[HPLC fingerprint analysis of flavonoids of phyllanthi fructus from different habitats].

PubMed

Wang, Fei; Wang, Shuai; Meng, Xian-sheng; Bao, Yong-rui; Zhu, Ying-huan

2014-11-01

To establish the HPLC fingerprint of flavonoids of Phyllanthi Fructus from different habitats. HPLC method was adopted. The flavonoids composition of Phyllanthi Fructus from 10 different habitats was determined on an Agilent C, chromatographic column with 0. 5% formic acid water (A)-acetonitrile (B) as the mobile phase in gradient elution under the wavelength of 254 nm. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus were established to evaluate the qualitiy of them. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus from 10 different habitats were established. 18 common peaks were found and the similarities of them were more than 0. 90 except the ones from Guangxi and Guangdong. The method is simple, accurate and repeatable. It can be used for research and quality control of the effective components in Phyllanthi Fructus.

Development of high performance liquid chromatography method for miconazole analysis in powder sample

NASA Astrophysics Data System (ADS)

Hermawan, D.; Suwandri; Sulaeman, U.; Istiqomah, A.; Aboul-Enein, H. Y.

2017-02-01

A simple high performance liquid chromatography (HPLC) method has been developed in this study for the analysis of miconazole, an antifungal drug, in powder sample. The optimized HPLC system using C8 column was achieved using mobile phase composition containing methanol:water (85:15, v/v), a flow rate of 0.8 mL/min, and UV detection at 220 nm. The calibration graph was linear in the range from 10 to 50 mg/L with r 2 of 0.9983. The limit of detection (LOD) and limit of quantitation (LOQ) obtained were 2.24 mg/L and 7.47 mg/L, respectively. The present HPLC method is applicable for the determination of miconazole in the powder sample with a recovery of 101.28 % (RSD = 0.96%, n = 3). The developed HPLC method provides short analysis time, high reproducibility and high sensitivity.

Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS.

PubMed

Liu, Shiming; Chen, Kaoshan; Schliemann, Willibald; Strack, Dieter

2005-01-01

A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC.

PubMed

López-Cervantes, J; Sánchez-Machado, D I; Gutiérrez-Coronado, M A; Ríos-Vázquez, N J

2006-10-01

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.

Pharmacokinetic study of arctigenin in rat plasma and organ tissue by RP-HPLC method.

PubMed

He, Fan; Dou, De-Qiang; Hou, Qiang; Sun, Yu; Kang, Ting-Guo

2013-01-01

A high-performance liquid chromatography (HPLC) technique was developed for the determination of arctigenin in plasma and various organs of rats after the oral administration of 30, 50 and 70 mgkg(-1) of arctigenin to the Sprague-Dawley rats. Results showed that the validated HPLC method was simple, fast, reproducible and suitable to the determination of arctigenin in rat plasma and organ tissue and one-compartmental model with zero-order absorption process can well describe the changes of arctigenin concentration in the plasma. The concentration of compound was highest in the spleen, less in the liver and the least in the lung.

High performance liquid chromatographic determination of caffeine in decaffeinated coffee, tea, and beverage products.

PubMed

Ashoor, S H; Seperich, G J; Monte, W C; Welty, J

1983-05-01

A method was developed for determining caffeine in decaffeinated coffee, tea, and beverage products by high performance liquid chromatography (HPLC). The HPLC system consisted of a Bio-Sil ODS-5S C18 column, methanol-water (25 + 75) mobile phase at 1 mL/min, and a UV detector. The method is simple and specific. Caffeine recoveries were 93.8-98.3% and coefficients of variation were 0.90-2.25%.

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves.

PubMed

Katekhaye, S; Kale, M S; Laddha, K S

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C(18) column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r(2)>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves.

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves

PubMed Central

Katekhaye, S; Kale, M. S.; Laddha, K. S.

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C18 column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves. PMID:23204626

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection †

PubMed Central

Karr, Dale B.; Waters, James K.; Emerich, David W.

1983-01-01

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. PMID:16346443

Simultaneous determination of all polyphenols in vegetables, fruits, and teas.

PubMed

Sakakibara, Hiroyuki; Honda, Yoshinori; Nakagawa, Satoshi; Ashida, Hitoshi; Kanazawa, Kazuki

2003-01-29

Polyphenols, which have beneficial effects on health and occur ubiquitously in plant foods, are extremely diverse. We developed a method for simultaneously determining all the polyphenols in foodstuffs, using HPLC and a photodiode array to construct a library comprising retention times, spectra of aglycons, and respective calibration curves for 100 standard chemicals. The food was homogenized in liquid nitrogen, lyophilized, extracted with 90% methanol, and subjected to HPLC without hydrolysis. The recovery was 68-92%, and the variation in reproducibility ranged between 1 and 9%. The HPLC eluted polyphenols with good resolution within 95 min in the following order: simple polyphenols, catechins, anthocyanins, glycosides of flavones, flavonols, isoflavones and flavanones, their aglycons, anthraquinones, chalcones, and theaflavins. All the polyphenols in 63 vegetables, fruits, and teas were then examined in terms of content and class. The present method offers accuracy by avoiding the decomposition of polyphenols during hydrolysis, the ability to determine aglycons separately from glycosides, and information on simple polyphenol levels simultaneously.

A simple, sensitive determination of ganciclovir in infant plasma by high-performance liquid chromatography with fluorescence detection.

PubMed

Yoshida, Terumitsu; Takahashi, Ryohei; Imai, Koichi; Uchida, Hiroshi; Arai, Yasutoshi; Oh-ishi, Tsutomu

2010-03-01

This study developed a simple and sensitive method using reversed-phase high-performance liquid chromatography (HPLC) for ganciclovir (GCV) plasma concentrations in cytomegalovirus infectious infants with hearing loss. The method involves a simple protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C(18) column (3 microm, 4.6 mm x 150 mm) with phosphate buffer (pH 2.5, 25 mM) containing 1% methanol-acetonitrile mixture (4:3, v/v) as a mobile phase at a 0.7 mL/min flow rate. GCV was detected using a fluorescence detection (lambdaex/em: 265/380 nm). The quantification limit was 0.025 microg/mL for 100 microL of plasma sample at which good intra- and inter-assay coefficient of variation values (< 4.96%) and recoveries (94.9-96.5%) were established.

Use of refractometry and colorimetry as field methods to rapidly assess antimalarial drug quality.

PubMed

Green, Michael D; Nettey, Henry; Villalva Rojas, Ofelia; Pamanivong, Chansapha; Khounsaknalath, Lamphet; Grande Ortiz, Miguel; Newton, Paul N; Fernández, Facundo M; Vongsack, Latsamy; Manolin, Ot

2007-01-04

The proliferation of counterfeit and poor-quality drugs is a major public health problem; especially in developing countries lacking adequate resources to effectively monitor their prevalence. Simple and affordable field methods provide a practical means of rapidly monitoring drug quality in circumstances where more advanced techniques are not available. Therefore, we have evaluated refractometry, colorimetry and a technique combining both processes as simple and accurate field assays to rapidly test the quality of the commonly available antimalarial drugs; artesunate, chloroquine, quinine, and sulfadoxine. Method bias, sensitivity, specificity and accuracy relative to high-performance liquid chromatographic (HPLC) analysis of drugs collected in the Lao PDR were assessed for each technique. The HPLC method for each drug was evaluated in terms of assay variability and accuracy. The accuracy of the combined method ranged from 0.96 to 1.00 for artesunate tablets, chloroquine injectables, quinine capsules, and sulfadoxine tablets while the accuracy was 0.78 for enterically coated chloroquine tablets. These techniques provide a generally accurate, yet simple and affordable means to assess drug quality in resource-poor settings.

High-Performance Liquid Chromatography in the Undergraduate Chemical Engineering Laboratory

ERIC Educational Resources Information Center

Frey, Douglas D.; Guo, Hui; Karnik, Nikhila

2013-01-01

This article describes the assembly of a simple, low-cost, high-performance liquid chromatography (HPLC) system and its use in the undergraduate chemical engineering laboratory course to perform simple experiments. By interpreting the results from these experiments students are able to gain significant experience in the general method of…

HPLC fingerprint analysis combined with chemometrics for pattern recognition of ginger.

PubMed

Feng, Xu; Kong, Weijun; Wei, Jianhe; Ou-Yang, Zhen; Yang, Meihua

2014-03-01

Ginger, the fresh rhizome of Zingiber officinale Rosc. (Zingiberaceae), has been used worldwide; however, for a long time, there has been no standard approbated internationally for its quality control. To establish an efficacious and combinational method and pattern recognition technique for quality control of ginger. A simple, accurate and reliable method based on high-performance liquid chromatography with photodiode array (HPLC-PDA) detection was developed for establishing the chemical fingerprints of 10 batches of ginger from different markets in China. The method was validated in terms of precision, reproducibility and stability; and the relative standard deviations were all less than 1.57%. On the basis of this method, the fingerprints of 10 batches of ginger samples were obtained, which showed 16 common peaks. Coupled with similarity evaluation software, the similarities between each fingerprint of the sample and the simulative mean chromatogram were in the range of 0.998-1.000. Then, the chemometric techniques, including similarity analysis, hierarchical clustering analysis and principal component analysis were applied to classify the ginger samples. Consistent results were obtained to show that ginger samples could be successfully classified into two groups. This study revealed that HPLC-PDA method was simple, sensitive and reliable for fingerprint analysis, and moreover, for pattern recognition and quality control of ginger.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS.

PubMed

Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom

2009-10-01

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.

A PLS-based extractive spectrophotometric method for simultaneous determination of carbamazepine and carbamazepine-10,11-epoxide in plasma and comparison with HPLC

NASA Astrophysics Data System (ADS)

Hemmateenejad, Bahram; Rezaei, Zahra; Khabnadideh, Soghra; Saffari, Maryam

2007-11-01

Carbamazepine (CBZ) undergoes enzyme biotransformation through epoxidation with the formation of its metabolite, carbamazepine-10,11-epoxide (CBZE). A simple chemometrics-assisted spectrophotometric method has been proposed for simultaneous determination of CBZ and CBZE in plasma. A liquid extraction procedure was operated to separate the analytes from plasma, and the UV absorbance spectra of the resultant solutions were subjected to partial least squares (PLS) regression. The optimum number of PLS latent variables was selected according to the PRESS values of leave-one-out cross-validation. A HPLC method was also employed for comparison. The respective mean recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 (±0.25)% and 103.00 (±0.09)% for PLS and 99.40 (±0.15)% and 102.20 (±0.02)%. The concentrations of CBZ and CBZE were also determined in five patients using the PLS and HPLC methods. The results showed that the data obtained by PLS were comparable with those obtained by HPLC method.

[Determination of triterpenoic acids in fruits of Ziziphus jujuba using HPLC-MS with polymeric ODS column].

PubMed

Zhang, Yong; Zhou, An; Xie, Xiao-Mei

2013-03-01

A simple and sensitive method has been developed to simultaneously determine betunilic acid, oleanolic acid and ursolic acid in the fruits of Ziziphus jujuba from different regions by HPLC-MS. This HPLC assay was performed on PAH polymeric C18 bonded stationary phase column with mobile phase contained acetonitrile-water (90: 10) and with negative ESI detection mode. The developed approach was characterized by short time consumption for chromatographic separation, high sensitivity and good reliability so as to meet the requirements for rapid analysis of large-batch fruits of Z. jujuba from different habitats.

Direct injection analysis of fatty and resin acids in papermaking process waters by HPLC/MS.

PubMed

Valto, Piia; Knuutinen, Juha; Alén, Raimo

2011-04-01

A novel HPLC-atmospheric pressure chemical ionization/MS (HPLC-APCI/MS) method was developed for the rapid analysis of selected fatty and resin acids typically present in papermaking process waters. A mixture of palmitic, stearic, oleic, linolenic, and dehydroabietic acids was separated by a commercial HPLC column (a modified stationary C(18) phase) using gradient elution with methanol/0.15% formic acid (pH 2.5) as a mobile phase. The internal standard (myristic acid) method was used to calculate the correlation coefficients and in the quantitation of the results. In the thorough quality parameters measurement, a mixture of these model acids in aqueous media as well as in six different paper machine process waters was quantitatively determined. The measured quality parameters, such as selectivity, linearity, precision, and accuracy, clearly indicated that, compared with traditional gas chromatographic techniques, the simple method developed provided a faster chromatographic analysis with almost real-time monitoring of these acids. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method.

PubMed

Choo, K S; Kim, I W; Jung, J K; Suh, Y G; Chung, S J; Lee, M H; Shim, C K

2001-06-01

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.

HPLC method for rapidly following biodiesel fuel transesterification reaction progress using a core-shell column.

PubMed

Allen, Samuel J; Ott, Lisa S

2012-07-01

There are a wide and growing variety of feedstocks for biodiesel fuel. Most commonly, these feedstocks contain triglycerides which are transesterified into the fatty acid alkyl esters (FAAEs) which comprise biodiesel fuel. While the tranesterification reaction itself is simple, monitoring the reaction progress and reaction products is not. Gas chromatography-mass spectrometry is useful for assessing the FAAE products, but does not directly address either the tri-, di-, or monoglycerides present from incomplete transesterification or the free fatty acids which may also be present. Analysis of the biodiesel reaction mixture is complicated by the solubility and physical property differences among the components of the tranesterification reaction mixture. In this contribution, we present a simple, rapid HPLC method which allows for monitoring all of the main components in a biodiesel fuel transesterification reaction, with specific emphasis on the ability to monitor the reaction as a function of time. The utilization of a relatively new, core-shell stationary phase for the HPLC column allows for efficient separation of peaks with short elution times, saving both time and solvent.

Current HPLC Methods for Assay of Nano Drug Delivery Systems.

PubMed

Tekkeli, Serife Evrim Kepekci; Kiziltas, Mustafa Volkan

2017-01-01

In nano drug formulations the mechanism of release is a critical process to recognize controlled and targeted drug delivery systems. In order to gain high bioavailability and specificity from the drug to reach its therapeutic goal, the active substance must be loaded into the nanoparticles efficiently. Therefore, the amount in biological fluids or tissues and the remaining amount in nano carriers are very important parameters to understand the potential of the nano drug delivery systems. For this aim, suitable and validated quantitation methods are required to determine released drug concentrations from nano pharmaceutical formulations. HPLC (High Performance Liquid Chromatography) is one of the most common techniques used for determination of released drug content out of nano drug formulations, in different physical conditions, over different periods of time. Since there are many types of HPLC methods depending on detector and column types, it is a challenge for the researchers to choose a suitable method that is simple, fast and validated HPLC techniques for their nano drug delivery systems. This review's goal is to compare HPLC methods that are currently used in different nano drug delivery systems in order to provide detailed and useful information for researchers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Simple and rapid high-performance liquid chromatographic method for the determination of aspartame and its metabolites in foods.

PubMed

Gibbs, B F; Alli, I; Mulligan, C N

1996-02-23

A method for the determination of aspartame (N-L-alpha-aspartyl-L-phenylalanine methyl ester) and its metabolites, applicable on a routine quality assurance basis, is described. Liquid samples (diet Coke, 7-Up, Pepsi, etc.) were injected directly onto a mini-cartridge reversed-phase column on a high-performance liquid chromatographic system, whereas solid samples (Equal, hot chocolate powder, pudding, etc.) were extracted with water. Optimising chromatographic conditions resulted in resolved components of interest within 12 min. The by-products were confirmed by mass spectrometry. Although the method was developed on a two-pump HPLC system fitted with a diode-array detector, it is straightforward and can be transformed to the simplest HPLC configuration. Using a single-piston pump (with damper), a fixed-wavelength detector and a recorder/integrator, the degradation of products can be monitored as they decompose. The results obtained were in harmony with previously reported tedious methods. The method is simple, rapid, quantitative and does not involve complex, hazardous or toxic chemistry.

Determination of rivaroxaban in patient's plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS).

PubMed

Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo Dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

2017-01-01

Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels.

Development and validation of an RP-HPLC method for the quantitation of odanacatib in rat and human plasma and its application to a pharmacokinetic study.

PubMed

Police, Anitha; Gurav, Sandip; Dhiman, Vinay; Zainuddin, Mohd; Bhamidipati, Ravi Kanth; Rajagopal, Sriram; Mullangi, Ramesh

2015-11-01

A simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 μL plasma aliquot with simple liquid-liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP18 using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 50.9-2037 ng/mL (r(2) = 0.994). The intra- and inter-day precisions were in the range of 2.06-5.11 and 5.84-13.1%, respectively, in rat plasma and 2.38-7.90 and 6.39-10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.

PubMed

Jia, Zhaofeng; Liang, Yujie; Ma, Bin; Xu, Xiao; Xiong, Jianyi; Duan, Li; Wang, Daping

2017-05-17

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

PubMed Central

Duarte, Ana Joana; Vieira, Luis

2013-01-01

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. PMID:27335677

Detection of honey adulteration with starch syrup by high performance liquid chromatography.

PubMed

Wang, Shaoqing; Guo, Qilei; Wang, Linlin; Lin, Li; Shi, Hailiang; Cao, Hong; Cao, Baosen

2015-04-01

According to saccharide profile comparison between starch syrups and pure honeys analysed through high performance liquid chromatography (HPLC), a characteristic peak was found at 15.25 min retention time in HPLC chromatogram of syrup, but no peak was observed at the same retention time in chromatogram of pure honeys. This characteristic peak for syrup was identified as an overlapping peak of oligosaccharides with more than 5 degree of polymerisation (DP) based on HPLC chromatogram comparison between starch syrup and a series of standard mono-, di- and oligosaccharides of 3-7 DP. Additionally syrup content correlated linearly with the height of the characteristic peak of syrup under different slope in two ranges 2.5-7.5% and 10-100%, respectively. Therefore, the characteristic peak at 15.25 min retention time can serve as a syrup indicator in HPLC analysis of the adulterated honeys. This new HPLC method for honey adulteration detection was further applied in an authenticity inspection on more than 100 commercial honeys. In addition to the improved accuracy of honey adulteration detection, the proposed HPLC method was simple, low cost and easy practice for honey product quality control by government department considering the popularity of HPLC device and technology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous Chloramphenicol and Florfenicol Determination by A Validated DLLME-HPLC-UV Method in Pasteurized Milk.

PubMed

Karami-Osboo, Rouhollah; Miri, Ramin; Javidnia, Katayoun; Kobarfard, Farzad

2016-01-01

The antibiotic residues in milk are a well-known serious problem and pose several health hazards to consumers. We have described a simple, rapid, and inexpensive DLLME-HPLC/UV technique for the extraction of chloramphenicol and florfenicol residues in milk samples. Under the optimum conditions, linearity of the method was observed over the range 0.02-0.85 µg/L with correlation coefficients > 0.999. The proposed method has been found to have a good limit of detection (signal to noise ratio = 3) for chloramphenicol (12.5 µg/Kg) and florfenicol (12.2 µg/Kg), and precision with relative standard deviation values under 15% (RSD, n = 3). Good recoveries (69.1-79.4%) were obtained for the extraction of the target analytes in milk samples. This simple and economic method has been applied for analyses of 15 real milk samples. Among all samples only one of them was contaminated to florfenicol; 62.4 µg/Kg and contamination to chloramphenicol was not detected.

Simultaneous Chloramphenicol and Florfenicol Determination by A Validated DLLME-HPLC-UV Method in Pasteurized Milk

PubMed Central

Karami-Osboo, Rouhollah; Miri, Ramin; Javidnia, Katayoun; Kobarfard, Farzad

2016-01-01

The antibiotic residues in milk are a well-known serious problem and pose several health hazards to consumers. We have described a simple, rapid, and inexpensive DLLME-HPLC/UV technique for the extraction of chloramphenicol and florfenicol residues in milk samples. Under the optimum conditions, linearity of the method was observed over the range 0.02-0.85 µg/L with correlation coefficients > 0.999. The proposed method has been found to have a good limit of detection (signal to noise ratio = 3) for chloramphenicol (12.5 µg/Kg) and florfenicol (12.2 µg/Kg), and precision with relative standard deviation values under 15% (RSD, n = 3). Good recoveries (69.1–79.4%) were obtained for the extraction of the target analytes in milk samples. This simple and economic method has been applied for analyses of 15 real milk samples. Among all samples only one of them was contaminated to florfenicol; 62.4 µg/Kg and contamination to chloramphenicol was not detected. PMID:27980571

Modified HPLC-ESI-MS Method for Glycated Hemoglobin Quantification Based on the IFCC Reference Measurement Procedure and Its Application for Quantitative Analyses in Clinical Laboratories of China.

PubMed

Song, Zhixin; Xie, Baoyuan; Ma, Huaian; Zhang, Rui; Li, Pengfei; Liu, Lihong; Yue, Yuhong; Zhang, Jianping; Tong, Qing; Wang, Qingtao

2016-09-01

The level of glycated hemoglobin (HbA1c ) has been recognized as an important indicator of long-term glycemic control. However, the HbA1c measurement is not currently included as a diagnostic determinant in China. Current study aims to assess a candidate modified International Federation of Clinical Chemistry reference method for the forthcoming standardization of HbA1c measurements in China. The HbA1c concentration was measured using a modified high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method. The modified method replaces the propylcyanide column with a C18 reversed-phase column, which has a lower cost and is more commonly used in China, and uses 0.1% (26.5 mmol/l) formic acid instead of trifluoroacetic acid. Moreover, in order to minimize matrix interference and reduce the running time, a solid-phase extraction was employed. The discrepancies between HbA1c measurements using conventional methods and the HPLC-ESI-MS method were clarified in clinical samples from healthy people and diabetic patients. Corresponding samples were distributed to 89 hospitals in Beijing for external quality assessment. The linearity, reliability, and accuracy of the modified HPLC-ESI-MS method with a shortened running time of 6 min were successfully validated. Out of 89 hospitals evaluated, the relative biases of HbA1c concentrations were < 8% for 74 hospitals and < 5% for 60 hospitals. Compared with other conventional methods, HbA1c concentrations determined by HPLC methods were similar to the values obtained from the current HPLC-ESI-MS method. The HPLC-ESI-MS method represents an improvement over existing methods and provides a simple, stable, and rapid HbA1c measurement with strong signal intensities and reduced ion suppression. © 2015 Wiley Periodicals, Inc.

Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography.

PubMed

Ghosh, Bidisha; Mitra, Joy; Chakraborty, Saikat; Bhattacharyya, Jagannath; Chakraborty, Anirban; Sen, Soumitra Kumar; Neerathilingam, Muniasamy

2015-01-01

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease "neurolathyrism", present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r2 > 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.

Simultaneous determination of dextromethorphan HBr and bromhexine HCl in tablets by first-derivative spectrophotometry.

PubMed

Tantishaiyakul, V; Poeaknapo, C; Sribun, P; Sirisuppanon, K

1998-06-01

A rapid, simple and direct assay procedure based on first-derivative spectrophotometry, using a zero-crossing and peak-to-base measurement at 234 and 324 nm, respectively, has been developed for the specific determination of dextromethorphan HBr and bromhexine HCl in tablets. Calibration graphs were linear with the correlation coefficients of 0.9999 for both analytes. The limit of detections were 0.033 and 0.103 microgram ml-1 for dextromethorphan HBr and bromhexine HCl, respectively. A HPLC method has been developed as the reference method. The results obtained by the first-derivative spectrophotometry were in good agreement with those found by the HPLC method.

Profiling of components of rhizoma et radix polygoni cuspidati by high-performance liquid chromatography with ultraviolet diode-array detector and ion trap/time-of-flight mass spectrometric detection.

PubMed

Fu, Jinfeng; Wang, Min; Guo, Huimin; Tian, Yuan; Zhang, Zunjian; Song, Rui

2015-01-01

Rhizoma et Radix Polygoni Cuspidati (Huzhang in Chinese, HZ) is a traditional medicinal plant in China. Many of the components of HZ have been proved to be bioactive while it is difficult to conduct a comprehensive chemical profiling of HZ as a consequence of the absence of efficient separation system and sensitive detective means. We developed a simple and effective method for comprehensive characterization of constituents in HZ. To develop a simple and effective method to characterize the components in HZ and provide useful information for subsequent metabolic studies of HZ. The components in HZ aqueous extract were characterized by using high performance liquid chromatography with UV diode-array detector (HPLC-DAD) and ion trap/time-of-flight mass spectrometric detection (HPLC-IT/TOF). Stilbenes, anthraquinones, gallates and tannins, naphthalenes and some other compounds were identified and confirmed by diagnostic fragment ions with accurate mass measurements, characteristic fragmentation pathways and relevant published literatures. Among the 238 constituents detected in HZ, a total number of 74 constituents were identified unambiguously or tentatively, including 29 compounds reported for the first time in HZ. The identification and structure elucidation of these chemicals provided essential data for quality control and further in vivo metabolic studies of HZ. Key words: Polygonum cuspidatum, HPLC-DAD, HPLC-IT/TOF, qualitative analysis.

A novel HPLC-MS/MS method for the simultaneous determination of astemizole and its major metabolite in dog or monkey plasma and application to pharmacokinetics.

PubMed

Back, Hyun-moon; Lee, Jong-Hwa; Chae, Jung-woo; Song, Byungjeong; Seo, Joung-Wook; Yun, Hwi-yeol; Kwon, Kwang-il

2015-10-10

Astemizole (AST), a second-generation antihistamine, is metabolized to desmethyl astemizole (DEA), and although it has been removed from the market for inducing QT interval prolongation, it has reemerged as a potential anticancer and antimalarial agent. This report describes a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the concentrations of AST and DEA in beagle dog and cynomolgus monkey plasma with simple preparation method and short retention time. Prior to HPLC analyses, the plasma samples were extracted with simple liquid-liquid extraction method. The isocratic mobile phase was 0.025% trifluoroacetic acid (TFA dissolved in acetonitrile) and 20 mM ammonium acetate (94:6) at a flow rate of 0.25 mL/min and diphenhydramine used as internal standard. In MS/MS analyses, precursor ions of the analytes were optimized as protonated molecular ions: [M+H](+). The lower limit of quantification of astemizole was 2.5 ng/mL in both species and desmethyl astemizole were 7.5 ng/mL and 10 ng/mL in dog and monkey plasma, respectively. The accuracy, precision, and stability of the method were in accordance with FDA guidelines for the validation of bioanalytical methods. Finally this validated method was successfully applied to a pharmacokinetic study in dogs and monkeys after oral administration of 10 mg/kg AST. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of hydroxyurea in human plasma by HPLC-UV using derivatization with xanthydrol.

PubMed

Legrand, Tiphaine; Rakotoson, Marie-Georgine; Galactéros, Frédéric; Bartolucci, Pablo; Hulin, Anne

2017-10-01

A simple and rapid high performance liquid chromatography (HPLC) method using ultraviolet (UV) detection was developed to determine hydroxyurea (HU) concentration in plasma sample after derivatization with xanthydrol. Two hundred microliters samples were spiked with methylurea (MeU) as internal standard and proteins were precipitated by adding methanol. Derivatization of HU and MeU was immediately performed by adding 0.02M xanthydrol and 1.5M HCl in order to obtain xanthyl-derivatives of HU and MeU that can be further separated using HPLC and quantified using UV detection at 240nm. Separation was achieved using a C18 column with a mobile phase composed of 20mM ammonium acetate and acetonitrile in gradient elution mode at a flow rate of 1mL/min. The total analysis time did not exceed 18min. The method was found linear from 5 to 400μM and all validation parameters fulfilled the international requirements. Between- and within-run accuracy error ranged from -4.7% to 3.2% and precision was lower than 12.8%. This simple method requires small volume samples and can be easily implemented in most clinical laboratories to develop pharmacokinetics studies of HU and to promote its therapeutic monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a sensitive high-performance liquid chromatographic method with simple extraction for simultaneous determination of huperzine A and huperzine B in the species containing lycopodium alkaloids.

PubMed

Zhang, Yanqing; Xie, Junbo; Chen, Wen-Qian; Zhou, Tian-Yan; Lu, Wei

2009-01-01

A sensitive HPLC method with simple extraction was developed for simultaneous determination of huperzine A (HupA) and huperzine B (HupB) in Huperzia serrata, H. crispata, H. miyoshiana, and Lycopodiastrum casuarinoides. In order to avoid conventional multiple-step and time-consuming sample preparation methods, direct reflux extraction with alkaline chloroform was adopted. The quantitative determination was conducted by reversed-phase HPLC with a photodiode array detector set at 308 nm. Separation was performed on a Luna C18 column (250 x 4.6 mm id, 5 microm) with methanol-0.2% aqueous acetic acid (18 + 82, v/v) mobile phase. The method was validated for accuracy, reproducibility, precision, and limits of detection and quantification. Quantification of the two active compounds in the samples was performed by this newly developed method, and the content of HupA and HupB varied substantially among four different species. The satisfactory results indicated that the developed method can readily be utilized for quality control of the species of Huperziaceae and Lycopodiaceae containing the two compounds.

Comparison of cyclosporine determinations in whole blood by three different methods. HPLC, /sup 125/I RIA and /sup 3/H RIA

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

Huang, W.Y.; Lipsey, A.I.; Cheng, M.H.

1987-04-01

The authors have analyzed and compared the cyclosporine concentrations in whole blood specimens from pediatric renal transplant patients using three different methods: high-performance liquid chromatography (HPLC) (5u C18 reverse-phase column), /sup 3/H radioimmunoassay (RIA), and /sup 125/I RIA (substituted /sup 3/H-tracer in Sandoz Kit with /sup 125/I tracer. Results obtained by the /sup 125/I RIA correlated well with results obtained by the /sup 3/H RIA. Both RIA methods had similar correlation with the HPLC method. The /sup 125/I RIA method showed higher sensitivity and greater precision than the /sup 3/H RIA method. The authors conclude that the /sup 125/I RIAmore » method can be used for cyclosporine determination in whole blood specimens. The use of the /sup 125/I RIA provides a simple and rapid method with higher counting efficiency and less background quenching than the /sup 3/H RIA method, which requires cumbersome liquid scintillation counting procedures.« less

Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in beer, red wine, and grape juice.

PubMed

Cao, Jiliang; Kong, Weijun; Zhou, Shujun; Yin, Lihui; Wan, Li; Yang, Meihua

2013-04-01

A simple, reliable, and low-cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC-FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC-FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)

PubMed Central

Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

2017-01-01

Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels. PMID:28170419

A simple and rapid electrophoretic method to characterize simple phenols, lignans, complex phenols, phenolic acids, and flavonoids in extra-virgin olive oil.

PubMed

Carrasco-Pancorbo, Alegria; Gómez-Caravaca, Ana Maria; Cerretani, Lorenzo; Bendini, Alessandra; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

2006-09-01

We have devised a simple and rapid capillary electrophoretic method which provides the analyst with a useful tool for the characterization of the polyphenolic fraction of extra-virgin olive oil. This method that uses a capillary with 50 microm id and a total length of 47 cm (40 cm to the detector) with a detection window of 100 x 200 microm, and a buffer solution containing 45 mM of sodium tetraborate pH 9.3 offers valuable information about all the families of compounds present in the polar fraction of the olive oil. The detection was carried out by UV absorption at 200, 240, 280, and 330 nm in order to facilitate the identification of the compounds. Concretely, the method permits the identification of simple phenols, lignans, complex phenols (isomeric forms of secoiridoids), phenolic acids, and flavonoids in the SPE-Diol extracts from extra-virgin olive oil in a short time (less than 10 min) and provides a satisfactory resolution. Peak identification was done by comparing both migration time and spectral data obtained from olive oil samples and standards (commercial or isolated (by HPLC-MS) standards), with spiked methanol-water extracts of olive oil with HPLC-collected compounds and commercially available standards at several concentration levels, studying the information of the electropherograms obtained at several wavelengths and also using the information previously reported.

Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study.

PubMed

S, Vijay Kumar; Dhiman, Vinay; Giri, Kalpesh Kumar; Sharma, Kuldeep; Zainuddin, Mohd; Mullangi, Ramesh

2015-09-01

A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C18 column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182-5035 ng/mL (r(2) = 0.995). The intra- and inter-day precisions were in the range of 1.41-11.2 and 3.66-8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.

HPLC-Assisted Automated Oligosaccharide Synthesis: Implementation of the Autosampler as a Mode of the Reagent Delivery.

PubMed

Pistorio, Salvatore G; Nigudkar, Swati S; Stine, Keith J; Demchenko, Alexei V

2016-10-07

The development of a useful methodology for simple, scalable, and transformative automation of oligosaccharide synthesis that easily interfaces with existing methods is reported. The automated synthesis can now be performed using accessible equipment where the reactants and reagents are delivered by the pump or the autosampler and the reactions can be monitored by the UV detector. The HPLC-based platform for automation is easy to setup and adapt to different systems and targets.

Rapid Development and Validation of Improved Reversed-Phase High-performance Liquid Chromatography Method for the Quantification of Mangiferin, a Polyphenol Xanthone Glycoside in Mangifera indica

PubMed Central

Naveen, P.; Lingaraju, H. B.; Prasad, K. Shyam

2017-01-01

Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica, is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica. RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography–mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica. SUMMARY The present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica. The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica. Abbreviations Used: M. indica: Mangifera indica, RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification. PMID:28539748

Rapid Development and Validation of Improved Reversed-Phase High-performance Liquid Chromatography Method for the Quantification of Mangiferin, a Polyphenol Xanthone Glycoside in Mangifera indica.

PubMed

Naveen, P; Lingaraju, H B; Prasad, K Shyam

2017-01-01

Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica , is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica . RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography-mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica . The present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica . The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica . Abbreviations Used: M. indica : Mangifera indica , RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification.

A simple analytical procedure to replace HPLC for monitoring treatment concentrations of chloramine-T on fish culture facilities

USGS Publications Warehouse

Dawson, Verdel K.; Meinertz, Jeffery R.; Schmidt, Larry J.; Gingerich, William H.

2003-01-01

Concentrations of chloramine-T must be monitored during experimental treatments of fish when studying the effectiveness of the drug for controlling bacterial gill disease. A surrogate analytical method for analysis of chloramine-T to replace the existing high-performance liquid chromatography (HPLC) method is described. A surrogate method was needed because the existing HPLC method is expensive, requires a specialist to use, and is not generally available at fish hatcheries. Criteria for selection of a replacement method included ease of use, analysis time, cost, safety, sensitivity, accuracy, and precision. The most promising approach was to use the determination of chlorine concentrations as an indicator of chloramine-T. Of the currently available methods for analysis of chlorine, the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method best fit the established criteria. The surrogate method was evaluated under a variety of water quality conditions. Regression analysis of all DPD colorimetric analyses with the HPLC values produced a linear model (Y=0.9602 X+0.1259) with an r2 value of 0.9960. The average accuracy (percent recovery) of the DPD method relative to the HPLC method for the combined set of water quality data was 101.5%. The surrogate method was also evaluated with chloramine-T solutions that contained various concentrations of fish feed or selected densities of rainbow trout. When samples were analyzed within 2 h, the results of the surrogate method were consistent with those of the HPLC method. When samples with high concentrations of organic material were allowed to age more than 2 h before being analyzed, the DPD method seemed to be susceptible to interference, possibly from the development of other chloramine compounds. However, even after aging samples 6 h, the accuracy of the surrogate DPD method relative to the HPLC method was within the range of 80–120%. Based on the data comparing the two methods, the U.S. Food and Drug Administration has concluded that the DPD colorimetric method is appropriate to use to measure chloramine-T in water during pivotal efficacy trials designed to support the approval of chloramine-T for use in fish culture.

Development of a rapid, simple and sensitive HPLC-FLD method for determination of rhodamine B in chili-containing products.

PubMed

Qi, Ping; Lin, Zhihao; Li, Jiaxu; Wang, ChengLong; Meng, WeiWei; Hong, Hong; Zhang, Xuewu

2014-12-01

In this work, a simple, rapid and sensitive analytical method for the determination of rhodamine B in chili-containing foodstuffs is described. The dye is extracted from samples with methanol and analysed without further cleanup procedure by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD). The influence of matrix fluorescent compounds (capsaicin and dihydrocapsaicin) on the analysis was overcome by the optimisation of mobile-phase composition. The limit of determination (LOD) and limit of quantification (LOQ) were 3.7 and 10 μg/kg, respectively. Validation data show a good repeatability and within-lab reproducibility with relative standard deviations <10%. The overall recoveries are in the range of 98-103% in chili powder and in the range of 87-100% in chili oil depending on the concentration of rhodamine B in foodstuffs. This method is suitable for the routine analysis of rhodamine B due to its sensitivity, simplicity, reasonable time and cost. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous determination of three polyphenols in rat plasma after orally administering hawthorn leaves extract by the HPLC method.

PubMed

Ying, Xixiang; Meng, Xiansheng; Wang, Siyuan; Wang, Dong; Li, Haibo; Wang, Bing; Du, Yang; Liu, Xun; Zhang, Wenjie; Kang, Tingguo

2012-01-01

A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C₁₈ analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6 : 1.5 : 18.5 : 74, v/v/v/v). The calibration curves were linear over the range of 2.5-500, 0.2-25 and 0.25-12.5 µg mL⁻¹ for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5-10  mL kg⁻¹.

Ultrasound-assisted extraction of azadirachtin from dried entire fruits of Azadirachta indica A. Juss. (Meliaceae) and its determination by a validated HPLC-PDA method.

PubMed

de Paula, Joelma Abadia Marciano; Brito, Lucas Ferreira; Caetano, Karen Lorena Ferreira Neves; de Morais Rodrigues, Mariana Cristina; Borges, Leonardo Luiz; da Conceição, Edemilson Cardoso

2016-01-01

Azadirachta indica A. Juss., also known as neem, is a Meliaceae family tree from India. It is globally known for the insecticidal properties of its limonoid tetranortriterpenoid derivatives, such as azadirachtin. This work aimed to optimize the azadirachtin ultrasound-assisted extraction (UAE) and validate the HPLC-PDA analytical method for the measurement of this marker in neem dried fruit extracts. Box-Behnken design and response surface methodology (RSM) were used to investigate the effect of process variables on the UAE. Three independent variables, including ethanol concentration (%, w/w), temperature (°C), and material-to-solvent ratio (gmL(-1)), were studied. The azadirachtin content (µgmL(-1)), i.e., dependent variable, was quantified by the HPLC-PDA analytical method. Isocratic reversed-phase chromatography was performed using acetonitrile/water (40:60), a flow of 1.0mLmin(-1), detection at 214nm, and C18 column (250×4.6mm(2), 5µm). The primary validation parameters were determined according to ICH guidelines and Brazilian legislation. The results demonstrated that the optimal UAE condition was obtained with ethanol concentration range of 75-80% (w/w), temperature of 30°C, and material-to-solvent ratio of 0.55gmL(-1). The HPLC-PDA analytical method proved to be simple, selective, linear, precise, accurate and robust. The experimental values of azadirachtin content under optimal UAE conditions were in good agreement with the RSM predicted values and were superior to the azadirachtin content of percolated extract. Such findings suggest that UAE is a more efficient extractive process in addition to being simple, fast, and inexpensive. Copyright © 2015 Elsevier B.V. All rights reserved.

[HPLC fingerprint of the antiarrhythmic fraction of Valeriana officinalis].

PubMed

Duan, Xue-Yun; Gong, Zhan-Feng; Chen, Shu-He; Fang, Ying; Liu, Yan-Wen

2009-06-01

To establish HPLC fingerprints of the Antiarrhythmic fraction of Valeriana officinalis. Agilent C18 (250 mm x 4.6 mm, 5 microm) column was used and the acetonitrile-water was chosen as the mobile phase in a gradient mode. The column temperature was 380 degrees C and the detection wavelength was 218 nm. The detection time was 70 min, and the flow rate was 1.0 mL/ min. Fifteen characteristic peaks were indicated in HPLC fingerprints. The relative retention time and the ranges of relative areas of the common peaks were also determined. This method is simple and accurate with a good reproducibility and provides a reference standard for the quality control of Valeriana officinalis.

Chip-based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids.

PubMed

Chang, Yuwei; Zhao, Chunxia; Wu, Zeming; Zhou, Jia; Zhao, Sumin; Lu, Xin; Xu, Guowang

2012-08-01

In this work a chip-based nano HPLC coupled MS (HPLC-chip/MS) method with a simple sample preparation procedure was developed for the flavonoid profiling of soybean. The analytical properties of the method including the linearity (R(2) , 0.992-0.995), reproducibility (RSD, 1.50-7.66%), intraday precision (RSD, 1.41-5.14%) and interday precision (RSD, 2.76-16.90%) were satisfactory. Compared with the conventional HPLC/MS method, a fast extraction and analysis procedure was applied and more flavonoids were detected in a single run. Additionally, 13 flavonoids in soybean seed were identified for the first time. The method was then applied to the profiling of six varieties of soybean sowed at the same place. A clear discrimination was observed among different cultivars, three isoflavones, accounting for nearly 80% of total flavonoid contents, were found increased in the spring soybeans compared with the summer cultivars. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fingerprint chromatogram analysis of Pseudostellaria heterophylla (Miq.) Pax root by high performance liquid chromatography.

PubMed

Han, Chao; Chen, Junhui; Chen, Bo; Lee, Frank Sen-Chun; Wang, Xiaoru

2006-09-01

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the fingerprinting of extracts from the root of Pseudostellaria heterophylla (Miq.) Pax. HPLC with gradient elution was performed on an authentic reference standard of powdered P. heterophylla (Miq.) Pax root and 11 plant samples of the root were collected from different geographic locations. The HPLC chromatograms have been standardized through the selection and identification of reference peaks and the normalization of retention times and peak intensities of all the common peaks. The standardized HPLC fingerprints show high stability and reproducibility, and thus can be used effectively for the screening analysis or quality assessment of the root or its derived products. Similarity index calculations based on cosine angle values or correlation methods have been performed on the HPLC fingerprints. As a group, the fingerprints of the P. heterophylla (Miq.) Pax samples studied are highly correlated with closely similar fingerprints. Within the group, the samples can be further divided into subgroups based on hierarchical clustering analysis (HCA). Sample grouping based on HCA coincides nicely with those based on the geographical origins of the samples. The HPLC fingerprinting techniques thus have high potential in authentication or source-tracing types of applications.

Determination of tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations by HPLC.

PubMed

Almeida, M M; Alves, J M P; Patto, D C S; Lima, C R R C; Quenca-Guillen, J S; Santoro, M I R M; Kedor-Hackmann, E R M

2009-12-01

A rapid HPLC method was developed for the assay of tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations. The validated method was applied for quantitative determination of these vitamins in simulated emulsion formulation. Samples were analysed directly on a RP-18 reverse phase column with UV detection at 222 nm. A mixture of methanol and isopropanol (25 : 75 v/v) was used as mobile phase. The retention time of tocopheryl acetate and ascorbyl tetraisopalmitate were 3.0 min and 5.9 min, respectively. Recovery was between 95% and 104%. In addition, the excipients did not interfere in the analysis. The method is simple, reproducible, selective and is suitable for routine analyses of commercial products.

Development and Validation of a Precise, Single HPLC Method for the Determination of Tolperisone Impurities in API and Pharmaceutical Dosage Forms.

PubMed

Raju, Thummala Veera Raghava; Seshadri, Raja Kumar; Arutla, Srinivas; Mohan, Tharlapu Satya Sankarsana Jagan; Rao, Ivaturi Mrutyunjaya; Nittala, Someswara Rao

2013-01-01

A novel, sensitive, stability-indicating HPLC method has been developed for the quantitative estimation of Tolperisone-related impurities in both bulk drugs and pharmaceutical dosage forms. Effective chromatographic separation was achieved on a C18 stationary phase with a simple mobile phase combination delivered in a simple gradient programme, and quantitation was by ultraviolet detection at 254 nm. The mobile phase consisted of a buffer and acetonitrile delivered at a flow rate 1.0 ml/min. The buffer consisted of 0.01 M potassium dihydrogen phosphate with the pH adjusted to 8.0 by using diethylamine. In the developed HPLC method, the resolution between Tolperisone and its four potential impurities was found to be greater than 2.0. Regression analysis showed an R value (correlation coefficient) of greater than 0.999 for the Tolperisone impurities. This method was capable of detecting all four impurities of Tolperisone at a level of 0.19 μg/mL with respect to the test concentration of 1000 μg/mL for a 10 µl injection volume. The tablets were subjected to the stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. Considerable degradation was found to occur in base hydrolysis, water hydrolysis, and oxidation. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 100%. The established method was validated and found to be linear, accurate, precise, specific, robust, and rugged.

Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method

PubMed Central

Chen, Xiao; Wan, Yanjian; Chen, Xi; Li, Yuanyuan

2018-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1–400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid. PMID:29342170

Determination of 2,4-Dichlorophenoxyacetic acid (2,4-D) in rat serum for pharmacokinetic studies with a simple HPLC method.

PubMed

Chen, Xiao; Zhang, Hongling; Wan, Yanjian; Chen, Xi; Li, Yuanyuan

2018-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1-400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid.

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 quantification of five major active components in capsules of the traditional Chinese medicine ‘Shu-Jin-Zhi-Tong’ by high performance liquid chromatography

PubMed Central

Yang, Xing-Xin; Zhang, Xiao-Xia; Chang, Rui-Miao; Wang, Yan-Wei; Li, Xiao-Ni

2011-01-01

A simple and reliable high performance liquid chromatography (HPLC) method has been developed for the simultaneous quantification of five major bioactive components in ‘Shu-Jin-Zhi-Tong’ capsules (SJZTC), for the purposes of quality control of this commonly prescribed traditional Chinese medicine. Under the optimum conditions, excellent separation was achieved, and the assay was fully validated in terms of linearity, precision, repeatability, stability and accuracy. The validated method was applied successfully to the determination of the five compounds in SJZTC samples from different production batches. The HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of SJZTC. PMID:29403711

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine.

PubMed

Makino, Yukiko

2012-03-01

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d-methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d-methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C18 MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH2 PO4-acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d-methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high-purity d-methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high-purity methamphetamine profiling. Copyright © 2011 John Wiley & Sons, Ltd.

[Determination of protopine and isocorydine in root of Dactylicapnos scandens by HPLC].

PubMed

Yan, Tian-qing; Yang, Yan-fang; Ai, Tie-min

2004-10-01

To establish a HPLC method for determination of protopine and isocorydine in root of Dactylicapnos scandens. The separation was performed in a PHENOMENEX-C18 column with a mobile phase of methanol-0.2% phosphoric acid (adjusted to pH 7.0 with triethylamine)(50:50), The detection wavelength was at 254 nm and the flow rate was 0.8 mL x min(-1). The average recovery of Protopine and Isocorydine was 97.9%, 98.6% respectively, and RSD 1.3%, 1.4%. This method is accurate, simple and reliable. It can be used for quality control of D. scandens.

A simple quantitative approach for the determination of long and medium chain lipids in bio-relevant matrices by high performance liquid chromatography with refractive index detection.

PubMed

Lee, Kathy Wai Yu; Porter, Christopher J H; Boyd, Ben J

2013-09-01

There is increasing attention in the literature towards understanding the behaviour of lipid-based drug formulations under digestion conditions using in vitro and in vivo methods. This necessitates a convenient method for quantitation of lipids and lipid digestion products. In this study, a simple and accessible method for the separation and quantitative determination of typical formulation and digested lipids using high performance liquid chromatography coupled to refractive index detection (HPLC-RI) is described. Long and medium chain lipids were separated and quantified in a biological matrix (gastrointestinal content) without derivatisation using HPLC-RI on C18 and C8 columns, respectively. The intra- and inter-assay accuracy was between 92% and 106%, and the assays were precise to within a coefficient of variation of less than 10% over the range of 0.1-2 mg/mL for both long and medium chain lipids. This method is also shown to be suitable for quantifying the lipolysis products collected from the gastrointestinal tract in the course of in vivo lipid digestion studies.

Quantitative determination of flavonoids by column high-performance liquid chromatography with mass spectrometry and ultraviolet absorption detection in Artemisia afra and comparative studies with various species of Artemisia plants.

PubMed

Avula, Bharathi; Wang, Yan-Hong; Smillie, Troy J; Mabusela, Wilfred; Vincent, Leszek; Weitz, Frans; Khan, Ikhlas A

2009-01-01

A simple and specific analytical method for the quantitative determination of flavonoids from the aerial parts of the Artemisia afra plant samples was developed. By column high-performance liquid chromatography (HPLC) with UV absorption and mass spectrometry (MS) detection, separation was achieved on a reversed-phase octadecylsilyl (C18) column with water, methanol, and acetonitrile, all containing 0.1% acetic acid, as the mobile phase. These methods were used to analyze various species of Artemisia plant samples. The wavelength used for quantification of flavonoids with the diode array detector was 335 nm. The limits of detection (LOD) by HPLC/MS were found to be 7.5, 7.5, 10, 2.0, and 2.0 ng/mL; and by LC-UV the LODs were 500, 500, 500, 300, and 300 ng/mL for apigenin, chrysoeriol, tamarixetin, acacetin, and genkwanin, respectively. The HPLC/MS method was found to be 50-150 times more sensitive than the HPLC-UV method. HPLC/MS coupled with an electrospray ionization interface is described for the identification and quantification of flavonoids in various plant samples. This method involved the use of the [M+H]+ ions of the compounds at mass-to-charge ratio of 1.0606, 301.0712, 317.0661, 285.0763, and 285.0763 (calculated mass), respectively, in the positive ion mode with extractive ion monitoring.

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.

A simple HPLC-DAD method for simultaneous detection of two organophosphates, profenofos and fenthion, and validation by soil microcosm experiment.

PubMed

Mahajan, Rishi; Chatterjee, Subhankar

2018-05-05

Indiscriminate use of two broad spectrum pesticides, profenofos and fenthion, in agricultural system, often results in their accumulation in a non-target niche and leaching into water bodies. The present study, therefore, aims at developing a simple and rapid HPLC method that allows simultaneous extraction and detection of these two pesticides, especially in run-off water. Extraction of the two pesticides from spiked water samples using dichloromethane resulted in recovery ranging between 80 and 90%. An HPLC run of 20 min under optimized chromatographic parameters (mobile phase: methanol (75%) and water (25%); flow rate of 0.8 ml min -1 ; diode array detector at wavelength 210 nm) resulted in a significant difference in retention times of two pesticides (4.593 min) which allows a window of opportunity to study any possible intermediates/transformants of the parent compounds while evaluating run-off waters from agricultural fields. The HPLC method developed allowed simultaneous detection of profenofos and fenthion with a single injection into the HPLC system with 0.0328 mg l -1 (32.83 ng ml -1 ) being the limit of detection (LOD) and 0.0995 mg l -1 (99.5 ng ml -1 ) as the limit of quantification (LOQ) for fenthion; for profenofos, LOD and LOQ were 0.104 mg l -1 (104.50 ng ml -1 ) and 0.316 mg l -1 (316.65 ng ml -1 ), respectively. The findings were further validated using the soil microcosm experiment that allowed simultaneous detection and quantification of profenofos and fenthion. The findings indicate towards the practical significance of the methodology developed as the soil microcosm experiment closely mimics the agricultural run-off water under natural environmental conditions.

New Stability Indicating RP-HPLC Method for the Estimation of Cefpirome Sulphate in Bulk and Pharmaceutical Dosage Forms.

PubMed

Rao, Kareti Srinivasa; Kumar, Keshar Nargesh; Joydeep, Datta

2011-01-01

A simple stability indicating reversed-phase HPLC method was developed and subsequently validated for estimation of Cefpirome sulphate (CPS) present in pharmaceutical dosage forms. The proposed RP-HPLC method utilizes a LiChroCART-Lichrosphere100, C18 RP column (250 mm × 4mm × 5 μm) in an isocratic separation mode with mobile phase consisting of methanol and water in the proportion of 50:50 % (v/v), at a flow rate 1ml/min, and the effluent was monitored at 270 nm. The retention time of CPS was 2.733 min and its formulation was exposed to acidic, alkaline, photolytic, thermal and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The described method was linear over a range of 0.5-200μg/ml. The percentage recovery was 99.46. F-test and t-test at 95% confidence level were used to check the intermediate precision data obtained under different experimental setups; the calculated value was found to be less than the critical value.

Direct quantification of lipopeptide biosurfactants in biological samples via HPLC and UPLC-MS requires sample modification with an organic solvent.

PubMed

Biniarz, Piotr; Łukaszewicz, Marcin

2017-06-01

The rapid and accurate quantification of biosurfactants in biological samples is challenging. In contrast to the orcinol method for rhamnolipids, no simple biochemical method is available for the rapid quantification of lipopeptides. Various liquid chromatography (LC) methods are promising tools for relatively fast and exact quantification of lipopeptides. Here, we report strategies for the quantification of the lipopeptides pseudofactin and surfactin in bacterial cultures using different high- (HPLC) and ultra-performance liquid chromatography (UPLC) systems. We tested three strategies for sample pretreatment prior to LC analysis. In direct analysis (DA), bacterial cultures were injected directly and analyzed via LC. As a modification, we diluted the samples with methanol and detected an increase in lipopeptide recovery in the presence of methanol. Therefore, we suggest this simple modification as a tool for increasing the accuracy of LC methods. We also tested freeze-drying followed by solvent extraction (FDSE) as an alternative for the analysis of "heavy" samples. In FDSE, the bacterial cultures were freeze-dried, and the resulting powder was extracted with different solvents. Then, the organic extracts were analyzed via LC. Here, we determined the influence of the extracting solvent on lipopeptide recovery. HPLC methods allowed us to quantify pseudofactin and surfactin with run times of 15 and 20 min per sample, respectively, whereas UPLC quantification was as fast as 4 and 5.5 min per sample, respectively. Our methods provide highly accurate measurements and high recovery levels for lipopeptides. At the same time, UPLC-MS provides the possibility to identify lipopeptides and their structural isoforms.

A Simple, Fast, Low Cost, HPLC/UV Validated Method for Determination of Flutamide: Application to Protein Binding Studies

PubMed Central

Esmaeilzadeh, Sara; Valizadeh, Hadi; Zakeri-Milani, Parvin

2016-01-01

Purpose: The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. Methods: Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. Results: Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r2 > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. Conclusion: Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide. PMID:27478788

Analytical Method Development and Validation for the Simultaneous Estimation of Abacavir and Lamivudine by Reversed-phase High-performance Liquid Chromatography in Bulk and Tablet Dosage Forms.

PubMed

Raees Ahmad, Sufiyan Ahmad; Patil, Lalit; Mohammed Usman, Mohammed Rageeb; Imran, Mohammad; Akhtar, Rashid

2018-01-01

A simple rapid, accurate, precise, and reproducible validated reverse phase high performance liquid chromatography (HPLC) method was developed for the determination of Abacavir (ABAC) and Lamivudine (LAMI) in bulk and tablet dosage forms. The quantification was carried out using Symmetry Premsil C18 (250 mm × 4.6 mm, 5 μm) column run in isocratic way using mobile phase comprising methanol: water (0.05% orthophosphoric acid with pH 3) 83:17 v/v and a detection wavelength of 245 nm and injection volume of 20 μl, with a flow rate of 1 ml/min. In the developed method, the retention times of ABAC and LAMI were found to be 3.5 min and 7.4 min, respectively. The method was validated in terms of linearity, precision, accuracy, limits of detection, limits of quantitation, and robustness in accordance with the International Conference on Harmonization guidelines. The assay of the proposed method was found to be 99% - 101%. The recovery studies were also carried out and mean % recovery was found to be 99% - 101%. The % relative standard deviation from reproducibility was found to be <2%. The proposed method was statistically evaluated and can be applied for routine quality control analysis of ABAC and LAMI in bulk and in tablet dosage form. Attempts were made to develop RP-HPLC method for simultaneous estimation of Abacavir and Lamivudine for the RP-HPLC method. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness were within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible. So the proposed methods can be used for the routine quality control analysis of Abacavir and Lamivudine in bulk drug as well as in formulations. Abbreviations Used: HPLC: High-performance liquid chromatography, UV: Ultraviolet, ICH: International Conference on Harmonization, ABAC: Abacavir, LAMI: Lamivudine, HIV: Human immunodeficiency virus, AIDS: Acquired immunodeficiency syndrome, NRTI: Nucleoside reverse transcriptase inhibitors, ARV: Antiretroviral, RSD: Relative standard deviation, RT: Retention time, SD: Standard deviation.

A Simple and Rapid HPLC-PDA MS Method for the Profiling of Citrus Peels and Traditional Italian Liquors.

PubMed

Guccione, Clizia; Bergonzi, Maria Camilla; Piazzini, Vieri; Bilia, Anna Rita

2016-07-01

A chromatographic method for the qualitative and quantitative characterization of peels and preparations based on different species of Citrus was developed in order to obtain a complete profile of the constituents, including flavonoids and protoalkaloids. Commercial peels of sweet orange, lemon, mandarin, and grapefruit were analyzed. Seventeen constituents including flavanones, flavones, polymethoxyflavones, and protoalkaloids were identified by HPLC-PDA, HPLC-MS, and HPLC-MS/MS using a comparison of retention times and UV-Vis and MS spectra with reference standards and literature data. The total amount of flavanones [neoeriocitrin (5), naringin (8) and hesperidin (9)] and polymethoxflavones [sinensetin (12), nobiletin (14), 3,5,6,7,8,3',4'-heptamethoxyflavone (15), and tangeretin (16)] was determined and expressed as naringin (8) or hesperidin (9), and sinensetin (12), respectively. The protoalkaloid synephrine was detected in all samples, except in grapefruit, but its content was lower than the limit of quantification. Qualitative and quantitative chemical profiles of three different Italian aromatic liquors ("Limoncello", "Arancello", and "Mandarinetto"), prepared according to traditional recipes, were also analyzed. Georg Thieme Verlag KG Stuttgart · New York.

[Research on chemical fingerprint chromatograms of Sinopodophyllum hexandrum].

PubMed

Wang, Ai-Hua; Liu, Guang-Xue; Xu, Feng; Shang, Ming-Ying; Cai, Shao-Qing

2013-10-01

To study the HPLC fingerprint of Sinopodophylli Fructus, compare the major chemical differences in fruit of Sinopodophyllum hexandrum as well as the roots and rhizomas of S. hexandrum, and provide scientific evidence for clinical application and quality control. HPLC fingerprint method was used to analyze 12 fruits of S. hexandrum. A total of 20 common peaks were confirmed, and 12 peaks in the HPLC fingerprint were identified. Furthermore, similarity evaluation method and clustering analysis method were introduced to compare HPLC chromatograms, between fruits and underground parts. In this paper, quercetin-3-O-beta-D-glucopyranosid, kaempferol-3-O-beta-D-glucopyranoside, 8-prenylquercetin and 8-prenylquercetin 3-methyl ether were firstly reported in Sinopodophylli Fructus. Among the existing fingerprint research, a total of 11 peaks were identified for the first time, containing 9 flavonoids and 2 lignans. The chemical constituents differed significantly in different medicinal parts of S. hexandrum. Prenylflavonoid compounds were the main constituents of Sinopodophylli Fructus. However, podophyllotoxin, flavonoids with simple substituent groups and flavonoid glycosides were the major ingredients in the roots and rhizomas of S. hexandrum. This method can be used for the quality control of Sinopodophylli Fructus and the roots and rhizomas of S. hexandrum. It has provided a reference for the pharmacodynamic differences of the two different parts.

Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

PubMed

Katharopoulos, Efstathios; Touloupi, Katerina; Touraki, Maria

2016-08-01

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC. Copyright © 2016 Elsevier B.V. All rights reserved.

A simple analytical procedure to replace HPLC for monitoring treatment concentrations of chloramine-T on fish culture facilities

USGS Publications Warehouse

Dawson, V.K.; Meinertz, J.R.; Schmidt, L.J.; Gingerich, W.H.

2003-01-01

Concentrations of chloramine-T must be monitored during experimental treatments of fish when studying the effectiveness of the drug for controlling bacterial gill disease. A surrogate analytical method for analysis of chloramine-T to replace the existing high-performance liquid chromatography (HPLC) method is described. A surrogate method was needed because the existing HPLC method is expensive, requires a specialist to use, and is not generally available at fish hatcheries. Criteria for selection of a replacement method included ease of use, analysis time, cost, safety, sensitivity, accuracy, and precision. The most promising approach was to use the determination of chlorine concentrations as an indicator of chloramine-T. Of the currently available methods for analysis of chlorine, the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method best fit the established criteria. The surrogate method was evaluated under a variety of water quality conditions. Regression analysis of all DPD colorimetric analyses with the HPLC values produced a linear model (Y=0.9602 X+0.1259) with an r2 value of 0.9960. The average accuracy (percent recovery) of the DPD method relative to the HPLC method for the combined set of water quality data was 101.5%. The surrogate method was also evaluated with chloramine-T solutions that contained various concentrations of fish feed or selected densities of rainbow trout. When samples were analyzed within 2 h, the results of the surrogate method were consistent with those of the HPLC method. When samples with high concentrations of organic material were allowed to age more than 2 h before being analyzed, the DPD method seemed to be susceptible to interference, possibly from the development of other chloramine compounds. However, even after aging samples 6 h, the accuracy of the surrogate DPD method relative to the HPLC method was within the range of 80-120%. Based on the data comparing the two methods, the U.S. Food and Drug Administration has concluded that the DPD colorimetric method is appropriate to use to measure chloramine-T in water during pivotal efficacy trials designed to support the approval of chloramine-T for use in fish culture. ?? 2003 Elsevier Science B.V. All rights reserved.

Simultaneous determination of flubendiamide its metabolite desiodo flubendiamide residues in cabbage, tomato and pigeon pea by HPLC.

PubMed

Paramasivam, M; Banerjee, Hemanta

2011-10-01

A sensitive and simple method for simultaneous analysis of flubendiamide and its metabolite desiodo flubendiamide in cabbage, tomato and pigeon pea has been developed. The residues were extracted with QuEChERS method followed by dispersive solid-phase extraction with primary secondary amine sorbent to remove co extractives, prior to analysis by HPLC coupled with UV-Vis detector. The recoveries of flubendiamide and desiodo flubendiamide were ranged from 85.1 to 98.5% and 85.9 to 97.1% respectively with relative standard deviations (RSD) less than 5% and sensitivity of 0.01 μg g(-1). The method offers a less expensive and safer alternative to the existing residue analysis methods for vegetables. © Springer Science+Business Media, LLC 2011

Comparison of micellar extraction combined with ionic liquid based vortex-assisted liquid-liquid microextraction and modified quick, easy, cheap, effective, rugged, and safe method for the determination of difenoconazole in cowpea.

PubMed

Chen, Xiaochu; Bian, Yanli; Liu, Fengmao; Teng, Peipei; Sun, Pan

2017-10-06

Two simple sample pretreatment for the determination of difenoconazole in cowpea was developed including micellar extraction combined with ionic liquid based vortex-assisted liquid-liquid microextraction (ME-IL-VALLME) prior to high performance liquid chromatography (HPLC), and modified quick, easy, cheap, effective, rugged, and safe method (QuEChERS) coupled with HPLC-MS/MS. In ME-IL-VALLME method, the target analyte was extracted by surfactant Tween 20 micellar solution, then the supernatant was diluted with 3mL water to decrease the solubility of micellar solution. Subsequently, the vortex-assisted liquid-liquid microextraction (VALLME) procedure was performed in the diluted extraction solution by using the ionic liquid of 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIM]PF 6 ) as the extraction solvent and Tween 20 as an emulsifier to enhance the dispersion of the water-immiscible ionic liquid into the aqueous phase. Parameters that affect the extraction have been investigated in both methods Under the optimum conditions, the limits of quantitation were 0.10 and 0.05mgkg -1 , respectively. And good linearity was achieved with the correlation coefficient higher than 0.9941. The relative recoveries ranged from 78.6 to 94.8% and 92.0 to 118.0% with the relative standard deviations (RSD) of 7.9-9.6% and 1.2-3.2%, respectively. Both methods were quick, simple and inexpensive. However, the ME-IL-VALLME method provides higher enrichment factor compared with conventional QuEChERS method. The ME-IL-VALLME method has a strong potential for the determination of difenoconazole in complex vegetable matrices with HPLC. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple and sensitive HPLC method with fluorescence detection for the measurement of ibuprofen in rat plasma: application to a long-lasting dosage form.

