira, Antonio R; Rao, Govind; Frey, Douglas D

2017-01-01

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Microchip electrospray: improvements in spray and signal stability during gradient elution by an inverted postcolumn makeup flow.

PubMed

Jung, Stephanie; Effelsberg, Uwe; Tallarek, Ulrich

2011-12-01

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.

Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids: Influence of the Complexity of the Matrix.

PubMed

Leitão, Gilda Guimarães; Pinto, Shaft Correa; de Oliveira, Danilo Ribeiro; Timoteo, Patrícia; Guimarães, Michelle Guedes; Cordova, Wilmer H Perera; Leitão, Suzana Guimarães

2015-11-01

Verbascoside is a phenylethanoid glycoside widely distributed in nature, especially among the order Lamiales, occurring in numerous plants that are constituents of folk medicine preparations. This natural compound, previously isolated by our group from the ethyl acetate extract of Lantana trifolia using the gradient approach in countercurrent chromatography, was now isolated from the butanol extract of the same plant and from Lippia alba f. intermedia (Verbenaceae) using countercurrent chromatography in either gradient or isocratic elution modes. The ethyl acetate extract of L. alba, rich in phenylethanoids and flavonoids, was fractionated using countercurrent chromatography in the step-gradient elution approach. The four-step solvent system was composed of n-hexane-ethyl acetate-n-butanol-water (4 : 10 : X : 10), where X = 1 (solvent system A), 3 (solvent system B), 5 (solvent system C), and 7 (solvent system D), and allowed for the isolation of verbascoside along with other phenylethanoids and flavonoids from both plants. Verbascoside and 2'-O-β-apiosylverbascoside were further isolated from the n-butanol extract of L. trifolia using the solvent system ethyl acetate-n-butanol-water 10 : 2 : 10 on an isocratic run. The difference in the complexity of the two plant extracts demanded different purification steps, which included a second high-speed countercurrent chromatography purification using the isocratic elution mode. Georg Thieme Verlag KG Stuttgart · New York.

Comprehensive two-dimensional liquid chromatography separations of pharmaceutical samples using dual Fused-Core columns in the 2nd dimension.

PubMed

Alexander, Anthony J; Ma, Lianjia

2009-02-27

This paper focuses on the application of RPLC x RPLC to pharmaceutical analysis and addresses the specific problem of separating co-eluting impurities/degradation products that maybe "hidden" within the peak envelope of the active pharmaceutical ingredient (API) and thus may escape detection by conventional methods. A comprehensive two-dimensional liquid chromatograph (LC x LC) was constructed from commercially available HPLC equipment. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30s total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates. The operation of the interface is demonstrated for the analysis of a 1mg/ml standard mixture containing 0.05% of a minor component. The practicality of using RPLC x RPLC for the analysis of actual co-eluting pharmaceutical degradation products, by exploiting pH-induced changes in selectivity, is also demonstrated using a three component mixture. This mixture (an API, an oxidation product of the API at 1.0%, w/w, and a photo degradant of the API at 0.5%, w/w) was used to assess the stability indicating nature of an established LC method for analysis of the API.

High performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry for V and Ni quantification as tetrapyrroles

NASA Astrophysics Data System (ADS)

Duyck, Christiane Béatrice; Saint'Pierre, Tatiana Dillenburg; Miekeley, Norbert; da Fonseca, Teresa Cristina Oliveira; Szatmari, Peter

2011-05-01

A method was developed for the determination of V and Ni as tetrapyrroles by High Performance Liquid Chromatography hyphenated to Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) using reversed phase and elution gradient. Chlorinated solvents and tetrahydrofuran were investigated as regard to separation time and ICP-MS detection efficiencies. The final elution gradient program started from pure methanol to a mixture of 20:80 (v/v) chloroform:methanol. External quantification of V and Ni with inorganic standards by flow injection ICP-MS, used online with HPLC, resulted in 95% of recoveries. The Limits of Detection for V during methanol elution and for Ni during the 20% chloroform gradient elution were evaluated by their minimum detectable concentrations, which were, respectively, 5 μg L - 1 and 8 μg L - 1 . The methodology was applied to polar and resin fractions separated from a Brazilian crude oil and a sediment extract from an oil-polluted area in the Guanabara Bay, Rio de Janeiro, Brazil. Vanadium as tetrapyrroles represented the totality of V content in the polar fraction, whereas Ni was in different polar forms in the resin and sediment extract.

Purification of bacteriophage M13 by anion exchange chromatography.

PubMed

Monjezi, Razieh; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2010-07-01

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious. Copyright 2010 Elsevier B.V. All rights reserved.

Fast and sensitive high performance liquid chromatography analysis of cosmetic creams for hydroquinone, phenol and six preservatives.

PubMed

Gao, Wenhui; Legido-Quigley, Cristina

2011-07-15

A fast and sensitive HPLC method for analysis of cosmetic creams for hydroquinone, phenol and six preservatives has been developed. The influence of sample preparation conditions and the composition of the mobile phase and elution mode were investigated to optimize the separation of the eight studied components. Final conditions were 60% methanol and 40% water (v/v) extraction of the cosmetic creams. A C18 column (100 mm × 2.1 mm) was used as the separation column and the mobile phase consisted of methanol and 0.05 mol/L ammonium formate in water (pH=3.0) with gradient elution. The results showed that complete separation of the eight studied components was achieved within 10 min, the linear ranges were 1.0-200 μg/mL for phenol, 0.1-150 μg/mL for sorbic acid, 2.0-200 μg/mL for benzoic acid, 0.5-200 μg/mL for hydroquinone, methyl paraben, ethyl paraben and propyl paraben, butyl paraben, and good linear correlation coefficient (≥0.9997) were obtained, the detection limit was in the range of 0.05-1.0 μg/mL, the average recovery was between 86.5% and 116.3%, and the relative standard deviation (RSD) was less than 5.0% (n=6). The method is easy, fast and sensitive, it can be employed to analyze component residues in cosmetic creams especially in a quality control setting. Copyright © 2011 Elsevier B.V. All rights reserved.

Prediction of the retention of s-triazines in reversed-phase high-performance liquid chromatography under linear gradient-elution conditions.

PubMed

D'Archivio, Angelo Antonio; Maggi, Maria Anna; Ruggieri, Fabrizio

2014-08-01

In this paper, a multilayer artificial neural network is used to model simultaneously the effect of solute structure and eluent concentration profile on the retention of s-triazines in reversed-phase high-performance liquid chromatography under linear gradient elution. The retention data of 24 triazines, including common herbicides and their metabolites, are collected under 13 different elution modes, covering the following experimental domain: starting acetonitrile volume fraction ranging between 40 and 60% and gradient slope ranging between 0 and 1% acetonitrile/min. The gradient parameters together with five selected molecular descriptors, identified by quantitative structure-retention relationship modelling applied to individual separation conditions, are the network inputs. Predictive performance of this model is evaluated on six external triazines and four unseen separation conditions. For comparison, retention of triazines is modelled by both quantitative structure-retention relationships and response surface methodology, which describe separately the effect of molecular structure and gradient parameters on the retention. Although applied to a wider variable domain, the network provides a performance comparable to that of the above "local" models and retention times of triazines are modelled with accuracy generally better than 7%. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a fast and selective UHPLC-DAD-QTOF-MS/MS method for the qualitative and quantitative assessment of destruxin profiles.

PubMed

Taibon, Judith; Sturm, Sonja; Seger, Christoph; Parth, Martin; Strasser, Hermann; Stuppner, Hermann

2014-11-01

A fast and selective ultrahigh-performance liquid chromatography diode array detector (UHPLC-DAD) method combined with an off-line solid phase extraction (SPE) protocol was established to monitor destruxins (dtxs), a secondary metabolite class of highly bioactive cyclic depsipeptides. Sample purification via SPE was tailored to remove both more polar and apolar matrix constituents by applying analyte class-selective washing and elution conditions. To separate and detect destruxin congeners an UHPLC-DAD system hyphenated to a quadrupole-time-of-flight (Q-TOF) hybrid mass spectrometer was utilized. Analyses were performed on a sub-2-μm-particle-size RP-18 column with an acidified (0.02% acetic acid) 12 min water/acetonitrile solvent gradient. In the dtx congener elution zone 22 chromatographic peaks were separated. Four of these were identified by comparison with reference materials as dtx A, dtx B, dtx E, and dtx E-diol; 16 were tentatively assigned as known or novel dtx congeners by the analysis of high resolution UHPLC-DAD-QTOF-MS/MS data recorded in the positive electrospray ionization (ESI) mode. The applicability of the UHPLC-DAD assay to investigate biological materials in a qualitative and quantitative manner was proven by the application of the platform to monitor the dtx production profile of three Metarhizium brunneum strain fungal culture broths.

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue.

PubMed

Sajic, Tatjana; Varesio, Emmanuel; Szanto, Ildiko; Hopfgartner, Gérard

2015-09-01

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K(+) as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue. Copyright © 2015 Elsevier Inc. All rights reserved.

A narrow open tubular column for high efficiency liquid chromatographic separation.

PubMed

Chen, Huang; Yang, Yu; Qiao, Zhenzhen; Xiang, Piliang; Ren, Jiangtao; Meng, Yunzhu; Zhang, Kaiqi; Juan Lu, Joann; Liu, Shaorong

2018-04-30

We report a great feature of open tubular liquid chromatography when it is run using an extremely narrow (e.g., 2 μm inner diameter) open tubular column: more than 10 million plates per meter can be achieved in less than 10 min and under an elution pressure of ca. 20 bar. The column is coated with octadecylsilane and both isocratic and gradient separations are performed. We reveal a focusing effect that may be used to interpret the efficiency enhancement. We also demonstrate the feasibility of using this technique for separating complex peptide samples. This high-resolution and fast separation technique is promising and can lead to a powerful tool for trace sample analysis.

Retention prediction of highly polar ionizable solutes under gradient conditions on a mixed-mode reversed-phase and weak anion-exchange stationary phase.

PubMed

Balkatzopoulou, P; Fasoula, S; Gika, H; Nikitas, P; Pappa-Louisi, A

2015-05-29

In the present work the retention of three highly polar and ionizable solutes - uric acid, nicotinic acid and ascorbic acid - was investigated on a mixed-mode reversed-phase and weak anion-exchange (RP/WAX) stationary phase in buffered aqueous acetonitrile (ACN) mobile phases. A U-shaped retention behavior was observed for all solutes with respect to the eluent organic modifier content studied in a range of 5-95% (v/v). This retention behavior clearly demonstrates the presence of a HILIC-type retention mechanism at ACN-rich hydro-organic eluents and an RP-like retention at aqueous-rich hydro-organic eluents. Hence, this column should be promising for application under both RP and HILIC gradient elution modes. For this reason, a series of programmed elution runs were carried out with increasing (RP) and decreasing (HILIC) organic solvent concentration in the mobile phase. This dual gradient process was successfully modeled by two retention models exhibiting a quadratic or a cubic dependence of the logarithm of the solute retention factor (lnk) upon the organic modifier volume fraction (φ). It was found that both models produced by gradient retention data allow the prediction of solute retention times for both types of programmed elution on the mixed-mode column. Four, in the case of the quadratic model, or five, in the case of the cubic model, initial HILIC- and RP-type gradient runs gave satisfactory retention predictions of any similar kind elution program, even with different flow rate, with an overall error of only 2.5 or 1.7%, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Predicting the chromatographic retention of polymers: application of the polymer model to poly(styrene/ethylacrylate)copolymers.

PubMed

Bashir, Mubasher A; Radke, Wolfgang

2012-02-17

The retention behavior of a range of statistical poly(styrene/ethylacrylate) copolymers is investigated, in order to determine the possibility to predict retention volumes of these copolymers based on a suitable chromatographic retention model. It was found that the composition of elution in gradient chromatography of the copolymers is closely related to the eluent composition at which, in isocratic chromatography, the transition from elution in adsorption to exclusion mode occurs. For homopolymers this transition takes place at a critical eluent composition at which the molar mass dependence of elution volume vanishes. Thus, similar critical eluent compositions can be defined for statistical copolymers. The existence of a critical eluent composition is further supported by the narrower peak width, indicating that the broad molar mass distribution of the samples does not contribute to the retention volume. It is shown that the existing retention model for homopolymers allows for correct quantitative predictions of retention volumes based on only three appropriate initial experiments. The selection of these initial experiments involves a gradient run and two isocratic experiments, one at the composition of elution calculated from first gradient run and second at a slightly higher eluent strength. Copyright © 2011 Elsevier B.V. All rights reserved.

Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

PubMed Central

Park, Sung-Soo; Maudsley, Stuart

2010-01-01

Global profiling of phosphoproteomes has proven a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically-relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography (TOC) can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH-gradient elution technique for enhanced phosphopeptide identification in conjunction with TOC. Using this process, we have demonstrated its superiority to the traditional ‘one-pot’ strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH-gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pH 9.2 and 9.4. High-resolution separation of multiply-phosphorylated peptides was primarily achieved using elution ranges > pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values. PMID:20946866

Method developments approaches in supercritical fluid chromatography applied to the analysis of cosmetics.

PubMed

Lesellier, E; Mith, D; Dubrulle, I

2015-12-04

Analyses of complex samples of cosmetics, such as creams or lotions, are generally achieved by HPLC. These analyses are often multistep gradients, due to the presence of compounds with a large range of polarity. For instance, the bioactive compounds may be polar, while the matrix contains lipid components that are rather non-polar, thus cosmetic formulations are usually oil-water emulsions. Supercritical fluid chromatography (SFC) uses mobile phases composed of carbon dioxide and organic co-solvents, allowing for good solubility of both the active compounds and the matrix excipients. Moreover, the classical and well-known properties of these mobile phases yield fast analyses and ensure rapid method development. However, due to the large number of stationary phases available for SFC and to the varied additional parameters acting both on retention and separation factors (co-solvent nature and percentage, temperature, backpressure, flow rate, column dimensions and particle size), a simplified approach can be followed to ensure a fast method development. First, suited stationary phases should be carefully selected for an initial screening, and then the other operating parameters can be limited to the co-solvent nature and percentage, maintaining the oven temperature and back-pressure constant. To describe simple method development guidelines in SFC, three sample applications are discussed in this paper: UV-filters (sunscreens) in sunscreen cream, glyceryl caprylate in eye liner and caffeine in eye serum. Firstly, five stationary phases (ACQUITY UPC(2)) are screened with isocratic elution conditions (10% methanol in carbon dioxide). Complementary of the stationary phases is assessed based on our spider diagram classification which compares a large number of stationary phases based on five molecular interactions. Secondly, the one or two best stationary phases are retained for further optimization of mobile phase composition, with isocratic elution conditions or, when necessary, two-step gradient elution. The developed methods were then applied to real cosmetic samples to assess the method specificity, with regards to matrix interferences, and calibration curves were plotted to evaluate quantification. Besides, depending on the matrix and on the studied compounds, the importance of the detector type, UV or ELSD (evaporative light-scattering detection), and of the particle size of the stationary phase is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Chiral separation with gradient elution isotachophoresis for future in situ extraterrestrial analysis.

PubMed

Danger, Grégoire; Ross, David

2008-10-01

The first results of chiral separations with the gradient elution isotachophoresis method are presented. As previously described, citrate is used in the run buffer as the leading ion and borate in the sample buffer as the terminating ion. Modulation of parameters such as electrolyte pH, pressure scan rate, chiral selector concentration, combinations of CD or the percentage of ampholytes provides an easy optimization of the separations. To perform fluorescent detection 5-carboxyfluorescein succinimidyl ester and two fluorogenic-labeling agents, fluorescamine (Fluram) and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, are used to label amino acids. With the 5-carboxyfluorescein amino acids, chiral separations are easily obtained using a neutral CD ((2-hydroxypropyl)-beta-CD) at a low concentration (2 mmol/L). With Fluram amino acids, the situation is more complicated due to the formation of diastereoisomers and due to weak interactions with the different CDs used. The use of the 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde-labeling agent solves the problems observed with the Fluram agent while retaining the fluorogenic properties. These first results demonstrate the simplicity and the feasibility of gradient elution isotachophoresis for chiral separations.

Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS.

PubMed

Zhu, Ming-Zhi; Li, Na; Wang, Yi-Tong; Liu, Ning; Guo, Ming-Quan; Sun, Bao-Qing; Zhou, Hua; Liu, Liang; Wu, Jian-Lin

2017-09-01

The usage of strong cation exchange (SCX) chromatography in proteomics is limited by its poor resolution and nonspecific hydrophobic interactions with peptides, which lead to peptide overlap across fractions and change of peptide retention, respectively. The application of high concentration of salt (up to 1000 mM) in SCX also restricted its use in online 2D SCX-RP LC. In the present research, we first exploited the chromatographic ability of online 2D SCX-RP LC by combination of acid, salt, and pH gradient, three relatively independent modes of eluting peptides from SCX column. 50% ACN was added to elution buffer for eliminating hydrophobic interactions between SCX matrix and peptides, and the concentration of volatile salt was reduced to 50 mM. Acid/salt/pH gradient showed superior resolution and sensitivity as well as uniform distribution across fractions, consequently leading to significant improvements in peptide and protein identification. 112 191 unique peptides and 7373 proteins were identified by acid/salt/pH fractionation, while 69 870 unique peptides and 4536 proteins were identified by salt elution, that is, 62.5 and 60.6% more proteins and unique peptides, respectively, identified by the former. Fraction overlap was also significantly minimized by acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more identification for acidic peptides and hydrophilic peptides.

Advantages of using tetrahydrofuran-water as mobile phases in the quantitation of cyclosporin A in monkey and rat plasma by liquid chromatography-tandem mass spectrometry.

PubMed

Li, Austin C; Li, Yinghe; Guirguis, Micheal S; Caldwell, Robert G; Shou, Wilson Z

2007-01-04

A new analytical method is described here for the quantitation of anti-inflammatory drug cyclosporin A (CyA) in monkey and rat plasma. The method used tetrahydrofuran (THF)-water mobile phases to elute the analyte and internal standard, cyclosporin C (CyC). The gradient mobile phase program successfully eluted CyA into a sharp peak and therefore improved resolution between the analyte and possible interfering materials compared with previously reported analytical approaches, where CyA was eluted as a broad peak due to the rapid conversion between different conformers. The sharp peak resulted from this method facilitated the quantitative calculation as multiple smoothing and large number of bunching factors were not necessary. The chromatography in the new method was performed at 30 degrees C instead of 65-70 degrees C as reported previously. Other advantages of the method included simple and fast sample extraction-protein precipitation, direct injection of the extraction supernatant to column for analysis, and elimination of evaporation and reconstitution steps, which were needed in solid phase extraction or liquid-liquid extraction reported before. This method is amenable to high-throughput analysis with a total chromatographic run time of 3 min. This approach has been verified as sensitive, linear (0.977-4000 ng/mL), accurate and precise for the quantitation of CyA in monkey and rat plasma. However, compared with the usage of conventional mobile phases, the only drawback of this approach was the reduced detection response from the mass spectrometer that was possibly caused by poor desolvation in the ionization source. This is the first report to demonstrate the advantages of using THF-water mobile phases to elute CyA in liquid chromatography.

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads.

PubMed

Creasy, Arch; Reck, Jason; Pabst, Timothy; Hunter, Alan; Barker, Gregory; Carta, Giorgio

2018-05-29

A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method showed good agreement with experimental elution curves for protein loads up to 40 mg/mL column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components. This article is protected by copyright. All rights reserved.

Development and validation of an RP-HPLC method for quantitative determination of vanillin and related phenolic compounds in Vanilla planifolia.

PubMed

Sinha, Arun Kumar; Verma, Subash Chandra; Sharma, Upendra Kumar

2007-01-01

A simple and fast method was developed using RP-HPLC for separation and quantitative determination of vanillin and related phenolic compounds in ethanolic extract of pods of Vanilla planifolia. Ten phenolic compounds, namely 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal were quantitatively determined using ACN, methanol, and 0.2% acetic acid in water as a mobile phase with a gradient elution mode. The method showed good linearity, high precision, and good recovery of compounds of interest. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.

Simultaneous concentration and purification through gradient deformation chromatography

NASA Technical Reports Server (NTRS)

Velayudhan, A.; Hendrickson, R. L.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

1995-01-01

Mobile-phase additives, commonly used to modulate absorbate retention in gradient elution chromatography, are usually assumed to be either linearly retained or unretained. Previous theoretical work from our laboratory has shown that these modulators, such as salts in ion-exchange and hydrophobic interaction chromatography and organic modifiers in reversed-phase chromatography, can absorb nonlinearly, giving rise to gradient deformation. Consequently, adsorbate peaks that elute in the vicinity of the head of the deformed gradient may exhibit unusual shapes, form shoulders, and/or be concentrated. These effects for a reversed-phase sorbent with aqueous acetonitrile (ACN) as the modulator are verified experimentally. Gradient deformation is demonstrated experimentally and agrees with simulations based on ACN isotherm parameters that are independently determined from batch equilibrium studies using the layer model. Unusual absorbate peak shapes were found experimentally for single-component injections of phenylalanine, similar to those calculated by the simulations. A binary mixture of tryptophan and phenylalanine is used to demonstrate simultaneous concentration and separation, again in agreement with simulations. The possibility of gradient deformation in ion-exchange and hydrophobic interaction chromatography is discussed.

Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I--elution of an unretained tracer.

PubMed

Kaczmarski, Krzysztof; Poe, Donald P; Guiochon, Georges

2010-10-15

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as "supercritical fluid chromatography" or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data. Copyright © 2010 Elsevier B.V. All rights reserved.

Recursion equations in predicting band width under gradient elution.

PubMed

Liang, Heng; Liu, Ying

2004-06-18

The evolution of solute zone under gradient elution is a typical problem of non-linear continuity equation since the local diffusion coefficient and local migration velocity of the mass cells of solute zones are the functions of position and time due to space- and time-variable mobile phase composition. In this paper, based on the mesoscopic approaches (Lagrangian description, the continuity theory and the local equilibrium assumption), the evolution of solute zones in space- and time-dependent fields is described by the iterative addition of local probability density of the mass cells of solute zones. Furthermore, on macroscopic levels, the recursion equations have been proposed to simulate zone migration and spreading in reversed-phase high-performance liquid chromatography (RP-HPLC) through directly relating local retention factor and local diffusion coefficient to local mobile phase concentration. This new approach differs entirely from the traditional theories on plate concept with Eulerian description, since band width recursion equation is actually the accumulation of local diffusion coefficients of solute zones to discrete-time slices. Recursion equations and literature equations were used in dealing with same experimental data in RP-HPLC, and the comparison results show that the recursion equations can accurately predict band width under gradient elution.

The group separation of the rare-earth elements and yttrium from geologic materials by cation-exchange chromatography

USGS Publications Warehouse

Crock, J.G.; Lichte, F.E.; Wildeman, T.R.

1984-01-01

Demand is increasing for the determination of the rare-earth elements (REE) and yttrium in geologic materials. Due to their low natural abundance in many materials and the interferences that occur in many methods of determination, a separation procedure utilizing gradient strong-acid cation-exchange chromatography is often used to preconcentrate and isolate these elements from the host-rock matrix. Two separate gradient strong-acid cation-exchange procedures were characterized and the major elements as well as those elements thought to provide the greatest interference for the determination of the REE in geologic materials were tested for separation from the REE. Simultaneous inductively coupled argon plasma-atomic emission spectroscopy (ICAP-AES) measurements were used to construct the chromatograms for the elution studies, allowing the elution patterns of all the elements of interest to be determined in a single fraction of eluent. As a rock matrix, U.S. Geological Survey standard reference BCR-1 basalt was digested using both an acid decomposition procedure and a lithium metaborate fusion. Hydrochloric and nitric acids were tested as eluents and chromatograms were plotted using the ICAP-AES data; and we observed substantial differences in the elution patterns of the REE and as well as in the solution patterns of Ba, Ca, Fe and Sr. The nitric acid elution required substantially less eluent to elute the REE and Y as a group when compared to the hydrochloric acid elution, and provided a clearer separation of the REE from interfering and matrix elements. ?? 1984.

Using contemporary liquid chromatography theory and technology to improve capillary gradient ion-exchange separations.

PubMed

Wouters, Bert; Broeckhoven, Ken; Wouters, Sam; Bruggink, Cees; Agroskin, Yury; Pohl, Christopher A; Eeltink, Sebastiaan

2014-11-28

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion. Copyright © 2014 Elsevier B.V. All rights reserved.

Very high pressure liquid chromatography using core-shell particles: quantitative analysis of fast gradient separations without post-run times.

PubMed

Stankovich, Joseph J; Gritti, Fabrice; Stevenson, Paul G; Beaver, Lois A; Guiochon, Georges

2014-01-17

Five methods for controlling the mobile phase flow rate for gradient elution analyses using very high pressure liquid chromatography (VHPLC) were tested to determine thermal stability of the column during rapid gradient separations. To obtain rapid separations, instruments are operated at high flow rates and high inlet pressure leading to uneven thermal effects across columns and additional time needed to restore thermal equilibrium between successive analyses. The purpose of this study is to investigate means to minimize thermal instability and obtain reliable results by measuring the reproducibility of the results of six replicate gradient separations of a nine component RPLC standard mixture under various experimental conditions with no post-run times. Gradient separations under different conditions were performed: constant flow rates, two sets of constant pressure operation, programmed flow constant pressure operation, and conditions which theoretically should yield a constant net heat loss at the column's wall. The results show that using constant flow rates, programmed flow constant pressures, and constant heat loss at the column's wall all provide reproducible separations. However, performing separations using a high constant pressure with programmed flow reduces the analysis time by 16% compared to constant flow rate methods. For the constant flow rate, programmed flow constant pressure, and constant wall heat experiments no equilibration time (post-run time) was required to obtain highly reproducible data. Copyright © 2013 Elsevier B.V. All rights reserved.

A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins.

PubMed

Rho, Hoon Suk; Hanke, Alexander Thomas; Ottens, Marcel; Gardeniers, Han J G E

2018-04-01

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro-column, pH gradient generation, and fraction collection in a single device. Using a sieve micro-valve, anion exchange particles were packed into a microchannel in order to realize a solid-phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro-column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and R-Phycoerythrin (R-PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development. © 2018 The Authors Electrophoresis Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Separation of proteins by hydrophobic interaction chromatography at low salt concentration.

PubMed

Kato, Yoshio; Nakamura, Koji; Kitamura, Takashi; Moriyama, Hiroyuki; Hasegawa, Masazumi; Sasaki, Hiroo

2002-09-20

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.

Separation of five compounds from leaves of Andrographis paniculata (Burm. f.) Nees by off-line two-dimensional high-speed counter-current chromatography combined with gradient and recycling elution.

PubMed

Zhang, Li; Liu, Qi; Yu, Jingang; Zeng, Hualiang; Jiang, Shujing; Chen, Xiaoqing

2015-05-01

An off-line two-dimensional high-speed counter-current chromatography method combined with gradient and recycling elution mode was established to isolate terpenoids and flavones from the leaves of Andrographis paniculata (Burm. f.) Nees. By using the solvent systems composed of n-hexane/ethyl acetate/methanol/water with different volume ratios, five compounds including roseooside, 5,4'-dihydroxyflavonoid-7-O-β-d-pyranglucuronatebutylester, 7,8-dimethoxy-2'-hydroxy-5-O-β-d-glucopyranosyloxyflavon, 14-deoxyandrographiside, and andrographolide were successfully isolated. Purities of these isolated compounds were all over 95% as determined by high-performance liquid chromatography. Their structures were identified by UV, mass spectrometry, and (1) H NMR spectroscopy. It has been demonstrated that the combination of off-line two-dimensional high-speed counter-current chromatography with different elution modes is an efficient technique to isolate compounds from complex natural product extracts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic separation of proteins in space

NASA Technical Reports Server (NTRS)

Brown, R. K.

1976-01-01

Commercially available and synthetic wide range and short range ampholytes used in the isoelectric focusing of proteins was analyzed by ion exchange chromatography. A pH gradient over the pH range 3.8 to 11.0 was used to elute the ampholytes from a column of a sulfonated polystyrene resin. The wide range ampholytes were resolved into some 60 to 70 ninhydrin positive components. The recovery obtained with the method was quantitative. Acid short range ampholytes have approximately 35 components which elute readily from the ion exchange resin. Basic short range ampholytes gave about 50 components, most of which eluted at alkaline pH.

Application of linear pH gradients for the modeling of ion exchange chromatography: Separation of monoclonal antibody monomer from aggregates.

PubMed

Kluters, Simon; Wittkopp, Felix; Jöhnck, Matthias; Frech, Christian

2016-02-01

The mobile phase pH is a key parameter of every ion exchange chromatography process. However, mechanistic insights into the pH influence on the ion exchange chromatography equilibrium are rare. This work describes a mechanistic model capturing salt and pH influence in ion exchange chromatography. The pH dependence of the characteristic protein charge and the equilibrium constant is introduced to the steric mass action model based on a protein net charge model considering the number of amino acids interacting with the stationary phase. This allows the description of the adsorption equilibrium of the chromatographed proteins as a function of pH. The model parameters were determined for a monoclonal antibody monomer, dimer, and a higher aggregated species based on a manageable set of pH gradient experiments. Without further modification of the model parameters the transfer to salt gradient elution at fixed pH is demonstrated. A lumped rate model was used to predict the separation of the monoclonal antibody monomer/aggregate mixture in pH gradient elution and for a pH step elution procedure-also at increased protein loadings up to 48 g/L packed resin. The presented model combines both salt and pH influence and may be useful for the development and deeper understanding of an ion exchange chromatography separation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid column heating method for subcritical water chromatography.

PubMed

Fogwill, Michael O; Thurbide, Kevin B

2007-01-19

A novel resistive heating method is presented for subcritical water chromatography (SWC) that provides higher column heating rates than those conventionally obtained from temperature-programmed gas chromatography (GC) convection ovens. Since the polarity of water reduces dramatically with increasing temperature, SWC employs column heating to achieve gradient elution. As such, the rate at which the mobile phase is heated directly impacts the magnitude of such gradients applied in SWC. Data from the current study demonstrate that the maximum column heating rate attainable in a typical SWC apparatus (i.e. using a GC convection oven) is around 10 degrees C/min, even at instrument oven settings of over three times this value. Conversely, by wrapping the separation column with ceramic insulation and a resistively heated wire, the column heating rates are increased five-fold. As a result, elution times can be greatly decreased in SWC employing gradients. Separations of standard alcohol test mixtures demonstrate that the retention time of the latest eluting component decreases by 35 to 50% using the prototype method. Additionally, solute retention times in this mode deviate by less than 1% RSD over several trials, which compares very well to those obtained using a conventional GC convection oven. Results suggest that the developed method can be a useful alternative heating technique in SWC.

Effect of suppressor current intensity on the determination of glyphosate and aminomethylphosphonic acid by suppressed conductivity ion chromatography.

PubMed

Dimitrakopoulos, Ioannis K; Thomaidis, Nikolaos S; Megoulas, Nikolaos C; Koupparis, Michael A

2010-05-28

This paper presents the application of ion chromatography with electrolytic eluent generation and mobile phase suppression for the direct conductimetric detection of glyphosate and its degradation product aminomethylphosphonic acid (AMPA). The compounds were separated on a Dionex AS18 anion exchange column with a 12-40 mM KOH step gradient from 9 to 9.5 min. The effect of the suppressor current intensity on the electrostatic interaction of these amphoteric compounds with the suppressor cation exchange membranes was evaluated. A suppressor current gradient technique was proposed for the limitation of peak broadening and baseline noise, in order to improve method sensitivity and detectability. It was observed that residual sample carbonates co-eluted with AMPA when a large injection loop was installed for the low level determination of both compounds in natural waters. For this reason, glyphosate was isocratically eluted using 33 mM KOH in order to decrease analysis time within 10 min and a column clean up step using 100 mM KOH was used to ensure retention time reproducibility. The developed method was applied to the analysis of drinking and natural water and it was further successfully applied to orange samples with slight modifications. Instrumental LOD for glyphosate was 0.24 microg/L, while method LOD was 0.54 microg/L for spring waters and 0.01 mg/kg for oranges using a 1000 microL direct loop injection of the sample. Intra-day and inter-day precision (as %RSD) for water samples was 4.6% and 12% at a spiking level of 2 microg/L, and the recovery ranged from 64% to 88% depending on sample conductivity. For orange samples, the inter-day precision was 1.4% at a spiking level of 4.4 mg/kg, while overall recovery was 103%. The developed method is direct, fast, sensitive and relatively inexpensive, and could be used as an ideal fast screening tool for the monitoring of glyphosate residues in water and fruit samples. Copyright 2010 Elsevier B.V. All rights reserved.

Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography.

PubMed

Stoll, Dwight R; Sajulga, Ray W; Voigt, Bryan N; Larson, Eli J; Jeong, Lena N; Rutan, Sarah C

2017-11-10

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension ( 1 D) column into the second dimension ( 2 D) column leads to severe 2 D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1 D mobile phase overwhelms the 2 D column with each injection of 1 D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2 D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2 D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4-80μL), loop filling level (25-100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (-20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of -1.0 and -4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real injection profiles obtained from 2D-LC interface valves are used to simulate the introduction of samples into the 2 D column. These profiles are highly asymmetric - simulation using simple rectangular pulses leads to peak widths that are far too narrow under many conditions. We believe the simulation approach developed here will be useful for addressing practical questions in the development of 2D-LC methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of new types of stationary phases for fast liquid chromatographic applications.

PubMed

Fekete, Szabolcs; Fekete, Jeno; Ganzler, Katalin

2009-12-05

The performance of a narrow bore silica based monolith column (5 cm x 2 mm) was compared to 5 cm long narrow bore (internal diameter < or = 2.1 mm) columns, packed with shell particles (2.7 microm) and totally porous sub-2 microm particles (1.5 microm, 1.7 microm and 1.9 microm) in gradient and isocratic elution separations of steroids. The highest peak capacity could be achieved with the column packed with 1.5 microm totally porous particles. The columns packed with porous 1.7 microm and shell 2.7 microm particles showed very similar capacity. The monolith column provided the lowest capacity during gradient elution. The plate height (HETP) of the 2.7 microm Ascentis Express column was very similar to the HETP obtained with 1.5 microm and 1.7 microm totally porous particles. The Chromolith monolithic column displayed an efficiency that is comparable to that of columns packed with spherical particles having their diameter between 3 microm and 4 microm. A kinetic plot analysis is presented to compare the theoretical analysis speed of different separation media. At 200 bar, the monolith column provided the highest performance when the required plate number was higher than 5000 (N>5000), however the efficiency drifted off faster in the range of N<5000 than in the case of packed columns. If the possibility of maximum performance was utilized (1000 bar for sub-2 microm particles, 600 bar for shell particles and 200 bar for monolith column) the monolith column would provide the poorest efficiency, while the column, packed with 1.5 microm particles offered the shortest impedance time.

Very high pressure liquid chromatography using fully porous particles: quantitative analysis of fast gradient separations without post-run times.

PubMed

Stankovich, Joseph J; Gritti, Fabrice; Stevenson, Paul G; Beaver, Lois Ann; Guiochon, Georges

2014-01-10

Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, producing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were performed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time. Copyright © 2013 Elsevier B.V. All rights reserved.

HPLC of the Polypeptides in a Hydrolyzate of Egg-White Lysozyme. An Experiment for the Undergraduate Biochemistry Laboratory.

ERIC Educational Resources Information Center

Richardson, W. S., III; Burns, L.

1988-01-01

Describes a simple high-performance liquid chromatography experiment for undergraduate biochemistry laboratories. The experiment illustrates the separation of polypeptides by a step gradient elution using a single pump instrument with no gradient attachments. Discusses instrumentation, analysis, a sample preparation, and results. (CW)

Demonstration of a strategy for product purification by high-gradient magnetic fishing: recovery of superoxide dismutase from unconditioned whey.

PubMed

Meyer, Andrea; Hansen, Dennis B; Gomes, Cláudia S G; Hobley, Timothy J; Thomas, Owen R T; Franzreb, Matthias

2005-01-01

A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high-gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.15-0.6 mg L(-1)) in whey. The first part of the process design consisted of ligand screening in which metal chelate supports charged with copper(II) ions were found to be the most suitable. The second stage involved systematic and sequential optimization of conditions for the following steps: product adsorption, support washing, and product elution. Next, the capacity of a novel high-gradient magnetic separator (designed for biotechnological applications) for trapping and holding magnetic supports was determined. Finally, all of the above elements were assembled to deliver a HGMF process for the isolation of SOD from crude sweet whey, which consisted of (i) binding SOD using Cu2+ -charged magnetic metal chelator particles in a batch reactor with whey; (ii) recovery of the "SOD-loaded" supports by high-gradient magnetic separation (HGMS); (iii) washing out loosely bound and entrained proteins and solids; (iv) elution of the target protein; and (v) recovery of the eluted supports from the HGMF rig. Efficient recovery of SOD was demonstrated at approximately 50-fold increased scale (cf magnetic rack studies) in three separate HGMF experiments, and in the best of these (run 3) an SOD yield of >85% and purification factor of approximately 21 were obtained.

Analytical strategy for the assessment of the protein glycation status in uremic patients by high-performance liquid chromatography.

PubMed

Floridi, A; Trizza, V; Paolotti, P; Lucarelli, C

1999-06-18

We propose a newly integrated procedure for the analysis of furosine (early glycation product) and pentosidine (glycoxidation end-product) in plasma proteins and the simultaneous assessment of advanced glycation end-product (AGE) peptides and free pentosidine in plasma. In order to determine furosine and protein-linked pentosidine, plasma proteins were hydrolyzed in 8 M HCl and each analyte was purified by solid-phase extraction. Furosine was determined by ion-pair RP-HPLC methodology with isocratic elution and spectrophotometric detection at 280 nm and pentosidine by ion-pair RP-HPLC by using gradient elution and fluorimetric detection at 335/385 nm. To assess free pentosidine concentration and simultaneously evaluate the AGE peptides, an aliquot of plasma sample was diluted and ultrafiltered by using Centricon 10 M(r) < or = 10,000) ultrafiltration membranes. Free pentosidine and AGE peptides were analysed by ion-pair RP-HPLC, by using gradient elution and fluorimetric detection at 385 nm upon excitation at 335 nm. The HPLC methodology has been successfully used for the determination of glycation and glycoxidation protein status in uremic patients.

Investigating desorption during ethanol elution to improve the quality and antioxidant activity of xylo-oligosaccharides from corn stalk.

PubMed

Zhang, Jie; Wang, Yue-Hai; Wei, Quan-Yuan; Du, Xiao-Jia; Qu, Yong-Shui

2018-02-01

As the most representative of lignocellulosic materials, corn stalk (CS) will be a great candidate to produce xylo-oligosaccharides (XOS). Owing to the high impurity content of the XOS produced by directly enzymatic hydrolysis of xylan extracted from CS, subsequent refining steps are essential. The present study was aimed to investigate desorption during ethanol elution to improve the quality and antioxidant activity of XOS from CS. The desorption was systematically investigated after optimizing the elution conditions. The results showed that it had an elution watershed when the volume ratio was 2:1. More interestingly, XOS had a obvious priorities of desorption during ethanol gradient elution. The highest purity of XOS was 98.12% from 30% ethanol eluate. Antioxidant activity assay showed that the highest radical scavenging activity of XOS was 89.89% obtained from 70% ethanol eluate at a concentration of 3 mg/mL, which could be used in antioxidant food, feed additives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel linear and step-gradient counter-current chromatography for bio-guided isolation and purification of cytotoxic podophyllotoxins from Dysosma versipellis (Hance).

PubMed

Yang, Zhi; Liu, Xiaoman; Wang, Kuiwu; Cao, Xiaoji; Wu, Shihua

2013-03-01

Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treatment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, four podophyllotoxin-like lignans including 4'-demethylpodophyllotoxin (1), α-peltatin (2), podophyllotoxin (3), β-peltatin (4) as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter-current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step-gradient elution can provide better resolution and save more time for the separation of photophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high-resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model.

PubMed

Wittkopp, Felix; Peeck, Lars; Hafner, Mathias; Frech, Christian

2018-04-13

Process development and characterization based on mathematic modeling provides several advantages and has been applied more frequently over the last few years. In this work, a Donnan equilibrium ion exchange (DIX) model is applied for modelling and simulation of ion exchange chromatography of a monoclonal antibody in linear chromatography. Four different cation exchange resin prototypes consisting of weak, strong and mixed ligands are characterized using pH and salt gradient elution experiments applying the extended DIX model. The modelling results are compared with the results using a classic stoichiometric displacement model. The Donnan equilibrium model is able to describe all four prototype resins while the stoichiometric displacement model fails for the weak and mixed weak/strong ligands. Finally, in silico chromatogram simulations of pH and pH/salt dual gradients are performed to verify the results and to show the consistency of the developed model. Copyright © 2018 Elsevier B.V. All rights reserved.

Quality by Design in the development of hydrophilic interaction liquid chromatography method with gradient elution for the analysis of olanzapine.

PubMed

Tumpa, Anja; Stajić, Ana; Jančić-Stojanović, Biljana; Medenica, Mirjana

2017-02-05

This paper deals with the development of hydrophilic interaction liquid chromatography (HILIC) method with gradient elution, in accordance with Analytical Quality by Design (AQbD) methodology, for the first time. The method is developed for olanzapine and its seven related substances. Following step by step AQbD methodology, firstly as critical process parameters (CPPs) temperature, starting content of aqueous phase and duration of linear gradient are recognized, and as critical quality attributes (CQAs) separation criterion S of critical pairs of substances are investigated. Rechtschaffen design is used for the creation of models that describe the dependence between CPPs and CQAs. The design space that is obtained at the end is used for choosing the optimal conditions (set point). The method is fully validated at the end to verify the adequacy of the chosen optimal conditions and applied to real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

UHPLC-UV method for the determination of flavonoids in dietary supplements and for evaluation of their antioxidant activities.

PubMed

Magiera, Sylwia; Baranowska, Irena; Lautenszleger, Anna

2015-01-01

A simple and rapid ultra-high performance liquid chromatographic (UHPLC) method coupled with an ultraviolet detector (UV) has been developed and validated for the separation and determination of 14 major flavonoids ((±)-catechin, (-)-epicatechin, glycitin, (-)-epicatechin gallate, rutin, quercitrin, hesperidine, neohesperidine, daidzein, glycitein, quercetin, genistein, hesperetin, and biochanin A) in herbal dietary supplements. The flavonoids have been separated on a Chromolith Fast Gradient Monolithic RP-18e column utilizing a mobile phase composed of 0.05% trifluoroacetic acid in water and acetonitrile in gradient elution mode. Under these conditions, flavonoids were separated in a 5 min run. The selectivity of the developed UHPLC-UV method was confirmed by comparison with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The validation parameters such as linearity, sensitivity, precision, and accuracy were found to be highly satisfactory. The optimized method was applied to determination of flavonoids in different dietary supplements. Additionally, the developed HPLC-UV method combined with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was used in the evaluation of antioxidant activity of the selected flavonoids. Copyright © 2014 Elsevier B.V. All rights reserved.

Gradient Scouting in Reversed-Phase HPLC Revisited

ERIC Educational Resources Information Center

Alcazar, A.; Jurado, J. M.; Gonzalez, A. G.

2011-01-01

Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio [delta]t/t[subscript G] (where [delta]t is the difference in the retention time between the last and the first peak and t[subscript G] is…

Development of pseudo-linear gradient elution for high-throughput resin selectivity screening in RoboColumn® Format.

PubMed

Kiesewetter, André; Menstell, Peter; Peeck, Lars H; Stein, Andreas

2016-11-01

Rapid development of chromatographic processes relies on effective high-throughput screening (HTS) methods. This article describes the development of pseudo-linear gradient elution for resin selectivity screening using RoboColumns ® . It gives guidelines for the implementation of this HTS method on a Tecan Freedom EVO ® robotic platform, addressing fundamental aspects of scale down and liquid handling. The creation of a flexible script for buffer preparation and column operation plus efficient data processing provided the basis for this work. Based on the concept of discretization, linear gradient elution was transformed into multistep gradients. The impact of column size, flow rate, multistep gradient design, and fractionation scheme on separation efficiency was systematically investigated, using a ternary model protein mixture. We identified key parameters and defined optimal settings for effective column performance. For proof of concept, we examined the selectivity of several cation exchange resins using various buffer conditions. The final protocol enabled a clear differentiation of resin selectivity on miniature chromatography column (MCC) scale. Distinct differences in separation behavior of individual resins and the influence of buffer conditions could be demonstrated. Results obtained with the robotic platform were representative and consistent with data generated on a conventional chromatography system. A study on antibody monomer/high molecular weight separation comparing MCC and lab scale under higher loading conditions provided evidence of the applicability of the miniaturized approach to practically relevant feedstocks with challenging separation tasks as well as of the predictive quality for larger scale. A comparison of varying degrees of robotic method complexity with corresponding effort (analysis time and labware consumption) and output quality highlights tradeoffs to select a method appropriate for a given separation challenge or analytical constraints. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1503-1519, 2016. © 2016 American Institute of Chemical Engineers.

An automated method for the determination of deoxyribonuclease activity as exemplified by fractionation of the components of the medicament Varidase®

PubMed Central

Locke, I. C.; Ramsey, M. P.; Hill, S. S.; Carpenter, B. G.

1993-01-01

The activity of most deoxyribonuclease enzymes can be monitored by measuring the change in absorbance at 260 nm which accompanies the breakdown of the double-stranded structure of native DNA. An automated method for determining deoxyribonuclease activity, based on such an absorbance change, which can overcome problems of inhibition arising from the presence of inorganic cations, is described. Variations in inorganic cation concentration is a particular problem when measuring the activity of chromatographic fractions eluted via a salt gradient. A comparison is made between the automated and a manual method for the assay of deoxyribonuclease active constituents, of the medicament ‘Varidase’, eluted from a Cellex-D (Bio-Rad Laboratories Ltd) anionic exchange resin using a 0.05-1.0 M sodium chloride gradient. PMID:18924962

Numerical modeling of the elution peak profiles of retained solutes in supercritical fluid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaczmarski, Krzysztof; Guiochon, Georges A

2011-01-01

In supercritical fluid chromatography (SFC), the significant expansion of the mobile phase along the column causes the formation of axial and radial gradients of temperature. Due to these gradients, the mobile phase density, its viscosity, its velocity, its diffusion coefficients, etc. are not constant throughout the column. This results in a nonuniform flow velocity distribution, itself causing a loss of column efficiency in certain cases, even at low flow rates, as they do in HPLC. At high flow rates, an important deformation of the elution profiles of the sample components may occur. The model previously used to account satisfactorily formore » the retention of an unsorbed solute in SFC is applied to the modeling of the elution peak profiles of retained compounds. The numerical solution of the combined heat and mass balance equations provides the temperature and the pressure profiles inside the column and values of the retention time and the band profiles of retained compounds that are in excellent agreement with independent experimental data for large value of mobile phase reduced density. At low reduced densities, the band profiles can strongly depend on the column axial distribution of porosity.« less

Concentration and fractionation of hydrophobic organic acid constituents from natural waters by liquid chromatography

USGS Publications Warehouse

Thurman, E.M.; Malcolm, R.L.

1979-01-01

A scheme is presented which used adsorption chromatography with pH gradient elution and size-exclusion chromatography to concentrate and separate hydrophobic organic acids from water. A review of chromatographic processes involved in the flow scheme is also presented. Organic analytes which appear in each aqueous fraction are quantified by dissolved organic carbon analysis. Hydrophobic organic acids in a water sample are concentrated on a porous acrylic resin. These acids usually constitute approximately 30-50 percent of the dissolved organic carbon in an unpolluted water sample and are eluted with an aqueous eluent (dilute base). The concentrate is then passed through a column of polyacryloylmorpholine gel, which separates the acids into high- and low-molecular-weight fractions. The high- and low-molecular-weight eluates are reconcentrated by adsorption chromatography, then are eluted with a pH gradient into strong acids (predominately carboxylic acids) and weak acids (predominately phenolic compounds). For standard compounds and samples of unpolluted waters, the scheme fractionates humic substances into strong and weak acid fractions that are separated from the low molecular weight acids. A new method utilizing conductivity is also presented to estimate the acidic components in the methanol fraction.

Advantages of novel BioMimeTM Sirolimus Eluting Coronary Stent system. Moving towards biomimicry.

PubMed

Upendra, K; Sanjeev, B

2012-02-01

Since the first reported use of percutaneous transluminal coronary angioplasty (PTCA), advancements in interventional cardiology arena have been fast paced. Within the last ten years, these developments have been exponential. Developers & clinicians are fast adapting from the learning curve awarded by the time course of DES evolution. In that light BioMimeTM Sirolimus Eluting Coronary Stent comes as a fresh thought in taking stents towards a biomimicry concept. The stent is built on an ultra-low strut thickness (65 µm) cobalt chromium stent platform, using an intelligent hybrid of close and open cells allowing for morphology mediated expansion, employs a well known anti-proliferative - Sirolimus that elutes from a biodegradable co-polymer formulation in 30 days and ensures high coating integrity and low coating thickness of 2 µm. The resultant stent demonstrates almost 100% endothelialization at 30 days in preclinical model and zero percent MACE >18 months in the primary efficacy and safety clinical study.

A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish.

PubMed

Guidi, Letícia Rocha; Santos, Flávio Alves; Ribeiro, Ana Cláudia S R; Fernandes, Christian; Silva, Luiza H M; Gloria, Maria Beatriz A

2017-01-15

The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg -1 ), suggesting possible contamination or illegal use of that antibiotic. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.

PubMed

Zong, Shi-Yu; Han, Han; Wang, Bing; Li, Ning; Dong, Tina Ting-Xia; Zhang, Tong; Tsim, Karl W K

2015-12-04

A reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLC(TM) HSS C18 column (100 mm × 2.1 mm, 1.8 μm) with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970) within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%-117.3%, with RSD < 6.18%. The developed UHPLC-ESI-MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329-2057 µg/g.

Study on pharmacokinetics of 3,4-divanillyltetrahydrofuran in rats by ultra-fast liquid chromatography/tandem mass spectrometry.

PubMed

Shan, Chen-Xiao; Cui, Xiao-Bing; Yu, Sheng; Chai, Chuan; Wen, Hong-Mei; Wang, Xin-Zhi; Sun, Xue

2016-01-01

3,4-Divanillyltetrahydrofuran is the main active ingredient of nettle root which can increase steroid hormones in the bloodstream for many of bodybuilders. To better understand its pharmacological activities, we need to determine its pharmacokinetic profiles. In this study, a rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed for the determination of 3,4-divanillyltetrahydrofuran in the plasma of rats. Chromatographic separation was performed on a C18 column at 40°C, with a gradient elution consisting of methanol and water containing 0.3% (v/v) formic acid at a flow rate of 0.8mL/min. The detection was performed using an electrospray triple-quadrupole MS/MS via positive ion multiple reaction monitoring mode. The lower limits-of-quantification determined were 0.5ng/mL. The intra- and inter-day precision (RSD%) was found to be within 15% and the accuracy (RE%) ranged from -4.0% to 7.0%. This simple yet sensitive method was fully validated and could be successfully applied to the study on pharmacokinetics of 3, 4-divanillyltetrahydrofuran. Copyright © 2015 Elsevier B.V. All rights reserved.

Ion chromatographic methods for the detection of starch hydrolysis products in ruminal digesta.

PubMed

Barsuhn, K; Kotarski, S F

1991-06-21

Dionex high-performance ion chromatographic methods were evaluated for separation and quantitation of plant sugars and starch digestion products in the ruminal digesta of cattle. Mono- and disaccharides were eluted from a Dionex CarboPac PA1 column with sodium hydroxide used isocratically or as a pH gradient. Maltooligosaccharides which had a degree of polymerization (DP) less than 30 glucose residues were eluted in 60 min by a sodium hydroxide eluent containing a sodium acetate gradient. Carbohydrates were detected amperometrically. Responses were linear (r2 greater than 0.99) for glucose, disaccharides and maltooligosaccharides (DP less than 8). Precipitation and solid-phase extraction methods were evaluated for clean-up of samples of feedstuffs, ruminal contents, and bacterial culture fluids. Perchloric acid precipitation hydrolyzed sucrose but did not affect recoveries of cellobiose, isomaltose or maltose. Ethanol in concentrations of 79 and 86% precipitated maltooligosaccharides having chain lengths larger than 14 and 9 glucose residues, respectively. Maltooligosaccharide recoveries from solid-phase extraction columns varied with maltooligosaccharide size and column packing. Recoveries were greater than 94% for short chains (DP less than 6) eluted from phenyl-substituted columns and variable for all oligosaccharides eluted from C18 columns. Applications of these methods are presented and include: (1) detection of sugars in ruminant feed, (2) monitoring changes in ruminal sugars after feeding and (3) monitoring changes in extracellular sugars and oligosaccharides in the culture fluids of the ruminal bacterium, Bacteroides ruminicola.

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection.

PubMed

Liu, E-Hu; Liu, Qun; Chu, Chu; Li, Ping

2011-10-01

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

PubMed

Di Rago, Matthew; Saar, Eva; Rodda, Luke N; Turfus, Sophie; Kotsos, Alex; Gerostamoulos, Dimitri; Drummer, Olaf H

2014-10-01

The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Hydroxylapatite chromatography.

PubMed

Broadhurst, A V

2001-05-01

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.

STUDIES ON THE DISTRIBUTION AND PHOSPHATE TURNOVER OF THE ACID-SOLUBLE PHOSPHORUS COMPOUNDS IN VARIOUS NORMAL AND NEOPLASTIC TISSUES OF RATS. I. A SEMI-MICRO GRADIENT ELUTION CHROMATOGRAPHY WITH DOWEX FORMATE AND IDENTIFICATION OF SOME PHOSPHATE ESTERS ON THE CHROMATOGRAM (ENGLISH TEXT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horie, S.; Terada, S.

A modified semi-micro gradient elution chromatography for the analysis of tissue acid-soluble phosphorus compounds is described. One to 3 g of tissue can be analyzed by this method. Liver, muscle, and thymus tissue of rats were analyzed and the chromatograms are illustrated. The distribution and turnover of the acid-soluble phosphorus compounds in rat liver were also studied by P/sup 32/ injection and the use of the semi-micro method. Glucose 6-phosphate and L- alpha -glycerophosphate were identified on the chromatogram, and a phosphorus compound containing aminoacid was separated from the dowex 1 formate non-adsorbable fraction. (Abstr. Japan Med., 1: No. 9,more » 1961)« less

Comprehensive two-dimensional chromatography with coupling of reversed phase high performance liquid chromatography and supercritical fluid chromatography.

PubMed

Stevenson, Paul G; Tarafder, Abhijit; Guiochon, Georges

2012-01-13

A 2D comprehensive chromatographic separation of blackberry sage fragrant oil was performed by using HPLC in the first dimension and SFC in the second. A C(18)-bonded silica column eluted with an ACN gradient was used in the HPLC dimension and an amino-bonded silica column eluted with ACN as a modifier in the SFC dimension. This 2D separation was completed in the off-line mode, the fractions from the HPLC column being collected and injected in the SFC column. The retention factors on the two columns have a -0.757 correlation coefficient. The method provides a practical peak capacity of 2400 in 280 min. The first eluted peaks in HPLC are the last ones eluted in SFC and vice versa. The results demonstrate that the coupling of an HPLC and an SFC separation have a great potential for 2D chromatographic separations. Copyright © 2011 Elsevier B.V. All rights reserved.

Application of quality by design concept to develop a dual gradient elution stability-indicating method for cloxacillin forced degradation studies using combined mixture-process variable models.

PubMed

Zhang, Xia; Hu, Changqin

2017-09-08

Penicillins are typical of complex ionic samples which likely contain large number of degradation-related impurities (DRIs) with different polarities and charge properties. It is often a challenge to develop selective and robust high performance liquid chromatography (HPLC) methods for the efficient separation of all DRIs. In this study, an analytical quality by design (AQbD) approach was proposed for stability-indicating method development of cloxacillin. The structures, retention and UV characteristics rules of penicillins and their impurities were summarized and served as useful prior knowledge. Through quality risk assessment and screen design, 3 critical process parameters (CPPs) were defined, including 2 mixture variables (MVs) and 1 process variable (PV). A combined mixture-process variable (MPV) design was conducted to evaluate the 3 CPPs simultaneously and a response surface methodology (RSM) was used to achieve the optimal experiment parameters. A dual gradient elution was performed to change buffer pH, mobile-phase type and strength simultaneously. The design spaces (DSs) was evaluated using Monte Carlo simulation to give their possibility of meeting the specifications of CQAs. A Plackett-Burman design was performed to test the robustness around the working points and to decide the normal operating ranges (NORs). Finally, validation was performed following International Conference on Harmonisation (ICH) guidelines. To our knowledge, this is the first study of using MPV design and dual gradient elution to develop HPLC methods and improve separations for complex ionic samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components.

PubMed

Xiang, Rong; Horváth, Csaba

2002-02-15

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.

[Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein].

PubMed

Li, Wan-yi; Kuang, Yu; Li, Ming-yuan; Yang, Yuan; Jiang, Zhong-hua; Yao, Feng; Chen, Chang-chun

2007-12-01

To optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae. Hap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis. The Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths. The pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.

Method development and application of offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis for comprehensive characterization of the saponins from Xueshuantong Injection.

PubMed

Yang, Wenzhi; Zhang, Jingxian; Yao, Changliang; Qiu, Shi; Chen, Ming; Pan, Huiqin; Shi, Xiaojian; Wu, Wanying; Guo, Dean

2016-09-05

Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chinese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated. We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically characterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36 saponin reference standards, high-accuracy MS and Fast-DDA-MS(2) data, elemental composition (C<80, H<120, O<50), double-bond equivalent (DBE 5-15), and searching an in-house library of Panax notoginseng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chemical composition of XSTI was achieved. The results obtained would benefit better understanding of the therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous products. Copyright © 2016. Published by Elsevier B.V.

Hyphenation of supercritical fluid chromatography with tandem mass spectrometry for fast determination of four aflatoxins in edible oil.

PubMed

Lei, Fang; Li, Chenglong; Zhou, Shuang; Wang, Dan; Zhao, Yunfeng; Wu, Yongning

2016-08-01

Aflatoxins (AFTs) are of great concern all over the world. Supercritical fluid chromatography (SFC) has the advantage of fast, high resolution and excellent compatibility with a broad range of organic solvents and samples, thus hyphenating SFC with tandem mass spectrometry (MS/MS) can be used for the easy and fast determination of AFTs in edible oils. Edible oil was spiked with isotope-labeled aflatoxin standards, diluted with hexane and extracted with acetonitrile. The extraction was directly loaded to an SFC apparatus and separated on a UPC(2) 2-EP column with CO2 -methanol gradient elution. A post-column make-up flow was introduced to facilitate mass spectrometry performance, and the mixture was analyzed by MS/MS with an electrospray ionization (ESI) source. The SFC conditions including separation column, modifier and sample solvent were optimized, and the four target aflatoxins were baseline separated. The ESI interface parameters were also investigated, implicating the make-up flow as a critical factor for sensitive determination by SFC-MS/MS. The LOQs for the AFTs were 0.05-0.12 μg L(-1) , while the RSDs were lower than 8.5%. Supercritical fluid chromatography was successfully coupled to tandem mass spectrometry to establish a simple, fast and sensitive method for the analysis of four aflatoxins in edible oil. This shows the combination of SFC-MS/MS has great potential in determination of trace contaminants in food. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of pharmaceutical and illicit drugs in oral fluid by ultra-high performance liquid chromatography-tandem mass spectrometry.

PubMed

Di Corcia, D; Lisi, S; Pirro, V; Gerace, E; Salomone, A; Vincenti, M

2013-05-15

A simple and extremely fast procedure for the quantitative determination in oral fluid samples of 44 substances, including the most common drugs of abuse and several pharmaceutical drugs, was developed and fully validated. Preliminary sample treatment was limited to protein precipitation. The resulting acetonitrile solution was directly injected into an ultra-high performance liquid chromatograph (UHPLC) equipped with a C18 column (100mm×2.1mm, 1.7μm). The mobile phase eluted with linear gradient (water/formic acid 5mM: acetonitrile/formic acid 5mM; v:v) from 98:2 to 0:100 in 5.0min, followed by isocratic elution at 100% B for 1.0min. The flow rate was 0.6mL/min and the total run time was 9.0min including re-equilibration at the initial conditions. The analytes were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The method proved to be simple, accurate, rapid and highly sensitive, allowing the simultaneous detection of all compounds. The ease of sample treatment, together with the wide range of detectable substances, all with remarkable analytical sensitivity, make this procedure ideal for the screening of large populations in several forensic and clinical contexts, whenever oral fluid sampling has to be preferred to blood sampling, as for example in short retrospective investigations. Copyright © 2013 Elsevier B.V. All rights reserved.

Fast "hyperlayer" separation development in sedimentation field flow fractionation.

PubMed

Kassab, James R; Cardot, Philippe J P; Zahoransky, Richard A; Battu, Serge

2005-11-05

Specific prototypes of sedimentation field flow fractionation devices (SdFFF) have been developed with relative success for cell sorting. However, no data are available to compare these apparatus with commercial ones. In order to compare with other devices mainly used for non-biological species, biocompatible systems were used for standard particle (latex: 3-10 microm of different size dispersities) separation development. In order to enhance size dependent separations, channels of reduced thickness were used (80 and 100 microm) and channel/carrier-phase equilibration procedures were necessary. For sample injection, the use of inlet tubing linked to the FFF accumulation wall, common for cell sorting, can be extended to latex species when they are eluted in the Steric Hyperlayer elution mode. It avoids any primary relaxation steps (stop flow injection procedure) simplifying series of elution processing. Mixtures composed of four different monodispersed latex beads can be eluted in 6 min with 100 microm channel thickness.

