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.

Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography-fluorescence detection.

PubMed

El-Maghrabey, Mahmoud H; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka

2014-11-01

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2'-furil to form fluorescent difurylimidazole derivatives that were separated on a C18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355nm, respectively. The method showed good linearity over the concentration ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Precolumn derivatization followed by liquid chromatographic separation and determination of tramiprosate in rat plasma by fluorescence detector: application to pharmacokinetics.

PubMed

Rao, R Nageswara; Maurya, Pawan K; Shinde, Dhananjay D; Khalid, Sara

2011-05-15

Alzheimer disease (AD) is characterized pathologically by extracellular amyloid deposits composed of amyloid β (Aβ) protein. A simple and rapid method using HPLC with fluorescence detector was developed and validated for determination of tramiprosate in rat plasma. Pre-column derivatization of the deproteinized rat plasma was carried out using o-phthaldialdehyde (OPA) as a fluorescent reagent in presence of 3-mercaptopropionic acid. The liquid chromatographic separation was achieved on a Kromasil C18 column using methanol:acetonitrile: 20 mM phosphate buffer pH 7.5 (8.0:17.5:74.5 v/v/v) as a mobile phase in an isocratic elution mode. The eluents were monitored by a fluorescence detector set at 330 and 450 nm of excitation and emission wavelength respectively. Vigabatrin was used as an internal standard. The method was linear within the range 30.0-1000.0 ng/mL. Design of experiments (DOE) was used to evaluate the robustness of the method. The developed method was applied to study the pharmacokinetics of tramiprosate in rats. Copyright © 2011. Published by Elsevier B.V.

Characterization of newfound natural luminescent properties of melamine, and development and validation of a method of high performance liquid chromatography with fluorescence detection for its determination in kitchen plastic ware.

PubMed

Finete, Virginia de Lourdes Mendes; Gouvêa, Marcos Martins; Marques, Flávia Ferreira de Carvalho; Netto, Annibal Duarte Pereira

2014-06-01

Experimental studies of the natural photoluminescence of melamine in aqueous solutions showed that its fluorescence intensity (at 250/365 nm) was appropriated for analytical purposes. The exploitation of such melamine property provided the basis of development of a new, simple, precise and accurate method based on high performance liquid chromatography with fluorescence detection (HPLC-Fluo) to determine melamine in kitchen plastic ware following aqueous extraction using a microwave oven. Optimization of analytical parameters such as solvent composition, pH and extraction conditions led to limits of detection and quantification of melamine of 0.0081 and 0.027 μg mL(-1), respectively, with a linear range up to 10 μg mL(-1). Sample extracts fortified with melamine at three concentration levels produced an average recovery of 98±6%, which was in agreement with the results achieved with a reference HPLC-UV method. Different samples of kitchen plastic ware analyzed by the developed and optimized method showed melamine concentrations in the aqueous extract up to 17 µg mL(-1), which corresponded to 86.0 mg kg(-1) in these utensils. The results obtained indicate that the use of kitchen plastic ware made of melamine can contaminate food with this compound after heating in a microwave oven. Copyright © 2014. Published by Elsevier B.V.

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...

Determination of vitamin E and carotenoid pigments by high performance liquid chromatography in shell of Chionoecetes opilio.

PubMed

Vilasoa-Martínez, María; Calaza-Ramos, Carina; López-Hernández, Julia; Lage-Yusty, María Asunción; Losada, Perfecto Paseiro; Rodríguez-Bernaldo de Quirós, Ana

2008-06-09

This study reports the optimization of a method for the determination of vitamin E and carotenoids in shells of Chionoecetes opilio samples by online HPLC coupled with UV-vis and fluorescence detectors. The carotenoids were determined with diode-array detector (lambda 450 nm) and vitamin E with fluorescence detection (lambda(ex) 288, lambda(em) 331 nm). Two extractions methods were compared, saponification followed by an extraction step and a simple extraction with acetone. The last one was selected because allows to determine all compounds. Linearity, precisions and recoveries achieved for all compounds were satisfactory. Mean concentrations (mg per 100 g dry weight) were; 23.3 for vitamin E, 9.49 for astaxanthin and 0.2 mg for beta-carotene.

Development and validation of a method for the determination of low-ppb levels of macrocyclic lactones in butter, using HPLC-fluorescence.

PubMed

Macedo, Fabio; Marsico, Eliane Teixeira; Conte-Júnior, Carlos Adam; de Resende, Michele Fabri; Brasil, Taila Figueiredo; Pereira Netto, Annibal Duarte

2015-07-15

An analytical method was developed and validated for the simultaneous determination of four macrocyclic lactones (ML) (abamectin, doramectin, ivermectin and moxidectin) in butter, using liquid chromatography with fluorescence detection. The method employed heated liquid-liquid extraction and a mixture of acetonitrile, ethyl acetate and water, with preconcentration and derivatization, to produce stable fluorescent derivatives. The chromatographic run time was <12.5 min, with excellent separation. The method validation followed international guidelines and employed fortified butter samples. The figures of merit obtained, e.g. recovery (72.4-106.5%), repeatability (8.8%), within-laboratory reproducibility (15.7%) and limits of quantification (0.09-0.16 μg kg(-1)) were satisfactory for the desired application. The application of the method to real samples showed that ML residues were present in six of the ten samples evaluated. The method proved to be simple, easy and appropriate for simultaneous determination of ML residues in butter. To our knowledge, this is the first method described for the evaluation of ML in butter. Copyright © 2015. Published by Elsevier Ltd.

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.

Measurement of H2S in vivo and in vitro by the monobromobimane method

PubMed Central

Shen, Xinggui; Kolluru, Gopi K.; Yuan, Shuai; Kevil, Christopher

2015-01-01

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with RP-HPLC. The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide-dibimane. The resultant fluorescent sulfide-dibimane (SDB) is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels. PMID:25725514

Measurement of H2S in vivo and in vitro by the monobromobimane method.

PubMed

Shen, Xinggui; Kolluru, Gopi K; Yuan, Shuai; Kevil, Christopher G

2015-01-01

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels. © 2015 Elsevier Inc. All rights reserved.

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.

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.

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.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

PubMed

Sorio, Daniela; De Palo, Elio Franco; Bertaso, Anna; Bortolotti, Federica; Tagliaro, Franco

2017-02-01

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

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.

A simple method for plasma total vitamin C analysis suitable for routine clinical laboratory use.

PubMed

Robitaille, Line; Hoffer, L John

2016-04-21

In-hospital hypovitaminosis C is highly prevalent but almost completely unrecognized. Medical awareness of this potentially important disorder is hindered by the inability of most hospital laboratories to determine plasma vitamin C concentrations. The availability of a simple, reliable method for analyzing plasma vitamin C could increase opportunities for routine plasma vitamin C analysis in clinical medicine. Plasma vitamin C can be analyzed by high performance liquid chromatography (HPLC) with electrochemical (EC) or ultraviolet (UV) light detection. We modified existing UV-HPLC methods for plasma total vitamin C analysis (the sum of ascorbic and dehydroascorbic acid) to develop a simple, constant-low-pH sample reduction procedure followed by isocratic reverse-phase HPLC separation using a purely aqueous low-pH non-buffered mobile phase. Although EC-HPLC is widely recommended over UV-HPLC for plasma total vitamin C analysis, the two methods have never been directly compared. We formally compared the simplified UV-HPLC method with EC-HPLC in 80 consecutive clinical samples. The simplified UV-HPLC method was less expensive, easier to set up, required fewer reagents and no pH adjustments, and demonstrated greater sample stability than many existing methods for plasma vitamin C analysis. When compared with the gold-standard EC-HPLC method in 80 consecutive clinical samples exhibiting a wide range of plasma vitamin C concentrations, it performed equivalently. The easy set up, simplicity and sensitivity of the plasma vitamin C analysis method described here could make it practical in a normally equipped hospital laboratory. Unlike any prior UV-HPLC method for plasma total vitamin C analysis, it was rigorously compared with the gold-standard EC-HPLC method and performed equivalently. Adoption of this method could increase the availability of plasma vitamin C analysis in clinical medicine.

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid.

PubMed

Anumula, K R; Dhume, S T

1998-07-01

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.

[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.

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.

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

PubMed Central

Zhu, Hao-Jie; Wang, Jun-Sheng; Patrick, Kennerly S.; Donovan, Jennifer L.; DeVane, C. Lindsay; Markowitz, John S.

2007-01-01

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (λex: 330 nm, λem: 460 nm). The lower limit of determination was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were ≤ 9.10 % and ≤ 7.58 %, respectively. The accuracy ranged between 92.59 % and 103.06 %. The method was successfully applied to the pharmacokinetic study of a subject who received a single oral dose (0.3 mg/kg) of immediate-release MPH and yielded consistent results with that of the GC-MS method. This method is the first HPLC assay with non-MS detection providing sufficient reliability and sensitivity for both pre-clinical and clinical studies of MPH. PMID:17804308

Spectrofluorometric determination of furosemide in some pharmaceutical product using acriflavine as a reagent

NASA Astrophysics Data System (ADS)

Qader, Aryan F.; Fakhre, Nabil A.

2017-09-01

Simple, inexpensive, rapid and sensitive determination of furosemide spectrofluorometrically was suggested using acriflavine as a new reagent. The method based on the quantitative quenching effect of furosemide on the native fluorescence of acriflavine in presence of Britton-Robinson buffer medium due to the reaction of furosemide with acriflavine to form an ion associated complex. The decrease of acriflavine fluorescence was observed at 505 nm after excitation at 265 nm. the florescence - concentration plot is rectilinear over the range of 2.0-10 µg/ml with correlation coefficient 0.9991 and detection limit 0.2 µg/ml. No interference was observed from the excipients that are commonly present in pharmaceutical formulations. The proposed method was determination of furosemide in some commercial tablets. The results were compared with that of HPLC method revealed with good agreements and no significant differences in the accuracy and precision.

Application of Fluorescence Spectrometry With Multivariate Calibration to the Enantiomeric Recognition of Fluoxetine in Pharmaceutical Preparations.

PubMed

Poláček, Roman; Májek, Pavel; Hroboňová, Katarína; Sádecká, Jana

2016-04-01

Fluoxetine is the most prescribed antidepressant chiral drug worldwide. Its enantiomers have a different duration of serotonin inhibition. A novel simple and rapid method for determination of the enantiomeric composition of fluoxetine in pharmaceutical pills is presented. Specifically, emission, excitation, and synchronous fluorescence techniques were employed to obtain the spectral data, which with multivariate calibration methods, namely, principal component regression (PCR) and partial least square (PLS), were investigated. The chiral recognition of fluoxetine enantiomers in the presence of β-cyclodextrin was based on diastereomeric complexes. The results of the multivariate calibration modeling indicated good prediction abilities. The obtained results for tablets were compared with those from chiral HPLC and no significant differences are shown by Fisher's (F) test and Student's t-test. The smallest residuals between reference or nominal values and predicted values were achieved by multivariate calibration of synchronous fluorescence spectral data. This conclusion is supported by calculated values of the figure of merit.

Analyses of procyanidins in foods using Diol phase HPLC

USDA-ARS?s Scientific Manuscript database

Separation of procyanidins using silica-based HPLC suffered from poor resolution for higher oligomers and low sensitivity due to the fluorescence quenching effects of methylene chloride in the mobile phase. Optimization of a published Diol-phase HPLC method resulted in near baseline separation for p...

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.

Measurement of menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

Mammals convert phylloquinone to MK-4, with menadione as a possible intermediate. We developed and validated a method measuring urinary menadione. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed. The mobile...

[Sensitive and selective HPLC methods with prechromatographic derivatization for the determination of cyclamate in foods].

PubMed

Rüter, J; Raczek, D I

1992-06-01

A sensitive and selective high pressure liquid chromatography (HPLC) procedure for the determination of sodium cyclamate in juices and preserves is presented. The method depends on the oxidation of cyclamate to cyclohexylamine, which then is converted prechromatographically into a fluorescent derivative. It is analyzed by HPLC on a C18:reversed-phase column and determined with fluorescence detection (excitation at 350 nm, emission at 440-650 nm). The detection limit of sodium cyclamate was 0.5-5 mg/kg, depending on the nature and dilution of the samples. The relative standard deviations thus obtained were +/- 1.0 to +/- 2.6%. The average recovery was 90%.

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

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.

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.

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.

A SIMPLE HPLC METHOD FOR DETECTING CARBARYL AND 1-NAPHTHOL IN BIOLOGICAL TISSUES.

EPA Science Inventory

Carbamates are a class of pesticide used in both agricultural and residential applications. A simple HPLC method for detecting Carb and its metabolite 1-naphthol (Naph) in tissues was developed to try to correlate tissue levels of carbaryl (Carb) (a prototypical carbamate) with c...