PubMed

Hassan, Ahmed Sheikh; Sapin, Anne; Ubrich, Nathalie; Maincent, Philippe; Bolzan, Claire; Leroy, Pierre

2008-10-01

A simple and sensitive high-performance liquid chromatography (HPLC) assay applied to the measurement of ibuprofen in rat plasma has been developed. Two parameters have been investigated to improve ibuprofen detectability using fluorescence detection: variation of mobile phase pH and the use of beta-cyclodextrin (beta-CD). Increasing the pH value from 2.5 to 6.5 and adding 5 mM beta-CD enhanced the fluorescence signal (lambda(exc) = 224 nm; lambda(em) = 290 nm) by 2.5 and 1.3-fold, respectively, when using standards. In the case of plasma samples, only pH variation significantly lowered detection and quantification limits, down to 10 and 35 ng/mL, respectively. Full selectivity was obtained with a single step for plasma treatment, that is, protein precipitation with acidified acetonitrile. The validated method was applied to a pharmacokinetic study of ibuprofen encapsulated in microspheres and subcutaneously administered to rats.

Rapid determination of minoxidil in human plasma using ion-pair HPLC.

PubMed

Zarghi, A; Shafaati, A; Foroutan, S M; Khoddam, A

2004-10-29

A rapid, simple and sensitive ion-pair high-performance liquid chromatography (HPLC) method has been developed for quantification of minoxidil in plasma. The assay enables the measurement of minoxidil for therapeutic drug monitoring with a minimum detectable limit of 0.5 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was performed on an analytical 150 x 4.6 mm i.d. microbondapak C18 column. The wavelength was set at 281 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (60:40, v/v) containing 2.5 mM sodium dodecyl sulphate adjusted to pH 3.5 at a flow rate of 1 ml/min. The column temperature was set at 50 degrees C. The calibration curve was linear over the concentration range 2-100 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame

PubMed Central

Ohtsuki, Takashi; Nakamura, Ryoichiro; Kubo, Satoru; Otabe, Akira; Oobayashi, Yoko; Suzuki, Shoko; Yoshida, Mika; Yoshida, Mitsuya; Tatebe, Chiye; Sato, Kyoko; Akiyama, Hiroshi

2016-01-01

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM. PMID:27015640

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame.

PubMed

Ohtsuki, Takashi; Nakamura, Ryoichiro; Kubo, Satoru; Otabe, Akira; Oobayashi, Yoko; Suzuki, Shoko; Yoshida, Mika; Yoshida, Mitsuya; Tatebe, Chiye; Sato, Kyoko; Akiyama, Hiroshi

2016-01-01

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.

A Simple, Fast, Low Cost, HPLC/UV Validated Method for Determination of Flutamide: Application to Protein Binding Studies.

PubMed

Esmaeilzadeh, Sara; Valizadeh, Hadi; Zakeri-Milani, Parvin

2016-06-01

The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r(2) > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide.

Simple method for the extraction and reversed-phase high-performance liquid chromatographic analysis of carotenoid pigments from red yeasts (Basidiomycota, Fungi).

PubMed

Weber, Roland W S; Anke, Heidrun; Davoli, Paolo

2007-03-23

A simple method for the extraction of carotenoid pigments from frozen wet cells of red yeasts (Basidiomycota) and their analysis by reversed-phase HPLC using a C(18) column and a water/acetone solvent system is described. Typical red yeast carotenoids belonging to an oxidative series from the monocyclic gamma-carotene to 2-hydroxytorularhodin and from the bicyclic beta-carotene to astaxanthin were separated. Pigment identity was confirmed by LC-atmospheric pressure chemical ionisation (APCI) mass spectrometry using similar chromatographic conditions.

[High performance liquid chromatogram (HPLC) determination of adenosine phosphates in rat myocardium].

PubMed

Miao, Yu; Wang, Cheng-long; Yin, Hui-jun; Shi, Da-zhuo; Chen, Ke-ji

2005-04-18

To establish method for the quantitative determination of adenosine phosphates in rat myocardium by optimized high performance liquid chromatogram (HPLC). ODS HYPERSIL C(18) column and a mobile phase of 50 mmol/L tribasic potassium phosphate buffer solution (pH 6.5), with UV detector at 254 nm were used. The average recovery rates of myocardial adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were 99%-107%, 96%-104% and 95%-119%, respectively; relative standard deviations (RSDs) of within-day and between-days were less than 1.5% and 5.1%, respectively. The method is simple, rapid and accurate, and can be used to analyse the adenosine phosphates in myocardium.

[Determination of glycyrrhizinic acid in biotransformation system by reversed-phase high performance liquid chromatography].

PubMed

Li, Hui; Lu, Dingqiang; Liu, Weimin

2004-05-01

A method for determining glycyrrhizinic acid in the biotransformation system by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The HPLC conditions were as follows: Hypersil C18 column (4.6 mm i.d. x 250 mm, 5 microm) with a mixture of methanol-water-acetic acid (70:30:1, v/v) as the mobile phase; flow rate at 1.0 mL/min; and UV detection at 254 nm. The linear range of glycyrrhizinic acid was 0.2-20 microg. The recoveries were 98%-103% with relative standard deviations between 0.16% and 1.58% (n = 3). The method is simple, rapid and accurate for determining glycyrrhizinic acid.

Quantitative estimation of itopride hydrochloride and rabeprazole sodium from capsule formulation.

PubMed

Pillai, S; Singhvi, I

2008-09-01

Two simple, accurate, economical and reproducible UV spectrophotometric methods and one HPLC method for simultaneous estimation of two component drug mixture of itopride hydrochloride and rabeprazole sodium from combined capsule dosage form have been developed. First developed method involves formation and solving of simultaneous equations using 265.2 nm and 290.8 nm as two wavelengths. Second method is based on two wavelength calculation, wavelengths selected for estimation of itopride hydrochloride was 278.0 nm and 298.8 nm and for rabeprazole sodium 253.6 nm and 275.2 nm. Developed HPLC method is a reverse phase chromatographic method using phenomenex C(18) column and acetonitrile: phosphate buffer (35:65 v/v) pH 7.0 as mobile phase. All developed methods obey Beer's law in concentration range employed for respective methods. Results of analysis were validated statistically and by recovery studies.

Quantitative Estimation of Itopride Hydrochloride and Rabeprazole Sodium from Capsule Formulation

PubMed Central

Pillai, S.; Singhvi, I.

2008-01-01

Two simple, accurate, economical and reproducible UV spectrophotometric methods and one HPLC method for simultaneous estimation of two component drug mixture of itopride hydrochloride and rabeprazole sodium from combined capsule dosage form have been developed. First developed method involves formation and solving of simultaneous equations using 265.2 nm and 290.8 nm as two wavelengths. Second method is based on two wavelength calculation, wavelengths selected for estimation of itopride hydrochloride was 278.0 nm and 298.8 nm and for rabeprazole sodium 253.6 nm and 275.2 nm. Developed HPLC method is a reverse phase chromatographic method using phenomenex C18 column and acetonitrile: phosphate buffer (35:65 v/v) pH 7.0 as mobile phase. All developed methods obey Beer's law in concentration range employed for respective methods. Results of analysis were validated statistically and by recovery studies. PMID:21394269

Separation, isolation and stereochemical assignment of imazalil enantiomers and their quantitation in an in vitro toxicity test.

PubMed

Casas, Mònica Escolà; Kretschmann, Andreas Christopher; Andernach, Lars; Opatz, Till; Bester, Kai

2016-06-24

A simple method for the separation of the enantiomers of the fungicide imazalil was developed. Racemic imazalil was separated into its enantiomers with an enantiomeric purity of 99% using HPLC-UV with an enantioselective column (permethylated cyclodextrin) operated in reversed phase mode (water with 0.2% trimethylamine and 0.08% acetic acid and methanol). The absolute configuration of the separated enantiomers was assigned and unequivocally confirmed by optical rotation as well as by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) combined with ab-initio calculations. The same enantioselective column was also used to develop an HPLC-MS/MS method for the quantification of imazalil enantiomers. The HPLC-MS/MS method reached limits of quantification (LOQs) of 0.025mg/mL with 5μL injections. This method was used to verify imazalil concentrations and enantiomeric fractions in samples from an in vitro test on effects on human steroidogenesis (H295R steroidogenesis assay). The quantification verified the stability of the enantiomers of imazalil during the in vitro tests. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2).

PubMed

Taamalli, Amani; Arráez-Román, David; Ibañez, Elena; Zarrouk, Mokhtar; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

2012-01-25

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique.

PubMed

Uchiyama, Kazuhisa; Kondo, Mari; Yokochi, Rika; Takeuchi, Yuri; Yamamoto, Atsushi; Inoue, Yoshinori

2011-07-01

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and Structure Elucidation of Forced Degradation Products of the Novel Propionic acid Derivative Loxoprofen: Development of Stability-Indicating Chromatographic Methods Validated as per ICH Guidelines.

PubMed

Eissa, Maya S; Abd El-Sattar, Osama I

2017-04-01

Loxoprofen sodium (LOX) is a recently developed novel propionic acid derivative. Owing to its instability under both hydrolytic and oxidative conditions, the development of simple, rapid and sensitive methods for its determination in the presence of its possible forced degradation products becomes essential. Two simple chromatographic methods, high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC), were developed associated with ultraviolet (UV) detection. In HPTLC-densitometric method, the separation of LOX from its degradation products was achieved using silica gel F254 plates and toluene:acetone:acetic acid (1.8:1.0:0.1, v/v/v) as the developing system followed by densitometric scanning at 220 nm. In the HPLC-UV method, the separation was performed using isocratic elution system with acetonitrile: 0.15% triethylamine (pH 2.2) (50:50, v/v) on C18 analytical column. The flow rate was optimized at 1.0 mL·min-1 and UV detection was achieved at 220 nm. Validation was performed in accordance with the International Conference on Harmonization guidelines and the method was perfectly applied for determination of LOX in its pharmaceutical preparation. The results obtained were statistically compared to those obtained after application of the official HPLC method, where no significant difference was found incompliance with precision and accuracy. Identification and characterization of the possible hydrolytic degradation product under alkaline conditions and that produced during oxidative degradation using hydrogen peroxide were structurally elucidated using infrared and mass spectrometry analyses. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC-DAD and HPLC-ESI-MS.

PubMed

Karioti, Anastasia; Bolognesi, Letizia; Vincieri, Franco Francesco; Bilia, Anna Rita

2010-09-21

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC-DAD-ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC-ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids. Copyright 2010 Elsevier B.V. All rights reserved.

Simultaneous Estimation of Withaferin A and Z-Guggulsterone in Marketed Formulation by RP-HPLC.

PubMed

Agrawal, Poonam; Vegda, Rashmi; Laddha, Kirti

2015-07-01

A simple, rapid, precise and accurate high-performance liquid chromatography (HPLC) method was developed for simultaneous estimation of withaferin A and Z-guggulsterone in a polyherbal formulation containing Withania somnifera and Commiphora wightii. The chromatographic separation was achieved on a Purosphere RP-18 column (particle size 5 µm) with a mobile phase consisting of Solvent A (acetonitrile) and Solvent B (water) with the following gradients: 0-7 min, 50% A in B; 7-9 min, 50-80% A in B; 9-20 min, 80% A in B at a flow rate of 1 mL/min and detection at 235 nm. The marker compounds were well separated on the chromatogram within 20 min. The results obtained indicate accuracy and reliability of the developed simultaneous HPLC method for the quantification of withaferin A and Z-guggulsterone. The proposed method was found to be reproducible, specific, precise and accurate for simultaneous estimation of these marker compounds in a combined dosage form. The HPLC method was appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin A and Z-guggulsterone. The method can be successively used for quantitative analysis of these two marker constituents in combination of marketed polyherbal formulation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Simultaneous determination of seven lignans in Justicia procumbens by high performance liquid chromatography-photodiode array detection using relative response factors.

PubMed

Luo, Zuliang; Kong, Weijun; Qiu, Feng; Yang, Meihua; Li, Qian; Wei, Riwei; Yang, Xiaoli; Qin, Jieping

2013-02-01

A simple and sensitive HPLC coupled with photodiode array (HPLC-PDA) method was developed for simultaneous determination of seven lignans in Justicia procumbens using relative response factors (RRFs). The chromatographic separation was performed on a Shiseido Capcell Pak C(18) column (250 × 4.6 mm id, 5 μm), a gradient elution of acetonitrile/water, and a photodiode array detector. The column temperature was maintained at 35°C and the detection wavelength was set at 256 nm. Chinensinaphthol methyl ether was selected as the reference compound for calculating the relative response factors of the lignans. It has shown that the RRFs for lignans are quite similar at 256 nm of detection under different analytical conditions (different columns and HPLC instruments). Using RRFs, not every lignan is needed as a reference standard, making the method ideal for rapid, routine analysis, especially for those laboratories where lignans standards are not readily available. An economic and practicable HPLC method using RRFs was established for the determination of seven lignans in J. procumbens. This method not only can determine multiple indexes in traditional Chinese medicines (TCMs) simultaneously, but also resolve the problem of lacking of chemical standards. It will be a good quality evaluation method and pattern for TCMs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A modified HPLC method improves the simultaneous determination of plasma kynurenine and tryptophan concentrations in patients following maintenance hemodialysis

PubMed Central

XIAO, CHENGGEN; CHEN, YUANHAN; LIANG, XINLING; XIE, ZHEN; ZHANG, MIN; LI, RUIZHAO; LI, ZHILIAN; FU, XIA; YU, XIYONG; SHI, WEI

2014-01-01

The ratio between plasma kynurenine (Kyn) and tryptophan (Trp) serves as a marker of indoleamine 2,3-dioxygenase, a critical immunomodulatory molecule. Simultaneous detection of the two markers may be performed using high-pressure liquid chromatography (HPLC). However, for uremic patients, the conventional detection method may be affected by a range of accumulated toxins. The current study aimed to establish a method for the simultaneous measurement of Kyn and Trp in patients following maintenance hemodialysis via HPLC-ultraviolet detection. The procedure involved the use of a SinoChrom ODS-BP C18 column (4.6×150 mm; inner diameter, 4.5 μm) and a mobile phase of 15 mmol/l sodium acetate acetic acid solution (containing 5% acetonitrile, pH 4.8). The modified method was verified using plasma samples from 10 healthy controls and 91 maintenance hemodialysis patients. The results demonstrated that the modified method was successful in simultaneously detecting the concentrations of Trp and Kyn in the healthy controls and maintenance hemodialysis patients. The method is simple, fast, accurate and suitable for clinical and research purposes in maintenance hemodialysis patients. PMID:24669249

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.

Validated HPLC-UV method for determination of naproxen in human plasma with proven selectivity against ibuprofen and paracetamol.

PubMed

Filist, Monika; Szlaska, Iwona; Kaza, Michał; Pawiński, Tomasz

2016-06-01

Estimating the influence of interfering compounds present in the biological matrix on the determination of an analyte is one of the most important tasks during bioanalytical method development and validation. Interferences from endogenous components and, if necessary, from major metabolites as well as possible co-administered medications should be evaluated during a selectivity test. This paper describes a simple, rapid and cost-effective HPLC-UV method for the determination of naproxen in human plasma in the presence of two other analgesics, ibuprofen and paracetamol. Sample preparation is based on a simple liquid-liquid extraction procedure with a short, 5 s mixing time. Fenoprofen, which is characterized by a similar structure and properties to naproxen, was first used as the internal standard. The calibration curve is linear in the concentration range of 0.5-80.0 µg/mL, which is suitable for pharmacokinetic studies following a single 220 mg oral dose of naproxen sodium. The method was fully validated according to international guidelines and was successfully applied in a bioequivalence study in humans. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Development and Validation of a Simple High Performance Liquid Chromatography/UV Method for Simultaneous Determination of Urinary Uric Acid, Hypoxanthine, and Creatinine in Human Urine.

PubMed

Wijemanne, Nimanthi; Soysa, Preethi; Wijesundara, Sulochana; Perera, Hemamali

2018-01-01

Uric acid and hypoxanthine are produced in the catabolism of purine. Abnormal urinary levels of these products are associated with many diseases and therefore it is necessary to have a simple and rapid method to detect them. Hence, we report a simple reverse phase high performance liquid chromatography (HPLC/UV) technique, developed and validated for simultaneous analysis of uric acid, hypoxanthine, and creatinine in human urine. Urine was diluted appropriately and eluted with C-18 column 100 mm × 4.6 mm with a C-18 precolumn 25 mm × 4.6 mm in series. Potassium phosphate buffer (20 mM, pH 7.25) at a flow rate of 0.40 mL/min was employed as the solvent and peaks were detected at 235 nm. Tyrosine was used as the internal standard. The experimental conditions offered a good separation of analytes without interference of endogenous substances. The calibration curves were linear for all test compounds with a regression coefficient, r 2 > 0.99. Uric acid, creatinine, tyrosine, and hypoxanthine were eluted at 5.2, 6.1, 7.2, and 8.3 min, respectively. Intraday and interday variability were less than 4.6% for all the analytes investigated and the recovery ranged from 98 to 102%. The proposed HPLC procedure is a simple, rapid, and low cost method with high accuracy with minimum use of organic solvents. This method was successfully applied for the determination of creatinine, hypoxanthine, and uric acid in human urine.

[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.

High performance liquid chromatography used for quality control of Achyranthis Radix.

PubMed

Zhao, Bing Tian; Jeong, Su Yang; Moon, Dong Cheul; Son, Kun Ho; Son, Jong Keun; Woo, Mi Hee

2012-08-01

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 μm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

NASA Technical Reports Server (NTRS)

Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

2002-01-01

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

Comparison of UV spectrophotometry and high performance liquid chromatography methods for the determination of repaglinide in tablets

PubMed Central

Dhole, Seema M.; Khedekar, Pramod B.; Amnerkar, Nikhil D.

2012-01-01

Background: Repaglinide is a miglitinide class of antidiabetic drug used for the treatment of type 2 diabetes mellitus. A fast and reliable method for the determination of repaglinide was highly desirable to support formulation screening and quality control. Objective: UV spectrophotometric and reversed-phase high performance liquid chromatography (RP-HPLC) methods were developed for determination of repaglinide in the tablet dosage form. Materials and Methods: The UV spectrum recorded between 200 400 nm using methanol as solvent and the wavelength 241 nm was selected for the determination of repaglinide. RP-HPLC analysis was carried out using Agilent TC-C18 (2) column and mobile phase composed of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the International Conference on Harmonization (ICH) guidelines. Results: The developed methods illustrated excellent linearity (r2 > 0.999) in the concentration range of 5-30 μg/ml and 5-50 μg/ml for UV spectrophotometric and HPLC methods, respectively. Precision (%R.S.D < 1.50) and mean recoveries were found in the range of 99.63-100.45% for UV spectrophotometric method and 99.71-100.25% for HPLC method which shows accuracy of the methods. Conclusion: The developed methods were found to be reliable, simple, fast, accurate and successfully used for the quality control of repaglinide as a bulk drug and in pharmaceutical formulations. PMID:23781481

An optimized high-performance liquid chromatography (HPLC) method for benzoylmesaconine determination in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) and its products

PubMed Central

Xie, Ying; Zhou, Hua; Wong, Yuen Fan; Liu, Zhongqiu; Xu, Hongxi; Jiang, Zhihong; Liu, Liang

2008-01-01

Background Benzoylmesaconine (BMA) is the main Aconitum alkaloid in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) with potent pharmacological activities, such as analgesia and anti-inflammation. The present study developed a simple and reliable method using BMA as a marker compound for the quality control of processed aconite roots and their products. Methods After extraction, a high-performance liquid chromatography (HPLC) determination of BMA was conducted on a RP-C18 column by gradient elution with acetonitrile and aqueous phase, containing 0.1% phosphoric acid adjusted with triethylamine to pH 3.0. Results A distinct peak profile was obtained and separation of BMA was achieved. Method validation showed that the relative standard deviations (RSDs) of the precision of BMA in all intra-day and inter-day assays were less than 1.36%, and that the average recovery rate was 96.95%. Quantitative analysis of BMA showed that the content of BMA varied significantly in processed aconite roots and their products. Conclusion This HPLC method using BMA as a marker compound is applicable to the quality control of processed aconite roots and their products. PMID:18513409

Profiling indomethacin impurities using high-performance liquid chromatography and nuclear magnetic resonance.

PubMed

Hess, S; Teubert, U; Ortwein, J; Eger, K

2001-12-01

The anti-inflammatory drug indomethacin was investigated regarding new related impurities. Therefore, related substances 2-9 were prepared by independent synthesis and physicochemically characterized. To determine indomethacin and its related substances, a new HPLC-UV method was developed and validated. Indomethacin and its impurities were eluted on a C(18) column with a mobile phase consisting of methanol and an aqueous solution of 0.2% phosphoric acid at a flow rate of 1.5 ml/min and were quantified by UV detection at 320 nm. Overall, the HPLC-UV method was simple and reliable for the detection of eight impurities in indomethacin. In addition to the HPLC-UV method, 1H nuclear magnetic resonance (NMR) was used to investigate indomethacin regarding impurities. For that purpose, related substances 2-9 were systematically added to indomethacin and investigated. The NMR method was found to be very useful for the identification of impurities in bulk substance without prior separation. Both HPLC-UV and NMR were used to analyze 38 batches of indomethacin available on the European market. The outcome was that 42% of the batches did not meet the compendial requirements although they met the specifications of current compendial methods. Some batches contained the previously undescribed impurity 8, while other batches contained by-products from two distinct synthetic routes. The methods presented herein are important contributions to the ongoing efforts to reduce impurities and therefore the risk of adverse side-effects in drugs that are no longer under patent protection.

HPLC determination of strychnine and brucine in rat tissues and the distribution study of processed semen strychni.

PubMed

Chen, Jun; Hou, Ting; Fang, Yun; Chen, Zhi-peng; Liu, Xiao; Cai, Hao; Lu, Tu-lin; Yan, Guo-jun; Cai, Bao-chang

2011-01-01

A simple and low-cost HPLC method with UV absorbance detection was developed and validated to simultaneously determine strychnine and brucine, the most abundant alkaloids in the processed Semen Strychni, in rat tissues (kidney, liver, spleen, lung, heart, stomach, small intestine, brain and plasma). The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. The LOQs were in the range of 0.039-0.050 µg/ml for different tissue or plasma samples. The extraction recoveries varied from 71.63 to 98.79%. The linear range was 0.05-2 µg/ml with correlation coefficient of over 0.991. The intra- and inter-day precision was less than 15%. Then the method was used to measure the tissue distribution of strychnine and brucine after intravenous administration of 1 mg/kg crude alkaloids fraction (CAF) extracted from the processed Semen Strychni. The results revealed that strychnine and brucine possessed similar tissue distribution characterization. The highest level was observed in kidney, while the lowest level was found in brain. It was indicated that kidney might be the primary excretion organ of prototype strychnine and brucine. It was also deduced that strychnine and brucine had difficulty in crossing the blood-brain barrier. Furthermore, no long-term accumulation of strychnine and brucine was found in rat tissues.

A sensitive determination method for carbendazim and thiabendazole in apples by solid-phase microextraction-high performance liquid chromatography with fluorescence detection.

PubMed

Hu, Yanxue; Yang, Xiumin; Wang, Chun; Zhao, Jin; Li, Weining; Wang, Zhi

2008-03-01

A new analytical method for the determination of carbendazim (MBC) and thiabendazole (TBZ) in apples is reported, based on solid-phase microextraction (SPME) coupling HPLC with fluorescence detection. The main SPME and HPLC experimental conditions were optimized. The apples were first blended and centrifuged. Then, an aliquot of the resulting solution was subjected to SPME on a 60 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre for 35 min at room temperature with the solution being stirred at 1100 rev min(-1). The extracted pesticides on the SPME fibre were desorbed in the mobile phase into the SPME/HPLC interface for HPLC analysis. The method was linear over the range 0.01-1 mg kg(-1) in apples for both MBC and TBZ, with detection limits of 0.005 and 0.003 mg kg(-1) and correlation coefficients of 0.9995 and 0.9998, respectively. The average recoveries for MBC and TBZ were 91.5 and 92.3% with the relative standard deviations (RSD) of 4.7 and 4.1% at the 0.1 mg kg(-1) level, and 94.6 and 96.1% with RSD of 3.3 and 3.8% at the 0.5 mg kg(-1) level, respectively. The method is simple, sensitive, organic solvent-free and is suitable for the determination of MBC and TBZ in apples.

Comparison of UV spectrophotometry and high performance liquid chromatography methods for the determination of repaglinide in tablets.

PubMed

Dhole, Seema M; Khedekar, Pramod B; Amnerkar, Nikhil D

2012-07-01

Repaglinide is a miglitinide class of antidiabetic drug used for the treatment of type 2 diabetes mellitus. A fast and reliable method for the determination of repaglinide was highly desirable to support formulation screening and quality control. UV spectrophotometric and reversed-phase high performance liquid chromatography (RP-HPLC) methods were developed for determination of repaglinide in the tablet dosage form. The UV spectrum recorded between 200 400 nm using methanol as solvent and the wavelength 241 nm was selected for the determination of repaglinide. RP-HPLC analysis was carried out using Agilent TC-C18 (2) column and mobile phase composed of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the International Conference on Harmonization (ICH) guidelines. The developed methods illustrated excellent linearity (r(2) > 0.999) in the concentration range of 5-30 μg/ml and 5-50 μg/ml for UV spectrophotometric and HPLC methods, respectively. Precision (%R.S.D < 1.50) and mean recoveries were found in the range of 99.63-100.45% for UV spectrophotometric method and 99.71-100.25% for HPLC method which shows accuracy of the methods. The developed methods were found to be reliable, simple, fast, accurate and successfully used for the quality control of repaglinide as a bulk drug and in pharmaceutical formulations.

Analysis and stability of retinol in plasma.

PubMed

Peng, Y M; Xu, M J; Alberts, D S

1987-01-01

A simple, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous measurement of retinol (ROH), 13-cis-retinoic acid (13-cRA), and 4-oxo-13-cRA. The average recovery of ROH from serum or plasma was 95%, and the precision of the assay was less than 5%. With this HPLC method, a series of studies was carried out to evaluate the stability of ROH in various matrices. ROH was stable under our HPLC assay conditions as well as in plasma- and in serum-enriched culture media; however, ROH was not stable in aqueous matrices. Serum or heparinized plasma may be routinely used for measurement of ROH concentrations, providing EDTA, oxalate, and citrate are not used as anticoagulants. Because of ROH stability, blood samples can be kept on ice in the dark for at least 24 hours prior to separation of plasma. In addition, plasma samples containing ROH can be stored for up to 1 year at -20 degrees C without loss of stability.

Determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine and its metabolites in human plasma and urine by column-switching high-performance liquid chromatography with ultraviolet detection.

PubMed

Ono, I; Matsuda, K; Kanno, S

1997-05-09

A simple, rapid and sensitive two column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (AY4166, I) and its seven metabolites in human plasma and urine. Measurements of I and its metabolites were carried out by two column-switching HPLC, because metabolites were classified into two groups according to their retention times. After purification of plasma samples using solid-phase extraction and direct dilution of urinary samples, I and each metabolite were injected into HPLC. The calibration graphs for plasma and urinary samples were linear in the ranges 0.1 to 10 microg ml(-1) and 0.5 to 50 microg ml(-1), respectively. Recoveries of I and its seven metabolites were over 88% by the standard addition method and the relative standard deviations of I and its metabolites were 1-6%.

Simultaneous extraction, identification and quantification of phenolic compounds in Eclipta prostrata using microwave-assisted extraction combined with HPLC-DAD-ESI-MS/MS.

PubMed

Fang, Xinsheng; Wang, Jianhua; Hao, Jifu; Li, Xueke; Guo, Ning

2015-12-01

A simple and rapid method was developed using microwave-assisted extraction (MAE) combined with HPLC-DAD-ESI-MS/MS for the simultaneous extraction, identification, and quantification of phenolic compounds in Eclipta prostrata, a common herb and vegetable in China. The optimized parameters of MAE were: employing 50% ethanol as solvent, microwave power 400 W, temperature 70 °C, ratio of liquid/solid 30 mL/g and extraction time 2 min. Compared to conventional extraction methods, the optimized MAE can avoid the degradation of the phenolic compounds and simultaneously obtained the highest yields of all components faster with less consumption of solvent and energy. Six phenolic acids, six flavonoid glycosides and one coumarin were firstly identified. The phenolic compounds were quantified by HPLC-DAD with good linearity, precision, and accuracy. The extract obtained by MAE showed significant antioxidant activity. The proposed method provides a valuable and green analytical methodology for the investigation of phenolic components in natural plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorescent HPLC for the detection of specific protein oxidation carbonyls - α-aminoadipic and γ-glutamic semialdehydes - in meat systems.

PubMed

Utrera, Mariana; Morcuende, David; Rodríguez-Carpena, Javier-Germán; Estévez, Mario

2011-12-01

Precise methodologies for the routine analysis of particular protein carbonyls are required in order to progress in this topic of increasing interest. The present paper originally describes the application of an improved method for the detection of α-aminoadipic and γ-glutamic semialdehydes in a meat system by using a derivatization procedure with p-amino-benzoic acid (ABA) followed by fluorescent high-performance liquid chromatography (HPLC). The method development comprises i) the description of a simple HPLC program which allows the efficient separation of the ABA and the key standard compounds and ii) the optimization of the procedure for the preparation of a meat sample in order to maximize the fluorescent signal for both protein carbonyls. Furthermore, the suitability of this method is evaluated by applying the technique to porcine burger patties. The present procedure enables an accurate and relatively fast analysis of both semialdehydes in meat samples in which they could play an interesting role as reliable indicators of protein oxidation. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Determination of protopine in Corydalis racemose by HPLC].

PubMed

Jiang, Xiazhi; Ye, Jinxia; Zeng, Jianwei; Zou, Xiuhong; Wu, Jinzhong

2010-09-01

To develop a HPLC method for determining the content of protopine in Corydalis racemose. Analysis was performed on a Gemini C18 column (4.6 mm x 250 mm, 5 microm) eluted with acetonitrile-water containing 0.8% triethylamine and 3% acetic acid acetum (20:80) as the mobile phase. The flow rate was 1.0 mL x min(-1). The detection wavelength was 289 nm. The average content of protopine in Herb of Racemose Corydalis was 0.905%. The calibration curve of protopine was linear between 0.124-1.36 microg (r = 0.9999). The average recovery was 98.49% with RSD 1.9%. This method is simple, reproducible and can be used to determine the content of protopine in C. racemose.

Optimisation of solid-phase microextraction coupled to HPLC-UV for the determination of organochlorine pesticides and their metabolites in environmental liquid samples.

PubMed

Torres Padrón, M E; Sosa Ferrera, Z; Santana Rodríguez, J J

2006-09-01

A solid-phase microextraction (SPME) procedure using two commercial fibers coupled with high-performance liquid chromatography (HPLC) is presented for the extraction and determination of organochlorine pesticides in water samples. We have evaluated the extraction efficiency of this kind of compound using two different fibers: 60-mum polydimethylsiloxane-divinylbenzene (PDMS-DVB) and Carbowax/TPR-100 (CW/TPR). Parameters involved in the extraction and desorption procedures (e.g. extraction time, ionic strength, extraction temperature, desorption and soaking time) were studied and optimized to achieve the maximum efficiency. Results indicate that both PDMS-DVB and CW/TPR fibers are suitable for the extraction of this type of compound, and a simple calibration curve method based on simple aqueous standards can be used. All the correlation coefficients were better than 0.9950, and the RSDs ranged from 7% to 13% for 60-mum PDMS-DVB fiber and from 3% to 10% for CW/TPR fiber. Optimized procedures were applied to the determination of a mixture of six organochlorine pesticides in environmental liquid samples (sea, sewage and ground waters), employing HPLC with UV-diode array detector.

A simple screening method using ion chromatography for the diagnosis of cerebral creatine deficiency syndromes.

PubMed

Wada, Takahito; Shimbo, Hiroko; Osaka, Hitoshi

2012-08-01

Cerebral creatine deficiency syndromes (CCDS) are caused by genetic defects in L-arginine:glycine amidinotransferase, guanidinoacetate methyltransferase or creatine transporter 1. CCDS are characterized by abnormal concentrations of urinary creatine (CR), guanidinoacetic acid (GA), or creatinine (CN). In this study, we describe a simple HPLC method to determine the concentrations of CR, GA, and CN using a weak-acid ion chromatography column with a UV detector without any derivatization. CR, GA, and CN were separated clearly with the retention times (mean ± SD, n = 3) of 5.54 ± 0.0035 min for CR, 6.41 ± 0.0079 min for GA, and 13.53 ± 0.046 min for CN. This new method should provide a simple screening test for the diagnosis of CCDS.

Rapid and simple immunochemical screening combined with hand-shaking extraction for thiamethoxam residue in agricultural products.

PubMed

Watanabe, Eiki; Kobara, Yuso; Miyake, Shiro

2013-06-01

With the aim of expanding the applicability of a kit-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide thiamethoxam, the ELISA was newly applied to three kinds of agricultural samples (green pepper, eggplant and spinach). To offer the ELISA as a screening analysis for thiamethoxam residues, a rapid and simple method of extraction by hand-shaking was used, and speed-up and simplification of the sample treatment before the ELISA analysis were examined. Finally, the validity of the ELISA combined with the proposed extraction method was verified against a reference high-performance liquid chromatography (HPLC) method using real-world agricultural samples. There were no marked matrix effects derived from green pepper and eggplant extracts. On the other hand, although the effect due to a pigment in spinach extract on the assay performance was significant, it was effectively avoided by increasing the dilution level of the spinach extract. For thiamethoxam-spiked samples, acceptable recoveries of 97.9-109.1% and coefficients of variation of 0.3-11.5% were obtained. Inspection of the validity of the ELISA by comparison with the reference HPLC method showed that the two analytical results were very similar, and a high correlation was found between them (r>0.997). The evaluated ELISA combined with hand-shaking extraction provided a rapid and simple screening analysis that was quantitative and reliable for the detection of thiamethoxam in complex agricultural products. © 2012 Society of Chemical Industry.

A simple one-step ultrasonic-assisted extraction and derivatization method coupling to high-performance liquid chromatographyfor the determination of ε-aminocaproic acid and amino acids in cosmetics.

PubMed

Du, Yuanqi; Xia, Ling; Xiao, Xiaohua; Li, Gongke; Chen, Xiaoguang

2018-06-15

Nowadays, the safety of cosmetics is a widespread concern. Amines are common cosmetic additives. Some of them such as amino acids are beneficial. Another kind of amines, however, ε-aminocaproic acid (EACA) is prohibited to add into cosmetics for its adverse reactions. In this study, a simple, rapid, sensitive and eco-friendly one-step ultrasonic-assisted extraction and derivatization (UAE-D) method was developed for determination of EACA and amino acids in cosmetics by coupling with high-performance liquid chromatography (HPLC). By using this sample preparation method, extraction and derivatization of EACA and amino acids were finished in one step in ultrasound field. During this procedure, 4-fluoro-7-nitrobenzofurazan (NBD-F)was applied as derivatization reagent. The extraction conditions including the amount of NBD-F, extraction and derivatization temperature, the ultrasonic vibration time and pH value of the aqueous phase were evaluated. Meanwhile, the extraction mechanism was investigated. Under optimized conditions, the method detection limits were 0.086-0.15 μg/L, and method quantitation limits were 0.29-0.47 μg/L with RSDs less than 3.7% (n = 3). The recoveries of EACA and amino acids obtained from cosmetic samples were in range from 76.9% to 122.3%. Amino acids were found in all selected samples and quantified in range from 1.9 ± 0.9 to 677.2 ± 17.9 μg/kg. And EACA was found and quantified with the contents of 1284.3 ± 22.1 μg/kg in a toner sample. This UAE-D-HPLC method shortened and simplified the sample pretreatment as well as enhanced the sensitivity of analytical method. In our record, only 10 min was needed for the total sample preparation process. And the method detection limits were two orders of magnitude less than literature reports. Furthermore, we reduced the consumption of solvent and minimized the usage of organic solvents, which made our method moving towards green analytical chemistry. In brief, our UAE-D-HPLC method is a simple, rapid, sensitive and eco-friendly analytical method for the determination of EACA and amino acids in cosmetics. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and validation of RP HPLC method to determine nandrolone phenylpropionate in different pharmaceutical formulations.

PubMed

Mukherjee, Jayanti; Das, Ayan; Chakrabarty, Uday Sankar; Sahoo, Bijay Kumar; Dey, Goutam; Choudhury, Hira; Pal, Tapan Kumar

2011-01-01

This study describes development and subsequent validation of a reversed phase high performance liquid chromatographic (RP-HPLC) method for the estimation of nandrolone phenylpropionate, an anabolic steroid, in bulk drug, in conventional parenteral dosage formulation and in prepared nanoparticle dosage form. The chromatographic system consisted of a Luna Phenomenex, CN (250 mm x 4.6 mm, 5 microm) column, an isocratic mobile phase comprising 10 mM phosphate buffer and acetonitrile (50:50, v/v) and UV detection at 240 nm. Nandrolone phenylpropionate was eluted about 6.3 min with no interfering peaks of excipients used for the preparation of dosage forms. The method was linear over the range from 0.050 to 25 microg/mL in raw drug (r2 = 0.9994). The intra-day and inter-day precision values were in the range of 0.219-0.609% and 0.441-0.875%, respectively. Limits of detection and quantitation were 0.010 microg/mL and 0.050 microg/mL, respectively. The results were validated according to International Conference on Harmonization (ICH) guidelines in parenteral and prepared nanoparticle formulation. The validated HPLC method is simple, sensitive, precise, accurate and reproducible.

[Direct determination of purine bases in tea by reversed-phase high performance liquid chromatography].

PubMed

Ding, M; Yang, H; Xiao, S; Chen, P

1999-09-01

A reversed-phase high performance liquid chromatographic(RP-HPLC) method for the direct determination of three purine bases(theobromin, theophyllin and caffeine) in tea was developed. An ODS column with Zorbax SB-C18(4.6 mm i.d. x 250 mm, 5 microns) was employed. The aqueous solution of methanol containing 0.05% of acetic acid and 0.25% of N,N-dimethylformamide(DMF) was used as eluent with a flow rate of 0.8 mL/min. In this method, the aqueous extract of tea can be injected into HPLC directly, but in current HPLC methods for purine bases the coexisted tea polyphenols must be pre-separated. The three purine bases in tea were separated without any interference from the coexisted tea polyphenols. This method is simple (without any special sample pretreatment) and sensitive with detection limits (S/N = 3) of 0.7, 0.9 and 1.8 mg/L for theobromin, theophyllin and caffeine respectively. The linear range of the calibration curve of peak area for the three purine bases were from 6 mg/L to 1,000 mg/L with a correlation coefficient (r) of 0.998-0.999.

[Determination of dimethyl fumarate in bakery food by d-SPE-HPLC-PDA].

PubMed

Yang, Jie; Luo, Mengtian; Feng, Di; Miao, Hong; Song, Shufeng; Zhao, Yunfeng

2015-05-01

To establish a simple and rapid pretreatment method with dispersive solid phase extraction ( d-SPE) by HPLC for determination of dimethyl fumarate in bakery foods. Dimethyl fumarate in samples was ultrasonically extracted by methanol, and cleaned up with d-SPE. Then, it was separated on C18 chromatographic column (4.6 mm x 25 mm, 5 μm) with a mixture of methanol--0.03 mol/L sodium acetate and 0.008 mol/L tetrabutyl ammonium bromide (40: 60, V/V) as mobile phase. The photodiode array detector was used in the determination under λ = 220 nm. In the linear range of 0.1 -25 μg/ml, the correlation coefficients was r > 0.999, and the average recoveries of the spiked samples were in the range of 82.8% - 107.5% with relative standard deviations (RSD) in the range of 3.30% - 7.30% (n = 6). The limit of detection ( LOD) was 0.4 mg/kg, and the limit of quantification was 1.0 mg/kg. The method is simple, rapid, sensitive and accurate, and suitable for determine dimethyl fumarate in bakery foods.

[Simultaneous determination of eleven components in Ginkgo biloba leaves by high performance liquid chromatography method].

PubMed

Lv, Jin-Li; Yang, Biao; Li, Meng-Xuan; Meng, Zhao-Qing; Ma, Shi-Ping; Wang, Zhen-Zhong; Ding, Gang; Huang, Wen-Zhe; Xiao, Wei

2017-03-01

To study Ginkgo biloba leaves in different producing area, we establish an HPLC method for the simultaneously determination of seven flavonoids glycosides and four biflavonoids in G. biloba leaves. The analysis was performed on an Agilent ZORBAX SB-C₁₈ column(4.6 mm×250 mm, 5 μm) wich acetonitrile, and 0.4% phosphoric acid as mobile phase at flow rate of 1 mL•min⁻¹ in a gradient edution, and the detection was carried out at 254 nm.The calibration curves of the seven flavonoids glycosides and four biflavonoids had a good linearitiy with good recoveries. The established HPLC method is simple, rapid, accurate, reliable, and sensitive, and can be applied to the identification and quality control of G. biloba leaves. Copyright© by the Chinese Pharmaceutical Association.

High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

PubMed

dos Santos, W D; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, O

2006-01-01

This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.

Quantifying fenbendazole and its metabolites in self-medicating wild red grouse Lagopus lagopus scoticus using an HPLC-MS-MS approach.

PubMed

Adam, Aileen; Webster, Lucy M I; Mullen, William; Keller, Lukas F; Johnson, Paul C D

2011-05-11

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC-MS-MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control. Copyright © 2011 Elsevier B.V. All rights reserved.

A combined simple bubbling method with high performance liquid chromatography purification strategy, higher radiochemical yield and purity and faster preparation of carbon-11-raclopride.

PubMed

Huang, Huacheng; Ning, Yanli; Zhang, Bucheng; Lou, Cen

2015-01-01

Carbon-11-raclopride (¹¹C-R) is a positron-emitting radiotracer successfully used for the study of cognitive control and widely applied in PET imaging. A simple automated preparation of ¹¹C-R by using the reaction of carbon-(11)-methyl triflate (¹¹C-MeOTF) or ¹¹C-methyl iodide (¹¹C-MeI) with demethylraclopride is described. Specifically we used a simple setup applied an additional "U" reaction vessel for ¹¹C-MeOTf compared with ¹¹C-MeI and assessed the influence of several solvents and of the amount of the percussor for ¹¹C-methylation of demethylraclopride by the bubbling method. The reversal of retention order between product and its precursor has been achieved for ¹¹C-R, enabling collection of the purified ¹¹C-R by using the HPLC column after shorter retention time. By the improved radiosynthesis and purification strategy, ¹¹C-R could be prepared with higher radiochemical yield than that of the previous studies. The yield for ¹¹C-MeOTf was 76% and for ¹¹C-CH3I >26% and with better radiochemical purity (>99% based on both ¹¹C-MeOTf and ¹¹C-MeI) as compared to the previously obtained purity of ¹¹C-R using HPLC method with acetonitrile as a part of mobile phase. Furthermore, by using ethanol as the organic modifier, residual solvent analysis prior to human injection could be avoided and ¹¹C-R could be injected directly following simple dilution and sterile filtration. Improved radiosynthesis and HPLC purification in combination with ethanol containing eluent, extremely shortened the time for preparation of ¹¹C-R, gave a higher radiochemical yield and purity for ¹¹C-R and can be used for multiple and faster synthesis of ¹¹C-R and probably for other ¹¹C-labeled radiopharmaceuticals.

Q Sepharose micro-column chromatography: A simple screening method for identifying beta thalassemia traits and hemoglobin E carriers.

PubMed

Wong, Peerapon; Sritippayawan, Suchila; Suwannakhon, Narutchala; Tapprom, Akamon; Deoisares, Rawisut; Sanguansermsri, Torpong

2016-11-01

For beta thalassemia control program in pregnancy, mass screening of the carrier state by determination of the hemoglobin (Hb) A 2 and Hb E proportions and mutation analysis is a preferred method for making prenatal diagnoses. Q Sepharose micro-column chromatography, developed for the determination of Hb A 2 and Hb E for screening purposes, was compared with high performance liquid chromatography (HPLC) to ascertain its relative accuracy and reliability. Results using Q Sepharose micro-column chromatography in 350 blood specimens, including 50 samples genetically proven to be beta thalassemia heterozygotes, were compared to HPLC for validation. An additional study was conducted to test a clinical application on a large-scale survey for beta thalassemia in 1581 pregnant women and their spouses. The mean (±SD) Hb A 2 proportions in the normal and genetically proven beta thalassemia heterozygotes were 2.70±0.40% and 6.30±1.23%, respectively, as determined by Q-Sepharose micro-column chromatography, and 2.65±0.31% and 5.37±0.96%, respectively, as determined by HPLC. The mean Hb E proportions in the Hb E heterozygotes were 23.25±4.13% and 24.72±3.5% as determined by Q Sepharose micro-column chromatography and HPLC, respectively. In the large-scale survey for beta thalassemia, 23 at risk couples were detected. Seven affected fetuses were identified by prenatal diagnosis. Q Sepharose micro-column chromatography was found to be reliable, reproducible and well-suited for large-scale surveys. Additionally, by being reusable and convenient, this simple and economical chromatography method may be an alternative means to screen for beta thalassemia and Hb E carriers in the mass population. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Magnetic solid-phase extraction of heterocyclic pesticides in environmental water samples using metal-organic frameworks coupled to high performance liquid chromatography determination.

PubMed

Ma, Jiping; Wu, Gege; Li, Shuang; Tan, Weiqiang; Wang, Xiaoyan; Li, Jinhua; Chen, Lingxin

2018-06-08

A simple method of magnetic solid-phase extraction (MSPE) coupled to high performance liquid chromatography (HPLC) was developed for the simultaneous extraction and determination of four kinds of heterocyclic pesticides (carbendazim, triadimefon, chlorfenapyr and fenpyroximate) in environmental water samples. Magnetic metal-organic frameworks (MOFs) of type MOF-5 were prepared and used as adsorbents of MSPE. Several main parameters influencing MSPE efficiency were investigated, including amount of magnetic MOF-5, sample solution pH, extraction time, salt concentration, type and volume of desorption solvents and desorption time. Under optimal conditions, the MSPE-HPLC method presented fast simple separation and analysis, and excellent linearity in the range of 0.3-500.0 μg/L for carbendazim and triadimefon, and 0.1-500.0 μg/L for chlorfenapyr and fenpyroximate, with correlation coefficients (r) higher than 0.9992. High sensitivity with limits of detection and quantification ranging from 0.04-0.11 μg/L and 0.13-0.35 μg/L, respectively, were achieved, as well as good precision with relative standard deviations of 2.98-7.11% (intra-day) and 3.31-7.12% (inter-day). Furthermore, the method was successfully applied to reservoir and Yellow River water samples, and satisfactory recoveries at three spiked concentration levels were between 80.20% and 108.33%.The magnetic MOF-5 composites based MSPE followed by HPLC proved promising for convenient and efficient determination of heterocyclic pesticides in environmental water samples. Copyright © 2018 Elsevier B.V. All rights reserved.

A simple HPLC method for simultaneous determination of lopinavir, ritonavir and efavirenz.

PubMed

Usami, Yoshiko; Oki, Tsuyoshi; Nakai, Masahiko; Sagisaka, Masafumi; Kaneda, Tsuguhiro

2003-06-01

We developed a simple HPLC method for the simultaneous determination of lopinavir (LPV), ritonavir (RTV) and efavirenz (EFV) to evaluate the efficiency of co-administration of LPV/RTV and EFV in Japanese patients enrolled in a clinical study. The monitoring of LPV plasma concentration is important because co-administration of LPV/RTV with EFV sometimes decreases plasma concentrations of LPV caused by EFV activation of cytochrome P-450 3A. A solution of acetonitrile, methanol and tetramethylammonium perchlorate (TMAP) in dilute aqueous trifluoroacetic acid (TFA) has been used as the mobile phase in a HPLC method to elute LPV and RTV. We found that a solvent ratio of 45 : 5 : 50 (v/v/v) of acetonitrile/methanol/0.02 M TMAP in 0.2% TFA optimized separation of LPV, RTV and EFV. A column temperature of 30 degrees C was necessary for the reproducibility of the analyses. Standard curves were linear in the range 0.060 to 24.06 micro g/ml for LPV, 0.010 to 4.16 micro g/ml for RTV, and 0.047 to 37.44 micro g/ml for EFV. Coefficients of variation (CVs) of LPV, RTV and EFV in intraday and interday assays ranged from 1.5 to 4.0%, 2.5 to 16.8% and 1.0 to 7.7%, respectively. Accuracies ranged from 100 to 110%, 101 to 116% and 99 to 106% for LPV, RTV and EFV, respectively. The extraction recoveries were 77-87, 77-83 and 81-91% for LPV, RTV and EFV, respectively.

Development and validation of stability indicating method for the quantitative determination of venlafaxine hydrochloride in extended release formulation using high performance liquid chromatography

PubMed Central

Kaur, Jaspreet; Srinivasan, K. K.; Joseph, Alex; Gupta, Abhishek; Singh, Yogendra; Srinivas, Kona S.; Jain, Garima

2010-01-01

Objective: Venlafaxine,hydrochloride is a structurally novel phenethyl bicyclic antidepressant, and is usually categorized as a serotonin–norepinephrine reuptake inhibitor (SNRI) but it has been referred to as a serotonin–norepinephrine–dopamine reuptake inhibitor. It inhibits the reuptake of dopamine. Venlafaxine HCL is widely prescribed in the form of sustained release formulations. In the current article we are reporting the development and validation of a fast and simple stability indicating, isocratic high performance liquid chromatographic (HPLC) method for the determination of venlafaxine hydrochloride in sustained release formulations. Materials and Methods: The quantitative determination of venlafaxine hydrochloride was performed on a Kromasil C18 analytical column (250 × 4.6 mm i.d., 5 μm particle size) with 0.01 M phosphate buffer (pH 4.5): methanol (40: 60) as a mobile phase, at a flow rate of 1.0 ml/min. For HPLC methods, UV detection was made at 225 nm. Results: During method validation, parameters such as precision, linearity, accuracy, stability, limit of quantification and detection and specificity were evaluated, which remained within acceptable limits. Conclusions: The method has been successfully applied for the quantification and dissolution profiling of Venlafaxine HCL in sustained release formulation. The method presents a simple and reliable solution for the routine quantitative analysis of Venlafaxine HCL. PMID:21814426

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC-Photodiode Array Detection Method.

PubMed

Kumar, Satyanshu; Singh, Raghuraj; Gajbhiye, Narendra; Dhanani, Tushar

2018-06-26

Background : Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective : The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods : The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results : The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions : The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera -enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights : A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera . Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera , crop improvement, bio-prospection studies and quality control.

Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 5600™ Mass Spectrometer.

PubMed

Deng, Shuang; Scott, David; Myers, Douglas; Garg, Uttam

2016-01-01

Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple protein precipitation, reverse phase C8 column chromatography, ion pairing with tributylamine, and long run time of 50 min to separate the two isomers (G3P and DHAP).

Validation of an HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface of pharmaceutical manufacturing equipments.

PubMed

Akl, Magda A; Ahmed, Mona A; Ramadan, Ahmed

2011-05-15

In pharmaceutical industry, an important step consists in the removal of possible drug residues from the involved equipments and areas. The cleaning procedures must be validated and methods to determine trace amounts of drugs have, therefore, to be considered with special attention. An HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface was developed and validated in order to control a cleaning procedure. Cotton swabs, moistened with extraction solution (50% water and 50% mobile phase), were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of 91.12, 93.8 and 98.7% for three concentration levels. The precision of the results, reported as the relative standard deviation (RSD), were below 1.5%. The method was validated over a concentration range of 1.15-6.92 μg ml(-1). Low quantities of drug residues were determined by HPLC-UV using a Hypersil ODS 5 μm (250×4.6 mm) at 50 °C with an acetonitrile:water:pH 7:pH 5 (39-55-5.5-0.5) mobile phase at flow rate of 1.5 ml min(-1), an injection volume of 20 μl and were detected at 254 nm. A simple, selective and sensitive HPLC-UV assay for the determination of ceftriaxone sodium residues on stainless steel surfaces was developed, validated and applied. Copyright © 2011 Elsevier B.V. All rights reserved.

Stress Degradation Studies on Varenicline Tartrate and Development of a Validated Stability-Indicating HPLC Method

PubMed Central

Pujeri, Sudhakar S.; Khader, Addagadde M. A.; Seetharamappa, Jaldappagari

2012-01-01

A simple, rapid and stability-indicating reversed-phase liquid chromatographic method was developed for the assay of varenicline tartrate (VRT) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm i.d. particle size is 5 μm) employing a mobile phase consisting of ammonium acetate buffer containing trifluoroacetic acid (0.02M; pH 4) and acetonitrile in gradient program mode with a flow rate of 1.0 mL min−1. The UV detector was operated at 237 nm while column temperature was maintained at 40 °C. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness and limit of quantification. The method was found to be simple, specific, precise and accurate. Selectivity of the proposed method was validated by subjecting the stock solution of VRT to acidic, basic, photolysis, oxidative and thermal degradation. The calibration curve was found to be linear in the concentration range of 0.1–192 μg mL−1 (R2 = 0.9994). The peaks of degradation products did not interfere with that of pure VRT. The utility of the developed method was examined by analyzing the tablets containing VRT. The results of analysis were subjected to statistical analysis. PMID:22396908

Composition of Indigo naturalis.

PubMed

Plitzko, Inken; Mohn, Tobias; Sedlacek, Natalie; Hamburger, Matthias

2009-06-01

A proposal for a European Pharmacopoeia monograph concerning Indigo naturalis has recently been published, whereby the indigo (1) and indirubin (2) content should be determined by HPLC-UV. This method was tested, but problems were seen with the dosage of indigo due to poor solubility. A quantitative assay for indigo based on (1)H-NMR was developed as an alternative. The HPLC and qNMR assays were compared with eight Indigo naturalis samples. The HPLC assay consistently gave much lower indigo concentrations because solubility was the limiting factor in sample preparation. In one sample, sucrose was identified by (1)H-NMR as an organic additive. Simple wet chemistry assays for undeclared additives such as sugars and starch were tested with artificially spiked Indigo naturalis samples to establish their limits of detection, and sulfate ash determinations were carried out in view of a better assessment of Indigo naturalis in a future European monograph.

Quantification of the Triazole Antifungal Compounds Voriconazole and Posaconazole in Human Serum or Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

PubMed

Molinelli, Alejandro R; Rose, Charles H

2016-01-01

Voriconazole and posaconazole are triazole antifungal compounds used in the treatment of fungal infections. Therapeutic drug monitoring of both compounds is recommended in order to guide drug dosing to achieve optimal blood concentrations. In this chapter we describe an HPLC-ESI-MS/MS method for the quantification of both compounds in human plasma or serum following a simple specimen preparation procedure. Specimen preparation consists of protein precipitation using methanol and acetonitrile followed by a cleanup step that involves filtration through a cellulose acetate membrane. The specimen is then injected into an HPLC-ESI-MS/MS equipped with a C18 column and separated over an acetonitrile gradient. Quantification of the drugs in the specimen is achieved by comparing the response of the unknown specimen to that of the calibrators in the standard curve using multiple reaction monitoring.

Spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of Curcuma aromatica.

PubMed

Liu, Meiqiong; Wu, Youjiao; Huang, Shushi; Liu, Huagang; Feng, Jie

2018-02-23

Curcuma aromatica is used as a traditional Chinese medicine, and it is mainly distributed in Guangxi, China. In this study, 10 batches of C. aromatica were collected from different origins in Guangxi. The fingerprints were established by HPLC technique to investigate the quality stability of C. aromatica. The spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of C. aromatica was assessed by similarity analysis, gray relational analysis and multiple linear regression analysis. From the results, the similarity values between each batch of C. aromatica and reference fingerprint were >0.880, indicating the good quality stability of the 10 batches of C. aromatica. Twenty common peaks were selected as the fingerprints to evaluate the quality and hypolipidemic effect of C. aromatica. The results of spectrum-effect relationship showed that peaks 10, 18, 13, 15 and 17 in the fingerprints were closely related to hypolipidemic effect. This study successfully established the spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of C. aromatica, which provided methods for quality control and more effectively studies on bioactive compounds of C. aromatica. It could also provide a new simple and effective method for utilizing the fingerprints to optimize the Chinese prescription and develop traditional Chinese medicine. Copyright © 2018 John Wiley & Sons, Ltd.

Comparison of the effects of Mylabris and Acanthopanax senticosus on promising cancer marker polyamines in plasma of a Hepatoma-22 mouse model using HPLC-ESI-MS.

PubMed

Wang, Qian; Wang, Yixiang; Liu, Ran; Yan, Xu; Li, Yujiao; Fu, Hui; Bi, Kaishun; Li, Qing

2013-02-01

A simple and sensitive method for the simultaneous determination of plasma concentrations of five polyamines in normal and Hepatoma-22 mice, and mice treated with Mylabris and Acanthopanax senticosus was developed by HPLC-ESI-MS. Male Kunming mice were divided into nine groups, a control group (inoculation without treatment), a positive group (Cyclophosphamide), treatment groups [Mylabris (4, 8, 16 mg/kg), Acanthopanax senticosus (6, 12, 24 g/kg)] and a normal group (without inoculation). Twenty-four hours after the last administration, plasma samples were collected. The derived polyamines were separated on a C(18) column by a gradient elution using methanol-water with excellent linearity within the range from 2.5 to 1000 ng/mL. Polyamines were confirmed as useful biochemical markers of hepatoma. The differences in anti-cancer therapeutic efficacy between Mylabris and Acanthopanax senticosus might contribute to the variability of polyamine levels in vivo. This HPLC-ESI-MS method was successfully applied to investigate the relationship between polyamines and cancer in mice and might be a useful method to test the activity of potential anti-tumor drugs. Copyright © 2012 John Wiley & Sons, Ltd.

A simple high performance liquid chromatography method for determination of rebamipide in rat urine.

PubMed

Cooper, Dustin L; Harirforoosh, Sam

2014-01-01

Rebamipide is a mucoprotective agent commonly used to prevent nonsteriodal anti-inflammatory drug-induced gastrointenstinal side effects [1]. Human plasma and urine analysis of rebamipide utilizing high performance liquid chromatography (HPLC) have been reported [2]. Recently, we reported on the plasma levels of rebamipide in presense or absence of celecoxib or diclofenac in rats [3] using a modified HPLC method of detection developed by Jeoung et al. [4]. To tailor the method towards use in urinary rebamipide extraction and analysis, the following modifications were made:•To compensate for high concentrations of rebamipide found in urine, a new rebamipide stock solution was prepared with a final concentration of 50,000 ng/mL.•Rat urine calibration standards were obtained within the range of 50-1000 ng/mL and 1000-50,000 ng/mL.•Plasma samples were replaced with urine samples.

Simultaneous Determination of Eight Hypotensive Drugs of Various Chemical Groups in Pharmaceutical Preparations by HPLC-DAD.

PubMed

Stolarczyk, Mariusz; Hubicka, Urszula; Żuromska-Witek, Barbara; Krzek, Jan

2015-01-01

A new sensitive, simple, rapid, and precise HPLC method with diode array detection has been developed for separation and simultaneous determination of hydrochlorothiazide, furosemide, torasemide, losartane, quinapril, valsartan, spironolactone, and canrenone in combined pharmaceutical dosage forms. The chromatographic analysis of the tested drugs was performed on an ACE C18, 100 Å, 250×4.6 mm, 5 μm particle size column with 0.0.05 M phosphate buffer (pH=3.00)-acetonitrile-methanol (30+20+50 v/v/v) mobile phase at a flow rate of 1.0 mL/min. The column was thermostatted at 25°C. UV detection was performed at 230 nm. Analysis time was 10 min. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The proposed method was successfully applied for the determination of the studied drugs in the selected combined dosage forms.

[Determination of content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes by RP-HPLC method].

PubMed

Han, Meihua; Chen, Jing; Chen, Shilin; Wang, Xiangtao

2009-05-01

To establish a RP-HPLC method for content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes. The separation was performed with a COSMOSIL 5 C18-MS-II column (4.6 mm x 250 mm, 5 mmicrom) using methanol-water (95:5) as the mobile phase and detected at 203 nm. The flow rate was 1.0 mL x min(-1) and 50 microL sample solution was injected for each time. The calibration curve was linear within the range 0.1-0.5 mg x mL(-1) (r = 0. 9999) , the intra-day RSD and inter-day RSD were less than 2% and the average recovery was between 101.44%-103.11% (n = 3). The method is simple, accurate, sensitive and applicable for determination of content and entrapment efficiency of 20 (S)-protopanaxadiol pharmacosomes.

Use of an online extraction liquid chromatography quadrupole time-of-flight tandem mass spectrometry method for the characterization of polyphenols in Citrus paradisi cv. Changshanhuyu peel.

PubMed

Tong, Chaoying; Peng, Mijun; Tong, Runna; Ma, Ruyi; Guo, Keke; Shi, Shuyun

2018-01-19

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC-DAD-QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction of Citrus paradisi cv. Changshanhuyu (Changshanhuyu) peel, OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of twenty-two secondary metabolites were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and nine of them were discovered in Changshanhuyu peel for the first time to our knowledge. It is concluded that the developed OLE-HPLC-DAD-QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

[Determination of biphenyl ether herbicides in water using HPLC with cloud-point extraction].

PubMed

He, Cheng-Yan; Li, Yuan-Qian; Wang, Shen-Jiao; Ouyang, Hua-Xue; Zheng, Bo

2010-01-01

To determine residues of multiple biphenyl ether herbicides simultaneously in water using high performance liquid chromatography (HPLC) with cloud-point extraction. The residues of eight biphenyl ether herbicides (including bentazone, fomesafen, acifluorfen, aclonifen, bifenox, fluoroglycofenethy, nitrofen, oxyfluorfen) in water samples were extracted with cloud-point extraction of Triton X-114. The analytes were separated and determined using reverse phase HPLC with ultraviolet detector at 300 nm. Optimized conditions for the pretreatment of water samples and the parameters of chromatographic separation applied. There was a good linear correlation between the concentration and the peak area of the analytes in the range of 0.05-2.00 mg/L (r = 0.9991-0.9998). Except bentazone, the spiked recoveries of the biphenyl ether herbicides in the water samples ranged from 80.1% to 100.9%, with relative standard deviations ranging from 2.70% to 6.40%. The detection limit of the method ranged from 0.10 microg/L to 0.50 microg/L. The proposed method is simple, rapid and sensitive, and can meet the requirements of determination of multiple biphenyl ether herbicides simultaneously in natural waters.

Aflatoxin B1 in eggs and chicken livers by dispersive liquid-liquid microextraction and HPLC.

PubMed

Amirkhizi, Behzad; Arefhosseini, Seyed Rafie; Ansarin, Masoud; Nemati, Mahboob

2015-01-01

A rapid, low-cost and simple technique has been developed for the determination of aflatoxin B1 (AFB1) in eggs and livers using high-performance liquid chromatography (HPLC) with UV detection. In this study, the presence of AFB1 was investigated in 150 eggs and 50 chicken livers from the local market of Tabriz, Iran. AFB1 was extracted with a mixture of acetonitrile:water (80:20) and cleaned up by dispersive liquid-liquid microextraction which is a very economical, fast and sensitive method. AFB1 was quantified by HPLC-UV without need for any complex derivatisation in samples to enhance the detection. The results showed that 72% of the liver and 58% of the egg samples were contaminated with AFB1 ranging from 0.30 to 16.36 µg kg (̶1). limit of detection and limit of quantification for AFB1 were 0.08 and 0.28 µg kg (̶ 1), respectively. The proposed method is suitable for fast analysing of AFB1 in egg and liver samples.

Flavylium chromophores as species markers for dragon's blood resins from Dracaena and Daemonorops trees.