Optimal conditions for elution of hepatitis B antigen after absorption onto colloidal silica.

PubMed Central

Pillot, J; Goueffon, S; Keros, R G

1976-01-01

Hepatitis B surface antigen (HBSAg) adsorbed from sera onto colloidal silica could be completely eluted through the use of 0.25% sodium deoxycholate in 0.01 M borax, pH 9.3, at 56 degrees C. The HBSAg recovered in the eluate represented 100% of that present in the original serum, and it was contaminated by only trace amounts of serum proteins (in decreasing amounts: beta-lipoprotein, immunoglobulin G, albumin). This preliminary step greatly facilitates purification of large amounts of HBSAg and provides small volumes of highly concentrated material for subsequent purification by density gradient centrifugation. PMID:9423

Optimized, Fast-Throughput UHPLC-DAD Based Method for Carotenoid Quantification in Spinach, Serum, Chylomicrons, and Feces.

PubMed

Eriksen, Jane N; Madsen, Pia L; Dragsted, Lars O; Arrigoni, Eva

2017-02-01

An improved UHPLC-DAD-based method was developed and validated for quantification of major carotenoids present in spinach, serum, chylomicrons, and feces. Separation was achieved with gradient elution within 12.5 min for six dietary carotenoids and the internal standard, echinenone. The proposed method provides, for all standard components, resolution > 1.1, linearity covering the target range (R > 0.99), LOQ < 0.035 mg/L, and intraday and interday RSDs < 2 and 10%, respectively. Suitability of the method was tested on biological matrices. Method precision (RSD%) for carotenoid quantification in serum, chylomicrons, and feces was below 10% for intra- and interday analysis, except for lycopene. Method accuracy was consistent with mean recoveries ranging from 78.8 to 96.9% and from 57.2 to 96.9% for all carotenoids, except for lycopene, in serum and feces, respectively. Additionally, an interlaboratory validation study on spinach at two institutions showed no significant differences in lutein or β-carotene content, when evaluated on four occasions.

Development of a fast liquid chromatography-tandem mass spectrometry method for the determination of endocrine-disrupting compounds in waters.

PubMed

Di Carro, Marina; Scapolla, Carlo; Liscio, Camilla; Magi, Emanuele

2010-09-01

A fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed to study five endocrine-disrupting compounds (4-n-nonylphenol, bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol) in water. Different columns were tested; the chromatographic separation of the analytes was optimized on a Pinnacle DB biphenylic column with a water-acetonitrile gradient elution, which allowed the separation of the selected endocrine-disrupting compounds (EDCs) in less than 6 min. Quantitative analysis was performed in selected reaction monitoring (SRM) mode; two transitions were chosen for each compound, using the most abundant for quantitation. Calibration curves using bisphenol A-d (16) as internal standard were drawn, showing good correlation coefficients (0.9993-0.9998). All figures of merit of the method were satisfactory; limits of detection were in the low pg range for all analytes. The method was then applied to the determination of the analytes in real water samples: to this aim, polar organic chemical integrative samplers (POCIS) were deployed in the influent and in the effluent of a drinking water treatment plant in Liguria (Italy). The EDC level was rather low in the influent and negligible in the outlet, reflecting the expected function of the treatment plant.

Elution of macrophage-bound immunoglobulins by temperature changes in vitro

PubMed Central

Tizard, I. R.

1972-01-01

Some mouse anti-sheep-erythrocyte macrophage cytophilic antibodies are more readily adsorbed to cells at 4° than at 37°. By treating cells with serum at 4°, washing them well and eluting bound material at 30°, it was found possible to isolate cytophilic immunoglobulins. These were found to have fast γ-mobility, but were heterogeneous upon preparative ultracentrifugation and gel-filtration. The macrophage eluate contained relatively high titres of anti-sheep-erythrocyte cytophilic antibodies. ImagesFIG. 1 PMID:5013911

Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

PubMed Central

Hey, Y; Dean, P D

1983-01-01

1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B. Images Fig. 1. PMID:6847623

Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

PubMed

Hey, Y; Dean, P D

1983-02-01

1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography.

PubMed

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Xu, Chunming; Ito, Yoichiro

2011-03-01

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, ¹HNMR and ¹³CNMR. Copyright © 2011 Elsevier B.V. All rights reserved.

Systematical Optimization of Reverse-phase Chromatography for Shotgun Proteomics

PubMed Central

Xu, Ping; Duong, Duc M.; Peng, Junmin

2009-01-01

Summary We report the optimization of a common LC/MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 μm internal diameter × 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC). The peptide mix was analyzed on a highly reproducible, automated nanoLC/MS/MS system with systematic adjustment of loading amount, flow rate, elution gradient range and length. Interestingly, the column was found to be almost saturated by loading ~1 μg of the sample. Whereas the optimal flow rate (~0.2 μl/min) and elution buffer range (13–32% of acetonitrile) appeared to be independent of the loading amount, the best gradient length varied according to the amount of samples: 160 min for 1 μg of the peptide mix, but 40 min for 10 ng of the same sample. The effect of these parameters on elution peptide peak width is evaluated. After full optimization, 1,012 proteins (clustered in 806 groups) with an estimated protein false discovery rate of ~3% were identified in 1 μg of yeast lysate in a single 160-min LC/MS/MS run. PMID:19566079

Extraction and preparation of high-aroma and low-caffeine instant green teas by the novel column chromatographic extraction method with gradient elution.

PubMed

Li, Qing-Rong; Wu, Min; Huang, Rui-Jie; Chen, Ya-Fei; Chen, Chan-Jian; Li, Hui; Ni, He; Li, Hai-Hang

2017-06-01

The lack of aroma and natural taste is a critical problem in production and consumption of instant green teas. A method to prepare instant green teas high in-natural-aroma and low-caffeine by the novel column chromatographic extraction with gradient elution is reported. This method simultaneously extracted aroma (or volatile) and non-aroma compounds from green tea. Green tea was loaded into columns with 2.0-fold of petroleum ether (PE): ethanol (8:2). After standing for 3 h until the aroma compounds dissolved, the column was sequentially eluted with 3.0-fold 40% ethanol and 3.5-fold water. The eluant was collected together and automatically separated into PE and ethanol aqueous phases. The aroma extracts was obtained by vacuum-evaporation of PE phase at 45 °C. The ethanol aqueous phase was vacuum-concentrated to aqueous and partially or fully decaffeinated with 4% or 9% charcoal at 70 °C. A regular instant green tea with epigallocatechin-3-gallate: caffeine of 3.5:1 and a low-caffeine instant green tea (less than 1% caffeine) with excellent aroma and taste were prepared, by combining the aroma and non-aroma extracts at a 1:10 ratio. This work provides a practical approach to solve the low-aroma and low-taste problems in the production of high quality instant green teas.

Centrifugal precipitation chromatography

PubMed Central

Ito, Yoichiro; Lin, Qi

2009-01-01

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. The countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation. PMID:19541553

Isolation and purification of series bioactive components from Hypericum Perforatum L. by high-speed counter-current chromatography

PubMed Central

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Ito, Yiochiro

2011-01-01

High-speed counter-current chromatography (HSCCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate–water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR. PMID:21306961

Chemical composition separation of a propylene-ethylene random copolymer by high temperature solvent gradient interaction chromatography.

PubMed

Liu, Yonggang; Phiri, Mohau Justice; Ndiripo, Anthony; Pasch, Harald

2017-11-03

A propylene-ethylene random copolymer was fractionated by preparative temperature rising elution fractionation (TREF). The structural heterogeneity of the bulk sample and its TREF fractions was studied by high temperature liquid chromatography with a solvent gradient elution from 1-decanol to 1,2,4-trichlorobenzene. HPLC alone cannot resolve those propylene-ethylene copolymers with high ethylene content in the bulk sample, due to their low weight fractions in the bulk sample and a small response factor of these components in the ELSD detector, as well as their broad chemical composition distribution. These components can only be detected after being separated and enriched by TREF followed by HPLC analysis. Chemical composition separations were achieved for TREF fractions with average ethylene contents between 2.1 and 22.0mol%, showing that copolymers with higher ethylene contents were adsorbed stronger in the Hypercarb column and eluted later. All TREF fractions, except the 40°C fraction, were relatively homogeneous in both molar mass and chemical composition. The 40°C fraction was rather broad in both molar mass and chemical composition distributions. 2D HPLC showed that the molar masses of the components containing more ethylene units were getting lower for the 40°C fraction. HPLC revealed and confirmed that co-crystallization influences the separation in TREF of the studied propylene-ethylene copolymer. Copyright © 2017 Elsevier B.V. All rights reserved.

Characteristic of theophylline imprinted monolithic column and its application for determination of xanthine derivatives caffeine and theophylline in green tea.

PubMed

Sun, Han-wen; Qiao, Feng-xia; Liu, Guang-yu

2006-11-17

Theophylline imprinted monolithic columns were designed and prepared for rapid separation of a homologous series of xanthine derivatives, caffeine, and theophylline by an in situ thermal-initiated copolymerization technique. Caffeine and theophylline were fully separated both under isocratic and gradient elutions on this kind of monolithic molecularly imprinted polymers (MIP) column. The broad peak showed in isocratic elution could be improved in gradient elution. Some chromatographic conditions such as mobile phase composition, flow rate, and the temperature on the retention times were investigated. Hydrogen bonding interaction and hydrophobic interaction played an important role in the retention and separation. The binding capacity was evaluated by static adsorption and Scatchard analysis, which showed that the dissociation constant (KD) and the maximum binding capacity (Qmax) were 1.50 mol/L, and 236 micromol/g for high affinity binding site, and 7.97 mol/L and 785 micromol/g for lower affinity binding site, respectively. Thermodynamic data (DeltaDeltaH and DeltaDeltaS) obtained by Van't Hoff plots revealed an enthalpy-controlled separation. The morphological characteristics of monolithic MIP were investigated by scanning electron microscope, which showed that both mesopores and macropores were formed in the monolith. The present monolithic MIP column was successfully applied for the quantitative determination of caffeine and theophylline in different kinds of green tea.

Porous polymer monolithic columns with gold nanoparticles as an intermediate ligand for the separation of proteins in reverse phase-ion exchange mixed mode

DOE PAGES

Terborg, Lydia; Masini, Jorge C.; Lin, Michelle; ...

2014-11-04

A new approach has been developed for the preparation of mixed-mode stationary phases to separate proteins. The pore surface of monolithic poly(glycidyl methacrylate- co-ethylene dimethacrylate) capillary columns was functionalized with thiols and coated with gold nanoparticles. The final mixed mode surface chemistry was formed by attaching, in a single step, alkanethiols, mercaptoalkanoic acids, and their mixtures on the free surface of attached gold nanoparticles. Use of these mixtures allowed fine tuning of the hydrophobic/hydrophilic balance. The amount of attached gold nanoparticles according to thermal gravimetric analysis was 44.8 wt.%. This value together with results of frontal elution enabled calculation ofmore » surface coverage with the alkanethiol and mercaptoalkanoic acid ligands. Interestingly, alkanethiols coverage in a range of 4.46–4.51 molecules/nm 2 significantly exceeded that of mercaptoalkanoic acids with 2.39–2.45 molecules/nm 2. The mixed mode character of these monolithic stationary phases was for the first time demonstrated in the separations of proteins that could be achieved in the same column using gradient elution conditions typical of reverse phase (using gradient of acetonitrile in water) and ion exchange chromatographic modes (applying gradient of salt in water), respectively.« less

Application of retention modelling to the simulation of separation of organic anions in suppressed ion chromatography.

PubMed

Zakaria, Philip; Dicinoski, Greg W; Ng, Boon Khing; Shellie, Robert A; Hanna-Brown, Melissa; Haddad, Paul R

2009-09-18

The ion-exchange separation of organic anions of varying molecular mass has been demonstrated using ion chromatography with isocratic, gradient and multi-step eluent profiles on commercially available columns with UV detection. A retention model derived previously for inorganic ions and based solely on electrostatic interactions between the analytes and the stationary phase was applied. This model was found to accurately describe the observed elution of all the anions under isocratic, gradient and multi-step eluent conditions. Hydrophobic interactions, although likely to be present to varying degrees, did not limit the applicability of the ion-exchange retention model. Various instrumental configurations were investigated to overcome problems associated with the use of organic modifiers in the eluent which caused compatibility issues with the electrolytically derived, and subsequently suppressed, eluent. The preferred configuration allowed the organic modifier stream to bypass the eluent generator, followed by subsequent mixing before entering the injection valve and column. Accurate elution prediction was achieved even when using 5-step eluent profiles with errors in retention time generally being less than 1% relative standard deviation (RSD) and all being less than 5% RSD. Peak widths for linear gradient separations were also modelled and showed good agreement with experimentally determined values.

MRI of gallstones with different compositions.

PubMed

Tsai, Hong-Ming; Lin, Xi-Zhang; Chen, Chiung-Yu; Lin, Pin-Wen; Lin, Jui-Che

2004-06-01

Gallstones are usually recognized on MRI as filling defects of hypointensity. However, they sometimes may appear as hyperintensities on T1-weighted imaging. This study investigated how gallstones appear on MRI and how their appearance influences the detection of gallstones. Gallstones from 24 patients who had MRI performed before the removal of the gallstones were collected for study. The gallstones were classified either as cholesterol gallstone (n = 4) or as pigment gallstone (n = 20) according to their gross appearance and based on analysis by Fourier transform infrared spectroscopy. MRI included three sequences: single-shot fast spin-echo T2-weighted imaging, 3D fast spoiled gradient-echo T1-weighted imaging, and in-phase fast spoiled gradient-echo T1-weighted imaging. The signal intensity and the detection rate of gallstones on MRI were further correlated with the character of the gallstones. On T1-weighted 3D fast spoiled gradient-echo images, most of the pigment gallstones (18/20) were hyperintense and all the cholesterol gallstones (4/4) were hypointense. The mean ratio of the signal intensity of gallstone to bile was (+/- standard deviation) 3.36 +/- 1.88 for pigment gallstone and 0.24 +/- 0.10 for cholesterol gallstone on the 3D fast spoiled gradient-echo sequence (p < 0.001). Combining the 3D fast spoiled gradient-echo and single-shot fast spin-echo sequences achieved the highest gallstone detection rate (96.4%). Based on the differences of signal intensity of gallstones, the 3D fast spoiled gradient-echo T1-weighted imaging was able to diagnose the composition of gallstones. Adding the 3D fast spoiled gradient-echo imaging to the single-shot fast spin-echo T2-weighted sequence can further improve the detection rate of gallstones.

High Performance Liquid Chromatography of Some Analgesic Compounds: An Instrumental Analysis Experiment.

ERIC Educational Resources Information Center

Haddad, Paul; And Others

1983-01-01

Background information, procedures, and results are provided for an experiment demonstrating techniques of solvent selection, gradient elution, pH control, and ion-pairing in the analysis of an analgesic mixture using reversed-phase liquid chromatography on an octadecylsilane column. Although developed using sophisticated/expensive equipment, less…

Stochastic approach for an unbiased estimation of the probability of a successful separation in conventional chromatography and sequential elution liquid chromatography.

PubMed

Ennis, Erin J; Foley, Joe P

2016-07-15

A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20-300, and 0.017-1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width ("gradient" conditions) and (ii) peak width increasing linearly with time ("isocratic/constant N" conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200-2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128-142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approach

Different elution modes and field programming in gravitational field-flow fractionation. III. Field programming by flow-rate gradient generated by a programmable pump.

PubMed

Plocková, J; Chmelík, J

2001-05-25

Gravitational field-flow fractionation (GFFF) utilizes the Earth's gravitational field as an external force that causes the settlement of particles towards the channel accumulation wall. Hydrodynamic lift forces oppose this action by elevating particles away from the channel accumulation wall. These two counteracting forces enable modulation of the resulting force field acting on particles in GFFF. In this work, force-field programming based on modulating the magnitude of hydrodynamic lift forces was implemented via changes of flow-rate, which was accomplished by a programmable pump. Several flow-rate gradients (step gradients, linear gradients, parabolic, and combined gradients) were tested and evaluated as tools for optimization of the separation of a silica gel particle mixture. The influence of increasing amount of sample injected on the peak resolution under flow-rate gradient conditions was also investigated. This is the first time that flow-rate gradients have been implemented for programming of the resulting force field acting on particles in GFFF.

Quantitative and pattern recognition analyses of magnoflorine, spinosin, 6'''-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen.

PubMed

Kim, Won Il; Zhao, Bing Tian; Zhang, Hai Yan; Lee, Je Hyun; Son, Jong Keun; Woo, Mi Hee

2014-01-01

Two rapid and simple HPLC methods with UV detector to determine three main compounds (magnoflorine, spinosin and 6'''-feruloyl spinosin) and evaporative light scattering detector (ELSD) to determine jujuboside A were developed for the chemical analyses of Zizyphi Semen. Magnoflorine, spinosin, and 6'''-feruloyl spinosin were separated with an YMC J'sphere ODS-H80 column (250 mm × 4.6 mm, 4 μm) by the gradient elution followed by the isocratic elution using methanol with 0.1 % formic acid and water with 0.1 % formic acid as the mobile phase. The flow rate was 1.0 mL/min. Jujuboside A was separated by HPLC-ELSD with YoungJinBioChrom Aegispak C18-L column (250 mm × 4.6 mm, 5 μm) column in a gradient elution using methanol with 0.1 % formic acid (A) and water with 0.1 % formic acid as the mobile phase. These two methods were fully validated with respect to linearity, precision, accuracy, stability, and robustness. These HPLC methods were applied successfully to quantify four compounds in a Zizyphi Semen extract. The HPLC analytical methods were validated for pattern recognition analysis by repeated analysis of 91 seed samples corresponding to 48 Zizyphus jujuba var. spinosa (J01-J48) and 43 Zizyphus mauritiana (M01-M43). The results indicate that these methods are suitable for a quality evaluation of Zizyphi Semen.

An analytical method to screen for six thyreostatic drug residues in the thyroid gland and muscle tissues of food producing animals by liquid chromatography with ultraviolet absorption detection and liquid chromatography/mass spectrometry.

PubMed

Asea, Philip E; MacNeil, James D; Boison, Joe O

2006-01-01

A method was developed and validated to screen for residues of the thyreostatic drugs, tapazole (TAP), mercaptobenzimidazole (MBI), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PrTU), and phenylthiouracil (PhTU) in bovine, equine, ovine, and porcine thyroid and muscle tissues at concentrations > or = 5 ng/g using 2-methoxy-mercaptobenzimidazole (MeMBI) and dimethylthiouracil (DMTU) as internal standards. In this method, the drugs were solvent extracted from thyroid and muscle tissue and cleaned up on an amino-propyl solid-phase extraction (SPE) cartridge. The unretained fraction containing TAP and MBI and the internal standard, MeMBI, was collected as Fraction 1. The retained fraction containing TU, MTU, PrTU, PhTU, and the internal standard, DMTU, was eluted with 3% acetic acid-isopropanol as Fraction 2. Fraction 1 was further cleaned up on an alumina B SPE cartridge and analyzed by gradient elution on a C18 high-performance liquid chromatography (HPLC) column with ultraviolet detection at wavelengths of 255 and 300 nm. Fraction 2 was taken to dryness, derivatized with 4-chloro-7-nitrobenzo-2-furazan at pH 8, and analyzed by gradient elution on a C18 LC column with mass spectrometry (MS) detection. Any "presumptive positive" test results were submitted for further analysis by LC/MS/MS. The validated method was applied to the analysis of over 300 thyroid tissue samples.

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

PubMed Central

Philipson, Lennart

1961-01-01

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s 20 o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained. PMID:13735276

A NEW HPLC METHOD FOR SEPARATION OF PHYTOPLANKTON PIGMENTS IN NATURAL SAMPLES

EPA Science Inventory

A new high-performance liquid chromatographic (HPLC) method was developed to analyze, in a single run, most polar and non-polar chlorophylls and carotenoids from marine phytoplankton. The method is based on a reverse-phase amide C16 (RP-amide C16) column and an elution gradient o...

Distribution and postprandial release of porcine peptide YY.

PubMed

Adrian, T E; Bacarese-Hamilton, A J; Smith, H A; Chohan, P; Manolas, K J; Bloom, S R

1987-04-01

Peptide YY (PYY), a thirty-six amino acid intestinal hormonal peptide with a tyrosine residue at each end (hence YY as Y represents tyrosine in the new peptide nomenclature), was found throughout the gastrointestinal tract of the pig. Concentrations were very low in the foregut (antrum, 3.4 +/- 0.3 pmol/g; duodenum, 1.1 +/- 1.5 pmol/g), higher in the distal small intestine (ileum, 100 +/- 13 pmol/g) and very high in the large bowel (descending colon, 270 +/- 45 pmol/g). Peptide YY was found to circulate in plasma and concentrations rose substantially in response to eating (fasting, 138 +/- 15 pmol/l; postprandial, 263 +/- 21 pmol/l; P less than 0.001). There was a small but significant portal/arterial gradient in postprandial PYY levels. More than 90% of the immunoreactive PYY in gut extracts eluted, on gel permeation chromatography, in an identical position to pure PYY standard, but small amounts of higher molecular weight material, possibly precursors, were detected. In contrast, plasma from fasting pigs contained a large proportion (60-70%) of these large molecular forms. These findings suggest that the putative pro-PYY may be cleared more slowly from the circulation than the 36 amino acid hormonal peptide. The high concentrations of immunoreactive PYY in the circulation of the young pig may reflect a species difference between pig and man or may indicate an important role for PYY in the developing animal.

A new method of analysis of peroxydisulfate using ion chromatography and its application to the simultaneous determination of peroxydisulfate and other common inorganic ions in a peroxydisulfate matrix.

PubMed

Khan, Nymul E; Adewuyi, Yusuf G

2011-01-21

A new method for the determination of peroxydisulfate using ion chromatography has been developed. Elution of peroxydisulfate was effected by isocratic elution using 200 mM NaOH at 40°C. A modification of the method using gradient elution was able to simultaneously determine other common inorganic ions (nitrate, nitrite, sulfate and chloride) down to significantly low concentrations in a peroxydisulfate matrix. The relative standard deviations (RSD) were in the range of 0.5-5%, for peak areas and <0.2% for peak retention times. The recoveries were between 95% and 120% for a concentration range of about 0.5-42 ppm. The limit of detection for peroxydisulfate ion was 0.2 ppm and for the other ions were ≤2×10(-2) ppm. The calibration curves were linear with slope and intercepts close to 1 and 0, respectively. Copyright © 2010 Elsevier B.V. All rights reserved.

Simple, rapid, and environmentally friendly method for the separation of isoflavones using ultra-high performance supercritical fluid chromatography.

PubMed

Wu, Wenjie; Zhang, Yuan; Wu, Hanqiu; Zhou, Weie; Cheng, Yan; Li, Hongna; Zhang, Chuanbin; Li, Lulu; Huang, Ying; Zhang, Feng

2017-07-01

Isoflavones are natural substances that exhibit hormone-like pharmacological activities. The separation of isoflavones remains an analytical challenge because of their similar structures. We show that ultra-high performance supercritical fluid chromatography can be an appropriate tool to achieve the fast separation of 12 common dietary isoflavones. Among the five tested columns the Torus DEA column was found to be the most effective column for the separation of these isoflavones. The impact of individual parameters on the retention time and separation factor was evaluated. These parameters were optimized to develop a simple, rapid, and green method for the separation of the 12 target analytes. It only took 12.91 min using gradient elution with methanol as an organic modifier and formic acid as an additive. These isoflavones were determined with limit of quantitation ranging from 0.10 to 0.50 μg/mL, which was sufficient for reliable determination of various matrixes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Characteristic fingerprint analysis and determination of main components on Andrographis paniculata extract by UPLC].

PubMed

Zhang, Hui-ye; Xu, Xiao-fei; Huang, Yong; Wamg, De-qin; Deng, Qiao-hua

2014-06-01

To establish an analytical method for characteristic fingerprint and determination of main components of Andrographis paniculata Extract by UPLC. The chromatographic conditions were Waters ACQUITY UPLC BEH-C18 (2. 1 mm x 0 mm,1.7 μm)by gradient elute using acetonitrile-water as mobile phase(0 -2 min,20% ~ 25% A;2 ~ 5 min,25% ~ 35% A;5 ~ 7 min,35% A;7 ~10 min,35%~ 55% A) at a flow rate of 0. 5 min/mL,detecting wavelength at 220 nm. Results:Contents of the andrographolide, neoandrographolide, 14-deoxyandrographolide and 14-deoxy-l11,12-didehydroandrographolide had good resolution with the correlation coefficients exceed 0. 9999 and the average percent recovery lied in 97. 2% to 103.9%, RSD was less than 3.0% (n = 6). The chromatograms of Andrographis paniculata Extract shared seven common peaks in which four of them were recognized by reference standard with the similarities over 0. 9. It is a fast,accurate and validated method,and can be useful in quality evaluations of Andrographis paniculata Extract.

Liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of the irreversible BTK inhibitor ibrutinib and its dihydrodiol-metabolite in plasma and its application in mouse pharmacokinetic studies.

PubMed

Rood, Johannes J M; van Hoppe, Stephanie; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

2016-01-25

A validated simple, fast and sensitive bio-analytical assay for ibrutinib and its dihydrodiol metabolite in human and mouse plasma was set up. Sample preparation was performed by protein precipitation, and addition of the respective deuterated internal standards, followed by LC-MS/MS analysis. Separation was performed on a 3.5 μm particle-size, bridged ethylene hybrid column with gradient elution by 0.1% v/v formic acid and acetonitrile. The full eluate was transferred to an electrospray interface in positive ionization mode, and subsequently analyzed by a triple quadrupole mass spectrometer by selected reaction monitoring. The assay was validated in a 5-5000 ng/ml calibration range. Both ibrutinib and dihydrodiol-ibrutinib were deemed stable under refrigerated or frozen storage conditions. At room temperature, ibrutinib showed a not earlier described instability, and revealed rapid degradation at 37 °C. Finally, the assay was used for a pharmacokinetic study of plasma levels in treated FVB mice. Copyright © 2015 Elsevier B.V. All rights reserved.

Quinolones and tetracyclines in aquaculture fish by a simple and rapid LC-MS/MS method.

PubMed

Guidi, Letícia Rocha; Santos, Flávio Alves; Ribeiro, Ana Cláudia S R; Fernandes, Christian; Silva, Luiza H M; Gloria, Maria Beatriz A

2018-04-15

The determination of antimicrobials in aquaculture fish is important to ensure food safety. Therefore, simple and fast multiresidue methods are needed. A liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of 14 antimicrobials (quinolones and tetracyclines) in fish. Antimicrobials were extracted with trichloroacetic acid and chromatographic separation was achieved with a C18 column and gradient elution (water and acetonitrile). The method was validated (Decision 2002/657/EC) and it was fit for the purpose. Linearities were established in the matrix and the coefficients of determination were ≥0.98. The method was applied to Nile tilapia and rainbow trout (n = 29) and 14% of them contained enrofloxacin at levels above the limit of quantification (12.53-19.01 µg.kg -1 ) but below the maximum residue limit (100 µg.kg -1 ). Even though prohibited in Brazil and other countries, this antimicrobial reached fish. Measures are needed to ascertain the source of this compound to warrant human safety. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fast profiling of anthocyanins in wine by desorption nano-electrospray ionization mass spectrometry.

PubMed

Hartmanova, Lucie; Ranc, Vaclav; Papouskova, Barbora; Bednar, Petr; Havlicek, Vladimir; Lemr, Karel

2010-06-18

Desorption electrospray ionization (DESI) mass spectrometry appears to be a useful technique applicable in different areas (e.g. analysis of pharmaceuticals, identification of biologically active compounds in tissues, imaging mass spectrometry). Its modification termed desorption nano-electrospray (nano-DESI) was tested for analysis of anthocyanins. Acidifying of samples and acidic spray liquid (methanol:water=75:25 with 0.2% HCOOH) were essential for obtaining good quality spectra. Profiles of main anthocyanins in wine samples, two vintages (2005 and 2007) of three cultivars (Alibernet, Neronet and Rubinet), were successfully acquired. They were in agreement with results of LC/MS experiments (anthocyanins isolated by solid phase extraction were separated by mu-HPLC with gradient elution and detected by ESI-MS). Nano-DESI-MS data also allowed to determine ratio of two cultivars (Neronet and Rubinet) in their mixture and to detect coloring of wine by tenturier or elderberry extract. Detection of main anthocyanins in slices of wine grape, chokeberries and elderberries or in a wine stain on cotton fabric is also presented. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Performances of CN-columns for the analysis of γ-oryzanol and its p-coumarate and caffeate derivatives by normal phase HPLC and a validated method of quantitation.

PubMed

D'Ambrosio, Michele

2013-06-15

γ-Oryzanol is an important phytochemical used in pharmaceutical, alimentary and cosmetic preparations. The present article, for the first time, discloses the performances of NP-HPLC in separating γ-oryzanol components and develops a validated method for its routine quantification. The analysis is performed on a cyanopropyl bonded column using the hexane/MTBE gradient elution and UV detection at 325 nm. The method allows: the separation of steryl ferulate, p-coumarate and caffeate esters, the separation of cis- from trans-ferulate isomers, the splitting of steroid moieties into saturated and unsaturated at the side chain. The optimised method provides excellent linear response (R(2)=0.99997), high precision (RSD<1.0%) and satisfactory accuracy (R(∗)=70-86%). In conclusion, the established method presents the details of the procedure and the experimental conditions in order to achieve the required precision and instrumental accuracy. The method is fast and sensitive and it could be a suitable tool for quality assurance and determination of origin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation of isoelectrically pure cholera toxin for crystallization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spangler, B.D.; Westbrook, E.M.

1989-01-01

We have determined that the failure of cholera toxin to crystallize well results from its isoelectric heterogeneity, which is probably due to a post-translational process such as deamidation of its B subunit. Every sample of cholera toxin we have examined from commercial or academic suppliers has been heterogeneous; heterogeneous cholera toxin does not crystallize satisfactorily. We have overcome this problem by using ion-exchange fast protein liquid chromatography (FPLC) to obtain an isoelectrically homogeneous species of cholera toxin. Homogeneous cholera toxin crystallizes readily, forming single, nonmosaic crystals suitable for x-ray diffraction studies. For this process, protein was applied to a MonoQmore » ion-exchange column, then eluted with an isocratic low salt buffer followed by a linear salt gradient (0-100 mM NaCl). Column fractions were analyzed on isoelectric focusing gels, and those fractions containing the desired homogeneous species were pooled and concentrated. Crystals formed within 24 to 48 hours in a MOPS/PEG buffer, which made use of slow isoelectric precipitation to induce crystallization. 23 refs., 6 figs.« less

Validation of an ultra-fast UPLC-UV method for the separation of antituberculosis tablets.

PubMed

Nguyen, Dao T-T; Guillarme, Davy; Rudaz, Serge; Veuthey, Jean-Luc

2008-04-01

A simple method using ultra performance LC (UPLC) coupled with UV detection was developed and validated for the determination of antituberculosis drugs in combined dosage form, i. e. isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF). Drugs were separated on a short column (2.1 mm x 50 mm) packed with 1.7 mum particles, using an elution gradient procedure. At 30 degrees C, less than 2 min was necessary for the complete separation of the three antituberculosis drugs, while the original USP method was performed in 15 min. Further improvements were obtained with the combination of UPLC and high temperature (up to 90 degrees C), namely HT-UPLC, which allows the application of higher mobile phase flow rates. Therefore, the separation of ISN, PYR and RIF was performed in less than 1 min. After validation (selectivity, trueness, precision and accuracy), both methods (UPLC and HT-UPLC) have proven suitable for the routine quality control analysis of antituberculosis drugs in combined dosage form. Additionally, a large number of samples per day can be analysed due to the short analysis times.

Isocratic and gradient impedance plot analysis and comparison of some recently introduced large size core-shell and fully porous particles.

PubMed

Vanderheyden, Yoachim; Cabooter, Deirdre; Desmet, Gert; Broeckhoven, Ken

2013-10-18

The intrinsic kinetic performance of three recently commercialized large size (≥4μm) core-shell particles packed in columns with different lengths has been measured and compared with that of standard fully porous particles of similar and smaller size (5 and 3.5μm, respectively). The kinetic performance is compared in both absolute (plot of t0 versus the plate count N or the peak capacity np for isocratic and gradient elution, respectively) and dimensionless units. The latter is realized by switching to so-called impedance plots, a format which has been previously introduced (as a plot of t0/N(2) or E0 versus Nopt/N) and has in the present study been extended from isocratic to gradient elution (where the impedance plot corresponds to a plot of t0/np(4) versus np,opt(2)/np(2)). Both the isocratic and gradient impedance plot yielded a very similar picture: the clustered impedance plot curves divide into two distinct groups, one for the core-shell particles (lowest values, i.e. best performance) and one for the fully porous particles (highest values), confirming the clear intrinsic kinetic advantage of core-shell particles. If used around their optimal flow rate, the core-shell particles displayed a minimal separation impedance that is about 40% lower than the fully porous particles. Even larger gains in separation speed can be achieved in the C-term regime. Copyright © 2013 Elsevier B.V. All rights reserved.

Binding mechanisms for histamine and agmatine ligands in plasmid deoxyribonucleic acid purifications.

PubMed

Sousa, Ângela; Pereira, Patrícia; Sousa, Fani; Queiroz, João A

2014-10-31

Histamine and agmatine amino acid derivatives were immobilized into monolithic disks, in order to combine the specificity and selectivity of the ligand with the high mass transfer and binding capacity offered by monolithic supports, to purify potential plasmid DNA biopharmaceuticals. Different elution strategies were explored by changing the type and salt concentration, as well as the pH, in order to understand the retention pattern of different plasmids isoforms The pVAX1-LacZ supercoiled isoform was isolated from a mixture of pDNA isoforms by using NaCl increasing stepwise gradient and also by ammonium sulfate decreasing stepwise gradient, in both histamine and agmatine monoliths. Acidic pH in the binding buffer mainly strengthened ionic interactions with both ligands in the presence of sodium chloride. Otherwise, for histamine ligand, pH values higher than 7 intensified hydrophobic interactions in the presence of ammonium sulfate. In addition, circular dichroism spectroscopy studies revealed that the binding and elution chromatographic conditions, such as the combination of high ionic strength with extreme pH values can reversibly influence the structural stability of the target nucleic acid. Therefore, ascending sodium chloride gradients with pH manipulation can be preferable chromatographic conditions to be explored in the purification of plasmid DNA biopharmaceuticals, in order to avoid the environmental impact of ammonium sulfate. Copyright © 2014. Published by Elsevier B.V.

HPLC-UV Method for the Identification and Screening of Hydroquinone, Ethers of Hydroquinone and Corticosteroids Possibly Used as Skin-Whitening Agents in Illicit Cosmetic Products.

PubMed

Gimeno, Pascal; Maggio, Annie-Françoise; Bancilhon, Marjorie; Lassu, Nelly; Gornes, Hervé; Brenier, Charlotte; Lempereur, Laurent

2016-03-01

Corticosteroids, hydroquinone and its ethers are regulated in cosmetics by the Regulation 1223/2009. As corticosteroids are forbidden to be used in cosmetics and cannot be present as contaminants or impurities, an identification of one of these illicit compounds deliberately introduced in these types of cosmetics is enough for market survey control. In order to quickly identify skin-whitening agents present in illegal cosmetics, this article proposes an HPLC-UV method for the identification and screening of hydroquinone, 3 ethers of hydroquinone and 39 corticosteroids that may be found in skin-whitening products. Two elution gradients were developed to separate all compounds. The main solvent gradient (A) allows the separation of 39 compounds among the 43 compounds considered in 50 min. Limits of detection on skin-whitening cosmetics are given. For compounds not separated, a complementary gradient elution (B) using the same solvents is proposed. Between 2004 and 2009, a market survey on "skin-whitening cosmetic" was performed on 150 samples and highlights that more than half of the products tested do not comply with the Cosmetic Regulation 1223/2009 (amending the Council Directive 76/768/EEC). © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Phase-space dependent critical gradient behavior of fast-ion transport due to Alfvén eigenmodes

DOE PAGES

Collins, C. S.; Heidbrink, W. W.; Podestà, M.; ...

2017-06-09

Experiments in the DIII-D tokamak show that many overlapping small-amplitude Alfv en eigenmodes (AEs) cause fast-ion transport to sharply increase above a critical threshold, leading to fast-ion density profile resilience and reduced fusion performance. The threshold is above the AE linear stability limit and varies between diagnostics that are sensitive to different parts of fast-ion phase-space. A comparison with theoretical analysis using the nova and orbit codes shows that, for the neutral particle diagnostic, the threshold corresponds to the onset of stochastic particle orbits due to wave-particle resonances with AEs in the measured region of phase space. We manipulated themore » bulk fast-ion distribution and instability behavior through variations in beam deposition geometry, and no significant differences in the onset threshold outside of measurement uncertainties were found, in agreement with the theoretical stochastic threshold analysis. Simulations using the `kick model' produce beam ion density gradients consistent with the empirically measured radial critical gradient and highlight the importance of including the energy and pitch dependence of the fast-ion distribution function in critical gradient models. The addition of electron cyclotron heating changes the types of AEs present in the experiment, comparatively increasing the measured fast-ion density and radial gradient. Our studies provide the basis for understanding how to avoid AE transport that can undesirably redistribute current and cause fast-ion losses, and the measurements are being used to validate AE-induced transport models that use the critical gradient paradigm, giving greater confidence when applied to ITER.« less

Phase-space dependent critical gradient behavior of fast-ion transport due to Alfvén eigenmodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, C. S.; Heidbrink, W. W.; Podestà, M.

Experiments in the DIII-D tokamak show that many overlapping small-amplitude Alfv en eigenmodes (AEs) cause fast-ion transport to sharply increase above a critical threshold, leading to fast-ion density profile resilience and reduced fusion performance. The threshold is above the AE linear stability limit and varies between diagnostics that are sensitive to different parts of fast-ion phase-space. A comparison with theoretical analysis using the nova and orbit codes shows that, for the neutral particle diagnostic, the threshold corresponds to the onset of stochastic particle orbits due to wave-particle resonances with AEs in the measured region of phase space. We manipulated themore » bulk fast-ion distribution and instability behavior through variations in beam deposition geometry, and no significant differences in the onset threshold outside of measurement uncertainties were found, in agreement with the theoretical stochastic threshold analysis. Simulations using the `kick model' produce beam ion density gradients consistent with the empirically measured radial critical gradient and highlight the importance of including the energy and pitch dependence of the fast-ion distribution function in critical gradient models. The addition of electron cyclotron heating changes the types of AEs present in the experiment, comparatively increasing the measured fast-ion density and radial gradient. Our studies provide the basis for understanding how to avoid AE transport that can undesirably redistribute current and cause fast-ion losses, and the measurements are being used to validate AE-induced transport models that use the critical gradient paradigm, giving greater confidence when applied to ITER.« less

Effect of ionization suppression by trace impurities in mobile phase water on the accuracy of quantification by high-performance liquid chromatography/mass spectrometry.

PubMed

Herath, H M D R; Shaw, P N; Cabot, P; Hewavitharana, A K

2010-06-15

The high-performance liquid chromatography (HPLC) column is capable of enrichment/pre-concentration of trace impurities in the mobile phase during the column equilibration, prior to sample injection and elution. These impurities elute during gradient elution and result in significant chromatographic peaks. Three types of purified water were tested for their impurity levels, and hence their performances as mobile phase, in HPLC followed by total ion current (TIC) mode of MS. Two types of HPLC-grade water produced 3-4 significant peaks in solvent blanks while LC/MS-grade water produced no peaks (although peaks were produced by LC/MS-grade water also after a few days of standing). None of the three waters produced peaks in HPLC followed by UV-Vis detection. These peaks, if co-eluted with analyte, are capable of suppressing or enhancing the analyte signal in a MS detector. As it is not common practice to run solvent blanks in TIC mode, when quantification is commonly carried out using single ion monitoring (SIM) or single or multiple reaction monitoring (SRM or MRM), the effect of co-eluting impurities on the analyte signal and hence on the accuracy of the results is often unknown to the analyst. Running solvent blanks in TIC mode, regardless of the MS mode used for quantification, is essential in order to detect this problem and to take subsequent precautions. Copyright (c) 2010 John Wiley & Sons, Ltd.

Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application.

PubMed

Kröner, Frieder; Hubbuch, Jürgen

2013-04-12

pH gradient protein separations are widely used techniques in the field of protein analytics, of which isoelectric focusing is the most well known application. The chromatographic variant, based on the formation of pH gradients in ion exchange columns is only rarely applied due to the difficulties to form controllable, linear pH gradients over a broad pH range. This work describes a method for the systematic generation of buffer compositions with linear titration curves, resulting in well controllable pH gradients. To generate buffer compositions with linear titration curves an in silico method was successfully developed. With this tool, buffer compositions for pH gradient ion exchange chromatography with pH ranges spanning up to 7.5 pH units were established and successfully validated. Subsequently, the buffer systems were used to characterize the elution behavior of 22 different model proteins in cation and anion exchange pH gradient chromatography. The results of both chromatographic modes as well as isoelectric focusing were compared to describe differences in between the methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Quality by design: a systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column.

PubMed

Kalariya, Pradipbhai D; Kumar Talluri, Murali V N; Gaitonde, Vinay D; Devrukhakar, Prashant S; Srinivas, Ragampeta

2014-08-01

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality-by-design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters--column temperature, gradient time, and flow rate using eight experiments--to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused-core C18 (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A semi-automatic microextraction in packed sorbent, using a digitally controlled syringe, combined with ultra-high pressure liquid chromatography as a new and ultra-fast approach for the determination of prenylflavonoids in beers.

PubMed

Gonçalves, João L; Alves, Vera L; Rodrigues, Fátima P; Figueira, José A; Câmara, José S

2013-08-23

In this work a highly selective and sensitive analytical procedure based on semi-automatic microextraction by packed sorbents (MEPS) technique, using a new digitally controlled syringe (eVol(®)) combined with ultra-high pressure liquid chromatography (UHPLC), is proposed to determine the prenylated chalcone derived from the hop (Humulus lupulus L.), xanthohumol (XN), and its isomeric flavonone isoxanthohumol (IXN) in beers. Extraction and UHPLC parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. Important parameters affecting MEPS performance, namely the type of sorbent material (C2, C8, C18, SIL, and M1), elution solvent system, number of extraction cycles (extract-discard), sample volume, elution volume, and sample pH, were evaluated. The optimal experimental conditions involves the loading of 500μL of sample through a C18 sorbent in a MEPS syringe placed in the semi-automatic eVol(®) syringe followed by elution using 250μL of acetonitrile (ACN) in a 10 extractions cycle (about 5min for the entire sample preparation step). The obtained extract is directly analyzed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and ACN (eluent B) in the gradient elution mode (10min total analysis). Under optimized conditions good results were obtained in terms of linearity within the established concentration range with correlation coefficients (R) values higher than 0.986, with a residual deviation for each calibration point below 12%. The limit of detection (LOD) and limit of quantification (LOQ) obtained were 0.4ngmL(-1) and 1.0ngmL(-1) for IXN, and 0.9ngmL(-1) and 3.0ngmL(-1) for XN, respectively. Precision was lower than 4.6% for IXN and 8.4% for XN. Typical recoveries ranged between 67.1% and 99.3% for IXN and between 74.2% and 99.9% for XN, with relative standard deviations %RSD no larger than 8%. The applicability of the proposed analytical procedure in commercial beers, revealed the presence of both target prenylchalcones in all samples being IXN the most abundant with concentration of between 0.126 and 0.200μgmL(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Large Airborne Full Tensor Gradient Data Inversion Based on a Non-Monotone Gradient Method

NASA Astrophysics Data System (ADS)

Sun, Yong; Meng, Zhaohai; Li, Fengting

2018-03-01

Following the development of gravity gradiometer instrument technology, the full tensor gravity (FTG) data can be acquired on airborne and marine platforms. Large-scale geophysical data can be obtained using these methods, making such data sets a number of the "big data" category. Therefore, a fast and effective inversion method is developed to solve the large-scale FTG data inversion problem. Many algorithms are available to accelerate the FTG data inversion, such as conjugate gradient method. However, the conventional conjugate gradient method takes a long time to complete data processing. Thus, a fast and effective iterative algorithm is necessary to improve the utilization of FTG data. Generally, inversion processing is formulated by incorporating regularizing constraints, followed by the introduction of a non-monotone gradient-descent method to accelerate the convergence rate of FTG data inversion. Compared with the conventional gradient method, the steepest descent gradient algorithm, and the conjugate gradient algorithm, there are clear advantages of the non-monotone iterative gradient-descent algorithm. Simulated and field FTG data were applied to show the application value of this new fast inversion method.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leighton, J.K.; Joyner, J.; Zamarripa, J.

Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of (35S)methionine-labeled lipoproteinsmore » secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.« less

Quantitative structure-retention relationships applied to development of liquid chromatography gradient-elution method for the separation of sartans.

PubMed

Golubović, Jelena; Protić, Ana; Otašević, Biljana; Zečević, Mira

2016-04-01

QSRR are mathematically derived relationships between the chromatographic parameters determined for a representative series of analytes in given separation systems and the molecular descriptors accounting for the structural differences among the investigated analytes. Artificial neural network is a technique of data analysis, which sets out to emulate the human brain's way of working. The aim of the present work was to optimize separation of six angiotensin receptor antagonists, so-called sartans: losartan, valsartan, irbesartan, telmisartan, candesartan cilexetil and eprosartan in a gradient-elution HPLC method. For this purpose, ANN as a mathematical tool was used for establishing a QSRR model based on molecular descriptors of sartans and varied instrumental conditions. The optimized model can be further used for prediction of an external congener of sartans and analysis of the influence of the analyte structure, represented through molecular descriptors, on retention behaviour. Molecular descriptors included in modelling were electrostatic, geometrical and quantum-chemical descriptors: connolly solvent excluded volume non-1,4 van der Waals energy, octanol/water distribution coefficient, polarizability, number of proton-donor sites and number of proton-acceptor sites. Varied instrumental conditions were gradient time, buffer pH and buffer molarity. High prediction ability of the optimized network enabled complete separation of the analytes within the run time of 15.5 min under following conditions: gradient time of 12.5 min, buffer pH of 3.95 and buffer molarity of 25 mM. Applied methodology showed the potential to predict retention behaviour of an external analyte with the properties within the training space. Connolly solvent excluded volume, polarizability and number of proton-acceptor sites appeared to be most influential paramateres on retention behaviour of the sartans. Copyright © 2015 Elsevier B.V. All rights reserved.

NEW SCX PEPTIDE ELUTION SCORE FOR PH/SALT-GRADIENT SCX CHROMATOGRAPHY IN 2D-NANO-LC/MSMS ANALYSIS OF PROTEIN DIGESTS

EPA Science Inventory

A new automated 2D-(SCX/RP)-nano-LC/MSMS method was developed. Separation of the peptides in the first LC dimension was the main focus of this work, and it was optimized using human serum albumin (HSA) and human lung cell lysate tryptic digests. Samples were reduced and alkylated...

Ultramicroelectrode Sensors and Detectors. Considerations of the Stability, Sensitivity, Reproducibility, and Mechanism of Ion Transport in Gas Phase Chromatography and in High Performance Liquid Phase Chromatography

DTIC Science & Technology

1988-07-15

solvents were used. For high performance liquid chromatographic studies, the DNA bases thymine, adenine, cytocine, uracil, and guanine (Aldrich...this experiment. The DNA bases guanine, adenine, cytocine, uracil, and thymine were detected for a gradient elution of a mixture of the bases in a

ANALYSIS OF PROTEIN DIGESTS BY nano- SCX/RP/MSMS WITH pH SALT GRADIENT SCX ELUTION

EPA Science Inventory

The objective of this study was to optimize chromatographic parameters for complex peptide mixture analyses using two dimensional nano-LC/MSMS system. It used a strong cation exchange (SCX) and reversed phase chromatography (RP). The SCX solvent system was designed to promote pep...

Characterization of Explosives Processing Waste Decomposition Due to Composting.

DTIC Science & Technology

1994-09-01

leachate were injected onto an Alltech RP-C 18/Anion column (150 mm x 4.6 mm ID) and were eluted at 1 mL/min using a complex ternary gradient of 0.015 M...the study because it is an agriculturally important legume; the seeds of this plant are also an important carbon sink. Thus, Glycine was advantageous

A Microwave Flow Detector for Gradient Elution Liquid Chromatography.

PubMed

Ye, Duye; Wang, Weizheng; Moline, David; Islam, Md Saiful; Chen, Feng; Wang, Pingshan

2017-10-17

This study presents a microwave flow detector technique for liquid chromatography (LC) application. The detector is based on a tunable microwave interferometer (MIM) with a vector network analyzer (VNA) for signal measurement and a computer for system control. A microstrip-line-based 0.3 μL flow cell is built and incorporated into the MIM. With syringe pump injection, the detector is evaluated by measuring a few common chemicals in DI water at multiple frequencies from 0.98 to 7.09 GHz. Less than 30 ng minimum detectable quantity (MDQ) is demonstrated. An algorithm is provided and used to obtain sample dielectric permittivity at each frequency point. When connected to a commercial HPLC system and injected with a 10 μL aliquot of 10 000 ppm caffeine DI-water solution, the microwave detector yields a signal-to-noise ratio (SNR) up to 10 under isocratic and gradient elution operations. The maximum sampling rate is 20 Hz. The measurements show that MIM tuning, aided by a digital tunable attenuator (DTA), can automatically adjust MIM operation to retain detector sensitivity when mobile phase changes. Furthermore, the detector demonstrates a capability to quantify coeluted vitamin E succinate (VES) and vitamin D 3 (VD 3 ).

Sub-1min separation in sequential injection chromatography for determination of synthetic water-soluble dyes in pharmaceutical formulation.

PubMed

Davletbaeva, Polina; Chocholouš, Petr; Bulatov, Andrey; Šatínský, Dalibor; Solich, Petr

2017-09-05

Sequential Injection Chromatography (SIC) evolved from fast and automated non-separation Sequential Injection Analysis (SIA) into chromatographic separation method for multi-element analysis. However, the speed of the measurement (sample throughput) is due to chromatography significantly reduced. In this paper, a sub-1min separation using medium polar cyano monolithic column (5mm×4.6mm) resulted in fast and green separation with sample throughput comparable with non-separation flow methods The separation of three synthetic water-soluble dyes (sunset yellow FCF, carmoisine and green S) was in a gradient elution mode (0.02% ammonium acetate, pH 6.7 - water) with flow rate of 3.0mLmin -1 corresponding with sample throughput of 30h -1 . Spectrophotometric detection wavelengths were set to 480, 516 and 630nm and 10Hz data collection rate. The performance of the separation was described and discussed (peak capacities 3.48-7.67, peak symmetries 1.72-1.84 and resolutions 1.42-1.88). The method was represented by validation parameters: LODs of 0.15-0.35mgL -1 , LOQs of 0.50-1.25mgL -1 , calibration ranges 0.50-150.00mgL -1 (r>0.998) and repeatability at 10.0mgL -1 of RSD≤0.98% (n=6). The method was used for determination of the dyes in "forest berries" colored pharmaceutical cough-cold formulation. The sample matrix - pharmaceuticals and excipients were not interfering with vis determination because of no retention in the separation column and colorless nature. The results proved the concept of fast and green chromatography approach using very short medium polar monolithic column in SIC. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasonic atomization and subsequent desolvation for monoclonal antibody (mAb) to the glycoprotein (GP) IIIa receptor into drug eluting stent.

PubMed

Wang, G X; Luo, L L; Yin, T Y; Li, Y; Jiang, T; Ruan, C G; Guidoin, R; Chen, Y P; Guzman, R

2010-01-01

An eluting-stent system with mAb dispersed in the PLLA (poly (L-lactic acid)) was validated in vitro. Specifically designed spray equipment based on the principle of ultrasonic atomization was used to produce a thin continuous PLLA (poly (L-lactic acid)) polymer coating incorporating monoclonal antibody (mAb). This PLLA coating was observed in light microscopy (LM) and scanning electron microscopy (SEM). The concentration of the monoclonal antibody (mAb) to the platelet glycoprotein (GP) IIIa receptor and the eluting rate were then measured by a radioisotope technique with (125)I-labelled GP IIIa mAb. An in vitro perfusion circuit was designed to evaluate the release rates at different velocities (10 or 20 ml min(-1)). The PLLA coating was thin and transparent, uniformly distributed on the surface of the stent. Three factors influenced its thickness: PLLA concentration, duration and gas pressure. The concentration of mAb was influenced by the duration of absorption and the concentration of the mAb solution; the maximum was 1662.23 + or - 38.83 ng. The eluting rate was fast for the first 2 h, then decreased slowly and attained 80% after 2 weeks. This ultrasonic atomization spray equipment and technological process to prepare protein eluting-stents were proved to be effective and reliable.

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; extraction of nitroaromatic compounds from water by polystyrene divinylbenzene cartridge and determination by high-performance liquid chromatography

USGS Publications Warehouse

Lindley, C.E.; Burkhardt, M.R.; DeRusseau, S.N.

1994-01-01

Organic explosives are determined in samples of ground water and surface water with emphasis on identifying and quantifying trinitrotoluene (TNT) metabolites. Water samples are filtered to remove suspended particulate material and passed through a polystyrene divinylbenzene-packed cartridge by a vacuum-extraction system. The target analytes subsequently are eluted with acetonitrile. A high-performance liquid chromatograph (HPLC) equipped with a photodiode-array detector is used for sample analysis. Analytes are separated on an octadecylsilane column using a methanol, water, and acetonitrile gradient elution. The compounds 2,4- and 2,6-dinitrotoluene are separated through an independent, isocratic elution. Method detection limits, on the basis of a 1-liter sample size, range from 0.11 to 0.32 microgram per liter. Recoveries averaged from 71 to 101 percent for 13 analytes in one set of HPLC-grade water fortified at about 1 microgram per liter. The method is limited to use by analysts experienced in handling explosive materials. (USGS)

Determination of water-soluble forms of oxalic and formic acids in soils by ion chromatography

NASA Astrophysics Data System (ADS)

Karicheva, E.; Guseva, N.; Kambalina, M.

2016-03-01

Carboxylic acids (CA) play an important role in the chemical composition origin of soils and migration of elements. The content of these acids and their salts is one of the important characteristics for agrochemical, ecological, ameliorative and hygienic assessment of soils. The aim of the article is to determine water-soluble forms of same carboxylic acids — (oxalic and formic acids) in soils by ion chromatography with gradient elution. For the separation and determination of water-soluble carboxylic acids we used reagent-free gradient elution ion-exchange chromatography ICS-2000 (Dionex, USA), the model solutions of oxalate and formate ions, and leachates from soils of the Kola Peninsula. The optimal gradient program was established for separation and detection of oxalate and formate ions in water solutions by ion chromatography. A stability indicating method was developed for the simultaneous determination of water-soluble organic acids in soils. The method has shown high detection limits such as 0.03 mg/L for oxalate ion and 0.02 mg/L for formate ion. High signal reproducibility was achieved in wide range of intensities which correspond to the following ion concentrations: from 0.04 mg/g to 10 mg/L (formate), from 0.1 mg/g to 25 mg/L (oxalate). The concentration of formate and oxalate ions in soil samples is from 0.04 to 0.9 mg/L and 0.45 to 17 mg/L respectively.

Two tools for applying chromatographic retention data to the mass-based identification of peptides during hydrogen/deuterium exchange experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Gershon, P D

2010-12-15

Two tools are described for integrating LC elution position with mass-based data in hydrogen-deuterium exchange (HDX) experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (nanoLC/MALDI-MS, a novel approach to HDX-MS). The first of these, 'TOF2H-Z Comparator', highlights peptides in HDX experiments that are potentially misidentified on the basis of mass alone. The program first calculates normalized values for the organic solvent concentration responsible for the elution of ions in nanoLC/MALDI HDX experiments. It then allows the solvent gradients for the multiple experiments contributing to an MS/MS-confirmed peptic peptide library to be brought into mutual alignment by iteratively re-modeling variables among LC parameters such as gradient shape, solvent species, fraction duration and LC dead time. Finally, using the program, high-probability chromatographic outliers can be flagged within HDX experimental data. The role of the second tool, 'TOF2H-XIC Comparator', is to normalize the LC chromatograms corresponding to all deuteration timepoints of all HDX experiments of a project, to a common reference. Accurate normalization facilitates the verification of chromatographic consistency between all ions whose spectral segments contribute to particular deuterium uptake plots. Gradient normalization in this manner revealed chromatographic inconsistencies between ions whose masses were either indistinguishable or separated by precise isotopic increments. Copyright © 2010 John Wiley & Sons, Ltd.

High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

PubMed

Madadkar, Pedram; Nino, Sergio Luna; Ghosh, Raja

2016-11-01

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution. Copyright © 2016 Elsevier B.V. All rights reserved.

Separation and quantitation of colour pigments of chili powder (Capsicum frutescens) by high-performance liquid chromatography-diode array detection.

PubMed

Cserháti, T; Forgács, E; Morais, M H; Mota, T; Ramos, A

2000-10-27

The performance of reversed-phase thin-layer (RP-TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) was compared for the separation and determination of the colour pigments of chili (Capsicum frutescens) powder using a wide variety of eluent systems. No separation of pigments was achieved in RP-TLC, however, it was established that tetrahydrofuran shows an unusually high solvent strength. RP-HPLC using water-methanol-acetonitrile gradient elution separated the chili pigments in many fractions. Diode array detection (DAD) indicated that yellow pigments are eluted earlier than the red ones and chili powder contains more yellow pigments than common paprika powders. It was established that the very different absorption spectra of pigments make the use of DAD necessary.

[HPLC specific chromatogram of Dendrobium officinale].

PubMed

Yan, Mei-Qiu; Chen, Su-Hong; Lv, Gui-Yuan; Zhou, Gui-Fen; Liu, Xia

2013-02-01

To establish the method of specific chromatogram analysis of ether extract of Dendrobium officinale for identification of D. officinale. Chromatographic separation was carried out at 30 degrees C on an Ultimate C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol and water containing 0.2% phosphoric acid in a gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 280 nm. The similarity evaluation system for chromatographic fingerprint of NPC (National Pharmacopoeia Committee) was adopted to specific chromatogram construction. The HPLC specific chromatogram of D. officinale was constructed with 6 common specific chromatographic peaks including naringenin as a reference peak. The method shows good precision and repeatability of relative retention time. It can be used to identify D. officinale.

Detection of malignant hepatic tumors with ferumoxides-enhanced MRI: comparison of five gradient-recalled echo sequences with different TEs.

PubMed

Matsuo, Masayuki; Kanematsu, Masayuki; Itoh, Kyo; Murakami, Takamichi; Maetani, Yoji; Kondo, Hiroshi; Goshima, Satoshi; Kako, Nobuo; Hoshi, Hiroaki; Konishi, Junji; Moriyama, Noriyuki; Nakamura, Hironobu

2004-01-01

The purpose of our study was to compare the detectability of malignant hepatic tumors on ferumoxides-enhanced MRI using five gradient-recalled echo sequences at different TEs. Ferumoxides-enhanced MRIs obtained in 31 patients with 50 malignant hepatic tumors (33 hepatocellular carcinomas, 17 metastases) were reviewed retrospectively by three independent offsite radiologists. T1-weighted gradient-recalled echo images with TEs of 1.4 and 4.2 msec; T2*-weighted gradient-recalled echo images with TEs of 6, 8, and 10 msec; and T2-weighted fast spin-echo images of livers were randomly reviewed on a segment-by-segment basis. Observer performance was tested using the McNemar test and receiver operating characteristic analysis for the clustered data. Lesion-to-liver contrast-to-noise ratio was also assessed. Mean lesion-to-liver contrast-to-noise ratios were negative and lower with gradient-recalled echo at 1.4 msec than with the other sequences. Sensitivity was higher (p < 0.05) with gradient-recalled echo at 6, 8, and 10 msec and fast spin-echo sequences (75-83%) than with gradient-recalled echo sequences at 1.4 and 4.2 msec (46-48%), and was higher (p < 0.05) with gradient-recalled echo sequence at 8 msec (83%) than with gradient-recalled echo at 6 msec and fast spin-echo sequences (75-78%). Specificity was comparably high with all sequences (95-98%). The area under the receiver operating characteristic curve (A(z)) was greater (p < 0.05) with gradient-recalled echo at 6, 8, and 10 msec and fast spin-echo sequences (A(z) = 0.91-0.93) than with gradient-recalled echo sequences at 1.4 and 4.2 msec (A(z) = 0.82-0.85). In the detection of malignant hepatic tumors, gradient-recalled echo sequences at 8 msec showed the highest sensitivity and had an A(z) value and lesion-to-liver contrast-to-noise ratio comparable with values from gradient-recalled echo sequences at 6 and 10 msec and fast spin-echo sequences.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, D-Trp7,9, mePhe8]-substance P (6-11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate.

PubMed

Cummings, J; MacLellan, A J; Mark, M; Jodrell, D I

1999-09-24

[Arg6, D-Trp7,9 mePhe8]-substance P (6-11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met11 (M2) and G[Met11(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (microBondapak C18, 30 cm X 2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40 degrees C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G-P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1-3 and G-P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1-3. Sample clean-up by solid-phase extraction using C2-bonded 40 microm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml-100 microg/ml) normally varied by < 10%, although at the highest concentrations of M1 and M2 studied (50 microg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.