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

PubMed Central

Zielonka, Jacek; Zielonka, Monika; Sikora, Adam; Adamus, Jan; Joseph, Joy; Hardy, Micael; Ouari, Olivier; Dranka, Brian P.; Kalyanaraman, Balaraman

2012-01-01

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants. PMID:22139901

40 CFR Appendix A to Subpart C of... - Alternative Testing Methods Approved for Analyses Under the Safe Drinking Water Act

Code of Federal Regulations, 2013 CFR

2013-07-01

... Chromatography/Mass Spectrometry (GC/MS) 525.3 24 Carbofuran High-performance liquid chromatography (HPLC) with... (HPLC) with Post-Column Derivatization and Fluorescence Detection 6651 B 6651 B 6651 B-00. Heptachlor... Spectrometry (GC/MS) 525.3 24 Oxamyl High-performance liquid chromatography (HPLC) with post-column...

Comparison of spectrofluorometric and HPLC methods for the characterization of fecal porphyrins in river otters.

PubMed

Taylor, C; Duffy, L K; Plumley, F G; Bowyer, R T

2000-09-01

A spectrofluorometric method (B. Grandchamp et al., 1980, Biochem. Biophys. Acta 629, 577-586) developed for the determination of amounts of uroporphyrin I (Uro I), coproporphyrin III (Copro III), and protoporphyrin IX (Proto IX) in skin fibroblasts was compared with a high-performance liquid chromatography (HPLC) method for the analysis of porphyrins in fecal samples of river otters (Lutra canadensis). Heptacarboxylate porphyrin I and coproporphyrin I, two porphyrins determined to be critical in defining the porphyrin profile in fecal samples of river otters with the HPLC method, contributed substantially to the calculation of the concentrations of Uro I and Copro III, respectively, in standard solutions of porphyrins with the spectrofluorometric method. Fluorescent components of the fecal matrix complicated the determination of the concentrations of Uro I, Copro III, and Proto IX with the spectrofluorometric method and resulted in erroneous values for the concentrations of these porphyrins compared with values determined with the HPLC method. These results indicate that the complexity of the sample, particularly with regard to the potential presence of interfering fluorescent compounds, as well as porphyrins additional to Uro I, Copro III, and Proto IX, should be considered prior to the application of the spectrofluorometric method. An alternative HPLC method developed for the rapid characterization of porphyrin profiles in fecal samples of river otters is described. Copyright 2000 Academic Press.

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.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

PubMed

Dai, Zhaolai; Wu, Zhenlong; Jia, Sichao; Wu, Guoyao

2014-08-01

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal). Copyright © 2014 Elsevier B.V. All rights reserved.

An HPLC Method for Microanalysis and Pharmacokinetics of Marine Sulfated Polysaccharide PSS-Loaded Poly Lactic-co-Glycolic Acid (PLGA) Nanoparticles in Rat Plasma

PubMed Central

Li, Peng-Li; Li, Chun-Xia; Xue, Yi-Ting; Li, Hai-Hua; Liu, Hong-Bing; He, Xiao-Xi; Yu, Guang-Li; Guan, Hua-Shi

2013-01-01

This study was aimed at developing a sensitive and selective HPLC method with postcolumn fluorescence derivatization for the detection of propylene glycol alginate sodium sulfate (PSS) in rat plasma. Plasma samples were prepared by a simple and fast ultrafiltration method. PSS was extracted from rat plasma with d-glucuronic acid as internal standard. Isocratic chromatographic separation was performed on a TSKgel G2500 PWxL column with the mobile phase of 0.1 M sodium sulfate at a flow rate of 0.5 mL/min. Analyte detection was achieved by fluorescence detection (FLD) at 250 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as postcolumn derivatizing reagent in an alkaline medium at 120 °C. The calibration curve was linear over a concentration range of 1–500 μg/mL, and the lower limit of detection (LLOD) was found to be 250 ng/mL. This validated method was applied successfully to the pharmacokinetic study of PSS and PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (PSS-NP) in rat plasma after a single intravenous (PSS only) and oral administration (PSS and PSS-NP). Significant differences in the main pharmacokinetic parameters of PSS and PSS-NP were observed. The relative bioavailability of PSS-NP was 190.10% compared with PSS which shows that PSS-NP can improve oral bioavailability. PMID:23549283

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.

A simple method for HPLC retention time prediction: linear calibration using two reference substances.

PubMed

Sun, Lei; Jin, Hong-Yu; Tian, Run-Tao; Wang, Ming-Juan; Liu, Li-Na; Ye, Liu-Ping; Zuo, Tian-Tian; Ma, Shuang-Cheng

2017-01-01

Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately. But the reference substances are costly. Thus, the relative retention (RR) method has been widely adopted in pharmacopoeias and literatures for characterizing HPLC behaviors of those reference substances unavailable. The problem is it is difficult to reproduce the RR on different columns due to the error between measured retention time (t R ) and predicted t R in some cases. Therefore, it is useful to develop an alternative and simple method for prediction of t R accurately. In the present study, based on the thermodynamic theory of HPLC, a method named linear calibration using two reference substances (LCTRS) was proposed. The method includes three steps, procedure of two points prediction, procedure of validation by multiple points regression and sequential matching. The t R of compounds on a HPLC column can be calculated by standard retention time and linear relationship. The method was validated in two medicines on 30 columns. It was demonstrated that, LCTRS method is simple, but more accurate and more robust on different HPLC columns than RR method. Hence quality standards using LCTRS method are easy to reproduce in different laboratories with lower cost of reference substances.

Comparison of HPLC & spectrophotometric methods for estimation of antiretroviral drug content in pharmaceutical products.

PubMed

Hemanth Kumar, A K; Sudha, V; Swaminathan, Soumya; Ramachandran, Geetha

2010-10-01

Simple and reliable methods to estimate drugs in pharmaceutical products are needed. In most cases, antiretroviral drug estimations are performed using a HPLC method, requiring expensive equipment and trained technicians. A relatively simple and accurate method to estimate antiretroviral drugs in pharmaceutical preparations is by spectrophotometric method, which is cheap and simple to use as compared to HPLC. We undertook this study to standardise methods for estimation of nevirapine (NVP), lamivudine (3TC) and stavudine (d4T) in single tablets/capsules by HPLC and spectrophotometry and to compare the content of these drugs determined by both these methods. Twenty tablets/capsules of NVP, 3TC and d4T each were analysed for their drug content by HPLC and spectrophotometric methods. Suitably diluted drug solutions were run on HPLC fitted with a C18 column using UV detection at ambient temperature. The absorbance of the diluted drug solutions were read in a spectrophotometer at 300, 285 and 270 nm for NVP, 3TC and d4T respectively. Pure powders of the drugs were used to prepare calibration standards of known drug concentrations, which was set up with each assay. The inter-day variation (%) of standards for NVP, 3TC and d4T ranged from 2.5 to 6.7, 2.1 to 7.7 and 6.2 to 7.7, respectively by HPLC. The corresponding values by spectrophotometric method were 2.7 to 4.7, 4.2 to 7.2 and 3.8 to 6.0. The per cent variation between the HPLC and spectrophotometric methods ranged from 0.45 to 4.49 per cent, 0 to 4.98 per cent and 0.35 to 8.73 per cent for NVP, 3TC and d4T,respectively. The contents of NVP, 3TC and d4T in the tablets estimated by HPLC and spectrophotometric methods were similar, and the variation in the amount of these drugs estimated by HPLC and spectrophotometric methods was below 10 per cent. This suggests that the spectrophotometric method is as accurate as the HPLC method for estimation of NVP, 3TC and d4T in tablet/capsule. Hence laboratories that do not have HPLC equipment can also undertake these drug estimations using spectrophotometer.

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

40 CFR Appendix A to Subpart C of... - Alternative Testing Methods Approved for Analyses Under the Safe Drinking Water Act

Code of Federal Regulations, 2014 CFR

2014-07-01

... Spectrometry (GC/MS) 525.3 24 Carbofuran High-performance liquid chromatography (HPLC) with post-column... (HPLC) with Post-Column Derivatization and Fluorescence Detection 6651 B 6651 B 6651 B-00, B-05... Chromatography/Mass Spectrometry (GC/MS) 525.3 24 Oxamyl High-performance liquid chromatography (HPLC) with post...

Quantification of aesculin in rabbit plasma and ocular tissues by high performance liquid chromatography using fluorescent detection: application to a pharmacokinetic study.

PubMed

Chen, QiuHong; Zeng, Ying; Kuang, JianChao; Li, Ye; Li, XiaoHui; Zheng, Yu; Hou, Hua; Hou, ShiXiang

2011-04-28

A simple and sensitive high performance liquid chromatography method with fluorescence detection (HPLC-FD) was described for the determination of aesculin (AL) at low concentrations in rabbit plasma and ocular tissues. After deproteinization by methanol using pazufloxacin mesilate (PM) as an internal standard (I.S.), supernatants were evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in mobile phase and a volume of 20μL was injected into the HPLC for analysis. Analytes were separated on an Ultimate XB-C18 column (250mm × 4.6mm i.d., 5μm particle size) and protected by a ODS guard column (10mm × 4.0mm i.d., 5μm particle size), using acetonitrile-0.1% triethylamine in water (adjusted to pH 3.0 using phosphoric acid) (12:88, v/v) as mobile phase with a flow rate of 1.0mL/min. The wavelengths of fluorescence detector (FD) were set at 344nm for excitation and 466nm for emission. The lower limit of quantitation (LOQ) for AL was 0.80ng/mL for plasma and vitreous body, 1.59ng/mL for aqueous humor, and 6.55ng/g for iris and 1.66ng/g for retina. The method was used in the study of AL concentrations in plasma and ocular tissues after topical administration of AL eye drops. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Sensitive, accurate and rapid detection of trace aliphatic amines in environmental samples with ultrasonic-assisted derivatization microextraction using a new fluorescent reagent for high performance liquid chromatography.

PubMed

Chen, Guang; Liu, Jianjun; Liu, Mengge; Li, Guoliang; Sun, Zhiwei; Zhang, Shijuan; Song, Cuihua; Wang, Hua; Suo, Yourui; You, Jinmao

2014-07-25

A new fluorescent reagent, 1-(1H-imidazol-1-yl)-2-(2-phenyl-1H-phenanthro[9,10-d]imidazol-1-yl)ethanone (IPPIE), is synthesized, and a simple pretreatment based on ultrasonic-assisted derivatization microextraction (UDME) with IPPIE is proposed for the selective derivatization of 12 aliphatic amines (C1: methylamine-C12: dodecylamine) in complex matrix samples (irrigation water, river water, waste water, cultivated soil, riverbank soil and riverbed soil). Under the optimal experimental conditions (solvent: ACN-HCl, catalyst: none, molar ratio: 4.3, time: 8 min and temperature: 80°C), micro amount of sample (40 μL; 5mg) can be pretreated in only 10 min, with no preconcentration, evaporation or other additional manual operations required. The interfering substances (aromatic amines, aliphatic alcohols and phenols) get the derivatization yields of <5%, causing insignificant matrix effects (<4%). IPPIE-analyte derivatives are separated by high performance liquid chromatography (HPLC) and quantified by fluorescence detection (FD). The very low instrumental detection limits (IDL: 0.66-4.02 ng/L) and method detection limits (MDL: 0.04-0.33 ng/g; 5.96-45.61 ng/L) are achieved. Analytes are further identified from adjacent peaks by on-line ion trap mass spectrometry (MS), thereby avoiding additional operations for impurities. With this UDME-HPLC-FD-MS method, the accuracy (-0.73-2.12%), precision (intra-day: 0.87-3.39%; inter-day: 0.16-4.12%), recovery (97.01-104.10%) and sensitivity were significantly improved. Successful applications in environmental samples demonstrate the superiority of this method in the sensitive, accurate and rapid determination of trace aliphatic amines in micro amount of complex samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC-Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma.

PubMed

El-Zaher, Asmaa A; Mahrouse, Marianne A

2013-01-01

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min(-1). A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20-200 ng mL(-1). The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.

Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process.

PubMed

Douša, Michal; Doubský, Jan; Srbek, Jan

2016-07-01

An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Selective identification and quantification of saccharin by liquid chromatography and fluorescence detection.