PubMed

Sousa, Micaela M; Melo, Maria J; Parola, A Jorge; Seixas de Melo, J Sérgio; Catarino, Fernando; Pina, Fernando; Cook, Frances E M; Simmonds, Monique S J; Lopes, João A

2008-10-31

A simple and rapid liquid chromatographic method with diode-array UV-vis spectrophotometric detection has been developed for the authentication of dragon's blood resins from Dracaena and Daemonorops trees. Using this method it was discovered that the flavylium chromophores, which contribute to the red colour of these resins, differ among the species and could be used as markers to differentiate among species. A study of parameters, such as time of extraction, proportion of MeOH and pH, was undertaken to optimise the extraction of the flavyliums. This method was then used to make extracts from samples of dragon's blood resin obtained from material of known provenance. From the samples analysed 7,6-dihydroxy-5-methoxyflavylium (dracorhodin), 7,4'-dihydroxy-5-methoxyflavylium (dracoflavylium) and 7,4'-dihydroxyflavylium were selected as species markers for Daemonorops spp., Dracaena draco and Dracaena cinnabari, respectively. The chromatograms from these samples were used to build an HPLC-DAD database. The ability to discriminate among species of dragon's blood using the single marker compounds was compared with a principal components analysis of the chromatograms in the HPLC-DAD database. The results from the HPLC-DAD method based on the presence of these flavylium markers was unequivocal. The HPLC-DAD method was subsequently applied to 37 samples of dragon blood resins from the historical samples in the Economic Botany Collection, Royal Botanic Gardens, Kew. The method identified anomalies in how samples in this collection had been labelled. It is clear that the method can be used to evaluate the provenance of samples used in different areas of cultural heritage. It also could be used to monitor the trade of endangered species of dragon's blood and the species being used in complex formulations of traditional Chinese medicine.

A Simple and Specific Stability- Indicating RP-HPLC Method for Routine Assay of Adefovir Dipivoxil in Bulk and Tablet Dosage Form.

PubMed

Darsazan, Bahar; Shafaati, Alireza; Mortazavi, Seyed Alireza; Zarghi, Afshin

2017-01-01

A simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of adefovir dipivoxil (ADV).The chromatographic separation was performed on a C 18 column using a mixture of acetonitrile-citrate buffer (10 mM at pH 5.2) 36:64 (%v/v) as mobile phase, at a flow rate of 1.5 mL/min. Detection was carried out at 260 nm and a sharp peak was obtained for ADV at a retention time of 5.8 ± 0.01 min. No interferences were observed from its stress degradation products. The method was validated according to the international guidelines. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 0.5-16 μg/mL; the regression coefficient was 0.9999and the linear regression equation was y = 24844x-2941.3. The detection (LOD) and quantification (LOQ) limits were 0.12 and 0.35 μg/mL, respectively. The results proved the method was fast (analysis time less than 7 min), precise, reproducible, and accurate for analysis of ADV over a wide range of concentration. The proposed specific method was used for routine quantification of ADV in pharmaceutical bulk and a tablet dosage form.

Stability of Cyclophosphamide in Extemporaneous Oral Suspensions

PubMed Central

Kennedy, Rachel; Groepper, Daniel; Tagen, Michael; Christensen, Robbin; Navid, Fariba; Gajjar, Amar; Stewart, Clinton F.

2010-01-01

Background Cyclophosphamide, an alkylating agent, is widely used for the treatment of many adult and pediatric malignancies. The stability of cyclophosphamide in aqueous- and methylcellulose-based oral suspending vehicles is currently unknown. Objectives The goals of this study were (1) to develop and validate a stability-indicating HPLC method to measure cyclophosphamide concentrations in simple syrup and Ora-Plus, and (2) to assess the 56-day chemical stability and physical appearance of cyclophosphamide in these suspensions at both room temperature and 4°C. Methods The i.v. formulation of cyclophosphamide was diluted to 20 mg/mL in normal saline, compounded 1:1 with either suspending vehicle, and stored in the dark in 3mL amber polypropylene oral syringes at 4°C and 22°C. Aliquots from each syringe were obtained on days 0, 3, 7, 14, 21, 28, 35, 42, 49, and 56 and assayed using the validated stability-indicating HPLC-UV method. A C18 analytical column was used to separate cyclophosphamide from the internal standard, ifosfamide, with a mobile phase of 21% acetonitrile in 79% sodium phosphate buffer. The suspension was examined for odor change, visually examined under normal fluorescent light for color change, and examined under a light microscope for evidence of microbial growth. Results Samples of cyclophosphamide in both simple syrup and Ora-Plus were stable when kept at 4°C for at least 56 days. At room temperature, cyclophosphamide in simple syrup and Ora-Plus had a shelf life of 8 and 3 days, respectively. No changes in color or odor or evidence of microbial growth were observed. Conclusion Cyclophosphamide can be extemporaneously prepared in simple syrup or Ora-Plus and stored at least 2 months under refrigeration without significant degradation. PMID:20103616

Rapid Discrimination for Traditional Complex Herbal Medicines from Different Parts, Collection Time, and Origins Using High-Performance Liquid Chromatography and Near-Infrared Spectral Fingerprints with Aid of Pattern Recognition Methods

PubMed Central

Fu, Haiyan; Fan, Yao; Zhang, Xu; Lan, Hanyue; Yang, Tianming; Shao, Mei; Li, Sihan

2015-01-01

As an effective method, the fingerprint technique, which emphasized the whole compositions of samples, has already been used in various fields, especially in identifying and assessing the quality of herbal medicines. High-performance liquid chromatography (HPLC) and near-infrared (NIR), with their unique characteristics of reliability, versatility, precision, and simple measurement, played an important role among all the fingerprint techniques. In this paper, a supervised pattern recognition method based on PLSDA algorithm by HPLC and NIR has been established to identify the information of Hibiscus mutabilis L. and Berberidis radix, two common kinds of herbal medicines. By comparing component analysis (PCA), linear discriminant analysis (LDA), and particularly partial least squares discriminant analysis (PLSDA) with different fingerprint preprocessing of NIR spectra variables, PLSDA model showed perfect functions on the analysis of samples as well as chromatograms. Most important, this pattern recognition method by HPLC and NIR can be used to identify different collection parts, collection time, and different origins or various species belonging to the same genera of herbal medicines which proved to be a promising approach for the identification of complex information of herbal medicines. PMID:26345990

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

PubMed Central

Moserová, Michaela; Kotrbová, Věra; Aimová, Dagmar; Šulc, Miroslav; Frei, Eva; Stiborová, Marie

2009-01-01

A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP. PMID:21217860

Quantitative estimation of gymnemagenin in Gymnema sylvestre extract and its marketed formulations using the HPLC-ESI-MS/MS method.

PubMed

Kamble, Bhagyashree; Gupta, Ankur; Patil, Dada; Janrao, Shirish; Khatal, Laxman; Duraiswamy, B

2013-02-01

Gymnema sylvestre, with gymnemic acids as active pharmacological constituents, is a popular ayurvedic herb and has been used to treat diabetes, as a remedy for cough and as a diuretic. However, very few analytical methods are available for quality control of this herb and its marketed formulations. To develop and validate a new, rapid, sensitive and selective HPLC-ESI (electrospray ionisation)-MS/MS method for quantitative estimation of gymnemagenin in G. sylvestre and its marketed formulations. HPLC-ESI-MS/MS method using a multiple reactions monitoring mode was used for quantitation of gymnemagenin. Separation was carried out on a Luna C-18 column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia). The developed method was validated as per International Conference on Harmonisation Guideline ICH-Q2B and found to be accurate, precise and linear over a relatively wide range of concentrations (5.280-305.920 ng/mL). Gymnemagenin contents were found from 0.056 ± 0.002 to 4.77 ± 0.59% w/w in G. sylvestre and its marketed formulations. The method established is simple, rapid, with high sample throughput, and can be used as a tool for quality control of G. sylvestre and its formulations. Copyright © 2012 John Wiley & Sons, Ltd.

Chemometrics enhanced HPLC-DAD performance for rapid quantification of carbamazepine and phenobarbital in human serum samples.

PubMed

Vosough, Maryam; Ghafghazi, Shiva; Sabetkasaei, Masoumeh

2014-02-01

This paper describes development and validation of a simple and efficient bioanalytical procedure for simultaneous determination of phenobarbital and carbamazepine in human serum samples using high performance liquid chromatography with photodiode-array detection (HPLC-DAD) regarding a fast elution methodology in less than 5 min. Briefly, this method consisted of a simple deproteinization step of serum samples followed by HPLC analysis on a Bonus-RP column using an isocratic mode of elution with acetonitrile/K2HPO4 (pH=7.5) buffer solution (45:55). Due to the presence of serum endogenous components as non-calibrated components in the sample, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS), has been applied on a set of absorbance matrices collected as a function of retention time and wavelengths. Acceptable resolution and quantification results were achieved in the presence of matrix interferences and the second-order advantage was fully exploited. The average recoveries for carbamazepine and phenobarbital were 89.7% and 86.1% and relative standard deviation values were lower than 9%. Additionally, computed elliptical joint confidence region (EJCR) confirmed the accuracy of the proposed method and indicated the absence of both constant and proportional errors in the predicted concentrations. The developed method enabled the determination of the analytes in different serum samples in the presence of overlapped profiles, while keeping experimental time and extraction steps at minimum. Finally, the serum concentration levels of carbamazepine in three time intervals were reported for morphine-dependents who had received carbamazepine for treating their neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

Determination of the ratio between mercaptalbumin and nonmercaptalbumin by HPLC with fluorescence probe specifically binding to albumin.

PubMed

Ohyama, Kaname; Kishikawa, Naoya; Matsuo, Aya; Imazato, Takahiro; Ueki, Yukitaka; Wada, Mitsuhiro; Nakashima, Kenichiro; Kuroda, Naotaka

2014-01-01

A simple and selective HPLC-fluorescence (FL) method with FL probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM), for simultaneous determination of mercaptalbumin (HMA) and nonmercaptalbumin (HNA1) was developed. After HMA and HNA1 were separated on an ion-exchange column, they were on-line and post-column mixed with DAPIM. The DAPIM-albumin complex produces FL (λex 370nm and λem 510nm); however, DAPIM solution never gives the FL. Based on this mechanism, selective determination of HMA and HNA1 were achieved without any pretreatment and interfering peak. The proposed method was applied to the measurement of HMA and HNA1 in human serum of healthy volunteers and diabetes mellitus patients. Copyright © 2013 Elsevier B.V. All rights reserved.

Paired-ion chromatography and high performance liquid chromatography of labetalol in feeds.

PubMed

Townley, E R; Ross, B

1980-11-01

A high performance liquid chromatographic (HPLC) method using reverse phase paired-ion chromatography and ultraviolet detection at 280 nm has been developed to determine labetalol, an alpha and beta adrenoceptor blocking agent, in Purina No. 5001 rodent chow. The method is simple and rapid, and demonstrates a separation technique applicable to other acidic and basic drugs. It requires only extraction of the drug with methanol--water--acetic acid (66 + 33 + 1) and separation of insoluble material by filtration before HPLC. Labetalol, is chromatographically separated from soluble feed components by means of a microBondapak C18 column and methanol--water--acetic acid (66 + 33 + 1) mobile phase, 0.005M with respect to sodium dioctylsulfosuccinate paired-ion reagent. Average recovery is 98.7% with a relative standard deviation of +/- 2.3% for the equipment described.

Simultaneous determination of bioactive compounds in Piper nigrum L. and a species comparison study using HPLC-PDA.

PubMed

Rao, Vidadala Rama Subba; Raju, Sagi Satyanarayana; Sarma, Vanka Umamaheswara; Sabine, Fouriner; Babu, Kothapalli Hari; Babu, Katragadda Suresh; Rao, Janaswamy Madhusudana

2011-08-01

Piper nigrum L. is a traditional medicine widely used in India for illnesses such as constipation, diarrhoea, earache, gangrene, heart disease, hernia, hoarseness, indigestion, insect bites, insomnia, joint pain, liver problems, lung disease, oral abscesses, sunburn, tooth decay and toothaches. In this study, six bioactive compounds, namely piperine (1), pellitorine (2), guineensine (3), pipnoohine (4), trichostachine (5) and piperonal (6) were quantified in different extracts of P. nigrum L. and compared with those of P. longum L. and P. chaba Hunter. To evaluate the quality of P. nigrum, a simple, accurate and precise HPLC-PDA method was developed for the simultaneous determination of the above-mentioned six compounds. The separation was achieved by Phenomenex Luna RP C(18) column (150 × 4.6 mm, 5 µm, Phenomenex Inc, CA, USA) with a binary gradient solvent system of water-acetonitrile, at a flow rate of 1.0 mL min(-1) and detected at 210, 232, 262 and 343 nm. All six calibration curves showed good linearity (R (2) > 0.9966). The method was reproducible with intra- and inter-day variations of less than 2% and 5%, respectively. The results demonstrated that this method is simple, reliable and suitable for the quality control of these plants.

Simple and sensitive determination of five quinolones in food by liquid chromatography with fluorescence detection.

PubMed

Ramos, Macarena; Aranda, Angela; Garcia, Elena; Reuvers, Thea; Hooghuis, Henny

2003-06-15

A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C(18) column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile-0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile-0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g(-1), except for sarafloxacin which had a limit of 10 ng g(-1). Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r(2) higher than 0.999 for all quinolones.

Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India

PubMed Central

Venkataramana, M.; Rashmi, R.; Uppalapati, Siva R.; Chandranayaka, S.; Balakrishna, K.; Radhika, M.; Gupta, Vijai K.; Batra, H. V.

2015-01-01

In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken. The generated mAb was further used to develop a simple, fast, and sensitive sandwich dot-ELISA (s-dot ELISA) method for detection of OTA from contaminated food grain samples. The limit of detection (LOD) of the developed enzyme-linked immunosorbent assay (ELISA) method was determined as 5.0 ng/mL of OTA. Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1. To assess the utility and reliability of the developed method, several field samples of maize, wheat and rice (n = 195) collected from different geographical regions of southern Karnataka region of India were evaluated for the OTA occurrence. Seventy two out of 195 samples (19 maize, 38 wheat, and 15 rice) were found to be contaminated by OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were negative for OTA presence by s-dot ELISA but found positive by HPLC. Although positive by HPLC, the amount of OTA in the three samples was found to be lesser than the accepted levels (>5 μg/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods. PMID:26074899

Novel Chromatographic Methods for Simultaneous Quantification of Fish and Wheat Germ Oils Mixture in Pharmaceutical Dosage Forms.

PubMed

El-Yazbi, Amira F; El-Hawiet, Amr

2017-05-01

Two simple, direct and environment-friendly chromatographic methods, high-performance liquid chromatography (HPLC) and high-performance thin layer chromatographic (HPTLC), were developed for the determination of a binary mixture of fish oil (FO) and wheat germ oil (WGO), for the first time, in their pharmaceutical dosage forms with no need for any sample pretreatment. The HPLC separation was carried out using C-18 stationary phase with mobile phase of 15% formic acid (pH 6), methanol and acetonitrile through gradient-elution, 1.5 mL min-1 flow-rate and detection at 215 nm for FO and 280 nm for WGO. HPTLC separation was carried out on silica-coated plates using diethyl ether-petroleum ether (0.5:9.5, v/v) as mobile phase. Detection was at 215 nm for FO and 240 nm for WGO. Regression analysis showed good linear relationship with r > 0.999 in the concentration-ranges of 0.2-2 mg mL-1 and 2.5-20 μg band-1 for WGO by HPLC and HPTLC methods, respectively, and 0.4-10 mg mL-1 and 25-200 μg band-1 for FO by HPLC and HPTLC methods, respectively. The methods were validated, showed good analytical performance and were successfully applied for the analysis of pharmaceutical formulations and synthetic mixtures of the analytes with good recoveries. Therefore, the two methods could be conveniently adopted for routine analysis of similar products in quality control laboratories of pharmaceutical industries especially that simultaneous determination of FO-WGO mixture has not been reported previously. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

L-Phenylalanine concentration in blood of phenylketonuria patients: a modified enzyme colorimetric assay compared with amino acid analysis, tandem mass spectrometry, and HPLC methods.

PubMed

De Silva, Veronica; Oldham, Charlie D; May, Sheldon W

2010-09-01

Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (Phe) to L-tyrosine, typically resulting from a deficiency in activity of a hepatic and renal enzyme L-phenylalanine hydroxylase. The disease is characterized by an increased concentration of Phe and its metabolites in body fluids. A modified assay based on an enzymatic-colorimetric methodology was developed for measuring blood Phe levels in PKU patients; this method is designed for use with undeproteinized samples and avoids the use of solvents or amphiphilic agents. Thus, the method could be suitable for incorporation into a simple home-monitoring device. We report here on a comparison of blood Phe concentrations in PKU patients measured in undeproteinized plasma using this enzyme colorimetric assay (ECA), with values determined by amino acid analysis (AAA) of deproteinized samples, and HPLC and tandem mass spectrometry (MS/MS) analyses of dried blood spot (DBS) eluates. Pearson correlation coefficients of 0.951, 0.976 and 0.988 were obtained when AAA-measured Phe concentrations were compared with the ECA-, HPLC- or MS/MS-measured values, respectively. A Bland-Altman analysis revealed that mean Phe concentrations determined using AAA were on average 65 μmol/L lower than values measured by our ECA. These results may be the result of minimizing the manipulations performed on the patient sample compared with AAA, HPLC, and MS/MS methods, which involve plasma deproteinization or DBS elution and derivatization. The results reported here confirm that Phe concentrations determined by our ECA method are comparable to those determined by other widely used methods for a broad range of plasma Phe concentrations.

A simple, sensitive and rapid isocratic reversed-phase high-performance liquid chromatography method for determination and stability study of curcumin in pharmaceutical samples

PubMed Central

Amanolahi, Farjad; Mohammadi, Ali; Kazemi Oskuee, Reza; Nassirli, Hooriyeh; Malaekeh-Nikouei, Bizhan

2017-01-01

Objective: This study was designed to develop and validate a new reversed-phase high-performance liquid chromatography (RP-HPLC) method based on Q2 (R1) International Conference on Harmonization (ICH) guideline for determination of curcumin in pharmaceutical samples. Materials and Methods: The HPLC instrument method was optimized with isocratic elution with acetonitrile: ammonium acetate (45:55, v/v, pH 3.5), C18 column (150 mm×4.6 mm×5 µm particle size) and a flow rate of 1 ml/min in ambient condition and total retention time of 17 min. The volume of injection was set at 20 µl and detection was recorded at 425 nm. The robustness of the method was examined by changing the mobile phase composition, mobile phase pH, and flow rate. Results: The method was validated with respect to precision, accuracy and linearity in a concentration range of 2-100 µg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.5 µg/ml, respectively. The percentage of recovery was 98.9 to 100.5 with relative standard deviation (RSD) < 0.638%. Conclusion: The method was found to be simple, sensitive and rapid for determination of curcumin in pharmaceutical samples and had enough sensitivity to detect degradation product of curcumin produced under photolysis and hydrolysis stress condition. PMID:29062806

Determination and confirmation of isopropyl p-toluenesulfonate in cosmetics by HPLC-diode array detector method and GC-MS.

PubMed

Tay, B Y P; Yung, S C; Teoh, T Y

2016-12-01

Isopropyl p-toluenesulfonate (IPTS) is a potentially genotoxic by-product formed during the esterification of palm oil-based palmitic and palm kernel oil-based myristic acid with isopropanol to produce isopropyl palmitate or isopropyl myristate. There are no methods described for the analysis of IPTS in cosmetic products. In this work, we have established a simple, precise and accurate method to determine the presence and level of IPTS in various finished cosmetic products which contain palm-based esters in their formulations. An Agilent 1200 series high-performance liquid chromatography (HPLC) unit using a diode-array detector (DAD) has been employed and optimized to detect IPTS in cosmetic products. For the separation, a reverse-phase Hypersil Gold C8 column (5 μm, 4.6 mm i.d. 250 mm) 5 mM tetrabutylammonium phosphate buffer 50 : 50, (v/v) solution in acetonitrile as mobile phase, in isocratic mode and a flow rate of 0.8 mL min -1 were used. A second method using a gas chromatography/mass selective detector GC-MSD was also developed to confirm the IPTS identity in the cosmetic products. Recoveries of IPTS from cosmetic matrices such as a lotion, cleansing milk and a cream ranged from 94.0% to 101.1% with <5% relative standard deviation (%RSD) showing good accuracy and repeatability of the method. The six-point calibration curves (determined over the range 0.5-50 μg mL -1 ) have a correlation coefficient of 0.9999 (based on HPLC peak area) and 0.9998 (based on HPLC peak height). The intra- and interday precisions (measured by the %RSD) of the method were <2% and <5%, respectively, indicating that the developed method is reliable, precise and reproducible. The detection and quantification limit of the method were found to be 0.5 μg mL -1 and 1.6 μg mL -1 , respectively. Analyses of 83 commercial cosmetics showed no presence of IPTS. The validation data indicated that this method was suitable for the quantitative analysis of IPTS in commercial cosmetics. This method is applicable for analyses of trace levels of IPTS in cosmetics and has the advantage of using only simple sample preparation steps. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Simple Quantification of Pentosidine in Human Urine and Plasma by High-Performance Liquid Chromatography

PubMed Central

Lee, Ji Sang; Chung, Yoon-Sok; Chang, Sun Young

2017-01-01

Pentosidine is an advanced glycation end-product (AGE) and fluorescent cross-link compound. A simple high-performance liquid chromatographic (HPLC) method was developed for the detection and quantification of pentosidine in human urine and plasma. The mobile phase used a gradient system to improve separation of pentosidine from endogenous peaks, and chromatograms were monitored by fluorescent detector set at excitation and emission wavelengths of 328 and 378 nm, respectively. The retention time for pentosidine was 24.3 min and the lower limits of quantification (LLOQ) in human urine and plasma were 1 nM. The intraday assay precisions (coefficients of variation) were generally low and found to be in the range of 5.19–7.49% and 4.96–8.78% for human urine and plasma, respectively. The corresponding values of the interday assay precisions were 9.45% and 4.27%. Accuracies (relative errors) ranged from 87.9% to 115%. Pentosidine was stable in a range of pH solutions, human urine, and plasma. In summary, this HPLC method can be applied in future preclinical and clinical evaluation of pentosidine in the diabetic patients. PMID:29181026

Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag.

PubMed

Song, Xuezheng; Lasanajak, Yi; Rivera-Marrero, Carlos; Luyai, Anthony; Willard, Margaret; Smith, David F; Cummings, Richard D

2009-12-15

Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by Pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core alpha1,3-fucose and core alpha1,2-xylose. After simple Fmoc deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.

Measurement of fumonisins in corn with a fiber optic fluoroimmunosensor

NASA Astrophysics Data System (ADS)

Thompson, Vicki S.; Maragos, Chris M.

1997-05-01

A fiber-optic immunosensor was used to determine concentrations of the mycotoxin fumonisin B1(FB1) in both spiked and naturally contaminated corn samples. Samples were extracted with a mixture of methanol/water. Two methods were used to prepare the methanolic corn extracts before introduction to the immunosensor: (1) simple dilution of the methanolic corn extract; or (2) affinity column cleanup. The sensor displayed an IC50 of 70 ng FB1/mL when toxin was introduced in phosphate buffered saline. Simple dilution of methanolic corn extracts yielded an assay with an IC50 equivalent to 25 (mu) gFB1/g corn and a limit of detection of 3.2 (mu) g/g corn, while affinity cleanup of corn extracts yielded an assay with an IC50 of 5 (mu) gFB1/g corn and a limit of detection of 0.4 (mu) gFB1/g corn. The difference in sensitivity between the two cleanup techniques was due to concentration of fumonisins obtained from the affinity cleanup procedure. Naturally contaminated corn samples were also analyzed after either simple dilution or affinity column cleanup. For comparison the naturally contaminated corn samples were analyzed with an HPLC method after isolation of the fumonisins with strong anion exchange (SAX) solid phase extraction cartridges. The SAX/HPLC method and the immunosensor method agreed well except when large amounts of other fumonisins (i.e. fumonisin B2) were present. This was due in part to the cross-reactivity of the monoclonal antibody with other fumonisins. The immunosensor has the potential to screen individual corn samples for fumonisins within six minutes, and is among the fastest of the currently available FB1 detection methods.

Combination of HPLC chromatogram and hypoglycemic effect identifies isoflavones as the principal active fraction of Belamcanda chinensis leaf extract in diabetes treatment.

PubMed

Chen, Yan; Wu, Chong-Ming; Dai, Rong-Ji; Li, Liang; Yu, Yu-Hong; Li, Yan; Meng, Wei-Wei; Zhang, Liang; Zhang, Yongqian; Deng, Yu-Lin

2011-02-15

In previous study, we demonstrated the hypoglycemic effect of aqueous extract of Belamcanda chinensis leaves in rats. Here, we separated the aqueous extract of B. chinensis leaves and investigated the spectrum-effect relationships between HPLC chromatograms and hypoglycemic activities of different isolates from B. chinensis leaf extract. Sequential solvent extraction with petroleum ether, chloroform, acetic ester and n-butanol provided several isolates showing similar hypoglycemic activities, making it difficult to discriminate the active fractions. Stepwise elution through HP20 macroporous resin by water, 40% and 95% ethanol provided isolates with distinct hypoglycemic activities, representing a simple, rapid and efficient preparative separation method. Combination of HPLC chromatogram and pharmacological effect targeted a hypoglycemic activity-related region in HPLC chromatogram. Each peak in this region was analyzed by UV spectrum scan. Most of them were flavonoids in which tectoridin and swertisin were known flavonoids with anti-diabetic activities. In together, this work provides a general model of combination of HPLC chromatography and pharmacological effect to study the spectrum-effect relationships of aqueous extract from B. chinensis leaves, which can be used to find principle components of B. chinensis on pharmacological activity. Copyright © 2011 Elsevier B.V. All rights reserved.

Screening and Analysis of the Marker Components in Ganoderma lucidum by HPLC and HPLC-MSn with the Aid of Chemometrics.

PubMed

Wu, Lingfang; Liang, Wenyi; Chen, Wenjing; Li, Shi; Cui, Yaping; Qi, Qi; Zhang, Lanzhen

2017-04-06

Ganoderma triterpenes (GTs) are the major secondary metabolites of Ganoderma lucidum , which is a popularly used traditional Chinese medicine for complementary cancer therapy. The present study was to establish a fingerprint evaluation system based on Similarity Analysis (SA), Cluster Analysis (CA) and Principal Component Analysis (PCA) for the identification and quality control of G. lucidum . Fifteen samples from the Chinese provinces of Hainan, Neimeng, Shangdong, Jilin, Anhui, Henan, Yunnan, Guangxi and Fujian were analyzed by HPLC-PAD and HPLC-MS n . Forty-seven compounds were detected by HPLC, of which forty-two compounds were tentatively identified by comparing their retention times and mass spectrometry data with that of reference compounds and reviewing the literature. Ganoderic acid B, 3,7,15-trihydroxy-11,23-dioxolanost-8,16-dien-26-oic acid, lucidenic acid A, ganoderic acid G, and 3,7-oxo-12-acetylganoderic acid DM were deemed to be the marker compounds to distinguish the samples with different quality according to both CA and PCA. This study provides helpful chemical information for further research on the anti-tumor activity and mechanism of action of G. lucidum . The results proved that fingerprints combined with chemometrics are a simple, rapid and effective method for the quality control of G. lucidum .

A novel, simple and inexpensive procedure for the simultaneous determination of iopamidol and p-aminohippuric acid for renal function assessment from plasma samples in awake rats.

PubMed

Rodríguez-Romero, Violeta; González-Villalva, Karla I; Reyes, José L; Franco-Bourland, Rebecca E; Guízar-Sahagún, Gabriel; Castañeda-Hernández, Gilberto; Cruz-Antonio, Leticia

2015-03-25

The purpose of the current study was to design, validate and implement a novel analytical method for the simultaneous plasma measurement of iopamidol and p-aminohippuric acid (PAH) to estimate renal function in awake rats. A reverse-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous measurement of iopamidol (for glomerular filtration rate estimation, GFR) and PAH (for tubular secretion determination, TS) was designed and validated using a C-18 column, 0.1M acetic acid-10% acetonitrile (90:10, v/v) as mobile phase, at a flow rate of 0.3 ml/min, and UV detection at 270 nm. Iopamidol (244.8 mg/kg) was administered intravenously followed immediately by sodium PAH (100 mg/kg) to healthy female Sprague-Dawley rats. Plasma samples obtained at 2.5, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min after drug administration were deproteinized with 2.5% trichloroacetic acid containing p-aminobenzoic acid as internal standard, and separated by the validated RP-HPLC method described above. The iopamidol and PAH chromatographic data were analyzed using a non-compartmental model. The results demonstrated that the RP-HPLC method was linear in ranges between 15-120 μg/ml and 2.5-120 μg/ml for iopamidol and PAH, respectively. Precision and accuracy were within 15% for both drugs. Recovery of iopamidol and PAH was 92% and 100%, respectively. Plasma iopamidol and PAH clearances in awake rats, estimates for GFR and TS, respectively, were 1.49±0.20 ml/min and 3.73±0.38 ml/min. In conclusion, the method here described is a simple and reliable procedure, for the simultaneous and time-saving determination of GFR and TS from plasma samples in the conscious rat. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid and sensitive method for determination of withaferin-A in human plasma by HPLC.

PubMed

Patial, Pankaj; Gota, Vikram

2011-02-01

To develop and validate a rapid and sensitive high-performance liquid chromatographic method for determination of withaferin-A in human plasma. Withaferin-A, the active molecule of a traditional Indian herb, has demonstrated several biological activities in preclinical models. A validated bioassay is not available for its pharmacokinetic evaluation. The chromatographic system used a reverse-phase C18 column with UV-visible detection at 225 nm. The mobile phase consisted of water and acetonitrile applied in a gradient flow. Withaferin-A was extracted by simple protein-precipitation technique. The calibration curve was linear in the concentration range of 0.05-1.6 µg/ml. The method has the desired sensitivity to detect the plasma concentration range of withaferin-A that is likely to show biological activity based on in vitro data. This is the first HPLC method ever described for the estimation of withaferin-A in human plasma which could be applied for pharmacokinetic studies.

Validated method for the analysis of goji berry, a rich source of zeaxanthin dipalmitate.

PubMed

Karioti, Anastasia; Bergonzi, Maria Camilla; Vincieri, Franco F; Bilia, Anna Rita

2014-12-31

In the present study an HPLC-DAD method was developed for the determination of the main carotenoid, zeaxanthin dipalmitate, in the fruits of Lycium barbarum. The aim was to develop and optimize an extraction protocol to allow fast, exhaustive, and repeatable extraction, suitable for labile carotenoid content. Use of liquid N2 allowed the grinding of the fruit. A step of ultrasonication with water removed efficiently the polysaccharides and enabled the exhaustive extraction of carotenoids by hexane/acetone 50:50. The assay was fast and simple and permitted the quality control of a large number of commercial samples including fruits, juices, and a jam. The HPLC method was validated according to ICH guidelines and satisfied the requirements. Finally, the overall method was validated for precision (% RSD ranging between 3.81 and 4.13) and accuracy at three concentration levels. The recovery was between 94 and 107% with RSD values <2%, within the acceptable limits, especially if the difficulty of the matrix is taken into consideration.

Determination of three steroidal saponins from Ophiopogon japonicus (Liliaceae) via high-performance liquid chromatography with mass spectrometry.

PubMed

Wang, Yongyi; Xu, Jinzhong; Qu, Haibin

2013-01-01

A simple and accurate analytical method was developed for simultaneous quantification of three steroidal saponins in the roots of Ophiopogon japonicus via high-performance liquid chromatography (HPLC) with mass spectrometry (MS) in this study. Separation was performed on a Tigerkin C(18) column and detection was performed by mass spectrometry. A mobile phase consisted of 0.02% formic acid in water (v/v) and 0.02% formic acid in acetonitrile (v/v) was used with a flow rate of 0.5 mL min(-1). The quantitative HPLC-MS method was validated for linearity, precision, repeatability, stability, recovery, limits of detection and quantification. This developed method provides good linearity (r >0.9993), intra- and inter-day precisions (RSD <4.18%), repeatability (RSD <5.05%), stability (RSD <2.08%) and recovery (93.82-102.84%) for three steroidal saponins. It could be considered as a suitable quality control method for O. japonicus.

Analysis of polymeric phenolics in red wines using different techniques combined with gel permeation chromatography fractionation.

PubMed

Guadalupe, Zenaida; Soldevilla, Alberto; Sáenz-Navajas, María-Pilar; Ayestarán, Belén

2006-04-21

A multiple-step analytical method was developed to improve the analysis of polymeric phenolics in red wines. With a common initial step based on the fractionation of wine phenolics by gel permeation chromatography (GPC), different analytical techniques were used: high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-mass spectrometry (MS), capillary zone electrophoresis (CZE) and spectrophotometry. This method proved to be valid for analyzing different families of phenolic compounds, such as monomeric phenolics and their derivatives, polymeric pigments and proanthocyanidins. The analytical characteristics of fractionation by GPC were studied and the method was fully validated, yielding satisfactory statistical results. GPC fractionation substantially improved the analysis of polymeric pigments by CZE, in terms of response, repeatability and reproducibility. It also represented an improvement in the traditional vanillin assay used for proanthocyanidin (PA) quantification. Astringent proanthocyanidins were also analyzed using a simple combined method that allowed these compounds, for which only general indexes were available, to be quantified.

The determination of antipyrine elimination in saliva by liquid chromatography.

PubMed

Gartzke, J; Jäger, H

1991-01-01

A simple, fast and reliable liquid chromatographic method for the determination of antipyrine in saliva is described. The elimination of antipyrine is a good indicator for general evaluation of the liver function for dispositional purposes for example in occupational and environmental medicine. The described LC method was compared with a more extensive photometric procedure. The results obtained from both methods show very good correlation. Only one measurement is necessary to determine the antipyrine clearance. Furthermore the antipyrine dosage can be minimized, because of the sensitivity of the HPLC-method.

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2005-04-01

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Simultaneous determination of 11 preservatives in cosmetics by high-performance liquid chromatography.

PubMed

Aoyama, Airin; Doi, Takahiro; Tagami, Takaomi; Kajimura, Keiji

2014-10-01

Preservatives prevent the growth of microorganisms in foods, pharmaceuticals and cosmetics. There exist numerous restrictions regarding the maximum allowable levels of preservatives in cosmetics. We analyzed 11 regulated preservatives in commercial cosmetics and manufacturers need to analyze their products for quality control purposes. However, methods used in previous studies to date have been inadequate for use by public institutions and manufacturers. Therefore, an effective, scalable method for the analysis of preservatives in cosmetics is required. We developed a novel method for the simultaneous determination of 11 regulated preservatives in cosmetics by high-performance liquid chromatography (HPLC). We applied the samples to a C18 column in a simple mobile phase (5 mmol/L ammonium formate solution and acetonitrile) with gradient elution at a flow rate of 1.0 mL/min at a single wavelength (230 nm). The correlation coefficients of the calibration curves were >0.997. The percent recoveries were 92.8-111.9% and the relative standard deviations were <4.3% (n = 6). The peak resolution for all preservatives was >1.9. Because of the simple conditions for isolation and complete separation, the HPLC method can be effectively applied to the analysis of preservatives in commercially retailed cosmetics. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient.

PubMed

Goutal, Sébastien; Auvity, Sylvain; Legrand, Tiphaine; Hauquier, Fanny; Cisternino, Salvatore; Chapy, Hélène; Saba, Wadad; Tournier, Nicolas

2016-05-10

In clinical practice, rifampicin exposure is estimated from its concentration in venous blood samples. In this study, we hypothesized that differences in rifampicin concentration may exist between arterial and venous plasma. An HPLC-UV method for determining rifampicin concentration in plasma using rifapentine as an internal standard was validated. The method, which requires a simple protein precipitation procedure as sample preparation, was performed to compare venous and arterial plasma kinetics after a single therapeutic dose of rifampicin (8.6 mg/kg i.v, infused over 30 min) in baboons (n=3). The method was linear from 0.1 to 40 μg mL(-1) and all validation parameters fulfilled the international requirements. In baboons, rifampicin concentration in arterial plasma was higher than in venous plasma. Arterial Cmax was 2.1±0.2 fold higher than venous Cmax. The area under the curve (AUC) from 0 to 120 min was ∼80% higher in arterial plasma, indicating a significant arteriovenous concentration gradient in early rifampicin pharmacokinetics. Arterial and venous plasma concentrations obtained 6h after rifampicin injection were not different. An important arteriovenous equilibration delay for rifampicin pharmacokinetics is reported. Determination in venous plasma concentrations may considerably underestimate rifampicin exposure to organs during the distribution phase. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of Carvedilol Enantiomers in Pharmaceutical Dosages by SBSE-HPLC Based on Diastereomer Formation.

PubMed

Taraji, Maryam; Talebpour, Zahra; Adib, Nuoshin; Karimi, Shima; Haghighi, Farideh; Aboul-Enein, Hassan Y

2015-09-01

A sensitive, selective and simple method for the simultaneous determination of carvedilol enantiomers in aqueous solution has been developed using stir bar sorptive extraction (SBSE) followed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. This method is based on the reaction of carvedilol enantiomers with (-)-menthyl chloroformate (MCF) after extraction by the SBSE method to produce diastereomeric derivatives. The separation was achieved by use of a C18 analytical column and the influence of mobile phase composition on the enantioseparation of carvedilol was studied. The applicability of two sorptive phases, poly(methyl methacrylate/ethyleneglycol dimethacrylate) (PA-EG) and polydimethylsiloxane, were tested for extraction of carvedilol enantiomers from aqueous samples. The obtained results showed excellent linear dynamic ranges and precisions for each of them. The least limit of detection for (S)- and (R)-carvedilol obtained 8 and 11 µg L(-1), respectively, using the PA-EG sorptive phase. Inter- and intra-mean recoveries were also satisfactory, ranging from 98 to 103%, with coefficient of variation in the range of 1-5% at three fortified levels using a PA-EG coated stir bar. The proposed SBSE (PA-EG)-MCF derivatization-HPLC-UV method was successfully applied to enantioselective analysis of carvedilol in water and pharmaceutical dosages, confirming the application of this method. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of Dithiocarbamate Fungicides in Vegetable Matrices Using HPLC-UV Followed by Atomic Absorption Spectrometry.

PubMed

Al-Alam, Josephine; Bom, Laura; Chbani, Asma; Fajloun, Ziad; Millet, Maurice

2017-04-01

A simple method combining ion-pair methylation, high-performance liquid chromatography (HPLC) analysis with detection at 272 nm and atomic absorption spectrometry was developed in order to determine 10 dithiocarbamate fungicides (Dazomet, Metam-sodium, Ferbam, Ziram, Zineb, Maneb, Mancozeb, Metiram, Nabam and Propineb) and distinguish ethylenbisdithiocarbamates (EBDTCs) Zineb, Maneb and Mancozeb in diverse matrices. This method associates reverse phase analysis by HPLC analysis with detection at 272 nm, with atomic absorption spectrometry in order to distinguish, with the same extraction protocol, Maneb, Mancozeb and Zineb. The limits of detection (0.4, 0.8, 0.5, 1.25 and 1.97) and quantification (1.18, 2.5, 1.52, 4.2 and 6.52) calculated in injected nanogram, respectively, for Dazomet, Metam-Na, dimethyldithiocarbamates (DMDTCs), EBDTCs and propylenebisdithiocarbamates (PBDTCs) justify the sensitivity of the method used. The coefficients of determination R2 were 0.9985, 0.9978, 0.9949, 0.988 and 0.9794, respectively, for Dazomet, Metam-Na, DMDTCs, EBDTCs and PBDTCs, and the recovery from fortified apple and leek samples was above 90%. Results obtained with the atomic absorption method in comparison with spectrophotometric analysis focus on the importance of the atomic absorption as a complementary specific method for the distinction between different EBDTCs fungicides. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Stability Indicating Reverse Phase HPLC Method for Estimation of Rifampicin and Piperine in Pharmaceutical Dosage Form.

PubMed

Shah, Umang; Patel, Shraddha; Raval, Manan

2018-01-01

High performance liquid chromatography is an integral analytical tool in assessing drug product stability. HPLC methods should be able to separate, detect, and quantify the various drug-related degradants that can form on storage or manufacturing, plus detect any drug-related impurities that may be introduced during synthesis. A simple, economic, selective, precise, and stability-indicating HPLC method has been developed and validated for analysis of Rifampicin (RIFA) and Piperine (PIPE) in bulk drug and in the formulation. Reversed-phase chromatography was performed on a C18 column with Buffer (Potassium Dihydrogen Orthophosphate) pH 6.5 and Acetonitrile, 30:70), (%, v/v), as mobile phase at a flow rate of 1 mL min-1. The detection was performed at 341 nm and sharp peaks were obtained for RIFA and PIPE at retention time of 3.3 ± 0.01 min and 5.9 ± 0.01 min, respectively. The detection limits were found to be 2.385 ng/ml and 0.107 ng/ml and quantification limits were found to be 7.228ng/ml and 0.325ng/ml for RIFA and PIPE, respectively. The method was validated for accuracy, precision, reproducibility, specificity, robustness, and detection and quantification limits, in accordance with ICH guidelines. Stress study was performed on RIFA and PIPE and it was found that these degraded sufficiently in all applied chemical and physical conditions. Thus, the developed RP-HPLC method was found to be suitable for the determination of both the drugs in bulk as well as stability samples of capsule containing various excipients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comparison of a fluorometric assay kit with high-performance liquid chromatography for the assessment of serum retinol concentration.

PubMed

Elom, Aglago Kouassivi; Imane, El Menchawy; Kaoutar, Benjeddou; Khalid, El Kari; Asmaa, El Hamdouchi; Mehdi, Azlaf; Noureddine, El Haloui; Hassan, Aguenaou

2015-06-01

Although high-performance liquid chromatography (HPLC) is the commonly used method for the analysis of retinol in biological samples, simple and rapid test kits are available. This study compared a rapid test kit (ICHECK Fluoro®) to HPLC for the assessment of serum retinol concentrations. For the analysis by HPLC, sample preparation included standard deproteinization and extraction phases. The analysis by ICHECK was performed by injecting serum into IEX reagent vials (n=89) and mixing manually for separation. After precipitation of the proteins, the vial was introduced into the chamber of the ICHECK Fluoro and analysed at 0 min (ICHECK0min) and 15 min later (ICHECK15min). Bland and Altman approach was applied to test the agreement between HPLC and ICHECK. Mean HPLC, ICHECK0min and ICHECK15min values were 421.2±106.0 µg/L, 423.1±118.3 µg/L and 413.2±107.6 µg/L, respectively. Retinol concentrations significantly decreased in the IEX solution over time (p<0.001). No significant proportional bias was observed between HPLC and ICHECK0min (r-0.038, p=0.73) and ICHECK15min (r=-0.024, p=0.82). Fixed biases (HPLC minus ICHECK) for ICHECK0min and ICHECK15min were respectively -1.9±23.1 µg/l (p=0.45) and 8.0±22.7 µg/l (p=0.002). ICHECK Fluoro may offer a reliable mean for assessing serum retinol for measurements performed with no significant time delay.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species.

PubMed

Chatterjee, Arnab; Kumar, Satyanshu; Chattopadhyay, Sunil K

2013-12-01

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.

HPLC/Fluorometric Detection of Carvedilol in Real Human Plasma Samples Using Liquid-Liquid Extraction.

PubMed

Yilmaz, Bilal; Arslan, Sakir

2016-03-01

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify carvedilol in human plasma using an isocratic system with fluorescence detection. The method included a single-step liquid-liquid extraction with diethylether and ethylacetate mixture (3 : 1, v/v). HPLC separation was carried out by reversed-phase chromatography with a mobile phase composed of 20 mM phosphate buffer (pH 7)-acetonitrile (65 : 35, v/v), pumped at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 240 nm (excitation) and 330 nm (emission). The calibration curve for carvedilol was linear from 10 to 250 ng/mL. Intra- and interday precision values for carvedilol in human plasma were <4.93%, and accuracy (relative error) was better than 4.71%. The analytical recovery of carvedilol from human plasma averaged out to 91.8%. The limits of detection and quantification of carvedilol were 3.0 and 10 ng/mL, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 25 mg carvedilol. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Validated RP-HPLC/DAD Method for the Quantification of Insect Repellent Ethyl 2-Aminobenzoate in Membrane-Moderated Matrix Type Monolithic Polymeric Device.

PubMed

Islam, Johirul; Zaman, Kamaruz; Chakrabarti, Srijita; Sharma Bora, Nilutpal; Mandal, Santa; Pratim Pathak, Manash; Srinivas Raju, Pakalapati; Chattopadhyay, Pronobesh

2017-07-01

A simple, accurate and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the estimation of ethyl 2-aminobenzoate (EAB) in a matrix type monolithic polymeric device and validated as per the International Conference on Harmonization guidelines. The analysis was performed isocratically on a ZORBAX Eclipse plus C18 analytical column (250 × 4.4 mm, 5 μm) and a diode array detector (DAD) using acetonitrile and water (75:25 v/v) as the mobile phase by keeping the flow-rate constant at 1.0 mL/min. Determination of EAB was not interfered in the presence of excipients. Inter- and intra-day relative standard deviations were not higher than 2%. Mean recovery was between 98.7 and 101.3%. Calibration curve was linear in the concentration range of 0.5-10 µg/mL. Limits of detection and quantification were 0.19 and 0.60 µg/mL, respectively. Thus, the present report put forward a novel method for the estimation of EAB, an emerging insect repellent, by using RP-HPLC technique. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rapid isocratic HPLC method and sample extraction procedures for measuring carotenoid, retinoid, and tocopherol concentrations in human blood and breast milk for intervention studies

USDA-ARS?s Scientific Manuscript database

Vitamin A deficiency is an important cause of death in the developing world. A variety of dietary interventions using provitamin A-carotenoid rich fruits and vegetables are being tested for their potential to alleviate vitamin A deficiency in several countries. Thus, there is a need for a simple, ...

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography.

PubMed

Chen, Lili; Liao, Linchuan; Zuo, Zhong; Yan, Youyi; Yang, Lin; Fu, Qiang; Chen, Yu; Hou, Junhong

2007-04-11

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 microm Zorbax Dikema C18 column (150 mm x 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine-acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 microg/ml in plasma and 0.007 and 0.14 microg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 microg/ml and nikethamide ranged from 1.25 to 106.8 microg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 microg/ml of lidocaine and 8.0 to 72.4 microg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.

Dithizone-functionalized solid phase extraction-displacement elution-high performance liquid chromatography-inductively coupled plasma mass spectrometry for mercury speciation in water samples.

PubMed

Yin, Yong-guang; Chen, Ming; Peng, Jin-feng; Liu, Jing-fu; Jiang, Gui-bin

2010-06-15

A novel and simple solid phase extraction (SPE)-high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) method was developed for determination of inorganic mercury (IHg), methylmercury MeHg and ethylmercury (EtHg) in water samples in the present work. The procedure involves pre-functionalization of the commercially available C18 SPE column with dithizone, loading water sample, displacement elution of mercury species by Na(2)S(2)O(3) solution, followed by HPLC-ICP-MS determination. Characterization and optimization of operation parameters of this new SPE procedure were discussed, including eluting reagent selection, concentration of eluting reagent, volume of eluting reagent, effect of NaCl and humic acid in sample matrix. At optimized conditions, the detection limits of mercury species for 100mL water sample were about 3ngL(-1) and the average recoveries were 93.7, 83.4, and 71.7% for MeHg, IHg and EtHg, respectively, by spiking 0.2microgL(-1) mercury species into de-ion water. Stability experiment reveals that both the dithizone-functionalized SPE cartridge and the mercury species incorporated were stable in the storage procedure. These results obtained demonstrate that SPE-HPLC-ICP-MS is a simple and sensitive technique for the determination of mercury species at trace level in water samples with high reproducibility and accuracy.

Determination of tylosin residues in pig tissues using high-performance liquid chromatography.

PubMed

De Liguoro, M; Anfossi, P; Angeletti, R; Montesissa, C

1998-06-01

In accordance with the maximum residue limit of 100 micrograms kg-1 established by EU legislation, a simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the measurement of tylosin residues in pig tissues (fat, kidney, liver and muscle). Tylosin, a macrolide antibiotic, is extracted with water-methanol and cleaned-up by solid-phase extraction (SPE) on cation-exchange cartridges using methanol elution. Tylosin was determined by reversed-phase HPLC with UV detection at 280 nm and the mean recovery from pig tissues fortified in the range 50-200 micrograms kg-1 was 70-85%, with intra- and inter-day RSDs in the ranges 3.4-9.1 and 3.9-10.1% respectively.

[Determination of benzyl glucosinolate in Lepidium meyenii from different regions by HPLC].

PubMed

Tang, Lin; Yin, Hong-jun; Si, Cong-cong; Hu, Xiao-yan; Long, Zheng-hai

2015-12-01

The content of benzyl isothiocyanate (BITC) which as the enzymatic hydrolysis product of benzyl glucosinolate through thioglucosidase was determined by HPLC. The content of benzyl isothiocyanate (BITC) which as the enzymatic hydrolysis product of benzyl glucosinolate through thioglucosidase was determined by HPLC. The chromatography condition was as follows: Kaseisorb LC ODS 2000 (4.6 mm x 150 mm, 5 min) column with the mobile phase of acetonitrile(A)-water( B) under gradient elution (0-5 min, 3%-8% A; 5-9 min, 8%-48% A; 9-23 min, 48%-62% A; 23-28 min, 62%-99% A); the flow rate was 1.0 mL x min(-1) with 10 microL injection volume; detection wavelength was 246 nm and temperature of column was 40 degrees C. The content of benzyl glucosinolate was in the range of 10.76-17.91 g x L(-1). The method is simple, accurate and good reproducibility which can be used for the determination of benzyl glucosinolate in Lepidium meyenii, effectively.

Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique

NASA Astrophysics Data System (ADS)

Li, Ai Ping; Peng, Jing Dong; Zhou, MingQiong; Zhang, Jin

2016-02-01

A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH 5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λex = λem = 315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L- 1 and the limit of detection (S/N = 3) was 0.05 μg L- 1. The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed.

Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique.

PubMed

Li, Ai Ping; Peng, Jing Dong; Zhou, MingQiong; Zhang, Jin

2016-02-05

A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λ(ex)=λ(em)=315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L(-1) and the limit of detection (S/N=3) was 0.05 μg L(-1). The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

A rapid liquid chromatography determination of free formaldehyde in cod.

PubMed

Storey, Joseph M; Andersen, Wendy C; Heise, Andrea; Turnipseed, Sherri B; Lohne, Jack; Thomas, Terri; Madson, Mark

2015-01-01

A rapid method for the determination of free formaldehyde in cod is described. It uses a simple water extraction of formaldehyde which is then derivatised with 2,4-dinitrophenylhydrazine (DNPH) to form a sensitive and specific chromophore for high-performance liquid chromatography (HPLC) detection. Although this formaldehyde derivative has been widely used in past tissue analysis, this paper describes an improved derivatisation procedure. The formation of the DNPH formaldehyde derivative has been shortened to 2 min and a stabilising buffer has been added to the derivative to increase its stability. The average recovery of free formaldehyde in spiked cod was 63% with an RSD of 15% over the range of 25-200 mg kg(-1) (n = 48). The HPLC procedure described here was also compared to a commercial qualitative procedure - a swab test for the determination of free formaldehyde in fish. Several positive samples were compared by both methods.

Simple and sensitive analysis of long-chain free fatty acids in milk by fluorogenic derivatization and high-performance liquid chromatography.

PubMed

Lu, Chi-Yu; Wu, Hsin-Lung; Chen, Su-Hwei; Kou, Hwang-Shang; Wu, Shou-Mei

2002-01-02

A highly sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of some important saturated and unsaturated fatty acids in milk, including lauric (dodecanoic), myristic (tetradecanoic), palmitic (hexadecanoic), stearic (octadecanoic), palmitoleic (hexadecenoic), oleic (octadecenoic), and linoleic acids (octadecadienoic acids). The fatty acids were fluorogenically derivatized with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) as their naphthoxyethyl derivatives. The resulting derivatives were separated by isocratic HPLC and monitored with a fluorometric detector (lambdaex = 235 nm, lambdaem = 350 nm). The fatty acids in milk were extracted with toluene, and the extract with the fatty acids was directly derivatized with NOEPES without solvent replacement. Determination of long-chain free fatty acids in milk is feasible by a standard addition method. A small amount of milk product, 10 microL, is sufficient for the analysis.

Improved HPLC method for determination of four PPIs, omeprazole, pantoprazole, lansoprazole and rabeprazole in human plasma.

PubMed

Noubarani, Maryam; Keyhanfar, Fariborz; Motevalian, Manijeh; Mahmoudian, Masoud

2010-01-01

To develop a simple and rapid HPLC method for measuring of four proton-pump inhibitors (PPIs), omeprazole (OPZ), pantoprazole (PPZ), lansoprazole (LPZ) and rabeprazole (RPZ) concentrations in human plasma. Following a single step liquid-liquid extraction analytes along with an internal standard (IS) were separated using an isocratic mobile phase of phosphate buffer (10 mM)/acetonitrile (53/47, v/v adjusted pH to 7.3 with triethylamine) at flow rate of 1 mL/min on reverse phase TRACER EXCEL 120 ODS-A column at room temperature. Total analytical run time for selected PPIs was 10 min. The assays exhibited good linearity (r(2)>0.99) over the studied range of 20 to 2500 ng/mL for OPZ, 20 to 4000 ng/mL for PPZ, 20 to 3000 ng/mL for LPZ and 20 to 1500 ng/mL for RPZ. The recovery of method was equal or greater than 80% and lower limit of quantification (LLOQ) was 20 ng/mL for four PPIs. Coefficient of variation and error at all of the intra-day and inter-day assessment were less than 9.2% for all compounds. The results indicated that this method is a simple, rapid, precise and accurate assay for determination of four PPIs concentrations in human plasma. This validated method is sensitive and reproducible enough to be used in pharmacokinetic studies and also is time- and cost-benefit when selected PPIs are desired to be analyzed.

Carrier mediated hollow fiber liquid phase microextraction combined with HPLC-UV for preconcentration and determination of some tetracycline antibiotics.

PubMed

Shariati, Shahab; Yamini, Yadollah; Esrafili, Ali

2009-02-01

In the present study, a simple and efficient preconcentration method was developed using carrier mediated three phase liquid phase microextraction prior to HPLC-UV for simultaneous extraction and determination of trace amounts of highly hydrophilic tetracycline antibiotics including tetracycline (TCN), oxytetracycline (OTCN) and doxycycline (DCN) in bovine milk, human plasma and water samples. For extraction, 11.0 mL of the aqueous sample containing TCNs and 0.05 M Na(2)HPO(4) (9.10.995). Finally, applicability of the proposed method was successfully confirmed by extraction and determination of the drugs in water and plasma samples as well as in bovine milk samples with low and high fat contents. Comparing to the traditional methods, the proposed method exhibits high sensitivity and high preconcentration factors as well as good precision. The extraction setup is simple and due to active transport of analytes, high cleanup effect and good selectivity are obtained in extraction process.

Development of an ATP measurement method suitable for xenobiotic treatment activated sludge biomass.

PubMed

Nguyen, Lan Huong; Chong, Nyuk-Min

2015-09-01

Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge's degradation of a xenobiotic, any disadvantageous effect on the sludge's performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R(2) of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD=0.00045mg/L, a LOQ=0.0015mg/L for HPLC measurement of ATP, a MDL=0.46mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4±2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid preconcentration method for the determination of azadirachtin-A and -B, nimbin and salannin in neem oil samples by using graphitised carbon solid phase extraction.

PubMed

Ramesh, A; Balasubramanian, M

1999-01-01

A simple and rapid method involving solid phase extraction and liquid chromatography for the determination of azadirachtin-A and -B, nimbin and salannin at nanogram levels in neem oil samples is presented. The neem oil samples are defatted and the compounds of interest extracted by mixing the sample with hexane and passing the hexane solution through a graphitised carbon black column. After washing the column with 2 ml of hexane, azadirachtin-A and -B, nimbin and salannin are eluted with 5 ml of acetonitrile and quantified using HPLC with UV detection. The recoveries of azadirachtin-A and -B, nimbin and salannin in fortified oil samples were 97.4-104.7%. The upper limit of quantification is up to 100 micrograms ml-1 without any additional clean-up and with little interference from lipids during the analysis by HPLC. The method was successfully applied to various neem oil samples collected from different locations in India.

Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine.

PubMed

de Oliveira, Anderson Rodrigo Moraes; Cesarino, Evandro José; Bonato, Pierina Sueli

2005-04-25

A simple and rapid solid-phase microextraction method was developed for the enantioselective analysis of ibuprofen in urine. The sampling was made with a polydimethylsiloxane-divinylbenzene coated fiber immersed in the liquid sample. After desorptioning from the fiber, ibuprofen enantiomers were analyzed by HPLC using a Chiralpak AD-RH column and UV detection. The mobile phase was made of methanol-pH 3.0 phosphoric acid solution (75:25, v/v), at a flow rate of 0.45 mL/min. The mean recoveries of SPME were 19.8 and 19.1% for (-)-R-ibuprofen and (+)-(S)-ibuprofen, respectively. The method was linear at the range of 0.25-25 microg/mL. Within-day and between-day assay precision and accuracy were below 15% for both ibuprofen enantiomers at concentrations of 0.75, 7.5 and 20 microg/mL. The method was tested with urine quality control samples and human urine fractions after administration of 200 mg rac-ibuprofen.

[Determination of phthalate plasticizers in foods by high performance liquid chromatography with gel permeation chromatographic clean-up].

PubMed

Zhang, Chunyu; Wang, Hui; Zhang, Xiaohui; Ma, Zhongqiang; Deng, Wanmei; Hu, Ke; Ding, Mingyu

2011-12-01

A method of gel permeation chromatography-high performance liquid chromatography (GPC-HPLC) was established for the simultaneous determination of 5 main phthalate plasticizers in foods (edible oil, instant noodles, fried pastries, Saqima, etc.). The samples were extracted with petroleum ether in an ultrasonator, purified by a GPC column, and analyzed by HPLC. The chromatographic separation was achieved on a Labtech-C18 column (250 mm x 4.6 mm, 5 microm) using acetonitrile and water mixture as the mobile phases in a gradient elution mode. The developed method exhibited a linear correlation coefficient of more than 0.997 and the detection limits of 3.25 - 13.4 microg/L. The spike recoveries were between 70.4% and 113.6% with the relative standard deviations (RSDs, n = 3) of 0.3% - 5.8% at the spiked level of 50 mg/L. This method is simple, rapid and practical, and can be used for the simultaneous determination of PAEs in grease food samples.

High Performance Liquid Chromatography-Diode Array Detector Method for the Simultaneous Determination of Five Compounds in the Pulp and Seed of Sea Buckthorn.

PubMed

Zhao, Lu; Wen, E; Upur, Halmuart; Tian, Shuge

2017-01-01

Sea buckthorn ( Hippophae rhamnoides L.) as a traditional Chinese medicinal plant has various uses in Xinjiang. A reversed-phase rapid-resolution liquid-chromatography method with diode array detector was developed for simultaneous determination of protocatechuic acid, rutin, quercetin, kaempferol, and isorhamnetin in the pulp and seed of sea buckthorn, a widely used traditional Chinese medicine for promoting metabolism and treating scurvy and other diseases. Compounds were separated on an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm; USA) with gradient elution using methanol and 0.4% phosphoric acid (v/v) at 1.0 mL/min. Detection wavelength was set at 280 nm. The fruits of wild sea buckthorn were collected from Wushi County in Aksu, Xinjiang Province. The RSD of precision test of the five compounds were in the range of 0.60-2.22%, and the average recoveries ranged from 97.36% to 101.19%. Good linearity between specific chromatographic peak and component qualities were observed in the investigated ranges for all the analytes ( R 2 > 0.9997). The proposed method was successfully applied to determine the levels of five active components in sea buckthorn samples from Aksu in Xinjiang. The proposed method is simple, fast, sensitive, accurate, and suitable for quantitative assessment of the pulp and seed of sea buckthorn. Quantitative analysis method of protocatechuic acid, rutin, quercetin, kaempferol, and isorhamnetin in the extract of sea buckthorn pulp and seed is developed by high-performance liquid chromatography (HPLC) diode array detection.This method is simple and accurate; has strong specificity, good precision, and high recovery rate; and provides a reliable basis for further development of the substances in the pulp and seed of sea buckthorn.The method is widely used for content determination of active ingredients or physiologically active components in traditional Chinese medicine and its preparation Abbreviation used: PR: protocatechuic acid, RU: rutin, QU: quercetin, KA: kaempferol, IS: isorhamnetin, HPLC: high-performance liquid chromatography, HPLC-DAD: high performance liquid chromatographydiode array detector, LOD: linearity and limit of detection, LOQ: limit of quantitation, RSD: relative standard deviation.

A simple, rapid and sensitive RP-HPLC-UV method for the simultaneous determination of sorafenib & paclitaxel in plasma and pharmaceutical dosage forms: Application to pharmacokinetic study.

PubMed

Khan, Ismail; Iqbal, Zafar; Khan, Abad; Hassan, Muhammad; Nasir, Fazle; Raza, Abida; Ahmad, Lateef; Khan, Amjad; Akhlaq Mughal, Muhammad

2016-10-15

A simple, economical, fast, and sensitive RP-HPLC-UV method has been developed for the simultaneous quantification of Sorafenib and paclitaxel in biological samples and formulations using piroxicam as an internal standard. The experimental conditions were optimized and method was validated according to the standard guidelines. The separation of both the analytes and internal standard was achieved on Discovery HS C18 column (250mm×4.6mm, 5μm) using Acetonitrile and TFA (0.025%) in the ratio of (65:35V/V) as the mobile phase in isocratic mode at a flow rate of 1ml/min, with a wavelength of 245nm and at a column oven temperature of 25°Cin a short run time of 12min. The limits of detection (LLOD) were 5 and 10ng/ml while the limits of quantification (LLOQ) were 10 and 15ng/ml for sorafenib and paclitaxel, respectively. Sorafenib, paclitaxel and piroxicam (IS) were extracted from biological samples by applying acetonitrile as a precipitating and extraction solvent. The method is linear in the range of 15-20,000ng/ml for paclitaxel and 10-5000ng/ml for sorafenib, respectively. The method is sensitive and reliable by considering both of its intra-day and inter-day co-efficient of variance. The method was successfully applied for the quantification of the above mentioned drugs in plasma. The developed method will be applied towards sorafenib and paclitaxel pharmacokinetics studies in animal models. Copyright © 2016 Elsevier B.V. All rights reserved.

Stability indicating simplified HPLC method for simultaneous analysis of resveratrol and quercetin in nanoparticles and human plasma.

PubMed

Kumar, Sandeep; Lather, Viney; Pandita, Deepti

2016-04-15

Resveratrol and quercetin are well-known polyphenolic compounds present in common foods, which have demonstrated enormous potential in the treatment of a wide variety of diseases. Owing to their exciting synergistic potential and combination delivery applications, we developed a simple and rapid RP-HPLC method based on isosbestic point detection. The separation was carried out on phenomenex Synergi 4μ Hydro-RP 80A column using methanol: acetonitrile (ACN): 0.1% phosphoric acid (60:10:30) as mobile phase. The method was able to quantify nanograms of analytes simultaneously on a single wavelength (269 nm), making it highly sensitive, rapid as well as economical. Additionally, forced degradation studies of resveratrol and quercetin were established and the method's applicability was evaluated on PLGA nanoparticles and human plasma. The analytes peaks were found to be well resolved in the presence of degradation products and excipients. The simplicity of the developed method potentializes its suitability for routine in vitro and in vivo analysis of resveratrol and quercetin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development and Validation of High Performance Liquid Chromatography Method for Determination Atorvastatin in Tablet

NASA Astrophysics Data System (ADS)

Yugatama, A.; Rohmani, S.; Dewangga, A.