A sensitive and accurate method for the determination of perfluoroalkyl and polyfluoroalkyl substances in human serum using a high performance liquid chromatography-online solid phase extraction-tandem mass spectrometry.

PubMed

Yu, Chang Ho; Patel, Bhupendra; Palencia, Marilou; Fan, Zhihua Tina

2017-01-13

A selective, sensitive, and accurate analytical method for the measurement of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in human serum, utilizing LC-MS/MS (liquid chromatography-tandem mass spectrometry), was developed and validated according to the Centers for Disease Control and Prevention (CDC) guidelines for biological sample analysis. Tests were conducted to determine the optimal analytical column, mobile phase composition and pH, gradient program, and cleaning procedure. The final analytical column selected for analysis was an extra densely bonded silica-packed reverse-phase column (Agilent XDB-C 8 , 3.0×100mm, 3.5μm). Mobile phase A was an aqueous buffer solution containing 10mM ammonium acetate (pH=4.3). Mobile phase B was a mixture of methanol and acetonitrile (1:1, v/v). The gradient program was programmed by initiating a fast elution (%B, from 40 to 65%) between 1.0 and 1.5min, followed by a slow elution (%B: 65-80%) in the period of 1.5-7.5min. The cleanup procedures were augmented by cleaning with (1) various solvents (isopropyl alcohol, methanol, acetonitrile, and reverse osmosis-purified water); (2) extensive washing steps for the autosampler and solid phase extraction (SPE) cartridge; and (3) a post-analysis cleaning step for the whole system. Under the above conditions, the resolution and sensitivity were significantly improved. Twelve target PFASs were baseline-separated (2.5-7.0min) within a 10-min of acquisition time. The limits of detection (LODs) were 0.01ng/mL or lower for all of the target compounds, making this method 5 times more sensitive than previously published methods. The newly developed method was validated in the linear range of 0.01-50ng/mL, and the accuracy (recovery between 80 and 120%) and precision (RSD<20%) were acceptable at three spiked levels (0.25, 2.5, and 25ng/mL). The method development and validation results demonstrated that this method was precise, accurate, and robust, with high-throughput (∼10min per sample); thus suitable for large-scale epidemiological studies. Published by Elsevier B.V.

Monitoring gradient profile on-line in micro- and nano-high performance liquid chromatography using conductivity detection.

PubMed

Zhang, Min; Chen, Apeng; Lu, Joann J; Cao, Chengxi; Liu, Shaorong

2016-08-19

In micro- or nano-flow high performance liquid chromatography (HPLC), flow-splitters and gradient elutions are commonly used for reverse phase HPLC separations. When a flow splitter was used at a high split-ratio (e.g., 1000:1 or higher), the actual gradient may deviate away from the programmed gradient. Sometimes, mobile phase concentrations can deviate by as much as 5%. In this work, we noticed that the conductivity (σ) of a gradient decreased with the increasing organic-solvent fraction (φ). Based on the relationship between σ and φ, a method was developed for monitoring gradient profile on-line to record any deviations in these HPLC systems. The conductivity could be measured by a traditional conductivity detector or a capacitively coupled contactless conductivity detector (C(4)D). The method was applied for assessing the performance of an electroosmotic pump (EOP) based nano-HPLC. We also observed that σ value of the gradient changed with system pressure; a=0.0175ΔP (R(2)=0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated. Copyright © 2016. Published by Elsevier B.V.

Fast and Cost-Effective Biochemical Spectrophotometric Analysis of Solution of Insect "Blood" and Body Surface Elution.

PubMed

Łoś, Aleksandra; Strachecka, Aneta

2018-05-09

Using insect hemolymph ("blood") and insect body surface elutions, researchers can perform rapid and cheap biochemical analyses to determine the insect's immunology status. The authors of this publication describe a detailed methodology for a quick marking of the concentration of total proteins and evaluation of the proteolytic system activity (acid, neutral, and alkaline proteases and protease inhibitors), as well as a methodology for quick "liver" tests in insects: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea and glucose concentration analyses. The meaning and examples of an interpretation of the results of the presented methodology for biochemical parameter determination are described for the example of honey bees.

Reduction of Solvent Effect in Reverse Phase Gradient Elution LC-ICP-MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, Patrick Allen

2005-12-17

Quantification in liquid chromatography (LC) is becoming very important as more researchers are using LC, not as an analytical tool itself, but as a sample introduction system for other analytical instruments. The ability of LC instrumentation to quickly separate a wide variety of compounds makes it ideal for analysis of complex mixtures. For elemental speciation, LC is joined with inductively coupled plasma mass spectrometry (ICP-MS) to separate and detect metal-containing, organic compounds in complex mixtures, such as biological samples. Often, the solvent gradients required to perform complex separations will cause matrix effects within the plasma. This limits the sensitivity ofmore » the ICP-MS and the quantification methods available for use in such analyses. Traditionally, isotope dilution has been the method of choice for LC-ICP-MS quantification. The use of naturally abundant isotopes of a single element in quantification corrects for most of the effects that LC solvent gradients produce within the plasma. However, not all elements of interest in speciation studies have multiple naturally occurring isotopes; and polyatomic interferences for a given isotope can develop within the plasma, depending on the solvent matrix. This is the case for reverse phase LC separations, where increasing amounts of organic solvent are required. For such separations, an alternative to isotope dilution for quantification would be is needed. To this end, a new method was developed using the Apex-Q desolvation system (ESI, Omaha, NE) to couple LC instrumentation with an ICP-MS device. The desolvation power of the system allowed greater concentrations of methanol to be introduced to the plasma prior to destabilization than with direct methanol injection into the plasma. Studies were performed, using simulated and actual linear methanol gradients, to find analyte-internal standard (AIS) pairs whose ratio remains consistent (deviations {+-} 10%) over methanol concentration ranges of 5%-35% (simulated) and 8%-32% (actual). Quadrupole (low resolution) and sector field (high resolution) ICP-MS instrumentation were utilized in these studies. Once an AIS pair is determined, quantification studies can be performed. First, an analysis is performed by adding both elements of the AIS pair post-column while performing the gradient elution without sample injection. A comparison of the ratio of the measured intensities to the atomic ratio of the two standards is used to determine a correction factor that can be used to account for the matrix effects caused by the mobile phase. Then, organic and/or biological molecules containing one of the two elements in the AIS pair are injected into the LC column. A gradient method is used to vary the methanol-water mixture in the mobile phase and to separate out the compounds in a given sample. A standard solution of the second ion in the AIS pair is added continuously post-column. By comparing the ratio of the measured intensities to the atomic ratio of the eluting compound and internal standard, the concentration of the injected compound can be determined.« less

Simple, high-yield purification of xanthine oxidase from bovine milk.

PubMed

Ozer, N; Müftüoglu, M; Ataman, D; Ercan, A; Ogüs, I H

1999-05-13

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

INVESTIGATION OF A MIXTURE CONTAINING ALPRAZOLAM, CODEINE AND PARACETAMOL USING THIN-LAYER AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHODS.

PubMed

Ciegis, Paulius; Zevzikovas, Andrejus; Zevzikoviene, Augusta; Nenortiene, Palma; Kazlauskiene, Daiva

2016-01-01

The increasing drug consumption in Lithuania and all over the world makes us think about the negative consequences - the risk of toxicity. Fast and accurate identification of material that caused the poisoning reduces the probability in death cases and makes easier to determine the main cause of death. The results have shown that the most appropriate systems of solvents for qualitative analysis by TLC method of the mixture consisting of alprazolam, codeine and paracetanol are: system "D" (trichloromethane : acetone : conc. ammonia = 55 : 40 : 5 (v/v/v)) and system "F" (trichloromethane : diethyl ether: isobutanol : conc. ammonia = 50 : 30 : 15 : 5 (v/v/v/v)). For qualitative analysis of the mixture consisting of alprazolam, codeine and paracetamol by HPLC method the chromatographic column ACE C18 (25 cm x 4.6 mm x 5 µm), gradient elution mode (mixture of 3% acetic acid and methanol and the flow rate 1 mL/min have been used. The injection volume was 10 pL. Photodiode array detector (210 - 240 nm range) has been used. UV absorption spectra of materials measured using photodiode array detector have been identical to those presented in the scientific literature.

Simultaneous Speciation Analysis of Arsenic, Chromium, and Selenium in the Bioaccessible Fraction for Realistic Risk Assessment of Food Safety.

PubMed

Sadiq, Nausheen W; Beauchemin, Diane

2017-12-19

A simple and fast method was developed for risk assessment of As, Cr, and Se in food, which is demonstrated here using three cooked and uncooked rice samples (basmati as well as organic white and brown rice). The bioaccessible fraction was first determined through online leaching of rice minicolumns (maintained at 37 °C) sequentially with artificial saliva, gastric juice, and intestinal juice while continuously monitoring potentially toxic elements (As, Cr, and Se) by inductively coupled plasma mass spectrometry (ICPMS). Then, a new ion chromatography method with online detection by ICPMS was developed for the simultaneous speciation analysis of As, Cr, and Se in the bioaccessible fraction to determine the portion of these elements that was actually toxic. Using gradient elution, four As species [As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid], two Cr species [Cr(III) and Cr(VI)], and two Se species [Se(IV) and Se(VI)] were separated within 12 min. The simultaneous speciation analysis of As, Cr, and Se revealed that the simple act of cooking can convert all of the carcinogenic Cr(VI) to the safer Cr(III).

Miniaturised medium pressure capillary liquid chromatography system with flexible open platform design using off-the-shelf microfluidic components.

PubMed

Li, Yan; Dvořák, Miloš; Nesterenko, Pavel N; Stanley, Roger; Nuchtavorn, Nantana; Krčmová, Lenka Kujovská; Aufartová, Jana; Macka, Mirek

2015-10-08

Trends towards portable analytical instrumentation of the last decades have not been equally reflected in developments of portable liquid chromatography (LC) instrumentation for rapid on-site measurements. A miniaturised medium pressure capillary LC (MPLC) system with gradient elution capability has been designed based on a flexible modular microfluidic system using primarily off-the-shelf low cost components to ensure wide accessibility to other analysts. The microfluidic platform was assembled on a breadboard and contained microsyringe pumps and switch valves, complemented with an injection valve and on-capillary detectors, all controlled by a PC. Four miniaturised microsyringe pumps, with 5, 20 and 100 μL syringe volume options, formed the basis of the pumping system. Two pairs of pumps were used for each mobile phase to create gradient elution capability. The two microsyringe pumps in each pairs were linked by two electrically operated microfluidic switching valves and both pairs of pumps were connected through a zero void volume cross-connector, thus providing a low hold-up volume for gradient formation. Sample was injected by a 20 nL nano-LC sampling valve, directly connected to a 18 cm long 100 μm i.d. Chromolith CapRod RP-18 monolithic capillary column. On-capillary LED-based UV-vis photometric detection was conducted through a piece of equal diameter fused silica capillary connected after the column. The performance of the portable LC system was evaluated theoretically and experimentally, including the maximum operating pressure, gradient mixing performance, and the performance of the detectors. The 5 μL microsyringe pump offered the best performance, with typical maximum operating pressures up to 11.4 ± 0.4 MPa (water) and gradient pumping repeatability of between 4 and 9% for gradients between 0.10% s(-1) and 0.33% s(-1). Test analytes of charged and uncharged dyes and pharmaceuticals of varying hydrophobicity showed typical RSD values of 0.7-1.4% and 3.3-4.8% in isocratic mode and 1.2-4.6% and 3.2-6.4% in gradient mode, respectively for retention time and peak area repeatability. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Liquid Chromatography at Critical Conditions: Balancing size exclusion and adsorption in nanopores

NASA Astrophysics Data System (ADS)

Abdulahad, Asem; Amos, Jeffrey; Ryu, Chang

2009-03-01

Liquid chromatography at critical condition (LCCC) is a measure to identify thermodynamic conditions, in which polymers elute independently of molar mass during high performance liquid chromatography. Under these critical conditions the entropic exclusions that dominate size exclusion chromatography (SEC) and the enthalpic adsorption that governs adsorption-based interaction chromatography (IC) are said to negate one another resulting in simultaneous elution of the polymer of different molecular weights. Using multiple C18-bonded silica columns with different average nanopore sizes (from 5 nm to 30 nm), we will study the LCCC conditions of PS in methylene chloride/acetonitrile solvent mixture at different temperature. In addition, we will show that the separation of polystyrene can be fine tuned using a refined temperature gradient interaction chromatography (TGIC) that employs multiple columns of varying pore size in sequence.

Purification of Bacteriophages Using Anion-Exchange Chromatography.

PubMed

Vandenheuvel, Dieter; Rombouts, Sofie; Adriaenssens, Evelien M

2018-01-01

In bacteriophage research and therapy, most applications ask for highly purified phage suspensions. The standard technique for this is ultracentrifugation using cesium chloride gradients. This technique is cumbersome, elaborate and expensive. Moreover, it is unsuitable for the purification of large quantities of phage suspensions.The protocol described here, uses anion-exchange chromatography to bind phages to a stationary phase. This is done using an FLPC system, combined with Convective Interaction Media (CIM ® ) monoliths. Afterward, the column is washed to remove impurities from the CIM ® disk. By using a buffer solution with a high ionic strength, the phages are subsequently eluted from the column and collected. In this way phages can be efficiently purified and concentrated.This protocol can be used to determine the optimal buffers, stationary phase chemistry and elution conditions, as well as the maximal capacity and recovery of the columns.

Bio-oil production of softwood and hardwood forest industry residues through fast and intermediate pyrolysis and its chromatographic characterization.

PubMed

Torri, Isadora Dalla Vecchia; Paasikallio, Ville; Faccini, Candice Schmitt; Huff, Rafael; Caramão, Elina Bastos; Sacon, Vera; Oasmaa, Anja; Zini, Claudia Alcaraz

2016-01-01

Bio-oils were produced through intermediate (IP) and fast pyrolysis (FP), using Eucalyptus sp. (hardwood) and Picea abies (softwood), wood wastes produced in large scale in Pulp and Paper industries. Characterization of these bio-oils was made using GC/qMS and GC×GC/TOFMS. The use of GC×GC provided a broader characterization of bio-oils and it allowed tracing potential markers of hardwood bio-oil, such as dimethoxy-phenols, which might co-elute in 1D-GC. Catalytic FP increased the percentage of aromatic hydrocarbons in P. abies bio-oil, indicating its potential for fuel production. However, the presence of polyaromatic hydrocarbons (PAH) draws attention to the need of a proper management of pyrolysis process in order to avoid the production of toxic compounds and also to the importance of GC×GC/TOFMS use to avoid co-elutions and consequent inaccuracies related to identification and quantification associated with GC/qMS. Ketones and phenols were the major bio-oil compounds and they might be applied to polymer production. Copyright © 2015 Elsevier Ltd. All rights reserved.

A rapid and accurate approach for prediction of interactomes from co-elution data (PrInCE).

PubMed

Stacey, R Greg; Skinnider, Michael A; Scott, Nichollas E; Foster, Leonard J

2017-10-23

An organism's protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome. Here we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, more predicted interactions at the same stringency, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2017a). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE , where usage instructions can be found. An example dataset and output are also provided for testing purposes. PrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics expertise to analyze high-throughput co-elution datasets.

Single-laboratory validation of a high-performance liquid chromatographic-diode array detector-fluorescence detector/mass spectrometric method for simultaneous determination of water-soluble vitamins in multivitamin dietary tablets.

PubMed

Chen, Pei; Atkinson, Renata; Wolf, Wayne R

2009-01-01

The purpose of this study was to develop a single-laboratory validated (SLV) method using high-performance liquid chromatography with different detectors [diode array detector (DAD); fluorescence detector (FLD); and mass spectrometry (MS)] for determination of 7 B-complex vitamins (B1-thiamin, B2-riboflavin, B3-nicotinamide, B6-pyridoxine, B9-folic acid, pantothenic acid, and biotin) and vitamin C in multivitamin/multimineral dietary supplements. The method involves the use of a reversed-phase octadecylsilyl column (4 microm, 250 x 2.0 mm id) and a gradient mobile phase profile. Gradient elution was performed at a flow rate of 0.25 mL/min. After a 5 min isocratic elution at 100% A (0.1% formic acid in water), a linear gradient to 50% A and 50% B (0.1% formic acid in acetonitrile) at 15 min was employed. Detection was performed with a DAD as well as either an FLD or a triple-quadrupole MS detector in the multiple reaction monitoring mode. SLV was performed using Standard Reference Material (SRM) 3280 Multivitamin/Multimineral Tablets, being developed by the National Institute of Standards and Technology, with support by the Office of Dietary Supplements of the National Institutes of Health. Phosphate buffer (10 mM, pH 2.0) extracts of the NIST SRM 3280 were analyzed by the liquid chromatographic (LC)-DAD-FLDIMS method. Following extraction, the method does not require any sample cleanup/preconcentration steps except centrifugation and filtration.

Single-Laboratory Validation of a High-Performance Liquid Chromatographic-Diode Array Detector-Fluorescence Detector/Mass Spectrometric Method for Simultaneous Determination of Water-Soluble Vitamins in Multivitamin Dietary Tablets

PubMed Central

Chen, Pei; Atkinson, Renata; Wolf, Wayne R.

2014-01-01

The purpose of this study was to develop a single-laboratory validated (SLV) method using high-performance liquid chromatography with different detectors [diode array detector (DAD); fluorescence detector (FLD); and mass spectrometry (MS)] for determination of 7 B-complex vitamins (B1-thiamin, B2-riboflavin, B3-nicotinamide, B6-pyridoxine, B9-folic acid, pantothenic acid, and biotin) and vitamin C in multivitamin/multimineral dietary supplements. The method involves the use of a reversed-phase octadecylsilyl column (4 µm, 250 × 2.0 mm id) and a gradient mobile phase profile. Gradient elution was performed at a flow rate of 0.25 mL/min. After a 5 min isocratic elution at 100% A (0.1% formic acid in water), a linear gradient to 50% A and 50% B (0.1% formic acid in acetonitrile) at 15 min was employed. Detection was performed with a DAD as well as either an FLD or a triple-quadrupole MS detector in the multiple reaction monitoring mode. SLV was performed using Standard Reference Material (SRM) 3280 Multivitamin/Multimineral Tablets, being developed by the National Institute of Standards and Technology, with support by the Office of Dietary Supplements of the National Institutes of Health. Phosphate buffer (10 mM, pH 2.0) extracts of the NIST SRM 3280 were analyzed by the liquid chromatographic (LC)-DAD-FLD/MS method. Following extraction, the method does not require any sample cleanup/preconcentration steps except centrifugation and filtration. PMID:19485230

Determination of pentachlorophenol in water and aquifer sediments by high-performance liquid chromatography

USGS Publications Warehouse

Goerlitz, D.F.

1981-01-01

Methods for the determination of pentachlorophenol (PCP) in water and aquifer sediments are presented. Reverse-phase high-performance liquid chromotography employing ion suppression and gradient elution is used. PCP can be determined directly in water at a lower limit of detection Of 0.2 micrograms per liter. For extracts of sediment, PCP can be determined to a lower limit of 1.0 micrograms per kilogram.

Purification and characterization of two isoenzymes of lipoxygenase from soybeans.

PubMed

Diel, E; Stan, H J

1978-01-01

A chromatographic procedure for the purification of two lipoxygenase isoenzymes (linoleate: O2 oxidoreductase, EC 1.13.11.12.) from soybean is described. The procedure for the purification of isoenzyme L-1 includes optimalized extraction, ammonium sulfate fractionation, heat treatment and gradient elution from a CM-Sephadex C-50 column. The purification of L-2 includes ammonium sulfate fractionation, gelfiltration on Sephadex G-150 and gradient elution from a DEAE-cellulose column. Both isoenzymes L-1 and L-2 appear homogeneous after Disc-PAGE. The isoelectric points are 5.6 for L-1 and 5.8 for L-2. Molecular weights are estimated as 100,000 for L-1 as well as L-2 applying three different methods. Both isoenzymes contain 0.9 mol iron per mol protien. The estimated turn over numbers are 8,200 mol linoleate per mol enzyme and min for L-1 and 3,100 for L-2. Amino acid compositions determined after acid hydrolysis show marked differences between L-1 and L-2, particularly with respect to the amino acids Lys, Phe, Ser, Gly and Leu. L-1 posesses a total of 9 cysteine molecules, 6 of which are present as disulfide bonds. L-2 posesses a total of 8 cysteine molecules with only one disulfide bond.

Gradient elution moving boundary electrophoresis enables rapid analysis of acids in complex biomass-derived streams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munson, Matthew S.; Karp, Eric M.; Nimlos, Claire T.

Biomass conversion processes such as pretreatment, liquefaction, and pyrolysis often produce complex mixtures of intermediates that are a substantial challenge to analyze rapidly and reliably. To characterize these streams more comprehensively and efficiently, new techniques are needed to track species through biomass deconstruction and conversion processes. Here, we present the application of an emerging analytical method, gradient elution moving boundary electrophoresis (GEMBE), to quantify a suite of acids in a complex, biomass-derived streams from alkaline pretreatment of corn stover. GEMBE offers distinct advantages over common chromatography-spectrometry analytical approaches in terms of analysis time, sample preparation requirements, and cost of equipment.more » As demonstrated here, GEMBE is able to track 17 distinct compounds (oxalate, formate, succinate, malate, acetate, glycolate, protocatechuate, 3-hydroxypropanoate, lactate, glycerate, 2-hydroxybutanoate, 4-hydroxybenzoate, vanillate, p-coumarate, ferulate, sinapate, and acetovanillone). The lower limit of detection was compound dependent and ranged between 0.9 and 3.5 umol/L. Results from GEMBE were similar to recent results from an orthogonal method based on GCxGC-TOF/MS. Altogether, GEMBE offers a rapid, robust approach to analyze complex biomass-derived samples, and given the ease and convenience of deployment, may offer an analytical solution for online tracking of multiple types of biomass streams.« less

Application of the artificial neural network in quantitative structure-gradient elution retention relationship of phenylthiocarbamyl amino acids derivatives.

PubMed

Tham, S Y; Agatonovic-Kustrin, S

2002-05-15

Quantitative structure-retention relationship(QSRR) method was used to model reversed-phase high-performance liquid chromatography (RP-HPLC) separation of 18 selected amino acids. Retention data for phenylthiocarbamyl (PTC) amino acids derivatives were obtained using gradient elution on ODS column with mobile phase of varying acetonitrile, acetate buffer and containing 0.5 ml/l of triethylamine (TEA). Molecular structure of each amino acid was encoded with 36 calculated molecular descriptors. The correlation between the molecular descriptors and the retention time of the compounds in the calibration set was established using the genetic neural network method. A genetic algorithm (GA) was used to select important molecular descriptors and supervised artificial neural network (ANN) was used to correlate mobile phase composition and selected descriptors with the experimentally derived retention times. Retention time values were used as the network's output and calculated molecular descriptors and mobile phase composition as the inputs. The best model with five input descriptors was chosen, and the significance of the selected descriptors for amino acid separation was examined. Results confirmed the dominant role of the organic modifier in such chromatographic systems in addition to lipophilicity (log P) and molecular size and shape (topological indices) of investigated solutes.

Analysis of isothiazolinone biocides in paper for food packaging by ultra-high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Lin, Q-B; Wang, T-J; Song, H; Li, B

2010-12-01

A novel and simple method to detect isothiazolinone-type biocides (2-methyl-3-isothiazolinone (MI), 5-chloro-2-methyl-3-isothiazolinone (CMI), 1,2-benzisothiazolinone (BIT) and 2-octyl-3-isothiazolinone (OIT)) in paper used for food packaging by ultrasonic extraction coupled with UPLC-MS/MS was developed. Parameters affecting process efficiency such as extraction solvents, UPLC mobile phase, gradient elution procedure and MS/MS conditions were studied to optimise the operating conditions. Using the optimised gradient elution procedure, the retention time was less than 6 min. The limits of detection (LODs) were found to be between 0.001 and 0.010 mg kg⁻¹, which was validated using actual concentrations. After diluting the standard solution with a blank matrix, the linear calibration curve ranges were 0.002-1.000 mg kg⁻¹ for BIT and OIT, 0.005-1.000 mg kg⁻¹ for MI, and 0.020-1.000 mg kg⁻¹ for CMI, with correlation coefficients higher than 0.9985 (n = 6). A good level of precision with a mean recovery greater than 81.3% and a relative standard deviation (RSD) less than 6.2% were also obtained. A methodology has been proposed for the analysis of isothiazolinones in paper.

Gradient elution moving boundary electrophoresis enables rapid analysis of acids in complex biomass-derived streams

DOE PAGES

Munson, Matthew S.; Karp, Eric M.; Nimlos, Claire T.; ...

2016-09-27

Biomass conversion processes such as pretreatment, liquefaction, and pyrolysis often produce complex mixtures of intermediates that are a substantial challenge to analyze rapidly and reliably. To characterize these streams more comprehensively and efficiently, new techniques are needed to track species through biomass deconstruction and conversion processes. Here, we present the application of an emerging analytical method, gradient elution moving boundary electrophoresis (GEMBE), to quantify a suite of acids in a complex, biomass-derived streams from alkaline pretreatment of corn stover. GEMBE offers distinct advantages over common chromatography-spectrometry analytical approaches in terms of analysis time, sample preparation requirements, and cost of equipment.more » As demonstrated here, GEMBE is able to track 17 distinct compounds (oxalate, formate, succinate, malate, acetate, glycolate, protocatechuate, 3-hydroxypropanoate, lactate, glycerate, 2-hydroxybutanoate, 4-hydroxybenzoate, vanillate, p-coumarate, ferulate, sinapate, and acetovanillone). The lower limit of detection was compound dependent and ranged between 0.9 and 3.5 umol/L. Results from GEMBE were similar to recent results from an orthogonal method based on GCxGC-TOF/MS. Altogether, GEMBE offers a rapid, robust approach to analyze complex biomass-derived samples, and given the ease and convenience of deployment, may offer an analytical solution for online tracking of multiple types of biomass streams.« less

Comparison of retention models for polymers 1. Poly(ethylene glycol)s.

PubMed

Bashir, Mubasher A; Radke, Wolfgang

2006-10-27

The suitability of three different retention models to predict the retention times of poly(ethylene glycol)s (PEGs) in gradient and isocratic chromatography was investigated. The models investigated were the linear (LSSM) and the quadratic solvent strength model (QSSM). In addition, a model describing the retention behaviour of polymers was extended to account for gradient elution (PM). It was found that all models are suited to properly predict gradient retention volumes provided the extraction of the analyte specific parameters is performed from gradient experiments as well. The LSSM and QSSM on principle cannot describe retention behaviour under critical or SEC conditions. Since the PM is designed to cover all three modes of polymer chromatography, it is therefore superior to the other models. However, the determination of the analyte specific parameters, which are needed to calibrate the retention behaviour, strongly depend on the suitable selection of initial experiments. A useful strategy for a purposeful selection of these calibration experiments is proposed.

A new strategy for fast radiofrequency CW EPR imaging: Direct detection with rapid scan and rotating gradients

PubMed Central

Subramanian, Sankaran; Koscielniak, Janusz W.; Devasahayam, Nallathamby; Pursley, Randall H.; Pohida, Thomas J.; Krishna, Murali C.

2007-01-01

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc. PMID:17350865

Focusing of relativistic electrons in dense plasma using a resistivity-gradient-generated magnetic switchyard.

PubMed

Robinson, A P L; Key, M H; Tabak, M

2012-03-23

A method for producing a self-generated magnetic focussing structure for a beam of laser-generated relativistic electrons using a complex array of resistivity gradients is proposed and demonstrated using numerical simulations. The array of resistivity gradients is created by using a target consisting of alternating layers of different Z material. This new scheme is capable of effectively focussing the fast electrons even when the source is highly divergent. The application of this technique to cone-guided fast ignition inertial confinement fusion is considered, and it is shown that it may be possible to deposit over 25% of the fast electron energy into a hot spot even when the fast electron divergence angle is very large (e.g., 70° half-angle).

Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination.

PubMed

Snauko, Marián; Berek, Dusan

2005-11-11

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.

UHPLC determination of catechins for the quality control of green tea.

PubMed

Naldi, Marina; Fiori, Jessica; Gotti, Roberto; Périat, Aurélie; Veuthey, Jean-Luc; Guillarme, Davy; Andrisano, Vincenza

2014-01-01

An ultra-high performance liquid chromatography (UHPLC) with UV detection method was developed for the fast quantitation of the most represented and biologically important green tea catechins and caffeine. UHPLC system was equipped with C18 analytical column (50mm×2.1mm, 1.8μm), utilizing a mobile phase composed of pH 2.5 triethanolamine phosphate buffer (0.1M) and acetonitrile in a gradient elution mode; under these conditions six major catechins and caffeine were separated in a 3min run. The method was fully validated in terms of precision, detection and quantification limits, linearity, accuracy, and it was applied to the identification and quantification of catechins and caffeine present in green tea infusions. In particular, commercially available green tea leaves samples of different geographical origin (Sencha, Ceylon Green and Lung Ching) were used for infusion preparations (water at 85°C for 15min). The selectivity of the developed UHPLC method was confirmed by comparison with UHPLC-MS/MS analysis. The recovery of the main six catechins and caffeine on the three analyzed commercial tea samples ranged from 94 to 108% (n=3). Limits of detection (LOD) were comprised in the range 0.1-0.4μgmL(-1). An orthogonal micellar electrokinetic (MEKC) method was applied for comparative purposes on selectivity and quantitative data. The combined use of the results obtained by the two techniques allowed for a fast confirmation on quantitative characterization of commercial samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of multiple constituents in Chinese medicinal prescription Shensong Yangxin capsule by ultra-fast liquid chromatography combined with quadrupole time-of-flight mass spectrometry.

PubMed

Liu, Minyan; Zhao, Shaohua; Wang, Yufeng; Liu, Ting; Li, Song; Wang, Hongtao; Tu, Pengfei

2015-02-01

A practical method using ultra-fast liquid chromatography in tandem with quadrupole time-of-flight mass spectrometry combined with dynamic background subtraction technology was developed for the rapid separation and identification of the complicated constituents in the Shensong Yangxin capsule (SSYX). The chromatographic separation was performed on a C18 column (2.1 × 100 mm, 2.6 μm) with a gradient elution program using methanol and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL min(-1). Accurate mass measurements of the molecular ions in the full scan and the characteristic fragment ions triggered by information-dependent acquisition provided reliable identification criteria. Thus, 99 compounds, including saponins, phenolic acids, tanshinones, lignans, terpenoids, alkaloids and flavonoids, were unambiguously or tentatively identified in 40 min by comparing their retention times and accurate mass measurements for each molecular ion and its subsequent fragment ions with those of authentic standards or literature data. Simultaneously, all the compounds were further assigned to the individual raw materials. In conclusion, these results will provide a basis for quality control and further study of SSYX, and the proposed technique based on high-resolution mass spectrometry would be expected to be adaptable to the analysis of complicated constituents in various complex matrices. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A fast random walk algorithm for computing the pulsed-gradient spin-echo signal in multiscale porous media.

PubMed

Grebenkov, Denis S

2011-02-01

A new method for computing the signal attenuation due to restricted diffusion in a linear magnetic field gradient is proposed. A fast random walk (FRW) algorithm for simulating random trajectories of diffusing spin-bearing particles is combined with gradient encoding. As random moves of a FRW are continuously adapted to local geometrical length scales, the method is efficient for simulating pulsed-gradient spin-echo experiments in hierarchical or multiscale porous media such as concrete, sandstones, sedimentary rocks and, potentially, brain or lungs. Copyright © 2010 Elsevier Inc. All rights reserved.

Separation and quantitative determination of 6α-hydroxycortisol and 6β-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection.

PubMed

Shibasaki, Hiromi; Okamoto, Sawako; Inoue, Risako; Okita, Misato; Yokokawa, Akitomo; Furuta, Takashi

2012-03-01

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6β-hydroxycortisol (6β-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6β-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C(18) plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and (B) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH/acetonitrile (2:3, v/v). 6α-OHF and 6β-OHF were well separated on an XTerra MS C(18) 5 μm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6β-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6β-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6β-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6β-OHF determined were in good agreement with the actual amounts added, the relative errors being -5.37% and -3.73% (gradient 2) and -5.69% and -3.96% (gradient 3) for both 6α-OHF and 6β-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6β-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6β-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6β-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.

Unusually high levels of bisphenol A (BPA) in thermal paper cash register receipts (CRs): development and application of a robust LC-UV method to quantify BPA in CRs.

PubMed

Babu, Sainath; Uppu, Sannihith N; Martin, Brittany; Agu, Ogad A; Uppu, Rao M

2015-01-01

We have developed a simple, reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of bisphenol A (BPA) in thermal paper cash register receipts (CRs). The method is suitable for analysis of other types of bisphenols and it involves an overnight extraction of CRs with acetonitrile (AN) at 50 °C followed by the HPLC analysis on a Supelcosil LC18 column (150 × 4.6 mm, particle size: 5 μ) using 50% AN in water as the mobile phase (5 min, isocratic). The composition of AN in the mobile phase changed to 100% over a 10 min period (linear gradient) and then held at 100% AN for 10 min (isocratic). The flow rate was set at 1 mL/min (injection volume: 20 μL) and the eluent was monitored at 234 nm. The authentic BPA eluted with a retention time of 5.9 min and gave a linear detector response in the concentration range of 0.23-50 mg/L. BPA in the CR extracts also eluted with the same retention and had identical absorbance properties as the standard. When CR extracts were co-injected with authentic BPA, they were resolved as a single peak. Further, GC/MS/EI analysis of authentic BPA and the HPLC-purified CR extracts have identical ion chromatograms and fragmentation of the molecular ion (m/z = 228). We have analyzed 170 CRs collected from 62 different vendors including supermarkets, fast food restaurants, gas stations and banking outlets. Almost all cash receipts (n = 168) showed the presence of BPA in the concentration range of 0.45-4.26% (M ± SD, 1.54 ± 0.73%).

High-performance cation-exchange chromatofocusing of proteins.

PubMed

Kang, Xuezhen; Frey, Douglas D

2003-03-28

Chromatofocusing using high-performance cation-exchange column packings, as opposed to the more commonly used anion-exchange column packings, is investigated with regard to the performance achieved and the range of applications possible. Linear or convex gradients in the range from pH 2.6 to 9 were formed using a variety of commercially available column packings that provide a buffering capacity in different pH ranges, and either polyampholytes or simple mixtures having a small number (three or fewer) of buffering species as the elution buffer. The resolutions achieved using cation-exchange or anion-exchange chromatofocusing were in general comparable, although for certain pairs of proteins better resolution could be achieved using one type of packing as compared to the other, evidently due to the way electrostatic charges are distributed on the protein surface. Several chromatofocusing methods were investigated that take advantage of the acid-base properties of commercially available cation-exchange column packings. These include the use of gradients with a composite shape, the use of very low pH ranges, and the use of elution buffers containing a single buffering species. The advantages of chromatofocusing over ion-exchange chromatography using a salt gradient at constant pH were illustrated by employing the former method and a cation-exchange column packing to separate beta-lactoglobulins A and B, which is a separation reported to be impossible using the latter method and a cation-exchange column packing. Trends in the apparent isoelectric points determined using cation- and anion-exchange chromatofocusing were interpreted using applicable theories. Results of this study indicate that cation-exchange chromatofocusing is a useful technique which is complementary to anion-exchange chromatofocusing and isoelectric focusing for separating proteins at both the analytical and preparative scales.

Improved optimization of polycyclic aromatic hydrocarbons (PAHs) mixtures resolution in reversed-phase high-performance liquid chromatography by using factorial design and response surface methodology.

PubMed

Andrade-Eiroa, Auréa; Diévart, Pascal; Dagaut, Philippe

2010-04-15

A new procedure for optimizing PAHs separation in very complex mixtures by reverse phase high performance (RPLC) is proposed. It is based on changing gradually the experimental conditions all along the chromatographic procedure as a function of the physical properties of the compounds eluted. The temperature and speed flow gradients allowed obtaining the optimum resolution in large chromatographic determinations where PAHs with very different medium polarizability have to be separated. Whereas optimization procedures of RPLC methodologies had always been accomplished regardless of the physico-chemical properties of the target analytes, we found that resolution is highly dependent on the physico-chemical properties of the target analytes. Based on resolution criterion, optimization process for a 16 EPA PAHs mixture was performed on three sets of difficult-to-separate PAHs pairs: acenaphthene-fluorene (for the optimization procedure in the first part of the chromatogram where light PAHs elute), benzo[g,h,i]perylene-dibenzo[a,h]anthracene and benzo[g,h,i]perylene-indeno[1,2,3-cd]pyrene (for the optimization procedure of the second part of the chromatogram where the heavier PAHs elute). Two-level full factorial designs were applied to detect interactions among variables to be optimized: speed flow, temperature of column oven and mobile-phase gradient in the two parts of the studied chromatogram. Experimental data were fitted by multivariate nonlinear regression models and optimum values of speed flow and temperature were obtained through mathematical analysis of the constructed models. An HPLC system equipped with a reversed phase 5 microm C18, 250 mm x 4.6mm column (with acetonitrile/water mobile phase), a column oven, a binary pump, a photodiode array detector (PDA), and a fluorimetric detector were used in this work. Optimum resolution was achieved operating at 1.0 mL/min in the first part of the chromatogram (until 45 min) and 0.5 mL/min in the second one (from 45 min to the end) and by applying programmed temperature gradient (15 degrees C until 30 min and progressively increasing temperature until reaching 40 degrees C at 45 min). (c) 2009 Elsevier B.V. All rights reserved.

Characterization of cellulose acetates according to DS and molar mass using two-dimensional chromatography.

PubMed

Ghareeb, Hewa Othman; Radke, Wolfgang

2013-11-06

A two-dimensional liquid chromatographic method (2D LC) was developed to analyze the heterogeneities of cellulose acetates (CA) in the DS-range DS=1.5-2.9 with respect to both, molar mass and degree of substitution (DS). The method uses gradient liquid chromatography (HPLC) as the first dimension in order to separate by DS followed by separation of the different fractions by size (SEC) in the second dimension. The 2D experiments revealed different correlations between gradient and SEC elution volume. These correlations might arise from differences in the synthetic conditions. The newly developed 2D LC separation therefore provides new insights into the heterogeneity of CAs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Contour Detection and Completion for Inpainting and Segmentation Based on Topological Gradient and Fast Marching Algorithms

PubMed Central

Auroux, Didier; Cohen, Laurent D.; Masmoudi, Mohamed

2011-01-01

We combine in this paper the topological gradient, which is a powerful method for edge detection in image processing, and a variant of the minimal path method in order to find connected contours. The topological gradient provides a more global analysis of the image than the standard gradient and identifies the main edges of an image. Several image processing problems (e.g., inpainting and segmentation) require continuous contours. For this purpose, we consider the fast marching algorithm in order to find minimal paths in the topological gradient image. This coupled algorithm quickly provides accurate and connected contours. We present then two numerical applications, to image inpainting and segmentation, of this hybrid algorithm. PMID:22194734

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

PubMed

Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2015-01-01

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation conditions for seven phenolic compounds was also achieved using reversed-phase HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and seven phenolic compounds could be separated and detected at 230 nm within 16 min. Copyright © 2014 Elsevier B.V. All rights reserved.

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase.

PubMed

Rodriguez-Aller, Marta; Guillarme, Davy; Beck, Alain; Fekete, Szabolcs

2016-01-25

The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼ 1%. Copyright © 2015 Elsevier B.V. All rights reserved.

Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column.

PubMed

Magda, Balázs; Márta, Zoltán; Imre, Tímea; Kalapos-Kovács, Bernadett; Klebovich, Imre; Fekete, Jenő; Szabó, Pál T

2015-01-01

The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation ("inverse gradient elution" on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, i.d.; 5 μm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r = 0.9987) over the concentration range from 1 to 1000 nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0-10.2% and -6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1 nM (0.446 ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides. Copyright © 2015. Published by Elsevier B.V.

Simultaneous determination of some ultraviolet-absorbing chemicals in sunscreen cosmetics using a high-performance liquid chromatography method.

PubMed

Liu, T; Wu, D

2011-10-01

A method of gradient elution high-performance liquid chromatography (HPLC) for simultaneous determination of 11 different ultraviolet-absorbing chemicals of phenylbenzlmldazole sulphonic acid, 4-aminobenzoic acid, benzophenone-4, benzophenone-3, isoamyl p-methoxycinnamate, 4-methylbenzylidene camphor, octocrylene, ethylhexyl methoxycinnamate, homosalate, ethylhexyl salicylate, methylene bis-benzotriazolyl tetramethylbutyl phenol was developed for the application to sunscreen cosmetic products. In this study, an Agilent SB-C18 analytical column (250 × 4.6 mm, 5 μm) was utilized and methanol, tetrahydrofuran and perchloric acid aqueous solution (0.2 mL HClO(4) + 300 mL H(2)O) were used for gradient elution at a total flow rate of 1.0 mL min(-1). The optimum conditions for 11 different ultraviolet-absorbing chemicals analyses were investigated. All calibration curves showed good linear regression with UV detection (311 nm) within test ranges. The correlation coefficients were better than 0.999 in all cases. The assay was simple, selective, convenient and reproducible and is suitable for the determination of ultraviolet-absorbing chemicals in commercial sunscreen cosmetic products. The use frequency of 11 different ultraviolet absorbents in 100 sunscreen cosmetics was investigated and statistically analysed. The ultraviolet absorbent of maximum use frequency was ethylhexyl methoxycinnamate. © 2011 The Authors. ICS © 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

PubMed

Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

2013-11-01

Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH

Direct injection ion chromatography for the control of chlorinated drinking water: simultaneous estimation of nine haloacetic acids and quantitation of bromate, chlorite and chlorate along with the major inorganic anions.

PubMed

Garcia-Villanova, Rafael J; Raposo Funcia, César; Oliveira Dantas Leite, M Vilani; Toruño Fonseca, Ivania M; Espinosa Nieto, Miguel; Espuelas India, Javier

2014-09-01

Most methods for the analysis of haloacetic acids published in recent years are based on ion chromatography with direct injection, employing a gradient elution with potassium hydroxide (KOH). This work reports the exploration of an alternative eluent, a buffer of sodium carbonate/sodium hydrogen carbonate, aimed at the simultaneous analysis of nine haloacetic acids along with bromate, chlorite and chlorate. The alternative of both a less alkaline eluent and a lower temperature of operation may prevent the partial decomposition of some of the haloacetic acids during the analytical process, especially the more vulnerable brominated ones. Gradient elution at temperature of 7 °C yielded the best results, with an acceptable separation of 17 analytes (which includes the major natural inorganic anions) and a good linearity. Precision ranges from 0.3 to 23.4 (% V.C.), and detection limits are within units of μg L⁻¹, except for tribromoacetic acid - somewhat high in comparison with those of the official methods. Nonetheless, with the basic instrumentation setup herein described, this method may be suitable for monitoring when the drinking water treatments are to be optimized. This is especially interesting for small communities or for developing/developed countries in which regulations on disinfection by-products others than trihalomethanes are being addressed.

Synthetic oligonucleotide separations by mixed-mode reversed-phase/weak anion-exchange liquid chromatography.

PubMed

Zimmermann, Aleksandra; Greco, Roberto; Walker, Isabel; Horak, Jeannie; Cavazzini, Alberto; Lämmerhofer, Michael

2014-08-08

Synthetic oligonucleotides gain increasing importance in new therapeutic concepts and as probes in biological sciences. If pharmaceutical-grade purities are required, chromatographic purification using ion-pair reversed-phase chromatography is commonly carried out. However, separation selectivity for structurally closely related impurities is often insufficient, especially at high sample loads. In this study, a "mixed-mode" reversed-phase/weak anion exchanger stationary phase has been investigated as an alternative tool for chromatographic separation of synthetic oligonucleotides with minor sequence variations. The employed mixed-mode phase shows great flexibility in method development. It has been run in various gradient elution modes, viz. one, two or three parameter (mixed) gradients (altering buffer pH, buffer concentration, and organic modifier) to find optimal elution conditions and gain further insight into retention mechanisms. Compared to ion-pair reversed-phase and mere anion-exchange separation, enhanced selectivities were observed with the mixed-mode phase for 20-23 nucleotide (nt) long oligonucleotides with similar sequences. Oligonucleotides differing by 1, 2 or 3 nucleotides in length could be readily resolved and separation factors for single nucleotide replacements declined in the order Cytosine (C)/Guanine (G)>Adenine (A)/Guanine∼Guanine/Thymine (T)>Adenine/Cytosine∼Cytosine/Thymine>Adenine/Thymine. Selectivities were larger when the modification was at the 3' terminal-end, declined when it was in the middle of the sequence and was smallest when it was located at the 5' terminus. Due to the lower surface area of the 200Å pore size mixed-mode stationary phase compared to the corresponding 100Å material, lower retention times with equal selectivities under milder elution conditions were achievable. Considering high sample loading capacities of the mixed-mode anion-exchanger phase, it should have great potential for chromatographic oligonucleotide separation and purification. Copyright © 2014 Elsevier B.V. All rights reserved.

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.

PubMed

Xu, Zhihao; Li, Jason; Zhou, Joe X

2012-01-01

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.

Development and application of a specially designed heating system for temperature-programmed high-performance liquid chromatography using subcritical water as the mobile phase.

PubMed

Teutenberg, T; Goetze, H-J; Tuerk, J; Ploeger, J; Kiffmeyer, T K; Schmidt, K G; Kohorst, W gr; Rohe, T; Jansen, H-D; Weber, H

2006-05-05

A specially designed heating system for temperature-programmed HPLC was developed based on experimental measurements of eluent temperature inside a stainless steel capillary using a very thin thermocouple. The heating system can be operated at temperatures up to 225 degrees C and consists of a preheating, a column heating and a cooling unit. Fast cycle times after a temperature gradient can be realized by an internal silicone oil bath which cools down the preheating and column heating unit. Long-term thermal stability of a polybutadiene-coated zirconium dioxide column has been evaluated using a tubular oven in which the column was placed. The packing material was stable after 50h of operation at 185 degrees C. A mixture containing four steroids was separated at ambient conditions using a mobile phase of 25% acetonitrile:75% deionized water and a mobile phase of pure deionized water at 185 degrees C using the specially designed heating system and the PBD column. Analysis time could be drastically reduced from 17 min at ambient conditions and a flow rate of 1 mL/min to only 1.2 min at 185 degrees C and a flow rate of 5 mL/min. At these extreme conditions, no thermal mismatch was observed and peaks were not distorted, thus underlining the performance of the developed heating system. Temperature programming was performed by separating cytostatic and antibiotic drugs with a temperature gradient using only water as the mobile phase. In contrast to an isocratic elution of this mixture at room temperature, overall analysis time could be reduced two-fold from 20 to 10 min.

Effect of the thermal environment on the efficiency of packed columns in supercritical fluid chromatography.

PubMed

Zauner, Jordan; Lusk, Ryan; Koski, Steven; Poe, Donald P

2012-11-30

When a packed column is operated at temperatures and pressures near the critical point in supercritical fluid chromatography, the thermal environment in which it is placed has a significant impact on retention and efficiency. We measured the retention factors, plate heights, and related parameters for elution of a test mixture of alkylbenzenes with 5% methanol/95% carbon dioxide mobile phase on a 250 mm × 4.6 mm i.d. column packed with 5-micron Luna-C18 particles. Separations were performed at outlet pressures from 100 to 150 bar and a column oven temperature of 323K. For a bare column thermostated with convective air, significant efficiency losses were observed for outlet pressures equal to or less than 120 bar. These large efficiency losses are attributed to radial temperature gradients. Addition of foam insulation resulted in significant improvements in efficiency. Operating the column in still air using a commercially available column heater provided the best overall performance, with no measurable efficiency loss over the entire range of pressures studied. A reduced plate height of 1.88 was obtained at an optimum flow rate of 3.0 mL/min at 100 bar outlet pressure and with the temperature of the incoming mobile phase set approximately 2.3K above the temperature of the column oven. Retention time repeatability for all three thermal conditions was equal to or less than 0.5% RSD. These results demonstrate that it is possible to perform fast, efficient separations with excellent repeatability using SFC under near-critical conditions if the thermal environment is optimized to minimize the generation of radial temperature gradients. Copyright © 2012 Elsevier B.V. All rights reserved.

A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl

2009-10-02

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this systemmore » using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.« less

Assessment study of ion-exchange chromatography combined with solution X-ray scattering measurement for protein characterization.

PubMed

Watanabe, Yasushi

2018-03-02

The performance of ion-exchange chromatography combined with small-angle X-ray scattering measurement was evaluated by characterization of the hen egg white lysozyme as a model protein. The X-ray transmittance was estimated using a micro-ionization chamber equipped with a sample cell holder for the real-time monitoring of the X-ray beam strength through the salt gradient elution. The radius of gyration of the eluted protein was estimated to be 1.50 ± 0.06 (n = 3) nm and 1.4 ± 0.1 nm as the value at the zero protein concentration. By using the X-ray transmittance values for the scattering intensity correction, the molecular weight of the eluted protein was estimated to be 15,200 ± 500 (n = 3) and 14,400 ± 200 as the value at the zero protein concentration. These values are close to those of the monomer of this protein. The ion-exchange chromatography combined with the small-angle X-ray scattering measurement system equipped with the X-ray transmittance monitor is a reliable method for protein characterization in solution. Copyright © 2018 Elsevier B.V. All rights reserved.

[Determination of 25 quinolones in cosmetics by liquid chromatography-tandem mass spectrometry].

PubMed

Lin, Li; Zhang, Yi; Tu, Xiaoke; Xie, Liqi; Yue, Zhenfeng; Kang, Haining; Wu, Weidong; Luo, Yao

2015-03-01

An analytical method was developed for the simultaneous determination of 25 quinolones, including danofloxacin mesylate, enrofloxacin, flumequine, oxloinic acid, ciprofloxacin, sarafloxacin, nalidixic acid, norfloxacin, and ofloxacin etc in cosmetics using direct extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Cosmetic sample was extracted by acidified acetonitrile, defatted by n-hexane and separated on Poroshell EC-C18 column with gradient elution program using acetonitrile and water (both containing 0. 1% formic acid) as the mobile phases and analyzed by LC-ESI-MS/MS under the positive mode using multiple reaction monitoring (MRM). The interference of matrix was reduced by the matrix-matched calibration standard curve. The method showed good linearities over the range of 1-200 mg/kg for the 25 quinolones with good linear correlation coefficients (r ≥ 0.999). The method detection limit of the 25 quinolones was 1.0 mg/kg, and the recoveries of all analytes in lotion, milky and cream cosmetics matrices ranged from 87.4% to 105% at the spiked levels of 1, 5 and 10 mg/kg with the relative standard deviations (RSD) of 4.54%-19.7% (n = 6). The results indicated that this method is simple, fast and credible, and suitable for the simultaneous determination of the quinolones in the above three types of cosmetics.

Ultrafast quantification of β-lactam antibiotics in human plasma using UPLC-MS/MS.

PubMed

Carlier, Mieke; Stove, Veronique; De Waele, Jan J; Verstraete, Alain G

2015-01-26

There is an increasing interest in monitoring plasma concentrations of β-lactam antibiotics. The objective of this work was to develop and validate a fast ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC-MS/MS) for simultaneous quantification of amoxicillin, cefuroxime, ceftazidime, meropenem and piperacillin with minimal turn around time. Sample clean-up included protein precipitation with acetonitrile containing 5 deuterated internal standards, and subsequent dilution of the supernatant with water after centrifugation. Runtime was only 2.5 min. Chromatographic separation was performed on a Waters Acquity UPLC system using a BEH C18 column (1.7 μm, 100 mm × 2.1 mm) applying a binary gradient elution of water and methanol both containing 0.1% formic acid and 2 mmol/L ammonium acetate on a Water TQD instrument in MRM mode. All compounds were detected in electrospray positive ion mode and could be quantified between 1 and 100 mg/L for amoxicillin and cefuroxime, between 0.5 and 80 mg/L for meropenem and ceftazidime, and between 1 and 150 mg/L for piperacillin. The method was validated in terms of precision, accuracy, linearity, matrix effect and recovery and has been compared to a previously published UPLC-MS/MS method. Copyright © 2014 Elsevier B.V. All rights reserved.

Ultra-performance liquid chromatography fingerprinting for quality control of Phragmitis rhizoma (Lugen) produced in Baiyangdian

PubMed Central

Li, Hong; Gao, Yu-Mei; Zhang, Jing; Wang, Lin; Wang, Xiao-Xin

2013-01-01

Objective: To establish an ultra-performance liquid chromatography (UPLC) fingerprinting method for quality control of Phragmitis rhizoma from Baiyangdian. Materials and Methods: Ultrasonic extraction with 70% methanol was performed on 10 samples of P. rhizoma collected from 10 different villages in Baiyangdian. The sample solutions were analyzed by Waters UPLC equipped with the ACQUITY UPLC BEH C18 column and photodiode array (PDA) detector, and gradient eluted with acetonitrile/water as the mobile phase. The flow rate was set to 0.1 mL/min; the column temperature was set to 25°C; and the detection wavelength was set to 285 nm. Results: The chromatograms of the 10 samples showed 27 common peaks, of which one was identified as the ferulic acid standard. The similarity indexes were all above 0.82. Hierarchical cluster analysis showed that the constituents and their quantities differed according to the diameter of the original plant, which is related to its age. Conclusion: The UPLC fingerprinting method had the advantages of being fast, accurate, and highly efficient; this indicated that it can be used for quality control of P. rhizoma produced in Baiyangdian. Also, the relation between the quality and diameter/age of the plant needs to be further investigated. PMID:24124278

Randomized comparison of Zotarolimus-Eluting Endeavor Sprint versus bare-metal stent implantation in uncertain drug-eluting stent candidates: rationale, design, and characterization of the patient population for the Zotarolimus-eluting Endeavor Sprint stent in uncertain DES candidates study.

PubMed

Valgimigli, Marco; Patialiakas, Athanasios; Thury, Attila; Colangelo, Salvatore; Campo, Gianluca; Tebaldi, Matteo; Ungi, Imre; Tondi, Stefano; Roffi, Marco; Menozzi, Alberto; de Cesare, Nicoletta; Garbo, Roberto; Meliga, Emanuele; Testa, Luca; Gabriel, Henrique M; Airoldi, Flavio; Ferlini, Marco; Liistro, Francesco; Dellavalle, Antonio; Vranckx, Pascal; Briguori, Carlo

2013-11-01

The use of drug-eluting stent (DES) instead of bare-metal stent (BMS) in patients at high stent thrombosis or bleeding risk as well as in those at low restenosis risk (ie, uncertain DES candidates) remains a matter of debate. Zotarolimus-Eluting Endeavor Sprint stent (E-ZES) (Santa Rosa, CA) is a hydrophilic polymer-based second-generation device with unique drug fast-release profile, which may allow for a shorter dual antiplatelet therapy (DAPT) duration without safety concerns. The primary objective is to assess whether E-ZES implantation followed by a shorter than currently recommended course of DAPT will decrease the incidence of 12-month major adverse cardiovascular events as compared with BMS in undefined DES recipients. Actual duration of DAPT regimen will be dictated by patients' characteristics and not by stent type and, as such, can be as short as 30 days after intervention in both stent groups. The ZEUS study is an open-label randomized clinical trial conducted at 20 clinical sites in Italy, Switzerland, Portugal, and Hungary. With 1,600 individuals, this study will have 85% power to detect a 33% difference in the primary end point consisting of the composite of death, nonfatal myocardial infarction, or target vessel revascularization. The ZEUS trial aims to assess whether the use of E-ZES, followed by a DAPT duration regimen based on patients' characteristics and not by stent type, is superior to conventional BMS implantation in undefined DES recipients who qualify for the presence of high thrombosis, bleeding, or low restenosis risk criteria. © 2013.

Combined solvent- and non-uniform temperature-programmed gradient liquid chromatography. I - A theoretical investigation.

PubMed

Gritti, Fabrice

2016-11-18

An new class of gradient liquid chromatography (GLC) is proposed and its performance is analyzed from a theoretical viewpoint. During the course of such gradients, both the solvent strength and the column temperature are simultaneously changed in time and space. The solvent and temperature gradients propagate along the chromatographic column at their own and independent linear velocity. This class of gradient is called combined solvent- and temperature-programmed gradient liquid chromatography (CST-GLC). The general expressions of the retention time, retention factor, and of the temporal peak width of the analytes at elution in CST-GLC are derived for linear solvent strength (LSS) retention models, modified van't Hoff retention behavior, linear and non-distorted solvent gradients, and for linear temperature gradients. In these conditions, the theory predicts that CST-GLC is equivalent to a unique and apparent dynamic solvent gradient. The apparent solvent gradient steepness is the sum of the solvent and temperature steepness. The apparent solvent linear velocity is the reciprocal of the steepness-averaged sum of the reciprocal of the actual solvent and temperature linear velocities. The advantage of CST-GLC over conventional GLC is demonstrated for the resolution of protein digests (peptide mapping) when applying smooth, retained, and linear acetonitrile gradients in combination with a linear temperature gradient (from 20°C to 90°C) using 300μm×150mm capillary columns packed with sub-2 μm particles. The benefit of CST-GLC is demonstrated when the temperature gradient propagates at the same velocity as the chromatographic speed. The experimental proof-of-concept for the realization of temperature ramps propagating at a finite and constant linear velocity is also briefly described. Copyright © 2016 Elsevier B.V. All rights reserved.

Intelligent peak deconvolution through in-depth study of the data matrix from liquid chromatography coupled with a photo-diode array detector applied to pharmaceutical analysis.

PubMed

Arase, Shuntaro; Horie, Kanta; Kato, Takashi; Noda, Akira; Mito, Yasuhiro; Takahashi, Masatoshi; Yanagisawa, Toshinobu

2016-10-21

Multivariate curve resolution-alternating least squares (MCR-ALS) method was investigated for its potential to accelerate pharmaceutical research and development. The fast and efficient separation of complex mixtures consisting of multiple components, including impurities as well as major drug substances, remains a challenging application for liquid chromatography in the field of pharmaceutical analysis. In this paper we suggest an integrated analysis algorithm functioning on a matrix of data generated from HPLC coupled with photo-diode array detector (HPLC-PDA) and consisting of the mathematical program for the developed multivariate curve resolution method using an expectation maximization (EM) algorithm with a bidirectional exponentially modified Gaussian (BEMG) model function as a constraint for chromatograms and numerous PDA spectra aligned with time axis. The algorithm provided less than ±1.0% error between true and separated peak area values at resolution (R s ) of 0.6 using simulation data for a three-component mixture with an elution order of a/b/c with similarity (a/b)=0.8410, (b/c)=0.9123 and (a/c)=0.9809 of spectra at peak apex. This software concept provides fast and robust separation analysis even when method development efforts fail to achieve complete separation of the target peaks. Additionally, this approach is potentially applicable to peak deconvolution, allowing quantitative analysis of co-eluted compounds having exactly the same molecular weight. This is complementary to the use of LC-MS to perform quantitative analysis on co-eluted compounds using selected ions to differentiate the proportion of response attributable to each compound. Copyright © 2016 Elsevier B.V. All rights reserved.

Poly(1-vinylimidazole) functionalized magnetic ion imprinted polymer for fast and selective extraction of trace gold in geological, environmental and biological samples followed by graphite furnace atomic absorption spectrometry detection

NASA Astrophysics Data System (ADS)

Zhao, Bingshan; He, Man; Chen, Beibei; Xu, Hongrun; Hu, Bin

2018-05-01

In this study, poly(1-vinylimidazole) functionalized gold ion imprinted polymer coated magnetic nanoparticles (MNPs@PVIM-Au-IIP) were prepared and characterized. The adsorption behaviors of the prepared MNPs@PVIM-Au-IIP toward gold ions (Au(III)) were studied, it was found that MNPs@PVIM-Au-IIP has good selectivity, high adsorption capacity (185.4 mg g-1) and fast adsorption kinetic for Au(III). Based on it, a new method of ion imprinted magnetic solid phase extraction (II-MSPE) coupled with graphite furnace atomic absorption spectrometry (GFAAS) detection was proposed for the analysis of trace Au(III) in real samples with complicated matrix. Factors affecting MSPE including sample pH, desorption reagent, elution concentration and volume, elution time, sample volume and adsorption time were optimized. With high enrichment factor of 100-fold, the detection limit of the proposed method is 7.9 ng L-1 for Au(III) with the relative standard deviation of 7.4% (c = 50 ng L-1, n = 7). In order to validate the accuracy of the proposed method, the Certified Reference Material of GBW07293 geological sample (platinpalladium ore) was analyzed, and the determined value was in good agreement with the certified value. The proposed II-MSPE-GFAAS method is simple, fast, selective, sensitive and has been successfully applied in the determination of trace Au in ore, sediment, environmental water and human urine samples with satisfactory results.

The Effect of Concomitant Fields in Fast Spin Echo Acquisition on Asymmetric MRI Gradient Systems

PubMed Central

Tao, Shengzhen; Weavers, Paul T.; Trzasko, Joshua D.; Huston, John; Shu, Yunhong; Gray, Erin M.; Foo, Thomas K.F.; Bernstein, Matt A.