PubMed

Bruno, Sergio N F; Cardoso, Carlos R; Maciel, Márcia Mosca A; Vokac, Lidmila; da Silva Junior, Ademário I

2014-09-15

High-pressure liquid chromatography with ultra-violet detection (HPLC-UV) is one of the most commonly used methods to identify and quantify saccharin in non-alcoholic beverages. However, due to the wide variety of interfering UV spectra in saccharin-containing beverage matrices, the same method cannot be used to measure this analyte accurately. We have developed a new, highly effective method to identify and quantify saccharin using HPLC with fluorescence detection (HPLC-FLD). The excitation wavelength (250 nm) and emission wavelength (440 nm) chosen increased selectivity for all matrices and ensured few changes were required in the mobile phase or other parameters. The presence of saccharin in non-diet beverages - a fraud commonly used to replace more expensive sucrose - was confirmed by comparing coincident peaks as well as the emission spectra of standards and samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Graphene oxide-coated stir bar sorptive extraction of trace aflatoxins from soy milk followed by high performance liquid chromatography-laser-induced fluorescence detection.

PubMed

Ma, Haiyan; Ran, Congcong; Li, Mengjiao; Gao, Jinglin; Wang, Xinyu; Zhang, Lina; Bian, Jing; Li, Junmei; Jiang, Ye

2018-04-01

Mycotoxins are potential food pollutants produced by fungi. Among them, aflatoxins (AFs) are the most toxic. Therefore, AFs were selected as models, and a sensitive, simple and green graphene oxide (GO)-based stir bar sorptive extraction (SBSE) method was developed for extraction and determination of AFs with high performance liquid chromatography-laser-induced fluorescence detector (HPLC-LIF). This method improved the sensitivity of AFs detection and solved the deposition difficulty of the direct use of GO as adsorbent. Several parameters including a spiked amount of NaCl, stirring rate, extraction time and desorption time were investigated. Under optimal conditions, the quantitative method had low limits of detection of 2.4-8.0 pg/mL, which were better than some reported AFs analytical methods. The developed method has been applied to soy milk samples with good recoveries ranging from 80.5 to 102.3%. The prepared GO-based SBSE can be used as a sensitive screening technique for detecting AFs in soy milk.

Determination of Urine Albumin by New Simple High-Performance Liquid Chromatography Method.

PubMed

Klapkova, Eva; Fortova, Magdalena; Prusa, Richard; Moravcova, Libuse; Kotaska, Karel

2016-11-01

A simple high-performance liquid chromatography (HPLC) method was developed for the determination of albumin in patients' urine samples without coeluting proteins and was compared with the immunoturbidimetric determination of albumin. Urine albumin is important biomarker in diabetic patients, but part of it is immuno-nonreactive. Albumin was determined by high-performance liquid chromatography (HPLC), UV detection at 280 nm, Zorbax 300SB-C3 column. Immunoturbidimetric analysis was performed using commercial kit on automatic biochemistry analyzer COBAS INTEGRA ® 400, Roche Diagnostics GmbH, Manheim, Germany. The HLPC method was fully validated. No significant interference with other proteins (transferrin, α-1-acid glycoprotein, α-1-antichymotrypsin, antitrypsin, hemopexin) was found. The results from 301 urine samples were compared with immunochemical determination. We found a statistically significant difference between these methods (P = 0.0001, Mann-Whitney test). New simple HPLC method was developed for the determination of urine albumin without coeluting proteins. Our data indicate that the HPLC method is highly specific and more sensitive than immunoturbidimetry. © 2016 Wiley Periodicals, Inc.

Measurement of Menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

Menadione may be an important metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed to measure menadione in urine. The mobile phase was composed of 95% methanol...

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).

An industry consensus study on an HPLC fluorescence method for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products.

PubMed

Shumow, Laura; Bodor, Alison

2011-07-05

This manuscript describes the results of an HPLC study for the determination of the flavan-3-ol monomers, (±)-catechin and (±)-epicatechin, in cocoa and plain dark and milk chocolate products. The study was performed under the auspices of the National Confectioners Association (NCA) and involved the analysis of a series of samples by laboratories of five member companies using a common method. The method reported in this paper uses reversed phase HPLC with fluorescence detection to analyze (±)-epicatechin and (±)-catechin extracted with an acidic solvent from defatted cocoa and chocolate. In addition to a variety of cocoa and chocolate products, the sample set included a blind duplicate used to assess method reproducibility. All data were subjected to statistical analysis with outliers eliminated from the data set. The percent coefficient of variation (%CV) of the sample set ranged from approximately 7 to 15%. Further experimental details are described in the body of the manuscript and the results indicate the method is suitable for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products and represents the first collaborative study of this HPLC method for these compounds in these matrices.

[The determination of the herbicide glyphosate and its chief metabolite aminomethylphosphonic acid (AMPA) in drinking water with the aid of HPLC].

PubMed

Gauch, R; Leuenberger, U; Müller, U

1989-01-01

A method for the determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) is described. With a detection limit of 0.02 microgram/l, the method suitably fulfills the requirements of the Swiss legislation (tolerance value of 0.1 micrograms/l water). The compounds are derivatized directly in the original water sample with 9-fluorenylmethyl chloroformate (FMOCC1) in order to obtain extractable and fluorescent derivatives. These are extracted with organic solvents and determined by HPLC using a fluorescence detector. Neither of the compounds could be detected in 151 tap water samples from the Canton of Berne.

[Determination of homocysteine level in plasma by high performance liquids chromatography with fluorescence detection].

PubMed

Da, Xu; Qian, Ling-Jia

2005-08-01

To establish a method for detection of plasma total homocysteine with HPLC. The chromatography analysis was carried out using a Symmetry Shield RP18. The mobile phase was sodium acetate (0.08 mol/L) and methanol (1%) and we utilized a HPLC system with fluorescence detection of plasma homocysteine derivatized from reaction with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). The average recoveries were 95.8 - 100.8% and the relative standard deviations were 1.2-2.0%. The results showed it to be a rapid and accurate method for the determination of homocysteine level in plasma.

Simultaneous determination of potassium guaiacolsulfonate, guaifenesin, diphenhydramine HCl and carbetapentane citrate in syrups by using HPLC-DAD coupled with partial least squares multivariate calibration.

PubMed

Dönmez, Ozlem Aksu; Aşçi, Bürge; Bozdoğan, Abdürrezzak; Sungur, Sidika

2011-02-15

A simple and rapid analytical procedure was proposed for the determination of chromatographic peaks by means of partial least squares multivariate calibration (PLS) of high-performance liquid chromatography with diode array detection (HPLC-DAD). The method is exemplified with analysis of quaternary mixtures of potassium guaiacolsulfonate (PG), guaifenesin (GU), diphenhydramine HCI (DP) and carbetapentane citrate (CP) in syrup preparations. In this method, the area does not need to be directly measured and predictions are more accurate. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of analytes could be obtained with HPLC-DAD coupled with PLS calibration. This method was tested by analyzing the synthetic mixture of PG, GU, DP and CP. As a comparison method, a classsical HPLC method was used. The proposed methods were applied to syrups samples containing four drugs and the obtained results were statistically compared with each other. Finally, the main advantage of HPLC-PLS method over the classical HPLC method tried to emphasized as the using of simple mobile phase, shorter analysis time and no use of internal standard and gradient elution. Copyright © 2010 Elsevier B.V. All rights reserved.

Validation of a LC-fluorescence method for determination of free captopril in human plasma, using a pre-column derivatization reaction with monobromobimane.

PubMed

Tache, Florentin; Farca, Alexandru; Medvedovici, Andrei; David, Victor

2002-05-15

Both derivatization of free captopril in human plasma samples using monobromobimane as fluorescent label and the corresponding HPLC-fluorescence detection (FLD) method were validated. Calibration curve for the fluorescent captopril derivative in plasma samples is linear in the ppb-ppm range with a detection limit of 4 ppb and an identification limit of 10 ppb (P%: 90; nu > or = 5). These methods were successfully applied on bioequivalence studies carried out on some marketed pharmaceutical formulations.

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.

Analysis and quality control of carbohydrates in therapeutic proteins with fluorescence HPLC

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

Liu, Kun; Huang, Jian; Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

Demonstration and Evaluation of Solid Phase Microextraction for the Assessment of Bioavailability and Contaminant Mobility

DTIC Science & Technology

2012-05-01

methods demonstrated that desorption into solvents suitable for subsequent chemical analysis (into acetonitrile for HPLC analysis or hexane for GC...SPME. Analysis by HPLC with EPA 8310 with fluorescent detection. a) surface water quality criteria (NRWQC) are given for comparison to detection... analysis ) or hexane (for PCB analysis ) was added to the inserts. The vials were then analyzed directly by HPLC (PAHs) or GC-ECD (PCBs). Fiber achieved

Unique anthranilic acid chemistry facilitates profiling and characterization of Ser/Thr-linked sugar chains following hydrazinolysis.

PubMed

Anumula, Kalyan Rao

2008-02-01

A novel method for the analysis of Ser/Thr-linked sugar chains was made possible by the virtue of unique anthranilic acid (AA, 2-aminobenzoic acid [2AA]) chemistry for labeling carbohydrates in aqueous salt solutions (K. R. Anumula, Anal. Biochem. 350 (2006) 1-23). The protocol for profiling of Ser/Thr carbohydrates by hydrazinolysis was made simple by eliminating intermediary isolation steps involved in a sample preparation such as desalting and various chromatographic purification schemes. A 6-h hydrazinolysis was carried out at 60 degrees C for O-linked oligosaccharides and at 95 degrees C for total oligosaccharides (N-linked with some O-linked). Following evaporation of hydrazine (<10 min), the oligosaccharides were N-acetylated and derivatized with AA in the same reaction mixture containing salts. Presumably, the glycosyl-hydrazines/hydrazones present in the mixture did not interfere with AA labeling. Because AA is the most fluorescent and highly reactive tag for labeling carbohydrates, the procedures described are suitable for the analysis of a limited amount of samples ( approximately 5 microg) by the current high-resolution high-performance liquid chromatography (HPLC) methods. HPLC conditions developed for the separation of O-linked sugar chains based on size on an amide column were satisfactory for quantitative profiling and characterization. Common O-linked sugar chains found in fetuin, equine chorionic gonadotropin, and glycophorin can be analyzed in less than 50 min. In addition, these fast profiling methods were comparable to profiling by PNGase F (peptide N-glycosidase from Flavobacterium meningosepticum) digestion in terms of time, effort, and simplicity and also were highly reproducible for routine testing. The procedures for the release of sugar chains by hydrazinolysis at the microgram level, labeling with fluorescent tag AA, and profiling by HPLC should be useful in characterization of carbohydrates found in glycoproteins.

[The occurrence of Fusarium varieties and their mycotoxins on silo corn. 1. A method for the determination of zearalenone in corn and corn silage by high performance liquid chromatography (HPLC) with fluorescence detection].

PubMed

Lepom, P

1988-09-01

A method for the determination of zearalenone in maize and maize silage was developed which distinguishes itself by the effective and fast cleaning of the extracts with the help of a silica gel minicolumn. The samples were extracted with chloroform/methanol (9 + 1) and cleaned on a silica gel minicolumn after acid-base partition. The zearalenone was quantitatively determined optionally by means of high-performance liquid chromatography (HPLC) with fluorescence detection (excitation wavelength 236 nm, emission filter 418 nm) or thin-layer chromatography (TLC), p-methoxybenzene diazonium fluoroborate and aluminium chloride were used as detection chemicals. The limits of detection are 0.01 mg/kg (HPLC) and 0.1 mg/kg resp. (TLC), the average recovery is 81%. The method was used for the determination of zearalenone in grain maize, CCM silage and silage from whole maize plants.

[Determination of cyclamate in complex matrix using HPLC after column derivatization with 4-fluoro-7-nitrobenzofurazan].

PubMed

Hauck, M; Köbler, H

1990-01-01

A method for the analysis of cyclamate in complex foodstuffs has been developed. This method is applicable in strongly coloured and protein-rich foodstuffs. The quantitative determination depends on oxidation of cyclamate to cyclohexylamine and derivatisation with 4-fluoro-7-nitrobenzofuran (NBD-F). The derivatives are analysed by HPLC on a C18: reversed-phase column, their minimal stability being 12 h. There are two possible methods of detection: (a) absorbance at 485 nm and (b) fluorescence with excitation at 485 nm and emission at 530 nm. The detection limit of cyclamate is 5 mg/kg foodstuff, with fluorescence detection 0.4 mg/kg. The recoveries are in the range of 88% to 104%.

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.

Improved Quantitation of Gluten in Wheat Starch for Celiac Disease Patients by Gel-Permeation High-Performance Liquid Chromatography with Fluorescence Detection (GP-HPLC-FLD).

PubMed

Scherf, Katharina Anne; Wieser, Herbert; Koehler, Peter

2016-10-12

Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD) patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem, a new sensitive method based on the sequential extraction of gliadins, glutenins, and gluten from WSt followed by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may be applied as confirmatory method to ELISA to quantitate gluten in WSt.