2018-03-01

Atorvastatin is the primary choice for dyslipidemia treatment. Due to patent expiration of atorvastatin, the pharmaceutical industry makes copy of the drug. Therefore, the development methods for tablet quality tests involving atorvastatin concentration on tablets needs to be performed. The purpose of this research was to develop and validate the simple atorvastatin tablet analytical method by HPLC. HPLC system used in this experiment consisted of column Cosmosil C18 (150 x 4,6 mm, 5 µm) as the stationary reverse phase chomatography, a mixture of methanol-water at pH 3 (80:20 v/v) as the mobile phase, flow rate of 1 mL/min, and UV detector at wavelength of 245 nm. Validation methods were including: selectivity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The results of this study indicate that the developed method had good validation including selectivity, linearity, accuracy, precision, LOD, and LOQ for analysis of atorvastatin tablet content. LOD and LOQ were 0.2 and 0.7 ng/mL, and the linearity range were 20 - 120 ng/mL.

Development of an HPLC method for determination of metabolic compounds in myocardial tissue.

PubMed

Volonté, M G; Yuln, G; Quiroga, P; Consolini, A E

2004-05-28

The determination of adenine nucleotides and creatine compounds has great importance in the characterization of ischemic myocardial injury and post-ischemic recovery. It was developed by an HPLC method for the quantification of creatine (Cr), creatine phosphate (CrP), hypoxanthine (HX), AMP, adenosine (Ad), ADP and ATP in isolated perfused rat hearts. The chromatographic conditions were: RP 18 column; mobile phase composed by KH(2)PO(4) (215 mM), tetrabutylammonium hydrogen sulfate (2.3mM), acetonitrile (4%) and KOH (1M 0.4%); flow rate 1 ml min(-1); temperature 25 degrees C; injection volume 20 microl; detection at 220 nm and height peak (HP) as the integration parameter. The method was validated by means of linearity and sensitivity evaluations, using calibration curves done with five concentration levels of each compound. The limits of quantification (LOQ) were also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. The purity of the peaks was established using enzymatic peak shift analysis with hexokinase and creatine kinase and also comparing HP at various wavelengths. Frozen hearts were homogenized with a mechanical homogenizer for 3 min at 0 degrees C added with 5 ml of 0.4N HCLO(4). After precipitation with 0.8 ml of 2M KOH the extract was shaked for 2 min and later centrifuged at 0 degrees C for 10 min. The supernatant was kept on ice, filtrated and injected into the HPLC system. The results show that the method for the determination of Cr, CrP, HX, AMP, Ad, ADP and ATP by HPLC here described has good linearity, LOQ, precision, specificity and is simple and rapid to perform.

Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

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

Yinfa, Ma.

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will bemore » described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.« less

Simultaneous estimation of lisofylline and pentoxifylline in rat plasma by high performance liquid chromatography-photodiode array detector and its application to pharmacokinetics in rat.

PubMed

Italiya, Kishan S; Sharma, Saurabh; Kothari, Ishit; Chitkara, Deepak; Mittal, Anupama

2017-09-01

Lisofylline (LSF) is an anti-inflammatory and immunomodulatory agent with proven activity in serious infections associated with cancer chemotherapy, hyperoxia-induced acute lung injury, autoimmune disorders including type-1 diabetes (T1DM) and islet rejection after islet transplantation. It is also an active metabolite of another anti-inflammatory agent, Pentoxifylline (PTX). LSF bears immense therapeutic potential in multiple pharmacological activities and hence appropriate and accurate quantification of LSF is very important. Although a number of analytical methods for quantification of LSF and PTX have been reported for pharmacokinetics and metabolic studies, each of these have certain limitations in terms of large sample volume required, complex extraction procedure and/or use of highly sophisticated instruments like LC-MS/MS. The aim of current study is to develop a simple reversed-phase HPLC method in rat plasma for simultaneous determination of LSF and PTX with the major objective of ensuring minimum sample volume, ease of extraction, economy of analysis, selectivity and avoiding use of instruments like LC-MS/MS to ensure a widespread application of the method. A simple liquid-liquid extraction method using methylene chloride as extracting solvent was used for extracting LSF and PTX from rat plasma (200μL). Samples were then evaporated, reconstituted with mobile phase and injected into HPLC coupled with photo-diode detector (PDA). LSF, PTX and 3-isobutyl 1-methyl xanthine (IBMX, internal standard) were separated on Inertsil® ODS (C18) column (250×4.6mm, 5μm) with mobile phase consisting of A-methanol B-water (50:50v/v) run in isocratic mode at flow rate of 1mL/min for 15min and detection at 273nm. The method showed linearity in the concentration range of 50-5000ng/mL with LOD of 10ng/mL and LLOQ of 50ng/mL for both LSF and PTX. Weighted linear regression analysis was also performed on the calibration data. The mean absolute recoveries were found to be 80.47±3.44 and 80.89±3.73% for LSF and PTX respectively. The method was successfully applied for studying the pharmacokinetics of LSF and PTX after IV bolus administration at dose of 25mg/kg in Wistar rat. In conclusion, a simple, sensitive, accurate and precise reversed-phase HPLC-UV method was established for simultaneous determination of LSF and PTX in rat plasma. Copyright © 2017 Elsevier B.V. All rights reserved.

Stability of cyclophosphamide in extemporaneous oral suspensions.

PubMed

Kennedy, Rachel; Groepper, Daniel; Tagen, Michael; Christensen, Robbin; Navid, Fariba; Gajjar, Amar; Stewart, Clinton F

2010-02-01

Cyclophosphamide, an alkylating agent, is widely used for the treatment of many adult and pediatric malignancies. The stability of cyclophosphamide in aqueous- and methylcellulose-based oral suspending vehicles is currently unknown. To develop and validate a stability-indicating high-performance liquid chromatography (HPLC) method to measure cyclophosphamide concentrations in simple syrup and Ora-Plus, and assess the 56-day chemical stability and physical appearance of cyclophosphamide in these suspensions at both room temperature (22 degrees C) and 4 degrees C. The intravenous formulation of cyclophosphamide was diluted to 20 mg/mL in NaCl 0.9%, compounded 1:1 with either suspending vehicle, and stored in the dark in 3-mL amber polypropylene oral syringes at 4 degrees C and 22 degrees C. Aliquots from each syringe were obtained on days 0, 3, 7, 14, 21, 28, 35, 42, 49, and 56 and assayed using the validated stability-indicating HPLC-UV method. A C18 analytical column was used to separate cyclophosphamide from the internal standard, ifosfamide, with a mobile phase of 21% acetonitrile in 79% sodium phosphate buffer. The suspension was examined for odor change, visually examined under normal fluorescent light for color change, and examined under a light microscope for evidence of microbial growth. Samples of cyclophosphamide in both simple syrup and Ora-Plus were stable when kept at 4 degrees C for at least 56 days. At room temperature, cyclophosphamide in simple syrup and Ora-Plus had a shelf life of 8 and 3 days, respectively. No changes in color or odor or evidence of microbial growth were observed. Cyclophosphamide can be extemporaneously prepared in simple syrup or Ora-Plus and stored for at least 2 months under refrigeration without significant degradation.

Chemiluminescent Determination of Oxamyl in Drinking Water and Tomato Using Online Postcolumn UV Irradiation in a Chromatographic System.

PubMed

Murillo Pulgarín, José A; García Bermejo, Luisa F; Durán, Armando Carrasquero

2018-03-07

High-performance liquid chromatography (HPLC) was used to separate oxamyl from other pesticides in drinking water and tomato paste. The eluate emerging from the column tail was mixed with an alkaline solution of Co 2+ in EDTA and irradiated with UV light to induce photolysis of the carbamate in order to obtain free radicals and other reactive species that oxidize luminol and produce chemiluminescence (CL) as a result. The intensity of the CL signal was monitored in the form of chromatographic peaks. Under the optimum operating conditions for the HPLC-UV-CL system, the analyte concentration was linearly related to peak area. The limit of detection as determined in accordance with the IUPAC criterion was 0.17 mg L -1 . Oxamyl was successfully extracted with recoveries of 88.7-103.1% from spiked tomato paste by using a simple QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation approach. Similar recoveries were obtained from drinking water samples spiked with oxamyl concentrations above the LOD. The proposed method is a simple, fast, accurate choice for quantifying this pesticide.

A simple approach to quantitative analysis using three-dimensional spectra based on selected Zernike moments.

PubMed

Zhai, Hong Lin; Zhai, Yue Yuan; Li, Pei Zhen; Tian, Yue Li

2013-01-21

A very simple approach to quantitative analysis is proposed based on the technology of digital image processing using three-dimensional (3D) spectra obtained by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). As the region-based shape features of a grayscale image, Zernike moments with inherently invariance property were employed to establish the linear quantitative models. This approach was applied to the quantitative analysis of three compounds in mixed samples using 3D HPLC-DAD spectra, and three linear models were obtained, respectively. The correlation coefficients (R(2)) for training and test sets were more than 0.999, and the statistical parameters and strict validation supported the reliability of established models. The analytical results suggest that the Zernike moment selected by stepwise regression can be used in the quantitative analysis of target compounds. Our study provides a new idea for quantitative analysis using 3D spectra, which can be extended to the analysis of other 3D spectra obtained by different methods or instruments.

Genetic and Chemical Profiling of Gymnema sylvestre Accessions from Central India: Its Implication for Quality Control and Therapeutic Potential of Plant

PubMed Central

Verma, Ashutosh Kumar; Dhawan, Sunita Singh; Singh, Seema; Bharati, Kumar Avinash; Jyotsana

2016-01-01

Background: Gymnema sylvestre, a vulnerable plant species, is mentioned in Indian Pharmacopeia as an antidiabetic drug Objective: Study of genetic and chemical diversity and its implications in accessions of G. sylvestre Materials and Methods: Fourteen accessions of G. sylvestre collected from Central India and assessment of their genetic and chemical diversity were carried out using ISSR (inter simple sequence repeat) and HPLC (high performance liquid chromatography) fingerprinting methods Results: Among the screened 40 ISSR primers, 15 were found polymorphic and collectively produced nine unique accession-specific bands. The maximum and minimum numbers of amplicones were noted for ISSR-15 and ISSR-11, respectively. The ISSR -11 and ISSR-13 revealed 100% polymorphism. HPLC chromatograms showed that accessions possess the secondary metabolites of mid-polarity with considerable variability. Unknown peaks with retention time 2.63, 3.41, 23.83, 24.50, and 44.67 were found universal type. Comparative hierarchical clustering analysis based on foresaid fingerprints indicates that both techniques have equal potential to discriminate accessions according to percentage gymnemic acid in their leaf tissue. Second approach was noted more efficiently for separation of accessions according to their agro-climatic/collection site Conclusion: Highly polymorphic ISSRs could be utilized as molecular probes for further selection of high gymnemic acid yielding accessions. Observed accession specific bands may be used as a descriptor for plant accessions protection and converted into sequence tagged sites markers. Identified five universal type peaks could be helpful in identification of G. sylvestre-based various herbal preparations. SUMMARY Nine accession specific unique bandsFive marker peaks for G. sylvestre.Suitability of genetic and chemical fingerprinting Abbreviations used: HPLC: High Performance Liquid Chromatography, ISSR: Inter Simple Sequence Repeats, CTAB: Cetyl Trimethylammonium Bromide, DNTP: Deoxynucleotide Triphosphates PMID:27761067

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing.

PubMed

AlKhalidi, Bashar A; Shtaiwi, Majed; AlKhatib, Hatim S; Mohammad, Mohammad; Bustanji, Yasser

2008-01-01

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.

A Stability-Indicating HPLC Method for the Determination of Memantine Hydrochloride in Dosage Forms through Derivatization with 1-Fluoro-2,4-dinitrobenzene

PubMed Central

Jalalizadeh, Hassan; Raei, Mahdi; Tafti, Razieh Fallah; Farsam, Hassan; Kebriaeezadeh, Abbas; Souri, Effat

2014-01-01

Memantine is chemically a tricyclic amine and is used for Parkinson’s disease and movement disorders. Although several HPLC methods with different derivatization reagents have been developed for the determination of memantine in biological fluids, there are some complications which limit the use of these methods in routine analysis of memantine in in vitro tests. We established a simple, sensitive, precise, and accurate HPLC method for the quantification of memantine in dosage forms. Pre-column derivatization of memantine was performed with 1-fluoro-2,4-dinitrobenzene and the reaction product was separated on a Nova-Pak C18 column. A mixture of acetonitrile and sodium dihydrogenphosphate (pH 2.5; 0.05 M) (70: 30, v/v) was used as the mobile phase. UV detection was performed at 360 nm. Forced degradation studies were performed on a powdered tablet sample of memantine hydro-chloride using acidic (0.1 M hydrochloric acid), basic (0.1 M sodium hydroxide), oxidative (10% hydrogen peroxide), thermal (105°C), photolytic, and humidity conditions. Good linearity (r2=0.999) was obtained over the range of 1–12 μg mL−1 of memantine hydrochloride with acceptable within-day and between-day precision values in the range of 0.05–0.95%. The proposed method was used for the assay determination and dissolution rate study of memantine dosage forms with excellent specificity. PMID:24959398

HPLC Plasma Assay of a Novel Anti-MRSA Compound, Kaempferol-3-O-Alpha-L-(2",3"-di-p-coumaroyl)rhamnoside, from Sycamore Leaves.

PubMed

Zhang, Yiguan; Valeriote, Frederick; Swartz, Kenneth; Chen, Ben; Hamann, Mark T; Rodenburg, Douglas L; McChesney, James D; Shaw, Jiajiu

2015-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that is resistant to current antibiotic therapy. Thus, there is an urgent need for novel antimicrobial agents that can effectively combat these new strains of drug-resistant "superbugs". Recently, fractionation of an extract from Platanus occidentalis (American sycamore) leaves produced an active kaempferol molecule, 3-O-alpha-L-(2",3"-di-p-coumaroyl)rhamnoside (KCR), in four isomeric forms; all four isomers exhibit potent anti-MRSA activity. In order to further the preclinical development of KCR as a new antibiotic class, we developed and validated a simple analytical method for assaying KCR plasma concentration. Because KCR will be developed as a new drug, although comprising four stereoisomers, the analytical method was devised to assay the total amount of all four isomers. In the present work, both a plasma processing procedure and an HPLC method have been developed and validated. Mouse plasma containing KCR was first treated with ethanol and then centrifuged. The supernatant was dried, suspended in ethanol, centrifuged, and the supernatant was injected into an HPLC system comprising a Waters C18, a mobile phase composing methanol, acetonitrile, and trifluoroacetic acid and monitored at 313 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 0.27 microg/mL, and high accuracy. In summary, this method allows a rapid analysis of KCR in the plasma samples for pharmacokinetics studies.

Simple, fast and reliable liquid chromatographic and spectrophotometric methods for the determination of theophylline in urine, saliva and plasma samples.

PubMed

Charehsaz, Mohammad; Gürbay, Aylin; Aydin, Ahmet; Sahin, Gönül

2014-01-01

In this study, a high-performance liquid chromatographic method (HPLC) and UV spectrophotometric method were developed, validated and applied for the determination of theophylline in biological fluids. Liquid- liquid extraction is performed for isolation of the drug and elimination of plasma and saliva interferences. Urine samples were applied without any extraction. The chromatographic separation was achieved on a C18 column by using 60:40 methanol:water as mobile phase under isocratic conditions at a flow rate of 0.75 mL/min with UV detection at 280 nm in HPLC method. UV spectrophotometric analysis was performed at 275 nm. the limit of quantification: 1.1 µg/mL for urine, 1.9 µg/mL for saliva, 3.1 µg/mL for plasma; recovery: 94.85% for plasma, 100.45% for saliva, 101.39% for urine; intra-day precision: 0.22-2.33%, inter-day precision: 3.17-13.12%. Spectrophotometric analysis results were as follows: the limit of quantitation: 5.23 µg/mL for plasma, 8.7 µg/mL for urine; recovery: 98.27% for plasma, 95.25% for urine; intra-day precision: 2.37 - 3.00%, inter-day precision: 5.43-7.91%. It can be concluded that this validated HPLC method is easy, precise, accurate, sensitive and selective for determination of theophylline in biological samples. Also spectrophotometric analysis can be used where it can be applicable.

HPLC Plasma Assay of a Novel Anti-MRSA Compound, Kaempferol-3-O-Alpha-L-(2",3"-di-p-coumaroyl)rhamnoside, from Sycamore Leaves

PubMed Central

Zhang, Yiguan; Valeriote, Frederick; Swartz, Kenneth; Chen, Ben; Hamann, Mark T.; Rodenburg, Douglas L.; McChesney, James D.

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that is resistant to current antibiotic therapy. Thus, there is an urgent need for novel antimicrobial agents that can effectively combat these new strains of drug-resistant “superbugs”. Recently, fractionation of an extract from Platanus occidentalis (American sycamore) leaves produced an active kaempferol molecule, 3-O-alpha-L-(2",3"-di-p-coumaroyl)rhamnoside (KCR), in four isomeric forms; all four isomers exhibit potent anti-MRSA activity. In order to further the preclinical development of KCR as a new antibiotic class, we developed and validated a simple analytical method for assaying KCR plasma concentration. Because KCR will be developed as a new drug, although comprising four stereoisomers, the analytical method was devised to assay the total amount of all four isomers. In the present work, both a plasma processing procedure and an HPLC method have been developed and validated. Mouse plasma containing KCR was first treated with ethanol and then centrifuged. The supernatant was dried, suspended in ethanol, centrifuged, and the supernatant was injected into an HPLC system comprising a Waters C18, a mobile phase composing methanol, acetonitrile, and trifluoroacetic acid and monitored at 313 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 0.27 µg/mL, and high accuracy. In summary, this method allows a rapid analysis of KCR in the plasma samples for pharmacokinetics studies. PMID:26434123

Development and validation of a rapid stability indicating HPLC-method using monolithic stationary phase and two spectrophotometric methods for determination of antihistaminic acrivastine in capsules

NASA Astrophysics Data System (ADS)

Gouda, Ayman A.; Hashem, Hisham; Jira, Thomas

2014-09-01

Simple, rapid and accurate high performance liquid chromatographic (HPLC) and spectrophotometric methods are described for determination of antihistaminic acrivastine in capsules. The first method (method A) is based on accurate, sensitive and stability indicating chromatographic separation method. Chromolith® Performance RP-18e column, a relatively new packing material consisting of monolithic rods of highly porous silica, was used as stationary phase applying isocratic binary mobile phase of ACN and 25 mM NaH2PO4 pH 4.0 in the ratio of 22.5:77.5 at flow rate of 5.0 mL/min and 40 °C. A diode array detector was used at 254 nm for detection. The elution time of acrivastine was found to be 2.080 ± 0.032. The second and third methods (methods B and C) are based on the oxidation of acrivastine with excess N-bromosuccinimide (NBS) and determination of the unconsumed NBS with, metol-sulphanilic acid (λmax: 520 nm) or amaranth dye (λmax: 530 nm). The reacted oxidant corresponds to the drug content. Beer’s law is obeyed over the concentration range 1.563-50, 2.0-20 and 1.0-10 μg mL-1 for methods A, B and C, respectively. The limits of detection and quantitation were 0.40, 0.292 and 0.113 μg mL-1 and 0.782, 0.973 and 0.376 μg mL-1 for methods A, B and C, respectively. The HPLC method was validated for system suitability, linearity, precision, limits of detection and quantitation, specificity, stability and robustness. Stability tests were done through exposure of the analyte solution for four different stress conditions and the results indicate no interference of degradants with HPLC-method. The proposed methods was favorably applied for determination of acrivastine in capsules formulation. Statistical comparison of the obtained results from the analysis of the studied drug to those of the reported method using t- and F-tests showed no significant difference between them.

Preliminary evaluation of monolithic column high-performance liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection for the determination of quetiapine in human body fluids.

PubMed

Bellomarino, Sara A; Brown, Allyson J; Conlan, Xavier A; Barnett, Neil W

2009-03-15

High-performance liquid chromatography (HPLC) with tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection methodology is reported for the determination of the atypical antipsychotic drug quetiapine and the observation of its major active and inactive metabolites in human urine and serum. The method uses a monolithic chromatographic column allowing high flow rates of 3 mLmin(-1) enabling rapid quantification. Flow injection analysis (FIA) with tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection and HPLC time of flight mass spectrometry (TOF-MS) were used for the determination of quetiapine in a pharmaceutical preparation to establish its suitability as a calibration standard. The limit of detection achieved with FIA was 2 x 10(-11) molL(-1) in simple aqueous solution. The limits of detection achieved with HPLC were 7 x 10(-8) and 2 x 10(-10) molL(-1) in urine and serum, respectively. The calibration range for FIA was between 5 x 10(-9) and 1 x 10(-6) molL(-1). The calibration ranges for HPLC were between 1 x 10(-7)-1 x 10(-4) and 1 x 10(-8)-1 x 10(-4) molL(-1) in urine and serum, respectively. The quetiapine concentrations in patient samples were found to be 3 x 10(-6) molL(-1) in urine and 7 x 10(-7) molL(-1) in serum. Without the need for preconcentration, the HPLC detection limits compared favourably with those in previously published methodologies. The metabolites were identified using HPLC-TOF-MS.

Determination of triterpene glycosides in sea cucumber (Stichopus japonicus) and its related products by high-performance liquid chromatography.

PubMed

Dong, Ping; Xue, Chang-Hu; Yu, Lin-Fang; Xu, Jie; Chen, Shi-Guo

2008-07-09

A creative and sensitive method has been developed for the determination of triterpene glycosides concentrations in sea cucumber ( Stichopus japonicus) and related products by using d-quinovose (6-deoxyglucose) as the measurement standard by reverse-phase high-performance liquid chromatography (HPLC) and variable-wavelength detection. d-quinovose, which is a unique monosaccharide in holostane triterpene glycosides, was liberated by acid hydrolysis and precolumn derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP). PMP-quinovose was analyzed by HPLC with 22% acetonitrile in 0.05 M KH2PO4 aquatic solution (pH 5.2) as mobile phase. The calibration curves of d-quinovose were linear within the range of 6.56-164 mg/L (r(2) > 0.995). The contents of triterpene glycosides in various S. japonicus products were determined after appropriate pretreatment methods. The concentration of triterpene glycosides was calculated by the formula C = C(qui) x alpha (alpha = 8.5). The result showed that this method was a simple, rapid, and stable method for the determination of triterpene glycosides in S. japonicus products.

Development of a rapid, simple assay of plasma total carotenoids

PubMed Central

2012-01-01

Background Plasma total carotenoids can be used as an indicator of risk of chronic disease. Laboratory analysis of individual carotenoids by high performance liquid chromatography (HPLC) is time consuming, expensive, and not amenable to use beyond a research laboratory. The aim of this research is to establish a rapid, simple, and inexpensive spectrophotometric assay of plasma total carotenoids that has a very strong correlation with HPLC carotenoid profile analysis. Results Plasma total carotenoids from 29 volunteers ranged in concentration from 1.2 to 7.4 μM, as analyzed by HPLC. A linear correlation was found between the absorbance at 448 nm of an alcohol / heptane extract of the plasma and plasma total carotenoids analyzed by HPLC, with a Pearson correlation coefficient of 0.989. The average coefficient of variation for the spectrophotometric assay was 6.5% for the plasma samples. The limit of detection was about 0.3 μM and was linear up to about 34 μM without dilution. Correlations between the integrals of the absorption spectra in the range of carotenoid absorption and total plasma carotenoid concentration gave similar results to the absorbance correlation. Spectrophotometric assay results also agreed with the calculated expected absorbance based on published extinction coefficients for the individual carotenoids, with a Pearson correlation coefficient of 0.988. Conclusion The spectrophotometric assay of total carotenoids strongly correlated with HPLC analysis of carotenoids of the same plasma samples and expected absorbance values based on extinction coefficients. This rapid, simple, inexpensive assay, when coupled with the carotenoid health index, may be useful for nutrition intervention studies, population cohort studies, and public health interventions. PMID:23006902

Comparative Validation of the Determination of Sofosbuvir in Pharmaceuticals by Several Inexpensive Ecofriendly Chromatographic, Electrophoretic, and Spectrophotometric Methods.

PubMed

El-Yazbi, Amira F

2017-07-01

Sofosbuvir (SOFO) was approved by the U.S. Food and Drug Administration in 2013 for the treatment of hepatitis C virus infection with enhanced antiviral potency compared with earlier analogs. Notwithstanding, all current editions of the pharmacopeias still do not present any analytical methods for the quantification of SOFO. Thus, rapid, simple, and ecofriendly methods for the routine analysis of commercial formulations of SOFO are desirable. In this study, five accurate methods for the determination of SOFO in pharmaceutical tablets were developed and validated. These methods include HPLC, capillary zone electrophoresis, HPTLC, and UV spectrophotometric and derivative spectrometry methods. The proposed methods proved to be rapid, simple, sensitive, selective, and accurate analytical procedures that were suitable for the reliable determination of SOFO in pharmaceutical tablets. An analysis of variance test with P-value > 0.05 confirmed that there were no significant differences between the proposed assays. Thus, any of these methods can be used for the routine analysis of SOFO in commercial tablets.

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography.

PubMed

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-11-01

To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 °C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 μm) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min(-1) at 210 nm and 230 nm detection. The injection volume was 10 μL, and the separation was carried out isothermally at 30 °C in a heated chamber. The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants.

Simultaneous determination of atorvastatin calcium and ramipril in capsule dosage forms by high-performance liquid chromatography and high-performance thin layer chromatography.

PubMed

Panchal, Hiral J; Suhagia, Bhanubhai N

2010-01-01

Two simple and accurate methods to determine atorvastatin calcium and ramipril in capsule dosage forms were developed and validated using HPLC and HPTLC. The HPLC separation was achieved on a Phenomenex Luna C18 column (250 x 4.6 mm id, 5 microm) in the isocratic mode using 0.1% phosphoric acid-acetonitrile (38 + 62, v/v), pH 3.5 +/- 0.05, mobile phase at a flow rate of 1 ml/min. The retention times were 6.42 and 2.86 min for atorvastatin calcium and ramipril, respectively. Quantification was achieved with a photodiode array detector set at 210 nm over the concentration range of 0.5-5 microg/mL for each, with mean recoveries (at three concentration levels) of 100.06 +/- 0.49% and 99.95 +/- 0.63% RSD for atorvastatin calcium and ramipril, respectively. The HPTLC separation was achieved on silica gel 60 F254 HPTLC plates using methanol-benzene-glacial acetic acid (19.6 + 80.0 + 0.4, v/v/v) as the mobile phase. The Rf values were 0.40 and 0.20 for atorvastatin calcium and ramipril, respectively. Quantification was achieved with UV densitometry at 210 nm over the concentration range of 50-500 ng/spot for each, with mean recoveries (at three concentration levels) of 99.98 +/- 0.75% and 99.87 +/- 0.83% RSD for atorvastatin calcium and ramipril, respectively. Both methods were validated according to International Conference on Harmonization guidelines and found to be simple, specific, accurate, precise, and robust. The mean assay percentages for atorvastatin calcium and ramipril were 99.90 and 99.55% for HPLC and 99.91 and 99.47% for HPTLC, respectively. The methods were successfully applied for the determination of atorvastatin calcium and ramipril in capsule dosage forms without any interference from common excipients.

Protein Adsorption and Its Role in Bacterial Film Development

DTIC Science & Technology

1989-06-27

only the secondary antibody conjugated to alkaline phosphatase was used. Combined Amino Acids as Measured by HPLC We are interested in a simple, direct...specific assay for chitin that relies on the lectin, wheat germ agglutinin (WGA). Lectins are a general class of proteins that bind to carbohydrates. The...protein; 2) a new method for measuring combined amino acids (includes proteins) in seawater was shown to measure higher concentration than the old

New method to measure the carbamoylating activity of nitrosoureas by electron paramagnetic resonance spectroscopy.

PubMed

Gadzheva, V; Ichimori, K; Raikov, Z; Nakazawa, H

1997-08-01

A new method for measuring the carbamoylating activity of nitrosoureas and isocyanates using electron paramagnetic resonance (EPR) spectroscopy is described. The extent and time course of carbamoylation reaction of chloroethyl isocyanate and a series of 9 nitrosoureas toward amino group of 4-amino-2,2,6,6-tetramethyl-piperidine-1-oxyl were examined with both the EPR method and the HPLC method which has been proposed by Brubaker et al. [Biochem. Pharmacol. 35:2359 (1986)]. Spin-labeled nitrosoureas we synthesized are included in this study since they have less toxicity or more efficiency than commercially available drug in some cases. The concentration of carbamoylated product was easily determined with the EPR spectra. There is a very high correlation (r = 0.982, t = 2.58, N = 10, p < 0.001) between the EPR and HPLC methods. Spin-labeled nitrosoureas showed lower carbamoylating activity than non-labeled analogues. The carbamoylating activity for these nitrosourea depended on the reactivity of isocyanate intermediate and almost independent of their half life. This rapid and simple EPR method is suitable for the detailed investigation of the rate and extent of carbamoylation reaction.

Liquid chromatography method to determine polyamines in thermosetting polymers.

PubMed

Dopico-García, M S; López-Vilariño, J M; Fernández-Martínez, G; González-Rodríguez, M V

2010-05-14

A simple, robust and sensitive analytical method to determine three polyamines commonly used as hardeners in epoxy resin systems and in the manufacture of polyurethane is reported. The studied polyamines are: one tetramine, TETA (triethylenetetramine), and two diamines, IPDA (Isophorone diamine) and TCD-diamine (4,7-methano-1H-indene-5,?-dimethanamine, octahydro-). The latter has an incompletely defined structure, and, as far as we know, has not been previously determined by other methods. All three polyamines contain primary amines; TETA also contains secondary amines. The analytical method involves derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, used for the first time for these compounds, followed by high performance liquid chromatography (HPLC) analysis with a fluorescence (FL) detector (lambda excitation 248nm, lambda emision 395nm). The HPLC-DAD-LTQ Orbitrap MS was used in order to provide structural information about the obtained derivatized compounds. The hybrid linear ion trap LTQ Orbitrap mass spectrometer has been introduced in recent years and provides a high mass accuracy. The structures of the derivatized analytes were identified from the protonated molecular ions [M+H](+) and corresponded to the fully labelled analytes. The following analytical parameters were determined for the method using the HPLC-FL: linearity, precision (2.5-10%), instrumental precision intraday (0.8-1.5%) and interday (2.9-6.3%), and detection limits (0.02-0.14mgL(-1)). The stability of stock solutions and derivatized compounds was also investigated. The method was applied to determine the amine free content in epoxy resin dust collected in workplaces. Copyright 2010 Elsevier B.V. All rights reserved.

Linear modeling of the soil-water partition coefficient normalized to organic carbon content by reversed-phase thin-layer chromatography.

PubMed

Andrić, Filip; Šegan, Sandra; Dramićanin, Aleksandra; Majstorović, Helena; Milojković-Opsenica, Dušanka

2016-08-05

Soil-water partition coefficient normalized to the organic carbon content (KOC) is one of the crucial properties influencing the fate of organic compounds in the environment. Chromatographic methods are well established alternative for direct sorption techniques used for KOC determination. The present work proposes reversed-phase thin-layer chromatography (RP-TLC) as a simpler, yet equally accurate method as officially recommended HPLC technique. Several TLC systems were studied including octadecyl-(RP18) and cyano-(CN) modified silica layers in combination with methanol-water and acetonitrile-water mixtures as mobile phases. In total 50 compounds of different molecular shape, size, and various ability to establish specific interactions were selected (phenols, beznodiazepines, triazine herbicides, and polyaromatic hydrocarbons). Calibration set of 29 compounds with known logKOC values determined by sorption experiments was used to build simple univariate calibrations, Principal Component Regression (PCR) and Partial Least Squares (PLS) models between logKOC and TLC retention parameters. Models exhibit good statistical performance, indicating that CN-layers contribute better to logKOC modeling than RP18-silica. The most promising TLC methods, officially recommended HPLC method, and four in silico estimation approaches have been compared by non-parametric Sum of Ranking Differences approach (SRD). The best estimations of logKOC values were achieved by simple univariate calibration of TLC retention data involving CN-silica layers and moderate content of methanol (40-50%v/v). They were ranked far well compared to the officially recommended HPLC method which was ranked in the middle. The worst estimates have been obtained from in silico computations based on octanol-water partition coefficient. Linear Solvation Energy Relationship study revealed that increased polarity of CN-layers over RP18 in combination with methanol-water mixtures is the key to better modeling of logKOC through significant diminishing of dipolar and proton accepting influence of the mobile phase as well as enhancing molar refractivity in excess of the chromatographic systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography

PubMed Central

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-01-01

Purpose: To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). Methods: The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 °C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 μm) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min−1 at 210 nm and 230 nm detection. The injection volume was 10 μL, and the separation was carried out isothermally at 30 °C in a heated chamber. Results: The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. Conclusion: This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants. PMID:26819933

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection.

PubMed

Celik, Saliha Esin; Ozyürek, Mustafa; Güçlü, Kubilay; Apak, Reşat

2010-07-26

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 microM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples. Copyright 2010 Elsevier B.V. All rights reserved.

Simple and rapid analysis of endocrine disruptors in liquid medicines and intravenous injection solutions by automated in-tube solid-phase microextraction/high performance liquid chromatography.

PubMed

Mitani, Kurie; Narimatsu, Shizuo; Izushi, Fumio; Kataoka, Hiroyuki

2003-07-14

A simple and rapid method was developed for analyzing contamination of endocrine disruptors in liquid medicines and intravenous injection solutions. Endocrine disrupting compounds such as bisphenol A (BPA), alkylphenols and phthalates were quantitated by on-line in-tube solid-phase microextraction coupled with high performance liquid chromatography (in-tube SPME/HPLC) with UV detection. The liquid medicines and intravenous injection solutions could be used directly without any pretreatment, and the BPA, alkylphenols and phthalates in these solutions were automatically analyzed. The limits of quantification for these compounds were 1-10 ng/ml. Recoveries of these compounds spiked to the intravenous injection solutions was over 80%, except for some phthalates. Di-n-butyl phthalate (DBP) was detected at a concentration of 7-60 ng/ml in most intravenous injection solutions in plastic containers, but it was not detected in solutions in glass bottles. Diethyl phthalate, di-n-propyl phthalate, DBP and di-2-ethylhexyl phthalate (DEHP) were also detected in syrup, lotion and eye drops in plastic containers. On the other hand, BPA and alkylphenols were not detected at all in these solutions. DEHP contamination from an administration set increased when total vitamin formulation was added to the infusion solution. DEHP was easily leached from polyvinyl chloride tubing by polysorbate 80. The in-tube SPME/HPLC method is simple, rapid and automatic, and it provides a useful tool for the screening and determination of endocrine disruptor contamination in liquid medicines and intravenous injection solutions.

Quantification of neutral human milk oligosaccharides by graphitic carbon HPLC with tandem mass spectrometry

PubMed Central

Bao, Yuanwu; Chen, Ceng; Newburg, David S.

2012-01-01

Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation. PMID:23068043

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Quantitative Clinical Diagnostic Analysis of Acetone in Human Blood by HPLC: A Metabolomic Search for Acetone as Indicator

PubMed Central

Akgul Kalkan, Esin; Sahiner, Mehtap; Ulker Cakir, Dilek; Alpaslan, Duygu; Yilmaz, Selehattin

2016-01-01

Using high-performance liquid chromatography (HPLC) and 2,4-dinitrophenylhydrazine (2,4-DNPH) as a derivatizing reagent, an analytical method was developed for the quantitative determination of acetone in human blood. The determination was carried out at 365 nm using an ultraviolet-visible (UV-Vis) diode array detector (DAD). For acetone as its 2,4-dinitrophenylhydrazone derivative, a good separation was achieved with a ThermoAcclaim C18 column (15 cm × 4.6 mm × 3 μm) at retention time (t R) 12.10 min and flowrate of 1 mL min−1 using a (methanol/acetonitrile) water elution gradient. The methodology is simple, rapid, sensitive, and of low cost, exhibits good reproducibility, and allows the analysis of acetone in biological fluids. A calibration curve was obtained for acetone using its standard solutions in acetonitrile. Quantitative analysis of acetone in human blood was successfully carried out using this calibration graph. The applied method was validated in parameters of linearity, limit of detection and quantification, accuracy, and precision. We also present acetone as a useful tool for the HPLC-based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis. PMID:27298750

Multivariate curve resolution-assisted determination of pseudoephedrine and methamphetamine by HPLC-DAD in water samples.

PubMed

Vosough, Maryam; Mohamedian, Hadi; Salemi, Amir; Baheri, Tahmineh

2015-02-01

In the present study, a simple strategy based on solid-phase extraction (SPE) with a cation exchange sorbent (Finisterre SCX) followed by fast high-performance liquid chromatography (HPLC) with diode array detection coupled with chemometrics tools has been proposed for the determination of methamphetamine and pseudoephedrine in ground water and river water. At first, the HPLC and SPE conditions were optimized and the analytical performance of the method was determined. In the case of ground water, determination of analytes was successfully performed through univariate calibration curves. For river water sample, multivariate curve resolution and alternating least squares was implemented and the second-order advantage was achieved in samples containing uncalibrated interferences and uncorrected background signals. The calibration curves showed good linearity (r(2) > 0.994).The limits of detection for pseudoephedrine and methamphetamine were 0.06 and 0.08 μg/L and the average recovery values were 104.7 and 102.3% in river water, respectively. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Online extraction-high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry for rapid flavonoid profiling of Fructus aurantii immaturus.

PubMed

Tong, Runna; Peng, Mijun; Tong, Chaoying; Guo, Keke; Shi, Shuyun

2018-03-01

Chemical profiling of natural products by high performance liquid chromatography (HPLC) was critical for understanding of their clinical bioactivities, and sample pretreatment steps have been considered as a bottleneck for analysis. Currently, concerted efforts have been made to develop sample pretreatment methods with high efficiency, low solvent and time consumptions. Here, a simple and efficient online extraction (OLE) strategy coupled with HPLC-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS) was developed for rapid chemical profiling. For OLE strategy, guard column inserted with ground sample (2 mg) instead of sample loop was connected with manual injection valve, in which components were directly extracted and transferred to HPLC-DAD-QTOF-MS/MS system only by mobile phase without any extra time, solvent, instrument and operation. By comparison with offline heat-reflux extraction for Fructus aurantii immaturus (Zhishi), OLE strategy presented higher extraction efficiency perhaps because of the high pressure and gradient elution mode. A total of eighteen flavonoids were detected according to their retention times, UV spectra, exact mass, and fragmentation ions in MS/MS spectra, and compound 9, natsudaidain-3-O-glucoside, was discovered in Zhishi for the first time. It is concluded that the developed OLE-HPLC-DAD-QTOF-MS/MS system offers new perspectives for rapid chemical profiling of natural products. Copyright © 2018. Published by Elsevier B.V.

Identification and Characterization of Asulam Impurities in Self Made Bulk Batch Synthesis and Quantification by RP-HPLC Method.

PubMed

Mahaboob Basha, D; Venkata Reddy, G; Gopi Krishna, Y; Kumara Swamy, B E; Vijay, Rajani

2018-04-19

The first approach of this research paper explores the simultaneous characterization and determination of theAsulam active ingredient and its associated nine impurities in bulk batch production by the gradient reverse-phase high-performance liquid chromatographic (RP-HPLC) method. The best separation from its potential impurities and reproducible method was achieved by selecting the Cosmosil C-18 (250 × 4.6 mm, 5 μm particle size) analytical column with a run time of 40 min. The pumping chromatographic mobile phase was composed of 0.1% formic acid in milli-Q water (pH ~2.72) and methanol (80 + 20, v/v). An ambient column-oven temperature and UV detection at 260 nm were used. For this broad resolution, a gradient program was employed at a flow rate of 1.20 mL/min. All potential related substances in Asulam bulk manufacturing were ascertained by mass, proton nuclear magnetic resonance, and infrared spectroscopy. The developed HPLC method was validated with respect to linearity (25.64-151.83 mg/L for Asulam and 0.71-16.29, 1.02-12.26, 1.01-20.29, 0.60-10.01, 1.04-16.65, 0.94-22.47, 0.93-16.60, 1.00-12.45, 1.00-12.45, and 0.71-12.17 mg/L for Impurities A to I with a correlation coefficient 0.999 for Asulam and all the impurities), precision (RSD, % for active analyte Asulam and impurities were ˂2%), accuracy (percent recovery for Asulam at two levels ranged from 99.28 to 99.35%, and for Impurities A to I, it was 93.44 to 101.41%), and specificity. Hence, this simple and reliable HPLC method was able to determine the purity of Asulam active analyte and the level of impurities in bulk batch synthesis. By using this quantified procedure, five self-made production batches were analyzed simultaneously.

77 FR 67282 - Dinotefuran; Pesticide Tolerances for Emergency Exemptions

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

... performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method is available. For the determination of residues of dinotefuran only, an HPLC/ultraviolet (UV) detection method is available. For the determination of only the metabolites (DN and UF), HPLC/MS and HPLC/MS/MS methods are available. These methods...

Development of a Simple Dipstick Assay for Operational Monitoring of DDT.

PubMed

Ismail, Hanafy M; Kumar, Vijay; Singh, Rudra P; Williams, Christopher; Shivam, Pushkar; Ghosh, Ayan; Deb, Rinki; Foster, Geraldine M; Hemingway, Janet; Coleman, Michael; Coleman, Marlize; Das, Pradeep; Paine, Mark J I

2016-01-01

Indoor residual spraying (IRS) of DDT is used to control visceral leishmaniasis (VL) in India. However, the quality of spraying is severely compromised by a lack of affordable field assays to monitor target doses of insecticide. Our aim was to develop a simple DDT insecticide quantification kit (IQK) for monitoring DDT levels in an operational setting. DDT quantification was based on the stoichiometric release of chloride from DDT by alkaline hydrolysis and detection of the released ion using Quantab chloride detection strips. The assay was specific for insecticidal p,p`-DDT (LoQ = 0.082 g/m2). Bostik discs were effective in post spray wall sampling, extracting 25-70% of active ingredient depending on surface. Residual DDT was sampled from walls in Bihar state in India using Bostik adhesive discs and DDT concentrations (g p,p`-DDT/m2) were determined using IQK and HPLC (n = 1964 field samples). Analysis of 161 Bostik samples (pooled sample pairs) by IQK and HPLC produced excellent correlation (R2 = 0.96; Bland-Altman bias = -0.0038). IQK analysis of the remaining field samples matched HPLC data in identifying households that had been under sprayed, in range or over sprayed. A simple dipstick assay has been developed for monitoring DDT spraying that gives comparable results to HPLC. By making laboratory-based analysis of DDT dosing accessible to field operatives, routine monitoring of DDT levels can be promoted in low- and middle- income countries to maximise the effectiveness of IRS.

A newly validated high-performance liquid chromatography method with diode array ultraviolet detection for analysis of the antimalarial drug primaquine in the blood plasma.

PubMed

Carmo, Ana Paula Barbosa do; Borborema, Manoella; Ribeiro, Stephan; De-Oliveira, Ana Cecilia Xavier; Paumgartten, Francisco Jose Roma; Moreira, Davyson de Lima

2017-01-01

Primaquine (PQ) diphosphate is an 8-aminoquinoline antimalarial drug with unique therapeutic properties. It is the only drug that prevents relapses of Plasmodium vivax or Plasmodium ovale infections. In this study, a fast, sensitive, cost-effective, and robust method for the extraction and high-performance liquid chromatography with diode array ultraviolet detection (HPLC-DAD-UV ) analysis of PQ in the blood plasma was developed and validated. After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm × 4.6mm i.d. × 5μm) as the stationary phase and a mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80) (45:55) as the mobile phase. The flow rate was 1.0mL·min-1, the oven temperature was 50OC, and absorbance was measured at 264nm. The method was validated for linearity, intra-day and inter-day precision, accuracy, recovery, and robustness. The detection (LOD) and quantification (LOQ) limits were 1.0 and 3.5ng·mL-1, respectively. The method was used to analyze the plasma of female DBA-2 mice treated with 20mg.kg-1 (oral) PQ diphosphate. By combining a simple, low-cost extraction procedure with a sensitive, precise, accurate, and robust method, it was possible to analyze PQ in small volumes of plasma. The new method presents lower LOD and LOQ limits and requires a shorter analysis time and smaller plasma volumes than those of previously reported HPLC methods with DAD-UV detection. The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.

Validated HPLC determination of 2-[(dimethylamino)methyl]cyclohexanone, an impurity in tramadol, using a precolumn derivatisation reaction with 2,4-dinitrophenylhydrazine.

PubMed

Medvedovici, Andrei; Albu, Florin; Farca, Alexandru; David, Victor

2004-01-27

A new method for the determination of 2-[(dimethylamino)methyl]cyclohexanone (DAMC) in Tramadol (as active substance or active ingredient in pharmaceutical formulations) is described. The method is based on the derivatisation of 2-[(dimethylamino)methyl]cyclohexanone with 2,4-dinitrophenylhydrazine (2,4-DNPH) in acidic conditions followed by a reversed-phase liquid chromatographic separation with UV detection. The method is simple, selective, quantitative and allows the determination of 2-[(dimethylamino)methyl]cyclohexanone at the low ppm level. The proposed method was validated with respect to selectivity, precision, linearity, accuracy and robustness.

Simultaneous isocratic HPLC determination of vigabatrin and gabapentin in human plasma by dansyl derivatization.

PubMed

Krivanek, Peter; Koppatz, Karl; Turnheim, Klaus

2003-06-01

A rapid and low-cost assay for simultaneous vigabatrin (VGA) and gabapentin (GBP) determination is described that can be performed with simple HPLC instrumentation. The method involves derivatization of the primary amine group of VGA and GBP with dansyl chloride followed by isocratic separation (column: microBondapak C-18, 10 microm, 300 x 3.9 mm; mobile phase: 50 mmol/L NaH(2)PO(4) in 40% acetonitrile) at 50 degrees C and fluorometric detection (excitation and emission wavelength: 318 and 510 nm, respectively) of the fluorescent product, which is stable for at least 7 days. Correlation coefficients of the calibration curves are >0.999 with a lower limit of detection of 0.3 microg/mL. Between- and within-run coefficients of variation are below 4.5%, and assay time is 15 minutes. This method may be used for therapeutic drug monitoring in the case of GBP and to control patient compliance in the case of VGA.

Quantitative Analysis of Apisin, a Major Protein Unique to Royal Jelly

PubMed Central

Arai, Yasuko; Kato, Kenji

2016-01-01

Apisin, a protein that is unique to royal jelly (RJ), is known to compose the greater part of the RJ proteins and to exist as a heterooligomer containing major royal jelly protein 1 and apisimin. However, few reports on the methods for quantifying apisin have been published. Thus, we attempted to quantify apisin using HPLC, a widely used analytical technique, as described below. Isoelectric precipitation and size-exclusion chromatography were used to obtain the purified protein, which was identified as apisin by SDS-PAGE and LC-MS analyses. The purified apisin was lyophilized and then used to generate a calibration curve to quantify apisin in RJ. The apisin content was fairly constant (i.e., 3.93 to 4.67 w/w%) in natural RJ. This study is the first to describe a simple, standardized method for quantifying apisin using HPLC and suggests that apisin can be used as a benchmark for future evaluations of RJ quality. PMID:27721892

Simple solution for a complex problem: proanthocyanidins, galloyl glucoses and ellagitannins fit on a single calibration curve in high performance-gel permeation chromatography.

PubMed

Stringano, Elisabetta; Gea, An; Salminen, Juha-Pekka; Mueller-Harvey, Irene

2011-10-28

This study was undertaken to explore gel permeation chromatography (GPC) for estimating molecular weights of proanthocyanidin fractions isolated from sainfoin (Onobrychis viciifolia). The results were compared with data obtained by thiolytic degradation of the same fractions. Polystyrene, polyethylene glycol and polymethyl methacrylate standards were not suitable for estimating the molecular weights of underivatized proanthocyanidins. Therefore, a novel HPLC-GPC method was developed based on two serially connected PolarGel-L columns using DMF that contained 5% water, 1% acetic acid and 0.15 M LiBr at 0.7 ml/min and 50 °C. This yielded a single calibration curve for galloyl glucoses (trigalloyl glucose, pentagalloyl glucose), ellagitannins (pedunculagin, vescalagin, punicalagin, oenothein B, gemin A), proanthocyanidins (procyanidin B2, cinnamtannin B1), and several other polyphenols (catechin, epicatechin gallate, epicallocatechin gallate, amentoflavone). These GPC predicted molecular weights represented a considerable advance over previously reported HPLC-GPC methods for underivatized proanthocyanidins. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous analysis of heparosan oligosaccharides by isocratic liquid chromatography with charged aerosol detection/mass spectrometry.

PubMed

Ji, Xiaohu; Hu, Guixin; Zhang, Qiongyan; Wang, Fengshan; Liu, Chunhui

2016-11-05

Uncovering the biological roles of heparosan oligosaccharides requires a simple and robust method for their separation and identification. We reported on systematic investigations of the retention behaviors of synthetic heparosan oligosaccharides on porous graphitic carbon (PGC) column by HPLC with charged aerosol detection. Oligosaccharides were strongly retained by PGC material in water-acetonitrile mobile phase, and eluted by trifluoroacetic acid occurring as narrow peaks. Addition of small fraction of methanol led to better selectivity of PGC to oligosaccharides than acetonitrile modifier alone, presumably, resulting from displacement of methanol to give different chemical environment at the PGC surface. Van't-Hoff plots demonstrated that retention behaviors highly depended on the column temperature and oligosaccharide moieties. By implementing the optimal MeOH content and temperature, a novel isocratic elution method was successfully developed for baseline resolution and identification of seven heparosan oligosaccharides using PGC-HPLC-CAD/MS. This approach allows for rapid analysis of heparosan oligosaccharides from various sources. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and quantitation of vitamins K1 and K3 in cosmetic products for facial skin protection.

PubMed

De Orsi, D; Giannini, G; Gagliardi, L; Carpani, I; Tonelli, D

2008-01-01

A simple and rapid analytical method was developed for the determination of vitamins K1 and K3 in facial anti-rash creams. The procedure is based on an ultrasonic extraction of the cosmetic sample with dimethylacetamide, in the presence of an internal standard, followed by HPLC separation. HPLC was performed using a C18 column and spectrophotometric detection at 333 nm. A linear gradient elution was carried out starting with 50% acetonitrile-methanol (75:25 v/v) and water up to 100% acetonitrile-methanol for 5 min. Linearity was established over the concentration range from 0.2 to 1.0 mg/ml for vitamin K1 and from 0.02 to 0.1 mg/ml for vitamin K3, with LOD values of 100 ng and 20 ng injected, respectively. The accuracy was verified by spiking experiments on model cosmetic samples. The proposed method has been successfully applied for the analysis of commercial samples of creams.

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

PubMed

Wan, Jun-Hui; Tian, Pei-Ling; Luo, Wei-Hao; Wu, Bing-Yi; Xiong, Fu; Zhou, Wan-Jun; Wei, Xiang-Cai; Xu, Xiang-Min

2012-07-15

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-β, β, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and β thalassemia) were found in the area ratio of α/pre-β+β applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/β and α/pre-β+β area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating β thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/β for β thalassemia carriers and 0.626 of α/pre-β+β for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work. Copyright © 2012 Elsevier B.V. All rights reserved.

A simple method for the enrichment of bisphenols using boron nitride.

PubMed

Fischnaller, Martin; Bakry, Rania; Bonn, Günther K

2016-03-01

A simple solid-phase extraction method for the enrichment of 5 bisphenol derivatives using hexagonal boron nitride (BN) was developed. BN was applied to concentrate bisphenol derivatives in spiked water samples and the compounds were analyzed using HPLC coupled to fluorescence detection. The effect of pH and organic solvents on the extraction efficiency was investigated. An enrichment factor up to 100 was achieved without evaporation and reconstitution. The developed method was applied for the determination of bisphenol A migrated from some polycarbonate plastic products. Furthermore, bisphenol derivatives were analyzed in spiked and non-spiked canned food and beverages. None of the analyzed samples exceeded the migration limit set by the European Union of 0.6mg/kg food. The method showed good recovery rates ranging from 80% to 110%. Validation of the method was performed in terms of accuracy and precision. The applied method is robust, fast, efficient and easily adaptable to different analytical problems. Copyright © 2015 Elsevier Ltd. All rights reserved.

High Performance Liquid Chromatography-Diode Array Detector Method for the Simultaneous Determination of Five Compounds in the Pulp and Seed of Sea Buckthorn

PubMed Central

Zhao, Lu; Wen, E; Upur, Halmuart; Tian, Shuge

2017-01-01

Context: Sea buckthorn (Hippophae rhamnoides L.) as a traditional Chinese medicinal plant has various uses in Xinjiang. Objective: A reversed-phase rapid-resolution liquid-chromatography method with diode array detector was developed for simultaneous determination of protocatechuic acid, rutin, quercetin, kaempferol, and isorhamnetin in the pulp and seed of sea buckthorn, a widely used traditional Chinese medicine for promoting metabolism and treating scurvy and other diseases. Settings and design: Compounds were separated on an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm; USA) with gradient elution using methanol and 0.4% phosphoric acid (v/v) at 1.0 mL/min. Detection wavelength was set at 280 nm. Materials and Methods: The fruits of wild sea buckthorn were collected from Wushi County in Aksu, Xinjiang Province. Statistical performances: The RSD of precision test of the five compounds were in the range of 0.60-2.22%, and the average recoveries ranged from 97.36% to 101.19%. Good linearity between specific chromatographic peak and component qualities were observed in the investigated ranges for all the analytes (R2 > 0.9997). Results: The proposed method was successfully applied to determine the levels of five active components in sea buckthorn samples from Aksu in Xinjiang. Conclusions: The proposed method is simple, fast, sensitive, accurate, and suitable for quantitative assessment of the pulp and seed of sea buckthorn. SUMMARY Quantitative analysis method of protocatechuic acid, rutin, quercetin, kaempferol, and isorhamnetin in the extract of sea buckthorn pulp and seed is developed by high-performance liquid chromatography (HPLC) diode array detection.This method is simple and accurate; has strong specificity, good precision, and high recovery rate; and provides a reliable basis for further development of the substances in the pulp and seed of sea buckthorn.The method is widely used for content determination of active ingredients or physiologically active components in traditional Chinese medicine and its preparation Abbreviation used: PR: protocatechuic acid, RU: rutin, QU: quercetin, KA: kaempferol, IS: isorhamnetin, HPLC: high-performance liquid chromatography, HPLC-DAD: high performance liquid chromatographydiode array detector, LOD: linearity and limit of detection, LOQ: limit of quantitation, RSD: relative standard deviation PMID:28216897

A new and simple HPLC method for determination of etamsylate in human plasma and its application to pharmacokinetic study in healthy adult male volunteers

PubMed Central

Helmy, Sally A.; El Bedaiwy, Heba M.

2013-01-01

A new and simple HPLC assay method was developed and validated for the determination of etamsylate in human plasma. After protein precipitation with 6% perchloric acid, satisfactory separation was achieved on a HyPURITY C18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase comprising 20 mM sodium dihydrogen phosphate-2 hydrate (pH was adjusted to 3.5 by phosphoric acid) and acetonitrile at a ratio of 95:5 v/v. The elution was isocratic at ambient temperature with a flow rate of 0.75 ml/min. Allopurinol was used as internal standard. The calibration curve was linear over the range from 0.25 to 20 μg/ml (r2 = 0.999). The limit of quantification for etamsylate in plasma was 0.25 μg/ml. The within day coefficient of variance (%CV) ranged from 3.9% to 10.2%, whereas the between-day %CV ranged from 3.1% to 8.7%. The assay method has been successfully used to estimate the pharmacokinetics of etamsylate after oral administration of a 500 mg tablet under fasting conditions to 24 healthy Egyptian human male volunteers. Various pharmacokinetic parameters including AUC0–t, AUC0–∞, Cmax, Tmax, t1/2, MRT, Cl/F, and Vd/F were determined from plasma concentration–time profile of etamsylate. PMID:24227961

Dispersive liquid-liquid microextraction followed by high-performance liquid chromatography-ultraviolet detection to determination of opium alkaloids in human plasma.

PubMed

Ahmadi-Jouibari, Toraj; Fattahi, Nazir; Shamsipur, Mojtaba; Pirsaheb, Meghdad

2013-11-01

A novel, simple, rapid and sensitive dispersive liquid-liquid microextraction method based on the solidification of floating organic drop (DLLME-SFO) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in human plasma. During the extraction procedure, plasma protein was precipitated by using a mixture of zinc sulfate solution and acetonitrile. Some effective parameters on extraction were studied and optimized. Under the optimum conditions (extraction solvent: 30.0 μl 1-undecanol; disperser solvent: 470 μl acetone; pH: 9; salt addition: 1%(w/v) NaCl and extraction time: 0.5 min), calibration curves are linear in the range of 1.5-1000 μgl(-1) and limit of detections (LODs) are in the range of 0.5-5 μgl(-1). The relative standard deviations (RSDs) for 100 μgl(-1) of morphine and codeine, 10.0 μgl(-1) of papaverine and 20.0 μgl(-1) of noscapine in diluted human plasma are in the range of 4.3-7.4% (n=5). Finally, the method was successfully applied in the determination of opium alkaloids in the actual human plasma samples. The relative recoveries of plasma samples spiked with alkaloids are 88-110.5%. The obtained results show that DLLME-SFO combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in human plasma. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells.

PubMed

Hawwa, Ahmed F; Millership, Jeff S; Collier, Paul S; McElnay, James C

2009-02-20

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

Determination of acrylamide and methacrylamide by normal phase high performance liquid chromatography and UV detection.

PubMed

Paleologos, E K; Kontominas, M G

2005-06-10

A method using normal phase high performance liquid chromatography (NP-HPLC) with UV detection was developed for the analysis of acrylamide and methacrylamide. The method relies on the chromatographic separation of these analytes on a polar HPLC column designed for the separation of organic acids. Identification of acrylamide and methacrylamide is approached dually, that is directly in their protonated forms and as their hydrolysis products acrylic and methacrylic acid respectively, for confirmation. Detection and quantification is performed at 200 nm. The method is simple allowing for clear resolution of the target peaks from any interfering substances. Detection limits of 10 microg L(-1) were obtained for both analytes with the inter- and intra-day RSD for standard analysis lying below 1.0%. Use of acetonitrile in the elution solvent lowers detection limits and retention times, without impairing resolution of peaks. The method was applied for the determination of acrylamide and methacrylamide in spiked food samples without native acrylamide yielding recoveries between 95 and 103%. Finally, commercial samples of french and roasted fries, cookies, cocoa and coffee were analyzed to assess applicability of the method towards acrylamide, giving results similar with those reported in the literature.

Preservative-free triamcinolone acetonide suspension developed for intravitreal injection.

PubMed

Bitter, Christoph; Suter, Katja; Figueiredo, Verena; Pruente, Christian; Hatz, Katja; Surber, Christian

2008-02-01

All commercially available triamcinolone acetonide (TACA) suspensions, used for intravitreal treatment, contain retinal toxic vehicles (e.g., benzyl alcohol, solubilizer). Our aim was to find a convenient and reproducible method to compound a completely preservative-free TACA suspension, adapted to the intraocular physiology, with consistent quality (i.e., proven sterility and stability, constant content and dose uniformity, defined particle size, and 1 year shelf life). We evaluated two published (Membrane-filter, Centrifugation) and a newly developed method (Direct Suspending) to compound TACA suspensions for intravitreal injection. Parameters as TACA content (HPLC), particle size (microscopy and laser spectrometry), sterility, and bacterial endotoxins were assessed. Stability testing (at room temperature and 40 degrees C) was performed: color and homogeneity (visually), particle size (microscopically), TACA content and dose uniformity (HPLC) were analyzed according to International Conference on Harmonisation guidelines. Contrary to the known methods, the direct suspending method is convenient, provides a TACA suspension, which fulfills all compendial requirements, and has a 2-year shelf life. We developed a simple, reproducible method to compound stable, completely preservative-free TACA suspensions with a reasonable shelf-life, which enables to study the effect of intravitreal TACA--not biased by varying doses and toxic compounds or their residues.

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…

[Simultaneous determination of 7 arsenic species in chicken muscle and chicken liver with high performance liquid chromatography-inductively coupled plasma mass spectrometry].

PubMed

Yang, Lijun; Hu, Qiaoru; Guo, Wei; Liu, Yumin; Song, Xiaohua; Zhang, Pengcheng

2011-05-01

A method for the simultaneous determination of 7 arsenic species was developed with high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The sample was extracted with artificial gastric juice. The HPLC separation was performed on an anion analytical column utilizing a gradient elution program of ammonium carbonate and water as the mobile phase. Identification and quantification were achieved by ICP-MS. Good linearities of 7 arsenic species were observed in the range from 1 microg/kg to 50 microg/kg with the correlation coefficients greater than 0.999. The average recoveries of 7 arsenic species spiked at the three levels of 1, 2 and 10 microg/kg ranged from 84.3% to 106.6% with the relative standard deviations of 1.4%-4.2%. The quantification limits of 7 arsenic species were 1 microg/kg. The method was proved to be good reproducibility, high sensitivity and simple preprocessing. This method is suitable for the simultaneous determination of 7 arsenic species in chicken muscle and chicken liver.

Determination of supplemental feeding needs for astaxanthin and canthaxanthin in salmonids by supramolecular solvent-based microextraction and liquid chromatography-UV/VIS spectroscopy.

PubMed

Caballo, Carmen; Costi, Esther María; Sicilia, María Dolores; Rubio, Soledad

2012-09-15

Development of simple and rapid analytical methods for predicting supplemental feeding requirements in aquaculture is a need to reduce production costs. In this article, a supramolecular solvent (SUPRAS) made up of decanoic acid (DeA) assemblies was proposed to simplify sample treatment in the total and individual determination of carotenoids (red-pink pigments) in farmed salmonids. The analytes were quantitatively extracted in a single step that spends a few minutes using a small volume of SUPRAS (i.e. 800 μL) and directly determined in extracts without the interference from fats or other matrix components. The methods based on the combination of microextraction with SUPRAS and photometry or HPLC-UV/VIS spectroscopy were developed for the determination of total and individual carotenoids, respectively. The applicability of the methods was demonstrated by analysing non-fortified and fortified samples of farmed Atlantic salmons and rainbow trouts. Recoveries obtained by photometry and HPLC-UV/VIS spectroscopy were within the intervals 98-104% and 94-106%, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns.

PubMed

Kumar, Ashwini; Kumar Malik, Ashok; Kumar Tewary, Dhananjay; Singh, Baldev

2008-02-01

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Tairabune, Tomohiko; Tomita, Takashi; Sanbe, Atsushi; Takeda, Rika; Kikuchi, Akihiko; Kudo, Kenzo

2017-03-25

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%-101.0%, with a coefficient of variation of 3.39%-8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1-51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility. Copyright © 2017 Elsevier Inc. All rights reserved.

Simultaneous determination of atorvastatin calcium, ezetimibe, and fenofibrate in a tablet formulation by HPLC.

PubMed

Patel, Archita; Macwana, Chhaya; Parmar, Vishal; Patel, Samir

2012-01-01

An accurate, simple, reproducible, and sensitive HPLC method was developed and validated for the simultaneous determination of atorvastatin calcium, ezetimibe, and fenofibrate in a tablet formulation. The analyses were performed on an RP C18 column, 150 x 4.60 mm id, 5 pm particle size. The mobile phase methanol-acetonitrile-water (76 + 13 + 11, v/v/v), was pumped at a constant flow rate of 1 mL/min. UV detection was performed at 253 nm. Retention times of atorvastatin calcium, ezetimibe, and fenofibrate were found to be 2.25, 3.68, and 6.41 min, respectively. The method was validated in terms of linearity, precision, accuracy, LOD, LOQ, and robustness. The response was linear in the range 2-10 microg/mL (r2 = 0.998) for atorvastatin calcium, 2-10 microg/mL (r2 = 0.998) for ezetimibe, and 40-120 microg/mL (r2 = 0.998) for fenofibrate. The developed method can be used for routine quality analysis of the drugs in the tablet formulation.

RP-HPLC determination of water-soluble vitamins in honey.

PubMed

Ciulu, Marco; Solinas, Silvia; Floris, Ignazio; Panzanelli, Angelo; Pilo, Maria I; Piu, Paola C; Spano, Nadia; Sanna, Gavino

2011-01-15

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B(2), riboflavin; vitamin B(3), nicotinic acid; vitamin B(5), pantothenic acid; vitamin B(9), folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg(-1)), we have observed a marked dependence of some of their concentrations (i.e. vitamin B(3) and vitamin B(5)) and the botanical origin of the honey. This insight might lead to important characterization features for this food item. Copyright © 2010 Elsevier B.V. All rights reserved.