2017-01-01

Purpose To investigate the effect of the asymmetric gradient concomitant fields (CF) with zeroth and first-order spatial dependence on fast/turbo spin-echo acquisitions, and to demonstrate the effectiveness of their real-time compensation. Methods After briefly reviewing the CF produced by asymmetric gradients, the effects of the additional zeroth and first-order CFs on these systems are investigated using extended-phase graph simulations. Phantom and in vivo experiments are performed to corroborate the simulation. Experiments are performed before and after the real-time compensations using frequency tracking and gradient pre-emphasis to demonstrate their effectiveness in correcting the additional CFs. The interaction between the CFs and prescan-based correction to compensate for eddy currents is also investigated. Results It is demonstrated that, unlike the second-order CFs on conventional gradients, the additional zeroth/first-order CFs on asymmetric gradients cause substantial signal loss and dark banding in fast spin-echo acquisitions within a typical brain-scan field of view. They can confound the prescan correction for eddy currents and degrade image quality. Performing real-time compensation successfully eliminates the artifacts. Conclusions We demonstrate that the zeroth/first-order CFs specific to asymmetric gradients can cause substantial artifacts, including signal loss and dark bands for brain imaging. These effects can be corrected using real-time compensation. PMID:28643408

The association between socioeconomic status and adult fast-food consumption in the U.S.

PubMed

Zagorsky, Jay L; Smith, Patricia K

2017-11-01

Health follows a socioeconomic status (SES) gradient in developed countries, with disease prevalence falling as SES rises. This pattern is partially attributed to differences in nutritional intake, with the poor eating the least healthy diets. This paper examines whether there is an SES gradient in one specific aspect of nutrition: fast-food consumption. Fast food is generally high in calories and low in nutrients. We use data from the 2008, 2010, and 2012 waves of the National Longitudinal Survey of Youth (NLSY79) to test whether adult fast-food consumption in the United States falls as monetary resources rise (n=8136). This research uses more recent data than previous fast-food studies and includes a comprehensive measure of wealth in addition to income to measure SES. We find little evidence of a gradient in adult fast-food consumption with respect to wealth. While adults in the highest quintile are 54.5% less likely to report fast-food consumption than those in the lowest quintile, adults in the second and third quintiles are no less likely to report fast food-food intake than the poorest. Contrary to popular belief, fast-food consumption rises as income rises from the lowest to middle quintiles. The variation in adult fast-food consumption across income and wealth groups is, however, small. Those in the wealthiest quintile ate about one less fast-food meal on average than those in the lowest quintile. Other factors play a bigger role in explaining fast-food consumption: reading ingredient labels is negatively associated while soda consumption and hours of work are positively associated with fast-food consumption. Copyright © 2017 Elsevier B.V. All rights reserved.

Gas amplified ionization detector for gas chromatography

DOEpatents

Huston, Gregg C.

1992-01-01

A gas-amplified ionization detector for gas chromatrography which possesses increased sensitivity and a very fast response time. Solutes eluding from a gas chromatographic column are ionized by UV photoionization of matter eluting therefrom. The detector is capable of generating easily measured voltage signals by gas amplification/multiplication of electron products resulting from the UV photoionization of at least a portion of each solute passing through the detector.

Determination of soluble bromine in an extra-high-pressure mercury discharge lamp by sodium hydroxide decomposition-suppressed ion chromatography.

PubMed

Mitsumata, Hiroshi; Mori, Toshio; Maeda, Tatsuo; Kita, Yoshiyuki; Kohatsu, Osamu

2006-02-01

We have established a simple method for assaying the quantity of soluble bromine in the discharge tubes of an extra-high-pressure mercury discharge lamp. Each discharge tube is destroyed in 5 ml of 10 mM sodium hydroxide, and the recovered sodium hydroxide solution is analyzed by suppressed-ion chromatography using gradient elution. We have clarified that this method can assay less than 1 microg of soluble bromine in a discharge tube.

Acoustic emission analysis of crack resistance and fracture behavior of 20GL steel having the gradient microstructure and strength

NASA Astrophysics Data System (ADS)

Nikulin, S.; Nikitin, A.; Belov, V.; Rozhnov, A.; Turilina, V.; Anikeenko, V.; Khatkevich, V.

2017-07-01

The crack resistances as well as fracture behavior of 20GL steel quenched with a fast-moving water stream and having gradient microstructure and strength are analyzed. Crack resistance tests with quenched and normalized flat rectangular specimens having different cut lengths loaded by three-point bending with acoustic emission measurements have been performed. The critical J-integral has been used as the crack resistance parameter of the material. Quenching with a fast moving water stream leads to gradient (along a specimen wall thickness) strengthening of steel due to highly refined gradient microstructure formation of the troostomartensite type. Quenching with a fast-moving water stream increases crack resistance Jc , of 20GL steel by a factor of ∼ 1.5. The fracture accrues gradually with the load in the normalized specimens while the initiated crack is hindered in the variable ductility layer and further arrested in the more ductile core in the quenched specimens.

Fast simultaneous analysis of caffeine, trigonelline, nicotinic acid and sucrose in coffee by liquid chromatography-mass spectrometry.

PubMed

Perrone, Daniel; Donangelo, Carmen Marino; Farah, Adriana

2008-10-15

A rapid liquid chromatography-mass spectrometry method for the simultaneous quantification of caffeine, trigonelline, nicotinic acid and sucrose in coffee was developed and validated. The method involved extraction with hot water, clarification with basic lead acetate and membrane filtration, followed by chromatographic separation using a Spherisorb(®) S5 ODS2, 5μm chromatographic column and gradient elution with 0.3% aqueous formic acid/methanol at a flow rate of 0.2mL/min. The electrospray ionization source was operated in the negative mode to generate sucrose ions and in the positive mode to generate caffeine, trigonelline and nicotinic acid ions. Ionization suppression of all analytes was found due to matrix effect. Calibrations curves prepared in green and roasted coffee extracts were linear with r(2)>0.999. Roasted coffee was spiked and recoveries ranged from 93.0% to 105.1% for caffeine, from 85.2% to 116.2% for trigonelline, from 89.6% to 113.5% for nicotinic acid and from 94.1% to 109.7% for sucrose. Good repeatibilities (RSD<5%) were found for all analytes in the matrix. The limit of detection (LOD), calculated on the basis of signal-to-noise ratios of 3:1, was 11.9, 36.4, 18.5 and 5.0ng/mL for caffeine, trigonelline, nicotinic acid and sucrose, respectively. Analysis of 11 coffee samples (regular or decaffeinated green, ground roasted and instant) gave results in agreement with the literature. The method showed to be suitable for different types of coffee available in the market thus appearing as a fast and reliable alternative method to be used for routine coffee analysis. Copyright © 2008 Elsevier Ltd. All rights reserved.

Quantitative determination of hederagenin in rat plasma and cerebrospinal fluid by ultra fast liquid chromatography-tandem mass spectrometry method.

PubMed

Yang, Xuemei; Li, Guoliang; Chen, Lingyun; Zhang, Cong; Wan, Xinxiang; Xu, Jiangping

2011-07-01

A rapid, sensitive and selective method was developed for the quantitative determination of hederagenin in rat plasma and cerebrospinal fluid (CSF) by ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). It has been successfully applied in a pharmacokinetic study of hederagenin in the central nervous system (CNS). Sample pretreatment involved a simple protein precipitation with methanol and a one-step extraction with ethyl acetate. Separation was carried out in a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d., 2.1 μm) column with gradient elution at a flow rate of 0.35 mL/min. The mobile phase was 5mM ammonium acetate and acetonitrile. Detection was performed in a triple-quadruple tandem mass spectrometer by multiple-reaction-monitoring mode via electrospray ionization. A linear calibration curve for hederagenin was obtained over a concentration range of 0.406 (lower limit of quantification, LLOQ) to 203 ng/mL (r² > 0.99) for both plasma and CSF. The intra-day and inter-day precision (relative standard deviation, RSD) values were less than 15%. At all quality control (QC) levels, the accuracy (relative error, RE) was within -9.0% and 11.1% for plasma and CSF, respectively. The pharmacokinetics results indicated that hederagenin could pass through the blood-brain barrier. This UFLC-MS/MS method demonstrates higher sensitivity and sample throughput than previous methods. It was also successfully applied to the pharmacokinetic study of hederagenin following oral administration of Fructus akebiae extract in rats. Copyright © 2011 Elsevier B.V. All rights reserved.

Analysis of perfluorinated chemicals in umbilical cord blood by ultra-high performance liquid chromatography/tandem mass spectrometry.

PubMed

Lien, Guang-Wen; Wen, Ting-Wen; Hsieh, Wu-Shiun; Wu, Kuen-Yuh; Chen, Chia-Yang; Chen, Pau-Chung

2011-03-15

Perfluorinated compounds (PFCs) can cross the placental barrier and enter fetal circulation. This study aimed at developing a fast and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry method for the determination of twelve perfluorinated compounds in cord blood. Samples were processed with protein precipitation using formic acid and methanol, mixed with stable isotope labeled standard, followed by sonication and centrifugation, and were analyzed using a Waters ACQUITY UPLC coupled with a Waters Quattro Premier XE triple-quadrupole mass spectrometer. The instrument was operated in selected reaction monitoring (SRM) with negative electrospray ionization. Using BEH C(18) column (2.1 mm×50 mm, 1.7 μm) with 10-mM N-methylmorpholine/methanol gradient elution provided a fast chromatographic separation (5.5 min) and sharp peaks. Intra- and inter-day calibration bias was less than 7% and intra- and inter-day calibration of relative standard deviations were within 0.02-8.22% for all the analytes and concentrations. The recoveries of PFCs spiked into bovine serum ranged from 85 to 104% with relative standard deviations from 0.02 to 6.37%. The limits of quantitation (LOQs), defined as a signal-to-noise ratio of ten, ranged from 0.15 to 3.1 ng/mL for the twelve PFCs. Perfluorooctanoic acid (PFOA), perfluorooctyl sulfonate (PFOS), perfluoroundecanoic acid (PFUA) and perfluorononanoic acid (PFNA) were detected in up to 68% of umbilical cord plasma (n=444) in Taiwan Birth Panel Study and the health effect of these chemicals on children developmental deserves further investigation. Copyright © 2011 Elsevier B.V. All rights reserved.

Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

PubMed

Ansermot, Nicolas; Brawand-Amey, Marlyse; Kottelat, Astrid; Eap, Chin B

2013-05-31

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis.

PubMed

Blanco López, S L; Moal, J; San Juan Serrano, F

2000-09-01

Reversed-phase HPLC was applied to obtain a sensitive and efficient means for quantitating nucleotides in the mussel Mytilus galloprovincialis. We obtained a good separation of adenylic, guanylic, uridylic and cytidylic nucleotides. Adenine nucleotides play a critical role in the regulation and integration of cellular metabolism; particularly in the mantle tissue in the mussel, they are involved in the regulation of the enzyme glycogen phosphorylase, a key enzyme in the transfer of bioenergetic reserves (glycogen) to gametogenic development; it is of great importance to have a measure of the concentrations in vivo during the reproductive cycle of the organism. Different elution conditions were tested: isocratic versus step gradient elution, different mobile phase pH and the type and proportion of ion-pairing agent added to the mobile phase. The best method was selected and the separation and accurate determination of adenine, citidine, guanine and uridine nucleotides was accomplished within a 20-min run, with UV-Vis detection (254 nm).

[Comparison of content of curdione, curcumol, germacrone and beta-elemene in different varieties of vinegar backed Rhizoma Curcuma].

PubMed

Jiang, Guofei; Lu, Tulin; Mao, Chunqin; Su, Tao; Sun, Xiaomin

2010-11-01

To establish a HPLC method for determination of 4 components in different varieties of vinegar backed Rhizoma Curcuma. The method was established by using an Elite Hypersil ODS2 column (4.6 mm x 250 mm, 5 microm). The mobile phase comprising acetonitrile (A) and water (B) was used to elute the targets in gradient elution mode. Flow rate and detection wavelength were set at 1 mL x min(-1) and 214 nm, respectively. The column temperature was 25 degrees C and the injection volume was 10 microL. All calibration curves showed good linearity with r > 0.999 5. Recoveries measured at three concentrations were in the range of 97.27% - 99.27% with RSD < 3%. The validated method is simple, reliable, and successfully applied to determine the contents of the selected compounds in vinegar backed Rhizoma Curcuma. The results of the determination showed that contents of the four components in vinegar backed Curcuma wenyujin were relatively high.

Transcription in Yeast: Separation and Properties of Multiple RNA Polymerases

PubMed Central

Adman, Ray; Schultz, Loren D.; Hall, Benjamin D.

1972-01-01

Four peaks of DNA-directed RNA polymerase activity are resolved by salt gradient elution of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named IA, IB, II, and III in order of elution, all appear to come from cell nuclei. Only enzyme II is sensitive to α-amanitin. All enzymes are more active with Mn++ than with Mg++ as divalent ion. Enzymes IB and II have salt optima in the range 0.05-0.10 M (NH4)2SO4, whereas enzyme III is maximally active at 0.20-0.25 M (NH4)2SO4. With optimal salt concentration and saturating DNA, the template preference ratio, activity on native calfthymus DNA divided by activity on denatured calf-thymus DNA, is 2.2 for IB, 0.4 for II, and 3.5 for III. None of the yeast polymerases was inhibited by rifamycin SV. Rifamycin AF/013 effectively inhibited polymerases IB, II, and III. PMID:4558656

Purification and stability characterization of a cell regulatory sialoglycopeptide inhibitor

NASA Technical Reports Server (NTRS)

Moos, P. J.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1995-01-01

Previous attempts to physically separate the cell cycle inhibitory and protease activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non-overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two-fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine-SDS-PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed.

[Study on HPLC fingerprint of 11 Taraxacum species in northeast of China].

PubMed

Zhu, Dan; Zhao, Xin; Xu, Qiao; Ning, Wei

2011-04-01

To study the RP-HPLC fingerprints of 11 plants in the genus Taraxacum for their quality control. The fingerprints were determined using an Agilent 1100 series instrument system. Chromatographic analyses were performed on a Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) analytical column,eluted with methanol and water containing 0.5% acetic acid as the mobile phases in gradient elution at the flow rate of 1.0 mL x min(-1). The detection wavelength was 323 nm. The temperature of column was 35 degrees C. Eleven species of Taraxacum in northeast of China were detected respectively. Twenty-five common peaks existed in 11 RP-HPLC fingerprints. By comparing the retention time and the on-line UV spectra, peaks No. 10, No. 12, No. 16 and No. 25 were identified as chlorogenic acid, caffeic acid, p-coumaroy acid and luteolin respectively. The analytical method with good precision and reproducibility can be useful in the quality control of Taraxacum plants.

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography.

PubMed

Beneito-Cambra, M; Ripoll-Seguer, L; Herrero-Martínez, J M; Simó-Alfonso, E F; Ramis-Ramos, G

2011-11-25

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater. Copyright © 2011 Elsevier B.V. All rights reserved.

Metabolism of (/sup 3/H)gibberellin A/sub 5/ by immature seeds of apricot (Prunus armeniaca L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Bottini, G.A.; Bottini, R.; Koshioka, M.

1987-01-01

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A/sub 5/ (GA/sub 5/) as 1- and 1,2-(/sup 3/H)GA/sub 5/ at doses 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free (/sup 3/H)GA-like metabolites were separated from the highly H/sub 2/O-soluble (/sup 3/H)metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted ..-->.. isocratic eluted reversed-phase C/sub 18/ high performance liquid chromatography (HPLC)-radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RCmore » peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of (/sup 3/H) GA/sub 5/ were identified as GA/sub 1/, GA/sub 3/, and GA/sub 6/ by GC-SIM. The major highly water soluble metabolite of (/sup 3/H)GA/sub 5/ at all levels of substrate GA/sub 5/ had chromatographic characteristics similar to authentic GA/sub 1/-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate (/sup 3/H)GA/sub 5/ feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA/sub 1/, GA/sub 3/, GA/sub 5/ methyl ester, GA/sub 6/, GA/sub 22/, GA/sub 29/ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA/sub 1/, GA/sub 3/, GA/sub 5/, and GA/sub 8/ (33, 11, 1, 0.1%, respectively) elute.« less

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

STUDIES ON FLUORESCENT ANTIBODY STAINING

PubMed Central

Goldstein, Gerald; Slizys, Irene S.; Chase, Merrill W.

1961-01-01

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules. PMID:13706641

Enzymic cross-linkage of monomeric extensin precursors in vitro. [Lycopersicon esculentum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Everdeen, D.S.; Kiefer, S.; Willard, J.J.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, the authors purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers inmore » the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. They therefore identified the cross-linking activity as extensin peroxidase.« less

3 alpha-Hydroxylated bile acid profiles in clinically normal cats, cats with severe hepatic lipidosis, and cats with complete extrahepatic bile duct occlusion.

PubMed

Center, S A; Thompson, M; Guida, L

1993-05-01

Concentrations of 3 alpha-hydroxylated bile acids were measured in serum and urine of clinically normal (healthy) cats (n = 6), cats with severe hepatic lipidosis (n = 9), and cats with complete bile duct occlusion (n = 4). Bile acid concentrations were measured by use of a gradient flow high-performance liquid chromatography procedure with an acetonitrile and ammonium phosphate mobile phase and an in-line postanalytic column containing 3 alpha-hydroxy-steroid dehydrogenase and a fluorescence detector. Specific identification of all bile acid peaks was not completed; unidentified moieties were represented in terms of their elution time (in minutes). Significant differences in serum and urine bile acid concentrations, quantitative and proportional, were determined among groups of cats. Cats with hepatic lipidosis and bile duct occlusion had significantly (P > or = 0.05) greater total serum and urine bile acids concentrations than did healthy cats. The proportion of hydrophobic bile acids in serum, those eluting at > or = 400 minutes, was 1.9% for healthy cats, 3.3% for cats with lipidosis, and 5.4% for bile duct-obstructed cats. Both groups of ill cats had a broader spectrum of unidentified late-eluting serum bile acids than did healthy cats; the largest spectrum developed in bile duct-occluded cats.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

PubMed Central

Groskreutz, Stephen R.; Weber, Stephen G.

2016-01-01

In this work we characterize the development of a method to enhance temperature-assisted on-column solute focusing (TASF) called two-stage TASF. A new instrument was built to implement two-stage TASF consisting of a linear array of three independent, electronically controlled Peltier devices (thermoelectric coolers, TECs). Samples are loaded onto the chromatographic column with the first two TECs, TEC A and TEC B, cold. In the two-stage TASF approach TECs A and B are cooled during injection. TEC A is heated following sample loading. At some time following TEC A’s temperature rise, TEC B’s temperature is increased from the focusing temperature to a temperature matching that of TEC A. Injection bands are focused twice on-column, first on the initial TEC, e.g. single-stage TASF, then refocused on the second, cold TEC. Our goal is to understand the two-stage TASF approach in detail. We have developed a simple yet powerful digital simulation procedure to model the effect of changing temperature in the two focusing zones on retention, band shape and band spreading. The simulation can predict experimental chromatograms resulting from spatial and temporal temperature programs in combination with isocratic and solvent gradient elution. To assess the two-stage TASF method and the accuracy of the simulation well characterized solutes are needed. Thus, retention factors were measured at six temperatures (25–75 °C) at each of twelve mobile phases compositions (0.05–0.60 acetonitrile/water) for homologs of n-alkyl hydroxylbenzoate esters and n-alkyl p-hydroxyphenones. Simulations accurately reflect experimental results in showing that the two-stage approach improves separation quality. For example, two-stage TASF increased sensitivity for a low retention solute by a factor of 2.2 relative to single-stage TASF and 8.8 relative to isothermal conditions using isocratic elution. Gradient elution results for two-stage TASF were more encouraging. Application of two-stage TASF increased peak height for the least retained solute in the test mixture by a factor of 3.2 relative to single-stage TASF and 22.3 compared to isothermal conditions for an injection four-times the column volume. TASF improved resolution and increased peak capacity; for a 12-minute separation peak capacity increased from 75 under isothermal conditions to 146 using single-stage TASF, and 185 for two-stage TASF. PMID:27836226

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment.

PubMed

Groskreutz, Stephen R; Weber, Stephen G

2016-11-25

In this work we characterize the development of a method to enhance temperature-assisted on-column solute focusing (TASF) called two-stage TASF. A new instrument was built to implement two-stage TASF consisting of a linear array of three independent, electronically controlled Peltier devices (thermoelectric coolers, TECs). Samples are loaded onto the chromatographic column with the first two TECs, TEC A and TEC B, cold. In the two-stage TASF approach TECs A and B are cooled during injection. TEC A is heated following sample loading. At some time following TEC A's temperature rise, TEC B's temperature is increased from the focusing temperature to a temperature matching that of TEC A. Injection bands are focused twice on-column, first on the initial TEC, e.g. single-stage TASF, then refocused on the second, cold TEC. Our goal is to understand the two-stage TASF approach in detail. We have developed a simple yet powerful digital simulation procedure to model the effect of changing temperature in the two focusing zones on retention, band shape and band spreading. The simulation can predict experimental chromatograms resulting from spatial and temporal temperature programs in combination with isocratic and solvent gradient elution. To assess the two-stage TASF method and the accuracy of the simulation well characterized solutes are needed. Thus, retention factors were measured at six temperatures (25-75°C) at each of twelve mobile phases compositions (0.05-0.60 acetonitrile/water) for homologs of n-alkyl hydroxylbenzoate esters and n-alkyl p-hydroxyphenones. Simulations accurately reflect experimental results in showing that the two-stage approach improves separation quality. For example, two-stage TASF increased sensitivity for a low retention solute by a factor of 2.2 relative to single-stage TASF and 8.8 relative to isothermal conditions using isocratic elution. Gradient elution results for two-stage TASF were more encouraging. Application of two-stage TASF increased peak height for the least retained solute in the test mixture by a factor of 3.2 relative to single-stage TASF and 22.3 compared to isothermal conditions for an injection four-times the column volume. TASF improved resolution and increased peak capacity; for a 12-min separation peak capacity increased from 75 under isothermal conditions to 146 using single-stage TASF, and 185 for two-stage TASF. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlations between quasi-coherent fluctuations and the pedestal evolution during the inter-edge localized modes phase on DIII-D

DOE PAGES

Diallo, A.; Groebner, R. J.; Rhodes, T. L.; ...

2015-05-15

Direct measurements of the pedestal recovery during an edge-localized mode cycle provide evidence that quasi-coherent fluctuations (QCFs) play a role in the inter-ELM pedestal dynamics. When using fast Thomson scattering measurements, we found that the pedestal density and temperature evolutions are probed on sub-millisecond time scales to show a fast recovery of the density gradient compared to the temperature gradient. The temperature gradient appears to provide a drive for the onset of quasi-coherent fluctuations (as measured with the magnetic probe and the density diagnostics) localized in the pedestal. The amplitude evolution of these QCFs tracks the temperature gradient evolution includingmore » its saturation. Such correlation suggests that these QCFs play a key role in limiting the pedestal temperature gradient. Moreover, the saturation of the QCFs coincides with the pressure gradient reaching the kinetic-ballooning mode (KBM) critical gradient as predicted by EPED1. Furthermore, linear microinstability analysis using GS2 indicates that the steep gradient is near the KBM threshold. Finally, the modeling and the observations together suggest that QCFs are consistent with dominant KBMs, although microtearing cannot be excluded as subdominant.« less

On-line pH modification of carbonate eluents using an electrolytic potassium hydroxide generator for ion chromatography.

PubMed

Novic, Milko; Liu, Yan; Avdalovic, Nebojsa; Pihlar, Boris

2002-05-31

Classical gradient elution, based on the application of a gradient pump used for mixing two or more prepared eluent components in pre-determined concentrations, was replaced by a chromatography system equipped with an isocratic pump and an electrolytic KOH generator. The isocratic pump delivered a constant concentration eluent composed of pure hydrogencarbonate solution. Carbonate ions, the main component of carbonate/hydrogencarbonate-based eluents, were formed by titration of hydrogencarbonate with KOH formed on-line in the electrolytic KOH generator. By changing the concentration of electrolytically-generated KOH, the eluent composition could be changed from pure hydrogencarbonate to a carbonate/hydrogencarbonate buffer, and finally to a carbonate/hydroxide-based eluent. The described system was tested to achieve pH-based changes of retention behavior of phosphate under constant inflow eluent composition conditions.

Separation and identification of corticosterone metabolites by liquid chromatography--electrospray ionization mass spectrometry.

PubMed

Miksík, I; Vylitová, M; Pácha, J; Deyl, Z

1999-04-16

High-performance liquid chromatography coupled to atmospheric pressure ionization-electrospray ionization mass spectrometry (API-ESI-MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C18 column (eluted with a methanol-water-acetic acid gradient) with identification using positive ion mode API-ESI-MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in avian intestines.

Isolation and identification of fatty acid amides from Shengli coal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ming-Jie Ding; Zhi-Min Zong; Ying Zong

Shengli coal, a Chinese brown coal, was extracted with carbon disulfide and the extract was gradiently eluted with n-hexane and ethyl acetate (EA)/n-hexane mixed solvents with different concentrations of EA in a silica gel-filled column. A series of fatty acid amides, including fourteen alkanamides (C{sub 15}-C{sub 28}) and three alkenamides (C{sub 18} and C{sub 22}), were isolated from the coal by this method and analyzed with a gas chromatography/mass spectrometry. 26 refs., 2 figs., 2 tabs.

Bioactivities and extraction optimization of crude polysaccharides from the fruits and leaves of Rubus chingii Hu.

PubMed

Zhang, Tian-Tian; Lu, Chuan-Li; Jiang, Jian-Guo; Wang, Min; Wang, Dong-Mei; Zhu, Wei

2015-10-05

Polysaccharides of Rubus chingii Hu fruit and leaf were extracted to compare their antioxidant, anti-inflammatory, and anticancer activities against breast cancer cells MCF-7 and liver cancer cells Bel-7402. Results showed that all the tested bioactivities of polysaccharides from leaf (L-Ps) were better than those of polysaccharides from fruit (F-Ps). Response surface methodology was then used to optimize the extraction conditions of polysaccharides from leaf. Additionally, polysaccharides from fruit and leaf were characterized and their contents of total sugars, proteins and uronic acid were compared. It was found that polysaccharides from fruit and leaf were similar in IR and UV absorption, but significantly different in contents of total sugars, protein and uronic acid. Their elution profiles of DEAE-Sepharose fast flow column were different too. The main peak of polysaccharides from fruit was eluted with 0.3 mol/l NaCl solution and the main peak of polysaccharides from leaf was eluted with deionized water. The differences between the two polysaccharides may be responsible for their differences in bioactivities. Further studies are required to explore their complete structural characteristics, structure-activity relationship and the mechanism of their activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fast ion beta limit measurements by collimated neutron detection in MST plasmas

NASA Astrophysics Data System (ADS)

Capecchi, William; Anderson, Jay; Bonofiglo, Phillip; Kim, Jungha; Sears, Stephanie

2015-11-01

Fast ion orbits in the reversed field pinch (RFP) are well ordered and classically confined despite magnetic field stochasticity generated by multiple tearing modes. Classical TRANSP modeling of a 1MW tangentially injected hydrogen neutral beam in MST deuterium plasmas predicts a core-localized fast ion density that can be up to 25% of the electron density and a fast ion beta of many times the local thermal beta. However, neutral particle analysis of an NBI-driven mode (presumably driven by a fast ion pressure gradient) shows mode-induced transport of core-localized fast ions and a saturated fast ion density. The TRANSP modeling is presumed valid until the onset of the beam-driven mode and gives an initial estimate of the volume-averaged fast ion beta of 1-2% (local core value up to 10%). A collimated neutron detector for fusion product profile measurements will be used to determine the spatial distribution of fast ions, allowing for a first measurement of the critical fast-ion pressure gradient required for mode destabilization. Testing/calibration data and initial fast-ion profiles will be presented. Characterization of both the local and global fast ion beta will be done for deuterium beam injection into deuterium plasmas for comparison to TRANSP predictions. Work supported by US DOE.

Development and validation of a sensitive and fast UPLC-MS/MS method for simultaneous determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-gancao decoction.

PubMed

Ji, Bin; Zhuo, Limeng; Yang, Bin; Wang, Yang; Li, Lin; Yu, Miao; Zhao, Yunli; Yu, Zhiguo

2017-04-15

Rapid, sensitive, selective and accurate UPLC-MS/MS method was developed and fully validated for simultaneous determination of cinnamaldehyde, cinnamic acid, 2-methoxy cinnamic acid, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin and isoliquiritin in rat plasma after oral administration of Guizhi-gancao decoction. Plasma samples were processed with a simple protein precipitation technique using acetonitrile, followed by chromatographic separation using a Thermo Hypersil GOLD C 18 column. A 11.0min linear gradient elution was used at a flow rate of 0.2mL/min with a mobile phase of 0.1% acetic acid containing 0.2mM ammonium acetate in water and acetonitrile. The analytes and internal standard, schisandrin, were detected using both positive and negative ion electrospray ionization in multiple reaction monitoring mode. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency of all the analytes was found to be >60%. Stability results showed that the analytes were stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of multiple compounds in rat plasma after oral administration of Guizhi-gancao decoction. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of a liquid chromatography-tandem mass spectrometry analytical method for the therapeutic drug monitoring of eight novel anticancer drugs.

PubMed

Herbrink, M; de Vries, N; Rosing, H; Huitema, A D R; Nuijen, B; Schellens, J H M; Beijnen, J H

2018-04-01

To support therapeutic drug monitoring of patients with cancer, a fast and accurate method for simultaneous quantification of the registered anticancer drugs afatinib, axitinib, ceritinib, crizotinib, dabrafenib, enzalutamide, regorafenib and trametinib in human plasma using liquid chromatography tandem mass spectrometry was developed and validated. Human plasma samples were collected from treated patients and stored at -20°C. Analytes and internal standards (stable isotopically labeled analytes) were extracted with acetonitrile. An equal amount of 10 mm NH 4 CO 3 was added to the supernatant to yield the final extract. A 2 μL aliquot of this extract was injected onto a C 18 -column, gradient elution was applied and triple-quadrupole mass spectrometry in positive-ion mode was used for detection. All results were within the acceptance criteria of the latest US Food and Drug Administration guidance and European Medicines Agency guidelines on method validation, except for the carry-over of ceritinib and crizotinib. These were corrected for by the injection order of samples. Additional stability tests were carried out for axitinib and dabrafenib in relation to their reported photostability. In conclusion, the described method to simultaneously quantify the eight selected anticancer drugs in human plasma was successfully validated and applied for therapeutic drug monitoring in cancer patients treated with these drugs. Copyright © 2017 John Wiley & Sons, Ltd.

Bio-Guided Isolation of Methanol-Soluble Metabolites of Common Spruce (Picea abies) Bark by-Products and Investigation of Their Dermo-Cosmetic Properties.

PubMed

Angelis, Apostolis; Hubert, Jane; Aligiannis, Nektarios; Michalea, Rozalia; Abedini, Amin; Nuzillard, Jean-Marc; Gangloff, Sophie C; Skaltsounis, Alexios-Leandros; Renault, Jean-Hugues

2016-11-21

Common spruce ( Picea abies L.) is a fast-growing coniferous tree, widely used in several countries for the production of sawn wood, timber and pulp. During this industrial exploitation, large quantities of barks are generated as waste materials. The aim of this study was the bio-guided investigation and the effective recovery of methanol-soluble metabolites of common spruce bark for the development of new dermo-cosmetic agents. The active methanol extract was initially fractionated by Centrifugal Partition Chromatography (CPC) using a triphasic solvent system in a step-gradient elution mode. All resulting fractions were evaluated for their antibacterial activity, antioxidant activity and their capability to inhibit tyrosinase, elastase and collagenase activity. In parallel, the chemical composition of each fraction was established by combining a 13 C-NMR dereplication approach and 2D-NMR analyses. As a result, fourteen secondary metabolites corresponding to stilbene, flavonoid and phenolic acid derivatives were directly identified in the CPC fractions. A high amount (0.93 g) of E -astringin was recovered from 3 g of crude extract in a single 125 min run. E -Astringin significantly induced the tyrosinase activity while E -piceid, taxifolin, and taxifolin-3'- O -glucopyranoside exhibited significant anti-tyrosinase activity. The above compounds showed important anti-collagenase and antimicrobial activities, thus providing new perspectives for potential applications as cosmetic ingredients.

Update of on-line coupled liquid chromatography - gas chromatography for the analysis of mineral oil hydrocarbons in foods and cosmetics.

PubMed

Biedermann, Maurus; Munoz, Celine; Grob, Koni

2017-10-27

On-line coupled high performance liquid chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID) is the most widely used method for the analysis of mineral oil hydrocarbons in food, food contact materials, tissues and cosmetics. With comprehensive two-dimensional gas chromatography (GCxGC), a tool became available for better establishing the elution sequence of the various types of hydrocarbons from the HPLC column used for isolating the mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). The performance of a heavily used HPLC column with reduced retention for MOAH was investigated to improve the robustness of the method. Updates are recommended that render the MOSH/MOAH separation less dependent of the state of the HPLC column and more correct in cases of highly refined mineral oil products of high molecular mass. Cyclohexyl cyclohexane (Cycy), used as internal standard, turned out to be eluted slightly after cholestane (Cho); apparently the size exclusion effect predominates the extra retention by ring number on the 60Å pore size silica gel. Hence, Cycy can be used to determine the end of the MOSH fraction. Long chain alkyl benzenes were eluted earlier than tri-tert. butyl benzene (Tbb). It is proposed to start the MOAH transfer immediately after the MOSH fraction and use a gradient causing breakthrough of dichloromethane (visible in the UV chromatogram) at a time suitable to elute perylene (Per) at the end of the fraction. In this way, a decrease in retention power of the HPLC column can be tolerated without adjustment of the MOAH fraction until some MOAH start being eluted into the MOSH fraction. This critical point can be checked either with di(2-ethylhexyl) benzene (DEHB) as a marker or the HPLC-UV chromatogram. Finally, based on new findings in rats and human tissues, it is recommended to integrate the MOSH and MOAH up to the retention time of the n-alkane C40. Copyright © 2017 Elsevier B.V. All rights reserved.

The performance of monotonic and new non-monotonic gradient ascent reconstruction algorithms for high-resolution neuroreceptor PET imaging.

PubMed

Angelis, G I; Reader, A J; Kotasidis, F A; Lionheart, W R; Matthews, J C

2011-07-07

Iterative expectation maximization (EM) techniques have been extensively used to solve maximum likelihood (ML) problems in positron emission tomography (PET) image reconstruction. Although EM methods offer a robust approach to solving ML problems, they usually suffer from slow convergence rates. The ordered subsets EM (OSEM) algorithm provides significant improvements in the convergence rate, but it can cycle between estimates converging towards the ML solution of each subset. In contrast, gradient-based methods, such as the recently proposed non-monotonic maximum likelihood (NMML) and the more established preconditioned conjugate gradient (PCG), offer a globally convergent, yet equally fast, alternative to OSEM. Reported results showed that NMML provides faster convergence compared to OSEM; however, it has never been compared to other fast gradient-based methods, like PCG. Therefore, in this work we evaluate the performance of two gradient-based methods (NMML and PCG) and investigate their potential as an alternative to the fast and widely used OSEM. All algorithms were evaluated using 2D simulations, as well as a single [(11)C]DASB clinical brain dataset. Results on simulated 2D data show that both PCG and NMML achieve orders of magnitude faster convergence to the ML solution compared to MLEM and exhibit comparable performance to OSEM. Equally fast performance is observed between OSEM and PCG for clinical 3D data, but NMML seems to perform poorly. However, with the addition of a preconditioner term to the gradient direction, the convergence behaviour of NMML can be substantially improved. Although PCG is a fast convergent algorithm, the use of a (bent) line search increases the complexity of the implementation, as well as the computational time involved per iteration. Contrary to previous reports, NMML offers no clear advantage over OSEM or PCG, for noisy PET data. Therefore, we conclude that there is little evidence to replace OSEM as the algorithm of choice for many applications, especially given that in practice convergence is often not desired for algorithms seeking ML estimates.

Chemical modification of protein A chromatography ligands with polyethylene glycol. I: Effects on IgG adsorption equilibrium, kinetics, and transport.

PubMed

Weinberg, Justin; Zhang, Shaojie; Crews, Gillian; Carta, Giorgio; Przybycien, Todd

2018-04-20

Chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) has been proposed as a strategy to increase the process selectivity and resin robustness by providing the ligand with a steric repulsion barrier against non-specific binding. This article comprises a comprehensive study of IgG adsorption and transport in Repligen CaptivA PriMAB resin with PEGylated ProA ligands that are modified using 5.2 and 21.5 kDa PEG chains. We studied the impact of the molecular weight of the PEG as well as the extent of PEGylation for the 5.2 kDa PEG modification. In all cases, PEGylation of ProA ligands decreases the resin average pore size, particle porosity, and static binding capacity for IgG proportional to the volume of conjugated PEG in the resin. Resin batch uptake experiments conducted in bulk via a stirred-tank system and with individual resin particles under confocal laser scanning microscopy suggests that PEGylation introduces heterogeneity into IgG binding kinetics: a fraction of the IgG binding sites are transformed from typical fast association kinetic behavior to slow kinetic behavior. pH gradient elution experiments of an IgG molecule on the modified resins show an increase in IgG elution pH for all modified resins, implying a decrease in IgG-ProA binding affinity on modification. Despite losses in static binding capacity for all resins with PEGylated ligands, the loss of dynamic binding capacity at 10% breakthrough (DBC 10% ) ranged more broadly from almost 0-47% depending on the PEG molecular weight and the extent of PEGylation. Minimal losses in DBC 10% were observed with a low extent of PEGylation with a smaller molecular weight PEG, while higher losses were observed at higher extents of PEGylation and with higher molecular weight PEG due to decreased static binding capacity and increased mass transfer resistance. This work provides insight into the practical implications for resin performance if PEGylation is considered as a strategy for selectivity enhancement in affinity chromatography with macromolecular ligands. Copyright © 2018 Elsevier B.V. All rights reserved.

Prediction of the chromatographic retention of acid-base compounds in pH buffered methanol-water mobile phases in gradient mode by a simplified model.

PubMed

Andrés, Axel; Rosés, Martí; Bosch, Elisabeth

2015-03-13

Retention of ionizable analytes under gradient elution depends on the pH of the mobile phase, the pKa of the analyte and their evolution along the programmed gradient. In previous work, a model depending on two fitting parameters was recommended because of its very favorable relationship between accuracy and required experimental work. It was developed using acetonitrile as the organic modifier and involves pKa modeling by means of equations that take into account the acidic functional group of the compound (carboxylic acid, protonated amine, etc.). In this work, the two-parameter predicting model is tested and validated using methanol as the organic modifier of the mobile phase and several compounds of higher pharmaceutical relevance and structural complexity as testing analytes. The results have been quite good overall, showing that the predicting model is applicable to a wide variety of acid-base compounds using mobile phases prepared with acetonitrile or methanol. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

PubMed Central

Strickler, M P; Neill, R J; Stone, M J; Hunt, R E; Brinkley, W; Gemski, P

1989-01-01

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity. Images PMID:2745678

ANOTHER LOOK AT THE FAST ITERATIVE SHRINKAGE/THRESHOLDING ALGORITHM (FISTA)*

PubMed Central

Kim, Donghwan; Fessler, Jeffrey A.

2017-01-01

This paper provides a new way of developing the “Fast Iterative Shrinkage/Thresholding Algorithm (FISTA)” [3] that is widely used for minimizing composite convex functions with a nonsmooth term such as the ℓ1 regularizer. In particular, this paper shows that FISTA corresponds to an optimized approach to accelerating the proximal gradient method with respect to a worst-case bound of the cost function. This paper then proposes a new algorithm that is derived by instead optimizing the step coefficients of the proximal gradient method with respect to a worst-case bound of the composite gradient mapping. The proof is based on the worst-case analysis called Performance Estimation Problem in [11]. PMID:29805242

Density functional theory for molecular and periodic systems using density fitting and continuous fast multipole method: Analytical gradients.

PubMed

Łazarski, Roman; Burow, Asbjörn Manfred; Grajciar, Lukáš; Sierka, Marek

2016-10-30

A full implementation of analytical energy gradients for molecular and periodic systems is reported in the TURBOMOLE program package within the framework of Kohn-Sham density functional theory using Gaussian-type orbitals as basis functions. Its key component is a combination of density fitting (DF) approximation and continuous fast multipole method (CFMM) that allows for an efficient calculation of the Coulomb energy gradient. For exchange-correlation part the hierarchical numerical integration scheme (Burow and Sierka, Journal of Chemical Theory and Computation 2011, 7, 3097) is extended to energy gradients. Computational efficiency and asymptotic O(N) scaling behavior of the implementation is demonstrated for various molecular and periodic model systems, with the largest unit cell of hematite containing 640 atoms and 19,072 basis functions. The overall computational effort of energy gradient is comparable to that of the Kohn-Sham matrix formation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Repeatability of gradient ultrahigh pressure liquid chromatography-tandem mass spectrometry methods in instrument-controlled thermal environments.

PubMed

Grinias, James P; Wong, Jenny-Marie T; Kennedy, Robert T

2016-08-26

The impact of viscous friction on eluent temperature and column efficiency in liquid chromatography is of renewed interest as the need for pressures exceeding 1000bar to use with columns packed with sub-2μm particles has grown. One way the development of axial and radial temperature gradients that arise due to viscous friction can be affected is by the thermal environment the column is placed in. In this study, a new column oven integrated into an ultrahigh pressure liquid chromatograph that enables both still-air and forced-air operating modes is investigated to find the magnitude of the effect of the axial thermal gradient that forms in 2.1×100mm columns packed with sub-2μm particles in these modes. Temperature increases of nearly 30K were observed when the generated power of the column exceeded 25W/m. The impact of the heating due to viscous friction on the repeatability of peak capacity, elution time, and peak area ratio to an internal standard for a gradient UHPLC-MS/MS method to analyze neurotransmitters was found to be limited. This result indicates that high speed UHPLC-MS/MS gradient methods under conditions of high viscous friction may be possible without the negative effects typically observed with isocratic separations under similar conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Modeling of the Dorsal Gradient across Species Reveals Interaction between Embryo Morphology and Toll Signaling Pathway during Evolution

PubMed Central

Koslen, Hannah R.; Chiel, Hillel J.; Mizutani, Claudia Mieko

2014-01-01

Morphogenetic gradients are essential to allocate cell fates in embryos of varying sizes within and across closely related species. We previously showed that the maternal NF-κB/Dorsal (Dl) gradient has acquired different shapes in Drosophila species, which result in unequally scaled germ layers along the dorso-ventral axis and the repositioning of the neuroectodermal borders. Here we combined experimentation and mathematical modeling to investigate which factors might have contributed to the fast evolutionary changes of this gradient. To this end, we modified a previously developed model that employs differential equations of the main biochemical interactions of the Toll (Tl) signaling pathway, which regulates Dl nuclear transport. The original model simulations fit well the D. melanogaster wild type, but not mutant conditions. To broaden the applicability of this model and probe evolutionary changes in gradient distributions, we adjusted a set of 19 independent parameters to reproduce three quantified experimental conditions (i.e. Dl levels lowered, nuclear size and density increased or decreased). We next searched for the most relevant parameters that reproduce the species-specific Dl gradients. We show that adjusting parameters relative to morphological traits (i.e. embryo diameter, nuclear size and density) alone is not sufficient to reproduce the species Dl gradients. Since components of the Tl pathway simulated by the model are fast-evolving, we next asked which parameters related to Tl would most effectively reproduce these gradients and identified a particular subset. A sensitivity analysis reveals the existence of nonlinear interactions between the two fast-evolving traits tested above, namely the embryonic morphological changes and Tl pathway components. Our modeling further suggests that distinct Dl gradient shapes observed in closely related melanogaster sub-group lineages may be caused by similar sequence modifications in Tl pathway components, which are in agreement with their phylogenetic relationships. PMID:25165818

Comparing monolithic and fused core HPLC columns for fast chromatographic analysis of fat-soluble vitamins.

PubMed

Kurdi, Said El; Muaileq, Dina Abu; Alhazmi, Hassan A; Bratty, Mohammed Al; Deeb, Sami El

2017-06-27

HPLC stationary phases of monolithic and fused core type can be used to achieve fast chromatographic separation as an alternative to UPLC. In this study, monolithic and fused core stationary phases are compared for fast separation of four fat-soluble vitamins. Three new methods on the first and second generation monolithic silica RP-18e columns and a fused core pentafluoro-phenyl propyl column were developed. Application of three fused core columns offered comparable separations of retinyl palmitate, DL-α-tocopheryl acetate, cholecalciferol and menadione in terms of elution speed and separation efficiency. Separation was achieved in approx. 5 min with good resolution (Rs > 5) and precision (RSD ≤ 0.6 %). Monolithic columns showed, however, a higher number of theoretical plates, better precision and lower column backpressure than the fused core column. The three developed methods were successfully applied to separate and quantitate fat-soluble vitamins in commercial products.

Experimental designs for modeling retention patterns and separation efficiency in analysis of fatty acid methyl esters by gas chromatography-mass spectrometry.

PubMed

Skartland, Liv Kjersti; Mjøs, Svein A; Grung, Bjørn

2011-09-23

The retention behavior of components analyzed by chromatography varies with instrumental settings. Being able to predict how changes in these settings alter the elution pattern is useful, both with regards to component identification, as well as with regards to optimization of the chromatographic system. In this work, it is shown how experimental designs can be used for this purpose. Different experimental designs for response surface modeling of the separation of fatty acid methyl esters (FAME) as function of chromatographic conditions in GC have been evaluated. Full factorial, central composite, Doehlert and Box-Behnken designs were applied. A mixture of 38 FAMEs was separated on a polar cyanopropyl substituted polysilphenylene-siloxane phase capillary column. The temperature gradient, the start temperature of the gradient, and the carrier gas velocity were varied in the experiments. The modeled responses, as functions of chromatographic conditions, were retention time, retention indices, peak widths, separation efficiency and resolution between selected peak pairs. The designs that allowed inclusion of quadratic terms among the predictors performed significantly better than factorial design. Box-Behnken design provided the best results for prediction of retention, but the differences between the central composite, Doehlert and Box-Behnken designs were small. Retention indices could be modeled with much better accuracy than retention times. However, because the errors of predicted tR of closely eluting peaks were highly correlated, models of resolution (Rs) that were based on retention time had errors in the same range as corresponding models based on ECL. Copyright © 2011 Elsevier B.V. All rights reserved.

Chiral separation and chemical profile of Dengzhan Shengmai by integrating comprehensive with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

PubMed

Sheng, Ning; Zheng, Hao; Xiao, Yao; Wang, Zhe; Li, Menglin; Zhang, Jinlan

2017-09-29

Chemical profile for Chinese medicine formulas composed of several herbs is always a challenge due to a big array of small molecules with high chemical diversity so much as isomers. The present paper develops a feasible strategy to characterize and identify complex chemical constituents of a four-herb traditional Chinese medicine formula, Denzhan Shenmai (DZSM) by integrating comprehensive two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC×LC-qTOF-MS) with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (MHC-qTOF-MS). DZSM was separated by C8×C18 HPLC column system for comprehensive two-dimensional liquid chromatography system and 283 compounds most of which belonged to phenolic acid, flavonoid, saponin and lignan families were characterized and identified within 75min. Some isomers and compounds at low level were analyzed on C8×Chiral HPLC column system for multiple heart-cutting two-dimensional liquid chromatography system with 1D and 2D optimized gradient elution program. These 1D cutting fractions were successively separated on 2D chiral chromatographic column under extended the 2D gradient elution time from 30s to 5.0min. 12 pairs of isomer compounds were separated with good resolution. The combination of LC×LC and MHC system provides a powerful technique for global chemical profiling of DZSM and provided feasible strategy for other complex systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

PubMed

Knob, Radim; Hanson, Robert L; Tateoka, Olivia B; Wood, Ryan L; Guerrero-Arguero, Israel; Robison, Richard A; Pitt, William G; Woolley, Adam T

2018-05-21

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

PubMed

Bhushan, Ravi; Nagar, Hariom

2015-03-01

Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.

[Determination of 49 drugs and 5 metabolites in drinking water samples using ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry].

PubMed

Wang, Shuo; Zhang, Xiangming; Zhang, Jing; Shao, Bing; Li, Shuming

2015-07-01

A method for the determination of 54 drugs in drinking water samples was developed by using ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI MS/MS). The target drugs in drinking water samples were enriched and cleaned-up by HLB solid-phase extraction (SPE) cartridges and then eluted with 5 mL methanol. The elute was collected, concentrated under a gentle stream of nitrogen gas, diluted with 0.4 mL 0.1% formic acid solution, and analyzed by UPLC-ESI MS/MS. The separation of the 54 drugs was performed on an ACQUITY UPLC™ BEH C18 column using mobile phases of 0.1% formic acid and methanol by gradient elution. The multiple reaction monitoring (MRM) mode was employed in mass spectrometry acquisition. The matrix-matched external standard calibration was used for quantitation. The results showed that the average recoveries of the drugs in ground water, tap water and surface water were 58.7%-104.4%, 53.1%-109.5%, and 50.7%-118.8%, respectively, and the corresponding relative standard deviations (RSD, n=6) were 0.3%-12.8%, 1.0%-15.5%, and 0.4%-19.3%, respectively. The method quantification limits (MQL) for target compounds were in the range of 0.002-5.000 ng/L. The developed method was applied to analyze the water samples from Beijing. The results showed that 26 drugs were detected in ground water samples.

Origin of bombesin-like peptides in human fetal lung.

PubMed

Yoshizaki, K; de Bock, V; Solomon, S

1984-02-27

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).

Preparation of magnetic core mesoporous shell microspheres with C18-modified interior pore-walls for fast extraction and analysis of phthalates in water samples.

PubMed

Li, Zhongbo; Huang, Danni; Fu, Chinfai; Wei, Biwen; Yu, Wenjia; Deng, Chunhui; Zhang, Xiangmin

2011-09-16

In this study, core-shell magnetic mesoporous microspheres with C18-functionalized interior pore-walls were synthesized through coating Fe(3)O(4) microspheres with a mesoporous inorganic-organic hybrid layer with a n-octadecyltriethoxysilane (C18TES) and tetraethyl orthosilicate (TEOS) as the silica source and cetyltrimethylammonia bromide (CTAB) as a template. The obtained C18-functionalized Fe(3)O(4)@mSiO(2) microspheres possess numerous C18 groups anchored in the interior pore-walls, large surface area (274.7 m(2)/g, high magnetization (40.8 emu/g) and superparamagnetism, uniform mesopores (4.1 nm), which makes them ideal absorbents for simple, fast, and efficient extraction and enrichment of hydrophobic organic compounds in water samples. Several kinds of phthalates were used as the model hydrophobic organic compounds to systematically evaluate the performance of the C18-functionalized Fe(3)O(4)@mSiO(2) microspheres in extracting hydrophobic molecules by using a gas chromatography-mass spectrometry. Various parameters, including eluting solvent, the amounts of absorbents, extraction time and elution time were optimized. Hydrophobic extraction was performed in the interior pore of magnetic mesoporous microspheres, and the materials had the anti-interference ability to macromolecular proteins, which was also investigated in the work. Under the optimized conditions, C18-functionalized Fe(3)O(4)@mSiO(2) microspheres were successfully used to analyze the real water samples. The results indicated that this novel method was fast, convenient and efficient for the target compounds and could avoid being interfered by macromolecules. Copyright © 2011 Elsevier B.V. All rights reserved.

One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel.

PubMed

Liu, Dan; Ma, Yan; Wang, Ye; Su, Zhiguo; Gu, Ming; Janson, Jan-Christer

2011-05-01

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross-linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0-240 mL, 0-25% B; 240-480 mL, 25-40% B; after 480 mL, 100% B. The purities of the collected corilagin and geraniin were 92.4 and 87.2%, and the corresponding yields were 88.0 and 76.8%, respectively. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fully automated and fast method using direct sample injection combined with fused-core column on-line SPE-HPLC for determination of ochratoxin A and citrinin in lager beers.

PubMed

Lhotská, Ivona; Šatínský, Dalibor; Havlíková, Lucie; Solich, Petr

2016-05-01

A new fast and sensitive method based on on-line solid-phase extraction on a fused-core precolumn coupled to liquid chromatography with fluorescence detection has been developed for ochratoxin A (OTA) and citrinin (CIT) determination in lager beer samples. Direct injection of 100 μL filtered beer samples into an on-line SPE-HPLC system enabled fast and effective sample extraction including separation in less than 6 min. Preconcentration of OTA and CIT from beer samples was performed on an Ascentis Express RP C18 guard column (5 × 4.6 mm), particle size 2.7 μm, with a mobile phase of methanol/0.5% aqueous acetic acid pH 2.8 (30:70, v/v) at a flow rate of 2.0 mL min(-1). The flow switch from extraction column to analytical column in back-flush mode was set at 2.0 min and the separation was performed on the fused-core column Ascentis Express Phenyl-Hexyl (100 × 4.6 mm), particle size 2.7 μm, with a mobile phase acetonitrile/0.5% aqueous acetic acid pH 2.8 in a gradient elution at a flow rate of 1.0 mL min(-1) and temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 335/497 nm. The accuracy of the method, defined as the mean recoveries of OTA and CIT from light and dark beer samples, was in the range 98.3-102.1%. The method showed high sensitivity owing to on-line preconcentration; LOQ values were found to be 10 and 20 ng L(-1) for OTA and CIT, respectively. The found values of OTA and CIT in all tested light, dark and wheat beer samples were significantly below the maximum tolerable limits (3.0 μg kg(-1) for OTA and 2000 μg kg(-1) for CIT) set by the European Union.

Facile Purification of Milligram to Gram Quantities of Condensed Tannins According to Mean Degree of Polymerization and Flavan-3-ol Subunit Composition.

PubMed

Brown, Ron H; Mueller-Harvey, Irene; Zeller, Wayne E; Reinhardt, Laurie; Stringano, Elisabetta; Gea, An; Drake, Christopher; Ropiak, Honorata M; Fryganas, Christos; Ramsay, Aina; Hardcastle, Emily E

2017-09-13

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1 H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1 H- 13 C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

DNA Polymerase in Virions of a Reptilian Type C Virus

PubMed Central

Twardzik, Daniel R.; Papas, Takis S.; Portugal, Frank H.

1974-01-01

A study was made of the DNA polymerase of reptilian type C virus isolated from Russell's viper spleen cells. Simultaneous detection experiments demonstrated the presence of 70S RNA and RNA-dependent DNA polymerase activity in reptilian type C virions. The endogenous activity was dependent on the addition of all four deoxynucleotide triphosphates and demonstrated an absolute requirement for a divalent cation. The reptilian viral DNA polymerase elutes from phosphocellulose at 0.22 M salt. In this respect, it is similar to the avian (avian myeloblastosis virus; AMV) viral enzyme but is different from the mammalian (Rauscher leukemia virus; RLV) viral enzyme which elutes at 0.4 M salt. The molecular weight of the viper DNA polymerase as estimated from glycerol gradient centrifugation is 109,000. It is a smaller enzyme than the AMV DNA polymerase (180,000 daltons) and somewhat larger than the RLV enzyme (70,000 daltons). A comparison of other properties of the type C reptilian DNA polymerase with the enzyme found in other type C oncogenic viruses is made. PMID:4129837

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation.

PubMed

Savary, B J

2001-08-01

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.

Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

PubMed

Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

2010-03-01

The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

Design and scaleup of downstream processing of monoclonal antibodies for cancer therapy: from research to clinical proof of principle.

PubMed

Horenstein, Alberto L; Crivellin, Federico; Funaro, Ada; Said, Marcela; Malavasi, Fabio

2003-04-01

Murine monoclonal antibodies (mAb) from cell culture supernatants have been purified in order to acquire clinical grade for in vivo cancer treatment. The starting material was purified by high performance liquid chromatography (HPLC) systems ranging from the analytical scale process to a scaleup to 1 g per batch. Three columns (Protein A affinity chromatography with single-step elution, hydroxyapatite (HA) chromatography followed by linear gradient elution and endotoxin removing-gel chromatography), exploiting different properties of the mAb were applied. The final batches of antibody were subjected to a large panel of tests for the purpose of evaluating the efficacy of the downstream processing. The resulting data have allowed us to determine the maximum number of times the column can be used and to precisely and thoroughly characterize antibody integrity, specificity, and potency according to in-house reference standards. The optimized bioprocessing is rapid, efficient, and reproducible. Not less importantly, all the techniques applied are characterized by costs which are affordable to medium-sized laboratories. They represent the basis for implementing immunotherapeutic protocols transferable to clinical medicine.

Fast ion stabilization of the ion temperature gradient driven modes in the Joint European Torus hybrid-scenario plasmas: a trigger mechanism for internal transport barrier formation

NASA Astrophysics Data System (ADS)

Romanelli, M.; Zocco, A.; Crisanti, F.; Contributors, JET-EFDA

2010-04-01

Understanding and modelling turbulent transport in thermonuclear fusion plasmas are crucial for designing and optimizing the operational scenarios of future fusion reactors. In this context, plasmas exhibiting state transitions, such as the formation of an internal transport barrier (ITB), are particularly interesting since they can shed light on transport physics and offer the opportunity to test different turbulence suppression models. In this paper, we focus on the modelling of ITB formation in the Joint European Torus (JET) [1] hybrid-scenario plasmas, where, due to the monotonic safety factor profile, magnetic shear stabilization cannot be invoked to explain the transition. The turbulence suppression mechanism investigated here relies on the increase in the plasma pressure gradient in the presence of a minority of energetic ions. Microstability analysis of the ion temperature gradient driven modes (ITG) in the presence of a fast-hydrogen minority shows that energetic ions accelerated by the ion cyclotron resonance heating (ICRH) system (hydrogen, nH,fast/nD,thermal up to 10%, TH,fast/TD,thermal up to 30) can increase the pressure gradient enough to stabilize the ITG modes driven by the gradient of the thermal ions (deuterium). Numerical analysis shows that, by increasing the temperature of the energetic ions, electrostatic ITG modes are gradually replaced by nearly electrostatic modes with tearing parity at progressively longer wavelengths. The growth rate of the microtearing modes is found to be lower than that of the ITG modes and comparable to the local E × B-velocity shearing rate. The above mechanism is proposed as a possible trigger for the formation of ITBs in this type of discharges.

Detection and Genotyping of Human Papillomavirus in Self-Obtained Cervicovaginal Samples by Using the FTA Cartridge: New Possibilities for Cervical Cancer Screening ▿

PubMed Central

Lenselink, Charlotte H.; de Bie, Roosmarie P.; van Hamont, Dennis; Bakkers, Judith M. J. E.; Quint, Wim G. V.; Massuger, Leon F. A. G.; Bekkers, Ruud L. M.; Melchers, Willem J. G.

2009-01-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF10-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening. PMID:19553570

Detection and genotyping of human papillomavirus in self-obtained cervicovaginal samples by using the FTA cartridge: new possibilities for cervical cancer screening.

PubMed

Lenselink, Charlotte H; de Bie, Roosmarie P; van Hamont, Dennis; Bakkers, Judith M J E; Quint, Wim G V; Massuger, Leon F A G; Bekkers, Ruud L M; Melchers, Willem J G

2009-08-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF(10)-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening.

Copper desorption from Gelidium algal biomass.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2007-04-01

Desorption of divalent copper from marine algae Gelidium sesquipedale, an algal waste (from agar extraction industry) and a composite material (the algal waste immobilized in polyacrylonitrile) was studied in a batch system. Copper ions were first adsorbed until saturation and then desorbed by HNO(3) and Na(2)EDTA solutions. Elution efficiency using HNO(3) increases as pH decreases. At pH=1, for a solid to liquid ratio S/L=4gl(-1), elution efficiency was 97%, 95% and 88%, the stoichiometric coefficient for the ionic exchange, 0.70+/-0.02, 0.73+/-0.05 and 0.76+/-0.06 and the selectivity coefficient, 0.93+/-0.07, 1.0+/-0.3 and 1.1+/-0.3, respectively, for algae Gelidium, algal waste and composite material. Complexation of copper ions by EDTA occurs in a molar proportion of 1:1 and the elution efficiency increases with EDTA concentration. For concentrations of 1.4, 0.88 and 0.57 mmoll(-1), the elution efficiency for S/L=4gl(-1), was 91%, 86% and 78%, respectively, for algae Gelidium, algal waste and composite material. The S/L ratio, in the range 1-20gl(-1), has little influence on copper recovery by using 0.1M HNO(3). Desorption kinetics was very fast for all biosorbents. Kinetic data using HNO(3) as eluant were well described by the mass transfer model, considering the average metal concentration in the solid phase and the equilibrium relationship given by the mass action law. The homogeneous diffusion coefficient varied between 1.0 x 10(-7)cm(2)s(-1) for algae Gelidium and 3.0 x 10(-7)cm(2)s(-1) for the composite material.

The multigrid preconditioned conjugate gradient method

NASA Technical Reports Server (NTRS)

Tatebe, Osamu

1993-01-01

A multigrid preconditioned conjugate gradient method (MGCG method), which uses the multigrid method as a preconditioner of the PCG method, is proposed. The multigrid method has inherent high parallelism and improves convergence of long wavelength components, which is important in iterative methods. By using this method as a preconditioner of the PCG method, an efficient method with high parallelism and fast convergence is obtained. First, it is considered a necessary condition of the multigrid preconditioner in order to satisfy requirements of a preconditioner of the PCG method. Next numerical experiments show a behavior of the MGCG method and that the MGCG method is superior to both the ICCG method and the multigrid method in point of fast convergence and high parallelism. This fast convergence is understood in terms of the eigenvalue analysis of the preconditioned matrix. From this observation of the multigrid preconditioner, it is realized that the MGCG method converges in very few iterations and the multigrid preconditioner is a desirable preconditioner of the conjugate gradient method.