HPLC-DAD-MS identification of bioactive secondary metabolites from Ferula communis roots.

PubMed

Arnoldi, Lolita; Ballero, Mauro; Fuzzati, Nicola; Maxia, Andrea; Mercalli, Enrico; Pagni, Luca

2004-06-01

A simple HPLC method was developed to distinguish between 'poisonous' and 'non-poisonous' chemotypes of Ferula communis. The method was performed on a C8 reverse phase analytical column using a binary eluent (aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition. The two chemotypes showed different fingerprints. The identification of five coumarins and eleven daucane derivatives by HPLC-diode array detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, was detected. Copyright 2004 Elsevier B.V.

FISH BILIARY POLYCYCLIC AROMATIC HYDROCARBON METABOLITES ESTIMATED BY FIXED-WAVELENGTH FLUORESCENCE: COMPARISON WITH HPLC-FLUORESCENT DETECTION

EPA Science Inventory

Fixed wavelength fluorescence (FF) was compared to high-performance liquid chromatography with fluorescence detection (HPLC-F) as an estimation of polycyclic aromatic hydrocarbon (PAH) exposure to fish. Two excitation/emission wavelength pairs were used to measure naphthalene- an...

Determination of fenoterol in human plasma by HPLC with fluorescence detection after derivatization.

PubMed

Meineke, Ingolf; Steinmetz, Hannelore; Kramer, Skaidrit; Gleiter, Christoph H

2002-06-20

A new method for the determination of fenoterol is described, which uses HPLC separation with fluorescence detection. Dobutamine is employed as an internal standard. The separation was achieved on a short reversed phase column with a mobile phase consisting of water, acetonitrile and methanol. Prior to chromatography both analytes are derivatized with 9-chloroformyl-carbazole. Isolation of the analytes from plasma is carried out by liquid-liquid extraction into 2-butanol after protein precipitation with acetonitrile. The method is capable of estimating fenoterol concentrations in the sub-nanogram per ml range with sufficient accuracy and precision. The determination of fenoterol can now be carried out in the average laboratory without radiolabelled material.

Demonstration and Evaluation of Solid Phase Microextraction for the Assessment of Bioavailability and Contaminant Mobility. ESTCP Cost and Performance Report

DTIC Science & Technology

2012-08-01

subsequent chemical analysis (into acetonitrile for high-performance liquid chromatography [ HPLC ] analysis or hexane for gas chromatography [GC... analysis ) is rapid and complete. In this work, PAHs were analyzed by Waters 2795 HPLC with fluorescent detection (USEPA Method 8310) and PCBs were...detection limits by direct water injection versus SPME with PDMS and coefficient of variation and correlation coefficient for SPME. Analysis by HPLC

Application of hollow fiber-supported liquid-phase microextraction coupled with HPLC for the determination of guaifenesin enantiomer-protein binding.

PubMed

Hatami, Mehdi; Farhadi, Khalil

2012-07-01

A hollow fiber liquid-phase microextraction technique coupled with high-performance liquid chromatography with fluorescence detection was employed for determination and evaluation of the binding characteristics of drugs to bovine serum albumin (BSA). Enantiomers of guaifenesin (an expectorant drug) were investigated as a model system. After optimization of some influencing parameters on microextraction, the proposed method was used for calculation of the target drug distribution coefficient between n-octanol and the buffer solution as well as study of drug-BSA binding in physiological conditions. The developed method shows a new, improved and simple procedure for determination of free drug concentration in biological fluids and the extent of drug-protein binding. Copyright © 2011 John Wiley & Sons, Ltd.

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

PubMed Central

Kamal, Abid; Khan, Washim; Ahmad, Sayeed; Ahmad, F. J.; Saleem, Kishwar

2015-01-01

Objective: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga. Materials and Methods: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase. Results: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin. Conclusions: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient. PMID:26681890

Differential coulometric oxidation following post column-switching high pressure liquid chromatography for fluorescence measurement of unmetabolized folic acid in human plasma.

PubMed

Bailey, Steven W; Ayling, June E

2013-11-08

Although many countries have fortified their grain supplies with folic acid (FA) to decrease the incidence of neural tube defects, others have not due to concerns that this synthetic folate might have some adverse effects. Persistent unmetabolized FA has been found even in plasma from fasted subjects. To facilitate measurement of low levels of folic acid in human plasma, post-column coulometric oxidative cleavage was used to convert poorly fluorescent FA into a highly fluorescent compound determined to be 6-formyl-pterin. To minimize sample work-up and maximize recovery, column-switching HPLC transferred a window of eluate containing the FA from the first column (C8) onto a second column (phenyl-hexyl). The pH of two mobile phases were adjusted to be above and then below a pK of the FA α-carboxyl group, thus promoting separation from compounds coeluting from the C8-column. This permitted sample preparation using only a simple high recovery protein precipitation. Definitive identification of FA in human plasma was accomplished by duplicate injections of sample with the electrochemical voltage set above and below its half-potential. The LOD (S/N=3) was 0.10 nM. The intra- and inter-assay CV's were 2.3% and 5%, respectively. Comparison of these results with those obtained by HPLC/MS/MS with stable isotope internal standard showed a slope of 1.00 ± 0.019. This simple, sensitive, and repeatable assay facilitates a more thorough investigation of the response of various human populations to folic acid intake. Post-column differential coulometric electrochemistry can expand the variety of compounds amenable to fluorescence detection. Copyright © 2013 Elsevier B.V. All rights reserved.

Two-dimensional fingerprinting approach for comparison of complex substances analysed by HPLC-UV and fluorescence detection.

PubMed

Ni, Yongnian; Liu, Ying; Kokot, Serge

2011-02-07

This work is concerned with the research and development of methodology for analysis of complex mixtures such as pharmaceutical or food samples, which contain many analytes. Variously treated samples (swill washed, fried and scorched) of the Rhizoma atractylodis macrocephalae (RAM) traditional Chinese medicine (TCM) as well as the common substitute, Rhizoma atractylodis (RA) TCM were chosen as examples for analysis. A combined data matrix of chromatographic 2-D HPLC-DAD-FLD (two-dimensional high performance liquid chromatography with diode array and fluorescence detectors) fingerprint profiles was constructed with the use of the HPLC-DAD and HPLC-FLD individual data matrices; the purpose was to collect maximum information and to interpret this complex data with the use of various chemometrics methods e.g. the rank-ordering multi-criteria decision making (MCDM) PROMETHEE and GAIA, K-nearest neighbours (KNN), partial least squares (PLS), back propagation-artificial neural networks (BP-ANN) methods. The chemometrics analysis demonstrated that the combined 2-D HPLC-DAD-FLD data matrix does indeed provide more information and facilitates better performing classification/prediction models for the analysis of such complex samples as the RAM and RA ones noted above. It is suggested that this fingerprint approach is suitable for analysis of other complex, multi-analyte substances.

Trace determination of lenalidomide in plasma by non-extractive HPLC procedures with fluorescence detection after pre-column derivatization with fluorescamine

PubMed Central

2013-01-01

Background Lenalidomide (LND) is a new potent drug used for treatment of multiple myeloma. For its pharmacokinetic studies and therapeutic monitoring, a proper analytical method was required. Results In this study, a non extractive and simple pre-column derivatization procedures have been proposed, for the for trace determination of lenalidomide (LND) in human plasma by HPLC with fluorescence detection. Plasma samples were treated with acetonitrile for protein precipitation then treated with copper acetate to form stable complexes with the biogenic amines and mask their interference with the derivatization reaction of LND. Treated plasma samples containing LND was derivatized with fluorescamine (FLC) in aqueous media at ambient temperature. Separation of the derivatized LND was performed on Hypersil BDS C18 column (250 × 4.6 mm, 5 μm particle size) using a mobile phase consisting of phosphate buffer (pH 4):methanol: tetrahydrofuran (70:10:20, v/v) at a flow rate of 1.0 mL/min. The derivatized samples were monitored at an emission wavelength of 495 nm after excitation at a wavelength of 382 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997, n = 9) was found between the peak area and LND concentrations in the range of 2–100 ng/mL. The limits of detection and quantitation were 0.8 and 2.30 ng/mL, respectively. The intra- and inter-assay precisions were satisfactory and the accuracy of the method was proved. The recovery of LND from the spiked human plasma was 99.30 ± 2.88. Conclusions The proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and a relatively short run-time (< 15 min). The results demonstrated that the method would have a great value when it is applied in the therapeutic monitoring and pharmacokinetic studies for LND. PMID:23497635

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

PubMed

Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

2009-11-01

Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry.

PubMed

Kakimoto, Kensaku; Toriba, Akira; Ohno, Takanori; Ueno, Mariko; Kameda, Takayuki; Tang, Ning; Hayakawa, Kazuichi

2008-05-15

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.

[Isolation and purification of seven catechin compounds from fresh tea leaves by semi-preparative liquid chromatography].

PubMed

Gong, Zhihong; Chen, Si; Gao, Jiangtao; Li, Meihong; Wang, Xiaxia; Lin, Jun; Yu, Xiaomin

2017-11-08

An effective and simple method was established to simultaneously purify seven tea catechins (gallocatechin (GC), epigallocatechin (EGC), catechin (C), epigallocatechin-3- O -gallate (EGCG), epicatechin (EC), epigallocatechin-3- O -(3- O -methyl)-gallate (EGCG3"Me) and epicatechin-3- O -gallate (ECG)) from fresh tea leaves by semi-preparative high performance liquid chromatography (HPLC). Fresh leaves of Tieguanyin tea were successively extracted with methanol and chloroform. Then crude catechins were precipitated from the aqueous fraction of chloroform extraction by adding lead subacetate. Crude catechins were used for the isolation of the seven target catechin compounds by semi-preparative HPLC. Methanol-water and acetonitrile-water were sequentially used as mobile phases. After two rounds of semi-preparative HPLC, all target compounds were achieved with high purities (>90%). The proposed method was tested on two additional tea cultivars and showed similar results. This method demonstrated a simple and efficient strategy based on solvent extraction, ion precipitation and semi-preparative HPLC for the preparation of multiple catechins from tea leaves.

An innovative approach to the analysis of 3-[4-(2-methylpropyl)phenyl]propanoic acid as an impurity of ibuprofen on a carbon-coated zirconia stationary phase.

PubMed

Kalafut, P; Kucera, R; Klimes, J; Sochor, J

2009-07-12

3-[4-(2-Methylpropyl)phenyl]propanoic acid has been introduced as impurity F to the European Pharmacopoeia in its Supplement 4.2. In contrast to other impurities, which are evaluated by HPLC, the content of impurity F is determined by gas chromatography after previous derivatization. Thus a novel reversed-phase HPLC method was developed to simplify the evaluation of pharmacopoeial impurity F of ibuprofen. Favourable properties of zirconia stationary phases were employed for this purpose. The HPLC separation was achieved on a Zr-CARB column (150 mm x 4.6mm i.d., 5 microm) using the mobile phase acetonitrile-phosphate buffer (pH 3.5, 25 mM) (38:62, v/v), temperature 80 degrees C and the flow rate 1.2 ml min(-1). The fluorescence detection was employed to enhance the sensitivity of the method. Optimal detection parameters were chosen on the basis of fluorescence spectra of the analytes. The excitation and emission wavelengths were 220 nm and 285 nm, respectively. The analysis was completed within 25 min. The subsequent validation of the method confirmed the applicability of method for the analytical assay of impurity F.

Binary Solvents Dispersive Liquid-Liquid Microextraction (BS-DLLME) Method for Determination of Tramadol in Urine Using High-Performance Liquid Chromatography.

PubMed

Kiarostami, Vahid; Rouini, Mohamad-Reza; Mohammadian, Razieh; Lavasani, Hoda; Ghazaghi, Mehri

2014-02-03

Tramadol is an opioid, synthetic analog of codeine and has been used for the treatment of acute or chronic pain may be abused. In this work, a developed Dispersive liquid liquid microextraction (DLLME) as binary solvents-based dispersive liquid-liquid microextraction (BS-DLLME) combined with high performance liquid chromatography (HPLC) with fluorescence detection (FD) was employed for determination of tramadol in the urine samples. This procedure involves the use of an appropriate mixture of binary extraction solvents (70 μL CHCl3 and 30 μL ethyl acetate) and disperser solvent (600 μL acetone) for the formation of cloudy solution in 5 ml urine sample comprising tramadol and NaCl (7.5%, w/v). After centrifuging, the small droplets of extraction solvents were precipitated. In the final step, the HPLC with fluorescence detection was used for determination of tramadol in the precipitated phase. Various factors on the efficiency of the proposed procedure were investigated and optimized. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found 0.2 and 0.9 μg/L, respectively. The relative standard deviations (RSD) for the extraction of 30 μg L of tramadol was found 4.1% (n = 6). The relative recoveries of tramadol from urine samples at spiking levels of 10, 30 and 60 μg/L were in the range of 95.6 - 99.6%. Compared with other methods, this method provides good figures of merit such as good repeatability, high extraction efficiency, short analysis time, simple procedure and can be used as microextraction technique for routine analysis in clinical laboratories.