[HPLC fingerprint chromatogram of Polygonum multiflorum from Guizhou].

PubMed

Li, Yan; Wang, Hui-Juan; Lin, Bing; Zhao, Zhi; Zhou, Ying

2012-12-01

To establish the fingerprint of Polygonum multiflorum from Guizhou and provide a standard for its quality control. HPLC analysis was performed on Agillent ZABAX-C18 (4.6 mm x 250 mm, 5 microm), gradient eluted composed of acetonitrile-0.4% water solution of phosphoric acid. Column temperature was set at 25 degrees C and the flow rate was 1 mL/min. The detection wavelength was 280 nm and the analysis time was 60 min. 9 common peaks were identified. The RSD of the relative retention time and the relative peak area were less than 3% in analyzing its precision, stability and repeatability of the common peaks, and the similarity of the 16 batches of sample was more than 0.9. The method is simple and reliable, and it can provide a standard and guidance for quality control of Polygonum multiflorum.

Offline solid-phase extraction for preconcentration of pharmaceuticals and personal care products in environmental water and their simultaneous determination using the reversed phase high-performance liquid chromatography method.

PubMed

G Archana; Dhodapkar, Rita; Kumar, Anupama

2016-09-01

The present study reports a precise and simple offline solid-phase extraction (SPE) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of five representative and commonly present pharmaceuticals and personal care products (PPCPs), a new class of emerging pollutants in the aquatic environment. The target list of analytes including ciprofloxacin, acetaminophen, caffeine benzophenone and irgasan were separated by a simple HPLC method. The column used was a reversed-phase C18 column, and the mobile phase was 1 % acetic acid and methanol (20:80 v/v) under isocratic conditions, at a flow rate of 1 mL min(-1). The analytes were separated and detected within 15 min using the photodiode array detector (PDA). The linearity of the calibration curves were obtained with correlation coefficients 0.98-0.99.The limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and ruggedness demonstrated the reproducibility, specificity and sensitivity of the developed method. Prior to the analysis, the SPE was performed using a C18 cartridge to preconcentrate the targeted analytes from the environmental water samples. The developed method was applied to evaluate and fingerprint PPCPs in sewage collected from a residential engineering college campus, polluted water bodies such as Nag river and Pili river and the influent and effluent samples from a sewage treatment plant (STP) situated at Nagpur city, in the peak summer season. This method is useful for estimation of pollutants present in microquantities in the surface water bodies and treated sewage as compared to nanolevel pollutants detected by mass spectrometry (MS) detectors.

A simple method for the determination of glyphosate and aminomethylphosphonic acid in seawater matrix with high performance liquid chromatography and fluorescence detection.

PubMed

Wang, Shu; Liu, Baomin; Yuan, Dongxing; Ma, Jian

2016-12-01

Glyphosate (GLYP) is an important herbicide which is also used as the phosphorus source for marine organisms. The wide applications of GLYP can lead to its accumulation in oceans and coastal waters, thus creating environmental issues. However, there is limited methods for detection of GLYP and its degradation product, aminomethylphosphonic acid (AMPA) in saline samples. Therefore, a simple and fast method for the quantification of GLYP and AMPA in seawater matrix has been developed based on the derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl), separation with high performance liquid chromatography (HPLC) and detection with fluorescence detector (FLD). In order to maximize sensitivity, the derivatization procedure was carefully optimized regarding concentration of FMOC-Cl, volume of borate buffer, pH of borate buffer, mixing and derivatization time. The derivatization reaction could be completed within 30min in seawater samples without any additional clean-up or desalting steps. Under the optimized conditions, the developed HPLC method showed a wide linear response (up to several mg/L, R 2 >0.99). The limits of detection were 0.60μg/L and 0.30μg/L for GLYP and AMPA in seawater matrix, respectively. The relative standard deviation was 14.0% for GLYP (1.00mg/L) and 3.1% for AMPA (100μg/L) in saline samples with three different operators (n=24). This method was applied to determine the concentration of GLYP and AMPA in seawater culture media and the recovery data indicated minimal matrix interference. Due to its simplicity, high reproducibility and successful application in seawater culture media analysis, this method is a potentially useful analytical technique for both marine research and environmental science. Copyright © 2016 Elsevier B.V. All rights reserved.

High-speed counter-current chromatography coupled online to high performance liquid chromatography-diode array detector-mass spectrometry for purification, analysis and identification of target compounds from natural products.

PubMed

Liang, Xuejuan; Zhang, Yuping; Chen, Wei; Cai, Ping; Zhang, Shuihan; Chen, Xiaoqin; Shi, Shuyun

2015-03-13

A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC-HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC-HPLC-diode array detector-mass spectrometry (HSCCC-HPLC-DAD-MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC-DAD-MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4',6'-dihydroxyhydrochalcone-2'-O-β-D-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

Simple saponification method for the quantitative determination of carotenoids in green vegetables.

PubMed

Larsen, Erik; Christensen, Lars P

2005-08-24

A simple, reliable, and gentle saponification method for the quantitative determination of carotenoids in green vegetables was developed. The method involves an extraction procedure with acetone and the selective removal of the chlorophylls and esterified fatty acids from the organic phase using a strongly basic resin (Ambersep 900 OH). Extracts from common green vegetables (beans, broccoli, green bell pepper, chive, lettuce, parsley, peas, and spinach) were analyzed by high-performance liquid chromatography (HPLC) for their content of major carotenoids before and after action of Ambersep 900 OH. The mean recovery percentages for most carotenoids [(all-E)-violaxanthin, (all-E)-lutein epoxide, (all-E)-lutein, neolutein A, and (all-E)-beta-carotene] after saponification of the vegetable extracts with Ambersep 900 OH were close to 100% (99-104%), while the mean recovery percentages of (9'Z)-neoxanthin increased to 119% and that of (all-E)-neoxanthin and neolutein B decreased to 90% and 72%, respectively.

Determination of Tinopal CBS-X in rice papers and rice noodles using HPLC with fluorescence detection and LC-MS/MS.

PubMed

Ko, Kyung Yuk; Lee, Chae A; Choi, Jae Chon; Kim, Meehye

2014-01-01

To date there have been no reports of methods to determine Tinopal CBS-X. We developed a rapid and simple method to determine the Tinopal CBS-X content in rice noodles and rice papers using HPLC equipped with fluorescence detection. Heating the rice noodles and rice papers to 80°C after adding 75% methanol solution induced the release of Tinopal CBS-X from processed rice products. Tinopal CBS-X was separated using an isocratic mobile phase comprising 50% acetonitrile/water containing 0.4% tetrabutyl ammonium hydrogen sulphate at pH 8.0. The samples suspected to be positive by HPLC analysis were then confirmed by LC-MS/MS analysis. This study also investigated the Tinopal CBS-X content of three rice noodle products and two rice papers. The limits of quantification for rice papers and rice noodles were 1.58 and 1.51 µg kg(-1), respectively, and their correlation curves showed good linearity with r(2) ≥ 0.9997 and ≥ 0.9998, respectively. Moreover, rice papers had recoveries of 70.3-83.3% with precision ranging from 5.0% to 7.9%, whereas rice noodles had slightly lower recoveries of 63.4-78.7% and precisions of 8.5-11.5%. Only one rice noodle product contained Tinopal CBS-X, at around 2.1 mg kg(-1), whereas it was not detected in four other samples. Consequently, Tinopal CBS-X from rice noodles and rice papers can be successfully detected using the developed pre-treatment and ion-pairing HPLC system coupled with fluorescence detection.

Development of an isocratic HPLC method for catechin quantification and its application to formulation studies.

PubMed

Li, Danhui; Martini, Nataly; Wu, Zimei; Wen, Jingyuan

2012-10-01

The aim of this study was to develop a simple, rapid and accurate isocratic HPLC analytical method to qualify and quantify five catechin derivatives, namely (+)-catechin (C), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG). To validate the analytical method, linearity, repeatability, intermediate precision, sensitivity, selectivity and recovery were investigated. The five catechin derivatives were completely separated by HPLC using a mobile phase containing 0.1% TFA in Milli-Q water (pH 2.0) mixed with methanol at the volume ratio of 75:25 at a flow rate of 0.8 ml/min. The method was shown to be linear (r²>0.99), repeatable with instrumental precision<2.0 and intra-assay precision<2.5 (%CV, percent coefficient of variation), precise with intra-day variation<1 and inter-day variation<2.5 (%CV, percent coefficient of variation) and sensitive (LOD<1 μg/mL and LOQ<3 μg/mL) over the calibration range for all five derivatives. Derivatives could be fully recovered in the presence of niosomal formulation (recovery rates>91%). Selectivity of the method was proven by the forced degradation studies, which showed that under acidic, basic, oxidation temperature and photolysis stresses, the parent drug can be separated from the degradation products by means of this analytical method. The described method was successfully applied in the in vitro release studies of catechin-loaded niosomes to manifest its utility in formulation characterization. Obtained results indicated that the drug release from niosomal formulations was a biphasic process and a diffusion mechanism regulated the permeation of catechin niosomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

NASA Technical Reports Server (NTRS)

Rounds, M. A.; Nielsen, S. S.; Mitchell, C. A. (Principal Investigator)

1993-01-01

The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.

Pipette-tip solid-phase extraction using poly(1-vinylimidazole-co-trimethylolpropane trimethacrylate) as a new molecularly imprinted polymer in the determination of avermectins and milbemycins in fruit juice and water samples.

PubMed

Teixeira, Roseane Andrade; Flores, Diego Hernando Ângulo; da Silva, Ricky Cássio Santos; Dutra, Flávia Viana Avelar; Borges, Keyller Bastos

2018-10-01

A simple HPLC method was developed for the determination of abamectin (ABA), eprinomectin (EPR), and moxidectin (MOX). Pipette-tip molecularly imprinted polymer solid-phase extraction (PT-MIP-SPE) using poly(1-vinylimidazole-co-trimethylolpropane trimethacrylate) as a selective adsorbent material was studied in detail, including the washing solvent, type and volume of eluent, pH, quantity of adsorbent material and sample volume. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, robustness and stability have been assessed and were within the recommended guidelines. The mean extraction recoveries/relative standard deviation for ABA 1b, EPR, ABA 1a and MOX were 98.77 ± 3.82%, 88.19 ± 2.57%, 110.54 ± 1.52% and 100.42 ± 0.59%, respectively. Finally, the results proved that PT-MIP-SPE coupled to HPLC-UV is an economical, simple and easy-to-perform technique, and presented a high potential for extraction of macrocyclic lactones in mineral water and grape and juice samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

Simultaneous determination of gabapentin, pregabalin, vigabatrin, and topiramate in plasma by HPLC with fluorescence detection.

PubMed

Martinc, Boštjan; Roškar, Robert; Grabnar, Iztok; Vovk, Tomaž

2014-07-01

Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) has been recognized as a useful tool in management of epilepsy. We developed a simple analytical method for simultaneous determination of four second generation AEDs, including gabapentin (GBP), pregabalin (PGB), vigabatrin (VGB), and topiramate (TOP). Analytes were extracted from human plasma using universal solid phase extraction, derivatized with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and analyzed by HPLC with fluorescence detection. Using mass spectrometry we confirmed that NBD-Cl reacts with sulfamate group of TOP similarly as with amine group of the other three analytes. The method is linear (r(2)>0.998) across investigated analytical ranges (0.375-30.0μg/mL for GBP, PGB, and VGB; 0.50-20.0μg/mL for TOP). Intraday and interday precision do not exceed 9.40%. The accuracy is from 95.6% to 106%. The recovery is higher than 80.6%, and the lower limit of quantification is at least 0.5μg/mL. The method is selective and robust. For TOP determination the method was compared to a previously published method and the results obtained by the two methods were in good agreement. The developed method is suitable for routine TDM. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps and traditional Chinese medicine Radix angelicae pubescentis using excitation-emission matrix fluorescence coupled with second-order calibration method

NASA Astrophysics Data System (ADS)

Wang, Li; Wu, Hai-Long; Yin, Xiao-Li; Hu, Yong; Gu, Hui-Wen; Yu, Ru-Qin

2017-01-01

A chemometrics-assisted excitation-emission matrix (EEM) fluorescence method is presented for simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps (SL) and traditional Chinese medicine Radix angelicae pubescentis (RAP). Using the strategy of combining EEM fluorescence data with second-order calibration method based on the alternating trilinear decomposition (ATLD) algorithm, the simultaneous quantification of umbelliferone and scopoletin in the two different complex systems was achieved successfully, even in the presence of potential interferents. The pretreatment is simple due to the "second-order advantage" and the use of "mathematical separation" instead of awkward "physical or chemical separation". Satisfactory results have been achieved with the limits of detection (LODs) of umbelliferone and scopoletin being 0.06 ng mL- 1 and 0.16 ng mL- 1, respectively. The average spike recoveries of umbelliferone and scopoletin are 98.8 ± 4.3% and 102.5 ± 3.3%, respectively. Besides, HPLC-DAD method was used to further validate the presented strategy, and t-test indicates that prediction results of the two methods have no significant differences. Satisfactory experimental results imply that our method is fast, low-cost and sensitive when compared with HPLC-DAD method.

Simultaneous detection and quantification of parecoxib and valdecoxib in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology.

PubMed

Saccomanni, G; Giorgi, M; Del Carlo, S; Manera, C; Saba, A; Macchia, M

2011-09-01

Parecoxib is the injectable prodrug of valdecoxib, a cicloxygenase-2 selective drug, currently used in human medicine. Recent studies have suggested both its excellent clinical effectiveness and wide safety profile. The aim of the present study was to develop and validate a new high-performance liquid chromatography (HPLC) with spectrofluorimetric detection method to quantify parecoxib and valdecoxib in canine plasma. Several parameters both in the extraction and the detection method were evaluated. The applicability of the method was determined by administering parecoxib to one dog: the protocol provided the expected pharmacokinetic results. The final mobile phase was acetonitrile: AcONH(4) (10 mM; pH 5.0) 55:45, v/v, with a flow rate of 0.4 mL min(-1), and excitation and emission wavelengths of 265 and 375 nm, respectively. The analytical column was a reverse-phase C18 ODS2 3-μm particle size. Protein precipitation in acidic medium followed by two successive liquid-liquid steps was carried out. The best extraction solvent was cyclohexane:Et(2)O (3:2, v/v) that gave recoveries ranging from 81.1% to 89.1% and from 94.8% to 103.6% for parecoxib and valdecoxib, respectively. The limits of quantification were 25 and 10 ng mL(-1) for parecoxib and valdecoxib, respectively. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC-mass spectrometry experiments. The other validation parameters were in agreement with the European Medicines Evaluation Agency and International Conference on Harmonisation guidelines. In conclusion, this method (extraction, separation and applied techniques) is simple and effective. This is the first time that use of a HPLC with spectrofluorimetric detection technique to simultaneously detect parecoxib and valdecoxib in plasma has been reported. This technique may have applications for pharmacokinetic studies.

RP-HPLC method for simultaneous estimation of vigabatrin, gamma-aminobutyric acid and taurine in biological samples.

PubMed

Police, Anitha; Shankar, Vijay Kumar; Narasimha Murthy, S

2018-02-15

Vigabatrin is used as first line drug in treatment of infantile spasms for its potential benefit overweighing risk of causing permanent peripheral visual field defects and retinal damage. Chronic administration of vigabatrin in rats has demonstrated these ocular events are result of GABA accumulation and depletion of taurine levels in retinal tissues. In vigabatrin clinical studies taurine plasma level is considered as biomarker for studying structure and function of retina. The analytical method is essential to monitor taurine levels along with vigabatrin and GABA. A RP-HPLC method has been developed and validated for simultaneous estimation of vigabatrin, GABA and taurine using surrogate matrix. Analytes were extracted from human plasma, rat plasma, retina and brain by simple protein precipitation method and derivatized by naphthalene 2, 3‑dicarboxaldehyde to produce stable fluorescent active isoindole derivatives. The chromatographic analysis was performed on Zorbax Eclipse AAA column using gradient elution profile and eluent was monitored using fluorescence detector. A linear plot of calibration curve was observed in concentration range of 64.6 to 6458, 51.5 to 5150 and 62.5 to 6258 ng/mL for vigabatrin, GABA and taurine, respectively with r 2  ≥ 0.997 for all analytes. The method was successfully applied for estimating levels of vigabatrin and its modulator effect on GABA and taurine levels in rat plasma, brain and retinal tissue. This RP-HPLC method can be applied in clinical and preclinical studies to explore the effect of taurine deficiency and to investigate novel approaches for alleviating vigabatrin induced ocular toxicity. Copyright © 2018. Published by Elsevier B.V.

An HPLC/UV method for the determination of RGH-1756 in dog and rat plasma.

PubMed

Terjéki, E; Kapás, M

2001-03-01

RGH-1756 (1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazo[2,1-b]-thiazolyl)-phenoxy]-butyl)-piperazine dimethansulphonate) is a novel atypical antipsychotic candidate of Gedeon Richter Ltd. A new HPLC method has been developed and validated for the quantitative determination of RGH-1756 in dog and rat plasma. The compound and the internal standard are extracted from the biological samples by a simple and fast liquid--liquid extraction method, using 1-chlorobutane. The recovery for RGH-1756 is about 90%. The extracts are analyzed by reversed phase HPLC (column: Supelcosil-LC-18-DB 250*4.6 mm, 5 microm, eluent:acetonitrile:methanol:0.2 molar ammonium-acetate 40:25:35, lambda=254 nm). The assay is specific for RGH-1756. The standard curves are linear in the range between 10 and 2000 ng ml(-1). The overall precision (expressed as CV%) and accuracy (expressed as bias%) of quality controls and calibration standards are within 15%. The validated lower limit of quantification is 10 ng/ml. No indications have been found for possible instabilities of RGH-1756 in plasma, in the extraction solvent, or after repeated thawing-freezing cycles. The method has been succesfully applied for the bioavailability studies of RGH-1756 in the two animal species. In these studies results of the inprocess method validation have shown the reliability of the method, too. CV% of quality controls in the rat study has been found between 7.4 and 10.0%, in the dog study between 4.1 and 12.5%. The bias has ranged from 0.4 to 3.8% and from -4.5 to 1.2% in the rat and dog study, respectively.

Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L.

PubMed

Prencipe, Francesco Pio; Brighenti, Virginia; Rodolfi, Margherita; Mongelli, Andrea; dall'Asta, Chiara; Ganino, Tommaso; Bruni, Renato; Pellati, Federica

2014-07-04

The study was aimed at developing a new analytical method for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. (hop), together with a simple extraction procedure. Different extraction techniques, including maceration, heat reflux extraction (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), were compared in order to obtain a high yield of the target analytes. Dynamic maceration for 30min with MeOH-HCOOH (99:1, v/v) as the extraction solvent provided the best result in terms of recovery of secondary metabolites. The analysis of hop constituents, including prenylflavonoids and prenylphloroglucinols (bitter acids), was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was used for the HPLC analysis, with a mobile phase composed of 0.25% formic acid in both water and acetonitrile, under gradient elution. The method validation was performed to show compliance with ICH guidelines. The validated technique was successfully applied to the phytochemical analysis of ten commercial cultivars and twenty-three wild Italian hop genotypes, thus demonstrating to be a reliable and useful tool for the comprehensive multi-component analysis of hop secondary metabolites. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Determination of linear aliphatic aldehydes in heavy metal containing waters by high-performance liquid chromatography using 2,4-dinitrophenylhydrazine derivatization.

PubMed

Lin, Yi-Liang; Wang, Po-Yen; Hsieh, Ling-Ling; Ku, Kuan-Hsuan; Yeh, Yun-Tai; Wu, Chien-Hou

2009-09-04

A simple and sensitive method is described for the determination of picomolar amounts of C(1)-C(9) linear aliphatic aldehydes in waters containing heavy metal ions. In this method, aldehydes were first derivatized with 2,4-dinitrophenylhydrazine (DNPH) at optimized pH 1.8 for 30 min and analyzed by HPLC with UV detector at 365 nm. Factors affecting the derivatization reaction of aldehydes and DNPH were investigated. Cupric ion, an example of heavy metals, is a common oxidative reagent, which may oxidize DNPH and greatly interfere with the determination of aldehydes. EDTA was used to effectively mask the interferences by heavy metal ions. The method detection limits for direct injection of derivatized most aldehydes except formaldehyde were of the order of 7-28 nM. The detection limit can be further lowered by using off-line C(18) adsorption cartridge enrichment. The recoveries of C(1)-C(9) aldehydes were 93-115% with a relative standard deviation of 3.6-8.1% at the 0.1 microM level for aldehydes. The HPLC-DNPH method has been applied for determining aldehyde photoproducts from Cu(II)-amino acid complex systems.

Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for levetiracetam

PubMed Central

Greiner-Sosanko, Elizabeth; Giannoutsos, Spiros; Lower, Darla R.; Virji, Mohamed A.; Krasowski, Matthew D.

2008-01-01

A high-performance liquid chromatography (HPLC) assay using ultraviolet detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10-hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders. PMID:17988451

Medicinal cannabis: Principal cannabinoids concentration and their stability evaluated by a high performance liquid chromatography coupled to diode array and quadrupole time of flight mass spectrometry method.

PubMed

Citti, Cinzia; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Vandelli, Maria Angela; Cannazza, Giuseppe

2016-09-05

In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UV detection (HPLC-UV) for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabis-based medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs. Copyright © 2016 Elsevier B.V. All rights reserved.

An improved method for assaying phosphocholine and glycerophosphocholine in mouse tissue.

PubMed

Murai, S; Saito, H; Shirato, R; Kawaguchi, T

2001-01-01

To measure the levels of phosphocholine (PCh) and glycerophosphocholine (GPCh) in the tissues and organs of mice, we developed a simple and rapid method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. Under our modifications of the separation procedure of Klein et al. [Neurochem. Int. 2 (1993) 293], PCh and GPCh in the hydrophilic phase of the homogenate samples were selectively hydrolyzed into free choline by alkaline phosphatase and a 0.4-N perchloric acid solution, respectively, and the resulting hydrolyzed mixtures were directly injected into the HPLC system for analysis. The present method permits PCh or GPCh assay within 5 min in one chromatographic run. Recoveries from tissue samples were 97% for PCh and 101% for GPCh. The percentages of the crossover reaction to the authentic PCh and GPCh were 0.4% and 3.8%, respectively. The within-run coefficients of variation for choline derived from PCh and GPCh in the tissue samples were 1.2% and 1.4%, respectively. The method is effective and has been applied to the measurement of PCh and GPCh levels in several tissues of mice.

Quantification of major flavonoids in carnation tissues (Dianthus caryophyllus) as a tool for cultivar discrimination.

PubMed

Galeotti, Francesco; Barile, Elisa; Lanzotti, Virginia; Dolci, Marcello; Curir, Paolo

2008-01-01

One flavone-C-glycoside and two flavonol-O-glycosides were recognized and isolated as the main flavonoidal components in nine different carnation cultivars, and their chemical structures have been determined by spectroscopic methods, including UV detection, MS and NMR. The distribution of these three compounds in flowers, leaves, stems, young sprouts, and roots of each cultivar was evaluated by a simple HPLC-UV method: the graphic representation of their content in the different tissues allows to identify and characterize unambiguously each considered carnation cultivar. The presented method could be an easy, inexpensive and reliable tool for carnation cultivar discrimination.

Development of a Simple Dipstick Assay for Operational Monitoring of DDT

PubMed Central

Ismail, Hanafy M.; Kumar, Vijay; Singh, Rudra P.; Williams, Christopher; Shivam, Pushkar; Ghosh, Ayan; Deb, Rinki; Foster, Geraldine M.; Hemingway, Janet; Coleman, Michael; Coleman, Marlize; Das, Pradeep; Paine, Mark J. I.

2016-01-01

Background Indoor residual spraying (IRS) of DDT is used to control visceral leishmaniasis (VL) in India. However, the quality of spraying is severely compromised by a lack of affordable field assays to monitor target doses of insecticide. Our aim was to develop a simple DDT insecticide quantification kit (IQK) for monitoring DDT levels in an operational setting. Methodology/ principle findings DDT quantification was based on the stoichiometric release of chloride from DDT by alkaline hydrolysis and detection of the released ion using Quantab chloride detection strips. The assay was specific for insecticidal p,p`-DDT (LoQ = 0.082 g/m2). Bostik discs were effective in post spray wall sampling, extracting 25–70% of active ingredient depending on surface. Residual DDT was sampled from walls in Bihar state in India using Bostik adhesive discs and DDT concentrations (g p,p`-DDT/m2) were determined using IQK and HPLC (n = 1964 field samples). Analysis of 161 Bostik samples (pooled sample pairs) by IQK and HPLC produced excellent correlation (R2 = 0.96; Bland-Altman bias = −0.0038). IQK analysis of the remaining field samples matched HPLC data in identifying households that had been under sprayed, in range or over sprayed. Interpretation A simple dipstick assay has been developed for monitoring DDT spraying that gives comparable results to HPLC. By making laboratory-based analysis of DDT dosing accessible to field operatives, routine monitoring of DDT levels can be promoted in low- and middle- income countries to maximise the effectiveness of IRS. PMID:26760773

A novel approach of periodate oxidation coupled with HPLC-FLD for the quantitative determination of 3-chloro-1,2-propanediol in water and vegetable oil.

PubMed

Hu, Zhixiong; Cheng, Peng; Guo, Mingli; Zhang, Weinong; Qi, Yutang

2013-07-10

A novel approach of periodate oxidation coupled with high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) for the quantitative determination of 3-chloro-1,2-propanediol (3-MCPD) has been established. The essence of this approach lies in the production of chloroacetaldehyde by the oxidization cleavage of 3-MCPD with sodium periodate and the HPLC analysis of chloroacetaldehyde monitored by an FLD detector after fluorescence derivatization with adenine. The experimental parameters relating to the efficiency of the derivative reaction such as concentration of adenine, chloroacetaldehyde reaction temperature, and time were studied. Under the optimized conditions, the proposed method can provide high sensitivity, good linearity (r(2) = 0.999), and repeatability (percent relative standard deviations between 2.57% and 3.44%), the limits of detection and quantification were 0.36 and 1.20 ng/mL, respectively, and the recoveries obtained for water samples were in the range 93.39-97.39%. This method has been successfully applied to the analysis of real water samples. Also this method has been successfully used for the analysis of vegetable oil samples after pretreatment with liquid-liquid extraction; the recoveries obtained by a spiking experiment with soybean oil ranged from 96.27% to 102.42%. In comparison with gas chromatography or gas chromatography-mass spectrometry, the proposed method can provide the advantages of simple instrumental requirement, easy operation, low cost, and high efficiency, thus making this approach another good choice for the sensitive determination of 3-MCPD.

Determination of metolcarb and diethofencarb in apples and apple juice by solid-phase microextraction-high performance liquid chromatography.

PubMed

Yang, Xiu-Min; Wang, Ou; Wang, Ming-Zhao; Hu, Yan-Xue; Li, Wei-Ning; Wang, Zhi

2008-09-01

A method for the determination of metolcarb and diethofencarb in apples and apple juice is developed using solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC). The experimental conditions of SPME, such as the kind of extraction fiber, extraction time, stirring rate, pH of the extracting solution, and desorption conditions are optimized. The SPME is performed on a 60 microm polydimethylsiloxane/divinylbenzene fiber for 40 min at room temperature with the solution being stirred at 1100 rpm. The extracted pesticides on the SPME fiber are desorbed in the mobile phase into SPME-HPLC interface for HPLC analysis. Separations are carried out on a Baseline C18 column (4.6 i.d. x 250 mm, 5.0 microm) with acetonitrile-water (55/45, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and photodiode-array detection at 210 nm. For apple samples, the method is linear for both metolcarb and diethofencarb in the range of 0.05-1.0 mg/kg (r > 0.99), with a detection limit (S/N = 3 ) of 15 and 5 microg/kg, respectively. For apple juice, the method is linear for both metholcarb and diethofencarb over the range of 0.05-1.0 mg/L (r > 0.99) with the detection limit (S/N = 3 ) of 15 and 3 microg/L, respectively. Excellent recovery and reproducibility values are achieved. The proposed method is shown to be simple, sensitive, and organic solvent-free, and is suitable for the determination of the two pesticides in apples and apple juice.

Development of an ion-pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro-2a cell line.

PubMed

Chen, Yunyun; Xing, Dongming; Wang, Wei; Ding, Yi; Du, Lijun

2007-06-01

The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post-ischemic recovery. An IP-HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro-2a cell line. The chromatographic conditions were: a Zorbax SB-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4 degrees C) in 1 mL of an ice-cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform. Copyright 2007 John Wiley & Sons, Ltd.

A simple isocratic HPLC method for the simultaneous determination of sinensetin, eupatorin, and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus extracts.

PubMed

Yam, Mun Fei; Mohamed, Elsnoussi Ali Hussin; Ang, Lee Fung; Pei, Li; Darwis, Yusrida; Mahmud, Roziahanim; Asmawi, Mohd Zaini; Basir, Rusliza; Ahmad, Mariam

2012-08-01

Orthosiphon stamineus extracts contain three flavonoids (3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20mM phosphate buffer (NaH(2)PO(4)) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052-250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048-0.368, 0.025-0.135, and 0.05-0.476 for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333-1.688, 0.722-1.055, and 0.548-1.819, respectively. The accuracy for intraday were 91.25%-103.38%, 94.32%-109.56%, and 92.85%-109.70% for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%-103.92%, 103.58%-104.57%, and 103.9%-105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract. Copyright © 2012. Published by Elsevier B.V.

Comparative Validation of the Determination of Sofosbuvir in Pharmaceuticals by Several Inexpensive Ecofriendly Chromatographic, Electrophoretic, and Spectrophotometric Methods.

PubMed

El-Yazbi, Amira F

2017-01-20

Sofosbuvir (SOFO) was approved by the U.S. Food and Drug Administration in 2013 for the treatment of hepatitis C virusinfection with enhanced antiviral potency compared with earlier analogs. Notwithstanding, all current editions of the pharmacopeias still do not present any analytical methods for the quantification of SOFO. Thus, rapid, simple, and ecofriendly methods for the routine analysis of commercial formulations of SOFO are desirable. In this study, five accurate methods for the determination of SOFO in pharmaceutical tablets were developed and validated. These methods include HPLC, capillary zone electrophoresis, HPTLC, and UV spectrophotometric and derivative spectrometry methods. The proposed methods proved to be rapid, simple, sensitive, selective, and accurate analytical procedures that were suitable for the reliable determination of SOFO in pharmaceutical tablets. An analysis of variance test with <em>P</em>-value &#x003E; 0.05 confirmed that there were no significant differences between the proposed assays. Thus, any of these methods can be used for the routine analysis of SOFO in commercial tablets.

HPLC method for determination of SN-38 content and SN-38 entrapment efficiency in a novel liposome-based formulation, LE-SN38.

PubMed

Xuan, Tong; Zhang, J Allen; Ahmad, Imran

2006-05-03

A simple HPLC method was developed for quantification of SN-38, 7-ethyl-10-hydroxycamptothecin, in a novel liposome-based formulation (LE-SN38). The chromatographic separation was achieved on an Agilent Zorbax SB-C18 (4.6 mmx250 mm, 5 microm) analytical column using a mobile phase consisting of a mixture of NaH2PO4 (pH 3.1, 25 mM) and acetonitrile (50:50, v/v). SN-38 was detected at UV wavelength of 265 nm and quantitatively determined using an external calibration method. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.05 and 0.25 microg/mL, respectively. The individual spike recovery of SN-38 ranged from 100 to 101%. The percent of relative standard deviation (%R.S.D.) of intra-day and inter-day analyses were less than 1.6%. The method validation results confirmed that the method is specific, linear, accurate, precise, robust and sensitive for its intended use. The current method was successfully applied to the determination of SN-38 content and drug entrapment efficiency in liposome-based formulation, LE-SN38 during early stage formulation development.

Validation and Uncertainty Estimation of an Ecofriendly and Stability-Indicating HPLC Method for Determination of Diltiazem in Pharmaceutical Preparations

PubMed Central

Sadeghi, Fahimeh; Navidpour, Latifeh; Bayat, Sima; Afshar, Minoo

2013-01-01

A green, simple, and stability-indicating RP-HPLC method was developed for the determination of diltiazem in topical preparations. The separation was based on a C18 analytical column using a mobile phase consisted of ethanol: phosphoric acid solution (pH = 2.5) (35 : 65, v/v). Column temperature was set at 50°C and quantitation was achieved with UV detection at 240 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The applied procedure was found to be linear in diltiazem concentration range of 0.5–50 μg/mL (r 2 = 0.9996). Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. The recoveries obtained (99.25%–101.66%) ensured the accuracy of the developed method. The degradation products as well as the pharmaceutical excipients were well resolved from the pure drug. The expanded uncertainty (5.63%) of the method was also estimated from method validation data. Accordingly, the proposed validated and sustainable procedure was proved to be suitable for routine analyzing and stability studies of diltiazem in pharmaceutical preparations. PMID:24163778

Easy, fast and environmental friendly method for the simultaneous extraction of the 16 EPA PAHs using magnetic molecular imprinted polymers (mag-MIPs).

PubMed

Villar-Navarro, Mercedes; Martín-Valero, María Jesús; Fernández-Torres, Rut Maria; Callejón-Mochón, Manuel; Bello-López, Miguel Ángel

2017-02-15

An easy and environmental friendly method, based on the use of magnetic molecular imprinted polymers (mag-MIPs) is proposed for the simultaneous extraction of the 16 U.S. EPA polycyclic aromatic hydrocarbons (PAHs) priority pollutants. The mag-MIPs based extraction protocol is simple, more sensitive and low organic solvent consuming compared to official methods and also adequate for those PAHs more retained in the particulate matter. The new proposed extraction method followed by HPLC determination has been validated and applied to different types of water samples: tap water, river water, lake water and mineral water. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous quantification of five steroid saponins from Dioscorea zingiberensis C.H.Wright in rat plasma by HPLC-MS/MS and its application to the pharmacokinetic studies

PubMed Central

Zhang, Xinxin; Li, Jing; Ito, Yoichiro; Sun, Wenji

2014-01-01

A simple, reliable and sensitive high-performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) was established for simultaneous analyses of the following 5 steroid saponins in rat plasma after the single dose administration of total steroid saponins extracted from the rhizome of Dioscorea zingiberensis C.H.Wright for the first time. Protodioscin, huangjiangsu A, zingiberensis new saponin, dioscin, and gracillin were quantified using ginsenoside Rb1 as the internal standard (IS). The plasma samples were pretreated by a single step acetonitrile-mediated protein precipitation. The chromatographic separation was performed on an Inersil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase composed of acetonitrile and water containing 0.1% formic acid under a gradient elution mode at 0.2 mL min−1 using a microsplit after the eluent from the HPLC apparatus. The quantification was accomplished on a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) in the positive ionization mode. The above five analytes were stable under sample storage and preparation conditions applied in the present study. The linearity, precision, accuracy, and recoveries of the analysis confirmed the requirements for quality-control purposes. After validation, this proposed method was successfully adopted to investigate the pharmacokinetic parameters of these five analytes. PMID:25201262

Synthesis of Fe3O4@CuS@Ni2P-CNTs magnetic nanocomposite for sonochemical-assisted sorption and pre-concentration of trace Allura Red from aqueous samples prior to HPLC-UV detection: CCD-RSM design.

PubMed

Asfaram, Arash; Ghaedi, Mehrorang; Abidi, Hassan; Javadian, Hamedreza; Zoladl, Mohammad; Sadeghfar, Fardin

2018-06-01

A simple procedure based on ultrasound-assisted (UA) dispersive micro solid phase extraction (D-μ-SPE) was applied for sorption of trace amount Allura Red (AR) in fruit juice and water samples. After loading process by UA-D-μ-SPE, the concentrated AR was eluted and monitored using high-performance liquid chromatography-ultraviolet -visible detector (HPLC-UV). The best operational conditions were obtained as follows: pH = 3.0, 8 mg of the sorbent, sonication time of 4.5 min and 0.16 mL of THF as elution solvent. Under the optimum operational conditions, the present method was acceptable for AR quantification in the range of 1.0-5000 ng mL -1 . The repeatability based on RSD with the amount of 1.67-3.18%, low LOD (0.198 ng mL -1 ) and LOQ (0.659 ng mL -1 ) were obtained. The UA-D-μ-SPE-HPLC-UV method was successfully applied for trace quantification of AR from water and commercial fruit juice samples supplied from local supermarkets, and acceptable relative recoveries over the range of 97.7-105.4% with RSDs ≤5.50% were obtained. Copyright © 2018 Elsevier B.V. All rights reserved.

Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification.

PubMed

Li, Hua-Bin; Jiang, Yue; Chen, Feng

2002-02-27

A simple and efficient method for the isolation and purification of lutein from the microalga Chlorella vulgaris was developed. Crude lutein was obtained by extraction with dichloromethane from the microalga after saponification. Partition values of lutein in the two-phase system of ethanol-water-dichloromethane at different ratios were measured by HPLC so as to assist the determination of an appropriate condition for washing water-soluble impurities in the crude lutein. Partition values of lutein in another two-phase system of ethanol-water-hexane at different ratios were also measured by HPLC for determining the condition for removing fat-soluble impurities. The water-soluble impurities in the crude lutein were removed by washing with 30% aqueous ethanol, and the fat-soluble impurities were removed by extraction with hexane. The final purity of lutein obtained was 90-98%, and the yield was 85-91%.

HPLC-SPE-NMR identification of a novel metabolite containing the benzo[c]oxepin skeleton from the endophytic fungus Pestalotiopsis virgatula culture.

PubMed

Kesting, Julie R; Staerk, Dan; Tejesvi, Mysore V; Kini, Kukkundoor R; Prakash, Harishchandra S; Jaroszewski, Jerzy W

2009-08-01

HPLC-SPE-NMR analysis of a crude extract of fermentation broth of cultured PESTALOTIOPSIS VIRGATULA isolate TC-320 from TERMINALIA CHEBULA Retz. (Combretaceae) disclosed the presence of a simple but unprecedented low-molecular-weight metabolite, 9-hydroxybenzo[ C]oxepin-3[1 H]-one, subsequently isolated by a targeted purification procedure. Georg Thieme Verlag KG Stuttgart.New York.

Determination of GABA and vigabatrin in human plasma by a rapid and simple HPLC method: correlation between clinical response to vigabatrin and increase in plasma GABA.

PubMed

Löscher, W; Fassbender, C P; Gram, L; Gramer, M; Hörstermann, D; Zahner, B; Stefan, H

1993-03-01

The novel antiepileptic drug vigabatrin (Sabril) acts by inhibiting degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), increasing the GABA concentrations in the brain. Because the GABA degrading enzyme GABA aminotransferase (GABA-T) is also present in peripheral tissues, including blood platelets, measurement of plasma GABA levels might be a useful indication of the pharmacological response to vigabatrin during therapeutic monitoring. However, because of the very low concentrations of GABA in plasma, the few methods available for plasma GABA analysis are time-consuming, difficult to perform and/or not selective enough because of potential interference with other plasma constituents. In the present study, a rapid, selective and sensitive amino acid analysis HPLC method has been developed for plasma GABA determination with fluorescence detection, using o-phthaldialdehyde as a precolumn derivatizing agent. By employing a 3 microns particle size reversed-phase column and a multi-step gradient system of two solvents, the very low endogenous concentration of GABA in human plasma could be reproducibly quantitated without interference of other endogenous compounds. Incubation of human plasma samples with GABA degrading enzyme(s) resulted in an almost total loss of the GABA peak, thus demonstrating the specificity of the method for GABA analysis. In addition to GABA and other endogenous amino acids, the HPLC method could be used to quantitate plasma levels of vigabatrin. Thus, this improved HPLC amino acid assay might be used to examine whether concomitant monitoring of plasma GABA and vigabatrin is useful for clinical purposes. This was examined in 20 epileptic patients undergoing chronic treatment with vigabatrin. The average plasma GABA level of these 20 patients did not differ significantly from non-epileptic controls. However, when epileptic patients were subdivided according to their clinical response to vigabatrin, vigabatrin responders had significantly higher GABA levels than nonresponders or controls. In contrast to the difference in plasma GABA, vigabatrin responders and nonresponders did not differ in dose or plasma level of vigabatrin. These data may indicate that determination of plasma GABA is a valuable non-invasive method for therapeutic monitoring in patients on medication with vigabatrin.

Development of a Simple RP-HPLC-UV Method for Determination of Azithromycin in Bulk and Pharmaceutical Dosage forms as an Alternative to the USP Method

PubMed Central

Ghari, Tayebeh; Kobarfard, Farzad; Mortazavi, Seyed Alireza

2013-01-01

The present study was designed to develop a simple, validated liquid chromatographic method for the analysis of azithromycin in bulk and pharmaceutical dosage forms using ultraviolet detector. The best stationary phase was determined as C18 column, 5 μm, 250 mm × 4.6 mm. Mobile phase was optimized to obtain a fast and selective separation of the drug. Flow rate was 1.5 mL/min, Wavelength was set at 210 nm and the volume of each injection was 500 μL. An isocratic methanol/buffer mobile phase at the ratio of 90:10 v/v gave the best separation and resolution. The proposed method was accurate, precise, sensitive, and linear over a wide range of concentration of azithromycin. The developed method has the advantage of using UV detector compared to the USP method in which electrochemical detector has been used. The validated method was successfully applied to the determination of azithromycin in bulk and pharmaceutical dosage forms. PMID:24250672

Determination of Carbonyl Compounds in Cork Agglomerates by GDME-HPLC-UV: Identification of the Extracted Compounds by HPLC-MS/MS.

PubMed

Brandão, Pedro Francisco; Ramos, Rui Miguel; Almeida, Paulo Joaquim; Rodrigues, José António

2017-02-08

A new approach is proposed for the extraction and determination of carbonyl compounds in solid samples, such as wood or cork materials. Cork products are used as building materials due to their singular characteristics; however, little is known about its aldehyde emission potential and content. Sample preparation was done by using a gas-diffusion microextraction (GDME) device for the direct extraction of volatile aldehydes and derivatization with 2,4-dinitrophenylhydrazine. Analytical determination of the extracts was done by HPLC-UV, with detection at 360 nm. The developed methodology proved to be a reliable tool for aldehyde determination in cork agglomerate samples with suitable method features. Mass spectrometry studies were performed for each sample, which enabled the identification, in the extracts, of the derivatization products of a total of 13 aldehydes (formaldehyde, acetaldehyde, furfural, propanal, 5-methylfurfural, butanal, benzaldehyde, pentanal, hexanal, trans-2-heptenal, heptanal, octanal, and trans-2-nonenal) and 4 ketones (3-hydroxy-2-butanone, acetone, cyclohexanone, and acetophenone). This new analytical methodology simultaneously proved to be consistent for the identification and determination of aldehydes in cork agglomerates and a very simple and straightforward procedure.

Determination of starting materials, intermediates, and subsidiary colors in the color additive Food Red No. 106 (Sulforhodamine B) using high-performance liquid chromatography.

PubMed

Tatebe, Chiye; Ohtsuki, Takashi; Fujita, Tsuyoshi; Nishiyama, Koji; Itoh, Sumio; Sugimoto, Naoki; Kubota, Hiroki; Tada, Atsuko; Sato, Kyoko; Akiyama, Hiroshi

2017-12-15

The main subsidiary color of structure in Food Red No. 106 (R106) was identified to be a desethyl derivative (R106-SubA). High-performance liquid chromatography (HPLC) was performed for the quantitative determination of benzaldehyde-2,4-disulfonic acid, N,N-diethyl-m-aminophenol, leuco acid, pyrone acid, R106-SubA, etc. in R106. An ammonium acetate solution (20mM) and acetonitrile:water (7:3) were used to stabilize the retention time of the HPLC analytes. The linearity of the calibration curves was in the range of 0.05-10μg/mL, with good correlation coefficients (R 2 >0.9983). The recoveries of impurities at levels 0.1%, 0.5% and 1% ranged from 94.2% to 106.6% with relative standard deviations of 0.1%-1.0%. While surveying commercial R106, the amounts obtained by area% determination were similar to those obtained by the calibration-curve determination. The area% determination by HPLC for the determinations of impurities in R106 is a simple and reliable method and can be applied in routine analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC with spectrofluorimetric detection.

PubMed

Vito, Virgina De; Saba, Alessandro; Lee, Hong-Ki; Owen, Helen; Poapolathep, Amnart; Giorgi, Mario

2016-01-25

Grapiprant, a novel pharmacologically active ingredient, acts as a selective EP4 receptor antagonist whose physiological ligand is prostaglandin E2 (PGE2). It is currently under development for use in humans and dogs for the control of pain and inflammation associated with osteoarthritis. The aim of the present study was to develop an easy and sensitive method to quantify grapiprant in canine plasma and to apply the method in a canine patient. Several parameters, both in the extraction and detection method were evaluated. The final mobile phase consisted of ACN:AcONH4 (20 mM) solution, pH 4 (70:30, v/v) at a flow rate of 1 mL/min. The elution of grapiprant and IS (metoclopramide) was carried out in isocratic mode through a Synergi Polar-RP 80A analytical column (150 mm × 4.6 mm). The best excitation and emission wavelengths were 320 and 365 nm, respectively. Grapiprant was extracted from the plasma using CHCl3, which gave a recovery of 88.1 ± 10.22% and a lower limit of quantification (LLOQ) of 10 ng/mL. The method was validated in terms of linearity, limit of detection (LOD), LLOQ, selectivity, accuracy and precision, extraction recovery, stability, and inter-laboratory cross validation, according to international guidelines. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte and IS, as confirmed by HPLC-MS experiments. In conclusion, this was a simple and effective method using HPLC-FL to detect grapiprant in plasma, which may be useful for future pharmacokinetic studies. Copyright © 2015 Elsevier B.V. All rights reserved.

HPLC-FLD determination of 4-nonylphenol and 4-tert-octylphenol in surface water samples.

PubMed

Cruceru, Ioana; Iancu, Vasile; Petre, Jana; Badea, Irinel Adriana; Vladescu, Luminita

2012-05-01

A simple, sensitive and reliable HPLC-FLD method for the routine determination of 4-nonylphenol, 4-NP and 4-tert-octylphenol, 4-t-OP content in water samples was developed. The method consists in a liquid-liquid extraction of the target analytes with dichloromethane at pH  3.0-3.5 followed by the HPLC-FLD analysis of the organic extract using a Zorbax Eclipse XDB C8 column, isocratic elution with a mixed solvent acetonitrile/water 65:35, at a flow rate of 1.0 mL/min and applying a column temperature of 40°C. The method was validated and then applied with good results for the determination of 4-NP and 4-t-OP in Ialomiţa River water samples collected each month during 2006. The concentration levels of 4-NP and 4-t-OP vary between 0.08-0.17 μg/L with higher values of 0.24-0.37 μg/L in the summer months for 4-NP, and frequently <0.05 μg/L but also between 0.06-0.09 μg/L with higher values of 0.12-0.16 μg/L in July and August for 4-t-OP and were strongly influenced by sesonial and anthropic factors. The method was also applied on samples collected over 2 years 2007 and 2008 from urban wastewaters discharged into sewage or directly into the rivers by economic agents located in 30 Romanian towns. Good results were obtained when the method was used for analysis of effluents discharged into surface waters by 16 municipal wastewater treatment plants, during the year 2008.

Validated Spectrophotometric and RP-HPLC-DAD Methods for the Determination of Ursodeoxycholic Acid Based on Derivatization with 2-Nitrophenylhydrazine.

PubMed

El-Kafrawy, Dina S; Belal, Tarek S; Mahrous, Mohamed S; Abdel-Khalek, Magdi M; Abo-Gharam, Amira H

2017-05-01

This work describes the development, validation, and application of two simple, accurate, and reliable methods for the determination of ursodeoxycholic acid (UDCA) in bulk powder and in pharmaceutical dosage forms. The carboxylic acid group in UDCA was exploited for the development of these novel methods. Method 1 is the colorimetric determination of the drug based on its reaction with 2-nitrophenylhydrazine hydrochloride in the presence of a water-soluble carbodiimide coupler [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride] and pyridine to produce an acid hydrazide derivative, which ionizes to yield an intense violet color with maximum absorption at 553 nm. Method 2 uses reversed-phase HPLC with diode-array detection for the determination of UDCA after precolumn derivatization using the same reaction mentioned above. The acid hydrazide reaction product was separated using a Pinnacle DB C8 column (4.6 × 150 mm, 5 μm particle size) and a mobile phase consisting of 0.01 M acetate buffer (pH 3)-methanol-acetonitrile (30 + 30 + 40, v/v/v) isocratically pumped at a flow rate of 1 mL/min. Ibuprofen was used as the internal standard (IS). The peaks of the reaction product and IS were monitored at 400 nm. Different experimental parameters for both methods were carefully optimized. Analytical performance of the developed methods were statistically validated for linearity, range, precision, accuracy, specificity, robustness, LOD, and LOQ. Calibration curves showed good linear relationships for concentration ranges 32-192 and 60-600 μg/mL for methods 1 and 2, respectively. The proposed methods were successfully applied for the assay of UDCA in bulk form, capsules, and oral suspension with good accuracy and precision. Assay results were statistically compared with a reference pharmacopeial HPLC method, and no significant differences were observed between proposed and reference methods.

Simultaneous determination of domperidone and Itopride in pharmaceuticals and human plasma using RP-HPLC/UV detection: Method development, validation and application of the method in in-vivo evaluation of fast dispersible tablets.

PubMed

Khan, Amjad; Iqbal, Zafar; Khadra, Ibrahim; Ahmad, Lateef; Khan, Abad; Khan, Muhammad Imran; Ullah, Zia; Ismail

2016-03-20

Domperidone and Itopride are pro-kinetic agents, regulating the gastric motility and are commonly prescribed as anti emetic drugs. In the present study a simple, rapid and sensitive RP-HPLC/UV method was developed for simultaneous determination of Domperidone and Itopride in pharmaceutical samples and human plasma, using Tenofavir as internal standard. Experimental conditions were optimized and method was validated according to the standard guidelines. Combination of water (pH 3.0) and acetonitrile (65:35 v/v) was used as mobile phase, pumped at the flow rate of 1.5 ml/min. Detector wavelength was set at 210 nm and column oven temperature was 40oC. Unlike conventional liquid-liquid extraction, simple precipitation technique was applied for drug extraction from human plasma using acetonitrile for deprotienation. The method showed adequate separation of both the analytes and best resolution was achieved using Hypersil BDS C8 column (150 mm × 4.6 mm, 5 μm). The method was quite linear in the range of 20-600 ng/ml. Recovery of the method was 92.31% and 89.82% for Domperidone and Itopride, respectively. Retention time of both the analytes and internal standard was below 15 min. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for Domperidone were 5 and 10 ng/ml while for Itopride was 12 and 15 ng/ml, respectively. The developed method was successfully applied for in-vivo analysis of fast dispersible tablets of Domperidone in healthy human volunteer. The proposed method was a part of formulation development study and was efficiently applied for determination of the two drugs in various pharmaceutical products and human plasma. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous determination of amphetamine derivatives in human urine after SPE extraction and HPLC-UV analysis.

PubMed

Soares, M E; Carvalho, M; Carmo, H; Remião, F; Carvalho, F; Bastos, M L

2004-03-01

Amphetamine derivatives are a class of compounds increasingly abused as recreational drugs in various regions of the world. Although d-amphetamine (AMPH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are among the most commonly used, the abuse of other designer drugs such as 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and 4-methylthioamphetamine (4-MTA) and their involvement in acute intoxications has been increasingly reported. There is evidence that abusers ingest these compounds either alone or in combination and the respective monitoring is important for both legal and health care purposes in hospital emergency. In the present study a simple and clean solid-phase extraction procedure from urine of AMPH and MDMA, and their major metabolites p-hydroxyamphetamine (OH-AMPH) and methylenedioxyamphetamine (MDA) and 2C-B and 4-MTA was developed. Analysis was performed by HPLC-UV and the precision of the technique was between 2.9 and 5.3% for all compounds. For the overall procedure, the precision values were between 3.3 and 5.9%. Recoveries obtained from spiked urines at three concentration levels were better than 84 +/- 4% for the six compounds. The limit of detection of the method for the compounds (between 5.3 and 84.0 ng) enables their identification in urine after ingestion of fatal and non-fatal doses. The main advantages of the present method lie in its simple, clean and reliable SPE extraction method of the six amphetamine derivatives from urine followed by their simultaneous detection and quantification by liquid chromatography with UV detection. Copyright 2004 John Wiley & Sons, Ltd.

Determination of blood cyanide by HPLC-MS.

PubMed

Tracqui, A; Raul, J S; Géraut, A; Berthelon, L; Ludes, B

2002-04-01

An original high-performance liquid chromatographic-mass spectrometric (HPLC-MS) procedure was developed for the determination of cyanide (CN) in whole blood. After the addition of K13C15N as internal standard, blood was placed in a microdiffusion device, the inner well of which was filled with a mixture of taurine (50mM in water)/naphthalene-2,3-dicarboxaldehyde (NDA, 10mM in methanol)/methanol/ concentrated (approximately 20%) ammonia solution (25:25:45:5, v/v). Concentrated H2SO4 was added to the blood sample, and the microdiffusion chamber was sealed. After 30 min of gentle agitation, 2 microL of the contents of the inner vial were pipetted and directly injected onto a NovaPak C18 HPLC column. Separation was performed by a gradient of acetonitrile in 2mM NH4COOH, pH 3.0 buffer (35-80% in 10 min). Detection was done with a Perkin-Elmer Sciex API-100 mass analyzer with an ionspray interface, operated in the negative ionization mode. MS data were collected as either TIC or SIM at m/z (299 + 191) and (301 + 193) for the derivatives formed with CN and 13C15N, respectively. Inspired by previous works dealing with the complexation of CN by NDA + taurine to form a 1-cyano [f] benzoisoindole derivative analyzed by HPLC-fluorimetry, this method appears simple, rapid, and extremely specific. Limits of detection and quantitation for blood CN are 5 and 15 ng/mL, respectively. The use of 13C15N as internal standard allows the quantitation of CN with elegance and accuracy in comparison with previously reported methods.

Radiotracer properties determined by high performance liquid chromatography: a potential tool for brain radiotracer discovery.

PubMed

Tavares, Adriana Alexandre S; Lewsey, James; Dewar, Deborah; Pimlott, Sally L

2012-01-01

Previously, development of novel brain radiotracers has largely relied on simple screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. We investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. Ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (P(m)); percentage of plasma protein binding (%PPB); and membrane partition coefficient (K(m)). Relationships between brain entry measurements (Log P, P(m) and %PPB) and in vivo brain percentage injected dose (%ID); and K(m) and specific binding in vivo (BP(ND)) were investigated. Log P values obtained using in silico packages and flask methods were compared with Log P values obtained using HPLC. The modelled associations with %ID were stronger for %PPB (r(2)=0.65) and P(m) (r(2)=0.77) than for Log P (r(2)=0.47) while 86% of BP(ND) variance was explained by K(m). Log P values were variable dependant on the methodology used. Log P should not be relied upon as a predictor of blood-brain barrier penetration during brain radiotracer discovery. HPLC measurements of permeability, %PPB and membrane interactions may be potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery. Copyright © 2012 Elsevier Inc. All rights reserved.

Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples

PubMed Central

Acharya, Miteshkumar; Lau-Cam, Cesar A.

2012-01-01

A simple reversed-phase HPLC method for measuring hepatic levels of acetaminophen- (APAP-) protein adduct following an overdose of APAP was developed. An aliquot of liver homogenate in phosphate-buffered saline pH 7.4 (PBS) was placed on a Nanosep centrifugal device, which was centrifuged to obtain a protein residue. This residue was incubated with a solution of p-aminobenzoic acid (PABA), the internal standard, and bacterial protease in PBS, transferred to a Nanosep centrifugal device, and centrifuged. A 100 μL portion of the filtrate was analyzed on a YMC-Pack ODS-AMQ C18 column, using 100 mM potassium dihydrogen phosphate-methanol-acetic acid (100 : 0.6 : 0.1) as the mobile phase, a flow rate of 1 mL/min, and photometric detection at 254 nm. PABA and APAP-cystein-S-yl (APAP-Cys) eluted at ~14.7 min and 22.7 min, respectively. Method linearity, based on on-column concentrations of APAP-Cys, was observed over the range 0.078–40 μg. Recoveries of APAP-Cys from spiked blank liver homogenates ranged from ~83% to 91%. Limits of detection and of quantification of APAP-Cys, based on column concentrations, were 0.06 μg and 0.14 μg, respectively. RSD values for interday and intraday analyses of a blank liver homogenate spiked with APAP-Cyst at three levels were, in all cases, ≤1.0% and <1.5%, respectively. The proposed method was found appropriate for comparing the antidotal properties of N-acetylcysteine and taurine in a rat model of APAP poisoning. PMID:22619591

A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography.

PubMed

Wang, Yu; Wang, Siming; Zhang, Lijiao; Zeng, Jie; Yang, Ruiyue; Li, Hongxia; Tang, Yueming; Chen, Wenxiang; Dong, Jun

2018-02-01

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FER HDL ) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.

Development and application of a HPLC method for eight sunscreen agents in suncare products.

PubMed

Peruchi, L M; Rath, S

2012-06-01

This work describes the development, validation and application of a simple and fast high-performance liquid chromatography-with diode array dectection (HPLC-DAD) method for the determination of eight sunscreen agents: benzophenone-3, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, homosalate (used in two isomeric forms), butyl methoxydibenzoylmethane, 4-methylbenzylidene camphor and ethylhexyl dimethyl PABA in sunscreen formulations. The separation of the eight sunscreen compounds was achieved using an ACE C18 column (250 × 4.6 mm, 5 μm), with a column temperature 20°C, and a mobile phase of 88 : 12 (v/v) methanol-water with isocratic elution. Column temperature strongly influences the retention time and resolution of the compounds. The flow rate was 1.0 mL min(-1) and quantitation was performed by external calibration at the maximum wavelength of each compound. The sample preparation was simple and consisted basically of sample dilution with methanol, centrifugation and filtration in syringe filters before quantitation. Total run time was 18 min. The method was validated according to the parameters: linear range, linearity, selectivity, intra-day and inter-day precision and accuracy. Ten samples of sunscreen emulsions commercially available in Brazil (SPF 30) from different manufacturers were analysed using the proposed method. The number of the sunscreen agents varied between one and five in a single sample. The concentrations of all compounds were in the range of 0.9-10% (w/w) and were in accordance with the current Brazilian legislation. © 2012 The Authors. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Simultaneous determination of four trace level endocrine disrupting compounds in environmental samples by solid-phase microextraction coupled with HPLC.

PubMed

Wang, Lingling; Zhang, Zhenzhen; Xu, Xu; Zhang, Danfeng; Wang, Fang; Zhang, Lei

2015-09-01

A simple, rapid, sensitive and effective method for the simultaneous determination of four endocrine disrupting compounds (EDCs) (bisphenol A (BPA), bisphenol F (BPF), bisphenol AF (BPAF) and bisphenol AP (BPAP)) in environment water samples based on solid-phase microextraction (SPME) coupled with high performance liquid chromatography (HPLC) was developed. Multi-wall carbon nanotubes (MWCNTs) adsorbents showed a good affinity to the target analytes. These compounds were rapidly extracted within 10 min. Various experimental parameters that could affect the extraction efficiencies had been investigated in detail. Under the optimum conditions, the enrichment factors of the method for the target EDCs were found to be 500. Satisfactory precision and accuracy of the method were obtained in a low concentration range of 2.0-500.0 ng mL(-1). The method detection limits were in the range of 0.10-0.30 ng mL(-1). The high pre-concentration rate and efficiency of the method ensure its successful application in extraction of trace EDCs from large volumes of environmental water samples. The extraction recoveries in real samples ranged from 85.3% to 102.5% with the relative standard deviations (n=5) less than 3.74%. Copyright © 2015 Elsevier B.V. All rights reserved.

[Simultaneous determination of 6 neonicotinoid residues in soil using DLLME-HPLC and UV].

PubMed

Sun, Bao-li; Shan, Hong; Li, Yan-hua; Zeng, Ya-ling; Shen, Xiu-li; Tong, Cheng-feng

2013-09-01

A simple, cheap and rugged method was developed for simultaneous deter mination of 6 neonicotinoid residues in soil, including imidacloprid, acetamiprid, thiamethoxam, thiacloprid, clothianidin and nitenpyram. The soil sample was produced by dispersive liquid-liquid micro-extraction (DLLME) after extracted by the mixed solution of acetonitrile and CH2Cl2 (2:1, phi). The analytes were separated by HPLC with Alltima C18 column (4.6 mm x 250 mm, 5 microm) and detected by PDA at 260 nm. External standard method was used for quantification. The results showed that good linearity was obtained with correlation coefficients between 0.9982 and 0.9999 in the range of 0.5-200 microg x L(-1). The limits of detection (LODs) were in the range between 0.0005 and 0.003 microg x mL(-1) (S/N = 3). The method was validated with five soil samples spiked at three fortification levels (0.05, 0.1, 1.0 mg x kg(-1)) and recoveries were in the range of 55.3%-95.6% with RSD of 1.4%-7.0%. The effect of clean-up was evaluated by UV spectra and demonstrated that the method established is effective. In conclusion, this method is competent for the simultaneous analysis of 6 neonicotinoid residues in soil.

Determination of tramadol hydrochloride in ampoule dosage forms by using UV spectrophotometric and HPLC-DAD methods in methanol and water media.

PubMed

Küçük, Aysel; Kadioğlu, Yücel

2005-02-01

Two newly developed simple and sensitive methods for determination of tramadol hydrochloride in ampoule dosage forms were described and validated. Measurements for spectrophotometric method were performed using UV-Vis Spectrophotometer in ranges of 200-400 nm. The solutions of standard and the samples were prepared in methanol and water media and the UV absorption spectrums of tramadol were monitored with maximum absorptions at 275 and 271 nm for both mediums, respectively. The standard calibration curves of tramadol were constructed by plotting absorbance vs. concentration in the concentration range with the final dilution of 10-100 microg ml-1. Reversed phase chromatography for HPLC method was conducted using a Phenomenex Bondclone C18 column with an isocratic mobile phase consisting of 25% acetonitrile in 75% 0.01 M phosphate buffer (pH 3). The effluent was monitored on a DAD detector at 218 nm. Linear response (r>0.99) was observed over the range of 0.5-40 microg ml-1 for methanol and water and run on six different occasions. The methods were applied successfully to pharmaceutical ampoule forms, but also for comparison in two different solvent media. Besides, it was completely validated and proven to be rugged.

[Preparation and applications of a supported liquid-liquid extraction column with a composite diatomite material].

PubMed

Bao, Jianmin; Ma, Zhishuang; Sun, Ying; Wang, Yongzun; Li, Youxin

2012-08-01

A rapid and special supported liquid-liquid extraction (SLE) column was developed with a composite diatomite material. The SLE column was evaluated by high performance liquid chromatography (HPLC) with acidic, neutral and alkaline compounds dissolved in water. Furthermore, some real complex samples were also analyzed by HPLC with the SLE method. The recoveries of benzoic acid (acidic), p-nitroaniline (alkaline) and 4-hydroxy-benzoic methyl ester (neutral) treated by the SLE column were 90.6%, 98.1% and 97.7%. However, the recoveries of the three compounds treated by traditional liquid-liquid extraction (LLE) method were 71.9%, 81.9% and 83.9%. The results showed that the SLE technique had higher recoveries than the traditional LLE method. The spiked recoveries of the complex samples, such as benzoic acid in Sprite and dexamethasone acetate, chlorphenamine maleate, indomethacin in bovine serum, were between 80% and 110% and the relative standard deviations (RSDs) were less than 15%. For biological specimen, the results could be accepted. Meantime, many disadvantages associated with traditional LLE method, such as emulsion formation, didn't occur using SLE column. The SLE column technique is a good sample preparation method with many advantages, such as rapid, simple, robust, easily automated, high recovery and high-throughput, which would be widely used in the future.