Dispersive micro solid phase extraction (DMSPE) using polymer anion exchange (PAX) as the sorbent followed by UPLC-MS/MS for the rapid determination of four bisphenols in commercial edible oils.

PubMed

Xian, Yanping; Wu, Yuluan; Dong, Hao; Guo, Xindong; Wang, Bin; Wang, Li

2017-09-29

The present work presents a novel and rapid analytical method for the simultaneous analysis of bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF) and bisphenol S (BPS) in edible oil based on dispersive micro solid phase extraction (DMSPE) for the first time followed by isotope dilution-ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The edible oil sample was dispersed by n-hexane and extracted with ammoniated methanol-water solution. Then the target analytes were dispersedly absorbed using the polymer anion exchange (PAX) as the sorbent and eluted by acidic methanol. After that, four bisphenols were separated on a C18 column by gradient elution with methanol and 0.05% ammonium hydroxide in water as mobile phase, detected by MS/MS under multiple reactions monitoring (MRM) mode and quantified by internal standard method. The PAX amounts, adsorption time, concentrations of formic acid in the elution solvent and volume of elution solvent for the DMSPE technique were optimized. The limit of detection and quantitation (LOD and LOQ), matrix effect, recovery and precision of the developed method were investigated. Results indicated that BPS and the rest three bisphenols displayed excellent linearity in the concentration ranges of 0.1-50μg/L and 0.5-250μg/L, respectively, with correlation coefficients (R 2 ) all larger than 0.998. Achieved MLODs (S/N=3) varied between 0.1-0.4μg/kg for all bisphenols. The mean recoveries at three spiked levels in edible oil were in the range of 87.3-108%. Intra-day precision (n=6) and inter-day precision (n=5) were <9% and <11%, respectively. This method is of rapid-and-simple pretreatment, accurate and sensitive, and suitable for the simultaneous determination of bisphenols in edible oil. Copyright © 2017. Published by Elsevier B.V.

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

PubMed Central

Murphy, Patrick J. M.

2014-01-01

Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes. PMID:25254496

[Simultaneous analysis of aromatic aldehydes and coumarins with high pressure liquid chromatography. Application to wines and brandies stored in oak barrels].

PubMed

Salagoity-Auguste, M H; Tricard, C; Sudraud, P

1987-04-17

Aromatic aldehydes (vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde) and coumarins (esculetin, umbelliferone, scopoletin and methylumbelliferone) are natural wood compounds. Storage of wines and brandies in oak barrels increases notably aldehydes and coumarins (particularly scopoletin) concentrations. These compounds were separated by high-performance liquid chromatography, on hydrocarbon bonded reversed-phase packings, with a water-acetonitrile elution gradient. They were first extracted from wines and brandies by diethyl ether and then injected on chromatographic column. A double detection was used to determine simultaneously aromatic aldehydes and coumarins by UV absorption and fluorescence respectively.

Microecology: Using Fast-Growing Filamentous Fungi to Study the Effects of Environmental Gradients on the Growth Patterns of Hyphae

ERIC Educational Resources Information Center

Delpech, Roger

2004-01-01

This paper describes some simple and rapid techniques for examining the growth responses of fungal hyphae cultivated on environmental gradients. The creation of such gradients using agar-based growth media in petri dishes is explained, along with recommendations for quantitative macroscopic and microscopic measurements. The intention is to provide…

Fat-suppressed three-dimensional fast spoiled gradient-recalled echo imaging: a modified FS 3D SPGR technique for assessment of patellofemoral joint chondromalacia.

PubMed

Wang, S F; Cheng, H C; Chang, C Y

1999-01-01

Fast fat-suppressed (FS) three-dimensional (3D) spoiled gradient-recalled echo (SPGR) imaging of 64 articular cartilage regions in 16 patellofemoral joints was evaluated to assess its feasibility in diagnosing patellofemoral chondromalacia. It demonstrated good correlation with arthroscopic reports and took about half of the examination time that FS 3D SPGR did. This modified, faster technique has the potential to diagnose patellofemoral chondromalacia with shorter examination time than FS 3D SPGR did.

High Efficiency, Low Distortion 3D Diffusion Tensor Imaging with Variable Density Spiral Fast Spin Echoes (3D DW VDS RARE)

PubMed Central

Frank, Lawrence R.; Jung, Youngkyoo; Inati, Souheil; Tyszka, J. Michael; Wong, Eric C.

2009-01-01

We present an acquisition and reconstruction method designed to acquire high resolution 3D fast spin echo diffusion tensor images while mitigating the major sources of artifacts in DTI - field distortions, eddy currents and motion. The resulting images, being 3D, are of high SNR, and being fast spin echoes, exhibit greatly reduced field distortions. This sequence utilizes variable density spiral acquisition gradients, which allow for the implementation of a self-navigation scheme by which both eddy current and motion artifacts are removed. The result is that high resolution 3D DTI images are produced without the need for eddy current compensating gradients or B0 field correction. In addition, a novel method for fast and accurate reconstruction of the non-Cartesian data is employed. Results are demonstrated in the brains of normal human volunteers. PMID:19778618

A general strategy for performing temperature-programming in high performance liquid chromatography--further improvements in the accuracy of retention time predictions of segmented temperature gradients.

PubMed

Wiese, Steffen; Teutenberg, Thorsten; Schmidt, Torsten C

2012-01-27

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed for systematic method development in high-temperature liquid chromatography (HT-HPLC). The ability to predict isothermal retention times based on temperature-gradient as well as isothermal input data was investigated. For a small temperature interval of ΔT=40°C, both approaches result in very similar predictions. Average relative errors of predicted retention times of 2.7% and 1.9% were observed for simulations based on isothermal and temperature-gradient measurements, respectively. Concurrently, it was investigated whether the accuracy of retention time predictions of segmented temperature gradients can be further improved by temperature dependent calculation of the parameter S(T) of the LES relationship. It was found that the accuracy of retention time predictions of multi-step temperature gradients can be improved to around 1.5%, if S(T) was also calculated temperature dependent. The adjusted experimental design making use of four temperature-gradient measurements was applied for systematic method development of selected food additives by high-temperature liquid chromatography. Method development was performed within a temperature interval from 40°C to 180°C using water as mobile phase. Two separation methods were established where selected food additives were baseline separated. In addition, a good agreement between simulation and experiment was observed, because an average relative error of predicted retention times of complex segmented temperature gradients less than 5% was observed. Finally, a schedule of recommendations to assist the practitioner during systematic method development in high-temperature liquid chromatography was established. Copyright © 2011 Elsevier B.V. All rights reserved.

Determination of reversed-phase high performance liquid chromatography based octanol-water partition coefficients for neutral and ionizable compounds: Methodology evaluation.

PubMed

Liang, Chao; Qiao, Jun-Qin; Lian, Hong-Zhen

2017-12-15

Reversed-phase liquid chromatography (RPLC) based octanol-water partition coefficient (logP) or distribution coefficient (logD) determination methods were revisited and assessed comprehensively. Classic isocratic and some gradient RPLC methods were conducted and evaluated for neutral, weak acid and basic compounds. Different lipophilicity indexes in logP or logD determination were discussed in detail, including the retention factor logk w corresponding to neat water as mobile phase extrapolated via linear solvent strength (LSS) model from isocratic runs and calculated with software from gradient runs, the chromatographic hydrophobicity index (CHI), apparent gradient capacity factor (k g ') and gradient retention time (t g ). Among the lipophilicity indexes discussed, logk w from whether isocratic or gradient elution methods best correlated with logP or logD. Therefore logk w is recommended as the preferred lipophilicity index for logP or logD determination. logk w easily calculated from methanol gradient runs might be the main candidate to replace logk w calculated from classic isocratic run as the ideal lipophilicity index. These revisited RPLC methods were not applicable for strongly ionized compounds that are hardly ion-suppressed. A previously reported imperfect ion-pair RPLC method was attempted and further explored for studying distribution coefficients (logD) of sulfonic acids that totally ionized in the mobile phase. Notably, experimental logD values of sulfonic acids were given for the first time. The IP-RPLC method provided a distinct way to explore logD values of ionized compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products.

PubMed

Collier, J W; Shah, R B; Bryant, A R; Habib, M J; Khan, M A; Faustino, P J

2011-02-20

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (L-T(4)) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250 mm × 3.9 mm) using a 0.01 M phosphate buffer (pH 3.0)-methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 μL and the column temperature was maintained at 28°C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r(2)>0.99) over the analytical range of 0.08-0.8 μg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for L-T(4) over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets. Published by Elsevier B.V.

Development and application of a validated HPLC method for the analysis of dissolution samples of levothyroxine sodium drug products

PubMed Central

Collier, J.W.; Shah, R.B.; Bryant, A.R.; Habib, M.J.; Khan, M.A.; Faustino, P.J.

2011-01-01

A rapid, selective, and sensitive gradient HPLC method was developed for the analysis of dissolution samples of levothyroxine sodium tablets. Current USP methodology for levothyroxine (l-T4) was not adequate to resolve co-elutants from a variety of levothyroxine drug product formulations. The USP method for analyzing dissolution samples of the drug product has shown significant intra- and inter-day variability. The sources of method variability include chromatographic interferences introduced by the dissolution media and the formulation excipients. In the present work, chromatographic separation of levothyroxine was achieved on an Agilent 1100 Series HPLC with a Waters Nova-pak column (250mm × 3.9mm) using a 0.01 M phosphate buffer (pH 3.0)–methanol (55:45, v/v) in a gradient elution mobile phase at a flow rate of 1.0 mL/min and detection UV wavelength of 225 nm. The injection volume was 800 µL and the column temperature was maintained at 28 °C. The method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, specificity, linearity, and analytical range. The standard curve was found to have a linear relationship (r2 > 0.99) over the analytical range of 0.08–0.8 µg/mL. Accuracy ranged from 90 to 110% for low quality control (QC) standards and 95 to 105% for medium and high QC standards. Precision was <2% at all QC levels. The method was found to be accurate, precise, selective, and linear for l-T4 over the analytical range. The HPLC method was successfully applied to the analysis of dissolution samples of marketed levothyroxine sodium tablets. PMID:20947276

Expression, purification, characterization and subcellular localization of the goose parvovirus rep1 protein.

PubMed

Chen, Zongyan; Li, Chuanfeng; Peng, Gaojing; Liu, Guangqing

2013-07-01

The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl β-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.

C18 solid-phase isolation and high-performance liquid chromatography/ultraviolet diode array determination of fully methoxylated flavones in citrus juices.

PubMed

Sendra, J M; Navarro, J L; Izquierdo, L

1988-09-01

A new analytical methodology for the determination of fully methoxylated flavones (FMFs) in citrus juices is described. Isolation of the FMFs is carried out by percolation of 30 mL of clarified citrus juice (to which tetramethyl-o-kaempferol is previously added as internal standard) through a C18 Sep-Pak cartridge, washing with 3 mL of water followed by 5 mL of water/acetonitrile (3:1), and selective elution of the retained FMFs with 5 mL of water/acetonitrile (9:11). Determination of the isolated FMFs is carried out by reversed-phase high-performance liquid chromatography (HPLC) and UV diode array detection (DAD). Signals at wavelengths 320, 335, and 345 nm (bandwidth 4 nm) are simultaneously acquired, stored, plotted, and integrated. The column used is a microbore (200 x 2.1-mm) Hypersil ODS 5 microns. Elution is in gradient mode, using a ternary mobile phase (water/acetonitrile/tetrahydrofuran). Column temperature is 40 degrees C. Recovery yields are nearly 100% for all the FMFs detected and identified: isosinensetin, hexamethyl-o-gossypetin, sinensetin, tetramethyl-o-isoscutellarein, hexamethyl-o-quercetagetin, nobiletin, tetramethyl-o-scutellarein, heptamethoxyflavone, and tangeretin. Chromatographic separation of the FMFs is extremely dependent upon the minor changes of the mobile phase composition and percentages, gradient rate, and temperature. The UV spectra (230 to 400 nm) of the FMFs obtained under chromatographic conditions are given. The FMFs relative response factors at 320, 335, and 345 nm and their concentrations in hand-squeezed and commercial concentrated orange and mandarin juices are tabulated. The FMF concentration differences found among samples are discussed.

Fast gradient screening of pharmaceuticals with 5 cm long, narrow bore reversed-phase columns packed with sub-3 μm core-shell and sub-2 μm totally porous particles.

PubMed

Fekete, Szabolcs; Fekete, Jeno

2011-04-15

The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d(p)=2.7 μm) and structured (ρ=0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation. Copyright © 2011 Elsevier B.V. All rights reserved.

Application of oil-water discrimination technology in fractured reservoirs using the differences between fast and slow shear-waves

NASA Astrophysics Data System (ADS)

Luo, Cong; Li, Xiangyang; Huang, Guangtan

2017-08-01

Oil-water discrimination is of great significance in the design and adjustment of development projects in oil fields. For fractured reservoirs, based on anisotropic S-wave splitting information, it becomes possible to effectively solve such problems which are difficult to deal with in traditional longitudinal wave exploration, due to the similar bulk modulus and density of these two fluids. In this paper, by analyzing the anisotropic character of the Chapman model (2009 Geophysics 74 97-103), the velocity and reflection coefficient differences between the fast and slow S-wave caused by fluid substitution have been verified. Then, through a wave field response analysis of the theoretical model, we found that water saturation causes a longer time delay, a larger time delay gradient and a lower amplitude difference between the fast and slow S-wave, while the oil case corresponds to a lower time delay, a lower gradient and a higher amplitude difference. Therefore, a new class attribute has been proposed regarding the amplitude energy of the fast and slow shear wave, used for oil-water distinction. This new attribute, as well as that of the time delay gradient, were both applied to the 3D3C seismic data of carbonate fractured reservoirs in the Luojia area of the Shengli oil field in China. The results show that the predictions of the energy attributes are more consistent with the well information than the time delay gradient attribute, hence demonstrating the great advantages and potential of this new attribute in oil-water recognition.

Separation of an associated 90K heat shock protein from the glucocorticoid receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller-Diener, A.; Kirsch, T.; Grove, B.

1986-05-01

A 90K heat shock protein(HSP), observed to copurify with the glucocorticoid receptor(GR), can be separated from the complex by 2 methods, allowing investigation of the role of HSP on kinase activity that was previously reported to be inherent to purified activated GR. Na/sub 2/MoO/sub 4/ stabilized unactivated rat hepatic GR complexes have been purified to >10,000-fold using a purification scheme that involves batchwise treatment of cytosol with phosphocellulose/DNAcellulose, elution from an affinity resin, gel filtration and ion exchange chromatography. Samples were subjected to 10-20% gradient SDS-PAGE. Proteins were transferred to nitrocellulose and blotted against monoclonal antibodies to GR(3A6), HSP ormore » nonspecific IgM/G. Immunoblots indicated that HSP was separated from unactivated GR complexes at the affinity step prior to elution of GR with active steroid. GR eluted from the resin with /sup 3/H Triamcinolone acetonide or /sup 3/H Dexamethasone mesylate had an apparent M/sub r/ = 94-96,000 for the steroid binding subunit and is recognized by 3A6. Purification of GR minus the affinity step resulted in copurification of HSP throughout the procedure. However, after Sephadex G75 filtration and subsequent incubation at 25/sup 0/C, 30 min., HSP was separated from activated (DNA binding) GR on DEAE cellulose-52. HSP did not enhance or inhibit /sup 32/P incorporation of the 94K steroid binding subunit nor did it affect phosphorylation of histones by GR.« less

On-line SPE-UHPLC method using fused core columns for extraction and separation of nine illegal dyes in chilli-containing spices.

PubMed

Khalikova, Maria A; Satínský, Dalibor; Smidrkalová, Tereza; Solich, Petr

2014-12-01

The presented work describes the development of a simple, fast and effective on-line SPE-UHPLC-UV/vis method using fused core particle columns for extraction, separation and quantitative analysis of the nine illegal dyes, most frequently found in chilli-containing spices. The red dyes Sudan I-IV, Sudan Red 7B, Sudan Red G, Sudan Orange G, Para Red, and Methyl Red were separated and analyzed in less than 9 min without labor-consuming pretreatment procedure. The chromatographic separation was performed on Ascentis Express RP-Amide column with gradient elution using mixture of acetonitrile and water, as a mobile phase at a flow rate of 1.0 mL min(-1) and 55°C of temperature. As SPE sorbent for cleanup and pre-concentration of illegal dyes short guard fused core column Ascentis Express F5 was used. The applicability of proposed method was proven for three different chilli-containing commercial samples. Recoveries for all compounds were between 90% and 108% and relative standard deviation ranged from 1% to 4% for within- and from 2% to 6% for between-day. Limits of detection showed lower values than required by European Union regulations and were in the range of 3.3-10.3 µg L(-1) for standard solutions, 5.6-235.6 µg kg(-1) for chilli-containing spices. Copyright © 2014 Elsevier B.V. All rights reserved.

Separation of cannabinoids on three different mixed-mode columns containing carbon/nanodiamond/amine-polymer superficially porous particles.

PubMed

Hung, Chuan-Hsi; Zukowski, Janusz; Jensen, David S; Miles, Andrew J; Sulak, Clayton; Dadson, Andrew E; Linford, Matthew R

2015-09-01

Three mixed-mode high-performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine-polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C18 ), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed-mode column (C18 ) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed-mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C18 ) mixed-mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and Validation of a Stability-indicating Reversed-phase UPLC-UV Method for the Assay of Imidacloprid and Estimation of its Related Compounds.

PubMed

Tian, Jingzhi; Rustum, Abu

2018-02-01

Imidacloprid is used as an active pharmaceutical ingredient (API) in veterinary drugs to control fleas and ticks for dogs and cats. Here we are reporting for the first time a validated stability-indicating reversed-phase UPLC-UV method for the assay of imidacloprid and estimation of its related compounds. The stability-indicating capability of this method has been demonstrated by a forced degradation study. All related compounds including processing impurities, imidacloprid API and degradates from stressed samples were well separated from each other. Structures of major degradates from forced degradation study were elucidated through UPLC-MS/MS and key degradation pathways were proposed from the proposed chemical structures of major degradates. The UPLC-UV method is carried out using an HSS T3 column (C18, 2.1 × 30 mm, 1.8 μm particle size) maintained at 30°C with mobile phase A (0.05% v/v of phosphoric acid in water) and mobile phase B (methanol/acetonitrile 75/25 v/v). Analytes are separated by a gradient elution and detected at 270 nm. The UPLC method is green and fast with only 6.5 min run time and about 3.5 ml mobile phase consumption for each sample analysis. The UPLC-UV method was validated according to ICH guidelines. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Sensitive method for the quantitative determination of proguanil and its metabolites in rat blood and plasma by liquid chromatography-mass spectrometry.

PubMed

Leveque, Nathalie L; Charman, William N; Chiu, Francis C K

2006-01-18

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.

Development and validation of an HPLC method for tetracycline-related USP monographs.

PubMed

Hussien, Emad M

2014-09-01

A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.

[Determination of 11 mycotoxins in baked foods and raw materials by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry].

PubMed

Li, Rong; He, Chunmei; Yang, Luqi; Wang, Yong; Zhang, Pengjie; Gao, Yongqing

2017-08-08

A method for the determination of 11 mycotoxins in baked foods and raw materials by ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-HRMS) is reported in this paper. The samples were extracted with 20 mL 90% (v/v) acetonitrile aqueous solution containing 1% (v/v) formic acid, and the extracts were salted out by 2.0 g MgSO 4 and 0.5 g NaCl, cleaned up by 300 mg C18. The analytes were carried out on a CORTECS C18 column (100 mm×2.1 mm, 1.6 μ m) by gradient elution with 2 mmol/L ammonium acetate with 0.1% (v/v) formic acid aqueous solution and 2 mmol/L ammonium acetate methanol with 0.1% (v/v) formic acid. The results showed that the 11 mycotoxins had good linear relationships in their respective mass concentration ranges. The correlation coefficients were not less than 0.9960 and the limits of quantitation (LOQs) were from 0.15 to 20.00 μ g/kg. The recoveries of the 11 mycotoxins in bread ranged from 64.38% to 122.61% with the relative standard deviations (RSDs) from 1.52% to 12.99% at three spiked levels ( n =6). The method is demonstrated to be simple, fast, highly sensitive, reliable and it is effective to detect common mycotoxins in baked foods and raw materials.

Determination of naphthalene-derived compounds in apples by ultra-high performance liquid chromatography-tandem mass spectrometry.

PubMed

Esparza, X; Moyano, E; Cosialls, J R; Galceran, M T

2013-06-11

Naphthylacetic acid, naphthyloxy acetic acid and naphthylacetamide belong to a group of synthetic substances known as "auxin-like" compounds which are used as growth regulators in vegetables and fruits due to their structure similarities with the indoleacetic acid, the most important plant auxin. This paper reports a selective, sensitive and fast ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of naphthylacetamide (NAD) and the isomers (α and β) of naphthylacetic acid (NAA) and naphthyloxy acetic (NOA) acid in apple samples. A baseline separation between the respective isomers was achieved using an RP-Amide column with gradient elution. The UHPLC-MS/MS method developed, using electrospray and selected reaction monitoring (SRM) acquisition mode led to a reliable determination of these family of compounds in apple samples at low quantitation levels, down to 1.0 μg kg(-1) and 0.25 μg kg(-1) respectively. For confirmation of NAA accurate mass measurement is proposed giving at these conditions quantitation limits of 10 μg kg(-1) for this compound. The UHPLC-MS/MS method developed was used for the analysis of apple samples harvested in three different apple fields from Lleida (Spain) during the blooming period. NAD and NAA were found in samples collected during 4-5 weeks after application at concentrations between the quantification limits and 43 μg kg(-1) and 24 μg kg(-1), respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC-APCI-MS.

PubMed

Talluri, Murali V N Kumar; Kalariya, Pradipbhai D; Dharavath, Shireesha; Shaikh, Naeem; Garg, Prabha; Ramisetti, Nageswara Rao; Ragampeta, Srinivas

2016-09-01

A novel ultra high performance liquid chromatography method development strategy was ameliorated by applying quality by design approach. The developed systematic approach was divided into five steps (i) Analytical Target Profile, (ii) Critical Quality Attributes, (iii) Risk Assessments of Critical parameters using design of experiments (screening and optimization phases), (iv) Generation of design space, and (v) Process Capability Analysis (Cp) for robustness study using Monte Carlo simulation. The complete quality-by-design-based method development was made automated and expedited by employing sub-2 μm particles column with an ultra high performance liquid chromatography system. Successful chromatographic separation of the Coenzyme Q10 from its biotechnological process related impurities was achieved on a Waters Acquity phenyl hexyl (100 mm × 2.1 mm, 1.7 μm) column with gradient elution of 10 mM ammonium acetate buffer (pH 4.0) and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Through this study, fast and organized method development workflow was developed and robustness of the method was also demonstrated. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance to the International Conference on Harmonization, Q2 (R1) guidelines. The impurities were identified by atmospheric pressure chemical ionization-mass spectrometry technique. Further, the in silico toxicity of impurities was analyzed using TOPKAT and DEREK software. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of five phthalides in rat plasma: Application to a comparative pharmacokinetic study of Huo Luo Xiao Ling Dan and herb-pair extract.

PubMed

Ma, Wen; Wang, Weihui; Peng, Yan; Bian, Qiaoxia; Wang, Nannan; Lee, David Y-W; Dai, Ronghua

2016-06-01

A fast, sensitive, and reliable ultra-high performance liquid chromatography with tandem mass spectrometry method has been developed and validated for the simultaneous quantitation and pharmacokinetic study of five phthalides (senkyunolide A, ligustilide, butylidenephthalide, 3-butylphthalide, and levistilide A) in rat plasma after oral administration of Huo Luo Xiao Ling Dan (HLXLD) or Angelica sinensis--Ligusticum chuanxiong herb pair (DG-CX) between normal and arthritis rats. After extraction from blood, the analytes and internal standard were subjected to ultra-high performance liquid chromatography with a Shim-pack XR-ODS column (75 × 3.0 mm(2) , 2.2 μm particles) and mobile phase was composed of methanol and water (containing 0.05% formic acid) under gradient elution conditions, with an electrospray ionization source in the positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.192-0.800 ng/mL for all the analytes. Satisfactory linearity, precision, accuracy, mean extraction recovery, and acceptable matrix effect have been achieved. The validated method was successfully applied to a comparative pharmacokinetic study of five bioactive components in rat plasma after oral administration of HLXLD or DG-CX alone, respectively, between normal and arthritic rats. The results showed that there were unlike characters of pharmacokinetics among different groups. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bare metal stents, durable polymer drug eluting stents, and biodegradable polymer drug eluting stents for coronary artery disease: mixed treatment comparison meta-analysis

PubMed Central

Toklu, Bora; Amoroso, Nicholas; Fusaro, Mario; Kumar, Sunil; Hannan, Edward L; Faxon, David P; Feit, Frederick

2013-01-01

Objective To compare the efficacy and safety of biodegradable polymer drug eluting stents with those of bare metal stents and durable polymer drug eluting stents. Design Mixed treatment comparison meta-analysis of 258 544 patient years of follow-up from randomized trials. Data sources and study selection PubMed, Embase, and Central were searched for randomized trials comparing any of the Food and Drug Administration approved durable polymer drug eluting stents (sirolimus eluting, paclitaxel eluting, cobalt chromium everolimus eluting, platinum chromium everolimus eluting, zotarolimus eluting-Endeavor, and zotarolimus eluting-Resolute) or biodegradable polymer drug eluting stents, with each other or against bare metal stents. Outcomes Long term efficacy (target vessel revascularization, target lesion revascularization) and safety (death, myocardial infarction, stent thrombosis). Landmark analysis at more than one year was evaluated to assess the potential late benefit of biodegradable polymer drug eluting stents. Results From 126 randomized trials and 258 544 patient years of follow-up, for long term efficacy (target vessel revascularization), biodegradable polymer drug eluting stents were superior to paclitaxel eluting stents (rate ratio 0.66, 95% credibility interval 0.57 to 0.78) and zotarolimus eluting stent-Endeavor (0.69, 0.56 to 0.84) but not to newer generation durable polymer drug eluting stents (for example: 1.03, 0.89 to 1.21 versus cobalt chromium everolimus eluting stents). Similarly, biodegradable polymer drug eluting stents were superior to paclitaxel eluting stents (rate ratio 0.61, 0.37 to 0.89) but inferior to cobalt chromium everolimus eluting stents (2.04, 1.27 to 3.35) for long term safety (definite stent thrombosis). In the landmark analysis after one year, biodegradable polymer drug eluting stents were superior to sirolimus eluting stents for definite stent thrombosis (rate ratio 0.29, 0.10 to 0.82) but were associated with increased mortality compared with cobalt chromium everolimus eluting stents (1.52, 1.02 to 2.22). Overall, among all stent types, the newer generation durable polymer drug eluting stents (zotarolimus eluting stent-Resolute, cobalt chromium everolimus eluting stents, and platinum chromium everolimus eluting stents) were the most efficacious (lowest target vessel revascularization rate) stents, and cobalt chromium everolimus eluting stents were the safest with significant reductions in definite stent thrombosis (rate ratio 0.35, 0.21 to 0.53), myocardial infarction (0.65, 0.55 to 0.75), and death (0.72, 0.58 to 0.90) compared with bare metal stents. Conclusions Biodegradable polymer drug eluting stents are superior to first generation durable polymer drug eluting stents but not to newer generation durable polymer stents in reducing target vessel revascularization. Newer generation durable polymer stents, and especially cobalt chromium everolimus eluting stents, have the best combination of efficacy and safety. The utility of biodegradable polymer stents in the context of excellent clinical outcomes with newer generation durable polymer stents needs to be proven. PMID:24212107

An on-line SPE-HPLC method for effective sample preconcentration and determination of fenoxycarb and cis, trans-permethrin in surface waters.

PubMed

Šatínský, Dalibor; Naibrtová, Linda; Fernández-Ramos, Carolina; Solich, Petr

2015-09-01

A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min. Copyright © 2015 Elsevier B.V. All rights reserved.

A fast mass spring model solver for high-resolution elastic objects

NASA Astrophysics Data System (ADS)

Zheng, Mianlun; Yuan, Zhiyong; Zhu, Weixu; Zhang, Guian

2017-03-01

Real-time simulation of elastic objects is of great importance for computer graphics and virtual reality applications. The fast mass spring model solver can achieve visually realistic simulation in an efficient way. Unfortunately, this method suffers from resolution limitations and lack of mechanical realism for a surface geometry model, which greatly restricts its application. To tackle these problems, in this paper we propose a fast mass spring model solver for high-resolution elastic objects. First, we project the complex surface geometry model into a set of uniform grid cells as cages through *cages mean value coordinate method to reflect its internal structure and mechanics properties. Then, we replace the original Cholesky decomposition method in the fast mass spring model solver with a conjugate gradient method, which can make the fast mass spring model solver more efficient for detailed surface geometry models. Finally, we propose a graphics processing unit accelerated parallel algorithm for the conjugate gradient method. Experimental results show that our method can realize efficient deformation simulation of 3D elastic objects with visual reality and physical fidelity, which has a great potential for applications in computer animation.

Ferritin (FER), isoferritins and aluminum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleming, J.; Cho, S.W.; Clauberg, M.

FER from Alzheimer's brain contains more Al. One source of Al is beverages. Of the several common beverages analyzed, Pepsi, sold in bottles but not in cans, contained the highest conc. of Al (10..mu..M). Male albino rats were fed 10..mu..M Al in drinking water for one year. They were then sacrificed and their brain homogenates were analyzed for FER, Al and several enzymes. The results: compared to controls, the homogenates of the Al fed rats had 276.5% more Al bound to Fer (114.2 +/- 25.3 g atoms/mol) and 30% less hexokinase activity (150 units/mg protein). Acetyl choline esterase and alkalinemore » ribonuclease levels remained unchanged. Isoelectrofocusing (pH 4-6.5) of human-brain FER yielded at least five bands. None corresponded with those from human liver FER or horse spleen FER. Horse spleen FER was applied to DEAE sephadex and eluted by NaCl-batchwise gradient. Five distinct fractions were obtained. The most acidic eluted last. It contained least Fe, tended to precipitate on standing and required less Al or Be to ppt. Thus, isoferritins may differ in their metal binding capacity and perhaps in their related physiological functions.« less

A chromatographic objective function to characterise chromatograms with unknown compounds or without standards available.

PubMed

Alvarez-Segura, T; Gómez-Díaz, A; Ortiz-Bolsico, C; Torres-Lapasió, J R; García-Alvarez-Coque, M C

2015-08-28

Getting useful chemical information from samples containing many compounds is still a challenge to analysts in liquid chromatography. The highest complexity corresponds to samples for which there is no prior knowledge about their chemical composition. Computer-based methodologies are currently considered as the most efficient tools to optimise the chromatographic resolution, and further finding the optimal separation conditions. However, most chromatographic objective functions (COFs) described in the literature to measure the resolution are based on mathematical models fitted with the information obtained from standards, and cannot be applied to samples with unknown compounds. In this work, a new COF based on the automatic measurement of the protruding part of the chromatographic peaks (or peak prominences) that indicates the number of perceptible peaks and global resolution, without the need of standards, is developed. The proposed COF was found satisfactory with regard to the peak purity criterion when applied to artificial peaks and simulated chromatograms of mixtures built using the information of standards. The approach was applied to mixtures of drugs containing unknown impurities and degradation products and to extracts of medicinal herbs, eluted with acetonitrile-water mixtures using isocratic and gradient elution. Copyright © 2015 Elsevier B.V. All rights reserved.

Multi-residue analysis method for analysis of pharmaceuticals using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS) in water sample

NASA Astrophysics Data System (ADS)

Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali

2013-11-01

In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.

On the electromagnetic scattering from infinite rectangular grids with finite conductivity

NASA Technical Reports Server (NTRS)

Christodoulou, C. G.; Kauffman, J. F.

1986-01-01

A variety of methods can be used in constructing solutions to the problem of mesh scattering. However, each of these methods has certain drawbacks. The present paper is concerned with a new technique which is valid for all spacings. The new method involved, called the fast Fourier transform-conjugate gradient method (FFT-CGM), represents an iterative technique which employs the conjugate gradient method to improve upon each iterate, utilizing the fast Fourier transform. The FFT-CGM method provides a new accurate model which can be extended and applied to the more difficult problems of woven mesh surfaces. The formulation of the FFT-conjugate gradient method for aperture fields and current densities for a planar periodic structure is considered along with singular operators, the formulation of the FFT-CG method for thin wires with finite conductivity, and reflection coefficients.

Chemometrics-enhanced high performance liquid chromatography-diode array detection strategy for simultaneous determination of eight co-eluted compounds in ten kinds of Chinese teas using second-order calibration method based on alternating trilinear decomposition algorithm.

PubMed

Yin, Xiao-Li; Wu, Hai-Long; Gu, Hui-Wen; Zhang, Xiao-Hua; Sun, Yan-Mei; Hu, Yong; Liu, Lu; Rong, Qi-Ming; Yu, Ru-Qin

2014-10-17

In this work, an attractive chemometrics-enhanced high performance liquid chromatography-diode array detection (HPLC-DAD) strategy was proposed for simultaneous and fast determination of eight co-eluted compounds including gallic acid, caffeine and six catechins in ten kinds of Chinese teas by using second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm. This new strategy proved to be a useful tool for handling the co-eluted peaks, uncalibrated interferences and baseline drifts existing in the process of chromatographic separation, which benefited from the "second-order advantages", making the determination of gallic acid, caffeine and six catechins in tea infusions within 8 min under a simple mobile phase condition. The average recoveries of the analytes on two selected tea samples ranged from 91.7 to 103.1% with standard deviations (SD) ranged from 1.9 to 11.9%. Figures of merit including sensitivity (SEN), selectivity (SEL), root-mean-square error of prediction (RMSEP) and limit of detection (LOD) have been calculated to validate the accuracy of the proposed method. To further confirm the reliability of the method, a multiple reaction monitoring (MRM) method based on LC-MS/MS was employed for comparison and the obtained results of both methods were consistent with each other. Furthermore, as a universal strategy, this new proposed analytical method was applied for the determination of gallic acid, caffeine and catechins in several other kinds of Chinese teas, including different levels and varieties. Finally, based on the quantitative results, principal component analysis (PCA) was used to conduct a cluster analysis for these Chinese teas. The green tea, Oolong tea and Pu-erh raw tea samples were classified successfully. All results demonstrated that the proposed method is accurate, sensitive, fast, universal and ideal for the rapid, routine analysis and discrimination of gallic acid, caffeine and catechins in Chinese tea samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Improved selectivity for high-performance liquid chromatographic determination of clonazepam in plasma of epileptic patients.

PubMed

Le Guellec, C; Gaudet, M L; Breteau, M

1998-11-20

We report a high-performance liquid chromatography method for clonazepam determination in plasma. The use of a synthetic silica-based stationary phase markedly improved clonazepam resolution compared to standard reversed-phase columns. A liquid-liquid extraction was used, associated with reversed-phase chromatography, gradient elution and ultraviolet detection. Accuracy and precision were satisfactory at therapeutic concentrations. Selectivity was studied for benzodiazepines or other antiepileptic drugs, with particular attention to newly marketed drugs i.e., gabapentine and vigabatrin. No interfering substance was evidenced. Under the conditions described, it was possible to quantify clonazepam at nanogram level even when carbamazepine was present at therapeutic concentrations.

A high-performance liquid chromatography micromethod for the simultaneous determination of vigabatrin and gabapentin in serum.

PubMed

Ratnaraj, N; Patsalos, P N

1998-08-01

A gradient high-performance liquid chromatography micromethod is described for the simultaneous quantitation of vigabatrin and gabapentin in human serum. Chromatography was performed using a 125- x 3-mm ID Hypersil BDS C-18 column with a 3-microm mini-bore, eluted with a gradient system comprised of phosphate buffer (pH 6.5)-acetonitrile-methanol-water at a flow rate of 0.45 ml/minute. The column eluent was monitored on a fluorescence detector using excitation and emission wavelengths of 340 and 440 nm, respectively. The lower limit of quantitation for vigabatrin and for gabapentin was 5 micromol/l, and the within-batch and between-batch coefficients of variation were <5%. No interference from commonly prescribed antiepileptic drugs (carbamazepine and its metabolite carbamazepine epoxide, oxcarbazepine and its metabolite 10-hydroxycarbazepine, ethosuximide, lamotrigine, phenobarbitone, phenytoin, primidone, and valproic acid) was observed; thus, the method can be used to monitor vigabatrin and gabapentin in patients on polytherapy antiepileptic drug regimens.

Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

1980-01-01

Isoenzymes of creatine kinase were separated by anion-exchange chromatography, with use of an elution gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and themore » bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creatine kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.« less

Simultaneous determination of phenylephrine hydrochloride, guaifenesin, and chlorpheniramine maleate in cough syrup by gradient liquid chromatography.

PubMed

Amer, Sawsan M; Abbas, Samah S; Shehata, Mostafa A; Ali, Nahed M

2008-01-01

A simple and reliable high-performance liquid chromatographic method was developed for the simultaneous determination of mixture of phenylephrine hydrochloride (PHENYL), guaifenesin (GUAIF), and chlorpheniramine maleate (CHLO) either in pure form or in the presence of methylparaben and propylparaben in a commercial cough syrup dosage form. Separation was achieved on a C8 column using 0.005 M heptane sulfonic acid sodium salt (pH 3.4 +/- 0.1) and acetonitrile as a mobile phase by gradient elution at different flow rates, and detection was done spectrophotometrically at 210 nm. A linear relationship in the range of 30-180, 120-1800, and 10-60 microg/mL was obtained for PHENYL, GUAIF, and CHLO, respectively. The results were statistically analyzed and compared with those obtained by applying the British Pharmacopoeia (2002) method and showed that the proposed method is precise, accurate, and can be easily applied for the determination of the drugs under investigation in pure form and in cough syrup formulations.

Shell and small particles; evaluation of new column technology.

PubMed

Fekete, Szabolcs; Fekete, Jeno; Ganzler, Katalin

2009-01-15

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 microm particles in gradient and isocratic elution separations of hormones (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone). Peak capacities around 140-150 could be achieved in 25 min with the 5 cm long columns. The Ascentis Express column (packed with 2.7 microm shell particles) showed similar efficiency to sub-2 microm particles under gradient conditions. Applying isocratic separation, the column of 2.7 microm shell particles had a reduced plate height minimum of approximately h=1.6. It was much smaller than obtained with totally porous particles (h approximately = 2.8). The impedance time also proved more favorable with 2.7 microm shell particles than with totally porous particles. The influence of extra-column volume on column efficiency was investigated. The extra-column dispersion of the chromatographic system may cause a shift of the HETP curves.

Novel diffusive gradients in thin films technique to assess labile sulfate in soil.

PubMed

Hanousek, Ondrej; Mason, Sean; Santner, Jakob; Chowdhury, Md Mobaroqul Ahsan; Berger, Torsten W; Prohaska, Thomas

2016-09-01

A novel diffusive gradients in thin films (DGT) technique for sampling labile soil sulfate was developed, based on a strong basic anion exchange resin (Amberlite IRA-400) for sulfate immobilization on the binding gel. For reducing the sulfate background on the resin gels, photopolymerization was applied instead of ammonium persulfate-induced polymerization. Agarose cross-linked polyacrylamide (APA) hydrogels were used as diffusive layer. The sulfate diffusion coefficient in APA gel was determined as 9.83 × 10(-6) ± 0.35 × 10(-6) cm(2) s(-1) at 25 °C. The accumulated sulfate was eluted in 1 mol L(-1) HNO3 with a recovery of 90.9 ± 1.6 %. The developed method was tested against two standard extraction methods for soil sulfate measurement. The obtained low correlation coefficients indicate that DGT and conventional soil test methods assess differential soil sulfate pools, rendering DGT a potentially important tool for measuring labile soil sulfate.

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection.

PubMed

Bishop, Michael Jason; Crow, Brian S; Kovalcik, Kasey D; George, Joe; Bralley, James A

2007-04-01

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (

Outcomes with various drug eluting or bare metal stents in patients with diabetes mellitus: mixed treatment comparison analysis of 22 844 patient years of follow-up from randomised trials

PubMed Central

Kumar, Sunil; Fusaro, Mario; Amoroso, Nicholas; Kirtane, Ajay J; Byrne, Robert A; Williams, David O; Slater, James; Cutlip, Donald E; Feit, Frederick

2012-01-01

Objectives To evaluate the efficacy and safety of currently used drug eluting stents compared with each other and compared with bare metal stents in patients with diabetes. Design Mixed treatment comparison meta-analysis. Data sources and study selection PubMed, Embase, and CENTRAL were searched for randomised clinical trials, until April 2012, of four durable polymer drug eluting stents (sirolimus eluting stents, paclitaxel eluting stents, everolimus eluting stents, and zotarolimus eluting stents) compared with each other or with bare metal stents for the treatment of de novo coronary lesions and enrolling at least 50 patients with diabetes. Primary outcomes Efficacy (target vessel revascularisation) and safety (death, myocardial infarction, stent thrombosis). Results From 42 trials with 22 844 patient years of follow-up, when compared with bare metal stents (reference rate ratio 1) all of the currently used drug eluting stents were associated with a significant reduction in target vessel revascularisation (37% to 69%), though the efficacy varied with the type of stent (everolimus eluting stents∼sirolimus eluting stents>paclitaxel eluting stents∼zotarolimus eluting stent>bare metal stents). There was about an 87% probability that everolimus eluting stents were the most efficacious compared with all others, though there were limited usable data for the zotarolimus eluting Resolute stent in patients with diabetes. Moreover, there was no increased risk of any safety outcome (including very late stent thrombosis) with any drug eluting stents compared with bare metal stents. There was about a 62% probability that the everolimus eluting stent was the safest stent for the outcome of “any” stent thrombosis. Conclusions Among patients with diabetes treated with coronary stents all currently available drug eluting stents were efficacious without compromising safety compared with bare metal stents. There were relative differences among the drug eluting stents, such that the everolimus eluting stent was the most efficacious and safe. PMID:22885395

Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

PubMed

Li, J; Cheong, K L; Zhao, J; Hu, D J; Chen, X Q; Qiao, C F; Zhang, Q W; Chen, Y W; Li, S P

2013-09-20

A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance. Copyright © 2013 Elsevier B.V. All rights reserved.

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids.

PubMed

Pappa-Louisi, A; Agrafiotou, P; Papachristos, K

2010-07-01

The combined effect of the ion-pairing reagent concentration, C(ipr), and organic modifier content, phi, on the retention under phi-gradient conditions at different constant C(ipr) was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D phi-gradient retention data obtained under the same phi-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C(ipr) values in the eluent. The second approach is based on a retention model describing the combined effect of C(ipr) and phi on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C(ipr) values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C(ipr) values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by phi-variation.

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

2002-01-01

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in wines.

PubMed

Silva, Catarina L; Gonçalves, João L; Câmara, José S

2012-08-20

A new approach based on microextraction by packed sorbent (MEPS) and reversed-phase high-throughput ultra high pressure liquid chromatography (UHPLC) method that uses a gradient elution and diode array detection to quantitate three biologically active flavonols in wines, myricetin, quercetin, and kaempferol, is described. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (selectivity, linearity, sensitivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters such as the type of sorbent material (C2, C8, C18, SIL, and C8/SCX), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, on the MEPS performance. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250μL) in five extraction cycle and in a short time period (about 5min for the entire sample preparation step). Under optimized conditions, excellent linearity (R(values)(2)>0.9963), limits of detection of 0.006μgmL(-1) (quercetin) to 0.013μgmL(-1) (myricetin) and precision within 0.5-3.1% were observed for the target flavonols. The average recoveries of myricetin, quercetin and kaempferol for real samples were 83.0-97.7% with relative standard deviation (RSD, %) lower than 1.6%. The results obtained showed that the most abundant flavonol in the analyzed samples was myricetin (5.8±3.7μgmL(-1)). Quercetin (0.97±0.41μgmL(-1)) and kaempferol (0.66±0.24μgmL(-1)) were found in a lower concentration. The optimized MEPS(C8) method was compared with a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis HLB) were used as reference. MEPS(C8) approach offers an attractive alternative for analysis of flavonols in wines, providing a number of advantages including highest extraction efficiency (from 85.9±0.9% to 92.1±0.5%) in the shortest extraction time with low solvent consumption, fast sample throughput, more environmentally friendly and easy to perform. Copyright © 2012 Elsevier B.V. All rights reserved.

Ultrasound image edge detection based on a novel multiplicative gradient and Canny operator.

PubMed

Zheng, Yinfei; Zhou, Yali; Zhou, Hao; Gong, Xiaohong

2015-07-01

To achieve the fast and accurate segmentation of ultrasound image, a novel edge detection method for speckle noised ultrasound images was proposed, which was based on the traditional Canny and a novel multiplicative gradient operator. The proposed technique combines a new multiplicative gradient operator of non-Newtonian type with the traditional Canny operator to generate the initial edge map, which is subsequently optimized by the following edge tracing step. To verify the proposed method, we compared it with several other edge detection methods that had good robustness to noise, with experiments on the simulated and in vivo medical ultrasound image. Experimental results showed that the proposed algorithm has higher speed for real-time processing, and the edge detection accuracy could be 75% or more. Thus, the proposed method is very suitable for fast and accurate edge detection of medical ultrasound images. © The Author(s) 2014.

Fast conjugate phase image reconstruction based on a Chebyshev approximation to correct for B0 field inhomogeneity and concomitant gradients.

PubMed

Chen, Weitian; Sica, Christopher T; Meyer, Craig H

2008-11-01

Off-resonance effects can cause image blurring in spiral scanning and various forms of image degradation in other MRI methods. Off-resonance effects can be caused by both B0 inhomogeneity and concomitant gradient fields. Previously developed off-resonance correction methods focus on the correction of a single source of off-resonance. This work introduces a computationally efficient method of correcting for B0 inhomogeneity and concomitant gradients simultaneously. The method is a fast alternative to conjugate phase reconstruction, with the off-resonance phase term approximated by Chebyshev polynomials. The proposed algorithm is well suited for semiautomatic off-resonance correction, which works well even with an inaccurate or low-resolution field map. The proposed algorithm is demonstrated using phantom and in vivo data sets acquired by spiral scanning. Semiautomatic off-resonance correction alone is shown to provide a moderate amount of correction for concomitant gradient field effects, in addition to B0 imhomogeneity effects. However, better correction is provided by the proposed combined method. The best results were produced using the semiautomatic version of the proposed combined method.

Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method.

PubMed

Vidović, Stojanka; Stojanović, Biljana; Veljković, Jelena; Prazić-Arsić, Ljiljana; Roglić, Goran; Manojlović, Dragan

2008-08-22

HPLC stability-indicating method has been developed for the simultaneous determination of some water-soluble vitamins (ascorbic acid, thiamine hydrochloride, riboflavin-5'-phosphate sodium, pyridoxine hydrochloride, nicotinamide, D(+)-panthenol) and two preservatives (methylparaben and sodium benzoate) in multivitamin syrup preparation. Water-soluble vitamins, preservatives and their degradants were separated on Zorbax SB-Aq (C(18)) (250 mm x 4.6 mm, 5 microm) column at an ambient temperature. Combined isocratic and gradient elution was performed with a mobile phase consisting of 0.0125 M hexane-1-sulfonic acid sodium salt in 0.1% (m/v) o-phosphoric acid, pH 2.4-2.5 (solvent A) and acetonitrile (solvent B) at the flow-rate 1 ml min(-1). Starting with solvent A an isocratic elution was performed for 15 min, then the composition was changed to 85% of A and 15% of B during the next 20 min and it was constant for 5 min, then the composition was changed to 70% of A and 30% of B during next 15 min and it was constant for 5 min and finally was changed to 100% of A as at the beginning of the elution. Detection was performed with diode array detector at 210, 230 and 254 nm. Multivitamin syrup preparation was subjected to stress testing (forced degradation) in order to demonstrate that degradants from the vitamins, preservatives and/or product excipients do not interfere with the quantification of vitamins and preservatives. Typical validation characteristics: selectivity, accuracy, precision, linearity, range, limit of quantification and limit of detection were evaluated for vitamins and preservatives.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

[Influences of the mobile phase constitution, salt concentration and pH value on retention characters of proteins on the metal chelate column].

PubMed

Li, R; Di, Z M; Chen, G L

2001-09-01

The effects of the nature and concentration of salts, pH value and competitive eluent in the mobile phase on the protein retention have been systematically investigated. A mathematical expression describing the protein retention in metal chelate chromatography has been derived. It is proposed that the eluting power of the salt solution can be expressed by the eluent strength exponent epsilon. According to the retention characters of protein under different chromatographic conditions, the interaction between the various metal chelate ligands and proteins is discussed. The protein retention on the metal chelate column is a cooperative interactions of coordination, electrostatic and hydrophobic interaction. For the strong combined metal column with proteins such as IDA-Cu, the coordination is the most important, and the electrostatic interaction is secondary in chromatographic process. However, for the weak combined metal columns with proteins such as IDA-Ni, IDA-Co and IDA-Zn, the electrostatic interaction between the metal chelate ligands and proteins is the chief one, while the coordination is the next in importance. When the mobile phase contains high concentration of salt which can't form complex with the immobilized metal, the hydrophobic interaction between the protein and stationary phase will be increased. As the interaction between the metal chelate ligand and proteins relates to chromatographic operating conditions closely, different elution processes may be selected for different metal chelate columns. The gradient elution is generally performed by the low concentration of salt or different pH for weakly combined columns with proteins, however the competitive elution procedure is commonly utilized for strongly combined column. The experiment showed that NH3 is an excellent competitive eluent. It isn't only give the efficient separation of proteins, but also has the advantages of cheapness, less bleeding of the immobilized metals and ease of controlling NH3 concentration. The interaction between the metal chelate ligand and proteins and the selectivity of metal chelate chromatography can be changed through changing chromatographic conditions.

Combining selection valve and mixing chamber for nanoflow gradient generation: Toward developing a liquid chromatography cartridge coupled with mass spectrometer for protein and peptide analysis.

PubMed

Chen, Apeng; Lu, Joann J; Gu, Congying; Zhang, Min; Lynch, Kyle B; Liu, Shaorong

2015-08-05

Toward developing a micro HPLC cartridge, we have recently built a high-pressure electroosmotic pump (EOP). However, we do not recommend people to use this pump to deliver an organic solvent directly, because it often makes the pump rate unstable. We have experimented several approaches to address this issue, but none of them are satisfactory. Here, we develop an innovative approach to address this issue. We first create an abruption (a dead-volume) within a fluid conduit. We then utilize an EOP to withdraw, via a selection valve, a train of eluent solutions having decreasing eluting power into the fluid conduit. When these solutions are further aspirated through the dead-volume, these solutions are partially mixed, smoothening concentration transitions between two adjacent eluent solutions. As these solutions are pushed back, through the dead-volume again, a smooth gradient profile is formed. In this work, we characterize this scheme for gradient formation, and we incorporate this approach with a high-pressure EOP, a nanoliter injection valve, and a capillary column, yielding a micro HPLC system. We then couple this micro HPLC with an electrospray ionization - mass spectrometer for peptide and protein separations and identifications. Copyright © 2015 Elsevier B.V. All rights reserved.

A fast, preconditioned conjugate gradient Toeplitz solver

NASA Technical Reports Server (NTRS)

Pan, Victor; Schrieber, Robert

1989-01-01

A simple factorization is given of an arbitrary hermitian, positive definite matrix in which the factors are well-conditioned, hermitian, and positive definite. In fact, given knowledge of the extreme eigenvalues of the original matrix A, an optimal improvement can be achieved, making the condition numbers of each of the two factors equal to the square root of the condition number of A. This technique is to applied to the solution of hermitian, positive definite Toeplitz systems. Large linear systems with hermitian, positive definite Toeplitz matrices arise in some signal processing applications. A stable fast algorithm is given for solving these systems that is based on the preconditioned conjugate gradient method. The algorithm exploits Toeplitz structure to reduce the cost of an iteration to O(n log n) by applying the fast Fourier Transform to compute matrix-vector products. Matrix factorization is used as a preconditioner.

Development and validation of a critical gradient energetic particle driven Alfven eigenmode transport model for DIII-D tilted neutral beam experiments

DOE PAGES

Waltz, Ronald E.; Bass, Eric M.; Heidbrink, William W.; ...

2015-10-30

Recent experiments with the DIII-D tilted neutral beam injection (NBI) varying the beam energetic particle (EP) source profiles have provided strong evidence that unstable Alfven eigenmodes (AE) drive stiff EP transport at a critical EP density gradient. Here the critical gradient is identified by the local AE growth rate being equal to the local ITG/TEM growth rate at the same low toroidal mode number. The growth rates are taken from the gyrokinetic code GYRO. Simulation show that the slowing down beam-like EP distribution has a slightly lower critical gradient than the Maxwellian. The ALPHA EP density transport code, used tomore » validate the model, combines the low-n stiff EP critical density gradient AE mid-core transport with the energy independent high-n ITG/TEM density transport model controling the central core EP density profile. For the on-axis NBI heated DIII-D shot 146102, while the net loss to the edge is small, about half the birth fast ions are transported from the central core r/a < 0.5 and the central density is about half the slowing down density. Lastly, these results are in good agreement with experimental fast ion pressure profiles inferred from MSE constrained EFIT equilibria.« less

Simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate in welding fume alkaline extracts by HPLC-ICP-MS.

PubMed

Ščančar, Janez; Berlinger, Balázs; Thomassen, Yngvar; Milačič, Radmila

2015-09-01

A novel analytical procedure was developed for the simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate by anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Linear gradient elution from 100% water to 100% 0.7 M NaCl was applied for chromatographic separation of metal species. In standard aqueous solution at neutral pH molybdate, tungstate and vanadate exist in several aqueous species, while chromate is present as a single CrO4(2-) species. Consequently, only chromate can be separated from this solution in a sharp chromatographic peak. For obtaining sharp chromatographic peaks for molybdate, tungstate and vanadate, the pH of aqueous standard solutions was raised to 12. At highly alkaline conditions single CrO4(2-), MoO4(2-) and WO4(2-) are present and were eluted in sharp chromatographic peaks, while VO4(3-) species, which predominates at pH 12 was eluted in slightly broaden peak. In a mixture of aqueous standard solutions (pH 12) chromate, molybdate, tungstate and vanadate were eluted at retention times from 380 to 420 s, 320 to 370 s, 300 to 350 s and 240 to 360 s, respectively. Eluted species were simultaneously detected on-line by ICP-MS recording m/z 52, 95, 182 and 51. The developed procedure was successfully applied to the analysis of leachable concentrations of chromate, molybdate, tungstate and vanadate in alkaline extracts (2% NaOH+3% Na2CO3) of manual metal arc (MMA) welding fumes loaded on filters. Good repeatability and reproducibility of measurement (RSD±3.0%) for the investigated species were obtained in both aqueous standard solutions (pH 12) and in alkaline extracts of welding fumes. Low limits of detection (LODs) were found for chromate (0.02 ng Cr mL(-1)), molybdate (0.1 ng Mo mL(-1)), tungstate (0.1 ng W mL(-1)) and vanadate (0.2 ng V mL(-1)). The accuracy of analytical procedure for the determination of chromate was checked by analysis of CRM 545, Cr(VI) in welding dust loaded on a filter. Good agreement between determined and reported certified values was obtained. For molybdate, tungstate and vanadate the assessment of accuracy was performed by spiking welding fume filters. Good recoveries for all investigated species (98-101%) confirmed the accuracy of the analytical procedure. Copyright © 2015 Elsevier B.V. All rights reserved.

A penalized linear and nonlinear combined conjugate gradient method for the reconstruction of fluorescence molecular tomography.

PubMed

Shang, Shang; Bai, Jing; Song, Xiaolei; Wang, Hongkai; Lau, Jaclyn

2007-01-01

Conjugate gradient method is verified to be efficient for nonlinear optimization problems of large-dimension data. In this paper, a penalized linear and nonlinear combined conjugate gradient method for the reconstruction of fluorescence molecular tomography (FMT) is presented. The algorithm combines the linear conjugate gradient method and the nonlinear conjugate gradient method together based on a restart strategy, in order to take advantage of the two kinds of conjugate gradient methods and compensate for the disadvantages. A quadratic penalty method is adopted to gain a nonnegative constraint and reduce the illposedness of the problem. Simulation studies show that the presented algorithm is accurate, stable, and fast. It has a better performance than the conventional conjugate gradient-based reconstruction algorithms. It offers an effective approach to reconstruct fluorochrome information for FMT.

Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography.

PubMed

Kazarian, Artaches A; Nesterenko, Pavel N; Soisungnoen, Phimpha; Burakham, Rodjana; Srijaranai, Supalax; Paull, Brett

2014-08-01

Liquid chromatographic assays were developed using a mixed-mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve-mediated column switching and was based upon a single high-performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion-exchange, (ii) mixed-mode interactions under an applied dual gradient (reversed-phase/ion-exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C18 column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed-mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well-resolved unknown peaks. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous gradient temperature Raman spectroscopy of n-6 DPA and DHA from -100 C to 20°C

USDA-ARS?s Scientific Manuscript database

One of the great unanswered questions with respect to biological science in general is the absolute necessity of DHA in fast signal processing tissues. N-6 DPA, with just one less diene, group, is fairly abundant in terrestrial food chains yet cannot substitute for DHA. Gradient Temperature Raman sp...

Analysis of iodinated quorum sensing peptides by LC-UV/ESI ion trap mass spectrometry.

PubMed

Janssens, Yorick; Verbeke, Frederick; Debunne, Nathan; Wynendaele, Evelien; Peremans, Kathelijne; De Spiegeleer, Bart

2018-02-01

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C 18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%).

Forced degradation and impurity profiling: recent trends in analytical perspectives.

PubMed

Jain, Deepti; Basniwal, Pawan Kumar

2013-12-01

This review describes an epigrammatic impression of the recent trends in analytical perspectives of degradation and impurities profiling of pharmaceuticals including active pharmaceutical ingredient (API) as well as drug products during 2008-2012. These recent trends in forced degradation and impurity profiling were discussed on the head of year of publication; columns, matrix (API and dosage forms) and type of elution in chromatography (isocratic and gradient); therapeutic categories of the drug which were used for analysis. It focuses distinctly on comprehensive update of various analytical methods including hyphenated techniques for the identification and quantification of thresholds of impurities and degradants in different pharmaceutical matrices. © 2013 Elsevier B.V. All rights reserved.

Application of liquid chromatography-electrospray ionization mass spectrometry for study of steroid-converting enzymes.

PubMed

Miksík, Ivan; Mikulíková, Katerina; Pácha, Jirí; Kucka, Marek; Deyl, Zdenek

2004-02-05

A high-performance liquid chromatography-atmospheric pressure ionization-electrospray ionization mass spectrometry (HPLC-API-ESI-MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol-water-acetic acid gradient) and identification of the steroids involved was done by API-ESI-MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).

UPLC-MS/MS determination of ptaquiloside and pterosin B in preserved natural water.

PubMed

Clauson-Kaas, Frederik; Hansen, Hans Christian Bruun; Strobel, Bjarne W

2016-11-01

The naturally occurring carcinogen ptaquiloside and its degradation product pterosin B are found in water leaching from bracken stands. The objective of this work is to present a new sample preservation method and a fast UPLC-MS/MS method for quantification of ptaquiloside and pterosin B in environmental water samples, employing a novel internal standard. A faster, reliable, and efficient method was developed for isolation of high purity ptaquiloside and pterosin B from plant material for use as analytical standards, with purity verified by 1 H-NMR. The chemical analysis was performed by cleanup and preconcentration of samples with solid phase extraction, before analyte quantification with UPLC-MS/MS. By including gradient elution and optimizing the liquid chromatography mobile phase buffer system, a total run cycle of 5 min was achieved, with method detection limits, including preconcentration, of 8 and 4 ng/L for ptaquiloside and pterosin B, respectively. The use of loganin as internal standard improved repeatability of the determination of both analytes, though it could not be employed for sample preparation. Buffering raw water samples in situ with ammonium acetate to pH ∼5.5 decisively increased sample integrity at realistic transportation and storing conditions prior to extraction. Groundwater samples collected in November 2015 at the shallow water table below a Danish bracken stand were preserved and analyzed using the above methods, and PTA concentrations of 3.8 ± 0.24 μg/L (±sd, n = 3) were found, much higher than previously reported. Graphical abstract Workflow overview of ptaquiloside determination.

A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma.

PubMed

Peer, Cody J; Spencer, Shawn D; VanDenBerg, Dustin A H; Pacanowski, Michael A; Horenstein, Richard B; Figg, William D

2012-01-01

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r(2)=0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range. Published by Elsevier B.V.

A Sensitive and Rapid Ultra HPLC-MS/MS Method for the Simultaneous Detection of Clopidogrel and its Derivatized Active Thiol Metabolite in Human Plasma

PubMed Central

Peer, Cody J.; Spencer, Shawn D.; VanDenBerg, Dustin A. H.; Pacanowski, Michael A.; Horenstein, Richard B.; Figg, William D.