Binary Solvents Dispersive Liquid—Liquid Microextraction (BS-DLLME) Method for Determination of Tramadol in Urine Using High-Performance Liquid Chromatography

PubMed Central

2014-01-01

Background Tramadol is an opioid, synthetic analog of codeine and has been used for the treatment of acute or chronic pain may be abused. In this work, a developed Dispersive liquid liquid microextraction (DLLME) as binary solvents-based dispersive liquid-liquid microextraction (BS-DLLME) combined with high performance liquid chromatography (HPLC) with fluorescence detection (FD) was employed for determination of tramadol in the urine samples. This procedure involves the use of an appropriate mixture of binary extraction solvents (70 μL CHCl3 and 30 μL ethyl acetate) and disperser solvent (600 μL acetone) for the formation of cloudy solution in 5 ml urine sample comprising tramadol and NaCl (7.5%, w/v). After centrifuging, the small droplets of extraction solvents were precipitated. In the final step, the HPLC with fluorescence detection was used for determination of tramadol in the precipitated phase. Results Various factors on the efficiency of the proposed procedure were investigated and optimized. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found 0.2 and 0.9 μg/L, respectively. The relative standard deviations (RSD) for the extraction of 30 μg L of tramadol was found 4.1% (n = 6). The relative recoveries of tramadol from urine samples at spiking levels of 10, 30 and 60 μg/L were in the range of 95.6 – 99.6%. Conclusions Compared with other methods, this method provides good figures of merit such as good repeatability, high extraction efficiency, short analysis time, simple procedure and can be used as microextraction technique for routine analysis in clinical laboratories. PMID:24495475

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS[S

PubMed Central

Kakiyama, Genta; Muto, Akina; Takei, Hajime; Nittono, Hiroshi; Murai, Tsuyoshi; Kurosawa, Takao; Hofmann, Alan F.; Pandak, William M.; Bajaj, Jasmohan S.

2014-01-01

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. PMID:24627129

Further aspects of ochratoxin A-cation interactions: complex formation with zinc ions and a novel analytical application of ochratoxin A-magnesium interaction in the HPLC-FLD system.

PubMed

Poór, Miklós; Kuzma, Mónika; Matisz, Gergely; Li, Yin; Perjési, Pál; Kunsági-Máté, Sándor; Kőszegi, Tamás

2014-04-10

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into different interactions of OTA at the molecular level. OTA is found worldwide in many foods and drinks. Moreover, it can also be detected in human and animal tissues and body fluids, as well. Therefore, the development of highly sensitive quantitative methods for the determination of OTA is of utmost importance. OTA most likely forms complexes with divalent cations, both in cells and body fluids. In the present study, the OTA-zinc interaction was investigated and compared to OTA-magnesium complex formation using fluorescence spectroscopy and molecular modeling. Our results show that zinc(II) ion forms a two-fold higher stable complex with OTA than magnesium(II) ion. In addition, based on the enhanced fluorescence emission of OTA in its magnesium-bound form, a novel RP-HPLC-fluorescence detector (FLD) method was also established. Our results highlight that the application of magnesium chloride in alkaline eluents results in an approximately two-fold increase in sensitivity using the HPLC-FLD technique.

Determination of blood sirolimus concentrations in liver and kidney transplant recipients using the Innofluor® fluorescence polarization immunoassay: Comparison with the microparticle enzyme immunoassay and high-performance liquid chromatography-ultraviolet method

PubMed Central

Bouzas, Lorena; Hermida, Jesús

2009-01-01

Background Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx® analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification. Methods Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor® Certican® FPIA (Seradyn Inc.) run on a TDx® analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx® analyser (Abbott Laboratories), and HPLC (UV detection) methods. Results The Innofluor® FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%–13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r≥0.900) than in the kidney transplant samples (r≥0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC. Conclusion The Innofluor® FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus. PMID:19242874

Detection of monohydroxylated polycyclic aromatic hydrocarbons in urine and particulate matter using LC separations coupled with integrated SPE and fluorescence detection or coupled with high-resolution time-of-flight mass spectrometry.

PubMed

Lintelmann, Jutta; Wu, Xiao; Kuhn, Evelyn; Ritter, Sebastian; Schmidt, Claudia; Zimmermann, Ralf

2018-05-01

A high-performance liquid chromatographic (HPLC) method with integrated solid-phase extraction for the determination of 1-hydroxypyrene and 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 μL urine, 100 μL of the sample was directly injected into the HPLC system. Integrated solid-phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core-shell column using a methanol gradient. For quantification, time-programmed fluorescence detection was used. Matrix-dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC-fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra-high-performance liquid chromatography pentafluorophenyl core-shell column and coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF-MS). The resulting method was used to demonstrate the applicability of LC-HR-TOF-MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter. Copyright © 2018 John Wiley & Sons, Ltd.

[Simultaneous determination of six fluorescent whitening agents in plastic and paper packaging materials by high performance liquid chromatography].

PubMed

Zhang, Juzhou; Ji, Shuilin; Cai, Huimei; Li, Jing; Wang, Yongxin; Wang, Jingqiu

2017-11-08

A novel analytical method was developed for the simultaneous determination of six fluorescent whitening agents (FWAs:FWA 135, FWA 184, FWA 185, FWA 199, FWA 378 and FWA 393) in paper and plastic food packaging materials by high performance liquid chromatography with fluorescence detection (HPLC-FLD). The sample was extracted with mixed solution of chloroform and acetonitrile (3:7, v/v), then cleaned up by HLB solid phase extraction column. Qualitative and quantitative analyses were carried out by HPLC. The sample was separated on a Phenomenex C18 column using acetonitrile and 5 mmol/L ammonium acetate aqueous solution as mobile phases. The results indicated that the linear range of FWA393 was 15-1500 μg/L and the linear ranges of the other five FWAs were 5-500 μg/L with correlation coefficients greater than 0.999. The recoveries in spiked samples were between 80.4% and 125.0% with RSDs ( n =6) of 1%-13%. Furthermore, this method was applied to analyze 12 samples in the market to verify the practicality of the method. The method showed the advantages of simplicity, high recovery and good precision, and is suitable for the detection of the six fluorescent whitening agents in food packaging materials.

A simple HPLC method for the comprehensive analysis of cis/trans (Z/E) geometrical isomers of carotenoids for nutritional studies

USDA-ARS?s Scientific Manuscript database

Geometrical isomers of carotenoids behave differently in aspects like stability towards oxidants, bioavailability, vitamin A activity and specificity for enzymes. The availability of HPLC methods for their detailed profiling is therefore advisable to expand our knowledge on their metabolism and biol...

Quantitation of polymethoxylated flavones in orange juice by high-performance liquid chromatography.

PubMed

Rouseff, R L; Ting, S V

1979-08-01

A quantitative high-performance liquid chromatographic (HPLC) procedure for the determination of the five major polymethoxylated flavones (PMFs) in orange juice has been developed. It employs a unique ternary solvent system with coupled UV-fluorescence detection. The dual detectors were employed to determine the presence of interfering substances and served as a cross check on quantitation. Stop flow UV and fluorescence scanning was used to identify peaks and determine the presence of impurities. Although all five citrus PMFs fluoresce, some HPLC fluorescence peaks were too small to be of much practical use. All five citrus PMFs could be quantitated satisfactorily with the fixed wavelength UV (313 nm) detector. The HPLC procedure has been used to evaluate each step in the preparation. The optimum extracting solvent was selected and one time consuming step was eliminated, as it was found to be unnecessary. HPLC values for nobiletin and sinensetin are in good agreement with the thin-layer chromatographic (TLC) values in the literature. HPLC values for the other three flavones were considerably lower than those reported in the literature. The HPLC procedure is considerably faster than the TLC procedure with equal or superior precision and accuracy.

Characterization of E 471 food emulsifiers by high-performance thin-layer chromatography-fluorescence detection.

PubMed

Oellig, Claudia; Brändle, Klara; Schwack, Wolfgang

2018-07-13

Mono- and diacylglycerol (MAG and DAG) emulsifiers, also known as food additive E 471, are widely used to adjust techno-functional properties in various foods. Besides MAGs and DAGs, E 471 emulsifiers additionally comprise different amounts of triacylglycerols (TAGs) and free fatty acids (FFAs). MAGs, DAGs, TAGs and FFAs are generally determined by high-performance liquid chromatography (HPLC) or gas chromatography (GC) coupled to mass selective detection, analyzing the individual representatives of the lipid classes. In this work we present a rapid and sensitive method for the determination of MAGs, DAGs, TAGs and FFAs in E 471 emulsifiers by high-performance thin-layer chromatography with fluorescence detection (HPTLC-FLD), including a response factor system for quantitation. Samples were simply dissolved and diluted with t-butyl methyl ether before a two-fold development was performed on primuline pre-impregnated LiChrospher silica gel plates with diethyl ether and n-pentane/n-hexane/diethyl ether (52:20:28, v/v/v) as the mobile phases to 18 and 75 mm, respectively. For quantitation, the plate was scanned in the fluorescence mode at UV 366/>400 nm, when the cumulative signal for each lipid class was used. Calibration was done with 1,2-distearin and amounts of lipid classes were calculated with response factors and expressed as monostearin, distearin, tristearin and stearic acid. Limits of detection and quantitation were 1 and 4 ng/zone, respectively, for 1,2-distearin. Thus, the HPTLC-FLD approach represents a simple, rapid and convenient screening alternative to HPLC and GC analysis of the individual compounds. Visual detection additionally enables an easy characterization and the direct comparison of emulsifiers through the lipid class pattern, when utilized as a fingerprint. Copyright © 2018 Elsevier B.V. All rights reserved.

A simple high performance liquid chromatography method for analyzing paraquat in soil solution samples.

PubMed

Ouyang, Ying; Mansell, Robert S; Nkedi-Kizza, Peter

2004-01-01

A high performance liquid chromatography (HPLC) method with UV detection was developed to analyze paraquat (1,1'-dimethyl-4,4'-dipyridinium dichloride) herbicide content in soil solution samples. The analytical method was compared with the liquid scintillation counting (LSC) method using 14C-paraquat. Agreement obtained between the two methods was reasonable. However, the detection limit for paraquat analysis was 0.5 mg L(-1) by the HPLC method and 0.05 mg L(-1) by the LSC method. The LSC method was, therefore, 10 times more precise than the HPLC method for solution concentrations less than 1 mg L(-1). In spite of the high detection limit, the UC (nonradioactive) HPLC method provides an inexpensive and environmentally safe means for determining paraquat concentration in soil solution compared with the 14C-LSC method.

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.

High-sensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule.

PubMed

Sato, Masaki; Ito, Yuji; Arima, Naomichi; Baba, Masanori; Sobel, Michael; Wakao, Masahiro; Suda, Yasuo

2009-07-01

To analyse the binding of sugar chains to proteins, viruses and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real-time, non-destructive detection system. Key to this method is linker compounds for immobilization of the sugar chains to the gold-coated chip for SPR. Also, well-designed fluorescent labelling reagents are essential when analysing the structure of trace amounts of sugar chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named 'f-mono', which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 microg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As a +2 Da larger MS peak ([M + H + 2](+) ion) was always associated with the theoretical MS peak ([M + H](+)) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold-coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.

Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard.

PubMed

Neville, David C A; Alonzi, Dominic S; Butters, Terry D

2012-04-13

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant. Copyright © 2012 Elsevier B.V. All rights reserved.

Bioanalytical Applications of Fluorescence Line-Narrowing and Non-Line-Narrowing Spectroscopy Interfaced with Capillary Electrophoresis and High-Performance Liquid Chromatography

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

Roberts, Kenneth Paul

Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are widely used analytical separation techniques with many applications in chemical, biochemical, and biomedical sciences. Conventional analyte identification in these techniques is based on retention/migration times of standards; requiring a high degree of reproducibility, availability of reliable standards, and absence of coelution. From this, several new information-rich detection methods (also known as hyphenated techniques) are being explored that would be capable of providing unambiguous on-line identification of separating analytes in CE and HPLC. As further discussed, a number of such on-line detection methods have shown considerable success, including Raman, nuclear magnetic resonancemore » (NMR), mass spectrometry (MS), and fluorescence line-narrowing spectroscopy (FLNS). In this thesis, the feasibility and potential of combining the highly sensitive and selective laser-based detection method of FLNS with analytical separation techniques are discussed and presented. A summary of previously demonstrated FLNS detection interfaced with chromatography and electrophoresis is given, and recent results from on-line FLNS detection in CE (CE-FLNS), and the new combination of HPLC-FLNS, are shown.« less

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.