Development of an Ion-Pairing Reagent and HPLC-UV Method for the Detection and Quantification of Six Water-Soluble Vitamins in Animal Feed.

PubMed

Kim, Ho Jin

2016-01-01

A novel and simple method for detecting six water-soluble vitamins in animal feed using high performance liquid chromatography equipped with a photodiode array detector (HPLC/PDA) and ion-pairing reagent was developed. The chromatographic peaks of the six water-soluble vitamins were successfully identified by comparing their retention times and UV spectra with reference standards. The mobile phase was composed of buffers A (5 mM PICB-6 in 0.1% CH3COOH) and B (5 mM PICB-6 in 65% methanol). All peaks were detected using a wavelength of 270 nm. Method validation was performed in terms of linearity, sensitivity, selectivity, accuracy, and precision. The limits of detection (LODs) for the instrument employed in these experiments ranged from 25 to 197 μg/kg, and the limits of quantification (LOQs) ranged from 84 to 658 μg/kg. Average recoveries of the six water-soluble vitamins ranged from 82.3% to 98.9%. Method replication resulted in intraday and interday peak area variation of <5.6%. The developed method was specific and reliable and is therefore suitable for the routine analysis of water-soluble vitamins in animal feed.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

PubMed

Kand'ár, Roman; Záková, Pavla; Jirosová, Jana; Sladká, Michaela

2009-01-01

The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Development and validation of a simple and robust method for arsenic speciation in human urine using HPLC/ICP-MS.

PubMed

Sen, Indranil; Zou, Wei; Alvaran, Josephine; Nguyen, Linda; Gajek, Ryszard; She, Jianwen

2015-01-01

In order to better distinguish the different toxic inorganic and organic forms of arsenic (As) exposure in individuals, we have developed and validated a simple and robust analytical method for determining the following six As species in human urine: arsenous (III) acid (As-III), As (V) acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine (AsB), and arsenocholine. In this method, human urine is diluted using a pH 5.8 buffer, separation is performed using an anion exchange column with isocratic HPLC, and detection is achieved using inductively coupled plasma-MS. The method uses a single mobile phase consisting of low concentrations of both phosphate buffer (5 mM) and ammonium nitrate salt (5 mM) at pH 9.0; this minimizes the column equilibration time and overcomes challenges with separation between AsB and As-III. In addition, As-III oxidation is prevented by degassing the sample preparation buffer at pH 5.8, degassing the mobile phase online at pH 9.0, and by the use of low temperature (-70 °C) and flip-cap airtight tubes for long term storage of samples. The method was validated using externally provided reference samples. Results were in agreement with target values at varying concentrations and successfully passed external performance test criteria. Internal QC samples were prepared and repeatedly analyzed to assess the method's long-term precision, and further analyses were completed on anonymous donor urine to assess the quality of the method's baseline separation. Results from analyses of external reference samples agreed with target values at varying concentrations, and results from precision studies yielded absolute CV values of 3-14% and recovery from 82 to 115% for the six As species. Analysis of anonymous donor urine confirmed the well-resolved baseline separation capabilities of the method for real participant samples.

Comparative evaluation of an ISO 3632 method and an HPLC-DAD method for safranal quantity determination in saffron.

PubMed

García-Rodríguez, M Valle; López-Córcoles, Horacio; Alonso, Gonzalo L; Pappas, Christos S; Polissiou, Moschos G; Tarantilis, Petros A

2017-04-15

The aim of this work was a comparison of the ISO 3632 (2011) method and an HPLC-DAD method for safranal quantity determination in saffron. Samples from different origins were analysed by UV-vis according to ISO 3632 (2011) and by HPLC-DAD. Both methods were compared, and there was no correlation between the safranal content obtained by UV-vis and HPLC-DAD. An over-estimation in the UV-vis experiment was observed, which was related to the cis-crocetin esters content, as well as other compounds. The results demonstrated that there was no relationship between ISO quality categories and safranal content using HPLC-DAD. Therefore, HPLC-DAD might be preferable to UV-vis for determining the safranal content and the classification of saffron for commercial purposes. In addition, HPLC-DAD was adequate for determining the three foremost parameters that define the quality of saffron (crocetin esters, picrocrocin and safranal); therefore, this approach could be included in the ISO 3632 method (2011). Copyright © 2016 Elsevier Ltd. All rights reserved.

A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations.

PubMed

Zmolek, Wesley; Bañas, Stefanie; Barfield, Robyn M; Rabuka, David; Drake, Penelope M

2016-10-01

Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and Validation of a Simple and Rapid UPLC-MS Assay for Valproic Acid and Its Comparison With Immunoassay and HPLC Methods.

PubMed

Zhao, Mingming; Li, Guofei; Qiu, Feng; Sun, Yaxin; Xu, Yinghong; Zhao, Limei

2016-04-01

Valproic acid (VPA), a widely used antiepileptic drug, has a narrow therapeutic range of 50-100 mcg/mL and shows large individual variability. It is very important to monitor the trough concentration of VPA using a reliable method. Therefore, the aim of this study was to develop and validate a rapid ultraperformance liquid chromatographic-mass spectrometry (UPLC-MS) method for quantification of VPA in human serum and to compare with fluorescence polarization immunoassay (FPIA), chemiluminescence microparticle immunoassay (CMIA), and high-performance liquid chromatography (HPLC) methods. The method included extraction of VPA in serum by deproteinization with acetonitrile. The analysis was performed using an EC-C18 column (2.7 μm, 4.6 × 50 mm) under isocratic conditions with a mobile phase of acetonitrile/water (containing 0.1% formic acid) (45/55, vol/vol) at a flow rate of 0.6 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer using an electrospary probe in the negative ionization mode. The method was validated by studies of selectivity, linearity, lower limit of quantification, accuracy, precision, recovery, matrix effect, and stability. Furthermore, all the 4 methods including FPIA, CMIA, and HPLC were subsequently used to assay the VPA concentration in 498 clinical serum samples collected from patients who received VPA. These methods were compared by Deming regression and Bland-Altman analysis. The retention time of VPA was 2.09 minutes. The calibration curve was linear over the concentration range of 1-200 mcg/mL, with a lower limit of quantification of 1 mcg/mL. The interday and intraday precision (RSD %) was less than 4.6% and 4.5%, respectively, and the accuracy (RE %) was below 7.9%. The recoveries and matrix effect of VPA at concentrations of 2, 50, and 160 mcg/mL met the requirement for the analysis of biological samples. No obvious degradation of VPA was observed under various storage conditions including room temperature for 12 hour, 3 freeze-thaw cycles, and -20°C for 3 months. Regression analysis showed that the correlation coefficients for the UPLC-MS versus FPIA, CMIA, and HPLC were 0.989, 0.988, and 0.987, respectively. The results of agreement tests between UPLC-MS and other methods showed that the mean difference of UPLC-MS and FPIA was -1.4 mcg/mL and 95% confidence interval of -7.7 to 4.9 mcg/mL, and the values for UPLC-MS and CMIA were -0.8 mcg/mL and -7.5 to 5.8 mcg/mL, for UPLC-MS and HPLC were 1.1 mcg/mL and -5.7 to 7.9 mcg/mL. The rapid UPLC-MS method we developed showed a good analytical performance required for therapeutic drug monitoring, leading to potential improvements in patient care and laboratory management. Compared with the FPIA, CMIA, and HPLC methods, the UPLC-MS method correlated well and displayed comparable VPA concentrations.

The use of dissolvable layered double hydroxide components in an in situ solid-phase extraction for chromatographic determination of tetracyclines in water and milk samples.

PubMed

Phiroonsoontorn, Nattaphorn; Sansuk, Sira; Santaladchaiyakit, Yanawath; Srijaranai, Supalax

2017-10-13

This research presents a simple and green in situ solid phase extraction (is-SPE) combined with high-performance liquid chromatography (HPLC) for the simultaneous analysis of tetracyclines (TCs) including tetracycline, oxytetracycline, and chlortetracycline. In is-SPE, TCs were efficiently extracted through the precipitation formation of dissolvable layered double hydroxides (LDHs) by mixing the LDH components such as magnesium and aluminum ions (both in metal chloride salts) thoroughly in an alkaline sample solution. After the centrifugation, the precipitate was completely dissolved with trifluoroacetic acid to release the enriched TCs, and then analyzed by HPLC. Under optimized conditions, this method gave good enrichment factors (EFs) of 41-93 with low limits of detection (LODs) of 0.7-6μg/L and limits of quantitation (LOQs) of 3-15μg/L. Also, the proposed method was successfully applied for the determination of TCs in water and milk samples with the recoveries ranging from 81.7-108.1% for water and 55.7-88.7% for milk. Copyright © 2017 Elsevier B.V. All rights reserved.

[Simultaneous determination of 4 diterpenoids in Rabdosia japonica var.glaucocalyx by HPLC-ESI-MS/MS and cluster analysis].

PubMed

Tian, Ting-Ting; Ma, Ying-Hua; Xie, Wei-Wei; Jin, Yi-Ran; Xu, Hui-Jun; Zhang, Lan-Tong; Du, Ying-Feng

2016-01-01

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of four major diterpenoids in Rabdosia japonica var.glaucocalyx, including glaucocalyxin A, oridonin, hebeirubesensin and enmenol. Analysis was performed on an Agilent ZORBAX SB-C18(4.6 mm×250 mm, 5 μm ) column eluted in a gradient program with methanol and water. The flow rate was 0.8 mL•min⁻¹. Multiple reaction monitoring (MRM) scanning mode was performed in negative ion switching mode to apply for the quantitative determination. The calibration curves for the above four compounds were linear in corresponding injection amount. The average recoveries of the compounds ranged from 92.40% to 105.9%, with RSDs of 1.7%-6.5%. The method is simple, rapid, accurate with good repeatability, which can provide a reference for overcalling evaluation the quality of R. japonica var.glaucocalyx. The result of cluster analysis- showed that the quality of R. japonica glaucocalyx var. greatly varied between areas and parts. Copyright© by the Chinese Pharmaceutical Association.

[Simultaneous determination of ephedrine hydrochloride, D-pseudoephedrine and amygdalin in xiao'er pingchuan qutan granule by HPLC].

PubMed

Yang, De-Bin; Tong, Yan; Ma, Zhen-Shan; Wang, Lin; Dong, Mei-Hong; Li, Yan-Ling; Wang, Jin-Yu

2013-03-01

To establish an HPLC method for the determination of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin in Xiao'er Pingchuan Qutan granule. Pheny ether chromatographic column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.1% phosphoric acid (containing 0.1% three ethylamine) (3:97) as the mobile phase. The UV detection wavelength was at 210 nm, with the flow rate of 1 mL x min(-1), and column temperature was at 35 degrees C. The linearity of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin ranged between 0.078 60-3.144 microg (r = 1.000 0), 0.103 4-2.068 microg (r = 0.999 7) and 0.430 5-3.157 microg (r = 0.999 8), respectively. Their average recoveries were 98.46% (RSD 1.1%), 103.0% (RSD 1.5%) and 97.15% (RSD 2.1%), respectively. The method is simple, stable and reliable that it can be used to determine the content of ephedrine hydrochloride, D-pseudo-ephedrine and amygdalin in Xiao'er Pingchuan Qutan granule.

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing.

PubMed

Kotani, Akira; Miyaguchi, Yuji; Tomita, Eiji; Takamura, Kiyoko; Kusu, Fumiyo

2004-03-24

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.

G-index: A new metric to describe dynamic refractive index effects in HPLC absorbance detection.

PubMed

Kraiczek, Karsten G; Rozing, Gerard P; Zengerle, Roland

2018-09-01

High performance liquid chromatography (HPLC) with a solvent gradient and absorbance detection is one of the most widely used methods in analytical chemistry. The observed absorbance baseline is affected by the changes in the refractive index (RI) of the mobile phase. Near the limited of detection, this complicates peak quantitation. The general aspects of these RI-induced apparent absorbance effects are discussed. Two different detectors with fundamentally different optics and flow cell concepts, a variable-wavelength detector equipped with a conventional flow cell and a diode-array detector equipped with a liquid core waveguide flow cell, are compared with respect to their RI behavior. A simple method to separate static - partly unavoidable - RI effects from dynamic RI effects is presented. It is shown that the dynamic RI behavior of an absorbance detector can be well described using a single, relatively easy-to-determine metric called the G-index. The G-index is typically in the order of a few seconds and its sign depends on the optical flow cell concept. Copyright © 2018 Elsevier B.V. All rights reserved.

Measurement of total polychlorinated biphenyls (PCBs) in catalytic combustion effluents using a simple HPLC procedure

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

Perera, S.P.; Jefferies, T.M.

1995-05-01

The production, use and toxicity of Polychlorinated biphenyls (PCBs) has been discussed in detail and increasing concern has been shown by the scientific community and by the public about the effectiveness of thermal incineration as an environmentally satisfactory means of destroying polychlorinated biphenyls. Therefore, a greater need for alternative, more efficient, low energy processes such as catalytic combustion, has been discussed. This paper is part of research to investigate the catalytic combustion of Aroclor 1242 in a packed bed reactor using chromia on an alumina support and other oxidation catalysts. It was essential for these studies to be able tomore » determine the PCB destruction efficiencies of the catalytic processes examined. The high efficiency of capillary GC columns permits the resolution of an Aroclor into a chromatogram consisting mainly of individual PCB congeners. The chromatogram may typically contain 50-150 peaks. Quantification requires each peak to be identified and individual response factors assigned before the amount of each congener present may be calculated. Response factors vary and the high cost of purchasing all individual PCB standards was prohibitive. A commonly adopted alternative method is to measure only the 6 or 7 individual PCB congeners available as a commercial mixture and convert the answer into a value for an Aroclor, not an accurate measure of the destruction efficiency of PCBs. This paper describes a simple quantitative HPLC method that measures all PCBs as a single peak. 14 refs., 3 figs., 1 tab.« less

Detection of Organophosphorus Pesticides in Wheat by Ionic Liquid-Based Dispersive Liquid-Liquid Microextraction Combined with HPLC

PubMed Central

Quan, Ji; Hu, Zeshu

2018-01-01

Food safety issues closely related to human health have always received widespread attention from the world society. As a basic food source, wheat is the fundamental support of human survival; therefore, the detection of pesticide residues in wheat is very necessary. In this work, the ultrasonic-assisted ionic liquid-dispersive liquid-liquid microextraction (DLLME) method was firstly proposed, and the extraction and analysis of three organophosphorus pesticides were carried out by combining high-performance liquid chromatography (HPLC). The extraction efficiencies of three ionic liquids with bis(trifluoromethylsulfonyl)imide (Tf2N) anion were compared by extracting organophosphorus in wheat samples. It was found that the use of 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([OMIM][Tf2N]) had both high enrichment efficiency and appropriate extraction recovery. Finally, the method was used for the determination of three wheat samples, and the recoveries of them were 74.8–112.5%, 71.8–104.5%, and 83.8–115.5%, respectively. The results show that the method proposed is simple, fast, and efficient, which can be applied to the extraction of organic matters in wheat samples. PMID:29854562

Fingerprint chromatogram analysis of extracts from the leaves of Tripterygium wilfordii Hook. F. by high performance liquid chromatography.

PubMed

Li, Ke; Wang, Shudong

2005-05-01

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the study of fingerprint chromatograms of extracts from the leaves of Tripterygium wilfordii Hook. F. (TWHF) and for controlling the quality of the herb. HPLC separation of the extracts was performed on a Lichrospher RP-18 column and detected by ultraviolet absorbance at 210 nm. The column temperature was maintained at 35 degrees C. A mobile phase composed of acetonitrile:H2O in the ratio of 39:61 (v/v) was found to be most suitable for this separation at a flow rate of 0.8 mL/min with isocratic elution. Under the chromatographic conditions described, the peak profile of the 10 components collected within 35 min made up the fingerprint of the extracts from leaves of TWHF with universal features. The fingerprint chromatograms had a good stability, precision, and reproducibility. The similarity of the extracts from leaves of TWHF collected in summer and winter was studied with triptolide as a reference peak. The method is suitable for differentiation of extracts from the leaves of TWHF, and can be used as a quality control method for this herb.

Phospholipid hydrolysis in a pharmaceutical emulsion assessed by physicochemical parameters and a new analytical method.

PubMed

Rabinovich-Guilatt, Laura; Dubernet, Catherine; Gaudin, Karen; Lambert, Gregory; Couvreur, Patrick; Chaminade, Pierre

2005-09-01

The aim of this work was to develop a simple high-performance liquid chromatography (HPLC) technique with evaporative light scattering detection (ELSD) for the separation and quantification of the major phospholipid (PL) and lysophospholipid (LPL) classes contained in a pharmaceutical phospholipid-based emulsion. In the established method, phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyeline (SM), lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were separated with a PVA-Sil stationary phase and a binary gradient from pure chloroform to methanol:water (94:6 v/v) at 3.4%/min. The ELSD detection was enhanced using 0.1% triethylamine and formic acid in each gradient mobile phases. Factors such as stationary phase and ELSD drift tube temperature were optimized, concluding in optimal temperatures of 25 degrees C for separation and 50 degrees C for evaporation. This HPLC-ELSD method was then applied to a PL-emulsion exposed to autoclaving and accelerated thermal conditions at 50 degrees C. Hydrolysis of PC and PE followed first-order kinetics, representing only 45% of the total lipid mass after 3 months. The chemical stability was correlated to commonly measured formulation physical and physico-chemical parameters such as droplet size, emulsion pH and zeta-potential.

The use of capillary electrophoresis as part of a specificity testing strategy for mitoguazone dihydrochloride HPLC methods.

PubMed

Thomson, C E; Gray, M R; Baxter, M P

1997-05-01

Capillary electrophoresis (CE) has been used as part of a validation experiment designed to prove the specificity of high performance liquid chromatography (HPLC) methods used for analysis of mitoguazone dihydrochloride drug substance. Data regarding accuracy, precision and sensitivity of the CE methods are presented as well as a comparison of results obtained from CE, HPLC and thin-layer chromatography (TLC) analysis of samples stressed under a variety of conditions. It was concluded that, not only were the HPLC methods being investigated specific, but that CE could potentially be used to replace HPLC for the routine assay of mitoguazone dihydrochloride.

Determination of alpha-hydroxy acids in cosmetic products by high-performance liquid chromatography with a narrow-bore column.

PubMed

Nicoletti, I; Corradini, C; Cogliandro, E; Cavazza, A

1999-08-01

This paper reports the results of a study carried out to develop a simple, rapid and sensitive method for the separation, identification and quantitative measurement of alpha-hydroxy acids in commercial cosmetics using high-performance liquid chromatography (HPLC). This method is successfully applied to the simultaneous identification and quantitative determination of glycolic, lactic, malic, tartaric and citric acids employing a reversed phase narrow-bore column under isocratic condition and UV detection. The method is validated by determining the precision of replicate analyses and accuracy by analyzing samples with and without adding know amount of the alpha-hydroxy acids. The procedure is suitable for routine analyses of commercial cosmetics.

Spectrophotometric estimation of ambroxol and cetirizine hydrochloride from tablet dosage form.

PubMed

Gowekar, N M; Pande, V V; Kasture, A V; Tekade, A R; Chandorkar, J G

2007-07-01

Fixed dose combination tablets containing ambroxol HCl and cetirizine HCl are clinically used as mucolytic and antiallergic. Several spectrophotometric and HPLC methods have been reported for simultaneous estimation of these drugs with other drugs. The drugs individually and in mixture obeys Beer's law over conc. range 1.2-4.4 microg/mL for cetirizine HCL and for ambroxol HCL 15-52 microg/mL at all five sampling wavelengths (correlation coeff. well above 0.995). The mean recoveries from tablet by standard addition method were 100.18% (+/-2.4) and 100.66 % (+/-2.31). The present work reports simple, accurate and precise spectrophotometric methods for their simultaneous estimation from tablet dosage form.

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples.

PubMed

Moein, Mohammad Mahdi; Javanbakht, Mehran; Akbari-Adergani, Behrouz

2011-04-01

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

PubMed Central

Aouri, Manel; Moradpour, Darius; Cavassini, Matthias; Mercier, Thomas; Buclin, Thierry; Csajka, Chantal; Telenti, Amalio; Rauch, Andri

2013-01-01

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy. PMID:23629707

Genetic and Chemical Profiling of Gymnema sylvestre Accessions from Central India: Its Implication for Quality Control and Therapeutic Potential of Plant.

PubMed

Verma, Ashutosh Kumar; Dhawan, Sunita Singh; Singh, Seema; Bharati, Kumar Avinash; Jyotsana

2016-07-01

Gymnema sylvestre , a vulnerable plant species, is mentioned in Indian Pharmacopeia as an antidiabetic drug. Study of genetic and chemical diversity and its implications in accessions of G. sylvestre . Fourteen accessions of G. sylvestre collected from Central India and assessment of their genetic and chemical diversity were carried out using ISSR (inter simple sequence repeat) and HPLC (high performance liquid chromatography) fingerprinting methods. Among the screened 40 ISSR primers, 15 were found polymorphic and collectively produced nine unique accession-specific bands. The maximum and minimum numbers of amplicones were noted for ISSR-15 and ISSR-11, respectively. The ISSR -11 and ISSR-13 revealed 100% polymorphism. HPLC chromatograms showed that accessions possess the secondary metabolites of mid-polarity with considerable variability. Unknown peaks with retention time 2.63, 3.41, 23.83, 24.50, and 44.67 were found universal type. Comparative hierarchical clustering analysis based on foresaid fingerprints indicates that both techniques have equal potential to discriminate accessions according to percentage gymnemic acid in their leaf tissue. Second approach was noted more efficiently for separation of accessions according to their agro-climatic/collection site. Highly polymorphic ISSRs could be utilized as molecular probes for further selection of high gymnemic acid yielding accessions. Observed accession specific bands may be used as a descriptor for plant accessions protection and converted into sequence tagged sites markers. Identified five universal type peaks could be helpful in identification of G. sylvestre -based various herbal preparations. Nine accession specific unique bandsFive marker peaks for G. sylvestre .Suitability of genetic and chemical fingerprinting Abbreviations used: HPLC: High Performance Liquid Chromatography, ISSR: Inter Simple Sequence Repeats, CTAB: Cetyl Trimethylammonium Bromide, DNTP: Deoxynucleotide Triphosphates.

High-pressure liquid chromatography analysis of antibiotic susceptibility disks.

PubMed Central

Hagel, R B; Waysek, E H; Cort, W M

1979-01-01

The analysis of antibiotic susceptibility disks by high-pressure liquid chromatography (HPLC) was investigated. Methods are presented for the potency determination of mecillinam, ampicillin, carbenicillin, and cephalothin alone and in various combinations. Good agreement between HPLC and microbiological data is observed for potency determinations with recoveries of greater than 95%. Relative standard deviations of lower than 2% are recorded for each HPLC method. HPLC methods offer improved accuracy and greater precision when compared to the standard microbiological methods of analysis for susceptibility disks. PMID:507793

HPLC-MS/MS method for the simultaneous determination of MB07133 and its metabolites, cytarabine and arabinofuranosyluracil, in rat plasma.

PubMed

Wang, Dan; Xiao, Qingqing; Yang, Wanqiu; Qian, Wei; Yang, Jin

2016-02-20

MB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100μL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi(®) fusion-RP column (150mm×4.6mm, 4μm) by a gradient-elution with a mobile phase consisting of ammonium formate (1mM) and methanol at a flow rate of 1mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100-50000ng/mL for MB07133, 2-1000ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18mg/kg i.v.). Copyright © 2015 Elsevier B.V. All rights reserved.

Chemometric quality inspection control of pyrantel pamoate, febantel and praziquantel in veterinary tablets by mid infrared spectroscopy

NASA Astrophysics Data System (ADS)

Piantavini, Mário S.; Pontes, Flávia L. D.; Uber, Caroline P.; Stremel, Dile P.; Sena, Marcelo M.; Pontarolo, Roberto

This paper describes the development and validation of a new multivariate calibration method based on diffuse reflectance mid infrared spectroscopy for direct and simultaneous determination of three veterinary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions, 3715-3150, 2865-2583, and 2298-1733 cm-1, preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centering. This model was built with five latent variables and provided root mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the three analytes. The method was validated according the appropriate regulations through the estimate of figures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these commercial products, which was not known a priori, was modeled by an experimental design that scanned the likely content range of the possible constituents. The results were verified with high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and were in agreement with the predicted values at 95% confidence level. The developed method presented the advantages of being simple, rapid, solvent free, and about ten times faster than the HPLC ones.

Salting-Out Assisted Liquid-Liquid Extraction for Quantification of Febuxostat in Plasma Using RP-HPLC and Its Pharmacokinetic Application.

PubMed

Tandel, Devang; Shah, Purvi; Patel, Kalpana; Thakkar, Vaishali; Patel, Kirti; Gandhi, Tejal

2016-11-01

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method using novel salting-out assisted liquid-liquid extraction technique has been developed for the quantitative determination of febuxostat (FEB), used for the treatment of gout, in rat plasma. The method was validated according to US FDA guideline. Separation was achieved using a Phenomenex Luna-C 18 (250 × 4.60 mm, 5 µm) column and mobile phase composed of potassium dihydrogen orthophosphate buffer 25 mM, adjusted to pH 6.8 with triethylamine:methanol in a ratio of 35:65 (v/v) showing retention time 5.56 and 8.86 min for FEB and internal standard, respectively. The optimal salting-out parameters; 1 mL of acetonitrile and 200 µL of 2 M ammonium acetate salt showed extraction recovery >90% for FEB from plasma. This extraction procedure afforded clear samples resulting in convenient and cost-saving procedure and showed good linear relationship (r > 0.9997) between peak area ratio and concentration from 0.3 to 20 µg/mL. The results of pharmacokinetic study showed that absorption profile of spherical agglomerate of FEB compared to marketed formulation was higher indicating greater systemic absorption. In conclusion, the developed SALLE-HPLC method with simple ultraviolet detection offered a number of advantages including good quantitative ability, wide linear range, high recovery, short analysis time as well as low cost. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

N,N-(2,4-dinitrophenyl)octylamine derivatives for the isolation, purification, and mass spectrometric characterization of oligosaccharides.

PubMed

Zhang, Y; Cedergren, R A; Nieuwenhuis, T J; Hollingsworth, R I

1993-02-01

A simple, sensitive method for the structural characterization of oligosaccharides by fast atom bombardment-mass spectrometry (FAB-MS) has been designed. Oligosaccharides are labeled with a uv chromophore (which also serves as a charge stabilizing group) and with a hydrophobic alkyl tail. The chromophore, a 2,4-dinitrophenyl group, aids uv detection during HPLC and stabilizes negative ion species formed during analysis by FAB-MS. The hydrophobic tail, provided by an octyl group, enhances the surface activity of the analytes and makes them amenable to separation by reverse-phase chromatography using a C18 bonded phase. This method was applied to the structural analysis of the components of a mixture of starch maltodextrins with a degree of polymerization 1-16, to the analysis of the structure of pure maltohexaose, and to a previously characterized oligosaccharide from a Rhizobium capsular polysaccharide. The method gave a good yield of [M-H]- anions for the derivatized compounds, which in most cases were detectable at a level of about 1 pmol. In the case of maltohexaose, four series of sequence anions corresponding to sequential loss of glycosyl residues from the reducing and nonreducing end by different mechanisms were observed. The mixture of derivatized malto-oligosaccharides could easily be separated by HPLC. Based on the relative proportions of the individual oligomers in the mixture calculated from HPLC analysis, even though the higher oligomers were present in amounts of about 0.1%, they could still be easily detected in mass spectra of the entire mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical Characterization of an Oil-in-Water Adjuvant Emulsion.

PubMed

Sun, Jenny; Remmele, Richard L; Sanyal, Gautam

2017-07-01

Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.

Chemometric quality inspection control of pyrantel pamoate, febantel and praziquantel in veterinary tablets by mid infrared spectroscopy.

PubMed

Piantavini, Mário S; Pontes, Flávia L D; Uber, Caroline P; Stremel, Dile P; Sena, Marcelo M; Pontarolo, Roberto

2014-05-05

This paper describes the development and validation of a new multivariate calibration method based on diffuse reflectance mid infrared spectroscopy for direct and simultaneous determination of three veterinary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions, 3715-3150, 2865-2583, and 2298-1733 cm(-1), preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centering. This model was built with five latent variables and provided root mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the three analytes. The method was validated according the appropriate regulations through the estimate of figures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these commercial products, which was not known a priori, was modeled by an experimental design that scanned the likely content range of the possible constituents. The results were verified with high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and were in agreement with the predicted values at 95% confidence level. The developed method presented the advantages of being simple, rapid, solvent free, and about ten times faster than the HPLC ones. Copyright © 2014 Elsevier B.V. All rights reserved.

Monitoring and evaluating the quality consistency of Compound Bismuth Aluminate tablets by a simple quantified ratio fingerprint method combined with simultaneous determination of five compounds and correlated with antioxidant activities.

PubMed

Liu, Yingchun; Liu, Zhongbo; Sun, Guoxiang; Wang, Yan; Ling, Junhong; Gao, Jiayue; Huang, Jiahao

2015-01-01

A combination method of multi-wavelength fingerprinting and multi-component quantification by high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was developed and validated to monitor and evaluate the quality consistency of herbal medicines (HM) in the classical preparation Compound Bismuth Aluminate tablets (CBAT). The validation results demonstrated that our method met the requirements of fingerprint analysis and quantification analysis with suitable linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ). In the fingerprint assessments, rather than using conventional qualitative "Similarity" as a criterion, the simple quantified ratio fingerprint method (SQRFM) was recommended, which has an important quantified fingerprint advantage over the "Similarity" approach. SQRFM qualitatively and quantitatively offers the scientific criteria for traditional Chinese medicines (TCM)/HM quality pyramid and warning gate in terms of three parameters. In order to combine the comprehensive characterization of multi-wavelength fingerprints, an integrated fingerprint assessment strategy based on information entropy was set up involving a super-information characteristic digitized parameter of fingerprints, which reveals the total entropy value and absolute information amount about the fingerprints and, thus, offers an excellent method for fingerprint integration. The correlation results between quantified fingerprints and quantitative determination of 5 marker compounds, including glycyrrhizic acid (GLY), liquiritin (LQ), isoliquiritigenin (ILG), isoliquiritin (ILQ) and isoliquiritin apioside (ILA), indicated that multi-component quantification could be replaced by quantified fingerprints. The Fenton reaction was employed to determine the antioxidant activities of CBAT samples in vitro, and they were correlated with HPLC fingerprint components using the partial least squares regression (PLSR) method. In summary, the method of multi-wavelength fingerprints combined with antioxidant activities has been proved to be a feasible and scientific procedure for monitoring and evaluating the quality consistency of CBAT.

Simultaneous determination of atorvastatin calcium and olmesartan medoxomil in a pharmaceutical formulation by reversed phase high-performance liquid chromatography, high-performance thin-layer chromatography, and UV spectrophotometric methods.

PubMed

Soni, Hiral; Kothari, Charmy; Khatri, Deepak; Mehta, Priti

2014-01-01

Validated RP-HPLC, HPTLC, and UV spectrophotometric methods have been developed for the simultaneous determination of atorvastatin calcium (ATV) and olmesartan medoxomil (OLM) in a pharmaceutical formulation. The RP-HPLC separation was achieved on a Kromasil C18 column (250 x 4.6 mm, 5 microm particle size) using 0.01 M potassium dihydrogen o-phosphate (pH 4 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1.5 mL/min. Quantification was achieved by UV detection at 276 nm. The HPTLC separation was achieved on precoated silica gel 60F254 plates using chloroform-methanol-acetonitrile (4 + 2+ 4, v/v/v) mobile phase. Quantification was achieved with UV detection at 276 nm. The UV-Vis spectrophotometric method was based on the simultaneous equation method that involves measurement of absorbance at two wavelengths, i.e., 255 nm (lambda max of OLM) and 246.2 nm (lambda max of ATV) in methanol. All three methods were validated as per International Conference on Harmonization guidelines. The proposed methods were simple, precise, accurate, and applicable for the simultaneous determination of ATV and OLM in a marketed formulation. The results obtained by applying the proposed methods were statistically analyzed and were found satisfactory.

Simultaneous quantification by HPLC of the phenolic compounds for the crude drug of Prunus serotina subsp. capuli.

PubMed

Rivero-Cruz, Blanca

2014-08-01

Prunus serotina Ehrenb. subsp. capuli (Cav.) McVaugh (Rosaceae), commonly known as "capulin", is a native North American tree, commercialized and used in folk medicine for the treatment of the hypertension, gastrointestinal illnesses, and cough. This work developed a suitable HPLC method for quantifying the major active constituents of the infusion of P. serotina, the most important preparation consumed by populations around the world. The analytical method was performed using a Fortis-RP column (150 mm × 4.6 mm; film thickness 5 µm). The mobile phase consisted of an isocratic acetate buffer solution (pH 2.7; A) and methanol (B) (65:35 v/v) at a flow rate of 1.0 mL min(-1). The proposed method was applied to the quantification of 1-3 in several samples of the leaves of P. serotina. The results indicated that amounts of 1-3 in the samples analyzed are uniform, and greater amounts of chlorogenic acid (2; 479.9 ± 33.6 µg g(-1), dry matter) along with hyperoside (1; 185.7 ± 55.3 µg g(-1), dry matter) were present. On the other hand, benzaldehyde (3; 118.2 ± 12.1 µg g(-1) dry matter) was found to be in lower concentration. A simple, sensitive, precise, and reproducible HPLC method for the simultaneous quantification of 1-3 in P. serotina was developed and validated. This is the first report on the quantification of 1-3 as active principles, and compound 1 was selected as a marker of P. serotina, which could be useful to guarantee the quality of the crude drug and herbal products.

Stability-indicating HPLC Method for Simultaneous Determination of Terbutaline Sulphate, Bromhexine Hydrochloride and Guaifenesin

PubMed Central

Porel, A.; Haty, Sanjukta; Kundu, A.

2011-01-01

The aim of the present study was the development and subsequent validation of a simple, precise and stability-indicating reversed phase HPLC method for the simultaneous determination of guaifenesin, terbutaline sulphate and bromhexine hydrochloride in the presence of their potential impurities in a single run. The photolytic as well as hydrolytic impurities were detected as 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]-ethanone from terbutaline, 2-methoxyphenol and an unknown impurity identified as (2RS)-3-(2-hydroxyphenoxy)-propane-1,2-diol from guaifenesin. The chromatographic separation of all the three active components and their impurities was achieved on Wakosil II column, using phosphate buffer (pH 3.0) and acetonitrile as mobile phase which was delivered initially in the ratio of 80:20 (v/v) for 18 min, then changed to 60:40 (v/v) for next 12 min, and finally equilibrated back to 80:20 (v/v) for 10 min. Other HPLC parameters were: Flow rate at 1.0 ml/min, detection wavelengths 248 and 280 nm, injection volume 10 μl. The calibration graphs plotted with five concentrations of each component were linear with a regression coefficient R2 >0.9999. The limit of detection and limit of quantitation were estimated for all the five impurities. The established method was then validated for linearity, precision, accuracy, and specificity and demonstrated to be applicable to the determination of the active ingredients in commercial and model cough syrup. No interference from the formulation excipients was observed. These results suggest that this LC method can be used for the determination of multiple active ingredients and their impurities in a cough and cold syrup. PMID:22131621

Stability-indicating HPLC Method for Simultaneous Determination of Terbutaline Sulphate, Bromhexine Hydrochloride and Guaifenesin.

PubMed

Porel, A; Haty, Sanjukta; Kundu, A

2011-01-01

The aim of the present study was the development and subsequent validation of a simple, precise and stability-indicating reversed phase HPLC method for the simultaneous determination of guaifenesin, terbutaline sulphate and bromhexine hydrochloride in the presence of their potential impurities in a single run. The photolytic as well as hydrolytic impurities were detected as 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]-ethanone from terbutaline, 2-methoxyphenol and an unknown impurity identified as (2RS)-3-(2-hydroxyphenoxy)-propane-1,2-diol from guaifenesin. The chromatographic separation of all the three active components and their impurities was achieved on Wakosil II column, using phosphate buffer (pH 3.0) and acetonitrile as mobile phase which was delivered initially in the ratio of 80:20 (v/v) for 18 min, then changed to 60:40 (v/v) for next 12 min, and finally equilibrated back to 80:20 (v/v) for 10 min. Other HPLC parameters were: Flow rate at 1.0 ml/min, detection wavelengths 248 and 280 nm, injection volume 10 μl. The calibration graphs plotted with five concentrations of each component were linear with a regression coefficient R(2) >0.9999. The limit of detection and limit of quantitation were estimated for all the five impurities. The established method was then validated for linearity, precision, accuracy, and specificity and demonstrated to be applicable to the determination of the active ingredients in commercial and model cough syrup. No interference from the formulation excipients was observed. These results suggest that this LC method can be used for the determination of multiple active ingredients and their impurities in a cough and cold syrup.

Development of a novel solid-phase extraction element for the detection of nonylphenol in the surface water of Hangzhou.

PubMed

Lou, Liping; Cheng, Guanghuan; Yang, Qiang; Xu, Xinhua; Hu, Baolan; Chen, Yingxu

2012-02-01

Nonylphenol (NP) is a representative environmental endocrine-disrupting chemical and persistent toxic pollutant. Previous studies have shown that the average concentration of NP in environmental waters was approximately tens to hundreds of ng L(-1) and it could even reach up to tens of μg L(-1). A simple, fast and accurate method employing a novel solid-phase extraction element named "Magic Chemisorber" (MC) followed by high-performance liquid chromatography (HPLC) using a fluorescence detector (FLD) was used for detecting NP. The most important parameters that affect the extraction process, including extraction time, desorption time, desorption solvent and repeatability, were optimized. The MC-HPLC method showed good linearity with concentrations of NP from 10 to 200 μg L(-1), a correlation coefficient of 0.9995 and the limit of detection (LOD) and limit of quantification (LOQ) of this method was 0.44 and 1.47 μg L(-1), respectively. Compared to commercial polydimethylsiloxane (PDMS) glass fiber, MC had both higher capacity and recovery and it could be used repeatedly. Using the MC-HPLC method we found that the concentration of NP in river water from Hangzhou city ranged from 8.54 ± 1.23 μg L(-1) (Qiantang River) to 65.77 ± 3.69 μg L(-1) (Tiesha River), which was similar to that of international regions heavily polluted with NP and higher than that of Bohai Bay, the Yellow River and the Pearl River Delta in China. This level of NP pollution is possibly related to the rapid development of the textile, printing and paper industries of Zhejiang province.

Rapid analysis of chlorinated anilines in environmental water samples using ultrasound assisted emulsification microextraction with solidification of floating organic droplet followed by HPLC-UV detection.

PubMed

Ramkumar, Abilasha; Ponnusamy, Vinoth Kumar; Jen, Jen-Fon

2012-08-15

The present study demonstrates a simple, rapid and efficient method for the determination of chlorinated anilines (CAs) in environmental water samples using ultrasonication assisted emulsification microextraction technique based on solidification of floating organic droplet (USAEME-SFO) coupled with high performance liquid chromatography-ultraviolet (HPLC-UV) detection. In this extraction method, 1-dodecanol was used as extraction solvent which is of lower density than water, low toxicity, low volatility, and low melting point (24 °C). After the USAEME, extraction solvent could be collected easily by keeping the extraction tube in ice bath for 2 min and the solidified organic droplet was scooped out using a spatula and transferred to another glass vial and allowed to thaw. Then, 10 μL of extraction solvent was diluted with mobile phase (1:1) and taken for HPLC-UV analysis. Parameters influencing the extraction efficiency, such as the kind and volume of extraction solvent, volume of sample, ultrasonication time, pH and salt concentration were thoroughly examined and optimized. Under the optimal conditions, the method showed good linearity in the concentration range of 0.05-500 ng mL(-1) with correlation coefficients ranging from 0.9948 to 0.9957 for the three target CAs. The limit of detection based on signal to noise ratio of 3 ranged from 0.01 to 0.1 ng mL(-1). The relative standard deviations (RSDs) varied from 2.1 to 6.1% (n=3) and the enrichment factors ranged from 44 to 124. The proposed method has also been successfully applied to analyze real water samples and the relative recoveries of environmental water samples ranged from 81.1 to 116.9%. Copyright © 2012 Elsevier B.V. All rights reserved.

Behavior of new complexes of tetrakis(4-methoxylphenyl)porphyrin with heavy rare earth elements in reversed-phase high performance liquid chromatography.

PubMed

Zhang, Jun-Feng; Wang, Hong; Hou, An-Xin; Wang, Chang-Fa; Zhang, Hua-Shan

2004-08-01

An HPLC method has been developed for the separation of new complexes of tetrakis(4-methoxylphenyl)porphyrin (TMOPP) with four heavy rare earth elements (RE = Y, Er, Tm, and Yb). The function of amine and acid in the mobile phase has been investigated and a reasonable explanation is presented. Successful separation of the RE-TMOPP-Cl complexes is accomplished in 10 min with a mobile phase consisting of methanol-water-acetic acid-triethanolamine. The detection limits (S/N= 3) for the four complexes are 0.01 microg/mL. This method is rapid, sensitive, and simple.

Analysis of carbendazim, benomyl, thiophanate methyl and 2,4-dichlorophenoxyacetic acid in fruits and vegetables after supercritical fluid extraction.

PubMed

Anastassiades, M; Schwack, W

1998-10-30

Simple methods for the analysis of carbendazim, benomyl and thiophanate methyl in fruits and vegetables and of 2,4-D in citrus fruits are presented. Sample preparation involves supercritical fluid extraction with carbon dioxide and further analysis is performed without any additional clean-up by GC-MS after derivatisation or directly by HPLC-diode array detection. The SFE methods presented are clearly faster and more cost effective than traditional solvent based approaches. The recoveries, detection limits and repeatabilities achieved, meet the needs of tolerance level monitoring of these compounds in fruits and vegetables.

Rapid and simple method for the determination of emodin in tartary buckwheat (Fagopyrum tataricum) by high-performance liquid chromatography coupled to a diode array detector.

PubMed

Peng, Lian-Xin; Wang, Jing-Bo; Hu, Li-Xue; Zhao, Jiang-Lin; Xiang, Da-Bing; Zou, Liang; Zhao, Gang

2013-01-30

A simple and rapid method for determining emodin, an active factor presented in tartary buckwheat (Fagopyrum tataricum), by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) has been developed. Emodin was separated from an extract of buckwheat on a Kromasil-ODS C(18) (250 mm × 4.6 mm × 5 μm) column. The separation is achieved within 15 min on the ODS column. Emodin can be quantified using an external standard method detecting at 436 nm. Good linearity is obtained with a correlation coefficient exceeding 0.9992. The limit of detection and the limit of quantification are 5.7 and 19 μg/L, respectively. This method shows good reproducibility for the quantification of the emodin with a relative standard deviation value of 4.3%. Under optimized extraction conditions, the recovery of emodin was calculated as >90%. The validated method is successfully applied to quantify the emodin in tartary buckwheat and its products.

Development and Application of an HPLC–UV Procedure to Determine Multiple Flavonoids and Phenolics in Acanthopanax Leaf Extracts

PubMed Central

Zhang, Xiaodan; Zhou, Xinqi; Liu, Xiangqian; Li, Xiaolong; Whang, Wankyunn

2016-01-01

Acanthopanax species are used in traditional medicine to treat numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) method was developed and validated for the determination of the flavonoid and phenolic content of Acanthopanax leaves. HPLC analysis was performed on an AKZO NOBEL Kromasil 100-5C18 column (250 × 4.6 mm, 5 µm) using a gradient elution of acetonitrile–water containing 0.2% formic acid with a flow rate of 1.0 mL/min, monitored at 320 nm. The method was linear over the range 1–500 µg/mL (determination coefficients R2 > 0.999). Satisfactory intraday and interday precision was achieved, with the relative standard deviation (RSD) <2.99%. The mean recoveries measured at the three concentrations were in the range of 90.11–104.83%, with RSD <2.91% for the targets. Twenty-four samples of Acanthopanax leaves from different species and locations were examined using this analytical method, and their chemical profiles provided information for the chemotaxonomic investigation. The established method is simple, rapid and reliable for the quality control of Acanthopanax leaves of various species from different collections. The complete phenolic and flavonoid profiles of Acanthopanax leaves of various species have been established. PMID:26711585

Isolation and quantification of Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms.

PubMed

Behboudi, S; Morein, B; Rönnberg, B

1995-12-01

In the iscom, multiple copies of antigen are attached by hydrophobic interaction to a matrix which is built up by Quillaja triterpenoid saponins and lipids. Thus, the iscom presents antigen in multimeric form in a small particle with a built-in adjuvant resulting in a highly immunogenic antigen formulation. We have designed a chloroform-methanol-water extraction procedure to isolate the triterpenoid saponins and lipids incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detecting their carbohydrate chains and by HPLC. The cholesterol and phosphatidylcholine in the lipid phase were quantitated by HPLC and a commercial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and lipids in their respective phases, while protein was detected in all phases after extraction. The protein content was determined by the method of Lowry and by amino acid analysis. Amino acid analysis was shown to be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and quantitate the triterpenoids and lipids added for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospective vaccines.

A rapid method for the extraction and analysis of carotenoids and other hydrophobic substances suitable for systems biology studies with photosynthetic bacteria.

PubMed

Bóna-Lovász, Judit; Bóna, Aron; Ederer, Michael; Sawodny, Oliver; Ghosh, Robin

2013-10-11

A simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%-100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.

HPLC-ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma.

PubMed

Mistri, Hiren N; Jangid, Arvind G; Pudage, Ashutosh; Shrivastav, Pranav

2008-03-15

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.

Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group; determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

USGS Publications Warehouse

Zimmerman, L.R.; Hostetler, K.A.; Thurman, E.M.

2000-01-01

Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: acetochlor ethanesulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The mean HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.50, and 2.0 mg/L (micrograms per liter) ranged from 84 to 112 percent, with relative standard deviations of 18 percent or less. The mean HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.20, and 2.0 mg/L ranged from 81 to 125 percent, with relative standard deviations of 20 percent or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 mg/L, whereas the LOQ using the HPLC/MS method was 0.05 mg/L. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water.

Quantitative and qualitative analysis of the novel antitumor 1,3,4-oxadiazole derivative (GLB) and its metabolites using HPLC-UV and UPLC-QTOF-MS

PubMed Central

Li, Pu; Wang, Xin; Li, Jian; Meng, Zhi-Yun; Li, Shu-Chun; Li, Zhong-Jun; Lu, Ying-Yuan; Ren, Hong; Lou, Ya-Qing; Lu, Chuang; Dou, Gui-Fang; Zhang, Guo-Liang

2015-01-01

Fructose-based 3-acetyl-2,3-dihydro-1,3,4-oxadiazole (GLB) is a novel antitumor agent and belongs to glycosylated spiro-heterocyclic oxadiazole scaffold derivative. This research first reported a simple, specific, sensitive and stable high performance liquid chromatography -ultraviolet detector (HPLC-UV) method for the quantitative determination of GLB in plasma. In this method, the chromatographic separation was achieved with a reversed phase C18 column. The calibration curve for GLB was linear at 300 nm. The lower limit of quantification was 10 ng/mL. The precision, accuracy and stability of the method were validated adequately. This method was successfully applied to the pharmacokinetic study in rats for detection of GLB after oral administration. Moreover, the structures of parent compound GLB and its two major metabolites M1 and M2 were identified in plasma using an ultra performance liquid chromatography- electrospray ionization-quadrupole-time of flight- mass spectrometry (UPLC-ESI-QTOF-MS) method. Our results indicated that the di-hydroxylation (M1) and hydroxylation (M2) of GLB are the major metabolites. In conclusion, the present study provided valuable information on an analytical method for the determination of GLB and its metabolites in rats, can be used to support further developing of this antitumor agent. PMID:26148672

A validated RP-HPLC method for simultaneous determination of propranolol and valsartan in bulk drug and gel formulation

PubMed Central

Imam, Syed Sarim; Ahad, Abdul; Aqil, Mohammed; Sultana, Yasmin; Ali, Asgar

2013-01-01

Objective: A simple, precise, and stability indicating high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of propranolol hydrochloride and valsartan in pharmaceutical dosage form. Materials and Methods: The method involves the use of easily available inexpensive laboratory reagents. The separation was achieved on Hypersil ODS C-18 column (250*4.6 mm, i.d., 5 μm particle size) with isocratic flow with UV detector. The mobile phase at a flow rate of 1.0 mL/min consisted of acetonitrile, methanol, and 0.01 M disodium hydrogen phosphate (pH 3.5) in the ratio of 50:35:15 v/v. Results: A linear response was observed over the concentration range 5-50 μg/mL of propranolol and the concentration range 4-32 μg/mL of valsartan. Limit of detection and limit of quantitation for propranolol were 0.27 μg/mL and 0.85 μg/mL, and for valsartan were 0.45 μg/mL and 1.39 μg/mL, respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for linearity, accuracy, precision, specificity, robustness. Conclusion: The analysis concluded that the method was selective for simultaneous estimation of propranolol and valsartan can be potentially used for the estimation of these drugs in combined dosage form. PMID:23559826

A rapid, one step preparation for measuring selected free plus SO2-bound wine carbonyls by HPLC-DAD/MS.

PubMed

Han, Guomin; Wang, Hua; Webb, Michael R; Waterhouse, Andrew L

2015-03-01

Carbonyl compounds are produced during fermentation and chemical oxidation during wine making and aging, and they are important to wine flavor and color stability. Since wine also contains these compounds as α-hydroxysulfonates as a result of their reaction with sulfur dioxide, an alkaline pre-treatment requiring oxygen exclusion has been used to release these bound carbonyls for analysis. By modifying the method to hydrolyze the hydroxysulfonates with heating and acid in the presence of 2,4-dinitrophenylhydrazine (DNPH), the carbonyl compounds are simultaneously and quickly released and derivatized, resulting in a simpler and more rapid method. In addition, the method avoids air exclusion complications during hydrolysis by the addition of sulfur dioxide. The method was optimized for temperature, reaction time, and the concentrations of DNPH, sulfur dioxide and acid. The hydrazones were shown to be stable for 10 h, adequate time for chromatographic analysis by HPLC-DAD/MS. This method is demonstrated for 2-ketoglutaric acid, pyruvic acid, acetoin and acetaldehyde, wine carbonyls of very different reactivities, and it offers good specificity, high recovery and low limits of detection. This new rapid, simple method is demonstrated for the measurement of carbonyl compounds in a range of wines of different ages and grape varieties. Copyright © 2014 Elsevier B.V. All rights reserved.

A method for the measurement of atmospheric HONO based on DNPH derivatization and HPLC analysis

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

Zhou, X.; Qiao, H.; Deng, G.

1999-10-15

A simple measurement technique was developed for atmospheric HONO based on aqueous scrubbing using a coil sampler followed by 2,4-dinitrophenylhydrazine (DNPH) derivatization and high-performance liquid chromatographic (HPLC) analysis. Quantitative sampling efficiency was obtained using a 1 mM phosphate buffer, pH 7.0, as the scrubbing solution at a gas sampling flow rate of 2 L min{sup {minus}1} and a liquid flow rate of 0.24 mL min{sup {minus}1}. Derivation of the scrubbed nitrous acid by DNPH was fast and was completed within 5 min in a derivatization medium containing 300 {micro}M DNPH and 8 mM HCI at 45 C. The azide derivativemore » was separated from DNPH reagent and carbonyl derivatives by reverse-phase HPLC and was detected with an UV detector at 309 nm. The detection limit is {le}5 pptv and may be lowered to 1 pptv with further DNPH purification. Interferences from NO, NO{sub 2} PAN, O{sub 3}, HNO{sub 3}, and HCHO were studied and found to be negligible. Ambient HONO concentration was measured simultaneously in downtown Albany, NY, by this method and by an ion chromatographic technique after sampling using a fritted bubbler. The results, from 70 pptv during the day to 1.7 ppbv in the early morning, were in very good agreement from the two techniques, within {+-} 20%.« less

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method.

PubMed

Zhang, Pei-Ting; Pan, Bi-Yan; Liao, Qiong-Feng; Yao, Mei-Cun; Xu, Xin-Jun; Wan, Jin-Zhi; Liu, Dan; Xie, Zhi-Yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method

PubMed Central

Zhang, Pei-ting; Pan, Bi-yan; Liao, Qiong-feng; Yao, Mei-cun; Xu, Xin-jun; Wan, Jin-zhi; Liu, Dan; Xie, Zhi-yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth. PMID:23738236

HPLC method for the pharmacokinetics and tissue distribution of taspine solution and taspine liposome after intravenous administrations to mice.

PubMed

Lu, Wen; He, Lang Chong; Zeng, Xian-Ming

2008-01-07

Taspine is a bioactive aporphine alkaloid, which has many potent pharmacological effects. A simple, rapid HPLC method to quantify taspine in mouse plasma and tissue homogenates containing either taspine solution or liposome was developed and validated. Sample preparation was achieved by liquid-liquid extraction with acetoacetate. Taspine was separated on a C(18) reversed phase HPLC column, and quantified by its absorbance at 245 nm. The pharmacokinetics and tissue distribution after intravenous administrations of taspine liposome (L-Ta) and taspine solution (Ta) to ICR mice were then compared. The area under the plasma concentration-time curve (AUC) was higher for L-Ta than for Ta. In contrast, the total body clearance (CL), apparent volume of distribution V(c) and plasma half-life for the distribution (t(1/2 alpha)) and elimination phase (t(1/2 beta)) were lower for L-Ta, in comparison to the respective parameter of Ta. The AUC values were higher in the lung than in other organs for both L-Ta and Ta. The AUC in the spleen, kidney and liver of L-Ta were higher than those of Ta. However, the heart and brain AUC of Ta was higher than that of L-Ta. It can thus be concluded that incorporation into liposomes prolonged taspine retention within the systemic circulation, increased its distribution to the spleen and liver but reduced its distribution to the heart and brain.

A novel approach for quantitation of nonderivatized sialic acid in protein therapeutics using hydrophilic interaction chromatographic separation and nano quantity analyte detection.

PubMed

Chemmalil, Letha; Suravajjala, Sreekanth; See, Kate; Jordan, Eric; Furtado, Marsha; Sun, Chong; Hosselet, Stephen

2015-01-01

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Tangential Flow Filtration of Hemoglobin

PubMed Central

Sun, Guoyong; Harris, David R.

2009-01-01

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells (bRBCs and hRBCs, respectively) via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P50), and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α-and β-globin chains. Taken together, our results demonstrate that HPLC grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers (HBOCs). PMID:19224583

Quantitation of the immunological adjuvants, monophosphoryl lipid A and Quil A in poly (lactic-co-glycolic acid) nanoparticles using high performance liquid chromatography with evaporative light scattering detection.

PubMed

Bobbala, Sharan; McDowell, Arlene; Hook, Sarah

2015-01-15

Monophosphoryl lipid A (MPL) and Quil A are two immunological adjuvants commonly used in vaccines. At present no simple, validated methods for the quantification of Quil A and MPL have been previously reported therefore the aim of the current study was to develop a simple, fast and validated method to quantify MPL and Quil A using high performance liquid chromatography evaporative light scattering detection (HPLC-ELSD). The HPLC-ELSD technique was carried out using a ZORBAX Eclipse XDB-C8 column (2.1×50 mm; particle size, 3.5 μm) in an isocratic elution mode at 25 °C. MPL was eluted at a retention time of 1.8 min with methanol-water as the mobile phase and a detector temperature of 75 °C. Quil A was resolved as three peaks with retention times of 4.1, 5.5 and 6.4 min with a detector temperature of 30 °C and with water-acetonitrile and 0.01% formic acid as the mobile phase. The nebulizer pressure and gain were set at 3.5 bar and 10, respectively. Calibration curves plotted for both the adjuvants had an R(2)>0.997. Accuracy, intra- and inter-day precision were within the accepted limits. The limit of detection for MPL and Quil A were calculated as 1.343 and 2.06 μg/mL, respectively. The limit of quantification was 2.445 for MPL and 8.97 μg/mL for Quil A. This analytical method was used to quantify the entrapment and in vitro release of MPL and Quil A in a poly lactic-co-glycolic acid (PLGA) nanoparticle vaccine. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of some cholesterol-lowering drugs in their binary mixture by novel spectrophotometric methods

NASA Astrophysics Data System (ADS)

Lotfy, Hayam Mahmoud; Hegazy, Maha Abdel Monem

2013-09-01

Four simple, specific, accurate and precise spectrophotometric methods manipulating ratio spectra were developed and validated for simultaneous determination of simvastatin (SM) and ezetimibe (EZ) namely; extended ratio subtraction (EXRSM), simultaneous ratio subtraction (SRSM), ratio difference (RDSM) and absorption factor (AFM). The proposed spectrophotometric procedures do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined, and the methods were validated and the specificity was assessed by analyzing synthetic mixtures containing the cited drugs. The four methods were applied for the determination of the cited drugs in tablets and the obtained results were statistically compared with each other and with those of a reported HPLC method. The comparison showed that there is no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.

PubMed

Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

2013-12-01

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. Copyright © 2013 Elsevier B.V. All rights reserved.

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase

PubMed Central

Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

2013-01-01

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25 °C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/Vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis–Menten kinetics showed that the Km values of UTL-5g were 2.07 mM with PLE and 0.37 mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. PMID:24126042

Comparison of UPLC and HPLC methods for determination of vitamin C.

PubMed

Klimczak, Inga; Gliszczyńska-Świgło, Anna

2015-05-15

Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehydroascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparations were compared. Both methods are rapid: total time of analysis was 15 and 6 min for HPLC and UPLC methods, respectively. The methods were validated in terms of linearity, instrument precision, limits of detection (LOD) and quantification (LOQ), accuracy and recovery. Intra- and inter-day instrument precisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% for UPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively (HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049 μg/mL for HPLC and 0.024 μg/mL for UPLC while LOQs were 0.149 and 0.073 μg/mL for HPLC and UPLC, respectively. These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in pharmaceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and consumes less eluent. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simple Approach for the Rapid Detection of Alternariol in Pear Fruit by Surface-Enhanced Raman Scattering with Pyridine-Modified Silver Nanoparticles.

PubMed

Pan, Ting-Tiao; Sun, Da-Wen; Pu, Hongbin; Wei, Qingyi

2018-03-07

A simple method based on surface-enhanced Raman scattering (SERS) was developed for the rapid determination of alternariol (AOH) in pear fruits using an easily prepared silver-nanoparticle (AgNP) substrate. The AgNP substrate was modified by pyridine to circumvent the weak affinity of the AOH molecules to the silver surface and to improve the sensitivity of detection. Quantitative analysis was performed in AOH solutions at concentrations ranging from 3.16 to 316.0 μg/L, and the limit of detection was 1.30 μg/L. The novel method was also applied to the detection of AOH residues in pear fruits purchased from the market and in pear fruits that were artificially inoculated with Alternaria alternata. AOH was not found in any of the fresh fruit, whereas it resided in the rotten and inoculated fruits. Finally, the SERS method was cross validated against HPLC. It was revealed that the SERS method has great potential utility in the rapid detection of AOH in pear fruits and other agricultural products.

Quantification of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

PubMed

Arning, Erland; Bottiglieri, Teodoro

2016-01-01

We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Determination of total GABA in CSF requires additional sample preparation in order to hydrolyze all the bound GABA in the sample to the free form. This requires hydrolyzing the sample by boiling in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90 % acetonitrile/0.1 % formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 nM to 1000 nM and 0.63 μM to 80 μM for free and total GABA, respectively.

Optimized HPLC method for analysis of polar and nonpolar heterocyclic amines in cooked meat products.

PubMed

Gibis, Monika

2009-01-01

A simple, precise, and specific column high-performance liquid chromatographic (HPLC) method with UV absorption diode array and fluorescence detection has been developed by optimizing a previously described method for the simultaneous quantification of 15 polar and nonpolar heterocyclic amines (HAs) in fried meat products. The HPLC determination could be improved due to the application of a silica-based reversed-phase column with octadecyl groups (TSK-gel Super ODS) and a particle size of 2 microm. The separation of HAs in the complex meat matrix was performed with a 21 min mobile phase gradient. The method was validated for instrumental precision, repeatability, and selectivity and compared with a previously published method. After liquid adsorption of the basic sample mixture on diatomaceous earth, HAs were extracted with ethyl acetate. For cleanup, solid-phase extraction (silica propylsulfonic acid and octadecyl cartridges) and different washing steps were applied. Both nonpolar and polar HAs were determined in one fraction. The calibration curves of all HAs were linear for the applied detection system (correlation coefficient = 0.990-0.995). The recoveries, with the exception of 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), were between 42 and 98% from meat samples spiked in a range of 1.5 to 3.3 ng/g for fluorescence-active and 4.3 to 8 ng/g for UV-active HAs. For quantification of HAs, the standard addition method was used for adjustment of different characteristics of HAs in the extraction. In fried meat samples (chicken breast and beef patties), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline(4,8-DiMelQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), norharmane, and harmane were found in a concentration range of 0.02 to 14.3 ng/g.

Determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

USGS Publications Warehouse

Hostetler, K.A.; Thurman, E.M.

2000-01-01

Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: alachlor ethanesulfonic acid (ESA); alachlor oxanilic acid; acetochlor ESA; acetochlor oxanilic acid; metolachlor ESA; and metolachlor oxanilic acid. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The average HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.5 and 2.0 ??g/l ranged from 84 to 112%, with relative standard deviations of 18% or less. The average HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.2 and 2.0 ??g/l ranged from 81 to 118%, with relative standard deviations of 20% or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 ??g/l, whereas the LOQ using the HPLC/MS method was at 0.05 ??g/l. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water. Copyright (C) 2000 Elsevier Science B.V.

Development and validation of a single robust HPLC method for the characterization of a pharmaceutical starting material and impurities from three suppliers using three separate synthetic routes.

PubMed

Sheldon, E M; Downar, J B

2000-08-15

Novel approaches to the development of analytical procedures for monitoring incoming starting material in support of chemical/pharmaceutical processes are described. High technology solutions were utilized for timely process development and preparation of high quality clinical supplies. A single robust HPLC method was developed and characterized for the analysis of the key starting material from three suppliers. Each supplier used a different process for the preparation of this material and, therefore, each suppliers' material exhibited a unique impurity profile. The HPLC method utilized standard techniques acceptable for release testing in a QC/manufacturing environment. An automated experimental design protocol was used to characterize the robustness of the HPLC method. The method was evaluated for linearity, limit of quantitation, solution stability, and precision of replicate injections. An LC-MS method that emulated the release HPLC method was developed and the identities of impurities were mapped between the two methods.

Evaluation of a novel semi-automated HPLC procedure for whole blood cyclosporin A confirms equivalence to adjusted monoclonal values from Abbott TDx.

PubMed

Roberts, Norman B; Dutton, John; Higgins, Gerald; Allars, Lesley

2005-01-01

The problem in the measurement of cyclosporin (CyA) is that the widely used immuno-based assays suffer from interference by metabolites present in unpredictable excess. To resolve this, the consensus view has been to develop more specific and robust procedures for the measurement of CyA alone in order to give values similar to those obtained by HPLC. We developed an alternative strategy based on Abbott poly- and monoclonal assays to derive an adjusted monoclonal value as an equivalent measurement to HPLC. We have now evaluated a recently developed semi-automated HPLC procedure and used it to test the validity of the adjusted monoclonal value. The automated HPLC procedure with online clean-up was optimised for the separation of CyA and internal standard CyD. The assay was simple to use, precise and gave good recovery of cyclosporin from whole blood. Comparisons with the more specific immunoassays Abbott AxSym and EMIT showed close agreement, whereas Abbott monoclonal values indicated up to 20% positive bias. In contrast, the adjusted monoclonal values gave good agreement with HPLC. Data obtained from HPLC linked to tandem mass spectrometry (MS) indicated closer agreement with Abbott monoclonal values than expected, suggesting some positive bias with MS. The benefit of using an adjusted monoclonal value is that a result equivalent to HPLC is obtained, as well as an indication of the concentration of metabolites from the Abbott polyclonal measurement.

HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora

PubMed Central

Yen, Feng-Lin; Chen, Pei-Chun; Wang, Moo-Chin; Lin, Chun-Nan; Lee, Chiang-Wen; Ko, Horng-Huey

2014-01-01

Phyla nodiflora is a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of this plant is necessary. In the present study, ten compounds, namely, 3,7,4′,5′-tetrahydroxy-3′-methoxyflavone (1), nodifloretin (2), 4′-hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4′-tetrahydroxy-3′-methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), and β-sitosterol were isolated from the methanolic extract of the aerial part of P. nodiflora (PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds 4, 5, and 7 showed strong antioxidant activity. To control the quality of P. nodiflora, a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4) and eupafolin (7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM. PMID:25140335

Optimization and validation of high-performance liquid chromatography method for analyzing 25-desacetyl rifampicin in human urine

NASA Astrophysics Data System (ADS)

Lily; Laila, L.; Prasetyo, B. E.

2018-03-01

A selective, reproducibility, effective, sensitive, simple and fast High-Performance Liquid Chromatography (HPLC) was developed, optimized and validated to analyze 25-Desacetyl Rifampicin (25-DR) in human urine which is from tuberculosis patient. The separation was performed by HPLC Agilent Technologies with column Agilent Eclipse XDB- Ci8 and amobile phase of 65:35 v/v methanol: 0.01 M sodium phosphate buffer pH 5.2, at 254 nm and flow rate of 0.8ml/min. The mean retention time was 3.016minutes. The method was linear from 2–10μg/ml 25-DR with a correlation coefficient of 0.9978. Standard deviation, relative standard deviation and coefficient variation of 2, 6, 10μg/ml 25-DR were 0-0.0829, 03.1752, 0-0.0317%, respectively. The recovery of 5, 7, 9μg/ml25-DR was 80.8661, 91.3480 and 111.1457%, respectively. Limits of detection (LoD) and quantification (LoQ) were 0.51 and 1.7μg/ml, respectively. The method has fulfilled the validity guidelines of the International Conference on Harmonization (ICH) bioanalytical method which includes parameters of specificity, linearity, precision, accuracy, LoD, and LoQ. The developed method is suitable for pharmacokinetic analysis of various concentrations of 25-DR in human urine.

Sensitive determination of estrogens in environmental waters treated with polymeric ionic liquid-based stir cake sorptive extraction and liquid chromatographic analysis.

PubMed

Chen, Lei; Mei, Meng; Huang, Xiaojia; Yuan, Dongxing

2016-05-15

A simple, sensitive and environmentally friendly method using polymeric ionic liquid-based stir cake sorptive extraction followed by high performance liquid chromatography with diode array detection (HPLC/DAD) has been developed for efficient quantification of six selected estrogens in environmental waters. To extract trace estrogens effectively, a poly (1-ally-3-vinylimidazolium chloride-co-ethylene dimethacrylate) monolithic cake was prepared and used as the sorbent of stir cake sorptive extraction (SCSE). The effects of preparation conditions of sorbent and extraction parameters of SCSE for estrogens were investigated and optimized. Under optimal conditions, the developed method showed satisfactory analytical performance for targeted analytes. Low limits of detection (S/N=3) and quantification limits (S/N=10) were achieved within the range of 0.024-0.057 µg/L and 0.08-0.19 µg/L, respectively. Good linearity of method was obtained for analytes with the correlation coefficients (R(2)) above 0.99. At the same time, satisfactory method repeatability and reproducibility was achieved in terms of intra- and inter-day precisions, respectively. Finally, the established SCSE-HPLC/DAD method was successfully applied for the determination of estrogens in different environmental water samples. Recoveries obtained for the determination of estrogens in spiked samples ranged from 71.2% to 108%, with RSDs below 10% in all cases. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

PubMed

Gupta, Shweta; Kesarla, Rajesh; Chotai, Narendra; Omri, Abdelwahab

2017-01-01

Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed-phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1-300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard) were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system.

PubMed

Yasuda, Makoto; Ota, Tatsuhiro; Morikawa, Atsushi; Mawatari, Ken-ichi; Fukuuchi, Tomoko; Yamaoka, Noriko; Kaneko, Kiyoko; Nakagomi, Kazuya

2013-09-01

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

Simultaneous analysis of insect repellent DEET, sunscreen oxybenzone and five relevant metabolites by reversed-phase HPLC with UV detection: application to an in vivo study in a piglet model.

PubMed

Kasichayanula, Sreeneeranj; House, James D; Wang, Tao; Gu, Xiaochen

2005-08-05

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

PubMed

Arning, Erland; Bottiglieri, Teodoro

2016-01-01

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.

[HPLC Specific Fingerprint of Alcohol Extract of Andrographis paniculata].

PubMed

Liu, Fei-fei; Fan, Chun-lin; Huang, Xiao-jun; Wang, Gui-yang; Wang, Ying; Ye, Wen-cai

2015-07-01

To establish the specific fingerprint of alcohol extract of Andrographis paniculata by HPLC. The analysis was performed on Cosmosil 5C18 -MS-II (250 mm x 4. 6 mm, 5 µm) column, with gradient phase consisting of acetonitrile and water at the flow rate of 1. 0 mL/min. The UV detection wavelength was set at 225 nm, and column temperature was 30 °C. The specific fingerprint chromatogram was established and seven common peaks were identified by comparison with the reference standards and LC-MS. The relative retention times were 1. 00 (No. 1, andrographolide), 1. 04 (No. 2, deoxyandrographoside), 1. 07 (No. 3, isoandrographolide), 1. 10 (No. 4, 14-deoxy-11, 12-didehydroandrographoside), 1. 50 (No. 5, neoandrographolide), 1. 75 ( No. 6, deoxyandrographolide) and 1. 79 (No. 7, dehydroandrographolide). The method is simple and reproducible with high precision, which can provide the basis for the quality control and evaluation of alcohol extract of Andrographis paniculata and its preparations.

Analysis of wax esters by silver-ion high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Vrkoslav, Vladimír; Urbanová, Klára; Háková, Matina; Cvačka, Josef

2013-08-09

Wax esters (WEs), esters of long-chain fatty acids and long-chain alcohols, were analysed by Ag-HPLC/APCI-MS/MS. Two ChromSpher Lipids columns connected in series (a total length of 50cm) and hexane-2-propanol-acetonitrile mobile phases were used to achieve good separation of the molecular species. The chromatographic behaviour of WEs was studied under optimised conditions: retention increased with the number of double bonds and with the temperature (15-35°C); retention times were affected by the double-bond position, trans isomers eluted earlier than cis isomers, and the WEs were partially separated depending on the aliphatic-chain length. The WEs provided simple APCI spectra with [M+H](+) ions, the MS/MS spectra showed fragments, which allowed their identification. The method was applied for an analysis of the WE mixtures from jojoba oil and human hair and the results were compared with analogous data from an optimised RP-HPLC system. Copyright © 2013 Elsevier B.V. All rights reserved.

Determination of free and esterified carotenoid composition in rose hip fruit by HPLC-DAD-APCI(+)-MS.

PubMed

Zhong, Lijie; Gustavsson, Karl-Erik; Oredsson, Stina; Głąb, Bartosz; Yilmaz, Jenny Lindberg; Olsson, Marie E

2016-11-01

Rose hip fruit, which contains high concentration of carotenoids is commonly used for different food products in Europe and it is considered to have medical properties. In this study, a simple, rapid and efficient HPLC-DAD-APCI(+)-MS method was developed and applied to identify and quantify the carotenoids in rose hip fruit of four rose species, including both unsaponified and saponified extract. In the unsaponified extract 23 carotenoid esters were detected, in which either rubixanthin ester or violaxanthin ester was the dominant component of the ester composition. In the saponified extract 21 carotenoids, including 11 xanthophylls and 10 carotenes were detected. This is the first time the total carotenoid composition, including the carotenoid esters in rose hip fruit were identified and quantified. This work reveals the potential of rose hip fruit to be utilized as a healthy dietary material and give chemical information for the possible future development in the pharmacology field. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrolyzable tannins with the hexahydroxydiphenoyl unit and the m-depsidic link: HPLC-DAD-MS identification and model synthesis.

PubMed

Arapitsas, Panagiotis; Menichetti, Stefano; Vincieri, Franco F; Romani, Annalisa

2007-01-10

This study was designed to develop efficient analytical tools for the difficult HPLC-DAD-MS identification of hydrolyzable tannins in natural tissue extracts. Throughout the study of the spectroscopic characteristics of properly synthesized stereodefined standards, it was observed that the UV-vis spectra of compounds with the m-depsidic link showed a characteristic shoulder at 300 nm, consistent with the simple glucogalloyl esters, whereas compounds with the hexahydroxydiphenoyl (HHDP) unit gave a diagnostic fragmentation pattern, caused by a spontaneous lactonization in the mass spectrometer. These observations were confirmed by HPLC-DAD-MS analyses of tannic acid and raspberry extracts, which are rich in hydrolyzable tannins with the m-depsidic link and the HHDP unit, respectively.

[Determination of acacetin in Xiangjuganmao Keli (no sweet) by HPLC].

PubMed

Bian, Jia-Hong; Qian, Kun; Xu, Xiang; Shen, Jun

2006-11-01

To establish a method for the determination of acacetin in Xiangjuganmao Keli (no sweet). Acacetin in powdered herb was extracted by ultrasonator with methanol and was hydrolyzed with hydrochloric acid. Separation was accomplished on an ODS reversed phase column (5 microm, 4.6 x 250 mm) with a mobile phase of methanol-water-acetic acid(350: 150: 2). The detective wavelength was at 340 nm. The method was accurate, the results were stable and reproducible. The linear range of calibration cure was within the concentration of 2.00 - 10.00 microg/ml (r = 0.9998). The average extraction recovery was 99.9% (n = 6), RSD = 0.41% (n = 6). The method is simple, convenient, sensitive, and reproducible for quality control of Xiangjuganmao Keli (no sweet).

Spectroscopic characterization and quantitative determination of atorvastatin calcium impurities by novel HPLC method

NASA Astrophysics Data System (ADS)

Gupta, Lokesh Kumar

2012-11-01

Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant.

PubMed

Gaber, Yasser; Akerman, Cecilia Orellana; Hatti-Kaul, Rajni

2014-01-01

N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant

PubMed Central

2014-01-01

Background N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Results Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Conclusion Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation. PMID:24914404

Simultaneous determination of chromones and coumarins in Radix Saposhnikoviae by high performance liquid chromatography with diode array and tandem mass detectors.

PubMed

Kim, Min Kyung; Yang, Dong-Hyug; Jung, Mihye; Jung, Eun Ha; Eom, Han Young; Suh, Joon Hyuk; Min, Jung Won; Kim, Unyong; Min, Hyeyoung; Kim, Jinwoong; Han, Sang Beom

2011-09-16

Methods using high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) were developed and validated for the simultaneous determination of 5 chromones and 6 coumarins: prim-O-glucosylcimifugin (1), cimifugin (2), nodakenin (3), 4'-O-β-d-glucosyl-5-O-methylvisamminol (4), sec-O-glucosylhamaudol (5), psoralen (6), bergapten (7), imperatorin (8), phellopterin (9), 3'-O-angeloylhamaudol (10) and anomalin (11), in Radix Saposhnikoviae. The separation conditions for HPLC-DAD were optimized using an Ascentis Express C18 (4.6 mm×100 mm, 2.7 μm particle size) fused-core column. The mobile phase was composed of 10% aqueous acetonitrile (A) and 90% acetonitrile (B) and the elution was performed under a gradient mode at a flow rate of 1.0 mL/min. The detection wavelength was set at 300 nm. The HPLC-DAD method yielded a base line separation of the 11 components in 50% methanol extract of Radix Saposhnikoviae with no interfering peaks detected. The HPLC-DAD method was validated in terms of linearity, accuracy and precision (intra- and inter-day), limit of quantification (LOQ), recovery, and robustness. Specific determination of the 11 components was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. This HPLC-MS/MS method was also validated by determining the linearity, limit of quantification, accuracy, and precision. Quantification of the 11 components in 51 commercial Radix Saposhnikoviae samples was successfully performed using the developed HPLC-DAD method. The identity, batch-to-batch consistency, and authenticity of Radix Saposhnikoviae were successfully monitored by the proposed HPLC-DAD and HPLC-MS/MS methods. Copyright © 2011 Elsevier B.V. All rights reserved.

76 FR 55329 - Receipt of Several Pesticide Petitions Filed for Residues of Pesticide Chemicals in or on Various...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-07

... (HPLC) method was validated for determination of dinotefuran, DN and UF in or on tomatoes and peppers... quantified after HPLC separation by tandem mass spectrometry (MS/MS) detection. Contact: Sidney Jackson, (703..., group 9 at 0.03 ppm. Adequate analytical methods (HPLC-fluorescence methods) are available for...

Quality Analysis of Chlorogenic Acid and Hyperoside in Crataegi fructus

PubMed Central

Weon, Jin Bae; Jung, Youn Sik; Ma, Choong Je

2016-01-01

Background: Crataegi fructus is a herbal medicine for strong stomach, sterilization, and alcohol detoxification. Chlorogenic acid and hyperoside are the major compounds in Crataegi fructus. Objective: In this study, we established novel high-performance liquid chromatography (HPLC)-diode array detection analysis method of chlorogenic acid and hyperoside for quality control of Crataegi fructus. Materials and Methods: HPLC analysis was achieved on a reverse-phase C18 column (5 μm, 4.6 mm × 250 mm) using water and acetonitrile as mobile phase with gradient system. The method was validated for linearity, precision, and accuracy. About 31 batches of Crataegi fructus samples collected from Korea and China were analyzed by using HPLC fingerprint of developed HPLC method. Then, the contents of chlorogenic acid and hyperoside were compared for quality evaluation of Crataegi fructus. Results: The results have shown that the average contents (w/w %) of chlorogenic acid and hyperoside in Crataegi fructus collected from Korea were 0.0438% and 0.0416%, respectively, and the average contents (w/w %) of 0.0399% and 0.0325%, respectively. Conclusion: In conclusion, established HPLC analysis method was stable and could provide efficient quality evaluation for monitoring of commercial Crataegi fructus. SUMMARY Quantitative analysis method of chlorogenic acid and hyperoside in Crataegi fructus is developed by high.performance liquid chromatography.(HPLC).diode array detectionEstablished HPLC analysis method is validated with linearity, precision, and accuracyThe developed method was successfully applied for quantitative analysis of Crataegi fructus sample collected from Korea and China. Abbreviations used: HPLC: High-performance liquid chromatography, GC: Gas chromatography, MS: Mass spectrometer, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation, RRT: Relative retention time, RPA: Relation peak area. PMID:27076744

Therapeutic drug monitoring of beta-lactam antibiotics - Influence of sample stability on the analysis of piperacillin, meropenem, ceftazidime and flucloxacillin by HPLC-UV.

PubMed

Pinder, Nadine; Brenner, Thorsten; Swoboda, Stefanie; Weigand, Markus A; Hoppe-Tichy, Torsten

2017-09-05

Therapeutic drug monitoring (TDM) is a useful tool to optimize antibiotic therapy. Increasing interest in alternative dosing strategies of beta-lactam antibiotics, e.g. continuous or prolonged infusion, require a feasible analytical method for quantification of these antimicrobial agents. However, pre-analytical issues including sample handling and stability are to be considered to provide valuable analytical results. For the simultaneous determination of piperacillin, meropenem, ceftazidime and flucloxacillin, a high performance liquid chromatography (HPLC) method including protein precipitation was established utilizing ertapenem as internal standard. Long-term stability of stock solutions and plasma samples were monitored. Furthermore, whole blood stability of the analytes in heparinized blood tubes was investigated comparing storage under ambient conditions and 2-8°C. A calibration range of 5-200μg/ml (piperacillin, ceftazidime, flucloxacillin) and 2-200μg/ml (meropenem) was linear with r 2 >0.999, precision and inaccuracy were <9% and <11%, respectively. The successfully validated HPLC assay was applied to clinical samples and stability investigations. At -80°C, plasma samples were stable for 9 months (piperacillin, meropenem) or 13 months (ceftazidime, flucloxacillin). Concentrations of the four beta-lactam antibiotics in whole blood tubes were found to remain within specifications for 8h when stored at 2-8°C but not at room temperature. The presented method is a rapid and simple option for routine TDM of piperacillin, meropenem, ceftazidime and flucloxacillin. Whereas long-term storage of beta-lactam samples at -80°C is possible for at least 9 months, whole blood tubes are recommended to be kept refrigerated until analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

PubMed

Chandra, A; Rana, J; Li, Y

2001-08-01

A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.

Simultaneous determination of six bioactive compounds in Evodiae Fructus by high-performance liquid chromatography with diode array detection.

PubMed

Tang, Xiaolong; Huang, Zhifang; Chen, Yan; Liu, Yunhua; Liu, Yuhong; Zhao, Junning; Yi, Jinhai

2014-02-01

A simple and reliable high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for the simultaneous determination of six bioactive components, rutaevine, limonin, evodiamine, rutaecarpine, N-formyldihydrorutaecarpine and dihydroevocarpine, in the traditional Chinese medicine Evodiae Fructus (Wuzhuyu in Chinese). HPLC separation was conducted on an Agilent Eclipse C18 column (4.6 × 150 mm, 5 µm) at 35°C with a mixture of mobile phase A [tetrahydrofuran-0.02% phosphoric acid (16 : 35)] and mobile phase B (acetonitrile) (gradient elution as follows: 0 min, 22% B; 23 min, 22% B; 24 min, 75% B) at a flow rate of 1 mL/min, and the DAD detection wavelength was set at 220 nm. A linear relationship within the range of investigated concentrations was observed for the six compounds, with correlation coefficients greater than 0.999. The average recovery yields of the six compounds ranged from 98.39 to 104.96%. The HPLC-DAD method was validated by its repeatability [relative standard deviation (RSD) < 2.0%] and intra-day and inter-day precision (RSD < 2.0%). The method was successfully applied to the simultaneous determination of the six previously mentioned components in Evodiae Fructus. It is the first report of a simultaneous qualitative and quantitative analysis for three classes of bioactive components in Wuzhuyu, including the indolequinazoline alkaloids, quinolone alkaloid and limonoids. Based on these results, it is suggested, for possible future revision of the Chinese Pharmacopoeia, that the total contents of evodiamine and rutaecarpine are not less than 0.15% and the total contents of rutaevine and limonin are not less than 0.50%.

Spectrophotometric method for quantitative determination of total anthocyanins and quality characteristics of roselle (Hibiscus sabdariffa).

PubMed

Sukwattanasinit, Tasamaporn; Burana-Osot, Jankana; Sotanaphun, Uthai

2007-11-01

A simple, rapid and cost-saving method for the determination of total anthocyanins in roselle has been developed. The method was based on pH-differential spectrophotometry. The calibration curve of the major anthocyanin in roselle, delphinidin 3-sambubioside (Dp-3-sam), was constructed by using methyl orange and their correlation factor. The reliability of this developed method was comparable to the direct method using standard Dp-3-sam and the HPLC method. Quality characteristics of roselle produced in Thailand were also reported. Its physical quality met the required specifications. The overall chemical quality was herein surveyed for the first time and it was found to be the important parameter corresponded to the commercial grading of roselle. Total contents of anthocyanins and phenolics were proportional to the antiradical capacity.

Different spectrophotometric methods applied for the analysis of simeprevir in the presence of its oxidative degradation product: Acomparative study

NASA Astrophysics Data System (ADS)

Attia, Khalid A. M.; El-Abasawi, Nasr M.; El-Olemy, Ahmed; Serag, Ahmed

2018-02-01

Five simple spectrophotometric methods were developed for the determination of simeprevir in the presence of its oxidative degradation product namely, ratio difference, mean centering, derivative ratio using the Savitsky-Golay filters, second derivative and continuous wavelet transform. These methods are linear in the range of 2.5-40 μg/mL and validated according to the ICH guidelines. The obtained results of accuracy, repeatability and precision were found to be within the acceptable limits. The specificity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. Furthermore, these methods were statistically comparable to RP-HPLC method and good results were obtained. So, they can be used for the routine analysis of simeprevir in quality-control laboratories.

[Determination of carbendazim and thiabendazole in tomatoes by solid-phase microextraction coupled with high performance liquid chromatography and fluorescence detection].

PubMed

Hu, Yanxue; Yang, Xiumin; Wang, Zhi; Wang, Chun; Zhao, Jin

2005-11-01

A novel method for the determination of carbendazim (MBC) and thiabendazole (TBZ) in tomatoes by solid-phase microextraction (SPME) coupled with high performance liquid chromatography (HPLC) and fluorescence detection was developed. The experimental conditions of SPME, including extraction fiber, extraction time, extraction temperature, desorption time, desorption solvent, desorption mode, pH value, organic solvent and ionic strength, and HPLC conditions were optimized. The SPME for MBC and TBZ was performed on a 65 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre for 50 min at room temperature with the solution being stirred at 1 100 r/min. The florescence detection was made at 315 nm with excitation wavelength at 280 nm. The method is linear for MBC and TBZ over the range assayed from 0.01 to 1.0 mg/kg tomatoes with the detection limits of 0.003 mg/kg and 0. 001 mg/kg and the correlation coefficients of 0.995 8 and 0.996 7, respectively. The average recoveries for MBC and TBZ were 83.5% and 85.6% with the relative standard deviations (RSDs) of 6.5% and 3.8%, respectively. The method is fast, simple, sensitive, solvent-free and suitable for the determination of MBC and TBZ in tomatoes.

Quantitation of zolpidem in biological fluids by electro-driven microextraction combined with HPLC-UV analysis.

PubMed

Yaripour, Saeid; Mohammadi, Ali; Esfanjani, Isa; Walker, Roderick B; Nojavan, Saeed

2018-01-01

In this study, for the first time, an electro-driven microextraction method named electromembrane extraction combined with a simple high performance liquid chromatography and ultraviolet detection was developed and validated for the quantitation of zolpidem in biological samples. Parameters influencing electromembrane extraction were evaluated and optimized. The membrane consisted of 2-ethylhexanol immobilized in the pores of a hollow fiber. As a driving force, a 150 V electric field was applied to facilitate the analyte migration from the sample matrix to an acceptor solution through a supported liquid membrane. The pHs of donor and acceptor solutions were optimized to 6.0 and 2.0, respectively. The enrichment factor was obtained >75 within 15 minutes. The effect of carbon nanotubes (as solid nano-sorbents) on the membrane performance and EME efficiency was evaluated. The method was linear over the range of 10-1000 ng/mL for zolpidem (R 2 >0.9991) with repeatability ( %RSD) between 0.3 % and 7.3 % ( n = 3). The limits of detection and quantitation were 3 and 10 ng/mL, respectively. The sensitivity of HPLC-UV for the determination of zolpidem was enhanced by electromembrane extraction. Finally, the method was employed for the quantitation of zolpidem in biological samples with relative recoveries in the range of 60-79 %.

Quantitation of zolpidem in biological fluids by electro-driven microextraction combined with HPLC-UV analysis

PubMed Central

Yaripour, Saeid; Mohammadi, Ali; Esfanjani, Isa; Walker, Roderick B.; Nojavan, Saeed

2018-01-01

In this study, for the first time, an electro-driven microextraction method named electromembrane extraction combined with a simple high performance liquid chromatography and ultraviolet detection was developed and validated for the quantitation of zolpidem in biological samples. Parameters influencing electromembrane extraction were evaluated and optimized. The membrane consisted of 2-ethylhexanol immobilized in the pores of a hollow fiber. As a driving force, a 150 V electric field was applied to facilitate the analyte migration from the sample matrix to an acceptor solution through a supported liquid membrane. The pHs of donor and acceptor solutions were optimized to 6.0 and 2.0, respectively. The enrichment factor was obtained >75 within 15 minutes. The effect of carbon nanotubes (as solid nano-sorbents) on the membrane performance and EME efficiency was evaluated. The method was linear over the range of 10-1000 ng/mL for zolpidem (R2 >0.9991) with repeatability ( %RSD) between 0.3 % and 7.3 % (n = 3). The limits of detection and quantitation were 3 and 10 ng/mL, respectively. The sensitivity of HPLC-UV for the determination of zolpidem was enhanced by electromembrane extraction. Finally, the method was employed for the quantitation of zolpidem in biological samples with relative recoveries in the range of 60-79 %. PMID:29805344

[Simultaneous Determination of Three Kinds of Effective Constituents in Cannabis Plants by Reversed-phase HPLC].

PubMed

Fu, Q; Shu, Z; Deng, K; Luo, X; Zeng, C G

2016-08-01

To establish a high performance liquid chromatographic （HPLC） method for simultaneous determination of three effective constituents, including tetrahydrocannabinol （THC）, cannabidiol （CBD） and cannabinol （CBN） in Cannabis plants. A C₁₈ column was used in this study, and acetonitrile-phosphate buffer （0.015 mol/L KH₂PO₄） was used as mobile phase at a flow rate of 1.0 mL/min. At a detection wavelength of 220 mm, UV absorption spectra were collected at the wavelength range of 190-400 nm, and the spectra and retention time were counted as qualitative evidence. THC, CBD and CBN could be well separated by this method. Three components had good linear relationship in the range of 0.4-40 μg/mL （ R ²≥0.999 3）. The recoveries were over 87%. The limits of detection were 1.8 ng, 2.0 ng and 1.3 ng, respectively. The relative standard deviation （RSD） were less than 5% for both inter-day and intra-day precisions. Reversed-phase HPLC method is simple, rapid and accurate, and it is suitable for the qualitative and quantitative detection of THC, CBD and CBN in Cannabis plants. Copyright© by the Editorial Department of Journal of Forensic Medicine

Evaluation of physicochemical properties as supporting information on quality control of raw materials and veterinary pharmaceutical formulations.

PubMed

Anacleto, Sara da Silva; Borges, Marcella Matos Cordeiro; de Oliveira, Hanna Leijoto; Vicente, Andressa Reis; de Figueiredo, Eduardo Costa; de Oliveira, Marcone Augusto Leal; Borges, Bárbara Juliana Pinheiro; de Oliveira, Marcelo Antonio; Borges, Warley de Souza; Borges, Keyller Bastos

2018-06-01

This study aimed to show that the physicochemical proprieties obtained by Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG), and scanning electronic microscopy (SEM) can be useful tools for evaluating the quality of active pharmaceutical ingredients (APIs) and pharmaceutical products. In addition, a simple, sensitive, and efficient method employing HPLC-DAD was developed for simultaneous determination of lidocaine (LID), ciprofloxacin (CFX) and enrofloxacin (EFX) in raw materials and in veterinary pharmaceutical formulations. Compounds were separated using a Gemini C 18 (250 mm × 4.6 mm, 5 µm) Phenomenex ® column, at a temperature of 25 °C, with a mobile phase containing 10 mM of phosphoric acid (pH 3.29): acetonitrile (85.7:14.3, v/v) and a flow rate of 1.5 mL/min. Physicochemical characterization by TG, FTIR, and SEM of raw materials of LID, CFX, and EFX provided information useful for the evaluation, differentiation, and qualification of raw materials. Finally, the HPLC method was proved to be useful for evaluation of raw material and finished products, besides satisfying the need for an analytical method that allows simultaneous determination of EFX, CFX, and LID, which can also be extended to other matrices and applications.

Identification and simultaneous quantification of five alkaloids in Piper longum L. by HPLC-ESI-MS(n) and UFLC-ESI-MS/MS and their application to Piper nigrum L.

PubMed

Liu, Hao-Long; Luo, Rong; Chen, Xiao-Qing; Ba, Yin-Ying; Zheng, Li; Guo, Wei-Wei; Wu, Xia

2015-06-15

A simple, effective and suitable UFLC-ESI-MS/MS method was firstly developed to simultaneously determine five characteristic constituents (piperine, piperlonguminine, Δα,β-dihydropiperlonguminine, pellitorine and piperanine) of Piper longum L. The total alkaloids of P. longum L. was prepared. The alkaloid contents of Piper nigrum L. and P. longum L. were compared. The analysis was carried out in multiple reaction monitoring scan mode. The method showed a good specificity, linearity (R(2)>0.995), stability (RSD<2.53%), repeatability (RSD<2.58%), and recovery (90.0-103.5%). The limits of detection and limits of quantification of five alkaloids were in the range of 0.02-0.03 and 0.05-0.10 ng/mL, respectively. The intra- and inter-day precision was less than 9.30% and 9.55%, respectively. The validation results confirmed that the method could simultaneously determine the target alkaloids in the sample. Furthermore, the identities of the alkaloids were verified by HPLC-ESI-MS/MS. Compared with P. nigrum, P. longum had lower piperine content but was enriched in the other four alkaloids. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Simultaneous determination of four common nonprotein nitrogen substances in urine by high performance liquid chromatography].

PubMed

Ma, Yuhua; Huang, Dongqun; Zhang, Rui; Xu, Shiru; Feng, Shun

2013-11-01

A high performance liquid chromatographic (HPLC) method was proposed to simultaneously determine four common nonprotein nitrogen substances, including creatine (Cr), creatinine (Cn), uric acid (Ua) and pseudouridine (Pu) in urine. After proteins being removed by acetone precipitation method, freeze drying and redissolving, the urine samples were analyzed by HPLC. Chromatographic separation was performed on a Waters RP18 Column (150 mm x 4.60 mm, 3.5 microm) in gradient elution mode using 10.0 mmol/L KH2PO4 solution (pH 4.78) and acetonitrile as mobile phases at a flow rate of 0.8 mL/min. The samples were detected at 220 nm. Rapid separation was achieved within 7 min. Under the optimized conditions, good linearities of four common nonprotein nitrogen substances were obtained in the range of 0.1-250 mg/L. The detection limits were 9.31 (Cr), 26.19 (Cn), 4.70 (Ua), an 6.30 (Pu) microg/L and the recoveries were in the range of 81%-111% with the relative standar deviations of 0.23%-2.78% (n = 3). The results demonstrate that this method is simple, rapid and accurate with good reproducibility, and can provide early diagnosis and preliminary judgment for type 2 diabetes mellitus (T2DM) patients with renal damage.

[Simultaneous determination of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid with HPLC].

PubMed

Zhang, Yu-Lin; Li, Yu-Ping

2013-10-01

To establish an HPLC method for determining the contents of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid. Agela Durashell RP-C18 (4. 6 mm x250 mm, 5 microm) was adopted, with acetonitrile-sodium phosphate buffer solution (0. 07 mol L-1 sodium phosphate solution with 17.5 mmol L-1 sodium dodecylsulfate adjusted to pH 6.0 with phosphoric acid solution) (30:70) as the mobile phase. The flow rate was 0. 9 mL min -1, the detection wavelength was 207 nm, and the column temperature was 25 degree C. Scopolamine hydrobromide, atropine sulfate, ephedrine hlvdrochloride and pseudoephedrine hydrochloride showed good linear relations with peak areas within the concentration range of 0. 021 21-1. 060 5 pg (r =0. 999 3) , 0. 011 14-0. 557 microg (r = 0. 999 6) , 0. 200 56-10. 028 microg (r =0. 999 7) and 0.070 33-3. 516 5 gg (r =0. 999 6), respectively, with the average recoveries of 101.9% , 99. 80%, 100. 3%, 100. 2% (n=6). The method was so quick, simple, highly reproducible and specific that it could be used as one of quality control methods of Zhichuanling oral liquid.

Development and Validation of an RP-HPLC Method for the Determination of Vinpocetine and Folic Acid in the Presence of a Vinpocetine Alkaline Degradation Product in Bulk and in Capsule Form.

PubMed

Elkady, Ehab F; Tammam, Marwa H; Mohamed, Ayman A

2017-05-01

An alkaline-forced degradation hydrolytic product of vinpocetine was prepared and characterized by 1H-NMR, FTIR spectroscopy, and MS. Subsequently, a simple, selective, and validated reversed-phase HPLC method was developed for the simultaneous estimation of vinpocetine and folic acid in the presence of a vinpocetine alkaline degradation product. Chromatographic separation was achieved using an isocratic mobile phase consisting of acetonitrile-0.02 M KH2PO4 [containing 0.2% (v/v) triethylamine and adjusted to pH 6 with orthophosphoric acid; (80 + 20, v/v)] at a flow rate of 0.9 mL/min at ambient temperature on a Eurospher II C18 (250 × 4.6 mm, 5 μm) column, with UV detection at 280 nm for folic acid and 230 nm for vinpocetine and its alkaline hydrolytic product. Linearity, accuracy, and precision were found to be acceptable over a concentration range of 12.5-200 μg/mL for vinpocetine and 1-16 μg/mL for folic acid. The proposed method was successfully applied for the determination of both drugs and a vinpocetine hydrolysis product in a laboratory-prepared mixture and in a capsule containing both drugs.

Rapid determination of polycyclic aromatic hydrocarbons in rainwater by liquid-liquid microextraction and LC with core-shell particles column and fluorescence detection.

PubMed

Vinci, Giuliana; Antonelli, Marta L; Preti, Raffaella

2013-02-01

Liquid-liquid microextraction coupled to LC with fluorescence detection for the determination of Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbons in rainwater has been developed. The optimization of the extraction method has involved several parameters, including the comparison between an ultrasonic bath and a magnetic stirrer as extractant apparatus, the choice of the extractant solvent, and the optimization of the extraction time. Liquid-liquid microextraction gave good results in terms of recoveries (from 73.6 to 102.8% in rainwater) and repeatability, with a very simple procedure and low solvent consumption. The reported chromatographic method uses a Core-Shell technology column, with particle size <3 μm instead of classical 5-μm particles column. The resulting backpressure was below 300 bar, allowing the use of a conventional HPLC system rather than the more expensive ultrahigh performance LC (UHPLC). An average decrease of 59% in run time and 75% in eluent consumption has been obtained, compared to classical HPLC methods, keeping good separation, sensitivity, and repeatability. The proposed conditions were successfully applied to the determinations of polycyclic aromatic hydrocarbons in genuine rainwater samples. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of extraction methods for ochratoxin A detection in cocoa beans employing HPLC.

PubMed

Mishra, Rupesh K; Catanante, Gaëlle; Hayat, Akhtar; Marty, Jean-Louis

2016-01-01

Cocoa is an important ingredient for the chocolate industry and for many food products. However, it is prone to contamination by ochratoxin A (OTA), which is highly toxic and potentially carcinogenic to humans. In this work, four different extraction methods were tested and compared based on their recoveries. The best protocol was established which involves an organic solvent-free extraction method for the detection of OTA in cocoa beans using 1% sodium hydrogen carbonate (NaHCO3) in water within 30 min. The extraction method is rapid (as compared with existing methods), simple, reliable and practical to perform without complex experimental set-ups. The cocoa samples were freshly extracted and cleaned-up using immunoaffinity column (IAC) for HPLC analysis using a fluorescence detector. Under the optimised condition, the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.62 and 1.25 ng ml(-1) respectively in standard solutions. The method could successfully quantify OTA in naturally contaminated samples. Moreover, good recoveries of OTA were obtained up to 86.5% in artificially spiked cocoa samples, with a maximum relative standard deviation (RSD) of 2.7%. The proposed extraction method could determine OTA at the level 1.5 µg kg(-)(1), which surpassed the standards set by the European Union for cocoa (2 µg kg(-1)). In addition, an efficiency comparison of IAC and molecular imprinted polymer (MIP) column was also performed and evaluated.

Fourth-derivative synchronous spectrofluorimetry and HPLC with fluorescence detection as two analytical techniques for the simultaneous determination of itopride and domperidone.

PubMed

Ibrahim, Fawzia; Nasr, Jenny Jeehan

2016-02-01

Two simple, rapid and sensitive methods, namely, fourth-derivative synchronous spectrofluorimetry (method I) and HPLC with fluorescence detection (method II) were developed for the simultaneous analysis of a binary mixture of itopride HCl (ITP) and domperidone (DOM) without prior separation. The first method was based on measuring the fourth derivative of the synchronous fluorescence spectra of the two drugs at Δλ = 40 nm in methanol. The different experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully optimized. Chromatographic separation was performed in < 6.0 min using a RP C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) with fluorescence detection at 344 nm after excitation at 285 nm. A mobile phase composed of a mixture of 0.02 M phosphate buffer with acetonitrile in a ratio of 55 : 45, pH 4.5, was used at a flow rate of 1 mL/min. Linearity ranges were found to be 0.1-2 µg/mL for ITP in both methods, whereas those for DOM were found to be 0.08-2 and 0.05-1.5 µg/mL in methods I and II, respectively. The proposed methods were successfully applied for the determination of the studied drugs in synthetic mixtures and laboratory-prepared tablets. Copyright © 2015 John Wiley & Sons, Ltd.

Optimization of a new method for extraction of cyanidin chloride and pelargonidin chloride anthocyanins with magnetic solid phase extraction and determination in fruit samples by HPLC with central composite design.

PubMed

Yari, Abdollah; Rashnoo, Saba

2017-11-01

Here, we are reporting a sensitive, simple and rapid method for the analysis of cyanidin chloride and pelargonidin chloride anthocyanins in cherry, sour cherry, pomegranate and barberry produced in Iran. The analytes were extracted with acetonitrile-hydrochloric acid (1% v/v) mixture under optimized pretreatment conditions. Clean-up of the extract from fruits was conducted by magnetic solid phase extraction using salicylic acid functionalized silica-coated magnetite nanoparticles (SCMNPs) as the adsorbent. The optimized conditions searched with central composite design. Working under optimum conditions specified as: SCMNPs modified with salicylic acid, sorbent contact time and sample 10min, mechanical stirring time 57.3min. HPLC with UV-detection was used for determination of the analytes. The limit of detection, LOD, obtained for the two anthocyanins were 0.02 and 0.03μgg -1 , respectively. The ranges of the spiked recoveries were 80.0-97.6 and 72.9-97.2%, with the relative standard deviations (RSD) of 2.1 and 2.5%, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

[Determination of alkylphenol and alkylphenolpolyethoxylates in brine by solid phase extraction and high performance liquid chromatography-mass spectrometry].

PubMed

Wang, Jincheng; Xiong, Li; Zhang, Haijun; Chen, Jiping

2011-12-01

A simple method based on solid phase extraction (SPE) coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) was developed for the determination of octylphenol (OP), nonylphenol (NP), octylphenol ethoxylates (OPEOs) and nonylphenol ethoxylates (NPEOs) in brine. The extraction and cleanup of brine samples were performed on C18 solid-phase extraction cartridges. The complete separation among OP, NP, OPEOs and NPEOs was achieved on a Hypersil GOLD analytical column with methanol-water as the mobile phase. The determination was achieved using HPLC-MS with electrospray ionization (ESI) in selected ion monitoring mode. The results showed that the average recoveries of target compounds were 59.6% - 104.4% and the corresponding relative standard deviations (RSDs, n = 3) were 1.0% - 13.5%. The instrumental limits of detection for the compounds were 0.08 - 3 microg/L. This method was applied to the analysis of the samples of seawater near Dalian coast. The results showed that both NP and NPEOs were detected in all samples and their concentrations in seaport and oil port were much higher than those in other sampling sites.

Optimisation for subcritical fluid extraction of 17-methyltestosterone with 1,1,1,2-tetrafluoroethane for HPLC analysis.

PubMed

Han, Yuqian; Ma, Qinchuan; Lu, Jie; Xue, Yong; Xue, Changhu

2012-12-15

A simple, rapid and sensitive method was developed for determination of 17α-methyltestosterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) extraction and high performance liquid chromatography (HPLC). Response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure 5 MPa, temperature 31°C, and co-solvent volume 3.35ml. The analysis was carried out on XDB-C(18) column (4.6 mm × 250 mm, 5 μm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 0.8 ml/min, temperature 30°C and wavelength 245 nm. Good linearity of detection was obtained for 17α-methyltestosterone between concentrations of 50-250 ng/ml, r(2)=0.999. The method was validated using samples fortified with 17α-methyltestosterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application.

PubMed

Chen, Feng; Hu, Zhe-Yi; Parker, Robert B; Laizure, S Casey

2017-06-01

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects. Copyright © 2016 John Wiley & Sons, Ltd.

Constant pressure mode extended simple gradient liquid chromatography system for micro and nanocolumns.

PubMed

Šesták, Jozef; Kahle, Vladislav

2014-07-11

Performing gradient liquid chromatography at constant pressure instead of constant flow rate has serious potential for shortening the analysis time and increasing the productivity of HPLC instruments that use gradient methods. However, in the constant pressure mode the decreasing column permeability during a long period of time negatively affects the repeatability of retention time. Thus a volume-based approach, in which the detector signal is plotted as a function of retention volume, must be taken into consideration. Traditional HPLC equipment, however, requires quite complex hardware and software modifications in order to work at constant pressure and in the volume-based mode. In this short communication, a low cost and easily feasible pressure-controlled extension of the previously described simple gradient liquid chromatography platform is proposed. A test mixture of four nitro esters was separated by 10-60% (v/v) acetone/water gradient and a high repeatability of retention volumes at 20MPa (RSD less than 0.45%) was realized. Separations were also performed at different values of pressure (20, 25, and 31MPa), and only small variations of the retention volumes (up to 0.8%) were observed. In this particular case, the gain in the analysis speed of 7% compared to the constant flow mode was realized at a constant pressure. Copyright © 2014 Elsevier B.V. All rights reserved.

Optimization of Robust HPLC Method for Quantitation of Ambroxol Hydrochloride and Roxithromycin Using a DoE Approach.

PubMed

Patel, Rashmin B; Patel, Nilay M; Patel, Mrunali R; Solanki, Ajay B

2017-03-01

The aim of this work was to develop and optimize a robust HPLC method for the separation and quantitation of ambroxol hydrochloride and roxithromycin utilizing Design of Experiment (DoE) approach. The Plackett-Burman design was used to assess the impact of independent variables (concentration of organic phase, mobile phase pH, flow rate and column temperature) on peak resolution, USP tailing and number of plates. A central composite design was utilized to evaluate the main, interaction, and quadratic effects of independent variables on the selected dependent variables. The optimized HPLC method was validated based on ICH Q2R1 guideline and was used to separate and quantify ambroxol hydrochloride and roxithromycin in tablet formulations. The findings showed that DoE approach could be effectively applied to optimize a robust HPLC method for quantification of ambroxol hydrochloride and roxithromycin in tablet formulations. Statistical comparison between results of proposed and reported HPLC method revealed no significant difference; indicating the ability of proposed HPLC method for analysis of ambroxol hydrochloride and roxithromycin in pharmaceutical formulations. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A novel approach for the quantitation of carbohydrates in mash, wort, and beer with RP-HPLC using 1-naphthylamine for precolumn derivatization.

PubMed

Rakete, Stefan; Glomb, Marcus A

2013-04-24

A novel universal method for the determination of reducing mono-, di-, and oligosaccharides in complex matrices on RP-HPLC using 1-naphthylamine for precolumn derivatization with sodium cyanoborhydride was established to study changes in the carbohydrate profile during beer brewing. Fluorescence and mass spectrometric detection enabled very sensitive analyses of beer-relevant carbohydrates. Mass spectrometry additionally allowed the identification of the molecular weight and thereby the degree of polymerization of unknown carbohydrates. Thus, carbohydrates with up to 16 glucose units were detected. Comparison demonstrated that the novel method was superior to fluorophore-assisted carbohydrate electrophoresis (FACE). The results proved the HPLC method clearly to be more powerful in regard to sensitivity and resolution. Analogous to FACE, this method was designated fluorophore-assisted carbohydrate HPLC (FAC-HPLC).

Development, Validation and Application of a Stability Indicating HPLC Method to Quantify Lidocaine from Polyethylene-co-Vinyl Acetate (EVA) Matrices and Biological Fluids.

PubMed

Bhusal, Prabhat; Sharma, Manisha; Harrison, Jeff; Procter, Georgina; Andrews, Gavin; Jones, David S; Hill, Andrew G; Svirskis, Darren

2017-09-01

An efficient and cost-effective quantification procedure for lidocaine by HPLC has been developed to estimate lidocaine from an EVA matrix, plasma, peritoneal fluid and intra-articular fluid (IAF). This method guarantees the resolution of lidocaine from the degradation products obtained from alkaline and oxidative stress. Chromatographic separation of lidocaine was achieved with a retention time of 7 min using a C18 column with a mobile phase comprising acetonitrile and potassium dihydrogen phosphate buffer (pH 5.5; 0.02 M) in the ratio of 26:74 at a flow rate of 1 mL min-1 with detection at 230 nm. Instability of lidocaine was observed to an oxidizing (0.02% H2O2) and alkaline environments (0.1 M NaOH). The calibration curve was found to be linear within the concentration range of 0.40-50.0 μg/mL. Intra-day and inter-day accuracy ranged between 95.9% and 99.1%, with precision (% RSD) below 6.70%. The limit of quantification and limit of detection were 0.40 μg/mL and 0.025 μg/mL, respectively. The simple extraction method described enabled the quantification of lidocaine from an EVA matrix using dichloromethane as a solvent. The assay and content uniformity of lidocaine within an EVA matrix were 103 ± 3.60% and 100 ± 2.60%, respectively. The ability of this method to quantify lidocaine release from EVA films was also demonstrated. Extraction of lidocaine from plasma, peritoneal fluid and IAF followed by HPLC analysis confirmed the utility of this method for ex vivo and in vivo studies where the calibration plot was found to be linear from 1.60 to 50.0 μg/mL. © Crown copyright 2017.

Phytochemical composition, antioxidant activity and HPLC fingerprinting profiles of three Pyrola species from different regions.

PubMed

Wang, Dongmei; He, Fengyuan; Lv, Zhenjiang; Li, Dengwu

2014-01-01

The present study was performed to investigate the variation of phytochemical composition, antioxidant activity and High Performance Liquid Chromatography (HPLC) fingerprinting profiles of three Pyrola species. Thirteen samples (eight P. decorata, three P. calliantha and two P. renifolia) were collected from different regions in China. The tannin, hyperoside and quercetin contents of all samples were determined by reverse-phase HPLC and varied within the range 9.77-34.75, 0.34-2.16 and 0.062-0.147 mg/g dry weigh, respectively. Total flavonoid content was evaluated and varied within the range 16.22-37.82 mg/g dry weight. Antioxidant activity was determined by DPPH assay, with IC50 ranging from 7.96 to 50.33 µg/ml, ABTS•+ and FRAP assay, within the range 612.66-1021.05 and 219.64-398.12 µmol equiv. Trolox/g, respectively. These results revealed that there were significant variations in phytochemical profiles and antioxidant activity among all samples. Due to the higher phytochemical content and significant antioxidant activity, P. calliantha was selected as the most valuable species, and the P. calliantha sample from Left banner of Alxa even possessed the strongest antioxidant activity among all the thirteen samples. Futhermore, Emei Mountain was proved to be the most suitable region for producing P. decorata. Moreover, in order to further evaluate the diversities and quality of Pyrola, HPLC fingerprint analysis coupled with hierarchical cluster and discrimination analyses were introduced to establish a simple, rapid and effective method for accurate identification, classification and quality assessment of Pyrola. Thirteen samples were divided into three groups consistent with their morphological classification. Two types of discriminant functions were generated and the ratio of discrimination was 100%. This method can identify different species of Pyrola and the same species from different regions of origin. Also, it can be used to compare and control the quality of Pyrola and other natural products prepared from them.

Phytochemical Composition, Antioxidant Activity and HPLC Fingerprinting Profiles of Three Pyrola Species from Different Regions

PubMed Central

Wang, Dongmei; He, Fengyuan; Lv, Zhenjiang; Li, Dengwu

2014-01-01

The present study was performed to investigate the variation of phytochemical composition, antioxidant activity and High Performance Liquid Chromatography (HPLC) fingerprinting profiles of three Pyrola species. Thirteen samples (eight P. decorata, three P. calliantha and two P. renifolia) were collected from different regions in China. The tannin, hyperoside and quercetin contents of all samples were determined by reverse-phase HPLC and varied within the range 9.77–34.75, 0.34–2.16 and 0.062–0.147 mg/g dry weigh, respectively. Total flavonoid content was evaluated and varied within the range 16.22–37.82 mg/g dry weight. Antioxidant activity was determined by DPPH assay, with IC50 ranging from 7.96 to 50.33 µg/ml, ABTS•+ and FRAP assay, within the range 612.66–1021.05 and 219.64–398.12 µmol equiv. Trolox/g, respectively. These results revealed that there were significant variations in phytochemical profiles and antioxidant activity among all samples. Due to the higher phytochemical content and significant antioxidant activity, P. calliantha was selected as the most valuable species, and the P. calliantha sample from Left banner of Alxa even possessed the strongest antioxidant activity among all the thirteen samples. Futhermore, Emei Mountain was proved to be the most suitable region for producing P. decorata. Moreover, in order to further evaluate the diversities and quality of Pyrola, HPLC fingerprint analysis coupled with hierarchical cluster and discrimination analyses were introduced to establish a simple, rapid and effective method for accurate identification, classification and quality assessment of Pyrola. Thirteen samples were divided into three groups consistent with their morphological classification. Two types of discriminant functions were generated and the ratio of discrimination was 100%. This method can identify different species of Pyrola and the same species from different regions of origin. Also, it can be used to compare and control the quality of Pyrola and other natural products prepared from them. PMID:24796694

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites.

PubMed

Xie, Li; Chen, Liqin; Gu, Pan; Wei, Lanlan; Kang, Xuejun

2018-03-01

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

A comparative uncertainty study of the calibration of macrolide antibiotic reference standards using quantitative nuclear magnetic resonance and mass balance methods.

PubMed

Liu, Shu-Yu; Hu, Chang-Qin

2007-10-17

This study introduces the general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of reference standards of macrolide antibiotics. Several qNMR experimental conditions were optimized including delay, which is an important parameter of quantification. Three kinds of macrolide antibiotics were used to validate the accuracy of the qNMR method by comparison with the results obtained by the high performance liquid chromatography (HPLC) method. The purities of five common reference standards of macrolide antibiotics were measured by the 1H qNMR method and the mass balance method, respectively. The analysis results of the two methods were compared. The qNMR is quick and simple to use. In a new medicine research and development process, qNMR provides a new and reliable method for purity analysis of the reference standard.

Simple and Rapid Determination of Ferulic Acid Levels in Food and Cosmetic Samples Using Paper-Based Platforms

PubMed Central

Tee-ngam, Prinjaporn; Nunant, Namthip; Rattanarat, Poomrat; Siangproh, Weena; Chailapakul, Orawon

2013-01-01

Ferulic acid is an important phenolic antioxidant found in or added to diet supplements, beverages, and cosmetic creams. Two designs of paper-based platforms for the fast, simple and inexpensive evaluation of ferulic acid contents in food and pharmaceutical cosmetics were evaluated. The first, a paper-based electrochemical device, was developed for ferulic acid detection in uncomplicated matrix samples and was created by the photolithographic method. The second, a paper-based colorimetric device was preceded by thin layer chromatography (TLC) for the separation and detection of ferulic acid in complex samples using a silica plate stationary phase and an 85:15:1 (v/v/v) chloroform: methanol: formic acid mobile phase. After separation, ferulic acid containing section of the TLC plate was attached onto the patterned paper containing the colorimetric reagent and eluted with ethanol. The resulting color change was photographed and quantitatively converted to intensity. Under the optimal conditions, the limit of detection of ferulic acid was found to be 1 ppm and 7 ppm (S/N = 3) for first and second designs, respectively, with good agreement with the standard HPLC-UV detection method. Therefore, these methods can be used for the simple, rapid, inexpensive and sensitive quantification of ferulic acid in a variety of samples. PMID:24077320

Determination of organophosphorus pesticides and their major degradation product residues in food samples by HPLC-UV.

PubMed

Peng, Guilong; He, Qiang; Lu, Ying; Mmereki, Daniel; Zhong, Zhihui

2016-10-01

A simple method based on dispersive solid-phase extraction (DSPE) and dispersive liquid-liquid microextraction method based on solidification of floating organic droplets (DLLME-SFO) was developed for the extraction of chlorpyrifos (CP), chlorpyrifos-methyl (CPM), and their main degradation product 3,5,6-trichloro-2-pyridinol (TCP) in tomato and cucumber samples. The determination was carried out by high performance liquid chromatography with ultraviolet detection (HPLC-UV). In the DSPE-DLLME-SFO, the analytes were first extracted with acetone. The clean-up of the extract by DSPE was carried out by directly adding activated carbon sorbent into the extract solution, followed by shaking and filtration. Under the optimum conditions, the proposed method was sensitive and showed a good linearity within a range of 2-500 ng/g, with the correlation coefficients (r) varying from 0.9991 to 0.9996. The enrichment factors ranged from 127 to 138. The limit of detections (LODs) were in the range of 0.12-0.68 ng/g, and the relative standard deviations (RSDs) for 50 ng/g of each analytes in tomato samples were in the range of 3.25-6.26 % (n = 5). The proposed method was successfully applied for the extraction and determination of the mentioned analytes residues in tomato and cucumber samples, and satisfactory results were obtained.

Determination of nonylphenol and nonylphenol ethoxylates in environmental solid samples by ultrasonic-assisted extraction and high performance liquid chromatography-fluorescence detection.

PubMed

Núñez, L; Turiel, E; Tadeo, J L

2007-04-06

A simple and rapid analytical method for the determination of nonylphenol (NP) and nonylphenol ethoxylates (NPEOx) in solid environmental samples has been developed. This method combines an ultrasonic-assisted extraction procedure in small columns and an enrichment step onto C(18) solid-phase extraction cartridges prior to separation using HPLC with fluorescence detection. Method optimization was carried out using soil samples fortified at different concentration levels (from 0.1 to 100 microg/g). Under optimum conditions, 2g of soil was placed in small glass columns and extraction was performed assisted by sonication (SAESC) at 45 degrees C in two consecutive steps of 15 min using a mixture of H(2)O/MeOH (30/70). The obtained extracts were collected, loaded onto 500 mg C(18) cartridges, and analytes were eluted with 3 x 1 ml of methanol and 1 ml of acetonitrile. Finally, sample extracts were evaporated under a nitrogen stream, redissolved in 500 microl H(2)O/AcN (50/50), and passed though a 0.45 microm nylon filter before final determination by HPLC-FL. The developed procedure allowed to achieve quantitative recoveries for NP and NPEOx, and was properly validated. Finally, the method was applied to the determination of these compounds in soils and other environmental solid samples such as sediments, compost and sludge.

Preparative isolation and characterization of some minor impurities of astaxanthin by high-performance liquid chromatography.

PubMed

Rao, R Nageswara; Alvi, S Naseeruddin; Rao, B Nageswara

2005-05-27

A simple and rapid preparative high-performance liquid chromatography (HPLC) method has been developed to isolate and characterize some minor impurities of astaxanthin using a normal-phase Lichrosorb silica column with n-hexane-acetone-tetrahydrofuran (90:2:8, v/v/v) as mobile-phase and detection at 475 nm. The isolated impurities were characterized as astacene, dehydro astacene and apoastaxanthinal by UV-vis, ESI-MS, 1H and 13C NMR spectroscopy and the molecular structures were assigned. The impurities collected using the developed conditions were over 98% pure.

Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats.

PubMed

Wang, Xiaofan; Zhao, Xu; Gu, Liqiang; Lv, Chunxiao; He, Bosai; Liu, Zhenzhen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui

2014-03-15

A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0-5.0ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative determination of regorafenib and its two major metabolites in human plasma with high-performance liquid chromatography and ultraviolet detection.

PubMed

Fujita, Kazuma; Miura, Masatomo; Shibata, Hiroyuki

2016-10-01

A simple, highly sensitive and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N-oxidemetabolite (M-2) and the desmethyl N-oxide metabolite (M-5) in human plasma. Regorafenib, M-2, M-5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH2 PO4 (pH 3.5)-acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M-2 and M-5 were 10 ng/mL for each analyte. A procedure using solid-phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra- and inter-day assays were <12.2% for regorafenib, <12.3% for M-2 and <15.1% for M-5. Accuracies for intra- and inter-day assays were <9.4% for regorafenib, <8.0% for M-2 and <12.8% for M-5 over a linear range from 10 to 10,000 ng/mL. This HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data.

PubMed

Mortera, Pablo; Zuljan, Federico A; Magni, Christian; Bortolato, Santiago A; Alarcón, Sergio H

2018-02-01

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20mmolL -1 phosphate buffer at pH 2.20) and a flow-rate of 1.0mLmin -1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0mmolL -1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of /sup 60/Co-irradiation on penicillin G procaine in veterinary mastitis products

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

Tsuji, K.; Goetz, J.F.; Vanmeter, W.

The effect of /sup 60/Co-irradation on penicillin G procaine in a peanut oil-based veterinary mastitis product was examined by reversed-phase high-performance liquid chromatography (HPLC). The HPLC method is capable of separating and quantifiying procaine, penicillin G, and various degradation compounds. Values obtained by the HPLC method on the product irradiated and stored at various temperatures correlated well with those of the microbiological assay. No significant decrease in the procaine was detected even after 4.0-Mrad irradiation. The HPLC method is applicable for analysis of other beta-lactam antibiotics.

High-performance liquid chromatography method for the determination of hydrogen peroxide present or released in teeth bleaching kits and hair cosmetic products.

PubMed

Gimeno, Pascal; Bousquet, Claudine; Lassu, Nelly; Maggio, Annie-Françoise; Civade, Corinne; Brenier, Charlotte; Lempereur, Laurent

2015-03-25

This manuscript presents an HPLC/UV method for the determination of hydrogen peroxide present or released in teeth bleaching products and hair products. The method is based on an oxidation of triphenylphosphine into triphenylphosphine oxide by hydrogen peroxide. Triphenylphosphine oxide formed is quantified by HPLC/UV. Validation data were obtained using the ISO 12787 standard approach, particularly adapted when it is not possible to make reconstituted sample matrices. For comparative purpose, hydrogen peroxide was also determined using ceric sulfate titrimetry for both types of products. For hair products, a cross validation of both ceric titrimetric method and HPLC/UV method using the cosmetic 82/434/EEC directive (official iodometric titration method) was performed. Results obtained for 6 commercialized teeth whitening products and 5 hair products point out similar hydrogen peroxide contain using either the HPLC/UV method or ceric sulfate titrimetric method. For hair products, results were similar to the hydrogen peroxide content using the cosmetic 82/434/EEC directive method and for the HPLC/UV method, mean recoveries obtained on spiked samples, using the ISO 12787 standard, ranges from 100% to 110% with a RSD<3.0%. To assess the analytical method proposed, the HPLC method was used to control 35 teeth bleaching products during a market survey and highlight for 5 products, hydrogen peroxide contents higher than the regulated limit. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecularly imprinted microspheres synthesized by a simple, fast, and universal suspension polymerization for selective extraction of the topical anesthetic benzocaine in human serum and fish tissues.

PubMed

Sun, Hui; Lai, Jia-Ping; Chen, Fang; Zhu, De-Rong

2015-02-01

A simple, fast, and universal suspension polymerization method was used to synthesize the molecularly imprinted microspheres (MIMs) for the topical anesthetic benzocaine (BZC). The desired diameter (10-20 μm) and uniform morphology of the MIMs were obtained easily by changing one or more of the synthesis conditions, including type and amount of surfactant, stirring rate, and ratio of organic to water phase. The MIMs obtained were used as a molecular-imprinting solid-phase-extraction (MISPE) material for extraction of BZC in human serum and fish tissues. The MISPE results revealed that the BZC in these biosamples could be enriched effectively after the MISPE operation. The recoveries of BZC on MIMs cartridges were higher than 90% (n = 3). Finally, an MISPE-HPLC method with UV detection was developed for highly selective extraction and fast detection of trace BZC in human serum and fish tissues. The developed method could also be used for the enrichment and detection of BZC in other complex biosamples.

RP-HPLC method development and validation for simultaneous estimation of atorvastatin calcium and pioglitazone hydrochloride in pharmaceutical dosage form.

PubMed

Peraman, Ramalingam; Mallikarjuna, Sasikala; Ammineni, Pravalika; Kondreddy, Vinod kumar

2014-10-01

A simple, selective, rapid, precise and economical reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous estimation of atorvastatin calcium (ATV) and pioglitazone hydrochloride (PIO) from pharmaceutical formulation. The method is carried out on a C8 (25 cm × 4.6 mm i.d., 5 μm) column with a mobile phase consisting of acetonitrile (ACN):water (pH adjusted to 6.2 using o-phosphoric acid) in the ratio of 45:55 (v/v). The retention time of ATV and PIO is 4.1 and 8.1 min, respectively, with the flow rate of 1 mL/min with diode array detector detection at 232 nm. The linear regression analysis data from the linearity plot showed good linear relationship with a correlation coefficient (R(2)) value for ATV and PIO of 0.9998 and 0.9997 in the concentration range of 10-80 µg mL(-1), respectively. The relative standard deviation for intraday precision has been found to be <2.0%. The method is validated according to the ICH guidelines. The developed method is validated in terms of specificity, selectivity, accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for simultaneous estimation of these drugs in marketed dosage forms. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Methods of Analysis by the U.S. Geological Survey Organic Geochemistry Research Group-Update and Additions to the Determination of Chloroacetanilide Herbicide Degradation Compounds in Water Using High-Performance Liquid Chromatography/Mass Spectrometry

USGS Publications Warehouse

Lee, E.A.; Kish, J.L.; Zimmerman, L.R.; Thurman, E.

2001-01-01

An analytical method using high-performance liquid chromatography/mass spectrometry (HPLC/MS) was developed by the U.S. Geological Survey in 1999 for the analysis of selected chloroacetanilide herbicide degradation compounds in water. These compounds were acetochlor ethane sulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. The HPLC/MS method was updated in 2000, and the method detection limits were modified accordingly. Four other degradation compounds also were added to the list of compounds that can be analyzed using HPLC/MS; these compounds were dimethenamid ESA, dimethenamid OXA, flufenacet ESA, and flufenacet OXA. Except for flufenacet OXA, good precision and accuracy were demonstrated for the updated HPLC/MS method in buffered reagent water, surface water, and ground water. The mean HPLC/MS recoveries of the degradation compounds from water samples spiked at 0.20 and 1.0 ?g/L (microgram per liter) ranged from 75 to 114 percent, with relative standard deviations of 15.8 percent or less for all compounds except flufenacet OXA, which had relative standard deviations ranging from 11.3 to 48.9 percent. Method detection levels (MDL's) using the updated HPLC/MS method varied from 0.009 to 0.045 ?g/L, with the flufenacet OXA MDL at 0.072 ?g/L. The updated HPLC/MS method is valuable for acquiring information about the fate and transport of the parent chloroacetanilide herbicides in water.

Analysis of formononetin from black cohosh (Actaea racemosa).

PubMed

Jiang, B; Kronenberg, F; Balick, M J; Kennelly, E J

2006-07-01

Black cohosh has been widely used as an herbal medicine for the treatment of symptoms related to menopause in America and Europe during the past several decades, but the bioactive constituents are still unknown. Formononetin is an isoflavone with known estrogen-like activity. This compound was first reported to be isolated from black cohosh in 1985, but subsequent research in 2002 using HPLC-PDA and LC-MS revealed no evidence to show the presence of formononetin in 13 populations of American black cohosh. A more recent report published in 2004 claimed to detect formononetin in an extract of black cohosh rhizomes using a TLC-fluorescent densitometry method. To further resolve these conflicting reports, we analyzed black cohosh roots and rhizomes for the presence of formononetin, using a combined TLC, HPLC-PDA and LC-MS method. We examined both methanolic and aqueous methanolic black cohosh extracts by HPLC-PDA and LC-MS methods, and did not detect formononetin in any extracts. We further determined the limits of detection of formononetin by HPLC-PDA and LC-MS. Our experimental results indicated that the sensitivity and accuracy of the HPLC-PDA and LC-MS methods for the analysis of formononetin were slightly higher than those of the reported fluorescent method, suggesting that the HPLC-PDA and LC-MS methods were reliable for the analysis of formononetin from black cohosh. We also repeated the reported TLC method to concentrate two fractions from a modern black cohosh sample and an 86-year-old black cohosh sample, respectively, and then analyzed these two fractions for formononetin using the HPLC-PDA and LC-MS method instead of the fluorescent method. Formononetin was not detected by HPLC-PDA or LC-MS. From the results of the present study it is not reasonable to attribute the estrogen-like activity of black cohosh extracts to formononetin.