2011-01-01

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix®) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3’-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2µm-C18 column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically-relevant concentration range of 0.01–50 ng/mL for parent clopidogrel and 0.1–150 ng/mL (r2= 0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) < ±12% and precision (%CV) of < ±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range. PMID:22169056

[Determination of three exogenous plant hormone residues in bean sprout by high performance liquid chromatography-quadrupole-time of flight mass spectrometry].

PubMed

Xie, Hanbing; Zhou, Mingying; Zhao, Haifeng; Wang, Yigang; Jiang, Wanfeng; Zhao, Shan

2014-05-01

This study was aimed to the establishment of an analytical method for the determination of three exogenous plant hormone residues in bean sprout by high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF-MS). The target compounds were gibberellins, 6-benzylaminopurine and parachlorophenoxyacetic acid. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used for sample preparation. The analytes were extracted with a solution containing 1% (v/v, if not specified) acetic, 50% ethanol, 49% acetonitrile, and cleaned-up by dispersive solid-phase extraction with diatomite dispersant, then degreased by hexane. The three target compounds were separated on an Eclipse Plus C18 column (100 mm x 3.0 mm, 1.8 microm) with mobile phases A (water containing 0.1% formic acid) and B (methanol) by gradient elution within 15 min, and detected under negative electrospray ionization (ESI) mode. The quantitative analysis was carried out by extracting the peak area with accurate mass. The confirmatory analysis of the target compounds was performed with the qualitative fragments. The results showed that the limits of quantification (LOQs, S/N = 10) for the three target compounds were from 5.0 microg/kg to 10 microg/kg. The respective mean recoveries were found to be in the range of 79.1%-96.1%, and the RSDs were 5.7%-10.4%. It was applicable to the analysis of the three exogenous plant hormones in bean sprout samples. This method is simple, fast and efficient.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

PubMed

Tölgyesi, Ádám; Barta, Enikő; Simon, Andrea; McDonald, Thomas J; Sharma, Virender K

2017-10-25

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L-4.95μg/L and 0.17μg/kg-2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

PubMed

Schmidt, A; Schreiner, M G; Mayer, H K

2017-06-02

As the formation of pyridoxal phosphate, the active cofactor of vitamin B 6 , is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B 6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B 2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B 6 and B 2 vitamers. In cow's milk, vitamin B 6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B 6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69μmolL -1 . Vitamin B 2 was present as riboflavin and its phosphate ester up to 12.86μmolL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

A Simple and High-Throughput Analysis of Amatoxins and Phallotoxins in Human Plasma, Serum and Urine Using UPLC-MS/MS Combined with PRiME HLB μElution Platform

PubMed Central

Zhang, Shuo; Zhao, Yunfeng; Li, Haijiao; Zhou, Shuang; Chen, Dawei; Zhang, Yizhe; Yao, Qunmei; Sun, Chengye

2016-01-01

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of fatal food poisoning in China. In the treatment of Amanita mushroom poisoning, an early and definite diagnosis is necessary for a successful outcome, which has prompted the development of protocols for the fast and confirmatory determination of amatoxins and phallotoxins in human biological fluids. For this purpose, a simple, rapid and sensitive multiresidue UPLC-MS/MS method for the simultaneous determination of α-amanitin, β-amanitin, γ-amanitin, phalloidin (PHD) and phallacidin (PCD) in human plasma, serum and urine was developed and validated. The diluted plasma, serum and urine samples were directly purified with a novel PRiME technique on a 96-well μElution plate platform, which allowed high-throughput sample processing and low reagent consumption. After purification, a UPLC-MS/MS analysis was performed using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. This method fulfilled the requirements of a validation test, with good results for the limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, intra- and inter-assay precision, recovery and matrix effects. All of the analytes were confirmed and quantified in authentic plasma, serum and urine samples obtained from cases of poisoning using this method. Using the PRiME μElution technique for quantification reduces labor and time costs and represents a suitable method for routine toxicological and clinical emergency analysis. PMID:27153089

Fast single run of vanilla fingerprint markers on microfluidic-electrochemistry chip for confirmation of common frauds.

PubMed

Avila, Mónica; Zougagh, Mohammed; Escarpa, Alberto; Ríos, Angel

2009-10-01

A new strategy based on the fast separation of the fingerprint markers of Vanilla planifolia extracts and vanilla-related samples on microfluidic-electrochemistry chip is proposed. This methodology allowed the detection of all required markers for confirmation of common frauds in this field. The elution order was strategically connected with sequential sample screening and analyte confirmation steps, where first ethyl vanillin was detected to distinguish natural from adultered samples; second, vanillin as prominent marker in V. planifolia, but frequently added in its synthetic form; and third, the final detection of the fingerprint markers (p-hydroxybenzaldehyde, vanillic acid, and p-hydroxybenzoic acid) of V. planifolia with confirmation purposes. The reliability of the proposed methodology was demonstrated in the confirmation the natural or non-natural origin of vanilla in samples using V. planifolia extracts and other selected food samples containing this flavor.

Phylogeny of Cyclic Nitramine-Degrading Psychrophilic Bacteria in Marine Sediment and Their Potential Role in the Natural Attenuation of Explosives

DTIC Science & Technology

2004-01-01

frigidimarina sp. nov., novel antarctic species with the ability to produce eicosapentaenoic acid (20:5x3) and grow anaerobically by dissimilatory Fe(III...11,25,29]. NDAB and MEDINA were determined on an AnionSep Ice-Ion-310 Fast organic acids HPLC column (6.5 150 mm, Cobert associates chromatog- raphy...products, St-Louis, MI) at 225 nm and 35 C. The mobile phase was 1.73 mM sulfuric acid at a flow rate of 0.6 ml/min. The elution times of MEDINA and

Development and validation of an UFLC-MS/MS assay for the absolute quantitation of nine notoginsenosides in rat plasma: Application to the pharmacokinetic study of Panax Notoginseng Extract.

PubMed

Zhou, Lijun; Xing, Rong; Xie, Lin; Rao, Tai; Wang, Qian; Ye, Wei; Fu, Hanxu; Xiao, Jingcheng; Shao, Yuhao; Kang, Dian; Wang, Guangji; Liang, Yan

2015-07-15

Notoginsenosides, the main active gradients of Chinese traditional medicine Panax notoginseng, possesses a variety of biological activities including antioxidant property, anti-hyperglycemic, anti-obese, etc. However, pharmacokinetic evaluation for notoginsenosides is still a formidable task due to their low concentrations and complex components in vivo. The summation of this work generated a rapid and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra fast liquid chromatographic-tandem mass spectrometric. After liquid-liquid extraction by n-butanol, notoginsenoside R1, Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1 and Re were simultaneously monitored in negative ionization mode after separating on a Thermo ODS C18 column (5mm 50mm×2.1mm) by a binary gradient elution, and all compounds were analyzed within 9min. Multiple reaction monitoring (MRM) was performed as follows: R1 (m/z 967.7→637.4), Rg3 (m/z 819.6→621.4), Rd (m/z 981.6→783.5), Rg2 (m/z 819.6→475.4), Rb2 (m/z 1113.4→783.4), Rf (m/z 835.6→475.4), Rg1 (m/z 835.6→637.6), Rb1 (m/z 1143.7→945.6), Re (m/z 981.6→637.4), internal standard (digoxin, m/z 815.5→779.4). Validation parameters (linearity, sensitivity, intra-and inter-assay precision and accuracy, recovery and matrix effect) were within acceptable ranges and biological extracts were stable during the entire storing and preparing process. This UFLC-MS/MS approach was further validated by being applied to the pharmacokinetic study for P. Notoginseng extract in rats, and the pharmacokinetic parameters were calculated by Winolin software. Thus, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins. Copyright © 2015 Elsevier B.V. All rights reserved.

Fermentation, isolation, purification and characterization of an antitubercular antibiotic from Streptomyces luridus MTCC 4402.

PubMed

Anuradha, S; Kumar, K Shravan; Bhama, S; Kishan, V

2016-09-01

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be a serious public health problem around the world, and it urges the need for development of new antitubercular drugs. An antibiotic producing strain, Streptomyces luridus (MTCC 4402) was earlier isolated from soil by our group. In this work, the phylogenic status was established by 16S rRNA gene sequence analysis. The strain was found to be active against clinically resistant strains. The culture was grown in shake flasks in a medium containing cornsteep liquor, glucose, CaCO(3), soyabean meal and starch. Antibiotic production reached maximum at the end of 72 h. and fermentation profile was obtained. The active compound was extracted into ethyl acetate and was subjected to activity guided purification by column chromatography using silica gel, TLC and HPLC methods. The pure compound eluted at 16.7 min. by gradient elution was subjected to (1)H, (13)C NMR and mass spectral analyses. The acquired data was compared with that of natural products’ data base and found to be a known antibiotic, spiramycin. The purified compound was studied for mutagenic, cytotoxicity, antitubercular activities. It was non mutagenic at the concentration of 1000 μg/mL, non cytotoxic and active as antitubercular agent at a concentration of 64 mg/mL and was comparable to rifampicin.

Chromatographic behavior of co-eluted plasma compounds and effect on screening of drugs by APCI-LC-MS(/MS): Applications to selected cardiovascular drugs.

PubMed

Tahboub, Yahya R

2014-12-01

Chromatographic behavior of co-eluted compounds from un-extracted drug-free plasma samples was studied by LC-MS and LC-MS/MS with positive APCI. Under soft gradient, total ion chromatogram (TIC) consisted of two major peaks separated by a constant lower intensity region. Early peak (0.15-0.4 min) belongs to polar plasma compounds and consisted of smaller mass ions ( m / z <250); late peak (3.6-4.6 min) belongs to thermally unstable phospholipids and consisted of fragments with m / z <300. Late peak is more sensitive to variations in chromatographic and MS parameters. Screening of most targeted cardiovascular drugs at levels lower than 50 ng/mL has been possible by LC-MS for drugs with retention factors larger than three. Matrix effects and recovery, at 20 and 200 ng/mL, were evaluated for spiked plasma samples with 15 cardiovascular drugs, by MRM-LC-MS/MS. Average recoveries were above 90% and matrix effects expressed as percent matrix factor (% MF) were above 100%, indicating enhancement character for APCI. Large uncertainties were significant for drugs with smaller masses ( m / z <250) and retention factors lower than two.

Simultaneous determination of plasma creatinine, uric acid, kynurenine and tryptophan by high-performance liquid chromatography: method validation and in application to the assessment of renal function.

PubMed

Zhao, Jianxing

2015-03-01

A high-performance liquid chromatography with ultraviolet detection method has been developed for the simultaneous determination of a set of reliable markers of renal function, including creatinine, uric acid, kynurenine and tryptophan in plasma. Separation was achieved by an Agilent HC-C18 (2) analytical column. Gradient elution and programmed wavelength detection allowed the method to be used to analyze these compounds by just one injection. The total run time was 25 min with all peaks of interest being eluted within 13 min. Good linear responses were found with correlation coefficient >0.999 for all analytes within the concentration range of the relevant levels. The recovery was: creatinine, 101 ± 1%; uric acid, 94.9 ± 3.7%; kynurenine, 100 ± 2%; and tryptophan, 92.6 ± 2.9%. Coefficients of variation within-run and between-run of all analytes were ≤2.4%. The limit of detection of the method was: creatinine, 0.1 µmol/L; uric acid, 0.05 µmol/L; kynurenine, 0.02 µmol/L; and tryptophan, 1 µmol/L. The developed method could be employed as a useful tool for the detection of chronic kidney disease, even at an early stage. Copyright © 2014 John Wiley & Sons, Ltd.

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

PubMed

Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

2015-05-01

Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC. Copyright © 2014 John Wiley & Sons, Ltd.

Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine.

PubMed

Humphrey, C D; Condon, C W; Cantey, J R; Pittman, F E

1979-03-01

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Optimal mixing rate in linear solvent strength gradient liquid chromatography. Balanced mixing program.

PubMed

Blumberg, Leonid M; Desmet, Gert

2016-12-09

The mixing rate (R ϕ ) is the temporal rate of increase in the solvent strength in gradient LC. The optimal R ϕ (R ϕ ,Opt ) is the one at which a required peak capacity of gradient LC analysis is obtained in the shortest time. The balanced mixing program is a one where, for better separation of early eluting solutes, the mixing ramp is preceded by a balanced isocratic hold of the duration depending on R ϕ . The improvement in the separation of the earlier eluites due to the balanced programming has been evaluated. The value of R ϕ ,Opt depends on the solvent composition range covered by the mixing ramp and on the column pressure conditions. The R ϕ ,Opt for a column operating at maximum instrumental pressure is different from R ϕ ,Opt for a column operating below the instrumental pressure limit. On the other hand, it has been shown that the difference in the R ϕ ,Opt values under different conditions is not very large so that a single default R ϕ previously recommended for gradient analyses without the isocratic hold also yields a good approximation to the shortest analysis time for all conditions in the balanced analyses. With or without the initial balance isocratic hold, the recommended default R ϕ is about 5%/t 0 (5% increase in the solvent strength per each t 0 -long increment in time) for small-molecule samples, and about an order of magnitude slower (0.5%/t 0 ) for protein samples. A discussion illustrating the use of the optimization criteria employed here for the techniques other than LSS gradient LC is included. Copyright © 2016 Elsevier B.V. All rights reserved.

Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

PubMed Central

Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

2015-01-01

A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation. PMID:26225356

MR imaging of the inner ear: comparison of a three-dimensional fast spin-echo sequence with use of a dedicated quadrature-surface coil with a gadolinium-enhanced spoiled gradient-recalled sequence.

PubMed

Naganawa, S; Ito, T; Fukatsu, H; Ishigaki, T; Nakashima, T; Ichinose, N; Kassai, Y; Miyazaki, M

1998-09-01

To prospectively evaluate the sensitivity and specificity of magnetic resonance (MR) imaging in the inner ear with a long echo train, three-dimensional (3D), asymmetric Fourier-transform, fast spin-echo (SE) sequence with use of a dedicated quadrature-surface phased-array coil to detect vestibular schwannoma in the cerebellopontine angle and the internal auditory canal. In 205 patients (410 ears) with ear symptoms, 1.5-T MR imaging was performed with unenhanced 3D asymmetric fast SE and gadolinium-enhanced 3D gradient-recalled (SPGR) sequences with use of a quadrature surface phased-array coil. The 3D asymmetric fast SE images were reviewed by two radiologists, with the gadolinium-enhanced 3D SPGR images used as the standard of reference. Nineteen lesions were detected in the 410 ears (diameter range, 2-30 mm; mean, 10.5 mm +/- 6.4 [standard deviation]; five lesions were smaller than 5 mm). With 3D asymmetric fast SE, sensitivity, specificity, and accuracy, respectively, were 100%, 99.5%, and 99.5% for observer 1 and 100%, 99.7%, and 99.8% for observer 2. The unenhanced 3D asymmetric fast SE sequence with a quadrature-surface phased-array coli allows the reliable detection of vestibular schwannoma in the cerebellopontine angle and internal auditory canal.

Fast conjugate phase image reconstruction based on a Chebyshev approximation to correct for B0 field inhomogeneity and concomitant gradients

PubMed Central

Chen, Weitian; Sica, Christopher T.; Meyer, Craig H.

2008-01-01

Off-resonance effects can cause image blurring in spiral scanning and various forms of image degradation in other MRI methods. Off-resonance effects can be caused by both B0 inhomogeneity and concomitant gradient fields. Previously developed off-resonance correction methods focus on the correction of a single source of off-resonance. This work introduces a computationally efficient method of correcting for B0 inhomogeneity and concomitant gradients simultaneously. The method is a fast alternative to conjugate phase reconstruction, with the off-resonance phase term approximated by Chebyshev polynomials. The proposed algorithm is well suited for semiautomatic off-resonance correction, which works well even with an inaccurate or low-resolution field map. The proposed algorithm is demonstrated using phantom and in vivo data sets acquired by spiral scanning. Semiautomatic off-resonance correction alone is shown to provide a moderate amount of correction for concomitant gradient field effects, in addition to B0 imhomogeneity effects. However, better correction is provided by the proposed combined method. The best results were produced using the semiautomatic version of the proposed combined method. PMID:18956462

X-PROP: a fast and robust diffusion-weighted propeller technique.

PubMed

Li, Zhiqiang; Pipe, James G; Lee, Chu-Yu; Debbins, Josef P; Karis, John P; Huo, Donglai

2011-08-01

Diffusion-weighted imaging (DWI) has shown great benefits in clinical MR exams. However, current DWI techniques have shortcomings of sensitivity to distortion or long scan times or combinations of the two. Diffusion-weighted echo-planar imaging (EPI) is fast but suffers from severe geometric distortion. Periodically rotated overlapping parallel lines with enhanced reconstruction diffusion-weighted imaging (PROPELLER DWI) is free of geometric distortion, but the scan time is usually long and imposes high Specific Absorption Rate (SAR) especially at high fields. TurboPROP was proposed to accelerate the scan by combining signal from gradient echoes, but the off-resonance artifacts from gradient echoes can still degrade the image quality. In this study, a new method called X-PROP is presented. Similar to TurboPROP, it uses gradient echoes to reduce the scan time. By separating the gradient and spin echoes into individual blades and removing the off-resonance phase, the off-resonance artifacts in X-PROP are minimized. Special reconstruction processes are applied on these blades to correct for the motion artifacts. In vivo results show its advantages over EPI, PROPELLER DWI, and TurboPROP techniques. Copyright © 2011 Wiley-Liss, Inc.

Compressed sensing with gradient total variation for low-dose CBCT reconstruction

NASA Astrophysics Data System (ADS)

Seo, Chang-Woo; Cha, Bo Kyung; Jeon, Seongchae; Huh, Young; Park, Justin C.; Lee, Byeonghun; Baek, Junghee; Kim, Eunyoung

2015-06-01

This paper describes the improvement of convergence speed with gradient total variation (GTV) in compressed sensing (CS) for low-dose cone-beam computed tomography (CBCT) reconstruction. We derive a fast algorithm for the constrained total variation (TV)-based a minimum number of noisy projections. To achieve this task we combine the GTV with a TV-norm regularization term to promote an accelerated sparsity in the X-ray attenuation characteristics of the human body. The GTV is derived from a TV and enforces more efficient computationally and faster in convergence until a desired solution is achieved. The numerical algorithm is simple and derives relatively fast convergence. We apply a gradient projection algorithm that seeks a solution iteratively in the direction of the projected gradient while enforcing a non-negatively of the found solution. In comparison with the Feldkamp, Davis, and Kress (FDK) and conventional TV algorithms, the proposed GTV algorithm showed convergence in ≤18 iterations, whereas the original TV algorithm needs at least 34 iterations in reducing 50% of the projections compared with the FDK algorithm in order to reconstruct the chest phantom images. Future investigation includes improving imaging quality, particularly regarding X-ray cone-beam scatter, and motion artifacts of CBCT reconstruction.

Tutorial: simulating chromatography with Microsoft Excel Macros.

PubMed

Kadjo, Akinde; Dasgupta, Purnendu K

2013-04-22

Chromatography is one of the cornerstones of modern analytical chemistry; developing an instinctive feeling for how chromatography works will be invaluable to future generation of chromatographers. Specialized software programs exist that handle and manipulate chromatographic data; there are also some that simulate chromatograms. However, the algorithm details of such software are not transparent to a beginner. In contrast, how spreadsheet tools like Microsoft Excel™ work is well understood and the software is nearly universally available. We show that the simple repetition of an equilibration process at each plate (a spreadsheet row) followed by discrete movement of the mobile phase down by a row, easily automated by a subroutine (a "Macro" in Excel), readily simulates chromatography. The process is readily understood by a novice. Not only does this permit simulation of isocratic and simple single step gradient elution, linear or multistep gradients are also easily simulated. The versatility of a transparent and easily understandable computational platform further enables the simulation of complex but commonly encountered chromatographic scenarios such as the effects of nonlinear isotherms, active sites, column overloading, on-column analyte degradation, etc. These are not as easily simulated by available software. Views of the separation as it develops on the column and as it is seen by an end-column detector are both available in real time. Excel 2010™ also permits a 16-level (4-bit) color gradation of numerical values in a column/row; this permits visualization of a band migrating down the column, much as Tswett may have originally observed, but in a numerical domain. All parameters of relevance (partition constants, elution conditions, etc.) are readily changed so their effects can be examined. Illustrative Excel spreadsheets are given in the Supporting Information; these are easily modified by the user or the user can write his/her own routine. Copyright © 2012 Elsevier B.V. All rights reserved.

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

PubMed

Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

2017-12-01

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination and validation of six sunscreen agents in suncare products by UPLC and HPLC.

PubMed

Lee, So-Mi; Jeong, Hye-Jin; Chang, Ih Seop

2008-01-01

Methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine are sunscreen agents that have hydrophobic behaviors in common. They were not normally assayed with the following four sunscreen agents that have hydrophilic behaviors in a single chromatographic run: ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. For that reason, methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine require much time in order to assay products with those materials. A rapid, selective, and reproducible determination method needs to be developed for the simultaneous examination of methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine with the sunscreen agents, ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. This new technique could reduce time in examining the sunscreen agents and be effective for quality control of suncare products. In this paper, the HPLC and UPLC system is used for developing the determination of the sunscreen agents. Several evaluations of some mixtures of eluents and columns were obtained for the optimal condition of separation. In HPLC, the optimal peak resolution was obtained through ethanol-water gradient elution and a 75-mm C18 column with a 3.5-microm-sized particle on a flow rate of 1.0 ml/min. In UPLC, the most distinctive peak resolution was obtained through methanol-water gradient elution and a 50-mm C18 column with a 1.7-microm-sized particle on a flow rate 0.4 ml/min. Both of those chromatographic determination methods could be used in the examination of six types of sunscreen agents without any interference from other product excipients in the agents. The proposed determination methods were validated for specificity, linearity, repeatability, system stability, intermediate precision, and accuracy. Consequently, HPLC and UPLC determination methods could be rapid, selective, and proper applications for the assay of sunscreen agents in suncare products.

Development of anion-exchange/reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry methods for the speciation of bio-available iodine and bromine from edible seaweed.

PubMed

Romarís-Hortas, Vanessa; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2012-05-04

Anion exchange high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry has been novelly applied to assess inorganic (iodide and iodate) and organic (3-iodotyrosine - MIT, and 3,5-diiodotyrosine - DIT) iodine species in a single chromatographic run. The optimized operating conditions (Dionex IonPac AS7, gradient elution with 175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase and flow rates within the 0.5-1.5 mL min(-1) range) have also been used to perform inorganic bromine speciation analysis (bromide and bromate). The developed method has been applied for determining the bio-available contents of iodine and bromine species in dialyzates from edible seaweed. Reverse phase high performance liquid chromatography (Zorbax Eclipse XDB-C8, gradient elution with 0.2% (m/m) acetic acid, and 0.2% (m/m) acetic acid in methanol, as mobile phases, and a constant flow rate of 0.75 mL min(-1)) also hyphenated with inductively coupled plasma-mass spectrometry was used to confirm the presence of organic iodine species (MIT and DIT) in the dialyzates. The verification of the presence of iodinated amino acids (MIT and DIT) in the extracts was also performed by reverse phase high performance liquid chromatography-electrospray ionization-mass spectrometry (LTQ Orbitrap). The developed methods have provided good repeatability (RSD values lower than 10% for both anion exchange and reverse phase separations) and analytical recoveries within the 90-105% range for all cases. The in vitro bio-availability method consisted of a simulated gastric and an intestinal digestion/dialysis (10 kDa molecular weight cut-off - MWCO) two-stage procedure. Iodide and MIT were the main bio-available species quantified, whereas bromide was the major bromine species found in the extracts. Copyright © 2012 Elsevier B.V. All rights reserved.

Stepwise pH-gradient elution for the preparative separation of natural anthraquinones by multiple liquid-liquid partition.

PubMed

Hynninen, P H; Räisänen, R

2001-01-01

Preparative-scale separation of substituted anthraquinones by multiple liquid-liquid partition was studied using isopropylmethyl ketone (IMK)/aqueous phosphate buffer (aq.) as the solvent system and the Hietala apparatus with 100 partition units as the partition equipment. The lower (aq.) phase was chosen as mobile, while the upper (IMK) phase remained stationary. Hence, the principle of stepwise pH-gradient elution could be utilized to separate the components in two complex mixtures of hydroxyanthraquinones and hydroxyanthraquinone carboxylic acids, isolated from the fungus Dermocybe sanguinea. In spite of the nonlinearity of the partition isotherms for these anthraquinones, implying deviations from the Nernst partition law, remarkable separations were achieved for the components in each mixture. Every anthraquinone carboxylic acid showed markedly irregular partition behavior, appearing in the effluent as two more or less resolved concentration zones. Such splitting was attributed to the formation of relatively stable sandwich-dimers, which were in a slow equilibrium with the monomers in the more nonpolar organic phase. At lower pH-values, only the polar monomers were distributed into the mobile aqueous phase and moved forward, whereas the nonpolar sandwich-dimers remained almost entirely in the stationary organic phase and lagged behind. When the pH of the mobile aqueous phase was raised high enough, the firmly linked dimers were monomerized and emerged from the apparatus as a second concentration profile. Intermolecular hydrogen bonding and pi-pi interaction between the pi-systems of two anthraquinone molecules in a parallel orientation were considered responsible for the nonlinear and markedly irregular partition behavior of the natural anthraquinones studied. The nonlinearity of the partition behavior of the hydroxyanthraquinones lacking the carboxyl group, appeared merely as excessive broadening of the experimental concentration profile, which impaired the resolution between the components only insignificantly. Thus, e.g. the main components, dermocybin and emodin, could both be obtained from Separation 1 in a purity of at least 99%.

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma.

PubMed

Wyss, R; Bucheli, F

1988-02-26

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.

A validated stability-indicating UPLC method for desloratadine and its impurities in pharmaceutical dosage forms.

PubMed

Rao, Dantu Durga; Satyanarayana, N V; Malleswara Reddy, A; Sait, Shakil S; Chakole, Dinesh; Mukkanti, K

2010-02-05

A novel stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of desloratadine in presence of its impurities and forced degradation products. The method was developed using Waters Aquity BEH C18 column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 280nm. The run time was 8min within which desloratadine and its five impurities were well separated. Desloratadine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Desloratadine was found to degrade significantly in oxidative and thermal stress conditions and stable in acid, base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of desloratadine in pharmaceutical dosage forms.

Diffusive gradients in thin films measurement of sulfur stable isotope variations in labile soil sulfate.

PubMed

Hanousek, Ondrej; Santner, Jakob; Mason, Sean; Berger, Torsten W; Wenzel, Walter W; Prohaska, Thomas

2016-11-01

A diffusive gradient in thin films (DGT) technique, based on a strongly basic anion exchange resin (Amberlite IRA-400), was successfully tested for 34 S/ 32 S analysis in labile soil sulfate. Separation of matrix elements (Na, K, and Ca) that potentially cause non-spectral interferences in 34 S/ 32 S analysis by MC ICP-MS (multi-collector inductively coupled plasma-mass spectrometry) during sampling of sulfate was demonstrated. No isotopic fractionation caused by diffusion or elution of sulfate was observed below a resin gel disc loading of ≤79 μg S. Above this threshold, fractionation towards 34 S was observed. The method was applied to 11 different topsoils and one mineral soil profile (0-100 cm depth) and compared with soil sulfate extraction by water. The S amount and isotopic ratio in DGT-S and water-extractable sulfate correlated significantly (r 2  = 0.89 and r 2  = 0.74 for the 11 topsoils, respectively). The systematically lower 34 S/ 32 S isotope ratios of the DGT-S were ascribed to mineralization of organic S.

Axial thermal gradients in microchip gas chromatography.

PubMed

Wang, Anzi; Hynynen, Sampo; Hawkins, Aaron R; Tolley, Samuel E; Tolley, H Dennis; Lee, Milton L

2014-12-29

Fabrication technologies for microelectromechanical systems (MEMS) allow miniaturization of conventional benchtop gas chromatography (GC) to portable, palm-sized microfabricated GC (μGC) devices, which are suitable for on-site chemical analysis and remote sensing. The separation performance of μGC systems, however, has not been on par with conventional GC. Column efficiency, peak symmetry and resolution are often compromised by column defects and non-ideal injections. The relatively low performance of μGC devices has impeded their further commercialization and broader application. In this work, the separation performance of μGC columns was improved by incorporating thermal gradient gas chromatography (TGGC). The analysis time was ∼20% shorter for TGGC separations compared to conventional temperature-programmed GC (TPGC) when a wide sample band was introduced into the column. Up to 50% reduction in peak tailing was observed for polar analytes, which improved their resolution. The signal-to-noise ratios (S/N) of late-eluting peaks were increased by 3-4 fold. The unique focusing effect of TGGC overcomes many of the previous shortcomings inherent in μGC analyses. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiresidue chromatographic method for the determination of macrolide residues in muscle by high-performance liquid chromatography with UV detection.

PubMed

Juhel-Gaugain, M; Anger, B; Laurentie, M

1999-01-01

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.

Magnetic Turbulence, Fast Magnetic Field line Diffusion and Small Magnetic Structures in the Solar Wind

NASA Astrophysics Data System (ADS)

Zimbardo, G.; Pommois, P.; Veltri, P.

2003-09-01

The influence of magnetic turbulence on magnetic field line diffusion has been known since the early days of space and plasma physics. However, the importance of ``stochastic diffusion'' for energetic particles has been challenged on the basis of the fact that sharp gradients of either energetic particles or ion composition are often observed in the solar wind. Here we show that fast transverse field line and particle diffusion can coexist with small magnetic structures, sharp gradients, and with long lived magnetic flux tubes. We show, by means of a numerical realization of three dimensional magnetic turbulence and by use of the concepts of deterministic chaos and turbulent transport, that turbulent diffusion is different from Gaussian diffusion, and that transport can be inhomogeneous even if turbulence homogeneously fills the heliosphere. Several diagnostics of field line transport and flux tube evolution are shown, and the size of small magnetic structures in the solar wind, like gradient scales and flux tube thickness, are estimated and compared to the observations.

Fast and Robust Registration of Multimodal Remote Sensing Images via Dense Orientated Gradient Feature

NASA Astrophysics Data System (ADS)

Ye, Y.

2017-09-01

This paper presents a fast and robust method for the registration of multimodal remote sensing data (e.g., optical, LiDAR, SAR and map). The proposed method is based on the hypothesis that structural similarity between images is preserved across different modalities. In the definition of the proposed method, we first develop a pixel-wise feature descriptor named Dense Orientated Gradient Histogram (DOGH), which can be computed effectively at every pixel and is robust to non-linear intensity differences between images. Then a fast similarity metric based on DOGH is built in frequency domain using the Fast Fourier Transform (FFT) technique. Finally, a template matching scheme is applied to detect tie points between images. Experimental results on different types of multimodal remote sensing images show that the proposed similarity metric has the superior matching performance and computational efficiency than the state-of-the-art methods. Moreover, based on the proposed similarity metric, we also design a fast and robust automatic registration system for multimodal images. This system has been evaluated using a pair of very large SAR and optical images (more than 20000 × 20000 pixels). Experimental results show that our system outperforms the two popular commercial software systems (i.e. ENVI and ERDAS) in both registration accuracy and computational efficiency.

Photothermal Nanocomposite Hydrogel Actuator with Electric-Field-Induced Gradient and Oriented Structure.

PubMed

Yang, Yang; Tan, Yun; Wang, Xionglei; An, Wenli; Xu, Shimei; Liao, Wang; Wang, Yuzhong

2018-03-07

Recent research of hydrogel actuators is still not sophisticated enough to meet the requirement of fast, reversible, complex, and robust reconfiguration. Here, we present a new kind of poly( N-isopropylacrylamide)/graphene oxide gradient hydrogel by utilizing direct current electric field to induce gradient and oriented distribution of graphene oxide into poly( N-isopropylacrylamide) hydrogel. Upon near-infrared light irradiation, the hydrogel exhibited excellent comprehensive actuation performance as a result of directional bending deformation, promising great potential in the application of soft actuators and optomechanical system.

Absolute change in fasting plasma glucose over 12 months is associated with 2-year and 5-year major adverse cardiovascular events in patients with drug-eluting stent implants.

PubMed

Kang, Dong Oh; Seo, Hong Seog; Choi, Byung Geol; Lee, Eunmi; Kim, Ji Park; Lee, Sun Ki; Im, Sung Il; Na, Jin Oh; Choi, Cheol Ung; Lim, Hong Euy; Kim, Jin Won; Kim, Eung Ju; Rha, Seung-Woon; Park, Chang Gyu; Oh, Dong Joo

2015-01-20

Major adverse cardiovascular events (MACEs) in patients with or without cardiovascular disease (CVD) are greatly affected by various factors associated with metabolism and inflammation. To determine which clinical parameters at treatment are associated with the development of 2-year and 5-year MACEs in high-risk patients with CVD who have undergone drug-eluting stent (DES) implantation. The present study involved a total of 432 patients who underwent percutaneous coronary intervention with DES. Variables representing the average and absolute amount of change in clinical parameters over the 12-month follow-up were assessed for association with 2-year and 5-year development of MACE. The study population was divided into quartiles for the variable showing the highest correlation to MACE development. Estimated incidence of 2-year and 5-year MACEs for each of the quartiles was determined by survival curve analysis, and subgroup analysis was performed for patients with diabetes and statin users. Absolute change in fasting plasma glucose (FPG) over 12 months showed the highest correlation with 2-year and 5-year MACE development. The estimated incidence of MACE increased with increasing quartiles for absolute change in FPG. The association between absolute change in FPG and MACE development exhibited a stronger relationship for the specific subgroups of patients with diabetes and statin users. Increases and decreases in FPG had a comparable contribution to MACE development. A greater absolute change in FPG over 12 months post-PCI is an independent risk factor for 2-year and 5-year MACE development in DES-implanted patients, especially in the diabetes and statin users. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Application of Snyder-Dolan classification scheme to the selection of "orthogonal" columns for fast screening of illicit drugs and impurity profiling of pharmaceuticals--I. Isocratic elution.

PubMed

Fan, Wenzhe; Zhang, Yu; Carr, Peter W; Rutan, Sarah C; Dumarey, Melanie; Schellinger, Adam P; Pritts, Wayne

2009-09-18

Fourteen judiciously selected reversed phase columns were tested with 18 cationic drug solutes under the isocratic elution conditions advised in the Snyder-Dolan (S-D) hydrophobic subtraction method of column classification. The standard errors (S.E.) of the least squares regressions of logk' vs. logk'(REF) were obtained for a given column against a reference column and used to compare and classify columns based on their selectivity. The results are consistent with those obtained with a study of the 16 test solutes recommended by Snyder and Dolan. To the extent these drugs are representative, these results show that the S-D classification scheme is also generally applicable to pharmaceuticals under isocratic conditions. That is, those columns judged to be similar based on the 16 S-D solutes were similar based on the 18 drugs; furthermore those columns judged to have significantly different selectivities based on the 16 S-D probes appeared to be quite different for the drugs as well. Given that the S-D method has been used to classify more than 400 different types of reversed phases the extension to cationic drugs is a significant finding.

A vortex-assisted MSPD method for the extraction of pesticide residues from fish liver and crab hepatopancreas with determination by GC-MS.

PubMed

Souza Caldas, Sergiane; Marian Bolzan, Cátia; Jaime de Menezes, Eliana; Laura Venquiaruti Escarrone, Ana; de Martinez Gaspar Martins, Camila; Bianchini, Adalto; Gilberto Primel, Ednei

2013-08-15

A method based on matrix solid-phase dispersion (MSPD) and gas chromatography-mass spectrometry to determine pesticides in fish liver and crab hepatopancreas was optimized. Ethyl acetate and acetonitrile were evaluated as elution solvents and their volumes were also checked. The best results were obtained with 1.0 g reused C18 as sorbent, using 5 mL acetonitrile as the elution solvent. Analytical recoveries ranged between 57 and 107% with RSD lower than 26% in fish liver and between 56 and 122% with RSD lower than 21% in crab hepatopancreas. The LOQ values for these compounds ranged from 0.05 to 0.5 mg kg(-1) for crab hepatopancreas and from 0.125 to 1.25 mg kg(-1) for fish liver. MPSD was shown to be easy and fast to use, with a clear advantage regarding costs because it does not need any expensive instrument. The proposed method was successfully applied to determine dimethoate, atrazine, clomazone, fenitrothion, malathion, fipronil and tebuconazole in fish liver and crab hepatopancreas samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Fertilizers mobilization in alluvial aquifer: laboratory experiments

NASA Astrophysics Data System (ADS)

Mastrocicco, M.; Colombani, N.; Palpacelli, S.

2009-02-01

In alluvial plains, intensive farming with conspicuous use of agrochemicals, can cause land pollution and groundwater contamination. In central Po River plain, paleo-channels are important links between arable lands and the underlaying aquifer, since the latter is often confined by clay sediments that act as a barrier against contaminants migration. Therefore, paleo-channels are recharge zones of particular interest that have to be protected from pollution as they are commonly used for water supply. This paper focuses on fertilizer mobilization next to a sand pit excavated in a paleo-channel near Ferrara (Italy). The problem is approached via batch test leaking and columns elution of alluvial sediments. Results from batch experiments showed fast increase in all major cations and anions, suggesting equilibrium control of dissolution reactions, limited availability of solid phases and geochemical homogeneity of samples. In column experiments, early elution and tailing of all ions breakthrough was recorded due to preferential flow paths. For sediments investigated in this study, dispersion, dilution and chemical reactions can reduce fertilizers at concentration below drinking standards in a reasonable time frame, provided fertilizer loading is halted or, at least, reduced. Thus, the definition of a corridor along paleo-channels is recommended to preserve groundwater quality.

Determination of adulteration of malachite green in green pea and some prepared foodstuffs by micellar liquid chromatography.

PubMed

Ashok, Vipin; Agrawal, Nitasha; Durgbanshi, Abhilasha; Esteve-Romero, Josep; Bose, Devasish

2014-01-01

A simple, fast, and robust micellar LC method was developed for the separation and identification of the nonpermitted color malachite green in green pea and some ready-to-eat foodstuffs. Malachite green (4-[(4-dimethylaminophenyl) phenyl-methyl]-N,N-dimethylaniline) is a hazardous dye that is used to treat fungal and protozoan infections in fish and is a common adulterant (coloring agent) in green pea and other green vegetables because of its green color. In the present work, malachite green was determined in various foodstuffs using a direct injection technique on an RP C18 column with isocratic elution. The optimum mobile phase consisted of 0.15 M sodium dodecyl sulfate (SDS), 6% pentanol buffered at pH 5. Detection was carried out at 620 nm. Malachite green was eluted in 9.2 min without any interference caused by endogenous compounds. Linearities (r > 0.9999), intraday and interday precision (RSD less than 1.00%) in micellar media, and robustness were studied for method validation. LOD and LOQ were 0.10 and 0.25 ppm, respectively. The simplicity of the developed method makes it useful for routine analysis in the area of food QC.

A new modeling strategy for third-order fast high-performance liquid chromatographic data with fluorescence detection. Quantitation of fluoroquinolones in water samples.

PubMed

Alcaráz, Mirta R; Bortolato, Santiago A; Goicoechea, Héctor C; Olivieri, Alejandro C

2015-03-01

Matrix augmentation is regularly employed in extended multivariate curve resolution-alternating least-squares (MCR-ALS), as applied to analytical calibration based on second- and third-order data. However, this highly useful concept has almost no correspondence in parallel factor analysis (PARAFAC) of third-order data. In the present work, we propose a strategy to process third-order chromatographic data with matrix fluorescence detection, based on an Augmented PARAFAC model. The latter involves decomposition of a three-way data array augmented along the elution time mode with data for the calibration samples and for each of the test samples. A set of excitation-emission fluorescence matrices, measured at different chromatographic elution times for drinking water samples, containing three fluoroquinolones and uncalibrated interferences, were evaluated using this approach. Augmented PARAFAC exploits the second-order advantage, even in the presence of significant changes in chromatographic profiles from run to run. The obtained relative errors of prediction were ca. 10 % for ofloxacin, ciprofloxacin, and danofloxacin, with a significant enhancement in analytical figures of merit in comparison with previous reports. The results are compared with those furnished by MCR-ALS.

Influence of amount of oxidizing slag discharged from stainless steelmaking process of electric arc furnace on elution behavior into fresh water

NASA Astrophysics Data System (ADS)

Yokoyama, S.; Shimomura, T.; Hisyamudin, M. N. N.; Takahashi, T.; Izaki, M.

2012-03-01

Fundamental study was carried out for provision for acidification of soil due to acid rain. The influence of weight of the additive slag on elution behavior of the slag into water was studied in this study. Elution experiment was carried out on a basis of JIS K 0058-1. Generally, the pH in the aqueous solution increased with an increase in weight of the additive slag. The pH converged to approximately eight. Calcium, magnesium and manganese, which were essential elements for plants, were eluted from the slag irrespective to elution condition. The eluted concentrations of Ca and Mg increased with an increase in weight of the additive slag. Silicon and zinc were also eluted depending on the conditions. Aluminum that was harmful for plants was not eluted from the used slag.

Experimental verification of the role of electron pressure in fast magnetic reconnection with a guide field

DOE PAGES

Fox, W.; Sciortino, F.; v. Stechow, A.; ...

2017-03-21

We report detailed laboratory observations of the structure of a reconnection current sheet in a two-fluid plasma regime with a guide magnetic field. We observe and quantitatively analyze the quadrupolar electron pressure variation in the ion-diffusion region, as originally predicted by extended magnetohydrodynamics simulations. The projection of the electron pressure gradient parallel to the magnetic field contributes significantly to balancing the parallel electric field, and the resulting cross-field electron jets in the reconnection layer are diamagnetic in origin. Furthermore, these results demonstrate how parallel and perpendicular force balance are coupled in guide field reconnection and confirm basic theoretical models ofmore » the importance of electron pressure gradients for obtaining fast magnetic reconnection.« less

Continuous gradient temperature Raman spectroscopy and differential scanning calorimetry of N-3DPA and DHA from -100 to 10°C

USDA-ARS?s Scientific Manuscript database

Docosahexaenoic acid (DHA, 22:6n-3) is exclusively utilized in fast signal processing tissues such as retinal, neural and cardiac. N-3 docosapentaenoic acid (n-3DPA, 22:5n-3), with just one less double bond, is also found in the marine food chain yet cannot substitute for DHA. Gradient Temperature R...

Turbulent transport stabilization by ICRH minority fast ions in low rotating JET ILW L-mode plasmas

NASA Astrophysics Data System (ADS)

Bonanomi, N.; Mantica, P.; Di Siena, A.; Delabie, E.; Giroud, C.; Johnson, T.; Lerche, E.; Menmuir, S.; Tsalas, M.; Van Eester, D.; Contributors, JET

2018-05-01

The first experimental demonstration that fast ion induced stabilization of thermal turbulent transport takes place also at low values of plasma toroidal rotation has been obtained in JET ILW (ITER-like wall) L-mode plasmas with high (3He)-D ICRH (ion cyclotron resonance heating) power. A reduction of the gyro-Bohm normalized ion heat flux and higher values of the normalized ion temperature gradient have been observed at high ICRH power and low NBI (neutral beam injection) power and plasma rotation. Gyrokinetic simulations indicate that ITG (ion temperature gradient) turbulence stabilization induced by the presence of high-energetic 3He ions is the key mechanism in order to explain the experimental observations. Two main mechanisms have been identified to be responsible for the turbulence stabilization: a linear electrostatic wave-fast particle resonance mechanism and a nonlinear electromagnetic mechanism. The dependence of the stabilization on the 3He distribution function has also been studied.

Microchip capillary gel electrophoresis using programmed field strength gradients for the ultra-fast analysis of genetically modified organisms in soybeans.

PubMed

Kim, Yun-Jeong; Chae, Joon-Seok; Chang, Jun Keun; Kang, Seong Ho

2005-08-12

We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11 s of the microchip. The MCGE-PFSG method was a program that changes the electric field strength during GMO analysis, and was also applied to the ultra-fast analysis of PCR products. Compared to MCGE using a conventional and constantly applied electric field, the MCGE-PFSG analysis generated faster results without the loss of resolving power and reproducibility for specific DNA fragments (100- and 250-bp DNA) of GM-soybeans. The MCGE-PFSG technique may prove to be a new tool in the GMO analysis due to its speed, simplicity, and high efficiency.

A general strategy for performing temperature-programming in high performance liquid chromatography--prediction of segmented temperature gradients.

PubMed

Wiese, Steffen; Teutenberg, Thorsten; Schmidt, Torsten C

2011-09-28

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed to predict retention times for segmented-temperature gradients based on temperature-gradient input data in liquid chromatography (LC) with high accuracy. The LES model assumes that retention times for isothermal separations can be predicted based on two temperature gradients and is employed to calculate the retention factor of an analyte when changing the start temperature of the temperature gradient. In this study it was investigated whether this approach can also be employed in LC. It was shown that this approximation cannot be transferred to temperature-programmed LC where a temperature range from 60°C up to 180°C is investigated. Major relative errors up to 169.6% were observed for isothermal retention factor predictions. In order to predict retention times for temperature gradients with different start temperatures in LC, another relationship is required to describe the influence of temperature on retention. Therefore, retention times for isothermal separations based on isothermal input runs were predicted using a plot of the natural logarithm of the retention factor vs. the inverse temperature and a plot of the natural logarithm of the retention factor vs. temperature. It could be shown that a plot of lnk vs. T yields more reliable isothermal/isocratic retention time predictions than a plot of lnk vs. 1/T which is usually employed. Hence, in order to predict retention times for temperature-gradients with different start temperatures in LC, two temperature gradient and two isothermal measurements have been employed. In this case, retention times can be predicted with a maximal relative error of 5.5% (average relative error: 2.9%). In comparison, if the start temperature of the simulated temperature gradient is equal to the start temperature of the input data, only two temperature-gradient measurements are required. Under these conditions, retention times can be predicted with a maximal relative error of 4.3% (average relative error: 2.2%). As an example, the systematic method development for an isothermal as well as a temperature gradient separation of selected sulfonamides by means of the adapted LES model is demonstrated using a pure water mobile phase. Both methods are compared and it is shown that the temperature-gradient separation provides some advantages over the isothermal separation in terms of limits of detection and analysis time. Copyright © 2011 Elsevier B.V. All rights reserved.

The effects of temperature gradient and growth rate on the morphology and fatigue properties of MAR-M246(Hf)

NASA Technical Reports Server (NTRS)

Schmidt, D. D.; Alter, W. S.; Hamilton, W. D.; Parr, R. A.

1989-01-01

MAR-M246(Hf) is a nickel based superalloy used in the turbopump blades of the Space Shuttle main engines. The effects are considered of temperature gradient (G) and growth rate (R) on the microstructure and fatigue properties of this superalloy. The primary dendrite arm spacings were found to be inversely proportional to both temperature gradient and growth rate. Carbide and gamma - gamma prime morphology trends were related to G/R ratios. Weibull analysis of fatigue results shows the characteristic life to be larger by a factor of 10 for the low gradient/fast rate pairing of G and R, while the reliability (beta) was lower.

Combined Yamamoto approach for simultaneous estimation of adsorption isotherm and kinetic parameters in ion-exchange chromatography.

PubMed

Rüdt, Matthias; Gillet, Florian; Heege, Stefanie; Hitzler, Julian; Kalbfuss, Bernd; Guélat, Bertrand

2015-09-25

Application of model-based design is appealing to support the development of protein chromatography in the biopharmaceutical industry. However, the required efforts for parameter estimation are frequently perceived as time-consuming and expensive. In order to speed-up this work, a new parameter estimation approach for modelling ion-exchange chromatography in linear conditions was developed. It aims at reducing the time and protein demand for the model calibration. The method combines the estimation of kinetic and thermodynamic parameters based on the simultaneous variation of the gradient slope and the residence time in a set of five linear gradient elutions. The parameters are estimated from a Yamamoto plot and a gradient-adjusted Van Deemter plot. The combined approach increases the information extracted per experiment compared to the individual methods. As a proof of concept, the combined approach was successfully applied for a monoclonal antibody on a cation-exchanger and for a Fc-fusion protein on an anion-exchange resin. The individual parameter estimations for the mAb confirmed that the new approach maintained the accuracy of the usual Yamamoto and Van Deemter plots. In the second case, offline size-exclusion chromatography was performed in order to estimate the thermodynamic parameters of an impurity (high molecular weight species) simultaneously with the main product. Finally, the parameters obtained from the combined approach were used in a lumped kinetic model to simulate the chromatography runs. The simulated chromatograms obtained for a wide range of gradient lengths and residence times showed only small deviations compared to the experimental data. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrathin, bioresorbable polymer sirolimus-eluting stents versus thin, durable polymer everolimus-eluting stents in patients undergoing coronary revascularisation (BIOFLOW V): a randomised trial.

PubMed

Kandzari, David E; Mauri, Laura; Koolen, Jacques J; Massaro, Joseph M; Doros, Gheorghe; Garcia-Garcia, Hector M; Bennett, Johan; Roguin, Ariel; Gharib, Elie G; Cutlip, Donald E; Waksman, Ron

2017-10-21

The development of coronary drug-eluting stents has included use of new metal alloys, changes in stent architecture, and use of bioresorbable polymers. Whether these advancements improve clinical safety and efficacy has not been shown in previous randomised trials. We aimed to examine the clinical outcomes of a bioresorbable polymer sirolimus-eluting stent compared with a durable polymer everolimus-eluting stent in a broad patient population undergoing percutaneous coronary intervention. BIOFLOW V was an international, randomised trial done in patients undergoing elective and urgent percutaneous coronary intervention in 90 hospitals in 13 countries (Australia, Belgium, Canada, Denmark, Germany, Hungary, Israel, the Netherlands, New Zealand, South Korea, Spain, Switzerland, and the USA). Eligible patients were those aged 18 years or older with ischaemic heart disease undergoing planned stent implantation in de-novo, native coronary lesions. Patients were randomly assigned (2:1) to either an ultrathin strut (60 μm) bioresorbable polymer sirolimus-eluting stent or to a durable polymer everolimus-eluting stent. Randomisation was via a central web-based data capture system (mixed blocks of 3 and 6), and stratified by study site. The primary endpoint was 12-month target lesion failure. The primary non-inferiority comparison combined these data from two additional randomised trials of bioresorbable polymer sirolimus-eluting stent and durable polymer everolimus-eluting stent with Bayesian methods. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT02389946. Between May 8, 2015, and March 31, 2016, 4772 patients were recruited into the study. 1334 patients met inclusion criteria and were randomly assigned to treatment with bioresorbable polymer sirolimus-eluting stents (n=884) or durable polymer everolimus-eluting stents (n=450). 52 (6%) of 883 patients in the bioresorbable polymer sirolimus-eluting stent group and 41 (10%) of 427 patients in the durable polymer everolimus-eluting stent group met the 12-month primary endpoint of target lesion failure (95% CI -6·84 to -0·29, p=0·0399), with differences in target vessel myocardial infarction (39 [5%] of 831 patients vs 35 [8%] of 424 patients, p=0·0155). The posterior probability that the bioresorbable polymer sirolimus-eluting stent is non-inferior to the durable polymer everolimus-eluting stent was 100% (Bayesian analysis, difference in target lesion failure frequency -2·6% [95% credible interval -5·5 to 0·1], non-inferiority margin 3·85%, n=2208). The outperformance of the ultrathin, bioresorbable polymer sirolimus-eluting stent over the durable polymer everolimus-eluting stent in a complex patient population undergoing percutaneous coronary intervention suggests a new direction in improving next generation drug-eluting stent technology. BIOTRONIK. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of Jamaican agro-industrial wastes. Part II, fatty acid profiling using HPLC: precolumn derivatization with phenacyl bromide.

PubMed

Bailey-Shaw, Y A; Golden, K D; Pearson, A G M; Porter, R B R

2012-09-01

This paper describes the determination of fatty acid composition of coffee, citrus and rum distillery wastes using reversed-phase high-performance liquid chromatography (RP-HPLC). Lipid extracts of the waste samples are derivatized with phenacyl bromide and their phenacyl esters are separated on a C8 reversed-phase column by using continuous gradient elution with water and acetonitrile. The presence of saturated and unsaturated fatty acids in quantifiable amounts in the examined wastes, as well as the high percentage recoveries, are clear indications that these wastes have potential value as inexpensive sources of lipids. The HPLC procedures described here could be adopted for further analysis of materials of this nature.

Qualitative and Quantitative Phytochemical Analysis of Different Extracts from Thymus algeriensis Aerial Parts.

PubMed

Boutaoui, Nassima; Zaiter, Lahcene; Benayache, Fadila; Benayache, Samir; Carradori, Simone; Cesa, Stefania; Giusti, Anna Maria; Campestre, Cristina; Menghini, Luigi; Innosa, Denise; Locatelli, Marcello

2018-02-20

This study was performed to evaluate the metabolite recovery from different extraction methods applied to Thymus algeriensis aerial parts. A high-performance liquid chromatographic method using photodiode array detector with gradient elution has been developed and validated for the simultaneous estimation of different phenolic compounds in the extracts and in their corresponding purified fractions. The experimental results show that microwave-assisted aqueous extraction for 15 min at 100 °C gave the most phenolics-enriched extract, reducing extraction time without degradation effects on bioactives. Sixteen compounds were identified in this extract, 11 phenolic compounds and five flavonoids, all known for their biological activities. Color analysis and determination of chlorophylls and carotenoids implemented the knowledge of the chemical profile of this plant.

Predicting ESI/MS Signal Change for Anions in Different Solvents.

PubMed

Kruve, Anneli; Kaupmees, Karl

2017-05-02

LC/ESI/MS is a technique widely used for qualitative and quantitative analysis in various fields. However, quantification is currently possible only for compounds for which the standard substances are available, as the ionization efficiency of different compounds in ESI source differs by orders of magnitude. In this paper we present an approach for quantitative LC/ESI/MS analysis without standard substances. This approach relies on accurately predicting the ionization efficiencies in ESI source based on a model, which uses physicochemical parameters of analytes. Furthermore, the model has been made transferable between different mobile phases and instrument setups by using a suitable set of calibration compounds. This approach has been validated both in flow injection and chromatographic mode with gradient elution.

Development of a gradient HPLC method for the simultaneous determination of sotalol and sorbate in oral liquid preparations using solid core stationary phase.

PubMed

Matysova, Ludmila; Zahalkova, Oxana; Klovrzova, Sylva; Sklubalova, Zdenka; Solich, Petr; Zahalka, Lukas

2015-01-01

A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100 × 4.6 mm, particles 2.7 μm) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3 mL min(-1) was used, and the injection volume was 5 µL. The UV/Vis absorbance detector was set to a wavelength of 237 nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7 mM) was used as the mobile phase buffer. The total analysis time was 4.5 min (+2.5 min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances.

Development of a Gradient HPLC Method for the Simultaneous Determination of Sotalol and Sorbate in Oral Liquid Preparations Using Solid Core Stationary Phase

PubMed Central

2015-01-01

A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100 × 4.6 mm, particles 2.7 μm) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3 mL min−1 was used, and the injection volume was 5 µL. The UV/Vis absorbance detector was set to a wavelength of 237 nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7 mM) was used as the mobile phase buffer. The total analysis time was 4.5 min (+2.5 min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances. PMID:25878920

Development and Validation of a Stability-Indicating HPLC Method for Imidapril and Its Degradation Products Using a Design of Experiment (DoE) Approach.

PubMed

Arumugam, Abiramasundari; Joshi, Amita; Vasu, Kamala K

2017-11-01

The present work focused on the application of design of experiment (DoE) principles to the development and optimization of a stability-indicating method (SIM) for the drug imidapril hydrochloride and its degradation products (DPs). The resolution of peaks for the DPs and their drug in a SIM can be influenced by many factors. The factors studied here were pH, gradient time, organic modifier, flow rate, molar concentration of the buffer, and wavelength, with the aid of a Plackett-Burman design. Results from the Plackett-Burman study conspicuously showed influence of two factors, pH and gradient time, on the analyzed response, particularly, the resolution of the closely eluting DPs (DP-5 and DP-6) and the retention time of the last peak. Optimization of the multiresponse processes was achieved through Derringer's desirability function with the assistance of a full factorial design. Separation was achieved using a C18 Phenomenex Luna column (250 × 4.6 mm id, 5 µm particle size) at a flow rate of 0.8 mL/min at 210 nm. The optimized mobile phase composition was ammonium-acetate buffer (pH 5) in pump A and acetonitrile-methanol (in equal ratio) in pump B with a run time of 40 min using a gradient method.

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography.

PubMed

Horie, Kanta; Ikegami, Tohru; Hosoya, Ken; Saad, Nabil; Fiehn, Oliver; Tanaka, Nobuo

2007-09-14

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.

Short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate.

PubMed

Hutta, Milan; Ráczová, Janka; Góra, Róbert; Pessl, Juraj

2015-08-21

Novel anion-exchange liquid chromatographic method with step gradient of aqueous EDTA(4-) based mobile phase elution has been developed to profile available Slovak soil humic substances and alkaline extracts of various soils. The method utilize short glass column (30mm×3mm) filled in with hydrolytically stable particles (60μm diameter) Separon HEMA-BIO 1000 having (diethylamino)ethyl functional groups. Step gradient was programmed by mixing mobile phase composed of aqueous solution of sodium EDTA (pH 12.0; 5mmolL(-1)) and mobile phase constituted of aqueous solution of sodium EDTA (pH 12.0, 500mmolL(-1)). The FLD of HSs was set to excitation wavelength 480nm and emission wavelength 530nm (λem). Separation mechanism was studied by use of selected aromatic acids related to humic acids with the aid of UV spectrophotometric detection at 280nm. The proposed method benefits from high ionic strength (I=5molL(-1)) of the end mobile phase buffer and provides high recovery of humic acids (98%). Accurate and reproducible profiling of studied humic substances, alkaline extracts of various types of soils enables straightforward characterization and differentiation of HSs in arable and forest soils. Selected model aromatic acids were used for separation mechanism elucidation. Copyright © 2015 Elsevier B.V. All rights reserved.

Simulation of concomitant magnetic fields on fast switched gradient coils used in advanced application of MRI

NASA Astrophysics Data System (ADS)

Salinas-Muciño, G.; Torres-García, E.; Hidalgo-Tobon, S.

2012-10-01

The process to produce an MR image includes nuclear alignment, RF excitation, spatial encoding, and image formation. To form an image, it is necessary to perform spatial localization of the MR signals, which is achieved using gradient coils. MRI requires the use of gradient coils that generate magnetic fields, which vary linearly with position over the imaging volume. Safety issues have been a motivation to study deeply the relation between the interaction of gradient magnetic field and the peripheral nerve stimulation. In this work is presented a numerical modeling between the concomitant magnetic fields produced by the gradient coils and the electric field induced in a cube with σ conductivity by the gradient field switching in pulse sequences as Eco planar Imaging (EPI), due to this kind of sequence is the most used in advance applications of magnetic resonance imaging as functional MRI, cardiac imaging or diffusion.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

PubMed

Yu, Shuizi Rachel; Burkhardt, Markus; Nowak, Matthias; Ries, Jonas; Petrásek, Zdenek; Scholpp, Steffen; Schwille, Petra; Brand, Michael

2009-09-24

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

Precision-controlled elution of a 82Sr/82Rb generator for cardiac perfusion imaging with positron emission tomography

NASA Astrophysics Data System (ADS)

Klein, R.; Adler, A.; Beanlands, R. S.; de Kemp, R. A.

2007-02-01

A rubidium-82 (82Rb) elution system is described for use with positron emission tomography. Due to the short half-life of 82Rb (76 s), the system physics must be modelled precisely to account for transport delay and the associated activity decay and dispersion. Saline flow is switched between a 82Sr/82Rb generator and a bypass line to achieve a constant-activity elution of 82Rb. Pulse width modulation (PWM) of a solenoid valve is compared to simple threshold control as a means to simulate a proportional valve. A predictive-corrective control (PCC) algorithm is developed which produces a constant-activity elution within the constraints of long feedback delay and short elution time. The system model parameters are adjusted through a self-tuning algorithm to minimize error versus the requested time-activity profile. The system is self-calibrating with 2.5% repeatability, independent of generator activity and elution flow rate. Accurate 30 s constant-activity elutions of 10-70% of the total generator activity are achieved using both control methods. The combined PWM-PCC method provides significant improvement in precision and accuracy of the requested elution profiles. The 82Rb elution system produces accurate and reproducible constant-activity elution profiles of 82Rb activity, independent of parent 82Sr activity in the generator. More reproducible elution profiles may improve the quality of clinical and research PET perfusion studies using 82Rb.

Precision-controlled elution of a 82Sr/82Rb generator for cardiac perfusion imaging with positron emission tomography.

PubMed

Klein, R; Adler, A; Beanlands, R S; Dekemp, R A

2007-02-07

A rubidium-82 ((82)Rb) elution system is described for use with positron emission tomography. Due to the short half-life of (82)Rb (76 s), the system physics must be modelled precisely to account for transport delay and the associated activity decay and dispersion. Saline flow is switched between a (82)Sr/(82)Rb generator and a bypass line to achieve a constant-activity elution of (82)Rb. Pulse width modulation (PWM) of a solenoid valve is compared to simple threshold control as a means to simulate a proportional valve. A predictive-corrective control (PCC) algorithm is developed which produces a constant-activity elution within the constraints of long feedback delay and short elution time. The system model parameters are adjusted through a self-tuning algorithm to minimize error versus the requested time-activity profile. The system is self-calibrating with 2.5% repeatability, independent of generator activity and elution flow rate. Accurate 30 s constant-activity elutions of 10-70% of the total generator activity are achieved using both control methods. The combined PWM-PCC method provides significant improvement in precision and accuracy of the requested elution profiles. The (82)Rb elution system produces accurate and reproducible constant-activity elution profiles of (82)Rb activity, independent of parent (82)Sr activity in the generator. More reproducible elution profiles may improve the quality of clinical and research PET perfusion studies using (82)Rb.