Comparison of gamma-oryzanol contents in crude rice bran oils from different sources by various determination methods.

PubMed

Yoshie, Ayano; Kanda, Ayato; Nakamura, Takahiro; Igusa, Hisao; Hara, Setsuko

2009-01-01

Although there are various determination methods for gamma -oryzanol contained in rice bran oil by absorptiometry, normal-phase HPLC, and reversed-phase HPLC, their accuracies and the correlations among them have not been revealed yet. Chloroform-containing mixed solvents are widely used as mobile phases in some HPLC methods, but researchers have been apprehensive about its use in terms of safety for the human body and the environment.In the present study, a simple and accurate determination method was developed by improving the reversed-phase HPLC method. This novel HPLC method uses methanol/acetonitrile/acetic acid (52/45/3 v/v/v), a non-chlorinated solvent, as the mobile phase, and shows an excellent linearity (y = 0.9527x + 0.1241, R(2) = 0.9974) with absorptiometry. The mean relative errors among the existing 3 methods and the novel method, determined by adding fixed amounts of gamma-oryzanol into refined rice salad oil, were -4.7% for the absorptiometry, -6.8% for the existing normal-phase HPLC, +4.6% for the existing reversed-phase HPLC, and -1.6% for the novel reversed-phase HPLC method. gamma -Oryzanol content in 12 kinds of crude rice bran oils obtained from different sources were determined by the four methods. The mean content of those oils were 1.75+/-0.18% for the absorptiometry, 1.29+/-0.11% for the existing normal-phase HPLC, 1.51+/-0.10% for the existing reversed-phase HPLC, and 1.54+/-0.19% for the novel reversed-phase HPLC method.

Determination of aristolochic acids by high-performance liquid chromatography with fluorescence detection.

PubMed

Wang, Yinan; Chan, Wan

2014-06-25

Nephrotoxic and carcinogenic aristolochic acids (AAs) are naturally occurring nitrophenanthrene carboxylic acids in the herbal genus Aristolochia. The misuse of AA-containing herbs in preparing slimming drugs has caused hundred of cases of kidney disease in Belgium women in a slimming regime in the early 1990s. Accumulating evidence also suggested that prolong dietary intake of AA-contaminated food is one of the major causes to the Balkan endemic nephropathy that was first observed in the late 1950s. Therefore, analytical methods of high sensitivity are extremely important for safeguarding human exposure to AA-containing herbal medicines, herbal remedies, and food composites. In this paper, we describe the development of a new high-performance liquid chromatography coupled fluorescence detector (HPLC-FLD) method for the sensitive determination of AAs. The method makes use of a novel cysteine-induced denitration reaction that "turns on" the fluorescence of AAs for fluorometric detections. Our results showed that the combination of cysteine-induced denitration and HPLC-FLD analysis allows for sensitive quantification of AA-I and AA-II at detection limits of 27.1 and 25.4 ng/g, respectively. The method was validated and has been successfully applied in quantifying AAs in Chinese herbal medicines.

Determination of Three Corticosteroids in the Biologic Matrix of Vitreous Humor by HPLC-tandem Mass Spectrometry: Method Development and Validation.

PubMed

Prieto, Esther; Vispe, Eugenio; Otín-Mallada, Sofía; Garcia-Martin, Elena; Polo-Llorens, Vicente; Fraile, José M; Pablo, Luis E; Mayoral, José A

2017-02-01

To develop a simple, specific, and rapid method to determine corticosteroid concentrations in vitreous humor. An analytical method based on high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS) with a simple extraction procedure was developed. New Zealand albino rabbits (n = 54) received a single (0.1 mL) intravitreal injection of dexamethasone (DXM, 0.1 mg), methylprednisolone (MP, 2 mg), or triamcinolone acetonide (TA, 10 mg). Eyes were enucleated and mean vitreous steroid levels were quantified at 12 h and 1, 2, 3, 7, and 14 days. Corticosteroids were extracted from the vitreous with acetonitrile, and TA was extracted with ethyl acetate, yielding high protein precipitation and clean solution samples. Vitreous samples were analyzed by isocratic HPLC-MS with mobile phase comprising acetonitrile and 2 mM ammonium formate buffer in water, pH 3.5. The linear range was 50-100,000 ng/g with a lower quantification limit of 45 ng/g for DXM and MP, and 50 ng/g for TA. Vitreous levels of DXM and MP were not detectable 14 days post-administration. Vitreous levels of TA were positive and stable throughout the study in both injected and control eyes. The HPLC-MS analytical method is an alternative to HPLC-MS/MS methods, sensitive enough for identifying and quantifying steroids in vitreous humor at a therapeutic dosage scale.

Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection.

PubMed

Liu, He; Zhang, Yupu; Sun, Yantao; Wang, Xue; Zhai, Yujuan; Sun, Ye; Sun, Shuo; Yu, Aimin; Zhang, Hanqi; Wang, Yinghua

2010-10-15

The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r>0.9998) in the concentration range of 0.010-5.0μgmL(-1) for arctiin and 0.025-7.5μgmL(-1) for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

A convenient ultrasound-assisted saponification for the simultaneous determination of vitamin E isomers in vegetable oil by HPLC with fluorescence detection.

PubMed

Yang, Yi; Lu, Dan; Yin, Shuo; Yang, Danni; Chen, Yaling; Li, Yongxin; Sun, Chengjun

2018-04-01

An efficient ultrasound-assisted saponification was developed for simultaneous determination of vitamin E isomers in vegetable oil by high-performance liquid chromatography with fluorescence detection. The samples were saponified ultrasonically with potassium hydroxide solution for only 7 min, then the analytes were extracted with ether. Vitamin E isomers were separated on a C 18 column at 25°C with a mobile phase of methanol/acetonitrile (81:19, v/v) at a flow rate of 0.8 mL/min. Fluorescence detection was operated at 290 nm of excitation wavelength and 327 nm of emission wavelength. Under the optimized conditions, good linearities over the range of 0.001-8.00 μg/mL (r > 0.999) were obtained. Mean recoveries of the method were 88.0-106%, with intra- and interday RSDs less than 11.8 and 12.8%, respectively. The detection limits and quantification limits of the method were 0.30-1.8 and 1.0-6.1 μg/kg, respectively. The recoveries of this method were much higher than that of the quick, easy, cheap, effective, rugged, and safe method and direct dilution method, but were similar to those of hot saponification. This proposed method provides reliable, simple, and rapid quantification of vitamin E isomers in vegetable oils. Five kinds of vegetable oils were analyzed, the quantification results were within the ranges reported by other authors. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chiral separation of the β2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

PubMed

Ullrich, Thomas; Wesenberg, Dirk; Bleuel, Corinna; Krauss, Gerd-Joachim; Schmid, Martin G; Weiss, Michael; Gübitz, Gerald

2010-10-01

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.

Development and validation of a method for the simultaneous extraction and separate measurement of oxytetracycline, florfenicol, oxolinic acid and flumequine from marine sediments.

PubMed

Norambuena, Luis; Gras, Nuri; Contreras, Sergio

2013-08-15

A simple and rapid method for the detection and extraction of oxolinic acid, flumequine, florfenicol and oxytetracycline from marine sediments was developed and validated. The analytes were extracted from the marine sediment using a solution of oxalic acid diluted in methanol with sonication before detection by HPLC using a diode-array detector (florfenicol and oxytetracycline) and fluorescence (oxolinic acid and flumequine). The quantification limits (QL) were 100 ng/g for oxytetracycline and florfenicol and 5 ng/g for oxolinic acid and flumequine. The coefficients of variation of the repeatability and intermediate precision were less than 10% in all of the analytes. The calibration curves were linear between 50 and 500 ng/ml for oxytetracycline and florfenicol and 1 and 20 ng/ml for oxolinic acid and flumequine. The recuperation rate for the analytes was above 86%. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Direct liquid chromatography method for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines.

PubMed

Piñeiro, Zulema; Cantos-Villar, Emma; Palma, Miguel; Puertas, Belen

2011-11-09

A validated HPLC method with fluorescence detection for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines is described. Detection conditions for both compounds were optimized (excitation at 279 and 278 and emission at 631 and 598 nm for hydroxytyrosol and tyrosol, respectively). The validation of the analytical method was based on selectivity, linearity, robustness, detection and quantification limits, repeatability, and recovery. The detection and quantification limits in red wines were set at 0.023 and 0.076 mg L(-1) for hydroxytyrosol and at 0.007 and 0.024 mg L(-1) for tyrosol determination, respectively. Precision values, both within-day and between-day (n = 5), remained below 3% for both compounds. In addition, a fractional factorial experimental design was developed to analyze the influence of six different conditions on analysis. The final optimized HPLC-fluorescence method allowed the analysis of 30 nonpretreated Spanish red wines to evaluate their hydroxytyrosol and tyrosol contents.

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.

Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

PubMed

Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J

2013-01-01

In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

1989-01-01

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Determination and removal of antibiotics in secondary effluent using a horizontal subsurface flow constructed wetland.

PubMed

Zhang, Chunhui; Ning, Ke; Zhang, Wenwen; Guo, Yuanjie; Chen, Jun; Liang, Chen

2013-04-01

Increased attention is currently being directed towards the potential negative effects of antibiotics and other PPCPs discharged into the aquatic environment via municipal WWTP secondary effluents. A number of analytical methods, such as high performance liquid chromatography technologies, including a high performance liquid chromatography-fluorescence method (HPLC-FLD), high performance liquid chromatography-UV detection method (HPLC-UV) and high performance liquid chromatography-mass spectrometry method (HPLC-MS), have been suggested as determination technologies for antibiotic residues in water. In this study, we implement a HPLC-MS/MS combined method to detect and analyze antibiotics in WWTP secondary effluent and apply a horizontal subsurface flow constructed wetland (CW) as an advanced wastewater treatment for removing antibiotics in the WWTP secondary effluent. The results show that there were 2 macrolides, 2 quinolones and 5 sulfas in WWTP secondary effluent among all the 22 antibiotics considered. After the CW advanced treatment, the concentration removal efficiencies and removal loads of 9 antibiotics were 53-100% and 0.004-0.7307 μg m(-2) per day, respectively.

Fluorescence polarization immunoassays for rapid, accurate, and sensitive determination of mycotoxins

USDA-ARS?s Scientific Manuscript database

Analytical methods for the determination of mycotoxins in foods are commonly based on chromatographic techniques (GC, HPLC or LC-MS). Although these methods permit a sensitive and accurate determination of the analyte, they require skilled personnel and are time-consuming, expensive, and unsuitable ...

A Liquid Chromatography-Mass Spectrometry Assay for Determination of Perampanel and Concomitant Antiepileptic Drugs in the Plasma of Patients with Epilepsy, Compared with A Fluorescent Hplc Assay.

PubMed

de Grazia, Ugo; D'Urso, Annachiara; Ranzato, Federica; De Riva, Valentina; Contarato, Giorgia; Billo, Giuseppe; Perini, Francesco; Galloni, Elisabetta

2018-05-09

Perampanel is a novel non-competitive selective antagonist at the postsynaptic ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) glutamate receptor, approved as an adjunctive agent for the treatment of partial-onset seizure with or without secondary generalization and for primary generalized tonic-clonic seizure in patients with epilepsy who are at least 12 years of age. Limited information is available about the clinical utility of therapeutic drug monitoring of perampanel and therapeutic ranges are so far not established. Therefore, perampanel titration should be performed especially in case of insufficient success of the drug. The authors developed a selective and sensitive LC-MS/MS assay to monitor perampanel concentrations in plasma which was compared to a commercially available HPLC kit with fluorescent detection. Perampanel and the internal standard were extracted from plasma samples by a simple protein precipitation. The method allows the simultaneous quantification of perampanel and several other antiepileptic drugs (AEDs). Data were evaluated according to EMA guidelines for bioanalytical method validation. Extraction recovery of perampanel from human plasma was consistently above 98%. No matrix effect was found. Analytical interferences by other AEDs were not observed. The method was linear in the range from 2.5 to 2800 ng/ml. Intra- and inter-assay reproducibility analyses demonstrated accuracy and precision within acceptance criteria. Data collected from 95 patients, given perampanel as their maintenance antiepileptic therapy, showed a very strong correlation between the two methods. The assay allows for highly sensitive and selective quantification of perampanel and concomitant antiepileptic drugs in patient plasma samples and can be easily implemented in clinical settings. Our findings are in agreement with previously published data in patients comedicated with enzyme inducer AEDs, but seem to indicate a possible interaction in patients treated with the enzyme inhibitor drug valproic acid (VPA).