Steroids

NASA Astrophysics Data System (ADS)

Frey, Felix J.; Frey, Brigitte M.; Benet, Leslie Z.

If a radioimmunoassay, a protein binding method, or a colorimetric assay for the assessment of a steroid level is replaced by high performance liquid chromatography (HPLC), the cost for the determination of a steroid level increases at least initially because one must acquire the new HPLC equipment. Therefore, if an older method provides the same results as the new, "advanced" HPLC method, the only advantage resulting from the introduction of a high performance chromatographic assay is that gained by the manufacturer in terms of greater sales. Thus, justification for the assessment of steroids by HPLC is only obtained if the quality and/or quantity of information gained is significantly increased as compared to that provided by the conventional methods. But this evidential relation, that more and better information justifies a higher price in any case, is no longer true in health care, with the birth some years ago of the categoric imperative for the reduction of costs in the medical sector. That is, each new technology introduced for health maintenance should demonstrate at least a stabilizing impact on total medical expenditures. Therefore, after reviewing the presently available HPLC methods for the clinically important steroids, we will consider whether HPLC analyses for these steroids can be recommended without violating this vox populi.

Determination of urine oxalate level in rats with renal calcium oxalate calculus by high-performance liquid chromatography.

PubMed

Cao, Qiu-shi; Ba, Yuan-ming; Luo, Jun-hua; Dai, Qi

2015-02-01

To establish a method of high-performance liquid chromatography (HPLC) for determining the urine oxalate levle in rats with renal calcium oxalate calculus. Totally 24 SPF Wistar healthy male rats were randomly divided into control group(n=12)and ethylene glycol (EG) group (n=12). Rats in EG group were administered intragastrically with 2% ammonium chloride (AC)2 ml/rat per day+1% ethylene glycol (EG), along with free access to drinking water.The control group was fed with deionized water, along with the intragastric administration of normal saline (1 ml per day). Twenty-eight days after modelling, the 24-hour urine samples were collected, and the urine oxalic acid levels were determined using HPLC and the results were compared with those of catalytic spectrophotometry using oxidation of methyl. During the HPLC, the samples were separated on Aglient 5TC-C18 (250×4.6 mm,5 Μm), eluted with mixture of methanol (0.1 mol/L) and ammonium acetate (15:85) at 1.2 ml/min, and detected at 314 nm, with the column temperature being 20 ℃. The standard curves of high and low concentrations of oxalic acid were y=5909.1x+378730, R² =0.9984 and y=7810.5x-16635, R² =0.9967,respectively. The lowest detectable concentration in this method was 5 Μg/ml. The linear high concentration range of oxalate stood at 62.50-2000.00 Μg/ml, and the linear low concentration range of oxalate stood at 6.25-100.00 Μg/ml. Its average recovery was 95.1%, and its within-day and day-to-day precisions were 3.4%-10.8% and 3.8%-9.4%. Both HPLC and catalytic spectrophotometry showed significantly higher urinary oxalic acid concentration and 24 h urine oxalate level in EG group compared with the control group [urinary oxalic acid concentration: (736.35 ± 254.52) Μg/ml vs.(51.56 ± 36.34) Μg/ml,(687.35 ± 234.53) Μg/ml vs.(50.24 ± 42.34) Μg/ml;24 h urine oxalate level: (11.23 ± 4.12)mg vs.(0.87 ± 0.45)mg,(9.89 ± 3.55)mg vs. (0.77 ± 0.65)mg; all P<0.01]. No statistically significant difference was observed in the results of urinary oxalate concentration and 24 h urine oxalate level between HPLC and potassium chromate oxidation of methyl red spectrophotometry (all P>0.05). HPLC is a simple, rapid, and precise method in detecting urine oxalate level in rats with renal calcium oxalate calculus, with high recovery rate.

HPLC determination of four active saponins from Panax notoginseng in rat serum and its application to pharmacokinetic studies.

PubMed

Li, Lie; Sheng, Yuxin; Zhang, Jinlan; Wang, Chuanshe; Guo, Dean

2004-12-01

Four main active saponins (ginsenosides Rg1, Rb1, Rd and notoginsenoside R1) in Panax notoginseng in rat serum after oral and intravenous administration of total saponins of P. notoginseng (PNS) to rats were determined using a simple and sensitive high-performance chromatographic method. The serum samples were pretreated with solid-phase extraction before analysis. The calibration curves for the four saponins were linear in the given concentration ranges. The intra-day and inter-day assay coefficients in serum were less than 10.0% and the recoveries of the method were higher than 80.0% in the high, middle and low concentrations. This method was applied to study the pharmacokinetics following oral and intravenous administration of PNS. Copyright 2004 John Wiley & Sons, Ltd.

Simultaneous kinetic spectrometric determination of three flavonoid antioxidants in fruit with the aid of chemometrics

NASA Astrophysics Data System (ADS)

Sun, Ruiling; Wang, Yong; Ni, Yongnian; Kokot, Serge

2014-03-01

A simple, inexpensive and sensitive kinetic spectrophotometric method was developed for the simultaneous determination of three anti-carcinogenic flavonoids: catechin, quercetin and naringenin, in fruit samples. A yellow chelate product was produced in the presence neocuproine and Cu(I) - a reduction product of the reaction between the flavonoids with Cu(II), and this enabled the quantitative measurements with UV-vis spectrophotometry. The overlapping spectra obtained, were resolved with chemometrics calibration models, and the best performing method was the fast independent component analysis (fast-ICA/PCR (Principal component regression)); the limits of detection were 0.075, 0.057 and 0.063 mg L-1 for catechin, quercetin and naringenin, respectively. The novel method was found to outperform significantly the common HPLC procedure.

Quantification of quercetin glycosides in 6 onion cultivars and comparisons of hydrolysis-HPLC and spectrophotometric methods in measuring total quercetin concentrations.

PubMed

Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

2010-03-01

This study was performed to purify and quantify quercetin glycosides (QG) and aglycone (free) quercetin (Q) in 6 selected onion cultivars and to compare analytical approaches based on high-performance liquid chromatography (HPLC) and spectrophotometry for the quantification of total quercetin (TQ) concentrations. Individual mono- and di-glycoside Q compounds were purified using a semipreparative HPLC and identified by comparing spectral data and by confirming corresponding peaks of QG and Q after incomplete enzyme-hydrolysis. Purified QG were quantified as Q by enzyme-hydrolysis/HPLC. TQ concentrations obtained from 20 onion bulbs with enzyme-hydrolysis/HPLC, no-hydrolysis/HPLC, and a spectrophotometric method without prior hydrolysis were significantly correlated (r(2)= 0.99) and were about 15% higher, identical, or 10% less than those concentrations by a standard acid-hydrolysis/HPLC method, respectively. During enzyme-hydrolysis of onion extracts, progressive reduction of the QG and formation of the corresponding mono-glycosides and Q were monitored using an analytical HPLC. TQ ranged from 83 to 330 microg/g F.W. in 6 selected cultivars of long-day or short-day onions. Q3,4'G and Q4'G were the 2 major compounds and comprised approximately between 94% and 97% of TQ in onions.

A high-performance liquid chromatography-electronic circular dichroism online method for assessing the absolute enantiomeric excess and conversion ratio of asymmetric reactions

NASA Astrophysics Data System (ADS)

Zhang, Xiang; Wang, Mingchao; Li, Li; Yin, Dali

2017-03-01

Asymmetric reactions often need to be evaluated during the synthesis of chiral compounds. However, traditional evaluation methods require the isolation of the individual enantiomer, which is tedious and time-consuming. Thus, it is desirable to develop simple, practical online detection methods. We developed a method based on high-performance liquid chromatography-electronic circular dichroism (HPLC-ECD) that simultaneously analyzes the material conversion ratio and absolute optical purity of each enantiomer. In particular, only a reverse-phase C18 column instead of a chiral column is required in our method because the ECD measurement provides a g-factor that describes the ratio of each enantiomer in the mixtures. We used our method to analyze the asymmetric hydrosilylation of β-enamino esters, and we discussed the advantage, feasibility, and effectiveness of this new methodology.

Polyamide as an efficient sorbent for simultaneous interface-free determination of three Sudan dyes in saffron and urine using high-performance liquid chromatography-ultra violet detection.

PubMed

Saeidi, Iman; Barfi, Behruz; Payrovi, Moazameh; Feizy, Javid; Sheibani, Hojat A; Miri, Mina; Ghollasi Moud, Farahnaz

2015-01-01

With polyamide (PA) as an efficient sorbent for solid phase extraction (SPE) of Sudan dyes II, III and Red 7B from saffron and urine, their determination by HPLC was performed. The optimum conditions for SPE were achieved using 7 mL methanol/water (1:9, v/v, pH 7) as the washing solvent and 3 mL tetrahydrofuran for elution. Good clean-up and high (above 90%) recoveries were observed for all the analytes. The optimized mobile phase composition for HPLC analysis of these compounds was methanol-water (70:30, v/v). The SPE parameters, such as the maximum loading capacity and breakthrough volume, were also determined for each analyte. The limits of detection (LODs), limits of quantification (LOQs), linear ranges and recoveries for the analytes were 4.6-6.6 microg/L, 13.0-19.8 microg/L, 13.0-5000 microg/L (r2>0.99) and 92.5%-113.4%, respectively. The precisions (RSDs) of the overall analytical procedure, estimated by five replicate measurements for Sudan II, III and Red 7B in saffron and urine samples were 2.3%, 1.8% and 3.6%, respectively. The developed method is simple and successful in the application to the determination of Sudan dyes in saffron and urine samples with HPLC coupled with UV detection.

Salting-out assisted liquid-liquid extraction combined with capillary HPLC for the determination of sulfonylurea herbicides in environmental water and banana juice samples.

PubMed

Gure, Abera; Lara, Francisco J; Moreno-González, David; Megersa, Negussie; del Olmo-Iruela, Monsalud; García-Campaña, Ana M

2014-09-01

A salting-out assisted liquid-liquid extraction (SALLE) combined with capillary high performance liquid chromatography with diode array detector (capillary HPLC-DAD) was proposed for extraction and determination of residues of nine sulfonylurea herbicides (SUHs) in environmental water and banana juice samples. Various parameters affecting the extraction process such as the type and volume of the organic solvent, sample volume, type and amount of salt, pH of the sample and vortex time were optimized. Under optimum conditions, matrix matched calibration curves were established using river water and banana juice samples. Good linear relationships as well as low limits of detection, LODs (0.4-1.3 and 3-13 µg/L) and quantification, LOQs (1.3-4.3 and 10-43 µg/L) were obtained in water and banana juice samples, respectively. The precision (intra- and inter-day) of the peak areas expressed as relative standard deviations (%, RSD), at two concentration levels were below 10 % in both matrices. Recoveries obtained from spiked environmental waters (river water and groundwater) and banana juice samples, at two concentration levels, ranged from 72 to 115%. The results of the analysis revealed that the proposed SALLE-capillary HPLC method is simple, rapid, cheap and environmentally friendly, being successfully applicable for the determination of SUH residues in waters and banana juices. Copyright © 2014 Elsevier B.V. All rights reserved.

Research on major antitumor active components in Zi-Cao-Cheng-Qi decoction based on hollow fiber cell fishing with high performance liquid chromatography.

PubMed

Li, Miaomiao; Hu, Shuang; Chen, Xuan; Wang, Runqin; Bai, Xiaohong

2018-02-05

Hollow fiber cell fishing (HFCF) based on hepatoma HepG-2 cells, human renal tubular ACHN cells or human cervical carcinoma HeLa cells, coupled with high-performance liquid chromatography (HPLC), was developed and employed to research the major active components in Zi-Cao-Cheng-Qi decoction both in vitro and in vivo. The research showed that the active components, such as hesperidin, magnolol, honokiol, shikonin, emodin and β,β'-dimethylacrylshikonin were screened out by HFCF based on the cancer cells in vitro, furthermore they can be absorbed into blood and reach in the target organ, and some of the active components can be fished by the cells and maintain effective concentrations. Before application of HFCF with HPLC, cell growth state, cell survival rate, positive effect on screening results binding between active centers on the fiber and target components, repeatability of retention times and relative peak areas of the target analytes were analysed and investigated. In short, HFCF with HPLC is a simple, inexpensive, effective, and reliable method that can be used in researching active components from traditional Chinese medicine (TCM) and its formula both in vitro and in vivo, elucidating preliminarily the TCM characteristics of multiple components and multiple targets, laying a foundation for expounding the antitumor efficacy material basis in TCM. Copyright © 2017 Elsevier B.V. All rights reserved.

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

PubMed Central

Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P.

2014-01-01

Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. PMID:24323699

76 FR 38033 - Cloquintocet-mexyl; Pesticide Tolerances

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-29

... enforcement methods are the High Performance Liquid Chromatography with Ultraviolet Detection (HPLC/UV) method REM 138.01 for determination of cloquintocet-mexyl (parent) and the HPLC/UV Method REM 138.10 for...

Simultaneous determination of selected biogenic amines in alcoholic beverage samples by isotachophoretic and chromatographic methods.

PubMed

Jastrzębska, Aneta; Piasta, Anna; Szłyk, Edward

2014-01-01

A simple and useful method for the determination of biogenic amines in beverage samples based on isotachophoretic separation is described. The proposed procedure permitted simultaneous analysis of histamine, tyramine, cadaverine, putrescine, tryptamine, 2-phenylethylamine, spermine and spermidine. The data presented demonstrate the utility, simplicity, flexibility, sensitivity and environmentally friendly character of the proposed method. The precision of the method expressed as coefficient of variations varied from 0.1% to 5.9% for beverage samples, whereas recoveries varied from 91% to 101%. The results for the determination of biogenic amines were compared with an HPLC procedure based on a pre-column derivatisation reaction of biogenic amines with dansyl chloride. Furthermore, the derivatisation procedure was optimised by verification of concentration and pH of the buffer, the addition of organic solvents, reaction time and temperature.

High-performance liquid chromatography as a technique to determine protein adsorption onto hydrophilic/hydrophobic surfaces.

PubMed

Huang, Tongtong; Anselme, Karine; Sarrailh, Segolene; Ponche, Arnaud

2016-01-30

The purpose of this study is to evaluate the potential of simple high performance liquid chromatography (HPLC) setup for quantification of adsorbed proteins on various type of plane substrates with limited area (<3 cm(2)). Protein quantification was investigated with a liquid chromatography chain equipped with a size exclusion column or a reversed-phase column. By evaluating the validation of the method according to guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), all the results obtained by HPLC were reliable. By simple adsorption test at the contact of hydrophilic (glass) and hydrophobic (polydimethylsiloxane: PDMS) surfaces, kinetics of adsorption were determined and amounts of adsorbed bovine serum albumin, myoglobin and lysozyme were obtained: as expected for each protein, the amount adsorbed at the plateau on glass (between 0.15 μg/cm(2) and 0.4 μg/cm(2)) is lower than for hydrophobic PDMS surfaces (between 0.45 μg/cm(2) and 0.8 μg/cm(2)). These results were consistent with bicinchoninic acid protein determination. According to ICH guidelines, both Reversed Phase and Size Exclusion HPLC can be validated for quantification of adsorbed protein. However, we consider the size exclusion approach more interesting in this field because additional informations can be obtained for aggregative proteins. Indeed, monomer, dimer and oligomer of bovine serum albumin (BSA) were observed in the chromatogram. On increasing the temperature, we found a decrease of peak intensity of bovine serum albumin as well as the fraction of dimer and oligomer after contact with PDMS and glass surface. As the surface can act as a denaturation parameter, these informations can have a huge impact on the elucidation of the interfacial behavior of protein and in particular for aggregation processes in pharmaceutical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

New Stability-Indicating RP-HPLC Method for Determination of Diclofenac Potassium and Metaxalone from their Combined Dosage Form

PubMed Central

Panda, Sagar Suman; Patanaik, Debasis; Ravi Kumar, Bera V. V.

2012-01-01

A simple, precise and accurate isocratic RP-HPLC stability-indicating assay method has been developed to determine diclofenac potassium and metaxalone in their combined dosage forms. Isocratic separation was achieved on a Hibar-C18, Lichrosphere-100® (250 mm × 4.6 mm i.d., particle size 5 μm) column at room temperature in isocratic mode, the mobile phase consists of methanol: water (80:20, v/v) at a flow rate of 1.0 ml/min, the injection volume was 20 μl and UV detection was carried out at 280nm. The drug was subjected to acid and alkali hydrolysis, oxidation, photolysis and heat as stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and system suitability. The method was linear in the drug concentration range of 2.5–30 μg/ml and 20–240 μg/ml for diclofenac potassium and metaxalone, respectively. The precision (RSD) of six samples was 0.83 and 0.93% for repeatability, and the intermediate precision (RSD) among six-sample preparation was 1.63 and 0.49% for diclofenac potassium and metaxalone, respectively. The mean recoveries were between 100.99–102.58% and 99.97–100.01% for diclofenac potassium and metaxalone, respectively. The proposed method can be used successfully for routine analysis of the drug in bulk and combined pharmaceutical dosage forms. PMID:22396909

Electrospun polystyrene/graphene nanofiber film as a novel adsorbent of thin film microextraction for extraction of aldehydes in human exhaled breath condensates.

PubMed

Huang, Jing; Deng, Hongtao; Song, Dandan; Xu, Hui

2015-06-09

In the current study, we introduced a novel polystyrene/graphene (PS/G) composite nanofiber film for thin film microextraction (TFME) for the first time. The PS/G nanofiber film was fabricated on the surface of filter paper by a facile electrospinning method. The morphology and extraction performance of the resultant composite film were investigated systematically. The PS/G nanofiber film exhibited porous fibrous structure, large surface area and strong hydrophobicity. A new thin film microextraction-high performance liquid chromatography (TFME-HPLC) method was developed for the determination of six aldehydes in human exhaled breath condensates. The method showed high enrichment efficiency and fast analysis speed. Under the optimal conditions, the linear ranges of the analytes were in the range of 0.02-30 μmol L(-1) with correlation coefficients above 0.9938, and the recoveries were between 79.8% and 105.6% with the relative standard deviation values lower than 16.3% (n=5). The limits of quantification of six aldehydes ranged from 13.8 to 64.6 nmol L(-1). The established method was successfully applied for the quantification of aldehyde metabolites in exhaled breath condensates of lung cancer patients and healthy people. Taken together, the TFME-HPLC method provides a simple, rapid, sensitive, cost-effective, non-invasion approach for the analysis of linear aliphatic aldehydes in human exhaled breath condensates. Copyright © 2015 Elsevier B.V. All rights reserved.

A Fast and Validated Reversed-Phase HPLC Method for Simultaneous Determination of Simvastatin, Atorvastatin, Telmisartan and Irbesartan in Bulk Drugs and Tablet Formulations

PubMed Central

Alhazmi, Hassan A.; Alnami, Ahmed M.; Arishi, Mohammed A. A.; Alameer, Raad K.; Al Bratty, Mohammed; Rehman, Zia ur; Javed, Sadique A.; Arbab, Ismail A.

2017-01-01

The aim of this study was to develop and validate a fast and simple reversed-phase HPLC method for simultaneous determination of four cardiovascular agents—atorvastatin, simvastatin, telmisartan and irbesartan in bulk drugs and tablet oral dosage forms. The chromatographic separation was accomplished by using Symmetry C18 column (75 mm × 4.6 mm; 3.5 μ) with a mobile phase consisting of ammonium acetate buffer (10 mM; pH 4.0) and acetonitrile in a ratio 40:60 v/v. Flow rate was maintained at 1 mL/min up to 3.5 min, and then suddenly changed to 2 mL/min till the end of the run (7.5 min). The data was acquired using ultraviolet detector monitored at 220 nm. The method was validated for linearity, precision, accuracy and specificity. The developed method has shown excellent linearity (R2 > 0.999) over the concentration range of 1–16 µg/mL. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.189–0.190 and 0.603–0.630 µg/mL, respectively. Inter-day and intra-day accuracy and precision data were recorded in the acceptable limits. The new method has successfully been applied for quantification of all four drugs in their tablet dosage forms with percent recovery within 100 ± 2%. PMID:29257120

HPLC-DAD-ELSD Combined Pharmacodynamics and Serum Medicinal Chemistry for Quality Assessment of Huangqi Granule

PubMed Central

Zhao, Yang; Gong, Xiao-Jian; He, Yan; Ma, Feng-Wei; Zhou, Mei; Zhao, Chao; Niu, Yi; Deng, Jie

2015-01-01

Objective To more scientifically and reasonably control the quality of Huangqi Granules, preliminary studies on the pharmacodynamics and serum pharmacochemistry of this medicine were performed. DPPH and MTT experiments showed that water extracts of Huangqi Granules had good antioxidant activity and increased immunity. Timed blood samples collected 5 min, 15 min, and 30 min after oral administration of a set amount of Huangqi Granules were collected and tested using UPLC-ESI-MS/MS. As a result, calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, and formononetin were found to exist in rat blood after dosing, indicating that the five chemical compounds might have pharmacological activity, and based on this result, they were designated biomarkers for quality control of Huangqi Granules. Consequently, a simple, rapid and efficient method was developed in the present study for the simultaneous determination of the five characteristic compounds in Huangqi Granules using HPLC-DAD-ELSD. Materials and Methods The separation was performed using an Agilent Hypersil ODS column (4.6 × 250 mm, 5 μm) at 30 ℃. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B) with a flow rate of 1 mL/min. The drift tube temperature of the ELSD system was set to 85 ℃, and the nitrogen pressure was 3.5 bar. Results All five characteristic compounds had good linear behavior with r2 values greater than 0.9972. The recoveries varied from 96.31% to 101.22%. Subsequently, the developed method was applied to evaluate the quality of Huangqi Granules from different batches, and hierarchical clustering analysis (HCA) was used to analyze the classification of the samples based on the values of the five compounds. Conclusion The established HPLC method combined with HCA proved to be effective to evaluate the quality of Huangqi Granules. PMID:25915040

Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue.

PubMed

Van Dam, Debby; Vermeiren, Yannick; Aerts, Tony; De Deyn, Peter Paul

2014-08-01

A fast and simple RP-HPLC method with electrochemical detection (ECD) and ion pair chromatography was developed, optimized and validated in order to simultaneously determine eight different biogenic amines and metabolites in post-mortem human brain tissue in a single-run analytical approach. The compounds of interest are the indolamine serotonin (5-hydroxytryptamine, 5-HT), the catecholamines dopamine (DA) and (nor)epinephrine ((N)E), as well as their respective metabolites, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxy-3-indoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). A two-level fractional factorial experimental design was applied to study the effect of five experimental factors (i.e. the ion-pair counter concentration, the level of organic modifier, the pH of the mobile phase, the temperature of the column, and the voltage setting of the detector) on the chromatographic behaviour. The cross effect between the five quantitative factors and the capacity and separation factors of the analytes were then analysed using a Standard Least Squares model. The optimized method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). Our human brain tissue sample preparation procedure is straightforward and relatively short, which allows samples to be loaded onto the HPLC system within approximately 4h. Additionally, a high sample throughput was achieved after optimization due to a total runtime of maximally 40min per sample. The conditions and settings of the HPLC system were found to be accurate with high intra and inter-assay repeatability, recovery and accuracy rates. The robust analytical method results in very low detection limits and good separation for all of the eight biogenic amines and metabolites in this complex mixture of biological analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of the desulfurization of phenothiazines for a sensitive detection method by high-performance liquid chromatography.

PubMed

Shimada, K; Mino, T; Nakajima, M; Wakabayashi, H; Yamato, S

1994-11-04

A simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of phenothiazine (PHE) is described. PHE is converted to diphenylamine (DIP) by desulfurization with Raney nickel catalyst. DIP is highly sensitive to electrochemical detection. The calibration graph for PHE quantification after desulfurization was linear between 0.1 and 2.0 ng per injection. The detection limit (signal-to-noise ratio = 3) of PHE after desulfurization was 10 pg, which is twenty times higher than that of the parent compound PHE. The proposed desulfurization technique was applied to other PHE-related compounds. The structural confirmation of the desulfurized product of PHE was carried out by LC-MS using atmospheric pressure chemical ionization.

Short communication: Determination of withdrawal time for oxytetracycline in different types of goats for milk consumption.

PubMed

Attaie, Rahmat; Bsharat, Mohammed; Mora-Gutierrez, Adela; Woldesenbet, Sela

2015-07-01

Antibiotics are widely used in animal husbandry and the presence of antibiotics in milk is a health hazard. The objective of this study was to determine residual amounts of oxytetracycline in fresh, aged, and pasteurized milk of 3 breeds of goats using HPLC analysis. It was also essential to determine the safe withdrawal period of oxytetracycline in lactating goats. The quantitative results obtained using the HPLC system were compared with the tolerance limit of oxytetracycline in milk in the United States. Fifteen milking does, 5 Nubians, 5 Alpines, and 5 LaManchas were randomly selected from the milking herd at the International Goat Research Center at Prairie View A&M University. A simple sample preparation and isocratic HPLC method using ultraviolet detection was used for analysis of milk samples. The HPLC results indicated that the withdrawal period of oxytetracycline in treated Alpine does was 82h (7 milking), whereas for Nubian does the period was 58h (5 milking), and for LaManchas the period was 72h (6 milking) after drug administration. The overall withdrawal period for all the treated goats of 3 breeds was 72h. Although these results indicated that the depletion rate of this antibiotic was faster in goats than the reported data for cows, the 96-h withdrawal period that is currently used for lactating cows is still necessary for these 3 breeds of goats. Additionally, our results indicated that oxytetracycline is not stable in goat milk at refrigeration temperature or during pasteurization and will decrease significantly. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Determination of very low levels of 5-(hydroxymethyl)-2-furaldehyde (HMF) in natural honey: comparison between the HPLC technique and the spectrophotometric white method.

PubMed

Truzzi, Cristina; Annibaldi, Anna; Illuminati, Silvia; Finale, Carolina; Rossetti, Monica; Scarponi, Giuseppe

2012-07-01

In this work we compared 2 official methods for the determination of HMF in honey, the spectrophotometric White method and the HPLC method (International Honey Commission) for the determination of HMF in unifloral honey and honeydew samples with a very low HMF content (<4 mg/kg), which is the most critical determination in terms of accuracy and precision of methods. In honey solutions, the limits of quantification for HPLC and White methods are 0.83 mg/L and 0.67 mg/L, respectively, and the linearity range is confirmed up to 20 mg/L for the HPLC method and up to 5 mg/L for the White method. In honeys with HMF >5 mg/kg, the molar extinction coefficient is 15369, lower than the literature value of 16830, and should be used for HMF determination. For samples with HMF content in the range 1-4 mg/kg the accuracy of the 2 methods is comparable both for unifloral and honeydew samples, whereas as regards precision, the HPLC method gives better results (3.5% compared with 6.4% for the White method). So, in general, the HPLC method seems to be more appropriate for the determination of HMF in honey in the range 1-4 mg/kg thanks to its greater precision, but for samples with a HMF content of less than 1 mg/kg the analyses are inaccurate for both methods. This work can help governmental and private laboratories that perform food analyses to choose the best method for the determination of HMF at very low levels in unifloral honey and honeydew samples. © 2012 Institute of Food Technologists®

Comparison of the BioRad Variant and Primus Ultra2 high-pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins.

PubMed

Gosselin, R C; Carlin, A C; Dwyre, D M

2011-04-01

Hemoglobin variants are a result of genetic changes resulting in abnormal or dys-synchronous hemoglobin chain production (thalassemia) or the generation of hemoglobin chain variants such as hemoglobin S. Automated high-pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies. In this study, we evaluated the performance of two HPLC methods used in the detection of common hemoglobin variants: Variant and Ultra2. There were 377 samples tested, 26% (99/377) with HbS, 8.5% (32/377) with HbC, 20.7% (78/377) with other hemoglobin variant or thalassemia, and 2.9% with increased hemoglobin A(1) c. The interpretations of each chromatograph were compared. There were no differences noted for hemoglobins A(0), S, or C. There were significant differences between HPLC methods for hemoglobins F, A(2), and A(1) c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). Both Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A(2), and between hemoglobins S and F using the Ultra2 HPLC method. © 2010 Blackwell Publishing Ltd.

Quantitative determination and sampling of azathioprine residues for cleaning validation in production area.

PubMed

Fazio, Tatiana Tatit; Singh, Anil Kumar; Kedor-Hackmann, Erika Rosa Maria; Santoro, Maria Inês Rocha Miritello

2007-03-12

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.

Determination of methylglyoxal in human blood plasma using fluorescence high performance liquid chromatography after derivatization with 1,2-diamino-4,5-methylenedioxybenzene.

PubMed

Ogasawara, Yuki; Tanaka, Ryo; Koike, Shin; Horiuchi, Yasue; Miyashita, Mitsuhiro; Arai, Makoto

2016-09-01

Methylglyoxal (MG) is a highly reactive dicarbonyl compound that promotes the non-enzymatic glycation of proteins to yield irreversible advanced glycated end products, leading to the cross-linking or degradation of proteins. The physiological relevance of MG currently remains unclear because its metabolic behavior has not yet been elucidated in detail. Although several labeling methods that require a HPLC system have been developed and used to measure MG, a standard method to analyze the content of MG in biological samples has not been established. We herein present a practical method based on HPLC with fluorescence detection to measure low MG levels. MG concentrations were also measured in human blood plasma using the present method in order to demonstrate its utility. A calibration curve was produced using freshly purified MG at concentrations ranging between 0.05 and 1.0μM. The intra-day and inter-day relative standard diviations of the method were 2.55% and 4.03%, respectively. The limit of detection and limit of quantification were 60fmol and 200fmol, respectively for MG with a 10-μl injection volume of the derivatized sample solution. When the optimized method was applied to human plasma, the resulting concentrations of MG in the plasma of healthy subjects (n=23) ranged between 0.024 and 0.258μM (mean±SD=0.098±0.066). Thus, the method developed herein is simple, sensitive, and easy to operate for the measurement of MG in biological samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis.

PubMed

Gobbo-Neto, Leonardo; Lopes, Norberto P

2008-02-27

Lychnophora ericoides Mart. (Asteraceae, Vernonieae) is a plant, endemic to Brazil, with occurrence restricted to the "cerrado" biome. Traditional medicine employs alcoholic and aqueous-alcoholic preparations of leaves from this species for the treatment of wounds, inflammation, and pain. Furthermore, leaves of L. ericoides are also widely used as flavorings for the Brazilian traditional spirit "cachaça". A method has been developed for the extraction and HPLC-DAD analysis of the secondary metabolites of L. ericoides leaves. This analytical method was validated with 11 secondary metabolites chosen to represent the different classes and polarities of secondary metabolites occurring in L. ericoides leaves, and good responses were obtained for each validation parameter analyzed. The same HPLC analytical method was also employed for online secondary metabolite identification by HPLC-DAD-MS and HPLC-DAD-MS/MS, leading to the identification of di- C-glucosylflavones, coumaroylglucosylflavonols, flavone, flavanones, flavonols, chalcones, goyazensolide, and eremantholide-type sesquiterpene lactones and positional isomeric series of chlorogenic acids possessing caffeic and/or ferulic moieties. Among the 52 chromatographic peaks observed, 36 were fully identified and 8 were attributed to compounds belonging to series of caffeoylferuloylquinic and diferuloylquinic acids that could not be individualized from each other.

Development of a low-cost method of analysis for the qualitative and quantitative analysis of butyltins in environmental samples.

PubMed

Bangkedphol, Sornnarin; Keenan, Helen E; Davidson, Christine; Sakultantimetha, Arthit; Songsasen, Apisit

2008-12-01

Most analytical methods for butyltins are based on high resolution techniques with complicated sample preparation. For this study, a simple application of an analytical method was developed using High Performance Liquid Chromatography (HPLC) with UV detection. The developed method was studied to determine tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT) in sediment and water samples. The separation was performed in isocratic mode on an ultra cyanopropyl column with a mobile phase of hexane containing 5% THF and 0.03% acetic acid. This method was confirmed using standard GC/MS techniques and verified by statistical paired t-test method. Under the experimental conditions used, the limit of detection (LOD) of TBT and DBT were 0.70 and 0.50 microg/mL, respectively. The optimised extraction method for butyltins in water and sediment samples involved using hexane containing 0.05-0.5% tropolone and 0.2% sodium chloride in water at pH 1.7. The quantitative extraction of butyltin compounds in a certified reference material (BCR-646) and naturally contaminated samples was achieved with recoveries ranging from 95 to 108% and at %RSD 0.02-1.00%. This HPLC method and optimum extraction conditions were used to determine the contamination level of butyltins in environmental samples collected from the Forth and Clyde canal, Scotland, UK. The values obtained severely exceeded the Environmental Quality Standard (EQS) values. Although high resolution methods are utilised extensively for this type of research, the developed method is cheaper in both terms of equipment and running costs, faster in analysis time and has comparable detection limits to the alternative methods. This is advantageous not just as a confirmatory technique but also to enable further research in this field.

Qualitative and quantitative analyses of Epimedium wushanense by high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry.

PubMed

Li, Hui-fang; Guan, Xiang-yu; Ye, Min; Xiang, Cheng; Lin, Chang-hu; Sun, Chao; Guo, De-an

2011-06-01

A new HPLC-DAD-ESI-MS(n) method was developed for rapid separation, characterization and quantitation of flavonoids in Epimedium wushanense, a popular Chinese herbal medicine. For qualitative identification, a total of 37 compounds were characterized from the underground and aerial parts of E. wushanense. Among them, 28 compounds were prenylated flavonoids, and 23 were confirmed by comparing with reference standards. For quantitative analysis, 12 major flavonoids including kaempferol glycosides, desmethylicaritin glycosides, and icaritin glycosides were simultaneously determined by HPLC/UV. Samples were separated on a Waters Symmetry C(18) column at 35 °C eluted with a gradient three-component mobile phase of acetonitrile, methanol, and water containing 0.03% v/v formic acid. All the flavonoids showed good linearity (r(2) ≥0.9997). The recoveries varied from 92.6 to 106.1% at three concentration levels. This method was applied to the determination of 20 samples of different geographical sources, harvesting time, and plant parts. Contents of the predominant flavonoid, epimedin C, ranged from 1.4 to 5.1% in aerial parts and 1.0 to 2.8% in underground parts. The methods established in this paper were simple and reliable and could be used for the quality control of E. wushanense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors.

PubMed

Qi, Lian-Wen; Yu, Qing-Tao; Li, Ping; Li, Song-Lin; Wang, Yu-Xia; Sheng, Liang-Hong; Yi, Ling

2006-11-17

A method, high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD), was developed to evaluate the quality of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins. The wavelength at 280 nm was chosen to determine six isoflavonoids: calycosin-7-O-beta-D-glucoside (1), ononin (2), (6alphaR, 11alphaR)-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (3), (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (4), calycosin (5), and formononetin (6); and ELSD connected after DAD was employed to determine four saponins: astragaloside IV (7), astragaloside II (8), astragaloside I (9), and acetylastragaloside I (10). This assay was fully validated with respect to precision, repeatability and accuracy. The proposed method was successfully applied to quantify the ten components in eleven samples from different localities in China; significant variations were demonstrated in the content of these compounds in the samples from different areas. This simple, rapid, low-cost and reliable HPLC-DAD-ELSD method is suitable for routine quantitative analysis and quality control of traditional Chinese medicines (TCMs) consisting of bioactive multi-components with different structures such as Radix Astragali.

Determination of captopril in biological samples by high-performance liquid chromatography with ThioGlo 3 derivatization.

PubMed

Aykin, N; Neal, R; Yusof, M; Ercal, N

2001-11-01

Captopril, a well-known angiotensin converting enzyme (ACE) inhibitor, is widely used for treatment of arterial hypertension. Recent studies suggest that it may also act as a scavenger of free radicals because of its thiol group. Therefore, the present study describes a rapid, sensitive and relatively simple method for the detection of captopril in biological tissues with reverse-phase HPLC. Captopril was first derivatized with ThioGlo 3 [3H-Naphto[2,1-b]pyran,9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl-3-oxo-)]. It was then detected by fluorescence-HPLC using an Astec C(18) column as the stationary phase and a water:acetonitrile:acetic acid:phosphoric acid mixture (50:50; 1 mL/L acids) as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for captopril was linear over a range of 10-2500 nM and the coefficient of variation acquired for the within- and between-run precision for captopril was 0.5 and 3.8%, respectively. The detection limit of captopril with this method was found to be 200 fmol/20 microL injection volume. Its relative recovery from biological samples was determined to the range from 93.3 to 105.3%. Based on these results, we believe that our method is advantageous for captopril determination. Copyright 2001 John Wiley & Sons, Ltd.

Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation.

PubMed

Shurbaji, Maher; Abu Al Rub, Mohamad H; Saket, Munib M; Qaisi, Ali M; Salim, Maher L; Abu-Nameh, Eyad S M

2010-01-01

A rapid, simple, and sensitive RP-HPLC analytical method was developed for the simultaneous determination of triclabendazole and ivermectin in combination using a C18 RP column. The mobile phase was acetonitrile-methanol-water-acetic acid (56 + 36 + 7.5 + 0.5, v/v/v/v) at a pH of 4.35 and flow rate of 1.0 mL/min. A 245 nm UV detection wavelength was used. Complete validation, including linearity, accuracy, recovery, LOD, LOQ, precision, robustness, stability, and peak purity, was performed. The calibration curve was linear over the range 50.09-150.26 microg/mL for triclabendazole with r = 0.9999 and 27.01-81.02 microg/mL for ivermectin with r = 0.9999. Calculated LOD and LOQ for triclabendazole were 0.03 and 0.08 microg/mL, respectively, and for ivermectin 0.07 and 0.20 microg/mL, respectively. The intraday precision obtained was 98.71% with RSD of 0.87% for triclabendazole and 100.79% with RSD 0.73% for ivermectin. The interday precision obtained was 99.51% with RSD of 0.35% for triclabendazole and 100.55% with RSD of 0.59% for ivermectin. Robustness was also studied, and there was no significant variation of the system suitability of the analytical method with small changes in experimental parameters.

[Determination of fluorescent whitening agents in plastic food contact materials by high performance liquid chromatography with fluorescence detector].

PubMed

Jiao, Yanna; Ding, Li; Zhu, Shaohua; Fu, Shanliang; Gong, Qiang; Li, Hui; Wang, Libing

2013-01-01

A method for the determination of fluorescent whitening agents in plastic food contact materials by high performance liquid chromatography (HPLC) with fluorescence detector was developed. The samples were extracted with trichloromethane by sonication for 30 min at 40 degrees C. The HPLC method was performed on a column of Eclipse XDB-C18 (250 mm x 4.6 mm, 5 microm) by gradient elution using 5 mmol/L ammonium acetate and acetonitrile as the mobile phases, and detected by the fluorescence detector at an excitation wavelength of 350 nm and an emission wavelength of 430 nm. The experimental results indicated that the four fluorescent whitening agents were separated well. The limits of detection (LOD) (S/N = 3) were 0.3, 0.1, 0.05, 0.14 mg/L, and the limits of quantification (LOQ) (S/N = 10) were 1.0, 0.4, 0.2, 0.5 mg/L for 1,4-bis (4-cyanostyryl) benzene (C. I. 199), 1,4-bis (2-benzoxazolyl) naphthalene (C. I. 367), 4,4'-bis(2-methoxystyryl) biphenyl (C. I. 378) and 2,5-thiophenediylbis (5-tert-butyl-1,3-benzoxazole) (C. I. 184), respectively. Good linearities with correlation coefficients (r2) not less than 0.991 were obtained. The proposed method is simple, accurate, sensitive and can meet the requirements of the routine determination of fluorescent whitening agents in entry-exit products.

Determination of itopride hydrochloride in human plasma by RP-HPLC with fluorescence detection and its use in bioequivalence study.

PubMed

Ma, Jing; Yuan, Li-Hua; Ding, Mei-Juan; Zhang, Jun; Zhang, Qing; Xu, Qun-Wei; Zhou, Xue-Min

2009-03-01

A sensitive, selective and simple method using a precipitation of protein with 10% perchloric acid, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of itopride hydrochloride in human plasma, using levofloxacin as the internal standard (IS). Chromatographic separation was obtained within 7.0 min using a reverse phase Hypersil BDS C(18) (250 mm x 4.6 mm, 5 microm) column and an isocratic mobile phase, constituting of a mixture of 0.1 mol/l ammonium acetate-methanol (30:70, v/v) flowing at 1.1 ml/min. The excitation and emission wavelengths were set at 304 and 344 nm, respectively. The method was validated over the concentration range of 5 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 5 ng/ml. The extractive recovery of itopride hydrochloride from the biological matrix was more than 80.77%. The intra-day accuracy of the drug containing serum samples was more than 82.94% with a precision of 2.81-4.37%. The inter-day accuracy was 82.91% or more, with a precision of 6.89-9.54%. The limit we have used (70-143%) is based on the local regulatory authority (SFDA). The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

New validated method for piracetam HPLC determination in human plasma.

PubMed

Curticapean, Augustin; Imre, Silvia

2007-01-10

The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.

[Determination of perfluoroalkyl acids in lamb liver by high performance liquid chromatography- tandem mass spectrometry combined with dispersive solid phase extraction].

PubMed

Zhu, Pingping; Yue, Zhenfeng; Zheng, Zongkun; Zhang, Yi; Li, Wenyin; Zhao, Fengjuan; Xiao Chengui; Bai, Runye; Lin, Wei

2015-05-01

A method was developed for the determination of 19 perfluoroalkyl acids (PFAs) in lamb liver by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) combined with dispersive solid phase extraction. The sample was extracted with acidified acetonitrile, and then cleaned-up by a mixture of N-propylethylenediamine (PSA), C18 and graphitized carbon black (GCB) sorbents. The 19 PFAs were analyzed by HPLC-MS/MS with a C18 chromatographic column, adopting the multiple reaction monitoring (MRM) mode with negative electrospray ionization. The effects of the dosages of hydrochloric acid and the sorbents on the recoveries of the 19 PFAs were studied. For accurate quantitative analysis, the isotope internal standard method was used. The calibration curves were linear with the correlation coefficients over 0.998 in the range of 0.05-20 µg/kg for the 19 PFAs. The limits of detection were 0.004-0.111 µg/kg. The limits of quantification were 0.012-0.370 µg/kg. The mean recoveries of the 19 PFAs at spiked levels of 0.5, 1.0, 2.0 µg/kg were in the range from 80% to 128% with the relative standard deviations of 0.31%-11.1%. The developed method is rapid, simple, accurate. It is suitable for the determination of the 19 PFAs in large quantities of lamb liver samples.

A high-performance liquid chromatography method for the serotonin release assay is equivalent to the radioactive method.

PubMed

Sono-Koree, N K; Crist, R A; Frank, E L; Rodgers, G M; Smock, K J

2016-02-01

The serotonin release assay (SRA) is considered the gold standard laboratory test for heparin-induced thrombocytopenia (HIT). The historic SRA method uses platelets loaded with radiolabeled serotonin to evaluate platelet activation by HIT immune complexes. However, a nonradioactive method is desirable. We report the performance characteristics of a high-performance liquid chromatography (HPLC) SRA method. We validated the performance characteristics of an HPLC-SRA method, including correlation with a reference laboratory using the radioactive method. Serotonin released from reagent platelets was quantified by HPLC using fluorescent detection. Results were expressed as % release and classified as positive, negative, or indeterminate based on previously published cutoffs. Serum samples from 250 subjects with suspected HIT were tested in the HPLC-SRA and with the radioactive method. Concordant classifications were observed in 230 samples (92%). Sera from 41 healthy individuals tested negative. Between-run imprecision studies showed standard deviation of <6 (% release) for positive, weak positive, and negative serum pools. Stability studies demonstrated stability after two freeze-thaw cycles or up to a week of refrigeration. The HPLC-SRA has robust performance characteristics, equivalent to the historic radioactive method, but avoids the complexities of working with radioactivity. © 2015 John Wiley & Sons Ltd.

HPLC and UPLC methods for the determination of zearalenone in noodles, cereal snacks and infant formula.

PubMed

Ok, Hyun Ee; Choi, Sung-Wook; Kim, Meehye; Chun, Hyang Sook

2014-11-15

High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were compared to validate a method for determination of zearalenone (ZON) in noodles, cereal snacks, and infant formulas. The limits of detection and quantification in HPLC and UPLC were found to be 4.0 and 13.0 μg kg(-1) and 2.5 and 8.3 μg kg(-1), respectively. The average recoveries of ZON by HPLC and UPLC ranged from 79.1% to 105.3% and from 85.1% to 114.5%, respectively. The measurement uncertainties of the two methods for ZON determination were within the maximum standard uncertainty. The two methods showed that the levels of ZON in 163 naturally contaminated samples ranged from 4.3 to 8.3 μg kg(-1) by HPLC and 3.1 to 17.6 μg kg(-1) by UPLC. These findings indicate that either method is suitable for the determination of ZON in noodles, cereal snacks, and infant formulas, but UPLC gives faster results with better sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quantification of allantoin in various Zea mays L. hybrids by RP-HPLC with UV detection.

PubMed

Maksimović, Z; Malenović, A; Jancić, B; Kovacević, N

2004-07-01

A RP-HPLC method for quantification of allantoin in silk of fifteen maize hybrids (Zea mays L., Poaceae) was described. Following extraction of the plant material with an acetone-water (7:3, VN) mixture, filtration and dilution, the extracts were analyzed without previous chemical derivatization. Separation and quantification were achieved using an Alltech Econosil C18 column under isocratic conditions at 40 degrees C. The mobile phase flow (20% methanol--80% water with 5 mM sodium laurylsulfate added at pH 2.5, adjusted with 85% orthophosphoric acid; pH of water phase was finally adjusted at 6.0 by addition of triethylamine) was maintained at 1.0 mL/min. Column effluent was monitored at 235 nm. This simple procedure afforded efficient separation and quantification of allantoin in plant material, without interference of polyphenols or other plant constituents of medium to high polarity, or similar UV absorption. Our study revealed that the silk of all investigated maize hybrids could be considered relatively rich in allantoin, covering the concentration range between 215 and 289 mg per 100 g of dry plant material.

Solid-phase microextraction coupled with high performance liquid chromatography: a complementary technique to solid-phase microextraction-gas chromatography for the analysis of pesticide residues in strawberries.

PubMed

Wang, Z; Hennion, B; Urruty, L; Montury, M

2000-11-01

Solid-phase microextraction coupled with high performance liquid chromatography has been studied for the analysis of methiocarb, napropamide, fenoxycarb and bupirimate in strawberries. The strawberries were blended and centrifuged. Then, an aliquot of the resulting extracting solution was subjected to solid-phase microextraction (SPME) on a 60 microns polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre for 45 min at room temperature. The extracted pesticides on the SPME fibre were desorbed into SPME/high performance liquid chromatography (HPLC) interface for HPLC analysis with diode-array detection (DAD). The method is organic solvent-free for the whole extraction process and is simple and easy to manipulate. The detection limits were shown to be at low microgram kg-1 level and the linear response covered the range from 0.05 to 2 mg kg-1 of pesticides in strawberries with a regression coefficient larger than 0.99. A good repeatability with RSDs between 2.92 and 9.25% was obtained, depending on compounds.

Chromatographic and Spectral Analysis of Two Main Extractable Compounds Present in Aqueous Extracts of Laminated Aluminum Foil Used for Protecting LDPE-Filled Drug Vials

PubMed Central

Akapo, Samuel O.; Syed, Sajid; Mamangun, Anicia; Skinner, Wayne

2009-01-01

Laminated aluminum foils are increasingly being used to protect drug products packaged in semipermeable containers (e.g., low-density polyethylene (LDPE)) from degradation and/or evaporation. The direct contact of such materials with primary packaging containers may potentially lead to adulteration of the drug product by extractable or leachable compounds present in the closure system. In this paper, we described a simple and reliable HPLC method for analysis of an aqueous extract of laminated aluminum foil overwrap used for packaging LDPE vials filled with aqueous pharmaceutical formulations. By means of combined HPLC-UV, GC/MS, LC/MS/MS, and NMR spectroscopy, the two major compounds detected in the aqueous extracts of the representative commercial overwraps were identified as cyclic oligomers with molecular weights of 452 and 472 and are possibly formed from poly-condensation of the adhesive components, namely, isophthalic acid, adipic acid, and diethylene glycol. Lower molecular weight compounds that might be associated with the “building blocks” of these compounds were not detected in the aqueous extracts. PMID:20140083

Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

PubMed

Zhang, Hongyan; Zhang, Yan; Wang, Shuo

2008-08-20

Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

Linear regression analysis and its application to multivariate chromatographic calibration for the quantitative analysis of two-component mixtures.

PubMed

Dinç, Erdal; Ozdemir, Abdil

2005-01-01

Multivariate chromatographic calibration technique was developed for the quantitative analysis of binary mixtures enalapril maleate (EA) and hydrochlorothiazide (HCT) in tablets in the presence of losartan potassium (LST). The mathematical algorithm of multivariate chromatographic calibration technique is based on the use of the linear regression equations constructed using relationship between concentration and peak area at the five-wavelength set. The algorithm of this mathematical calibration model having a simple mathematical content was briefly described. This approach is a powerful mathematical tool for an optimum chromatographic multivariate calibration and elimination of fluctuations coming from instrumental and experimental conditions. This multivariate chromatographic calibration contains reduction of multivariate linear regression functions to univariate data set. The validation of model was carried out by analyzing various synthetic binary mixtures and using the standard addition technique. Developed calibration technique was applied to the analysis of the real pharmaceutical tablets containing EA and HCT. The obtained results were compared with those obtained by classical HPLC method. It was observed that the proposed multivariate chromatographic calibration gives better results than classical HPLC.

A simple and sensitive method for the determination of fibric acids in the liver by liquid chromatography.

PubMed

Karahashi, Minako; Fukuhara, Hiroto; Hoshina, Miki; Sakamoto, Takeshi; Yamazaki, Tohru; Mitsumoto, Atsushi; Kawashima, Yoichi; Kudo, Naomi

2014-01-01

Fibrates are used in biochemical and pharmacological studies as bioactive tools. Nevertheless, most studies have lacked information concerning the concentrations of fibric acids working inside tissues because a simple and sensitive method is not available for their quantitation. This study aimed to develop a simple and sensitive bioanalytical method for the quantitation of clofibric, bezafibric and fenofibric acids in samples of very small portions of tissues. Fibric acids were extracted into n-hexane-ethyl acetate from tissue homogenates (10 mg of liver, kidney or muscle) or serum (100 µL) and were derivatized with 4-bromomethyl-6,7-dimethoxycoumarin, followed by HPLC with fluorescence detection. These compounds were separated isocratically on a reversed phase with acetonitrile-water. Standard analytical curves were linear over the concentration range of 0.2-20 nmol/10 mg of liver. Precision and accuracy were within acceptable limits. Recovery from liver homogenates ranged from 93.03 to 112.29%. This method enabled the quantitation of fibric acids in 10 mg of liver from rats treated with clofibric acid, bezafibric acid or fenofibrate. From these analytical data, it became clear that there was no large difference in ratio of acyl-CoA oxidase 1 (Acox1) mRNA level to fibric acid content in the liver among the three fibric acids, suggesting that these three fibric acids have similar potency to increase expression of the Acox1 gene, which is a target of peroxisome proliferator-activated receptor α. Thus, the proposed method is a simple, sensitive and reliable tool for the quantitation of fibric acids working in vivo inside livers.

Genetic, epigenetic, and HPLC fingerprint differentiation between natural and ex situ populations of Rhodiola sachalinensis from Changbai Mountain, China.

PubMed

Zhao, Wei; Shi, Xiaozheng; Li, Jiangnan; Guo, Wei; Liu, Chengbai; Chen, Xia

2014-01-01

Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R2 = 0.98 for genetic and epigenetic distance; R2 = 0.90 for DNA methylation level and altitude; R2 = -0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis.

Genetic, Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain, China

PubMed Central

Zhao, Wei; Shi, Xiaozheng; Li, Jiangnan; Guo, Wei; Liu, Chengbai; Chen, Xia

2014-01-01

Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R2 = 0.98 for genetic and epigenetic distance; R2 = 0.90 for DNA methylation level and altitude; R2 = –0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis. PMID:25386983

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

PubMed

Yang, Ting; Yang, Yi-Hua; Yang, Ju-Yun; Chen, Ben-Mei; Duan, Ju-Ping; Yu, Shu-Yi; Ouyang, Hong-Tao; Cheng, Jun-Ping; Chen, Yu-Xiang

2008-01-01

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts

PubMed Central

Saidan, Noor Hafizoh; Aisha, Abdalrahim F.A.; Hamil, Mohd Shahrul Ridzuan; Majid, Amin Malik Shah Abdul; Ismail, Zhari

2015-01-01

Background: Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. Objective: The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. Materials and Methods: The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 μm, 3 × 150 mm) with 18 min separation time. Results: The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. Conclusion: The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products. PMID:25598631

A green ultrasonic-assisted liquid-liquid microextraction based on deep eutectic solvent for the HPLC-UV determination of ferulic, caffeic and cinnamic acid from olive, almond, sesame and cinnamon oil.

PubMed

Khezeli, Tahere; Daneshfar, Ali; Sahraei, Reza

2016-04-01

A simple, inexpensive and sensitive ultrasonic-assisted liquid-liquid microextraction method based on deep eutectic solvent (UALLME-DES) was used for the extraction of three phenolic acids (ferulic, caffeic and cinnamic) from vegetable oils. In a typical experiment, deep eutectic solvent as green extraction solvent was added to n-hexane (as a typical oil medium) containing target analytes. Subsequently, the extraction was accelerated by sonication. After the extraction, phase separation (DES rich phase/n-hexane phase) was performed by centrifugation. DES rich phase (lower phase) was withdrawn by a micro-syringe and submitted to isocratic reverse-phase HPLC with UV detection. Under optimum conditions obtained by response surface methodology (RSM) and desirability function (DF), the method has good linear calibration ranges (between 1.30 and 1000 µg L(-1)), coefficients of determination (r(2)>0.9949) and low limits of detection (between 0.39 and 0.63 µg L(-1)). This procedure was successfully applied to the determination of target analytes in olive, almond, sesame and cinnamon oil samples. The relative mean recoveries ranged from 94.7% to 104.6%. Copyright © 2015 Elsevier B.V. All rights reserved.

Pharmacokinetics and tissue distribution study of Praeruptorin D from Radix peucedani in rats by high-performance liquid chromatography (HPLC).

PubMed

Liang, Taigang; Yue, Wenyan; Du, Xue; Ren, Luhui; Li, Qingshan

2012-01-01

Praeruptorin D (PD), a major pyranocoumarin isolated from Radix Peucedani, exhibited antitumor and anti-inflammatory activities. The aim of this study was to investigate the pharmacokinetics and tissue distribution of PD in rats following intravenous (i.v.) administration. The levels of PD in plasma and tissues were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The biosamples were treated by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) and osthole was used as the internal standard (IS). The chromatographic separation was accomplished on a reversed-phase C(18) column using methanol-water (75:25, v/v) as mobile phase at a flow rate of 0.8 mL/min and ultraviolet detection wave length was set at 323 nm. The results demonstrate that this method has excellent specificity, linearity, precision, accuracy and recovery. The pharmacokinetic study found that PD fitted well into a two-compartment model with a fast distribution phase and a relative slow elimination phase. Tissue distribution showed that the highest concentration was observed in the lung, followed by heart, liver and kidney. Furthermore, PD can also be detected in the brain, which indicated that PD could cross the blood-brain barrier after i.v. administration.

Estimation of the quantification uncertainty from flow injection and liquid chromatography transient signals in inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Laborda, Francisco; Medrano, Jesús; Castillo, Juan R.

2004-06-01

The quality of the quantitative results obtained from transient signals in high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) and flow injection-inductively coupled plasma mass spectrometry (FI-ICPMS) was investigated under multielement conditions. Quantification methods were based on multiple-point calibration by simple and weighted linear regression, and double-point calibration (measurement of the baseline and one standard). An uncertainty model, which includes the main sources of uncertainty from FI-ICPMS and HPLC-ICPMS (signal measurement, sample flow rate and injection volume), was developed to estimate peak area uncertainties and statistical weights used in weighted linear regression. The behaviour of the ICPMS instrument was characterized in order to be considered in the model, concluding that the instrument works as a concentration detector when it is used to monitorize transient signals from flow injection or chromatographic separations. Proper quantification by the three calibration methods was achieved when compared to reference materials, although the double-point calibration allowed to obtain results of the same quality as the multiple-point calibration, shortening the calibration time. Relative expanded uncertainties ranged from 10-20% for concentrations around the LOQ to 5% for concentrations higher than 100 times the LOQ.

ATR-FTIR spectroscopy for the determination of Na4EDTA in detergent aqueous solutions.

PubMed

Suárez, Leticia; García, Roberto; Riera, Francisco A; Diez, María A

2013-10-15

Fourier transform infrared spectroscopy in the attenuated total reflectance mode (ATR-FTIR) combined with partial last square (PLS) algorithms was used to design calibration and prediction models for a wide range of tetrasodium ethylenediaminetetraacetate (Na4EDTA) concentrations (0.1 to 28% w/w) in aqueous solutions. The spectra obtained using air and water as a background medium were tested for the best fit. The PLS models designed afforded a sufficient level of precision and accuracy to allow even very small amounts of Na4EDTA to be determined. A root mean square error of nearly 0.37 for the validation set was obtained. Over a concentration range below 5% w/w, the values estimated from a combination of ATR-FTIR spectroscopy and a PLS algorithm model were similar to those obtained from an HPLC analysis of NaFeEDTA complexes and subsequent detection by UV absorbance. However, the lowest detection limit for Na4EDTA concentrations afforded by this spectroscopic/chemometric method was 0.3% w/w. The PLS model was successfully used as a rapid and simple method to quantify Na4EDTA in aqueous solutions of industrial detergents as an alternative to HPLC-UV analysis which involves time-consuming dilution and complexation processes. © 2013 Elsevier B.V. All rights reserved.

[HPLC fingerprint of flavonoids in Sophora flavescens and determination of five components].

PubMed

Ma, Hong-Yan; Zhou, Wan-Shan; Chu, Fu-Jiang; Wang, Dong; Liang, Sheng-Wang; Li, Shao

2013-08-01

A simple and reliable method of high-performance liquid chromatography with photodiode array detection (HPLC-DAD) was developed to evaluate the quality of a traditional Chinese medicine Sophora flavescens through establishing chromatographic fingerprint and simultaneous determination of five flavonoids, including trifolirhizin, maackiain, kushenol I, kurarinone and sophoraflavanone G. The optimal conditions of separation and detection were achieved on an ULTIMATE XB-C18 column (4.6 mm x 250 mm, 5 microm) with a gradient of acetonitrile and water, detected at 295 nm. In the chromatographic fingerprint, 13 peaks were selected as the characteristic peaks to assess the similarities of different samples collected from different origins in China according to similarity evaluation for chromatographic fingerprint of traditional chinese medicine (2004AB) and principal component analysis (PCA) were used in data analysis. There were significant differences in the fingerprint chromatograms between S. flavescens and S. tonkinensis. Principal component analysis showed that kurarinone and sophoraflavanone G were the most important component. In quantitative analysis, the five components showed good regression (R > 0.999) with linear ranges, and their recoveries were in the range of 96.3% - 102.3%. This study indicated that the combination of quantitative and chromatographic fingerprint analysis can be readily utilized as a quality control method for S. flavescens and its related traditional Chinese medicinal preparations.

Evaluation of bioactive flavonoids and antioxidant activity in Pericarpium Citri Reticulatae (Citrus reticulata 'Chachi') during storage.

PubMed

Fu, Manqin; Xu, Yujuan; Chen, Yulong; Wu, Jijun; Yu, Yuanshan; Zou, Bo; An, Kejing; Xiao, Gengsheng

2017-09-01

A simple and accurate method using high performance liquid chromatography (HPLC) with dual wavelength detection was developed to simultaneously determine the contents of one flavanone glycoside (hesperidin) and five polymethoxylated flavones (PMFs: sinensetin, 4',5,7,8-tetramethoxyflavone, nobiletin, tangeretin and 5-O-desmethyl nobiletin) in Pericarpium Citri Reticulatae (Citrus reticulata 'Chachi') ('Chachi' PCR). By modifying the mobile phase compositions and detection wavelengths, an optimal HPLC condition was obtained, under which the calibration curves of all six compounds exhibited good linearity (R 2 >0.99). For all the tested compounds, the relative standard deviation (RSD) was less than 4%, and the accuracy ranged from 97.58 to 103.2%. The developed method was successfully applied to monitor the changes in the contents of six flavonoids in 'Chachi' PCR during storage at 25°C, over a three year period. Color parameters and antioxidant capacity were also determined to evaluate the sample quality. The contents of hesperidin decreased while all the polymethoxylated flavones and antioxidant activities increased throughout the storage period, demonstrating that polymethoxylated flavones could be used as indices for the quality change of Chachi' PCR during storage. The results from this study suggest that the longer storage periods increased the quality of PCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection.

PubMed

Ishihara, K; Fukutake, M; Asano, T; Mizuhara, Y; Wakui, Y; Yanagisawa, T; Kamei, H; Ohmori, S; Kitada, M

2001-04-05

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20x3.9 mm I.D.) and analytical column (Symmetry C18, 75x4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile-water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40 degrees C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.

Quantification of underivatised amino acids on dry blood spot, plasma, and urine by HPLC-ESI-MS/MS.

PubMed

Giordano, Giuseppe; Di Gangi, Iole Maria; Gucciardi, Antonina; Naturale, Mauro

2012-01-01

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting.

A new magnetic nanodiamond/graphene oxide hybrid (Fe3O4@ND@GO) material for pre-concentration and sensitive determination of sildenafil in alleged herbal aphrodisiacs by HPLC-DAD system.

PubMed

Yilmaz, Erkan; Ulusoy, Halil İbrahim; Demir, Özge; Soylak, Mustafa

2018-05-01

A sensitive analytical methodology was investigated to concentrate and determine of sildenafil citrate (SLC) present at trace level in herbal supplementary products. The proposed method is based on simple and sensitive pre-concentration of SLC by using magnetic solid phase extraction with new developed magnetic nanodiamond/graphene oxide hybrid (Fe 3 O 4 @ND@GO) material as a sorbent. Experimental variables affecting the extraction efficiency of SLC like; pH, sample volume, eluent type and volume, extraction time and amount of adsorbent were studied and optimized in detail. Determination of sildenafil citrate after magnetic solid phase extraction (MSPE) was carried out by HPLC-DAD system. The morphology, composition, and properties of the synthesized hybrid material was characterized by Fourier transform infrared spectrometry (FT-IR), Raman spectrometry (Raman), X-ray diffraction spectrometry (XRD), scanning electron microscopy (SEM), mapping photographs, zeta potential analyzer, and BET surface area analysis. Under optimized conditions, linear range was ranged from 5.00 to 250.00 ng mL -1 with R 2 of 0.9952. The limit of detection (LOD) was 1.49 ng mL -1 and the recoveries at two spiked levels were ranged from 94.0 to 104.1% with the relative standard deviation (RSD) < 7.1% (n = 5). The enhancement factor (EF) was 86.9. The results show that the combination MSPE with HPLC-DAD is a suitable and sensitive method for the determination of SLC in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid, cost-effective and accurate quantification of Yucca schidigera Roezl. steroidal saponins using HPLC-ELSD method.

PubMed

Tenon, Mathieu; Feuillère, Nicolas; Roller, Marc; Birtić, Simona

2017-04-15

Yucca GRAS-labelled saponins have been and are increasingly used in food/feed, pharmaceutical or cosmetic industries. Existing techniques presently used for Yucca steroidal saponin quantification remain either inaccurate and misleading or accurate but time consuming and cost prohibitive. The method reported here addresses all of the above challenges. HPLC/ELSD technique is an accurate and reliable method that yields results of appropriate repeatability and reproducibility. This method does not over- or under-estimate levels of steroidal saponins. HPLC/ELSD method does not require each and every pure standard of saponins, to quantify the group of steroidal saponins. The method is a time- and cost-effective technique that is suitable for routine industrial analyses. HPLC/ELSD methods yield a saponin fingerprints specific to the plant species. As the method is capable of distinguishing saponin profiles from taxonomically distant species, it can unravel plant adulteration issues. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.