Comparison of intracellular drug retention, DNA damage and cytotoxicity of derivatives of doxorubicin and daunorubicin in a human colon adenocarcinoma cell line (LoVo).

PubMed

Belvedere, G; Suarato, A; Geroni, C; Giuliani, F C; D'Incalci, M

1989-11-01

Formation of DNA single strand breaks (SSB) was assayed by alkaline elution in LoVo cells treated with doxorubicin, daunorubicin and six derivatives of these drugs modified either in the chromophore or the sugar. Seven compounds showed a biphasic relationship (initial increase and then a decrease) for the formation of DNA-SSB over the concentration range 0.05-10 micrograms/ml. At a drug concentration in the range causing an increase of DNA damage very fast repair of DNA-SSB was observed for 4'-deoxydoxorubicin and 4-demethoxydaunorubicin; the kinetics of DNA-SSB investigated after drug removal at a drug concentration reducing DNA-SSB showed a time dependent increase of DNA damage for both drugs although with different patterns. 4'-Deoxydoxorubicin reduced the effect of radiations on the rate of elution of DNA in a way resembling the formation of DNA interstrand cross links (ISC) at concentrations at which DNA-SSB were reduced. DNA-ISC were not produced by chemical reactions occurring during sample processing for alkaline elution and this derivative was not metabolized by LoVo cells. The IC50 of the anthracyclines were on a several log range, though for most of the derivatives the cytotoxicity curve showed a plateau at growth inhibition of about 15-30% at increasing intracellular drug levels. A relationship between DNA damage and cytotoxicity was observed only in a very small range of DNA-SSB. It is likely that the different effects of these anthracyclines on the formation of DNA-SSB depend on a qualitatively different interaction between drug-DNA and topoisomerase II when the drug concentration is raised.

Determination of T-2 toxin, HT-2 toxin, and three other type A trichothecenes in layer feed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)--comparison of two sample preparation methods.

PubMed

Bernhardt, Katrin; Valenta, Hana; Kersten, Susanne; Humpf, Hans-Ulrich; Dänicke, Sven

2016-05-01

A sensitive method for the simultaneous determination of T-2 toxin, HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol in layer feed using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry in the positive ionization mode (LC-ESI-MS/MS) is described. Two fast and easy clean-up methods-with BondElut Mycotoxin and MycoSep 227 columns, respectively-were tested. The separation of the toxins was conducted on a Pursuit XRs Ultra 2.8 HPLC column using 0.13 mM ammonium acetate as eluent A and methanol as eluent B. Detection of the mycotoxins was carried out in the multiple reaction monitoring (MRM) mode using ammonium adducts as precursor ions. Quantification of all analytes was performed with d3-T-2 toxin as an internal standard. The clean-up method with MycoSep 227 columns gave slightly better results for layer feed compared to the method using BondElut Mycotoxin columns (MycoSep 227: recovery between 50 and 63%, BondElut Mycotoxin: recovery between 32 and 67%) and was therefore chosen as the final method. The limits of detection ranged between 0.9 and 7.5 ng/g depending on the mycotoxin. The method was developed for the analysis of layer feed used at carry-over experiments with T-2 toxin in laying hens. For carry-over experiments, it is necessary that the method includes not only T-2 toxin but also the potential metabolites in animal tissues HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol which could naturally occur in cereals used as feed stuff as well.

Lesson from the stoichiometry determination of the cohesin complex: a short protease mediated elution increases the recovery from cross-linked antibody-conjugated beads.

PubMed

Holzmann, Johann; Fuchs, Johannes; Pichler, Peter; Peters, Jan-Michael; Mechtler, Karl

2011-02-04

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analyses-such as the stoichiometry determination of protein complexes-have become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periods-as performed in standard on-bead digestion-led to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with a 1:1:1:1 stoichiometry. Furthermore, using isoform specific reference peptides, we determined the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. In summary, we show that the protease elution protocol increases the recovery from affinity beads and is compatible with quantitative measurements such as the stoichiometry determination of protein complexes.

Drug-eluting versus bare-metal coronary stents: where are we now?

PubMed

Amoroso, Nicholas S; Bangalore, Sripal

2012-11-01

Drug-eluting stents have dramatically reduced the risk of restenosis, but concerns of an increased risk of stent thrombosis have provided uncertainty about their use. Recent studies have continued to show improved procedural and clinical outcomes with drug-eluting stents both in the setting of acute coronary syndromes and stable coronary artery disease. Newer generation drug-eluting stents (especially everolimus-eluting stents) have been shown to be not only efficacious but also safe with reduced risk of stent thrombosis when compared with bare-metal stents, potentially changing the benchmark for stent safety from bare-metal stents to everolimus-eluting stents. While much progress is being made in the development of bioabsorbable polymer stents, nonpolymer stents and bioabsorbable stent technology, it remains to be seen whether these stents will have superior safety and efficacy outcomes compared with the already much improved rates of revascularization and stent thrombosis seen with newer generation stents (everolimus-eluting stents and resolute zotarolimus-eluting stents).

Mass Spectral Investigation on Toxins. I. Isolation, Purification, and Characterization of Hepatotoxins from Freshwater Blue-Green Algae (Cyanobacteria) by High-Performance Liquid Chromatography and Fast Atom Bombardment Mass Spectrometric Techniques.

DTIC Science & Technology

1986-09-01

analysis ’" methods in environmental samples. The hepatotoxins from laboratory cultures of M. aeruginosa Strain 7820,15 Anabena flos- aguae (A. 4flos...flos- aguae S-23-g-1l (8 lug) F1 The results from the amino acid analysis using the Llqui-Mat Analyzer are listed in Table 2. The elution times of the...Runnegar, M.T.C., and Huynh, V.L. Effec- tiveness of Activated Carbon in the Removal of Algal Toxin from Potable Water Supplies: A Pilot Plant

Biodegradable-polymer drug-eluting stents vs. bare metal stents vs. durable-polymer drug-eluting stents: a systematic review and Bayesian approach network meta-analysis.

PubMed

Kang, Si-Hyuck; Park, Kyung Woo; Kang, Do-Yoon; Lim, Woo-Hyun; Park, Kyung Taek; Han, Jung-Kyu; Kang, Hyun-Jae; Koo, Bon-Kwon; Oh, Byung-Hee; Park, Young-Bae; Kandzari, David E; Cohen, David J; Hwang, Seung-Sik; Kim, Hyo-Soo

2014-05-01

The aim of this study was to compare the safety and efficacy of biodegradable-polymer (BP) drug-eluting stents (DES), bare metal stents (BMS), and durable-polymer DES in patients undergoing coronary revascularization, we performed a systematic review and network meta-analysis using a Bayesian framework. Study stents included BMS, paclitaxel-eluting (PES), sirolimus-eluting (SES), endeavor zotarolimus-eluting (ZES-E), cobalt-chromium everolimus-eluting (CoCr-EES), platinium-chromium everolimus-eluting (PtCr-EES), resolute zotarolimus-eluting (ZES-R), and BP biolimus-eluting stents (BP-BES). After a systematic electronic search, 113 trials with 90 584 patients were selected. The principal endpoint was definite or probable stent thrombosis (ST) defined according to the Academic Research Consortium within 1 year. Biodegradable polymer-biolimus-eluting stents [OR, 0.56; 95% credible interval (CrI), 0.33-0.90], SES (OR, 0.53; 95% CrI, 0.38-0.73), CoCr-EES (OR, 0.34; 95% CrI, 0.23-0.52), and PtCr-EES (OR, 0.31; 95% CrI, 0.10-0.90) were all superior to BMS in terms of definite or probable ST within 1 year. Cobalt-chromium everolimus-eluting stents demonstrated the lowest risk of ST of all stents at all times after stent implantation. Biodegradable polymer-biolimus-eluting stents was associated with a higher risk of definite or probable ST than CoCr-EES (OR, 1.72; 95% CrI, 1.04-2.98). All DES reduced the need for repeat revascularization, and all but PES reduced the risk of myocardial infarction compared with BMS. All DESs but PES and ZES-E were superior to BMS in terms of ST within 1 year. Cobalt-chromium everolimus-eluting stents was safer than any DES even including BP-BES. Our results suggest that not only the biodegradability of polymer, but the optimal combination of stent alloy, design, strut thickness, polymer, and drug all combined determine the safety of DES.

Metal elution from Ni- and Fe-based alloy reactors under hydrothermal conditions.

PubMed

Faisal, Muhammad; Quitain, Armando T; Urano, Shin-Ya; Daimon, Hiroyuki; Fujie, Koichi

2004-05-20

Elution of metals from Ni- and Fe-based alloy (i.e. Inconel 625 and SUS 316) under hydrothermal conditions was investigated. Results showed that metals could be eluted even in a short contact time. At subcritical conditions, a significant amount of Cr was extracted from SUS 316, while only traces of Ni, Fe, Mo, and Mn were eluted. In contrast, Ni was removed in significant amounts compared to Cr when Inconel 625 was tested. Several factors including temperature and contact time were found to affect elution behavior. The presence of air in the fluid even promoted elution under subcritical conditions.

Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking.

PubMed

Nowak, Matthias; Machate, Anja; Yu, Shuizi Rachel; Gupta, Mansi; Brand, Michael

2011-02-01

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.

Concentration of perrhenate and pertechnetate solutions

DOEpatents

Knapp, F.F.; Beets, A.L.; Mirzadeh, S.; Guhlke, S.

1998-03-17

A method is described for preparing a concentrated solution of a carrier-free radioisotope which includes the steps of: (a) providing a generator column loaded with a composition containing a parent radioisotope; (b) eluting the generator column with an eluent solution which includes a salt of a weak acid to elute a target daughter radioisotope from the generator column in a first eluate; (c) eluting a cation-exchange column with the first eluate to exchange cations of the salt for hydrogen ions and to elute the target daughter radioisotope and a weak acid in a second eluate; (d) eluting an anion-exchange column with the second eluate to trap and concentrate the target daughter radioisotope and to elute the weak acid solution therefrom; and (e) eluting the concentrated target daughter radioisotope from the anion-exchange column with a saline solution. 1 fig.

Concentration of perrhenate and pertechnetate solutions

DOEpatents

Knapp, Furn F.; Beets, Arnold L.; Mirzadeh, Saed; Guhlke, Stefan

1998-01-01

A method of preparing a concentrated solution of a carrier-free radioisotope which includes the steps of: a. providing a generator column loaded with a composition containing a parent radioisotope; b. eluting the generator column with an eluent solution which includes a salt of a weak acid to elute a target daughter radioisotope from the generator column in a first eluate. c. eluting a cation-exchange column with the first eluate to exchange cations of the salt for hydrogen ions and to elute the target daughter radioisotope and a weak acid in a second eluate; d. eluting an anion-exchange column with the second eluate to trap and concentrate the target daughter radioisotope and to elute the weak acid solution therefrom; and e. eluting the concentrated target daughter radioisotope from the anion-exchange column with a saline solution.

B0 concomitant field compensation for MRI systems employing asymmetric transverse gradient coils.

PubMed

Weavers, Paul T; Tao, Shengzhen; Trzasko, Joshua D; Frigo, Louis M; Shu, Yunhong; Frick, Matthew A; Lee, Seung-Kyun; Foo, Thomas K-F; Bernstein, Matt A

2018-03-01

Imaging gradients result in the generation of concomitant fields, or Maxwell fields, which are of increasing importance at higher gradient amplitudes. These time-varying fields cause additional phase accumulation, which must be compensated for to avoid image artifacts. In the case of gradient systems employing symmetric design, the concomitant fields are well described with second-order spatial variation. Gradient systems employing asymmetric design additionally generate concomitant fields with global (zeroth-order or B 0 ) and linear (first-order) spatial dependence. This work demonstrates a general solution to eliminate the zeroth-order concomitant field by applying the correct B 0 frequency shift in real time to counteract the concomitant fields. Results are demonstrated for phase contrast, spiral, echo-planar imaging (EPI), and fast spin-echo imaging. A global phase offset is reduced in the phase-contrast exam, and blurring is virtually eliminated in spiral images. The bulk image shift in the phase-encode direction is compensated for in EPI, whereas signal loss, ghosting, and blurring are corrected in the fast-spin echo images. A user-transparent method to compensate the zeroth-order concomitant field term by center frequency shifting is proposed and implemented. This solution allows all the existing pulse sequences-both product and research-to be retained without any modifications. Magn Reson Med 79:1538-1544, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Development and validation of a critical gradient energetic particle driven Alfven eigenmode transport model for DIII-D tilted neutral beam experiments

NASA Astrophysics Data System (ADS)

Waltz, R. E.; Bass, E. M.; Heidbrink, W. W.; VanZeeland, M. A.

2015-11-01

Recent experiments with the DIII-D tilted neutral beam injection (NBI) varying the beam energetic particle (EP) source profiles have provided strong evidence that unstable Alfven eigenmodes (AE) drive stiff EP transport at a critical EP density gradient [Heidbrink et al 2013 Nucl. Fusion 53 093006]. Here the critical gradient is identified by the local AE growth rate being equal to the local ITG/TEM growth rate at the same low toroidal mode number. The growth rates are taken from the gyrokinetic code GYRO. Simulation show that the slowing down beam-like EP distribution has a slightly lower critical gradient than the Maxwellian. The ALPHA EP density transport code [Waltz and Bass 2014 Nucl. Fusion 54 104006], used to validate the model, combines the low-n stiff EP critical density gradient AE mid-core transport with the Angioni et al (2009 Nucl. Fusion 49 055013) energy independent high-n ITG/TEM density transport model controling the central core EP density profile. For the on-axis NBI heated DIII-D shot 146102, while the net loss to the edge is small, about half the birth fast ions are transported from the central core r/a  <  0.5 and the central density is about half the slowing down density. These results are in good agreement with experimental fast ion pressure profiles inferred from MSE constrained EFIT equilibria.

A blind deconvolution method based on L1/L2 regularization prior in the gradient space

NASA Astrophysics Data System (ADS)

Cai, Ying; Shi, Yu; Hua, Xia

2018-02-01

In the process of image restoration, the result of image restoration is very different from the real image because of the existence of noise, in order to solve the ill posed problem in image restoration, a blind deconvolution method based on L1/L2 regularization prior to gradient domain is proposed. The method presented in this paper first adds a function to the prior knowledge, which is the ratio of the L1 norm to the L2 norm, and takes the function as the penalty term in the high frequency domain of the image. Then, the function is iteratively updated, and the iterative shrinkage threshold algorithm is applied to solve the high frequency image. In this paper, it is considered that the information in the gradient domain is better for the estimation of blur kernel, so the blur kernel is estimated in the gradient domain. This problem can be quickly implemented in the frequency domain by fast Fast Fourier Transform. In addition, in order to improve the effectiveness of the algorithm, we have added a multi-scale iterative optimization method. This paper proposes the blind deconvolution method based on L1/L2 regularization priors in the gradient space can obtain the unique and stable solution in the process of image restoration, which not only keeps the edges and details of the image, but also ensures the accuracy of the results.

Relationship between lipoprotein lipase activity and plasma sex steroid level in obese women.

PubMed

Iverius, P H; Brunzell, J D

1988-09-01

In obese women (n = 16) at their weight, fasting adipose tissue lipoprotein lipase (LPL) activity, obtained by elution with serum and heparin at 4 degrees and 37 degrees C, was inversely correlated to plasma estradiol levels (r = -0.724; P = 0.002) and (r = -0.641; P = 0.010), respectively. Furthermore, fasting postheparin plasma LPL activity during a heparin infusion, showed an even stronger inverse correlation to plasma estradiol when measured at 60 min (r = -0.815; P less than 0.001). None of the above parameters was correlated to the body mass index. Postprandial LPL activity in postheparin plasma, measured 10 min after a heparin injection, showed a strong positive correlation with plasma free testosterone (r = 0.780; P = 0.001). Neither of these parameters was correlated with the body mass index. The origin of this LPL activity is presently unknown but could conceivably represent a pool of LPL from skeletal muscle. Since it has been shown convincingly that estrogen decreases adipose tissue LPL activity in the rat, the present studies strongly suggest that estradiol is a major negative regulator of fasting adipose tissue LPL activity in women.

On the Convergence Analysis of the Optimized Gradient Method.

PubMed

Kim, Donghwan; Fessler, Jeffrey A

2017-01-01

This paper considers the problem of unconstrained minimization of smooth convex functions having Lipschitz continuous gradients with known Lipschitz constant. We recently proposed the optimized gradient method for this problem and showed that it has a worst-case convergence bound for the cost function decrease that is twice as small as that of Nesterov's fast gradient method, yet has a similarly efficient practical implementation. Drori showed recently that the optimized gradient method has optimal complexity for the cost function decrease over the general class of first-order methods. This optimality makes it important to study fully the convergence properties of the optimized gradient method. The previous worst-case convergence bound for the optimized gradient method was derived for only the last iterate of a secondary sequence. This paper provides an analytic convergence bound for the primary sequence generated by the optimized gradient method. We then discuss additional convergence properties of the optimized gradient method, including the interesting fact that the optimized gradient method has two types of worstcase functions: a piecewise affine-quadratic function and a quadratic function. These results help complete the theory of an optimal first-order method for smooth convex minimization.

On the Convergence Analysis of the Optimized Gradient Method

PubMed Central

Kim, Donghwan; Fessler, Jeffrey A.

2016-01-01

This paper considers the problem of unconstrained minimization of smooth convex functions having Lipschitz continuous gradients with known Lipschitz constant. We recently proposed the optimized gradient method for this problem and showed that it has a worst-case convergence bound for the cost function decrease that is twice as small as that of Nesterov’s fast gradient method, yet has a similarly efficient practical implementation. Drori showed recently that the optimized gradient method has optimal complexity for the cost function decrease over the general class of first-order methods. This optimality makes it important to study fully the convergence properties of the optimized gradient method. The previous worst-case convergence bound for the optimized gradient method was derived for only the last iterate of a secondary sequence. This paper provides an analytic convergence bound for the primary sequence generated by the optimized gradient method. We then discuss additional convergence properties of the optimized gradient method, including the interesting fact that the optimized gradient method has two types of worstcase functions: a piecewise affine-quadratic function and a quadratic function. These results help complete the theory of an optimal first-order method for smooth convex minimization. PMID:28461707

Reduction of characteristic RL time for fast, efficient magnetic levitation

NASA Astrophysics Data System (ADS)

Li, Yuqing; Feng, Guosheng; Wang, Xiaofeng; Wu, Jizhou; Ma, Jie; Xiao, Liantuan; Jia, Suotang

2017-09-01

We demonstrate the reduction of characteristic time in resistor-inductor (RL) circuit for fast, efficient magnetic levitation according to Kirchhoff's circuit laws. The loading time is reduced by a factor of ˜4 when a high-power resistor is added in series with the coils. By using the controllable output voltage of power supply and voltage of feedback circuit, the loading time is further reduced by ˜ 3 times. The overshoot loading in advance of the scheduled magnetic field gradient is equivalent to continuously adding a resistor without heating. The magnetic field gradient with the reduced loading time is used to form the upward magnetic force against to the gravity of the cooled Cs atoms, and we obtain an effectively levitated loading of the Cs atoms to a crossed optical dipole trap.

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography.

PubMed

Gu, Dongyu; Lazo-Portugal, Rodrigo; Fang, Chen; Wang, Zhantong; Ma, Ying; Knight, Martha; Ito, Yoichiro

2018-06-15

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A 565 /A 280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ) 6 γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of hexavalent chromium concentration in industrial waste incinerator stack gas by using a modified ion chromatography with post-column derivatization method.

PubMed

Miyake, Yuichi; Tokumura, Masahiro; Iwazaki, Yuta; Wang, Qi; Amagai, Takashi; Horii, Yuichi; Otsuka, Hideyuki; Tanikawa, Noboru; Kobayashi, Takeshi; Oguchi, Masahiro

2017-06-16

An ion chromatography with post-column derivatization with 1,5-diphenylcarbazide (IC-DPC) analytical method was modified to enable measurement of trace-level hexavalent chromium (Cr(VI)) in air. One of the difficulties in determining trace levels of Cr(VI) in air with conventional IC-DPC methods is co-elution of the solvent and ion peaks due to high concentrations of ionic compounds in the extract. However, by using gradient elution rather than isocratic elution we were able to fully resolve the Cr(VI) ion peak from the solvent peak without the need for diluting the extract, which would have reduced the minimum quantifiable level of the method. With this method, we were able to detect Cr(VI) in air at concentrations of 5.3ng/m 3 (assuming a sampling volume of 1m 3 and a final solution volume of 10mL). Recovery tests at three different concentrations of Cr(VI) (50, 250, 1000ng) were performed with or without fly ash; recovery rates at all the concentrations of Cr(VI), with or without fly ash, ranged from 68% to 110% (mean±relative standard deviation, 96%±11%), and there were no differences in recovery rates with respect to the presence or absence of fly ash. Finally, we used the developed method to determine the concentration of Cr(VI) in stack gases collected from eight industrial waste incinerators located in Japan. The concentration of Cr(VI) in the stack gases ranged from below the method quantification limit to 3100ng/m 3 . The highest concentrations of Cr(VI) detected in the stack gases were two to three orders of magnitude higher than that in ambient air in Japan. Copyright © 2017 Elsevier B.V. All rights reserved.

Observation of Critical-Gradient Behavior in Alfvén-Eigenmode-Induced Fast-Ion Transport.

PubMed

Collins, C S; Heidbrink, W W; Austin, M E; Kramer, G J; Pace, D C; Petty, C C; Stagner, L; Van Zeeland, M A; White, R B; Zhu, Y B

2016-03-04

Experiments in the DIII-D tokamak show that fast-ion transport suddenly becomes stiff above a critical threshold in the presence of many overlapping small-amplitude Alfvén eigenmodes (AEs). The threshold is phase-space dependent and occurs when particle orbits become stochastic due to resonances with AEs. Above threshold, equilibrium fast-ion density profiles are unchanged despite increased drive, and intermittent fast-ion losses are observed. Fast-ion Dα spectroscopy indicates radially localized transport of the copassing population at radii that correspond to the location of midcore AEs. The observation of stiff fast-ion transport suggests that reduced models can be used to effectively predict alpha profiles, beam ion profiles, and losses to aid in the design of optimized scenarios for future burning plasma devices.

EVALUATION OF FACTORS IN THE ELUTION OF HYDROCORTISONE FROM PAPER CHROMATOGRAMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganis, F.M.; Hendrickson, M.W.; Giunta, P.D.

An assessment was made of a number of variable factors which affect the recovery of hydrocortisone from eluted filter paper chromatographic fractions. Factors tested included time of elution, sample concentration, rinsing of eluting fractions and pre-washing of the filter paper. It was noted that a 50 mu g sample could be quantitatively recovered after a 15-minute elution time from a pre-washed filter paper fraction. The results were subjected to a statistical analysis and were found to be highly significant. (auth)

Everolimus-eluting stents in interventional cardiology

PubMed Central

Townsend, Jacob C; Rideout, Phillip; Steinberg, Daniel H

2012-01-01

Bare metal stents have a proven safety record, but limited long-term efficacy due to in-stent restenosis. First-generation drug-eluting stents successfully countered the restenosis rate, but were hampered by concerns about their long-term safety. Second generation drug-eluting stents have combined the low restenosis rate of the first generation with improved long-term safety. We review the evolution of drug-eluting stents with a focus on the safety, efficacy, and unique characteristics of everolimus-eluting stents. PMID:22910420

Column switching combined with hydrophilic interaction chromatography-tandem mass spectrometry for the analysis of saxitoxin analogues, and their biosynthetic intermediates in dinoflagellates.

PubMed

Cho, Yuko; Tsuchiya, Shigeki; Yoshioka, Renpei; Omura, Takuo; Konoki, Keiichi; Oshima, Yasukatsu; Yotsu-Yamashita, Mari

2016-11-25

Hydrophilic-interaction chromatography (HILIC) is reportedly useful for the analysis of saxitoxin (STX) analogues, collectively known as paralytic shellfish toxins. Column switching and two-step gradient elution using HILIC combined with mass spectrometry enabled the simultaneous analysis of the 15 primary STX analogues and their biosynthetic intermediates, arginine, Int-A', and Int-C'2, and the shunt product, Cyclic-C'. Crude extracts of toxin-producing dinoflagellates can be injected without any treatment except filtration. Enrichment of the compounds using this method was highly reproducible with respect to retention times (% RSD was under 1%) and highly sensitive (limits of detection (LODs) were in the range 0.9 (Int-C'2) - 116 (C3) μM) in terms of avoiding matrix effects associated with co-eluting substances. Validation studies demonstrated acceptable performance of this method for specificity, repeatability, linearity and recovery. A comparison of the quantitative results for STX analogues in Alexandrium tamarense using HPLC with post-column fluorescent derivatization and the column-switching HILIC-MS method revealed good agreement. The presence of Int-A', Int-C'2, and Cyclic-C' in toxic dinoflagellate species with different toxin profiles was confirmed using this method. Our data support the hypothesis that the early stages of the STX biosynthesis and shunt pathways are the same in dinoflagellates and cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

[Determination of 11 industrial antioxidants in the aqueous simulants by ultra-performance liquid chromatography-tandem mass spectrometry].

PubMed

Fan, Sai; Zou, Jianhong; Li, Liping; Zhang, Nan; Liu, Wei; Li, Bing; Zhao, Xudong; Wu, Guohua; Xue, Ying; Zhao, Rong

2014-09-01

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed to identify and determine 11 industrial antioxidants in the aqueous simulants. A ProElut PLS SPE column was used for the enrichment, and an ACQUITY UPLC BEH C18 UPLC column (100 mm x 2.1 mm, 1.7 μm) was used for separation by the gradient elution with pure water and acetonitrile as the mobile phases. The MS/MS detection was performed with an electrospray ionization (ESI) source in negative mode. The external standard method was used for quantitation in the present study. The linear ranges of the 11 analytes were from 5.0 to 100 μg/L. The coefficients of correlation were greater than 0.995. The recoveries of blank aqueous simulants fortified with the 11 analytes at the levels of 5.0, 10.0 and 20.0 μg/L were 61.4% to 109.4% with the relative standard deviations varied from 3.9% to 18.2% (n = 6). The LODs and LOQs of the 11 analytes in aqueous simulants were 0.2-1.0 μg/L and 0.5-3.0 μg/L, respectively. This method is highly sensitive and accurate, and can be applied to the determination of the 11 trace industrial antioxidants in the aqueous simulants.

Novel dispersive micro-solid-phase extraction combined with ultrahigh-performance liquid chromatography-high-resolution mass spectrometry to determine morpholine residues in citrus and apples.

PubMed

Chen, Dawei; Miao, Hong; Zou, Jianhong; Cao, Pei; Ma, Ning; Zhao, Yunfeng; Wu, Yongning

2015-01-21

This paper presents a new analytical method for the determination of morpholine residues in citrus and apples using a novel dispersive micro-solid-phase extraction (DMSPE), followed by ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Samples were extracted with 1% formic acid in acetonitrile/water (1:1, v/v) and then cleaned up using the DMSPE procedure. Morpholine from the extract was adsorbed to a polymer cation exchange sorbent and eluted with ammonium hydroxide/acetonitrile (3:97, v/v) through a 1 mL syringe with a 0.22 μm nylon syringe filter. All of the samples were analyzed by UHPLC-HRMS/MS on a Waters Acquity BEH hydrophilic interaction chromatography column using 0.1% formic acid and 4 mM ammonium formate in water/acetonitrile as the mobile phase with gradient elution. The method showed good linearity (R(2) > 0.999) in the range of 1-100 μg/L for the analyte. The limit of detection and limit of quantitation values of morpholine were 2 and 5 μg/kg, respectively. The average recoveries of morpholine from the citrus and apple samples spiked at three different concentrations (5, 20, and 100 μg/kg) were in a range from 78.4 to 102.7%.

Removal of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product quality.

PubMed

Rodrigues, Teresa; Alves, Ana; Lopes, António; Carrondo, Manuel J T; Alves, Paula M; Cruz, Pedro E

2008-10-01

We have investigated the role of the retroviral lipid bilayer and envelope proteins in the adsorption of retroviral vectors (RVs) to a Fractogel DEAE matrix. Intact RVs and their degradation components (envelope protein-free vectors and solubilized vector components) were adsorbed to this matrix and eluted using a linear gradient. Envelope protein-free RVs (Env(-)) and soluble envelope proteins (gp70) eluted in a significantly lower range of conductivities than intact RVs (Env(+)) (13.7-30 mS/cm for Env(-) and gp70 proteins vs. 47-80 mS/cm for Env(+)). The zeta (zeta)-potential of Env(+) and Env(-) vectors was evaluated showing that envelope proteins define the pI of the viral particles (pI (Env(+)) < 2 versus 3 < pI (Env(-)) < 4) and that Env(+) and Env(-) vectors have similar zeta-potentials within pH 5 and 8. The results presented herein indicate that the adsorption of retroviral particles occurs through multi-point interaction of the envelope proteins with the cationic groups on the chromatographic matrix. The strength of this adsorption is thus dependent on the amount of envelope protein present in the viral lipid bilayer. In conclusion, AEXc enables the separation of gp70 proteins as well as envelope protein-free vectors constituting a significant improvement to the quality of retroviral preparations for gene therapy applications.

Selection of an Aptamer Antidote to the Anticoagulant Drug Bivalirudin

PubMed Central

Martin, Jennifer A.; Parekh, Parag; Kim, Youngmi; Morey, Timothy E.; Sefah, Kwame; Gravenstein, Nikolaus; Dennis, Donn M.; Tan, Weihong

2013-01-01

Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care. PMID:23483901

Highly crosslinked polymeric monoliths for reversed-phase capillary liquid chromatography of small molecules.

PubMed

Liu, Kun; Tolley, H Dennis; Lee, Milton L

2012-03-02

Seven crosslinking monomers, i.e., 1,3-butanediol dimethacrylate (1,3-BDDMA), 1,4-butanediol dimethacrylate (1,4-BDDMA), neopentyl glycol dimethacrylate (NPGDMA), 1,5-pentanediol dimethacrylate (1,5-PDDMA), 1,6-hexanediol dimethacrylate (1,6-HDDMA), 1,10-decanediol dimethacrylate (1,10-DDDMA), and 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), were used to synthesize highly cross-linked monolithic capillary columns for reversed-phase liquid chromatography (RPLC) of small molecules. Dodecanol and methanol were chosen as "good" and "poor" porogenic solvents, respectively, for these monoliths, and were investigated in detail to provide insight into the selection of porogen concentration using 1,12-DoDDMA. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was conducted for all of the monoliths. Gradient elution of alkylbenzenes and alkylparabens provided high resolution separations. Optimized monoliths synthesized from all seven crosslinking monomers showed high permeability. Several of the monoliths demonstrated column efficiencies in excess of 50,000 plates/m. Monoliths with longer alkyl-bridging chains showed very little shrinking or swelling in solvents of different polarities. Column preparation was highly reproducible; the relative standard deviation (RSD) values (n=3) for run-to-run and column-to-column were less than 0.25% and 1.20%, respectively, based on retention times of alkylbenzenes. Copyright © 2012 Elsevier B.V. All rights reserved.

Carotenoids from Foods of Plant, Animal and Marine Origin: An Efficient HPLC-DAD Separation Method.

PubMed

Strati, Irini F; Sinanoglou, Vassilia J; Kora, Lintita; Miniadis-Meimaroglou, Sofia; Oreopoulou, Vassiliki

2012-12-19

Carotenoids are important antioxidant compounds, present in many foods of plant, animal and marine origin. The aim of the present study was to describe the carotenoid composition of tomato waste, prawn muscle and cephalothorax and avian (duck and goose) egg yolks through the use of a modified gradient elution HPLC method with a C 30 reversed-phase column for the efficient separation and analysis of carotenoids and their cis -isomers. Elution time was reduced from 60 to 45 min without affecting the separation efficiency. All- trans lycopene predominated in tomato waste, followed by all- trans -β-carotene, 13- cis -lutein and all- trans lutein, while minor amounts of 9- cis -lutein, 13- cis -β-carotene and 9- cis -β-carotene were also detected. Considering the above findings, tomato waste is confirmed to be an excellent source of recovering carotenoids, especially all- trans lycopene, for commercial use. Xanthophylls were the major carotenoids of avian egg yolks, all- trans lutein and all- trans zeaxanthin in duck and goose egg yolk, respectively. In the Penaeus kerathurus prawn, several carotenoids (zeaxanthin, all- trans -lutein, canthaxanthin, cryptoxanthin, optical and geometrical astaxanthin isomers) were identified in considerable amounts by the same method. A major advantage of this HPLC method was the efficient separation of carotenoids and their cis -isomers, originating from a wide range of matrices.

HPLC determination of olanzapine and carbamazepine in their nicotinamide cocrystals and investigation of the dissolution profiles of cocrystal tablet formulations.

PubMed

Renkoğlu, Pelin; Çelebier, Mustafa; Arıca-Yegin, Betül

2015-05-01

Cocrystals have recently gained importance in the pharmaceutical industry. In this study, olanzapine and carbamazepine cocrystals were synthesized by using nicotinamide as cocrystal forming agent to achieve improvements in the physicochemical characteristics of the active ingredients. An HPLC method was developed to determine the amount, thus, the stoichiometric ratios of olanzapine and carbamazepine in the synthesized cocrystals. Olanzapine:nicotinamide and olanzapine tablet formulations were prepared and the developed HPLC method was applied successfully in order to compare the dissolution profiles of these formulations. An ACE 5 CN, 25 cm × 4.6 mm, 5 µm column was used and a gradient elution program was performed for simultaneous determination of olanzapine, carbamazepine and nicotinamide. Phosphate buffer (pH 5.0, 25 mM) and methanol was used in a ratio from 80:20 to 70:30 while the flow rate was 1 mL min(-1) for the elution of the compounds within 12 min. In conclusion, two different aims were achieved, the first one was to indicate the stoichiometric ratios of the active ingredients olanzapine and carbamazepine with nicotinamide in their cocrystals, and the second one was the comparison of the dissolution profiles of the olanzapine and olanzapine:nicotinamide cocrystal formulations. It was found that the cocrystal formulation with nicotinamide improved the dissolution profile of olanzapine.

Reaction-Mediated Desorption of Macromolecules: Novel Phenomenon Enabling Simultaneous Reaction and Separation.

PubMed

Isakari, Yu; Kishi, Yuhi; Yoshimoto, Noriko; Yamamoto, Shuichi; Podgornik, Aleš

2018-02-02

Combining chemical reaction with separation offers several advantages. In this work possibility to induce spontaneous desorption of adsorbed macromolecules, once being PEGylated, through adjustment of the reagent composition is investigated. Bovine serum albumin (BSA) and activated oligonucleotide, 9T, are used as the test molecules and 20 kDa linear activated PEG is used for their PEGylation. BSA solid-phase PEGylation is performed on Q Sepharose HP. Distribution coefficient of BSA and PEG-BSA as a function of NaCl is determined using linear gradient elution (LGE) experiments and Yamamoto model. According to the distribution coefficient the selectivity between BSA and PEG - BSA of around 15 is adjusted by using NaCl. Spontaneous desorption of PEG - BSA is detected with no presence of BSA. However, due to a rather low selectivity, also desorption of BSA occurred at high elution volume. A similar procedure is applied for activated 9T oligonucleotide, this time using monolithic CIM QA disk monolithic column for adsorption. Selectivity of over 2000 is obtained by proper adjustment of PEG reagent composition. High selectivity enables spontaneous desorption of PEG-9T without any desorption of activated 9T. Both experiments demonstrates that reaction-mediated desorption of macromolecules is possible when the reaction conditions are properly tuned. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of gymnemagenin in rat plasma using high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics after oral administration of Gymnema sylvestre extract.

PubMed

Kamble, Bhagyashree; Gupta, Ankur; Patil, Dada; Khatal, Laxman; Janrao, Shirish; Moothedath, Ismail; Duraiswamy, Basavan

2013-05-01

A sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the determination of gymnemagenin (GMG), a triterpene sapogenin from Gymnema sylvestre, in rat plasma using withaferin A as the internal standard (IS). Plasma samples were simply extracted using liquid-liquid extraction with tetra-butyl methyl ether. Chromatographic separation was performed on Luna C(18) column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia) at a flow rate of 0.8 mL/min. GMG and IS were eluted at 4.64 and 4.36 min, ionized in negative and positive mode, respectively, and quantitatively estimated using multiple reaction monitoring (MRM) mode. Two MRM transitions were selected at m/z 505.70 → 455.5 and m/z 471.50 → 281.3 for GMG and IS, respectively. The assay was linear over the concentration range of 5.280-300.920 ng/mL. The mean plasma extraction recoveries for GMG and IS were found to be 80.92 ± 8.70 and 55.63 ± 0.76%, respectively. The method was successfully applied for the determination of pharmacokinetic parameters of GMG after oral administration of G. sylvestre extract. Copyright © 2012 John Wiley & Sons, Ltd.

Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography.

PubMed

Raweerith, Rutai; Ratanabanangkoon, Kavi

2003-11-01

A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.

Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC-MS/MS.

PubMed

Sun, Wei; Jiang, Zhe-li; Zhou, Lei; Chen, Rui-min; Wang, Zhe; Li, Wan-shu; Jiang, Shuo-min; Hu, Guo-xin; Chen, Rui-jie

2015-02-15

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of pirfenidone was at 1.39 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 186.2→92.1 for pirfenidone and m/z 237.1→194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5-2000 ng/mL with a lower limit of quantitation (LLOQ) of 5 ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4-84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0mg/kg pirfenidone in rats. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

NASA Technical Reports Server (NTRS)

Song, Q.; Putcha, L.; Harm, D. L. (Principal Investigator)

2001-01-01

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75-1400 ng/ml for PMZ and 2.5-1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within +/- 11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett-Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.

Antioxidant and antiproliferative activities of protein hydrolysate from the rhizomes of Zingiberaceae plants.

PubMed

Inthuwanarud, Kanok; Sangvanich, Polkit; Puthong, Songchan; Karnchanatat, Aphichart

2016-11-01

Plant proteins have been investigated for their antioxidant activities, but there are still no reports detailing the antioxidant activity levels of plants in the Zingiberaceae family, which are popular food agents and used in folklore medicine. In this study, the crude rhizome protein extract and associated pepsin/pancreatin protein hydrolysate of 15 plants in the Zingiberaceae family were screened using the DPPH method for antioxidant activity. The protein hydrolysate of C. zedoaria possessed the highest antioxidant activity (IC 50 of 25.7±6.3µg/mL), which was close to that of the reference ascorbic acid (IC 50 of 22.3±1.8µg/mL). After enrichment by Q Sepharose ion exchange chromatography using a five step elution gradient of increasing NaCl concentration (0, 0.25, 0.5, 0.75 and 1M), the fraction eluting in the 0.5M NaCl (F50) showed the highest antioxidant activity (IC 50 of 41.78±2.9µg/mL), and was found to have weak in vitro cytotoxicity against the HEP-G2 and SW620 cell lines (IC 50 of 200.8±11.8 and 241.0±9.3µg/mL, respectively), but not the BT474, CHAGO and KATO-3 cell lines. F50 had an estimated molecular weight by MALDI-TOF mass spectrometry of 12,400-12,800 Da.

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

PubMed

Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Kramberger, Petra; Tover, Andres; Berger, Eva; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

2017-02-01

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10 12 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10 11 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10 9 particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size-exclusion chromatography.

PubMed

Miller, N T; Feibush, B; Karger, B L

1984-12-21

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure identical to Si-(CH2)3-O-(CH2-CH2-O)n-R, where n = 1, 2, 3 and R = methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25 degrees C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[eta]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.

Preparative separation and purification of epigallocatechin gallate from green tea extracts using a silica adsorbent containing β-cyclodextrin.

PubMed

Lai, Shih-Ming; Gu, Jhe-Yu; Huang, Bing-Hao; Chang, Chieh-Ming J; Lee, Wen-Lung

2012-03-01

A silica adsorbent containing β-cyclodextrin (β-CD) has been developed and used for the separation and purification of epigallocatechin gallate (EGCG) from the green tea extracts. The batch adsorption experiments demonstrated that, the β-CD bonded silica adsorbent possessed excellent adsorption equilibrium capacity (> 55 mg/g adsorbent) and adsorption ratio (>95%) for EGCG compared to the other tea catechins and caffeine. The excellent adsorption capacity and selectivity for EGCG are attributed to the specific interactions between β-CD and EGCG. The preparative separation and purification performance of EGCG on the β-CD bonded silica column (220 mm L × 15 mm i.d., 40-63 μm) was then evaluated. The column was operated in the polar organic mode using methanol/acetonitrile/acetic acid as the mobile phase and eluted under a three-step gradient elution program. The sample was dissolved in acetonitrile and loaded on a preparative scale of about 0.8 mg/g adsorbent. Under the optimal chromatographic conditions, the target compound, EGCG, being the most retained species, was obtained at a purity of about 90% with a recovery of about 90%. The productivity of EGCG was about 6 mg per injection, which can be further increased by scaling-up the chromatographic system. Copyright © 2012 Elsevier B.V. All rights reserved.

[Rapid screening and identification of 22 allergenic disperse dyes in ecological textiles by high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry].

PubMed

Niu, Zengyuan; Luo, Xin; Ye, Xiwen; Xiu, Xiaoli; Zhang, Li; Wang, Xin; Chen, Jing

2015-10-01

A rapid screening method based on high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry (HPLC-LTQ/Orbitrap MS) for 22 disperse dyes in ecological textiles has been established. The target compounds were extracted by pyridine/water (1:1, v/v) by shaking extraction in 90 degrees C water bath. The extracts were then separated by a CAPCELL PAK C18 column (100 mm x 2.0 mm, 5 μm) using gradient elution with acetonitrile-5 mmol/L ammonium acetate containing 0.01% (v/v) formic acid as mobile phases, and finally analyzed by HPLC-LTQ/Orbitrap in positive and negative ESI modes. The retention time and accurate mass of parent ion were used for fast screening of 22 disperse dyes, while the confirmatory analysis was obtained by fragments generated by collision-induced dissociation (CID) MS/MS. Target analysis exhibited high mass accuracy (< 5 x 10(-6)). Each target showed a good linearity in its own concentration range and the correlation coefficient was higher than 0.99. The LOQs were 0.125-2.5 mg/kg. Except for Disperse Yellow 49, the average recoveries of most disperse dyes at three spiked levels were 65%-120%, and the relative standard deviations (n = 6) were less than 15%. The method was applied for screening 40 different kinds of textiles, and Disperse Orange 37/76 was detected in one of them. With high selectivity and strong anti-jamming ability, this method is simple, rapid, accurate, and it can be used for the inspection of disperse dyes in textiles.

Determination of mycotoxins in different food commodities by ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry.

PubMed

Beltrán, Eduardo; Ibáñez, María; Sancho, Juan Vicente; Hernández, Félix

2009-06-01

A rapid multianalyte-multiclass method with little sample manipulation has been developed for the simultaneous determination of eleven mycotoxins in different food commodities by using ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC/MS/MS). Toxins were extracted from the samples with acetonitrile/water (80:20, v/v) 0.1% HCOOH and, after a two-fold dilution with water, directly injected into the system. Thanks to the fast high-resolution separation of UHPLC, the eleven mycotoxins were separated by gradient elution in only 4 min. The method has been validated in three food matrices (maize kernels, dry pasta (wheat), and eight-multicereal babyfood (wheat, maize, rice, oat, barley, rye, sorghum, millet)) at four different concentration levels. Satisfactory recoveries were obtained (70-110%) and precision (expressed as relative standard deviation) was typically below 15% with very few exceptions. Quantification of samples was carried out with matrix-matched standards calibration. The lowest concentration successfully validated in sample was as low as 0.5 microg/kg for aflatoxins and ochratoxin A in babyfood, and 20 microg/kg for the rest of the selected mycotoxins in all matrices tested. Deoxynivalenol could be only validated at 200 microg/kg, due the poor sensitivity for this mycotoxin analysis. With only two exceptions (HT-2 and deoxynivalenol), the limits of detection (LODs), estimated for a signal-to-noise ratio of 3 from the chromatograms of samples spiked at the lowest level validated, varied between 0.1 and 1 microg/kg in the three food matrices tested. The method was applied to the analysis of different kinds of samples. Positive findings were confirmed by acquiring two transitions (Q quantification, q confirmation) and evaluating the Q/q ratio. Copyright (c) 2009 John Wiley & Sons, Ltd.

Determination of green, blue and yellow artificial food colorants and their abuse in herb-coloured green Easter beers on tap.

PubMed

Stachová, Ivana; Lhotská, Ivona; Solich, Petr; Šatínský, Dalibor

2016-07-01

Beer is one of the most popular alcoholic beverages worldwide. For consumer acceptance, significant factors are its taste, flavour and colour. This study determines selected synthetic green, blue and yellow food colorants in popular Easter herb-coloured green beers on tap produced in breweries on Holy Thursday. The abuse of beer colouring with Tartrazine (E 102), Quinoline yellow (E 104), Sunset yellow (E 110), Patent blue (E 131), Indigo carmine (E 132), Brilliant blue FCF (E 133), Green S (E 142) and Fast green FCF (E 143) was assessed in 11 green beer samples purchased in local restaurants. HPLC was used for the separation and detection of artificial colorants with diode-array detection and a Chromolith Performance CN 100 × 4.6 mm column with guard pre-column Chromolith CN 5 × 4.6 mm. Separation was performed in gradient elution with mobile phase containing methanol-aqueous 2% ammonium acetate at pH 7.0. The study showed that eight beers (70%) marketed in the Czech Republic contained artificial colorants (Tartrazine and Brilliant blue FCF). The concentration of colorants found in analysed green herb-coloured beers ranged from 1.58 to 3.49 mg l(-)(1) for Tartrazine, 0.45-2.18 mg l(-)(1) for Brilliant blue, while Indigo carmine was detected only once at concentration 2.36 mg l(-)(1). Only three beers showed no addition of the synthetic colorants. However, the levels of artificial colorants found in beers marketed in the Czech region were very low and did not show a serious risk for consumers' health.

Post-column infusion study of the 'dosing vehicle effect' in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples.

PubMed

Shou, Wilson Z; Naidong, Weng

2003-01-01

It has become increasingly popular in drug development to conduct discovery pharmacokinetic (PK) studies in order to evaluate important PK parameters of new chemical entities (NCEs) early in the discovery process. In these studies, dosing vehicles are typically employed in high concentrations to dissolve the test compounds in dose formulations. This can pose significant problems for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of incurred samples due to potential signal suppression of the analytes caused by the vehicles. In this paper, model test compounds in rat plasma were analyzed using a generic fast gradient LC/MS/MS method. Commonly used dosing vehicles, including poly(ethylene glycol) 400 (PEG 400), polysorbate 80 (Tween 80), hydroxypropyl beta-cyclodextrin, and N,N-dimethylacetamide, were fortified into rat plasma at 5 mg/mL before extraction. Their effects on the sample analysis results were evaluated by the method of post-column infusion. Results thus obtained indicated that polymeric vehicles such as PEG 400 and Tween 80 caused significant suppression (> 50%, compared with results obtained from plasma samples free from vehicles) to certain analytes, when minimum sample cleanup was used and the analytes happened to co-elute with the vehicles. Effective means to minimize this 'dosing vehicle effect' included better chromatographic separations, better sample cleanup, and alternative ionization methods. Finally, a real-world example is given to illustrate the suppression problem posed by high levels of PEG 400 in sample analysis, and to discuss steps taken in overcoming the problem. A simple but effective means of identifying a 'dosing vehicle effect' is also proposed. Copyright 2003 John Wiley & Sons, Ltd.

A comparative pharmacokinetic study of three flavonoids and three anthraquinones in normal and gastrointestinal motility disorders rat plasma after the oral administration of Wei-Chang-Shu tablet using high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Ren, Yan; Zhao, Weiwei; Zhao, Juanjuan; Chen, Xiangming; Yu, Chen; Liu, Mengan

2017-11-01

A simple, fast and reliable high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification and pharmacokinetic study of three flavonoids (liquiritigenin, isoliquiritigenin and formononetin) and three anthraquinones (emodin, rhein and aloe-emodin), which are the bioactive ingredients of Wei-Chang-Shu tablet found in rat plasma. After extraction by liquid-liquid extraction with ethyl acetate, chromatographic separation was achieved on an Agilent Zorbax SB-C 18 column (4.6 × 150 mm, 5 μm) at a flow rate of 1 mL/min by gradient elution using 0.1% aqueous acetic acid and acetonitrile. The detection was performed using a triple quadrupole mass spectrometer equipped with electrospray ionization source in the negative ionization and selected reaction monitoring mode. Method validation was performed in terms of specificity, carryover, linearity (r > 0.99), intra-/inter-day precision (1.0-10.1%), accuracy (relative error, <7.6%), stability (0.6-13.2%), extract recovery (74.9-91.9%) and matrix effect (89.1-109%). The lower limits of quantification of the six analytes varied from 0.92 to 10.4 ng/mL. The validated method was successfully applied to compare the pharmacokinetic properties of Wei-Chang-Shu tablet in normal rats and in rats with gastrointestinal motility disorders. The results indicated that there were obvious differences in the pharmacokinetic behavior between normal and model rats. This study will be helpful in the clinical application of Wei-Chang-Shu tablet. Copyright © 2017 John Wiley & Sons, Ltd.

Development of an ultra-fast liquid chromatography-tandem mass spectrometry method for simultaneous determination of seven flavonoids in rat plasma: Application to a comparative pharmacokinetic investigation of Ginkgo biloba extract and single pure ginkgo flavonoids after oral administration.

PubMed

Wang, Tianyang; Xiao, Jie; Hou, Huiping; Li, Pei; Yuan, Ziyue; Xu, Huarong; Liu, Ran; Li, Qing; Bi, Kaishun

2017-08-15

For deeper pharmacokinetic investigation and further curative application of ginkgo flavonoids, a delicate, efficient and precise UFLC-MS/MS technique for synchronous quantitation of seven flavonoids, apigenin, luteolin, naringenin, quercetin, diosmetin, kaempferol and isorhamnetin in rat plasma has been established. After mixing with the internal standard (IS) linarin, bio-samples were pretreated via ethyl acetate for liquid-liquid extraction, then isolated at 0.2ml/min flow rate on a Venusil MP C 18 chromatographic column (100mm×2.1mm, 3μm) by means of gradient elution. 0.1% formic acid-water and methanol system was used as the mobile phase. Mass spectrometric inspection was conducted on a 4000Q UFLC-MS/MS system with turbo ion spray source in patterns of negative ion and multiple reaction-monitoring (MRM). All calibration curves proved favorable linearity (R 2 ≥0.9918) in linear ranges. Intra-day and inter-day precisions didn't exceed 14.0% for all the analytes, and the accuracy was within 6.9%. Extraction recoveries of analytes and IS were less than ±15.0% of nominal concentrations. This method has been under thorough and firm verification for a comparative pharmacokinetic research after gavage between Ginkgo biloba extract and single pure ginkgo flavonoids. The results demonstrated that there're evident pharmacokinetic discrepancies, and possible structural influences were innovatively proposed. Generally, substitution with 3-hydroxylation, a double bond in ring C, ring B methoxylation often confer longer onset period. The existence of ring B catechol group gives rise to faster clearance. Copyright © 2017. Published by Elsevier B.V.

Simple and validated UHPLC-MS/MS analysis of nimodipine in plasma and cerebrospinal fluid of patients with subarachnoid haemorrhage.

PubMed

Mohamed, Susan; Riva, Roberto; Contin, Manuela

2016-08-15

We present a simple, fast and validated method for the determination of nimodipine in plasma and cerebrospinal fluid (CSF) of patients with subarachnoid haemorrhage using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma or CSF 250μL aliquots were pretreated with acetonitrile spiked with lacosamide as internal standard. The chromatographic separation was performed on a Fusion (3μm) 50×2.0mm I.D. column with gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.35mL/min. The MS/MS ion transitions were 419.1→343 for nimodipine and 251.1→91 for the internal standard. The linearity was determined from 2.0 to 40.0ng/mL in plasma and 40.0-800.0pg/mL in CSF. The lower limit of quantitation (LLOQ) of nimodipine was 0.4ng/mL in plasma and 40pg/mL in CSF. The mean recovery for nimodipine was ≥75% in plasma and ≥90% in CSF at all three considered concentrations. Intra- and interassay precision and accuracy were ≤15% at all quality control concentrations in plasma and CSF. The method was applied to measure plasma and CSF concentrations of nimodipine in a series of patients with subarachnoid haemorrhage treated with intravenous nimodipine. The present procedure, omitting time-consuming liquid-liquid extraction and drying steps, is faster, simpler and cheaper than published LC-MS/MS analytical methods for nimodipine in plasma and the first validated one for nimodipine in CSF. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast automated online xylanase activity assay using HPAEC-PAD.

PubMed

Cürten, Christin; Anders, Nico; Juchem, Niels; Ihling, Nina; Volkenborn, Kristina; Knapp, Andreas; Jaeger, Karl-Erich; Büchs, Jochen; Spiess, Antje C

2018-01-01

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Development of SPME-LC-MS method for screening of eight beta-blockers and bronchodilators in plasma and urine samples.

PubMed

Goryński, Krzysztof; Kiedrowicz, Alicja; Bojko, Barbara

2016-08-05

The current work describes the development and validation of a simple, efficient, and fast method using solid phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS) for the concomitant measurement of eight beta-blockers and bronchodilators in plasma and urine. The presented assay enables quantitative determination of acebutolol, atenolol, fenoterol, nadolol, pindolol, procaterol, sotalol, and timolol. In this work, samples were prepared on a high-throughput platform using the 96-well plate format of the thin film solid phase microextraction (TFME) system, and a biocompatible extraction phase made of hydrophilic-lipophilic balance particles. Analytes were separated on a pentafluorophenyl column (100mm×2.1mm, 3μm) by gradient elution using an UPLC Nexera coupled with an LCMS-8060 mass spectrometer. The mobile phase consisted of water-acetonitrile (0.1% formic acid) at a flow rate of 0.4mLmin(-1). The linearity of the method was checked within therapeutic blood-plasma concentrations, and shown to adequately reflect typically expected concentrations of future study samples. Post-extraction addition experiments showed that the matrix effect ranged in plasma from 98% for procaterol to 115% for nadolol, and in urine, from 85% for nadolol and pindolol to 119% for atenolol. The method was successfully validated using Food and Drug Administration (FDA) guidelines, and met all acceptance criteria for bioanalytical assays at five concentration levels for all selected drugs. The final protocol can be successfully applied for monitoring concentrations of the selected drugs in both plasma and urine matrices obtained from patients or athletes. Copyright © 2016 Elsevier B.V. All rights reserved.

Pharmacokinetic differences of mifepristone between sexes in animals.

PubMed

Chen, Wenge; Cheng, Yunlong; Chen, Jianzhong; Chen, Jiahang; Jiang, Kai; Zhou, Yuyang; Jia, Lee

2018-05-30

Mifepristone (RU486) is developed originally as a contraceptive used by hundreds of millions of women world-wide, and also reported as a safe and long-term psychotic depressant, or as a cancer chemotherapeutic agent used by both sexes. In our preliminary study aimed at developing mifepristone as a cancer metastatic chemopreventive, we coincidentally observed that blood mifepristone concentrations in female rats seem to be higher than those in male ones post administration. To substantiate if the pharmacokinetic differences between sexes exist, we established a fast UPLC-MS/MS method to determine mifepristone concentrations in plasma, and analyzed blood concentrations of mifepristone over time in rats and dogs of both sexes. Mifepristone in plasma or incubation liquid was recovered by liquid-liquid extraction using 1 mL of ethyl acetate. Chromatographic separation was performed on a C 18 column at 35 °C, with a gradient elution consisting of methanol and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. And pharmacokinetic parameters such as elimination half-life, and mean residence time were calculated by using the non-compartmental pharmacokinetics data analysis software. In this work, administrations of mifepristone to rats and beagle dogs revealed that the plasma concentrations of mifepristone (AUC, C max ) were significantly higher (P < 0.05) in females than that in males. In vitro liver microsomal incubation experiments showed that the metabolic rate of mifepristone in males was higher than that in females, which was consistent with the results of in vivo experiments. In general, we first found the sex-related differences about pharmacokinetic properties of mifepristone and revealed the metabolism difference of hepatic microsomal enzyme is the main reason. Copyright © 2018 Elsevier B.V. All rights reserved.

[Analyzing crude/processed root of Polygonum multiflorum from different habitats by UPLC fingerprint and mode identification methods].

PubMed

Xiao, Rong; Lin, Yan; Lei, Si-Min; Zhang, Ying; Huang, Jie; Xia, Bo-Hou; Li, Chun; Liao, Duan-Fang; Wu, Ping; Lin, Li-Mei

2017-06-01

To establish a content determination method for 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) of the crude/processed root of Polygonum multiflorum from different habitats in China and set up the fingerprint by using UPLC. Various samples were pretreated by macro-porous resin. Then UPLC analysis was performed on Waters ACQUITY UPLC@BEH C18 chromatographic column (2.1 mm×50 mm, 1.7 μm) at (25±5) ℃. A binary gradient elution system was composed of acetonitrile (phase A) and 0.5% acetic acid solution (phase B). Detection was performed at the wavelength of 254 nm, and the mobile flow rate was set at 0.3 mL•min⁻¹. Results showed that the yield of extraction of the 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside from root of P. multiflorum was all over 25.0% after macro-porous resin separation; an exclusive UPLC fingerprint method of the crude/processed root of P. multiflorum from different habitats was successfully set up and 17 chromatographic peaks were calibrated. Cluster analysis can not entirely distinguish the crude one from the processed one, while principal component analysis absolutely can. 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside is the composition that has largest differences in variable importance in projection (VIP) between crude and processed root of P. multiflorum. The separating method can gain high-purity 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside, and the determination method is simple, sensitive, reliable and can be used in fast identifying the crude/processed root of P. multiflorum or as a method for overall quality control of root of P. multiflorum. Copyright© by the Chinese Pharmaceutical Association.

Large-volume injection of sample diluents not miscible with the mobile phase as an alternative approach in sample preparation for bioanalysis: an application for fenspiride bioequivalence.

PubMed

Medvedovici, Andrei; Udrescu, Stefan; Albu, Florin; Tache, Florentin; David, Victor

2011-09-01

Liquid-liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion via UHPLC-MS/MS applicable to personalized antibiotic dosing research.

PubMed

Javorska, Lenka; Krcmova, Lenka Kujovska; Solich, Petr; Kaska, Milan

2017-08-05

Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC-MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100×4.6mm, 2.7μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5min. The method was found to be linear in the range of 2-60μM (or 0.5-10μM) for serum, 0.27-10μM (or 2-60μM) for peritoneal/pleural effusion and 25-300μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS-MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

PubMed

Zhao, Yue; Liu, Guowen; Angeles, Aida; Hamuro, Lora L; Trouba, Kevin J; Wang, Bonnie; Pillutla, Renuka C; DeSilva, Binodh S; Arnold, Mark E; Shen, Jim X

2015-04-15

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 μm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 μg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a validated HPLC method for the simultaneous determination of flavonoids in Cuscuta chinensis Lam. by ultra-violet detection

PubMed Central

2012-01-01

Background Cuscuta species known as dodder, have been used in traditional medicine of eastern and southern Asian countries as liver and kidney tonic. Flavonoids are considered as the main biologically active constituents in Cuscuta plants especially in C. chinensis Lam. Objective In the present study, a fast, simple and reliable method for the simultaneous determination and quantization of C. chinensis flavonols including hyperoside, rutin, isorhamnetin and kaempferol has been developed. Materials and methods The chromatographic separation was carried out on a reversed phase ACE 5 C18 with eluting at a flow rate of 1 ml/min using a gradient with O-phosphoric acid 0.25% : acetonitrile for 42 min. UV spectra were collected across the range of 200–900 nm, extracting 360 nm for the chromatograms. The method was validated according to linearity, selectivity, precision, recovery, LOD and LOQ. Results The method was selective for determination of rutin, hyperoside, isorhamnetin and kampferol. The calibration graphs of flavonols were linear with r2 > 0.999. RSDs% of intra- and inter-day precisions were found 1.3&3.4 for rutin, 1.5&2.8 for hyperoside, 1.3&3.3 for isorhamnetin and 1.7 & 2.9 for kaempferol which were satisfactory. LODs and LOQs were calculated as 1.73 & 8.19 for rutin, 0.09 & 4.19 for hyperoside, 2.09 & 6.3 for isorhamnetin and 0.18 & 0.56 for kaempferol. The recovery averages of above-mentioned flavonols were 90.3%, 97.4%, 98.7% and 90.0%, respectively. Conclusion The simplicity of the method makes it highly valuable for quality control of C. chinensis according to quantization of flavonols. PMID:23352257

Deflection and Distortion of CME internal magnetic flux rope due to the interaction with a structured solar wind

NASA Astrophysics Data System (ADS)

Shiota, D.; Iju, T.; Hayashi, K.; Fujiki, K.; Tokumaru, M.; Kusano, K.