HPLC with fluorescence detection assay of perampanel, a novel AMPA receptor antagonist, in human plasma for clinical pharmacokinetic studies.

PubMed

Mano, Yuji; Takenaka, Osamu; Kusano, Kazutomi

2015-10-01

Perampanel (Fycompa®), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial-onset seizures. To support therapeutic drug monitoring, a simple high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C18 column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile-water-acetic acid-sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0-500 ng/mL) and this could be extended to 25 µg/mL by 50-fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug-free blank human plasma from six individuals and no interference was observed with co-medications tested. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. Copyright © 2015 John Wiley & Sons, Ltd.

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: comparison of sub-2 microm particles and conventional C18 columns.

PubMed

Zhang, Hong; Chen, Si; Lu, Yanbin; Dai, Zhiyuan

2010-07-01

A simple and effective multi-residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi-residue separations on four columns packed with 5 microm or sub-2 microm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub-2 microm particle-sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre-treatment procedure. Good recoveries (71-98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub-2 microm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 microg/kg.

Determination of ochratoxin A in wine by means of immunoaffinity and aminopropyl solid-phase column cleanup and fluorometric detection.

PubMed

Longobardi, Francesco; Iacovelli, Vito; Catucci, Lucia; Panzarini, Giuseppe; Pascale, Michelangelo; Visconti, Angelo; Agostiano, Angela

2013-02-27

A new analytical method for the determination of ochratoxin A (OTA) in red wine has been developed by using a double-extract cleanup and a fluorometric measurement after spectral deconvolution. Wine samples were diluted with a solution containing 1% polyethylene glycol and 5% sodium hydrogencarbonate, filtered, and purified by immunoaffinity and aminopropyl solid-phase column. OTA contents in the purified extract were determined by a spectrofluorometer (excitation wavelength, 330 nm; emission wavelength, 470 nm) after deconvolution of fluorescence spectra. Average recoveries from wine samples spiked with OTA at levels ranging from 0.5 to 3.0 ng/mL were 94.5-105.4% with relative standard deviations (RSD) of <15% (n = 4). The limit of detection (LOD) was 0.2 ng/mL, and the total time of analysis was 30 min. The developed method was tested on 18 red wine samples (naturally contaminated and spiked with OTA at levels ranging from 0.4 to 3.0 ng/mL) and compared with AOAC Official Method 2001.01, based on immunoaffinity column cleanup and HPLC with fluorescence detector. A good correlation (r(2) = 0.9765) was observed between OTA levels obtained with the two methods, highlighting the reliability of the proposed method, the main advantage of which is the simple OTA determination by a benchtop fluorometer with evident reductions of cost and time of analysis.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

PubMed

Llambias, E B; Luo, J

1996-01-01

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Determination of ethoxyquin by high-performance liquid chromatography with fluorescence detection and its application to the survey of residues in food products of animal origin.

PubMed

Aoki, Yoichi; Kotani, Akira; Miyazawa, Naomi; Uchida, Kazunari; Igarashi, Yu; Hirayama, Norio; Hakamata, Hideki; Kusu, Fumiyo

2010-01-01

An analytical method using HPLC with fluorescence detection (HPLC-FL) has been developed and applied for the survey of residue levels of ethoxyquin in a variety of food products of animal origin. HPLC was performed using a silica octadecylsilane column, butylhydroxytoluene-acetonitrile-water (0.05 + 800 + 200, v/v/v) mobile phase, and detection at excitation and emission wavelengths of 370 and 415 nm, respectively. HPLC/MS was used to confirm whether a chromatographic peak was ethoxyquin. The LOQ of the foods was 0.01 microg/g, except for pig fat and cow's milk, and the RSD (n=6) at 0.1 microg/mL of the standard solution was 1.12%. The accuracy of the calculated data of the standard solution was within the range of 94.0 to 101.2%. Recoveries of ethoxyquin from the food products of cattle, pigs, chickens, and salmon were more than 71.0% with an RSD of < 9.3%, except for chicken liver at different concentration levels, including the lower LOQ, the maximum residue limit (MRL), and in some tissues, twice the MRL. Residue levels of ethoxyquin in 33 commercially available food products of animal origin that were purchased on the west side of the Tokyo metropolitan area were surveyed. Contents of ethoxyquin residues in three chicken fat samples by the HPLC-FL method were 0.08, 0.03, and 0.04 microg/g, all less than the MRL (5 microg/g).

Quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair by HPLC with fluorescence detection: a biological monitoring method to evaluate the exposure to PAHs.

PubMed

Toriba, Akira; Kuramae, Yayoi; Chetiyanukornkul, Thaneeya; Kizu, Ryoichi; Makino, Tsunehisa; Nakazawa, Hiroyuki; Hayakawa, Kazuichi

2003-01-01

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair. Fifteen kinds of PAHs classified as priority pollutants by the US EPA were quantified with four perdeuterated PAHs as internal standards. After 50 mg hair samples were washed with n-hexane to remove external contamination of PAHs, the samples were digested in 2.5 M sodium hydroxide. The digests were extracted with n-hexane and then analyzed by HPLC. Eleven kinds of PAHs were identified in hair samples of 20 subjects, and 10 kinds of PAHs were eventually quantified using the internal standards. For anthracene, chrysene and benzo[k]fluoranthene, significant differences were observed between smokers and non-smokers. Although benzo[b]fluoranthene, dibenz[a,h]anthracene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were observed in the particulates of indoor and outdoor air, they were not detected in all hair samples. The analysis of PAHs in human hair should be useful as a new biomarker to evaluate the exposure to PAHs.

Synthesis of the flavonoid 3',4',5'-trimethoxyflavonol and its determination in plasma and tissues of mice by HPLC with fluorescence detection.

PubMed

Britton, Robert G; Fong, Isabel; Saad, Shaban; Brown, Karen; Steward, William P; Gescher, Andreas; Sale, Stewart

2009-04-01

3',4',5'-Trimethoxyflavonol (TMFol) was synthesized as a potential colorectal cancer chemopreventive agent. An HPLC method for determination for TMFol in murine plasma and tissues was developed and validated using human plasma. Analyte was separated (C(18) column; fluorescence detection 330nm excitation, 440nm emission) using 69% methanol and 0.1M ammonium acetate buffer (pH 5.1) as mobile phase. The method was linear for 50-2500ng/ml plasma and 0.05-10microg/g tissue (r>0.99). TMFol was recovered from plasma or tissues using solid phase columns or organic solvent protein precipitation, respectively. Recovery at low, medium and high concentrations was 97.6-107.3%, with inter- and intra-day coefficients of variation of <10%. The lower limit of quantitation for plasma was 50ng/ml. The method was applied to measure steady-state TMFol plasma and tissue levels in mice which received dietary TMFol (0.2%).

Immunoassay screening of lysergic acid diethylamide (LSD) and its confirmation by HPLC and fluorescence detection following LSD ImmunElute extraction.

PubMed

Grobosch, T; Lemm-Ahlers, U

2002-04-01

In all, 3872 urine specimens were screened for lysergic acid diethylamide (LSD) using the CEDIA DAU LSD assay. Forty-eight samples, mainly from psychiatric patients or drug abusers, were found to be LSD positive, but only 13 (27%) of these could be confirmed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) following immunoaffinity extraction (IAE). Additional analysis for LSD using the DPC Coat-a-Count RIA was performed to compare the two immunoassay screening methods. Complete agreement between the DPC RIA assay and HPLC-FLD results was observed at concentrations below a cutoff concentration of 500 pg/mL. Samples that were LSD positive in the CEDIA DAU assay but not confirmed by HPLC-FLD were also investigated for interfering compounds using REMEDI HS drug-profiling system. REMEDI HS analysis identified 15 compounds (parent drugs and metabolites) that are believed to cross-react in the CEDIA DAU LSD assay: ambroxol, prilocaine, pipamperone, diphenhydramine, metoclopramide, amitriptyline, doxepine, atracurium, bupivacaine, doxylamine, lidocaine, mepivacaine, promethazine, ranitidine, and tramadole. The IAE/HPLC-FLD combination is rapid, easy to perform and reliable. It can reduce costs when standard, rather than more advanced, HPLC equipment is used, especially for labs that perform analyses for LSD infrequently. The chromatographic analysis of LSD, nor-LSD, and iso-LSD is not influenced by any of the tested cross-reacting compounds even at a concentration of 100 ng/mL.

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction.

PubMed

Irakli, Maria N; Samanidou, Victoria F; Papadoyannis, Ioannis N

2011-06-01

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selected retinoids: determination by isocratic normal-phase HPLC.

PubMed

Klvanova, J; Brtko, J

2002-09-01

Retinol (ROL), retinal (RAL) and retinoic acid (RA) are physiologically active forms of vitamin A. All-trans retinoic acid (ATRA) can be formed by oxidation from all-trans retinal (ATRAL). Isomerization of RA is considered to be an important metabolic pathway of retinoids. RA isomers transactivate various response pathways via their cognate nuclear receptors that act as ligand inducible transcription factors. The aim of this study was to establish a rapid and simple method for determination of ATRA, 13-cis retinoic acid (13CRA) and ATRAL by HPLC. In our laboratory, we slightly modified the method of Miyagi et al. (2001) and separated ATRA, 13CRA and ATRAL by simple isocratic normal phase HPLC. Both retinoic acid isomers and ATRAL were eluted within 13 min and all components were well resolved. The coefficients of variation (C.V.) for RAs and RAL were from 3.0 to 5.4 %.

Extraction and determination of biogenic amines in fermented sausages and other meat products using reversed-phase-HPLC.

PubMed

Straub, B; Schollenberger, M; Kicherer, M; Luckas, B; Hammes, W P

1993-09-01

A convenient method is described for the analysis of biogenic amines (BA) by means of reversed-phase-HPLC. The method is characterized by multi-channel UV detection (diodearray), subsequent post-column derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid, and fluorescence detection. For the analysis of meat products and especially fermented sausages an optimized perchloric acid extraction process was introduced to determine putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. BA recoveries from meat ranged between 96 and 113% with a detection limit for amines of 0.5 mg/kg.

4-Pyridoxic Acid in the Spent Dialysate: Contribution to Fluorescence and Optical Monitoring

PubMed Central

Kalle, Sigrid; Tanner, Risto; Arund, Jürgen; Tomson, Ruth; Luman, Merike; Fridolin, Ivo

2016-01-01

Aim In this work we estimated the contribution of the fluorescence of 4-pyridoxic acid (4-PA) to the total fluorescence of spent dialysate with the aim of evaluating the on-line monitoring of removal of this vitamin B-6 metabolite from the blood of patients with end-stage renal disease (ESRD). Methods Spectrofluorometric analysis of spent dialysate, collected from hemodialysis and hemodiafiltration sessions of 10 patients receiving regularly pyridoxine injections after dialysis treatment, was performed in the range of Ex/Em 220–500 nm. 4-PA in dialysate samples was identified and quantified using HPLC with fluorescent and MS/MS detection. Results Averaged HPLC chromatogram of spent dialysate had many peaks in the wavelength region of Ex320/Em430 nm where 4-PA was the highest peak with contribution of 42.2±17.0% at the beginning and 47.7±18.0% in the end of the dialysis. High correlation (R = 0.88–0.95) between 4-PA concentration and fluorescence intensity of spent dialysate was found in the region of Ex310-330/Em415-500 nm, respectively. Conclusion 4-PA elimination from the blood of ESRD patients can be potentially followed using monitoring of the fluorescence of the spent dialysate during dialysis treatments. PMID:27598005

Automated dispersive liquid-liquid microextraction coupled to high performance liquid chromatography - cold vapour atomic fluorescence spectroscopy for the determination of mercury species in natural water samples.

PubMed

Liu, Yao-Min; Zhang, Feng-Ping; Jiao, Bao-Yu; Rao, Jin-Yu; Leng, Geng

2017-04-14

An automated, home-constructed, and low cost dispersive liquid-liquid microextraction (DLLME) device that directly coupled to a high performance liquid chromatography (HPLC) - cold vapour atomic fluorescence spectroscopy (CVAFS) system was designed and developed for the determination of trace concentrations of methylmercury (MeHg + ), ethylmercury (EtHg + ) and inorganic mercury (Hg 2+ ) in natural waters. With a simple, miniaturized and efficient automated DLLME system, nanogram amounts of these mercury species were extracted from natural water samples and injected into a hyphenated HPLC-CVAFS for quantification. The complete analytical procedure, including chelation, extraction, phase separation, collection and injection of the extracts, as well as HPLC-CVAFS quantification, was automated. Key parameters, such as the type and volume of the chelation, extraction and dispersive solvent, aspiration speed, sample pH, salt effect and matrix effect, were thoroughly investigated. Under the optimum conditions, linear range was 10-1200ngL -1 for EtHg + and 5-450ngL -1 for MeHg + and Hg 2+ . Limits of detection were 3.0ngL -1 for EtHg + and 1.5ngL -1 for MeHg + and Hg 2+ . Reproducibility and recoveries were assessed by spiking three natural water samples with different Hg concentrations, giving recoveries from 88.4-96.1%, and relative standard deviations <5.1%. Copyright © 2017 Elsevier B.V. All rights reserved.