2016-12-01

CMEs are the most violent driver of geospace disturbances, and therefore their arrival to the Earth position is an important factor in space weather forecast. The dynamics of CME propagation is strongly affected by the interaction with background solar wind. To understand the interaction between a CME and background solar wind, we performed three-dimensional MHD simulations of the propagation of a CME with internal twisted magnetic flux rope into a structured bimodal solar wind. We compared three different cases in which an identical CME is launched into an identical bimodal solar wind but the launch dates of the CME are different. Each position relative to the boundary between slow and fast solar winds becomes almost in the slow wind stream region, almost in the fast wind stream region, or in vicinity of the boundary of the fast and slow solar wind stream (that grows to CIR). It is found that the CME is most distorted and deflected eastward in the case near the CIR, in contrast to the other two cases. The maximum strength of southward magnetic field at the Earth position is also highest in the case near CIR. The results are interpreted that the dynamic pressure gradient due to the back reaction from pushing the ahead slow wind stream and due to the collision behind fast wind stream hinders the expansion of the CME internal flux rope into the direction of the solar wind velocity gradient. As a result, the expansion into the direction to the velocity gradient is slightly enhanced and results in the enhanced deflection and distortion of the CME and its internal flux rope. These results support the pileup accident hypothesis proposed by Kataoka et al. (2015) to form unexpectedly geoeffective solar wind structure.

The Role of Nonlinear Gradients in Parallel Imaging: A k-Space Based Analysis.

PubMed

Galiana, Gigi; Stockmann, Jason P; Tam, Leo; Peters, Dana; Tagare, Hemant; Constable, R Todd

2012-09-01

Sequences that encode the spatial information of an object using nonlinear gradient fields are a new frontier in MRI, with potential to provide lower peripheral nerve stimulation, windowed fields of view, tailored spatially-varying resolution, curved slices that mirror physiological geometry, and, most importantly, very fast parallel imaging with multichannel coils. The acceleration for multichannel images is generally explained by the fact that curvilinear gradient isocontours better complement the azimuthal spatial encoding provided by typical receiver arrays. However, the details of this complementarity have been more difficult to specify. We present a simple and intuitive framework for describing the mechanics of image formation with nonlinear gradients, and we use this framework to review some the main classes of nonlinear encoding schemes.

Precision control of eluted activity from a Sr/Rb generator for cardiac positron emission tomography.

PubMed

Klein, R; Adler, A; Beanlands, R S; deKemp, R A

2004-01-01

A rubidium-82 (/sup 82/Rb) elution system is described for use with clinical positron emission tomography. The system is self-calibrating with 1.4% repeatability, independent of generator activity and elution flow rate. Saline flow is switched between a /sup 82/Sr//sup 82/Rb generator and a bypass line to achieve a constant activity elution of /sup 82/Rb. In the present study, pulse width modulation (PWM) of a solenoid valve is compared to simple threshold control as a means to simulate a proportional valve. A predictive-corrective control algorithm is developed which produces a constant activity elution within the constraints of long feedback delay and short elution time. Accurate constant-activity elutions of 10-70% of the total generator activity were demonstrated using the threshold comparison control. The adaptive-corrective control of the PWM valve provided a substantial improvement in precision of the steady-state output.

Method for enhanced accuracy in predicting peptides using liquid separations or chromatography

DOEpatents

Kangas, Lars J.; Auberry, Kenneth J.; Anderson, Gordon A.; Smith, Richard D.

2006-11-14

A method for predicting the elution time of a peptide in chromatographic and electrophoretic separations by first providing a data set of known elution times of known peptides, then creating a plurality of vectors, each vector having a plurality of dimensions, and each dimension representing the elution time of amino acids present in each of these known peptides from the data set. The elution time of any protein is then be predicted by first creating a vector by assigning dimensional values for the elution time of amino acids of at least one hypothetical peptide and then calculating a predicted elution time for the vector by performing a multivariate regression of the dimensional values of the hypothetical peptide using the dimensional values of the known peptides. Preferably, the multivariate regression is accomplished by the use of an artificial neural network and the elution times are first normalized using a transfer function.

Short infrared laser pulses increase cell membrane fluidity

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Cantu, Jody C.; Ibey, Bennett L.; Beier, Hope T.

2017-02-01

Short infrared laser pulses induce a variety of effects in cells and tissues, including neural stimulation and inhibition. However, the mechanism behind these physiological effects is poorly understood. It is known that the fast thermal gradient induced by the infrared light is necessary for these biological effects. Therefore, this study tests the hypothesis that the fast thermal gradient induced in a cell by infrared light exposure causes a change in the membrane fluidity. To test this hypothesis, we used the membrane fluidity dye, di-4-ANEPPDHQ, to investigate membrane fluidity changes following infrared light exposure. Di-4-ANEPPDHQ fluorescence was imaged on a wide-field fluorescence imaging system with dual channel emission detection. The dual channel imaging allowed imaging of emitted fluorescence at wavelengths longer and shorter than 647 nm for ratiometric assessment and computation of a membrane generalized polarization (GP) value. Results in CHO cells show increased membrane fluidity with infrared light pulse exposure and this increased fluidity scales with infrared irradiance. Full recovery of pre-infrared exposure membrane fluidity was observed. Altogether, these results demonstrate that infrared light induces a thermal gradient in cells that changes membrane fluidity.

Field assisted sintering of refractory carbide ceramics and fiber reinforced ceramic matrix composites

NASA Astrophysics Data System (ADS)

Gephart, Sean

The sintering behaviors of silicon carbide (SiC) and boron carbide (B4C) based materials were investigated using an emerging sintering technology known as field assisted sintering technology (FAST), also known as spark plasma sintering (SPS) and pulse electric current sintering (PECS). Sintering by FAST utilizes high density electric current, uniaxial pressure, and relatively high heating rate compared to conventional sintering techniques. This effort investigated issues of scaling from laboratory FAST system (25 ton capacity) to industrial FAST system (250 ton capacity), as well as exploring the difference in sintering behavior of single phase B4C and SiC using FAST and conventional sintering techniques including hot-pressing (HP) and pressure-less sintering (PL). Materials were analyzed for mechanical and bulk properties, including characterization of density, hardness, fracture toughness, fracture (bend) strength, elastic modulus and microstructure. A parallel investigation was conducted in the development of ceramic matrix composites (CMC) using SiC powder impregnation of fiber compacts followed by FAST sintering. The FAST technique was used to sinter several B4C and SiC materials to near theoretical density. Preliminary efforts established optimized sintering temperatures using the smaller 25 ton laboratory unit, targeting a sample size of 40 mm diameter and 8 mm thickness. Then the same B4C and SiC materials were sintered by the larger 250 ton industrial FAST system, a HP system, and PL sintering system with a targeted dense material geometry of 4 x 4 x 0.315 inches3 (101.6 x 101.6 x 8 mm3). The resulting samples were studied to determine if the sintering dynamics and/or the resulting material properties were influenced by the sintering technique employed. This study determined that FAST sintered ceramic materials resulted in consistently higher averaged values for mechanical properties as well as smaller grain size when compared to conventionally sintered materials. While FAST sintered materials showed higher average values, in general they also showed consistently larger variation in the scattered data and consequently larger standard deviation for the resulting material properties. In addition, dynamic impact testing (V50 test) was conducted on the resulting materials and it was determined that there was no discernable correlation between observed mechanical properties of the ceramic materials and the resulting dynamic testing. Another study was conducted on the sintering of SiC and carbon fiber reinforced SiC ceramic matrix composites (CMC) using FAST. There has been much interest recently in fabricating high strength, low porosity SiC CMC.s for high temperature structural applications, but the current methods of production, namely chemical vapor infiltration (CVI), melt infiltration (MI), and polymer infiltration and pyrolysis (PIP), are considered time consuming and involve material related shortcomings associated with their respective methodologies. In this study, SiC CMC.s were produced using the 25 ton laboratory unit with a target sample size of 40 mm diameter and 3 mm thickness, as well as on the larger 250 ton industrial FAST system targeting a sample size of 101.6 x 101.6 x 3 mm3 to investigate issues associated with scaling. Several sintering conditions were explored including: pressure of 35-65 MPa, temperature of 1700-1900°C, and heating rates between 50-400°C/min. The SiC fibers used in this study were coated using chemical vapor deposition (CVD) with boron nitride (BN) and pyrolytic carbon to act as a barrier layer and preserve the integrity of the fibers during sintering. Then the barrier coating was coated by an outer layer of SiC to enhance the bonding between the fibers and the SiC matrix. Microstructures of the sintered samples were examined by FE-SEM. Mechanical properties including flexural strength-deflection and stress-strain were characterized using 4-point bend testing. Tensile testing was performed on the larger 101.6 x 101.6 x 3 mm samples. The microstructures of samples sintered using the 25 ton laboratory FAST system showed a reduction in porosity and good adhesion between the fiber-fiber and fiber-matrix interface. The microstructures of samples sintered on the 250 ton industrial FAST system showed a reduction in porosity, but there was visible reaction of the fiber and fiber coatings with the surrounding matrix. Additionally, there was significant radial cracking of the fibers visible in the microstructures. There is gap in the understanding of sintering behavior between laboratory and industrial scale FAST systems. The vast majority of publications on FAST sintering have been primarily focused on small sample geometries (20 mm diameter, less than 3 mm thick). A study was coordinated to investigate the thermal properties during heating and cooling using a 250 ton industrial FAST system at 900°C using B4C and SiC materials inside the graphite die assembly. The thermal properties were then compared to the resulting material properties of the identically sintered B4C and SiC to approximately 94% relative density, at a temperature of 1950°C, pressure of 45 MPa, 10 minute hold, and heated at a rate of 100°C/min. The study determined that at 900°C there were significant thermal gradients within the system for the examined materials, and that these gradients correlated well with the material property difference of the samples sintered at higher temperatures where the gradients are presumably larger due to an increase in radiative heat loss. The observed temperatures throughout the graphite were significantly different between B4C and SiC. These temperatures also correlated well with the material properties of the sintered products which showed more substantial variation for B4C when compared to SiC which was overall less affected by thermal gradients. This was attributed to the intrinsic thermal conductivity difference between the two subject materials which was manifested as thermal gradients throughout the material and graphite die assembly. Additionally, both the observed temperature gradients throughout the graphite die assembly and the difference in temperature reading between the optical pyrometer and thermocouples were significantly larger for the 250 ton FAST system than previous publications have demonstrated experimentally or via modeling of smaller laboratory scale systems. The findings from this work showed that relative to conventional sintering methods, the FAST process demonstrated comparable or improved material and mechanical properties with a significantly shorter processing cycle. However, the results demonstrated on the 25 ton laboratory scale unit were significantly different compared to results for the same materials sintered using the 250 ton industrial scale unit. The temperature gradients observed on the 250 ton FAST unit were significantly larger than previous reports on smaller FAST units. This result showed future efforts to scale up the FAST sintering process while maintaining similar results will require careful attention to minimizing temperature gradients. This could potentially be achieved by reducing radiative heat loss during processing and/or optimizing the graphite die design and implementing heat spreaders in specific locations dependent on the host material.s thermal and electrical properties as well as the sample geometry.

Direct enantioseparation of nitrogen-heterocyclic pesticides on cellulose-based chiral column by high-performance liquid chromatography.

PubMed

Chai, Tingting; Yang, Wenwen; Qiu, Jing; Hou, Shicong

2015-01-01

The enantiomeric separation of eight pesticides including bitertanol (), diclobutrazol (), fenbuconazole (), triticonazole (), imazalil (), triapenthenol (), ancymidol (), and carfentrazone-ethyl () was achieved, using normal-phase high-performance liquid chromatography on two cellulosed-based chiral columns. The effects of isopropanol composition from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated. Satisfactory resolutions were obtained for bitertanol (), triticonazole (), imazalil () with the (+)-enantiomer eluted first and fenbuconazole () with the (-)-enantiomer eluted first on Lux Cellulose-2 and Lux Cellulose-3. (+)-Enantiomers of diclobutrazol () and triapenthenol () were first eluted on Lux Cellulose-2. (-)-Carfentrazone-ethyl () were eluted first on Lux Cellulose-2 and Lux Cellulose-3 with incomplete separation. Reversed elution orders were obtained for ancymidol (7). (+)-Ancymidol was first eluted on Lux Cellulose-2 while on Lux Cellulose-3 (-)-ancymidol was first eluted. The results of the elution order at different column temperatures suggested that column temperature did not affect the optical signals of the enantiomers. These results will be helpful to prepare and analyze individual enantiomers of chiral pesticides. © 2014 Wiley Periodicals, Inc.

Isolation and characterization of Chinese standard fulvic acid sub-fractions separated from forest soil by stepwise elution with pyrophosphate buffer.

PubMed

Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P

2015-03-04

XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.

Isolation and Characterization of Chinese Standard Fulvic Acid Sub-fractions Separated from Forest Soil by Stepwise Elution with Pyrophosphate Buffer

PubMed Central

Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P.

2015-01-01

XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants. PMID:25735451

Clinical outcomes in metabolic syndrome.

PubMed

Bhatheja, Rohit; Bhatt, Deepak L

2006-01-01

Metabolic syndrome is a clustering of cardiovascular risk factors. Its definition is the presence of any 3 of the following: obesity, hypertriglyceridemia, low high-density lipoprotein, hypertension, and impaired fasting glucose. The development of coronary artery disease is the most dreaded complication of this disease. In the United States, Mexican Americans and African American women are the most affected. Management of this syndrome includes physical exercise, weight loss, and effective drug treatment of dyslipidemia, high blood pressure, and impaired fasting blood glucose. Because of the increasing prevalence of obesity and diabetes, there is a rise in fatal and nonfatal cardiovascular events. With the development of effective antiplatelet medication and newer drug-eluting stents, percutaneous coronary intervention has become an effective revascularization strategy for those with coronary artery disease. Rates of stent restenosis and target-lesion revascularization have been reduced. Oral hypoglycemic drugs like thiazolidinediones improve insulin resistance and may have a favorable effect in those with metabolic syndrome. Diagnosis and appropriate management of metabolic syndrome are challenges as the presence of risk factors predates the coronary event.

Identification, isolation, and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Nicotiana alata.

PubMed

Jahnen, W; Batterham, M P; Clarke, A E; Moritz, R L; Simpson, R J

1989-05-01

S-Gene-associated glycoproteins (S-glycoproteins) from styles of Nicotiana alata, identified by non-equilibrium two-dimensional electrophoresis, were purified by cation exchange fast protein liquid chromatography with yields of 0.5 to 8 micrograms of protein per style, depending on the S-genotype of the plant. The method relies on the highly basic nature of the S-glycoproteins. The elution profiles of the different S-glycoproteins from the fast protein liquid chromatography column were characteristic of each S-glycoprotein, and could be used to establish the S-genotype of plants in outbreeding populations. In all cases, the S-genotype predicted from the style protein profile corresponded to that predicted from DNA gel blot analysis using S-allele-specific DNA probes and to that established by conventional breeding tests. Amino-terminal sequences of five purified S-glycoproteins showed a high degree of homology with the previously published sequences of N. alata and Lycopersicon esculentum S-glycoproteins.

Monitoring/Verification using DMS: TATP Example

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephan Weeks; Kevin Kyle

Field-rugged and field-programmable differential mobility spectrometry (DMS) networks provide highly selective, universal monitoring of vapors and aerosols at detectable levels from persons or areas involved with illicit chemical/biological/explosives (CBE) production. CBE sensor motes used in conjunction with automated fast gas chromatography with DMS detection (GC/DMS) verification instrumentation integrated into situational operations management systems can be readily deployed and optimized for changing application scenarios. The feasibility of developing selective DMS motes for a 'smart dust' sampling approach with guided, highly selective, fast GC/DMS verification analysis is a compelling approach to minimize or prevent the use of explosives or chemical and biologicalmore » weapons in terrorist activities. Two peroxide-based liquid explosives, triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD), are synthesized from common chemicals such as hydrogen peroxide, acetone, sulfuric acid, ammonia, and citric acid (Figure 1). Recipes can be readily found on the Internet by anyone seeking to generate sufficient quantities of these highly explosive chemicals to cause considerable collateral damage. Detection of TATP and HMTD by advanced sensing systems can provide the early warning necessary to prevent terror plots from coming to fruition. DMS is currently one of the foremost emerging technologies for the separation and detection of gas-phase chemical species. This is due to trace-level detection limits, high selectivity, and small size. DMS separates and identifies ions at ambient pressures by utilizing the non-linear dependence of an ion's mobility on the radio frequency (rf) electric field strength. GC is widely considered to be one of the leading analytical methods for the separation of chemical species in complex mixtures. Advances in the technique have led to the development of low-thermal-mass fast GC columns. These columns are capable of completing runs in less than 3 minutes. Fast GC columns are also more compact than their traditional counterparts. An earlier collaborative effort involving these authors optimized a handheld, fast GC/DMS, equipped with a non-rad ionization source, for the detection of TATP (Figure 2). The unit combines the separation capabilities of GC with the selectivity of DMS. Analytes are identified both by their elution time from the column and by the characteristic response in the DMS spectrum. Analysis times required to obtain results for these analytes are approximately 80 seconds for TATP and 160 seconds for HMTD (Figure 3). The limit of detection for both TATP and HMTD is approximately 1 ng/{micro}L. Substances that could interfere with the detection of peroxide-based explosives have been studied. Both the GC elution time and the DMS spectral peak locations were unique and do not hinder the detection of either TATP or HMTD.« less

ELUTION OF URANIUM VALUES FROM ION EXCHANGE RESINS

DOEpatents

Kennedy, R.H.

1959-11-24

A process is described for eluting complex uranium ions absorbed on ion exchange resins. The resin is subjected to the action of an aqueous eluting solution contuining sulfuric acid and an alkali metal, ammonium, or magnesium chloride or nitrate, the elution being carried out until the desired amount of the uranium is removed from the resin.

A reason for intermittent fasting to suppress the awakening of dormant breast tumors.

PubMed

Lankelma, Jan; Kooi, Bob; Krab, Klaas; Dorsman, Josephine C; Joenje, Hans; Westerhoff, Hans V

2015-01-01

For their growth, dormant tumors, which lack angiogenesis may critically depend on gradients of nutrients and oxygen from the nearest blood vessel. Because for oxygen depletion the distance from the nearest blood vessel to depletion will generally be shorter than for glucose depletion, such tumors will contain anoxic living tumor cells. These cells are dangerous, because they are capable of inducing angiogenesis, which will "wake up" the tumor. Anoxic cells are dependent on anaerobic glucose breakdown for ATP generation. The local extracellular glucose concentration gradient is determined by the blood glucose concentration and by consumption by cells closer to the nearest blood vessel. The blood glucose concentration can be lowered by 20-40% during fasting. We calculated that glucose supply to the potentially hazardous anoxic cells can thereby be reduced significantly, resulting in cell death specifically of the anoxic tumor cells. We hypothesize that intermittent fasting will help to reduce the incidence of tumor relapse via reducing the number of anoxic tumor cells and tumor awakening. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Prosthetic component segmentation with blur compensation: a fast method for 3D fluoroscopy.

PubMed

Tarroni, Giacomo; Tersi, Luca; Corsi, Cristiana; Stagni, Rita

2012-06-01

A new method for prosthetic component segmentation from fluoroscopic images is presented. The hybrid approach we propose combines diffusion filtering, region growing and level-set techniques without exploiting any a priori knowledge of the analyzed geometry. The method was evaluated on a synthetic dataset including 270 images of knee and hip prosthesis merged to real fluoroscopic data simulating different conditions of blurring and illumination gradient. The performance of the method was assessed by comparing estimated contours to references using different metrics. Results showed that the segmentation procedure is fast, accurate, independent on the operator as well as on the specific geometrical characteristics of the prosthetic component, and able to compensate for amount of blurring and illumination gradient. Importantly, the method allows a strong reduction of required user interaction time when compared to traditional segmentation techniques. Its effectiveness and robustness in different image conditions, together with simplicity and fast implementation, make this prosthetic component segmentation procedure promising and suitable for multiple clinical applications including assessment of in vivo joint kinematics in a variety of cases.

[Content comparison of diterpenoid pigments in different processed products of Gardenia jasminoides].

PubMed

Zhang, Cun; Xiao, Yong-Qing; Li, Li; Li, Gui-Liu; Pang, Zhen

2008-11-01

To compare the contents of diterpenoid pigments in the different processed products of Gardenia jasminoides. The separation of Crocin 1, Crocin 2, Crocin 3, Crocetin were determined simultaneously by HPLC on a kromasil C18 column at 35 degrees C with the m methanol-acetonitrile-0.3% formic acid anhydrous in gradient elution as the mobile phrase. The detection wavelength was set at 440 nm and the flow rate was 1.0 mL x min(-1). The obtained linearity of the four components was better over 0.9995 and the average recoveries were 97.77%, 100.05% , 98.40%, 101.02%, respectively. The method is simple, accurate with good reproducibility. The results showed that the remarkable variation regulations appear among the different processed products.

[Contents of diterpenoid pigments in didifferent parts of fruit from Gardenia jasminoides].

PubMed

Zhang, Cun; Xiao, Yongqing; Li, Li; Li, Wen; Yin, Xiaojie

2009-06-01

To compare the contents of diterpenoid pigments among the fruit, seed, and pericarp from Gardenia jasminoides. The separation of Crocin 1, Crocin 2, Crocin 3 were carried out simultaneously on a Kromasil C18 column at 35 degrees C with the methanol-acetonitrile-0.3% formic acid anhydrous in gradient elution as the mobile phrases. The detection wavelength was set at 440 nm and the flow rate was 1.0 mL x min(-1). The obtained linearity of the three components was better over 0.999 5 and the average recoveries were 97.50%, 98.76%, 97.67%, respectively. The results exhibited the remarkable variation among the different parts of the fruit. The contents of three constituents in seed were higher than in pericarp.

[Study on the fingerprint of Morus alba from different habitats by HPLC].

PubMed

Chen, Cheng; Li, Hong-Bo; Wang, Liu-Ping; Li, Yun-Rong; Xin, Ning

2012-12-01

To establish HPLC fingerprint of Morus alba from different habitats by HPLC and provide basis for its quality control. HPLC analysis was performed on an Agilent XDB C18 Column (4.6 mm x 250 mm, 5 microm), gradient eluted composed of acetonitrile and 0.3% phosphate acid. The column temperature was set at 35 degrees C and the flow rate was 0.5 mL/min. The detective wavelength was 290 nm. The HPLC fingerprint for 10 batches of Morus alba was studied on their similarity. There were twelve common peaks in the fingerprint. The similarity of 7 batches was above 0.9 and the other batches had low similarity. The HPLC fingerprint can be used for quality control of Morus alba with high characteristics and specificity.

Determination of para red, Sudan dyes, canthaxanthin, and astaxanthin in animal feeds using UPLC.

PubMed

Hou, Xiaolin; Li, Yonggang; Wu, Guojuan; Wang, Lei; Hong, Miao; Wu, Yongnin

2010-01-01

A simple high-performance liquid chromatography method was developed for quantitative determination of para red, Sudan I, Sudan II, Sudan III, Sudan IV, canthaxanthin, and astaxanthin in feedstuff. The sample was extracted using acetonitrile and cleaned up on a C(18) SPE column. The residues were analyzed using ultra-performance liquid chromatography coupled to a diode array detector at 500 nm. The mobile phase was acetonitrile-formic acid-water with a gradient elution condition. The external standard curves were calibrated. The mean recoveries of the seven colorants were 62.7-91.0% with relative standard deviation 2.6-10.4% (intra-day) and 4.0-13.2% (inter-day). The detection limits were in the range of 0.006-0.02 mg/kg.

[Simultaneous determination of five active constitutents in Xiaochaihu Tang by HPLC].

PubMed

Liu, Qingchun; Zhao, Junning; Yan, Liangchun; Yi, Jinhai; Song, Jun

2010-03-01

To establish a HPLC-PDA method for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang. A Symmetry Shield RP18 (4.6 mm x 250 mm, 5.0 microm) was used with a mobile phase of acetonitrile-0.01% H3PO4 in gradient elution. The detection wavelength was 251 nm,the flow rate was 0.45 mL x min(-1) and the column temperature was maintained at 30 degrees C. The accuracy, precision, sensitivity, specificity and linearity of this method met the requirements. The contents of the five effective fractions were determined simultaneously. The method is rapid,simple and accurate and it can be suitable for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang simultaneously.

[UPLC characteristic chromatographic profile of Poria].

PubMed

Zhang, Qi; Wang, Zhenzhong; Xiao, Wei; Zhang, Liangqi; Bi, Kaishun; Jia, Ying

2012-04-01

To establish a UPLC characteristic chromatographic profile analysis method to quickly assess Poria quality and provide basis fro controlling Poria quality. The UPLC characteristic chromatographic profiles of fifteen batches of Poria were determined by ACQUITY UPLC, with HSS T3 Column (2.1 mm x 100 mm, 1.8 microm) eluted with the mobile phases of water containing 0.05% phosphoric acid and acetonitrile in gradient mode. The detection wavelength was set at 243 nm. The common mode of the UPLC characteristic chromatographic profile was set up. There were 20 common peaks, seven of which were identified, and the similar degrees of the fifteen samples to the common mode were between 0.787 and 0.974. The method was so time-saving that it can be used for the quality control of Poria.

The Purification and Concentration of Hog Cholera Virus*

PubMed Central

Cunliffe, H. R.; Rebers, P. A.

1968-01-01

Partial purification of hog cholera virus (HCV) using a simple batch-type chromatographic procedure with magnetic ferric oxide (MFO) is described. Infectious HCV was adsorbed from isotonic solutions to MFO and was eluted under conditions of low ionic strength and high pH. Aqueous solutions of 0.01 M sodium cyanide or 0.0003 M ammonium hydroxide effectively dissociated MFO-HCV complexes. The data indicate that 50 to 100% of the original HCV infectivity was recovered concomitant with a 90 to 95% reduction of extraneous organic nitrogen. MFO-purified HCV was concentrated by density gradient type centrifugations in buffered solutions of cesium chloride and sucrose. Prolonged isodensity centrifugations of concentrated MFO-purified HCV indicated a buoyant density of 1.14 to 1.15 gm/ml for the strain of virus used. PMID:15846899

Speciation of inorganic selenium and selenoamino acids by an HPLC-UV-HG-AFS system.

PubMed

Ipolyi, I; Corns, W; Stockwell, P; Fodor, P

2001-01-01

For the on-line speciation of selenocystine (SeCys), selenomethionine (SeMet), selenoethionine (SeEt), selenite (Se(IV)) and selenate (Se(VI)), a high-performance liquid chromatography-UV irradiation-hydride generation-atomic fluorescence spectro- metric method is described. Separation was carried out on a conventional reversed-phase C18 column modified with didodecyl- dimethylammonium bromide with gradient elution applying two concentrations of ammonium acetate as the mobile phase. UV irradiation and hydride generation parameters were optimized. The obtained detection limits for SeCys, SeMet, SeEt, Se(IV) and Se(VI) were 0.31, 0.43, 0.7, 0.44 and 0.32 ng ml(-1), respectively, using a 100-microl loop. The method was tested with spiked mineral water and two volunteers' urine samples.

Speciation of inorganic selenium and selenoamino acids by an HPLC-UV-HG-AFS system

PubMed Central

Ipolyi, I.; Corns, W.; Stockwell, P.; Fodor, P.

2001-01-01

For the on-line speciation of selenocystine (SeCys), selenomethionine (SeMet), selenoethionine (SeEt), selenite (Se(IV)) and selenate (Se(VI)), a high-performance liquid chromatography-UV irradiation-hydride generation-atomic fluorescence spectro- metric method is described. Separation was carried out on a conventional reversed-phase C18 column modified with didodecyl- dimethylammonium bromide with gradient elution applying two concentrations of ammonium acetate as the mobile phase. UV irradiation and hydride generation parameters were optimized. The obtained detection limits for SeCys, SeMet, SeEt, Se(IV) and Se(VI) were 0.31, 0.43, 0.7, 0.44 and 0.32 ng ml−1, respectively, using a 100-wl loop. The method was tested with spiked mineral water and two volunteers' urine samples. PMID:18924707

[Determination of buprofezin, methamidophos, acephate, and triazophos residues in Chinese tea samples by gas chromatography].

PubMed

Zhang, Shuiba; Yi, Jun; Ye, Jianglei; Zheng, Wenhui; Cai, Xueqin; Gong, Zhenbin

2004-03-01

A method has been developed for the simultaneous determination of buprofezin, methamidophos, acephate and triazophos residues in Chinese tea samples. The pesticide residues were extracted from tea samples with a mixture of ethyl acetate and n-hexane (50:50, v/v) at 45 degrees C. The extracts were subsequently treated with a column packed with 40 mg of active carbon by gradient elution with ethyl acetate and n-hexane. Buprofenzin and the three organophosphorus pesticides were analyzed by gas chromatography using a DB-210 capillary column and a nitrogen-phosphorus detector. The recoveries for spiked standards were 73.4%-96.9%. The relative standard deviations were all within 4.63%. The limits of quantitation (3sigma) in the tea samples were about 7.0-12.0 microg/kg.

Development and validation of stability indicating the RP-HPLC method for the estimation of related compounds of guaifenesin in pharmaceutical dosage forms.

PubMed

Reddy, Sunil Pingili; Babu, K Sudhakar; Kumar, Navneet; Sekhar, Y V V Sasi

2011-10-01

A stability-indicating gradient reverse phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of related substances of guaifenesin in pharmaceutical formulations. The baseline separation for guaifenesin and all impurities was achieved by utilizing a Water Symmetry C18 (150 mm × 4.6 mm) 5 μm column particle size and a gradient elution method. The mobile phase A contains a mixture of 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 90:10 v/v, while the mobile phase B contains 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 10:90 v/v, respectively. The flow rate of the mobile phase was 0.8 ml/min with a column temperature of 25°C and detection wavelength at 273 nm. Guaifenesin was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.

Development and validation of stability indicating the RP-HPLC method for the estimation of related compounds of guaifenesin in pharmaceutical dosage forms

PubMed Central

Reddy, Sunil Pingili; Babu, K. Sudhakar; Kumar, Navneet; Sekhar, Y. V. V. Sasi

2011-01-01

Aim and background: A stability-indicating gradient reverse phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of related substances of guaifenesin in pharmaceutical formulations. Materials and methods: The baseline separation for guaifenesin and all impurities was achieved by utilizing a Water Symmetry C18 (150 mm × 4.6 mm) 5 μm column particle size and a gradient elution method. The mobile phase A contains a mixture of 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 90:10 v/v, while the mobile phase B contains 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 10:90 v/v, respectively. The flow rate of the mobile phase was 0.8 ml/min with a column temperature of 25°C and detection wavelength at 273 nm. Results: Guaifenesin was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Conclusion: The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness. PMID:23781462

Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate.

PubMed

Chitturi, Sreenivasa Rao; Somannavar, Yallappa Somappa; Peruri, Badarinadh Gupta; Nallapati, Sreenivas; Sharma, Hemant Kumar; Budidet, Shankar Reddy; Handa, Vijay Kumar; Vurimindi, Hima Bindu

2011-04-28

This paper proposes a simple and selective RP-HPLC method for the determination of process impurities and degradation products (degradants) of atazanavir sulfate (ATV) drug substance. Chromatographic separation was achieved on Ascentis(®) Express C8, (150mm×4.6mm, 2.7μm) column thermostated at 30°C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02M) and ACN at a flow rate of 1.0ml/min. A photodiode array (PDA) detector set at 250nm was used for detection. Stress testing (forced degradation) of ATV was carried out under acidic, alkaline, oxidative, photolytic, thermal and humidity conditions. In presence of alkali, ATV transformed into cyclized products and the order of degradation reaction is determined by the method of initial rates. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (lod and loq), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV. Copyright © 2011 Elsevier B.V. All rights reserved.

Metallic Limus-Eluting Stents Abluminally Coated with Biodegradable Polymers: Angiographic and Clinical Comparison of a Novel Ultra-Thin Sirolimus Stent Versus Biolimus Stent in the DESTINY Randomized Trial.

PubMed

Lemos, Pedro A; Abizaid, Alexandre A C; Meireles, George C; Sarmento-Leite, Rogério; Prudente, Mauricio; Cantarelli, Marcelo; Dourado, Adriano D; Mariani, Jose; Perin, Marco A; Costantini, Costantino; Costa, Ricardo A; Costa, José Ribamar; Chamie, Daniel; Campos, Carlos A; Ribeiro, Expedito

2015-12-01

To evaluate the outcomes of patients treated with a new drug-eluting stent formulation with low doses of sirolimus, built in an ultra-thin-strut platform coated with biodegradable abluminal coating. This study is a randomized trial that tested the main hypothesis that the angiographic late lumen loss of the novel sirolimus-eluting stent is noninferior compared with commercially available biolimus-eluting stent. A final study population comprising 170 patients with one or two de novo lesions was randomized in the ratio 2:1 for sirolimus-eluting stent or biolimus-eluting stent, respectively. The primary endpoint was 9-month angiographic in-stent late lumen loss. Adverse clinical events were prospectively collected for 1 year. After 9 months, the novel sirolimus-eluting stent was shown noninferior compared with the biolimus stent for the primary endpoint (angiographic in-stent late lumen loss: 0.20 ± 0.29 mm vs. 0.15 ± 0.20 mm, respectively; P value for noninferiority <0.001). The 1-year incidence of death, myocardial infarction, repeat revascularization, and stent thrombosis remained low and not significantly different between the groups. The present randomized trial demonstrates that the tested novel sirolimus-eluting stent was angiographically noninferior in comparison with a last-generation biolimus-eluting stent. © 2015 John Wiley & Sons Ltd.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

PubMed

Jakobsen, Simon S; Christensen, Jan H; Verdier, Sylvain; Mallet, Claude R; Nielsen, Nikoline J

2017-09-05

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, k w , above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

An optimized method for elution of enteroviral RNA from a cellulose-based substrate.

PubMed

Li, Yan; Yoshida, Hiromu; Wang, Lu; Tao, Zexin; Wang, Haiyan; Lin, Xiaojuan; Xu, Aiqiang

2012-12-01

The Flinders Technology Australia (FTA) Elute Card is a commercial product that facilitates the collection, transport, archiving and processing of nucleic acids from a wide variety of biological samples at room temperature. While the cards have been designed so that sterile/deionized water can elute DNA easily, they are not suitable for some less stable RNAs. This study was undertaken to determine the optimal conditions such as the buffer type, buffer pH and incubation temperature for the elution of enteroviral RNA from FTA Elute Cards prior to quantitative analysis using real-time PCR (qPCR) or consensus degenerate hybrid oligonucleotide primer VP1 RT-semi nested PCR (CODEHOP VP1 RT-snPCR). TE-1 (pH 8.0), rather than sterile water, was the best buffer for high efficiency elution of enteroviral RNA at 95°C. However, as the estimated recovery rate of viral RNA eluted from the cards averaged to be only 6.1%, enterovirus assays using FTA elution should be considered qualitative, especially at low virus titers, and therefore the results of the assay should be interpreted carefully. Copyright © 2012 Elsevier B.V. All rights reserved.

Exploring doxorubicin localization in eluting TiO2 nanotube arrays through fluorescence correlation spectroscopy analysis.

PubMed

De Santo, Ilaria; Sanguigno, Luigi; Causa, Filippo; Monetta, Tullio; Netti, Paolo A

2012-11-07

Drug elution properties of TiO(2) nanotube arrays have been largely investigated by means of solely macroscopic observations. Controversial elution performances have been reported so far and a clear comprehension of these phenomena is still missing as a consequence of a lack of molecular investigation methods. Here we propose a way to discern drug elution properties of nanotubes through the evaluation of drug localization by Fluorescence Correlation Spectroscopy (FCS) analysis. We verified this method upon doxorubicin elution from differently loaded TiO(2) nanotubes. Diverse elution profiles were obtained from nanotubes filled by soaking and wet vacuum impregnation methods. Impregnated nanotubes controlled drug diffusion up to thirty days, while soaked samples completed elution in seven days. FCS analysis of doxorubicin motion in loaded nanotubes clarified that more than 90% of drugs dwell preferentially in inter-nanotube spaces in soaked samples due to decorrelation in a 2D fashion, while a 97% fraction of molecules showed 1D mobility ascribable to displacements along the nanotube vertical axis of wet vacuum impregnated nanotubes. The diverse drug localizations inferred from FCS measurements, together with distinct drug-surface interaction strengths resulting from diverse drug filling techniques, could explain the variability in elution kinetics.

Multiscale interaction between a large scale magnetic island and small scale turbulence

NASA Astrophysics Data System (ADS)

Choi, M. J.; Kim, J.; Kwon, J.-M.; Park, H. K.; In, Y.; Lee, W.; Lee, K. D.; Yun, G. S.; Lee, J.; Kim, M.; Ko, W.-H.; Lee, J. H.; Park, Y. S.; Na, Y.-S.; Luhmann, N. C., Jr.; Park, B. H.

2017-12-01

Multiscale interaction between the magnetic island and turbulence has been demonstrated through simultaneous two-dimensional measurements of turbulence and temperature and flow profiles. The magnetic island and turbulence can mutually interact via coupling between the electron temperature (T e ) gradient, the T e turbulence, and the poloidal flow. The T e gradient altered by the magnetic island steepens outside and flattens inside the island. The T e turbulence can appear in increased T e gradient regions. The combined effects of the T e gradient and the poloidal flow shear determines the two-dimensional distribution of the T e turbulence. When the poloidal vortex flow forms, it can maintain the steepest T e gradient and the magnetic island acts more like an electron heat transport barrier. Interestingly, when the T e gradient, the T e turbulence, and the vortex flow shear increase beyond critical levels, the magnetic island turns into a fast electron heat transport channel, which directly leads to the minor disruption.

Application of Snyder-Dolan Classification Scheme to the Selection of “Orthogonal” Columns for Fast Screening for Illicit Drugs and Impurity Profiling of Pharmaceuticals - I. Isocratic Elution

PubMed Central

Fan, Wenzhe; Zhang, Yu; Carr, Peter W.; Rutan, Sarah C.; Dumarey, Melanie; Schellinger, Adam P.; Pritts, Wayne

2011-01-01

Fourteen judiciously selected reversed-phase columns were tested with 18 cationic drug solutes under the isocratic elution conditions advised in the Snyder-Dolan (S-D) hydrophobic subtraction method of column classification. The standard errors (S.E.) of the least squares regressions of log k′ vs. log k′REF were obtained for a given column against a reference column and used to compare and classify columns based on their selectivity. The results are consistent with those obtained with a study of the 16 test solutes recommended by Snyder and Dolan. To the extent that these drugs are representative these results show that the S-D classification scheme is also generally applicable to pharmaceuticals under isocratic conditions. That is, those columns judged to be similar based on the S-D 16 solutes were similar based on the 18 drugs; furthermore those columns judged to have significantly different selectivities based on the 16 S-D probes appeared to be quite different for the drugs as well. Given that the S-D method has been used to classify more than 400 different types of reversed phases the extension to cationic drugs is a significant finding. PMID:19698948

Phase-unwrapping algorithm by a rounding-least-squares approach

NASA Astrophysics Data System (ADS)

Juarez-Salazar, Rigoberto; Robledo-Sanchez, Carlos; Guerrero-Sanchez, Fermin

2014-02-01

A simple and efficient phase-unwrapping algorithm based on a rounding procedure and a global least-squares minimization is proposed. Instead of processing the gradient of the wrapped phase, this algorithm operates over the gradient of the phase jumps by a robust and noniterative scheme. Thus, the residue-spreading and over-smoothing effects are reduced. The algorithm's performance is compared with four well-known phase-unwrapping methods: minimum cost network flow (MCNF), fast Fourier transform (FFT), quality-guided, and branch-cut. A computer simulation and experimental results show that the proposed algorithm reaches a high-accuracy level than the MCNF method by a low-computing time similar to the FFT phase-unwrapping method. Moreover, since the proposed algorithm is simple, fast, and user-free, it could be used in metrological interferometric and fringe-projection automatic real-time applications.

Segmentation of hand radiographs using fast marching methods

NASA Astrophysics Data System (ADS)

Chen, Hong; Novak, Carol L.

2006-03-01

Rheumatoid Arthritis is one of the most common chronic diseases. Joint space width in hand radiographs is evaluated to assess joint damage in order to monitor progression of disease and response to treatment. Manual measurement of joint space width is time-consuming and highly prone to inter- and intra-observer variation. We propose a method for automatic extraction of finger bone boundaries using fast marching methods for quantitative evaluation of joint space width. The proposed algorithm includes two stages: location of hand joints followed by extraction of bone boundaries. By setting the propagation speed of the wave front as a function of image intensity values, the fast marching algorithm extracts the skeleton of the hands, in which each branch corresponds to a finger. The finger joint locations are then determined by using the image gradients along the skeletal branches. In order to extract bone boundaries at joints, the gradient magnitudes are utilized for setting the propagation speed, and the gradient phases are used for discriminating the boundaries of adjacent bones. The bone boundaries are detected by searching for the fastest paths from one side of each joint to the other side. Finally, joint space width is computed based on the extracted upper and lower bone boundaries. The algorithm was evaluated on a test set of 8 two-hand radiographs, including images from healthy patients and from patients suffering from arthritis, gout and psoriasis. Using our method, 97% of 208 joints were accurately located and 89% of 416 bone boundaries were correctly extracted.

A nano ultra-performance liquid chromatography-high resolution mass spectrometry approach for global metabolomic profiling and case study on drug-resistant multiple myeloma.

PubMed

Jones, Drew R; Wu, Zhiping; Chauhan, Dharminder; Anderson, Kenneth C; Peng, Junmin

2014-04-01

Global metabolomics relies on highly reproducible and sensitive detection of a wide range of metabolites in biological samples. Here we report the optimization of metabolome analysis by nanoflow ultraperformance liquid chromatography coupled to high-resolution orbitrap mass spectrometry. Reliable peak features were extracted from the LC-MS runs based on mandatory detection in duplicates and additional noise filtering according to blank injections. The run-to-run variation in peak area showed a median of 14%, and the false discovery rate during a mock comparison was evaluated. To maximize the number of peak features identified, we systematically characterized the effect of sample loading amount, gradient length, and MS resolution. The number of features initially rose and later reached a plateau as a function of sample amount, fitting a hyperbolic curve. Longer gradients improved unique feature detection in part by time-resolving isobaric species. Increasing the MS resolution up to 120000 also aided in the differentiation of near isobaric metabolites, but higher MS resolution reduced the data acquisition rate and conferred no benefits, as predicted from a theoretical simulation of possible metabolites. Moreover, a biphasic LC gradient allowed even distribution of peak features across the elution, yielding markedly more peak features than the linear gradient. Using this robust nUPLC-HRMS platform, we were able to consistently analyze ~6500 metabolite features in a single 60 min gradient from 2 mg of yeast, equivalent to ~50 million cells. We applied this optimized method in a case study of drug (bortezomib) resistant and drug-sensitive multiple myeloma cells. Overall, 18% of metabolite features were matched to KEGG identifiers, enabling pathway enrichment analysis. Principal component analysis and heat map data correctly clustered isogenic phenotypes, highlighting the potential for hundreds of small molecule biomarkers of cancer drug resistance.

Echo-Planar Imaging for a 9.4 Tesla Vertical-Bore Superconducting Magnet Using an Unshielded Gradient Coil.

PubMed

Kodama, Nao; Kose, Katsumi

2016-10-11

Echo-planar imaging (EPI) sequences were developed for a 9.4 Tesla vertical standard bore (~54 mm) superconducting magnet using an unshielded gradient coil optimized for live mice imaging and a data correction technique with reference scans. Because EPI requires fast switching of intense magnetic field gradients, eddy currents were induced in the surrounding metallic materials, e.g., the room temperature bore, and this produced serious artifacts on the EPI images. We solved the problem using an unshielded gradient coil set of proper size (outer diameter = 39 mm, inner diameter = 32 mm) with time control of the current rise and reference scans. The obtained EPI images of a phantom and a plant sample were almost artifact-free and demonstrated the promise of our approach.

A novel technique for real-time estimation of edge pedestal density gradients via reflectometer time delay data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, L., E-mail: zeng@fusion.gat.com; Doyle, E. J.; Rhodes, T. L.

2016-11-15

A new model-based technique for fast estimation of the pedestal electron density gradient has been developed. The technique uses ordinary mode polarization profile reflectometer time delay data and does not require direct profile inversion. Because of its simple data processing, the technique can be readily implemented via a Field-Programmable Gate Array, so as to provide a real-time density gradient estimate, suitable for use in plasma control systems such as envisioned for ITER, and possibly for DIII-D and Experimental Advanced Superconducting Tokamak. The method is based on a simple edge plasma model with a linear pedestal density gradient and low scrape-off-layermore » density. By measuring reflectometer time delays for three adjacent frequencies, the pedestal density gradient can be estimated analytically via the new approach. Using existing DIII-D profile reflectometer data, the estimated density gradients obtained from the new technique are found to be in good agreement with the actual density gradients for a number of dynamic DIII-D plasma conditions.« less

Combined use of heat and saline tracer to estimate aquifer properties in a forced gradient test

NASA Astrophysics Data System (ADS)

Colombani, N.; Giambastiani, B. M. S.; Mastrocicco, M.

2015-06-01

Usually electrolytic tracers are employed for subsurface characterization, but the interpretation of tracer test data collected by low cost techniques, such as electrical conductivity logging, can be biased by cation exchange reactions. To characterize the aquifer transport properties a saline and heat forced gradient test was employed. The field site, located near Ferrara (Northern Italy), is a well characterized site, which covers an area of 200 m2 and is equipped with a grid of 13 monitoring wells. A two-well (injection and pumping) system was employed to perform the forced gradient test and a straddle packer was installed in the injection well to avoid in-well artificial mixing. The contemporary continuous monitor of hydraulic head, electrical conductivity and temperature within the wells permitted to obtain a robust dataset, which was then used to accurately simulate injection conditions, to calibrate a 3D transient flow and transport model and to obtain aquifer properties at small scale. The transient groundwater flow and solute-heat transport model was built using SEAWAT. The result significance was further investigated by comparing the results with already published column experiments and a natural gradient tracer test performed in the same field. The test procedure shown here can provide a fast and low cost technique to characterize coarse grain aquifer properties, although some limitations can be highlighted, such as the small value of the dispersion coefficient compared to values obtained by natural gradient tracer test, or the fast depletion of heat signal due to high thermal diffusivity.

Comparison of methods for the determination of NO-O3-NO2 fluxes and chemical interactions over a bare soil

NASA Astrophysics Data System (ADS)

Stella, P.; Loubet, B.; Laville, P.; Lamaud, E.; Cazaunau, M.; Laufs, S.; Bernard, F.; Grosselin, B.; Mascher, N.; Kurtenbach, R.; Mellouki, A.; Kleffmann, J.; Cellier, P.

2011-08-01

Tropospheric ozone (O3) is a known greenhouse gas responsible for impacts on human and animal health and ecosystem functioning. In addition, O3 plays an important role in tropospheric chemistry, together with nitrogen oxides. Flux measurements of these trace gases are a major issue to establish their atmospheric budget and evaluate the ozone impact onto the biosphere. In this study, ozone, nitric oxide (NO) and nitrogen dioxide (NO2) fluxes were measured using the aerodynamic gradient method over a bare soil in an agricultural field. Vertical mixing ratio profile measurements were performed with fast response sensors. It was demonstrated that corrections of the aerodynamic gradient for chemical reactions between O3-NO-NO2 appeared to be negligible for O3 fluxes, whereas they accounted for about 10 % on average of the NO and NO2 fluxes. The flux uncertainties were mainly due to uncertainties of the friction velocity. In addition, the use of fast response sensors allowed to reduce the remaining part of the flux uncertainty. The aerodynamic gradient and eddy-covariance methods gave similar O3 fluxes (within 4 %). The chamber NO fluxes were up to 70 % lower than the aerodynamic gradient fluxes probably caused by either the spatial heterogeneity of the soil NO emissions or the environmental perturbation due to the chamber.

Real-time distortion correction of spiral and echo planar images using the gradient system impulse response function.

PubMed

Campbell-Washburn, Adrienne E; Xue, Hui; Lederman, Robert J; Faranesh, Anthony Z; Hansen, Michael S

2016-06-01

MRI-guided interventions demand high frame rate imaging, making fast imaging techniques such as spiral imaging and echo planar imaging (EPI) appealing. In this study, we implemented a real-time distortion correction framework to enable the use of these fast acquisitions for interventional MRI. Distortions caused by gradient waveform inaccuracies were corrected using the gradient impulse response function (GIRF), which was measured by standard equipment and saved as a calibration file on the host computer. This file was used at runtime to calculate the predicted k-space trajectories for image reconstruction. Additionally, the off-resonance reconstruction frequency was modified in real time to interactively deblur spiral images. Real-time distortion correction for arbitrary image orientations was achieved in phantoms and healthy human volunteers. The GIRF-predicted k-space trajectories matched measured k-space trajectories closely for spiral imaging. Spiral and EPI image distortion was visibly improved using the GIRF-predicted trajectories. The GIRF calibration file showed no systematic drift in 4 months and was demonstrated to correct distortions after 30 min of continuous scanning despite gradient heating. Interactive off-resonance reconstruction was used to sharpen anatomical boundaries during continuous imaging. This real-time distortion correction framework will enable the use of these high frame rate imaging methods for MRI-guided interventions. Magn Reson Med 75:2278-2285, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Real-time distortion correction of spiral and echo planar images using the gradient system impulse response function

PubMed Central

Campbell-Washburn, Adrienne E; Xue, Hui; Lederman, Robert J; Faranesh, Anthony Z; Hansen, Michael S

2015-01-01

Purpose MRI-guided interventions demand high frame-rate imaging, making fast imaging techniques such as spiral imaging and echo planar imaging (EPI) appealing. In this study, we implemented a real-time distortion correction framework to enable the use of these fast acquisitions for interventional MRI. Methods Distortions caused by gradient waveform inaccuracies were corrected using the gradient impulse response function (GIRF), which was measured by standard equipment and saved as a calibration file on the host computer. This file was used at runtime to calculate the predicted k-space trajectories for image reconstruction. Additionally, the off-resonance reconstruction frequency was modified in real-time to interactively de-blur spiral images. Results Real-time distortion correction for arbitrary image orientations was achieved in phantoms and healthy human volunteers. The GIRF predicted k-space trajectories matched measured k-space trajectories closely for spiral imaging. Spiral and EPI image distortion was visibly improved using the GIRF predicted trajectories. The GIRF calibration file showed no systematic drift in 4 months and was demonstrated to correct distortions after 30 minutes of continuous scanning despite gradient heating. Interactive off-resonance reconstruction was used to sharpen anatomical boundaries during continuous imaging. Conclusions This real-time distortion correction framework will enable the use of these high frame-rate imaging methods for MRI-guided interventions. PMID:26114951

Stent Thrombosis With Drug-Eluting Stents and Bioresorbable Scaffolds: Evidence From a Network Meta-Analysis of 147 Trials.

PubMed

Kang, Si-Hyuck; Chae, In-Ho; Park, Jin-Joo; Lee, Hak Seung; Kang, Do-Yoon; Hwang, Seung-Sik; Youn, Tae-Jin; Kim, Hyo-Soo

2016-06-27

This study sought to perform a systematic review and network meta-analysis to compare the relative safety and efficacy of contemporary DES and BVS. To improve outcomes of patients undergoing percutaneous coronary revascularization, there have been advances in the design of drug-eluting stents (DES), including the development of drug-eluting bioresorbable vascular scaffolds (BVS). Prospective, randomized, controlled trials comparing bare-metal stents (BMS), paclitaxel-eluting stents (PES), sirolimus-eluting stents (SES), Endeavor zotarolimus-eluting stents (E-ZES), cobalt-chromium (CoCr) everolimus-eluting stents (EES), platinum-chromium (PtCr)-EES, biodegradable polymer (BP)-EES, Resolute zotarolimus-eluting stents (R-ZES), BP biolimus-eluting stents (BP-BES), hybrid sirolimus-eluting stents (H [Orsiro]-SES), polymer-free sirolimus- and probucol-eluting stents, or BVS were searched in online databases. The primary endpoint was definite or probable stent thrombosis at 1 year. A total of 147 trials including 126,526 patients were analyzed in this study. All contemporary DES were superior to BMS and PES in terms of definite or probable stent thrombosis at 1 year. CoCr-EES, PtCr-EES, and H-SES were associated with significantly lower risk than BVS. CoCr-EES and H-SES were superior to SES and BP-BES. The risk of myocardial infarction was significantly lower with H-SES than with BVS. There were no significant differences regarding all-cause or cardiac mortality. Contemporary devices including BVS showed comparably low risks of repeat revascularization. Contemporary DES, including biocompatible DP-DES, BP-DES, and polymer-free DES, showed a low risk of definite or probable stent thrombosis at 1 year. BVS had an increased risk of device thrombosis compared with CoCr-EES, PtCr-EES, and H-SES. Data from extended follow-up are warranted to confirm the long-term safety of contemporary coronary devices. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Differential expression and elution behavior of basic 7S globulin among cultivars under hot water treatment of soybean seeds.

PubMed

Fujiwara, Keigo; Cabanos, Cerrone; Toyota, Kenji; Kobayashi, Yasunori; Maruyama, Nobuyuki

2014-06-01

Basic 7S globulin (Bg7S), which accumulates in mature soybean (Glycine max) seeds, is an extracellular matrix protein. A large amount of Bg7S is synthesized de novo and is eluted from soybean seeds when immersed in 50-60°C water (hot water treatment, HWT). However, the Bg7S elution mechanism remains unclear. Under HWT, the seeds probably undergo heat stress and flooding stress. To obtain fundamental knowledge related to how Bg7S is eluted from hot-water-treated seeds, this study compared Bg7S elution among soybean cultivars having different flooding tolerance during pre-germination. The amounts of Bg7S eluted from seeds varied significantly among cultivars. Elution was suppressed by seed coats regarded as preventing the leakage of seed contents by rapid water imbibition. Furthermore, Bg7S expression levels differed among cultivars, although the difference did not result from any variation in Bg7S promoter sequences. However, the expression levels of Bg7S under HWT were not associated with the flooding tolerance level. Immunoelectron microscopy revealed that the Bg7S accumulated in the intercellular space of hot-water-treated seeds. Plasma membrane shrinkage was observed. The main proteins eluted from seeds under HWT were located in the extracellular space. This study clarified the mechanism of Bg7S elution from seeds under HWT. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Arsenic-containing fatty acids and hydrocarbons in marine oils - determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS.

PubMed

Sele, Veronika; Sloth, Jens J; Holmelid, Bjarte; Valdersnes, Stig; Skov, Kasper; Amlund, Heidi

2014-04-01

Arsenolipids are the major arsenic species present in marine oils. Several structures of arsenolipids have been elucidated the last 5 years, demonstrating the chemical complexity of this trace element in the marine environment. Several commercial fish oils and marine oils, ranging in total arsenic concentrations from 1.6 to 12.5 mg kg(-1) oil, were analyzed for arsenolipids using reversed-phase high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The arsenolipids were quantified using three different arsenic-containing calibration standards; dimethylarsinate (DMA), triphenylarsinoxide (Ph₃AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C₂₁H₄₃AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time-resolved response factors for the arsenolipids. Isotopes of germanium ((74)Ge) and indium ((115)In) were suited as internal standards for arsenic, and were used for verification of the arsenic signal response factors during the gradient elution. Dimethylarsinate was the most suitable calibration standard for the quantification of arsenolipids, with recoveries between 91% and 104% compared to total arsenic measurements in the same extracts. A range of marine oils was investigated, including oils of several fish species, cod liver and seal, as well as three commercial fish oils. The AsHCs - C₁₇H₃₈AsO, C₁₉H₄₂AsO and C₂₃H₃₈AsO - were identified as the major arsenolipids in the extracts of all oils by HPLC coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). Minor amounts of two arsenic-containing fatty acids (AsFAs) (C₂₃H₃₈AsO₃ and C₂₄H₃₈AsO₃) were also detected in the oils. The sum of the AsHCs and the AsFAs determined in the present study accounted for 17-42% of the total arsenic in the oils. Copyright © 2014 Elsevier B.V. All rights reserved.

Update on the everolimus-eluting coronary stent system: results and implications from the SPIRIT clinical trial program

PubMed Central

Kirchner, R Michael; Abbott, J Dawn

2009-01-01

Drug-eluting stents (DES) have had a major impact in interventional cardiology. Compared to bare metal stents, they significantly reduce restenosis and the need for target vessel revascularization. Four DES are available in the US, the first-generation sirolimus-eluting (Cypher®) and paclitaxel-eluting (Taxus®) stents and later approved second-generation everolimus-eluting (Xience V®) and zotarolimus-eluting (Endeavor®) stents. The Xience V stent was approved on the basis of clinical efficacy and safety data from 3 studies in the SPIRIT clinical trial program. Within this trial series, the Xience V was superior to its bare metal stent counterpart, the Vision® stent, and noninferior to the paclitaxel-eluting stent for target vessel failure at 9 months. This review provides a comprehensive assessment of the data derived from both the pre- and post-approval randomized controlled trials and registry studies of Xience V that comprise the SPIRIT clinical trial program including recently published mid-term outcomes. The implications of the results in terms of interventional practice will be discussed. PMID:20057901

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

PubMed

Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

2013-04-12

An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling zone and mobile phase were identified as the main parameters affecting TCZR performance. In contrast to conventional systems, which rely on cooling the whole column to effect elution and permit only batch-wise operation, TCZR chromatography generates sharp concentrated elution peaks without tailing effects and appears ideally suited for continuous operation. Copyright © 2013 Elsevier B.V. All rights reserved.

Turbulent Transport of Fast Ions in the Large Plasma Device (LAPD)

NASA Astrophysics Data System (ADS)

Zhou, Shu; Heidbrink, William; McWilliams, Roger; Boehmer, Heinrich; Carter, Troy; Popovich, Pavel; Tripathi, Shreekrishna; Vincena, Steve; Jenko, Frank

2010-11-01

Due to gyroradius averaging and drift-orbit averaging, the transport of fast ions by microturbulence is often smaller than for thermal ions. In this experiment, Strong drift wave turbulence is observed in LAPD on gradients produced by a plate obstacle. Energetic lithium ions orbit through the turbulent region. Scans with a collimated analyzer and with probes give detailed profiles of the fast ion spatial distribution and of the fluctuating fields. The fast-ion transport decreases rapidly with increasing fast-ion gyroradius. Unlike the diffusive transport caused by Coulomb collisions, in this case the turbulent transport is non-diffusive. Analysis and simulation suggest that the fast ions interact ballistically with stationary two-dimensional electrostatic turbulence. The energy dependence of the transport is well explained by gyro-averaging theory. In new experiments, different sources and obstacles alter the drift-wave turbulence to modify the nature of the transport.

Pore networks and polymer rearrangement on a drug-eluting stent as revealed by correlated confocal Raman and atomic force microscopy.

PubMed

Biggs, Kevin B; Balss, Karin M; Maryanoff, Cynthia A

2012-05-29

Drug release from and coating morphology on a CYPHER sirolimus-eluting coronary stent (SES) during in vitro elution were studied by correlated confocal Raman and atomic force microscopy (CRM and AFM, respectively). Chemical surface and subsurface maps of the SES were generated in the same region of interest by CRM and were correlated with surface topography measured by AFM at different elution times. For the first time, a direct correlation between drug-rich regions and the coating morphology was made on a drug-eluting medical device, linking drug release with pore formation, pore throats, and pore networks. Drug release was studied on a drug-eluting stent (DES) system with a multicomponent carrier matrix (poly(n-butyl methacrylate) [PBMA] and poly(ethylene-co-vinyl acetate) [PEVA]). The polymer was found to rearrange postelution because confluence of the carrier polymer matrix reconstituted the voids created by drug release.

Fast optimal wavefront reconstruction for multi-conjugate adaptive optics using the Fourier domain preconditioned conjugate gradient algorithm.

PubMed

Vogel, Curtis R; Yang, Qiang

2006-08-21

We present two different implementations of the Fourier domain preconditioned conjugate gradient algorithm (FD-PCG) to efficiently solve the large structured linear systems that arise in optimal volume turbulence estimation, or tomography, for multi-conjugate adaptive optics (MCAO). We describe how to deal with several critical technical issues, including the cone coordinate transformation problem and sensor subaperture grid spacing. We also extend the FD-PCG approach to handle the deformable mirror fitting problem for MCAO.

Reconstruction for limited-projection fluorescence molecular tomography based on projected restarted conjugate gradient normal residual.

PubMed

Cao, Xu; Zhang, Bin; Liu, Fei; Wang, Xin; Bai, Jing

2011-12-01

Limited-projection fluorescence molecular tomography (FMT) can greatly reduce the acquisition time, which is suitable for resolving fast biology processes in vivo but suffers from severe ill-posedness because of the reconstruction using only limited projections. To overcome the severe ill-posedness, we report a reconstruction method based on the projected restarted conjugate gradient normal residual. The reconstruction results of two phantom experiments demonstrate that the proposed method is feasible for limited-projection FMT. © 2011 Optical Society of America

A new solution-adaptive grid generation method for transonic airfoil flow calculations

NASA Technical Reports Server (NTRS)

Nakamura, S.; Holst, T. L.

1981-01-01

The clustering algorithm is controlled by a second-order, ordinary differential equation which uses the airfoil surface density gradient as a forcing function. The solution to this differential equation produces a surface grid distribution which is automatically clustered in regions with large gradients. The interior grid points are established from this surface distribution by using an interpolation scheme which is fast and retains the desirable properties of the original grid generated from the standard elliptic equation approach.