[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.

Pyridylamination as a means of analyzing complex sugar chains

PubMed Central

Hase, Sumihiro

2010-01-01

Herein, I describe pyridylamination for versatile analysis of sugar chains. The reducing ends of the sugar chains are tagged with 2-aminopyridine and the resultant chemically stable fluorescent derivatives are used for structural/functional analysis. Pyridylamination is an effective “operating system” for increasing sensitivity and simplifying the analytical procedures including mass spectrometry and NMR. Excellent separation of isomers is achieved by reversed-phase HPLC. However, separation is further improved by two-dimensional HPLC, which involves a combination of reversed-phase HPLC and size-fractionation HPLC. Moreover, a two-dimensional HPLC map is also useful for structural analysis. I describe a simple procedure for preparing homogeneous pyridylamino sugar chains that is less laborious than existing techniques and can be used for functional analysis (e.g., sugar-protein interaction). This novel approach was applied and some of the results are described: i) a glucosyl-serine type sugar chain found in blood coagulation factors; ii) discovery of endo-β-mannosidase (EC 3.2.1.152) and a new type plant α1,2-l-fucosidase; and iii) novel substrate specificity of a cytosolic α-mannosidase. Moreover, using homogeneous sugar chains of a size similar to in vivo substrates we were able to analyze interactions between sugar chains and proteins such as enzymes and lectins in detail. Interestingly, our studies reveal that some enzymes recognize a wider region of the substrate than anticipated. PMID:20431262

Advanced glycated end-products (AGE) during haemodialysis treatment: discrepant results with different methodologies reflecting the heterogeneity of AGE compounds.

PubMed

Henle, T; Deppisch, R; Beck, W; Hergesell, O; Hänsch, G M; Ritz, E

1999-08-01

There has been much recent interest in accumulation of advanced glycation end-products (AGE) in uraemic patients. Analysis of AGE has been difficult, because commonly used methodologies, i.e. immunodetection assays or fluorescence measurements, reflect group reactivity and are not specific for chemically defined substances. Some investigators measured individual AGE compounds, e.g. pentosidine, carboxymethyllysine, pyrraline or imidazolone, but a systematic assessment of known compounds using specific HPLC methods in diabetic and non-diabetic end-stage renal disease (ESRD) patients during treatment has not been performed. For the present study, the concentrations of early and late products of the Maillard reaction in plasma and ultrafiltrate were monitored during high-flux dialysis sessions in diabetic and non-diabetic patients. AGE were analysed by fluorescence spectroscopy and size exclusion chromatography with fluorescence detection. Specific HPLC methods were used to quantify the Amadori product fructoselysine and the AGE compounds pentosidine and pyrraline in acid or enzymatic hydrolysates. Using size exclusion chromatography, we confirmed a similar fluorescent peak distribution for diabetic and non-diabetic ESRD patients. Main fractions were found at approximately 70, approximately 14 and <2 kDa, confirming results obtained by other authors. In diabetic patients, the fluorescence intensity of the low molecular weight fraction was higher. Uraemic patients differed from controls mainly by the fluorescence of the low molecular weight fraction. The peak spectrum in ultrafiltrates was similar to that in plasma regarding low molecular weight fractions and the 14 kDa peak, but no protein-bound fluorescence was found at 70 kDa. HPLC analysis revealed a significant reduction of plasma pentosidine during high-flux dialysis in non-diabetic (from 9.1+/-5.1 to 8.5+/-4.7 pmol/mg protein; P<0.05) and diabetic patients (from 10.0+/-9.1 to 6.8+/-4.0 pmol/mg protein; P<0.05). In contrast, plasma fructoselysine showed only a non-significant trend to decrease in diabetic (from 3.24+/-0.88 to 3.05+/-0.77 nmol/mg protein) and non-diabetic patients (from 2.69+/-0.52 to 2.56+/-0.50 nmol/mg protein). Pyrraline, a nonfluorescent late AGE product derived from reaction of 3-deoxyglucosone with lysine, could not be detected (detection limit approximately 40 pmol/mg protein). Comparing HPLC and size exclusion analysis, it was found that pentosidine accumulated in the range of low molecular weight substances and was removed by high-flux dialysis. High-flux dialysis reduces the plasma concentration of fluorescent AGE compounds, i.e. pentosidine, but the Amadori product fructoselysine is not removed, indicating that this compound is protein associated.

Measurement of Menadione in Urine by HPLC

PubMed Central

Rajabi, Ala Al; Peterson, James; Choi, Sang Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-01-01

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C30 column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H2O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R2) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Research support: USDA ARS Cooperative Agreement 58-1950-7-707. PMID:20719580

Measurement of menadione in urine by HPLC.

PubMed

Al Rajabi, Ala; Peterson, James; Choi, Sang-Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-09-15

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Simultaneous multi-mycotoxin determination in nutmeg by ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection.

PubMed

Kong, Wei-Jun; Liu, Shu-Yu; Qiu, Feng; Xiao, Xiao-He; Yang, Mei-Hua

2013-05-07

A simple and sensitive analytical method based on ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with high performance liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection (USLE-IAC-HPLC-PCD-FLD) has been developed for simultaneous multi-mycotoxin determination of aflatoxins B1, B2, G1, G2 (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) in 13 edible and medicinal nutmeg samples marketed in China. AFs and OTA were extracted from nutmeg samples by ultrasonication using a methanol : water (80 : 20, v/v) solution, followed by an IAC clean-up step. Different USL extraction conditions, pre-processing ways for nutmeg sample and clean-up columns for mycotoxins, as well as HPLC-PCD-FLD parameters (mobile phase, column temperature, elution procedure, excitation and emission wavelengths) were optimized. This method, which was appraised for analyzing nutmeg samples, showed satisfactory results with reference to limits of detection (LODs) (from 0.02 to 0.25 μg kg(-1)), limits of quantification (LOQs) (from 0.06 to 0.8 μg kg(-1)), linear ranges (up to 30 ng mL(-1) for AFB1, AFG1 and OTA and 9 ng mL(-1) for AFB2 and AFG2), intra- and inter-day variability (all <2%) and average recoveries (from 79.6 to 90.8% for AFs and from 93.6 to 97.3% for OTA, respectively). The results of the application of developed method in nutmeg samples have elucidated that four samples were detected with contamination of AFs and one with OTA. AFB1 was the most frequently found mycotoxin in 30.8% of nutmeg samples at contamination levels of 0.73-16.31 μg kg(-1). At least two different mycotoxins were co-occurred in three samples, and three AFs were simultaneously detected in one sample.

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.

[Determination of emamectin benzoate residue in vegetables by high performance liquid chromatography with fluorescence detection].

PubMed

Zhang, Yan; Wu, Yinliang; Hu, Jiye; Wang, Hongwei; Pan, Canping; Liu, Fengmao

2008-01-01

A method was developed for the determination of emamectin benzoate residue in cabbage and mushroom using solid-phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection. The sample was extracted with ethyl acetate. Further cleanup was performed on a propylsulfonic acid solid phase extraction cartridge, followed by the derivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. The amount of derivatized emamectin benzoate was determined by fluorescence detector after separation by HPLC. The detection limit was 0.10 microg/kg for cabbage and mushroom samples. The recoveries of emamectin benzoate in cabbage and mushroom samples were 78.6%-84.9%. The inter-day relative standard deviation (RSD) and intra-day RSD were 2.7%-6.0% and 3.1%-8.9%, respectively, at the fortified levels of 1.0-20.0 microg/kg. The calibration curve of emamectin benzoate in vegetables at the concentration range of 0.002 mg/L to 0.10 mg/L was linear (r = 0.9999).

Measurement of pyrimidine (6-4) photoproducts in DNA by a mild acidic hydrolysis-HPLC fluorescence detection assay.

PubMed

Douki, T; Voituriez, L; Cadet, J

1995-03-01

Pyrimidine (6-4) pyrimidone photoproducts constitute one of the major classes of DNA lesions induced by far-UV irradiation. However, their biological role remains difficult to assess partly because of the lack of a specific and sensitive assay for monitoring their formation in DNA. Here is presented a measurement method based on the release of the (6-4) base adducts from DNA followed by an HPLC separation associated with a sensitive and specific fluorescence detection. The quantitative and mechanistic aspects of the chemical hydrolysis, based on the use of hydrogen fluoride stabilized in pyridine, were investigated, using dinucleoside monophosphate (6-4) photoproducts as model compounds. The final hydrolysis products were isolated and characterized by UV, fluorescence, mass, and 1H NMR spectroscopies. Application of the assay to far-UV irradiated calf thymus DNA provided information on the sequence effect on the rate of formation of three of the four possible bipyrimidine (6-4) photoproducts.

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.

Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader.

PubMed

Mandalakis, Manolis; Stravinskaitė, Austėja; Lagaria, Anna; Psarra, Stella; Polymenakou, Paraskevi

2017-07-01

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2  = 0.9998) across the entire calibration range (0.05-240 pg μL -1 ). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg μL -1 ) and quantification (0.132 pg μL -1 ). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L -1 ) measured in seawater samples by HPLC and fluorescence microplate reader.

Assessment of phytoplankton class abundance using fluorescence excitation-emission matrix by parallel factor analysis and nonnegative least squares

NASA Astrophysics Data System (ADS)

Su, Rongguo; Chen, Xiaona; Wu, Zhenzhen; Yao, Peng; Shi, Xiaoyong

2015-07-01

The feasibility of using fluorescence excitation-emission matrix (EEM) along with parallel factor analysis (PARAFAC) and nonnegative least squares (NNLS) method for the differentiation of phytoplankton taxonomic groups was investigated. Forty-one phytoplankton species belonging to 28 genera of five divisions were studied. First, the PARAFAC model was applied to EEMs, and 15 fluorescence components were generated. Second, 15 fluorescence components were found to have a strong discriminating capability based on Bayesian discriminant analysis (BDA). Third, all spectra of the fluorescence component compositions for the 41 phytoplankton species were spectrographically sorted into 61 reference spectra using hierarchical cluster analysis (HCA), and then, the reference spectra were used to establish a database. Finally, the phytoplankton taxonomic groups was differentiated by the reference spectra database using the NNLS method. The five phytoplankton groups were differentiated with the correct discrimination ratios (CDRs) of 100% for single-species samples at the division level. The CDRs for the mixtures were above 91% for the dominant phytoplankton species and above 73% for the subdominant phytoplankton species. Sixteen of the 85 field samples collected from the Changjiang River estuary were analyzed by both HPLC-CHEMTAX and the fluorometric technique developed. The results of both methods reveal that Bacillariophyta was the dominant algal group in these 16 samples and that the subdominant algal groups comprised Dinophyta, Chlorophyta and Cryptophyta. The differentiation results by the fluorometric technique were in good agreement with those from HPLC-CHEMTAX. The results indicate that the fluorometric technique could differentiate algal taxonomic groups accurately at the division level.

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

PubMed

Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective classification and detection of malignant, premalignant and healthy conditions. The method is extremely sensitive to detect proteins with limit of detection of the order of femto-moles. The HPLC-LIF combined with PCA as a potential proteomic method for the diagnosis of oral cancer and cervical cancer has been discussed in this paper. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

[Determination of formaldehyde and acetaldehyde in packaging paper by dansylhydrazine derivatization-high performance liquid chromatography-fluorescence detection].

PubMed

Gong, Shuguo; Liang, Yong; Tang, Liyun; Huang, Ping; Dai, Yunhui

2017-07-08

A high performance liquid chromatography with fluorescence detection (HPLC-FLD) method was developed for the simultaneous determination of formaldehyde and acetaldehyde in packaging paper by dansylhydrazine (DNSH) derivatization. The samples were extracted by derivatization reagent for 30 min, and derived for 24 h. After purifying treatment with a PSA/C18 cartridge, a Diamonsil ® C18 column (150 mm×4.6 mm, 5 μ m) was used as stationary phase for separation, the mixtures of acetic acid aqueous solution (pH 2.55)-acetonitrile were used as mobile phases by gradient elution, and the excitation and emission wavelengths were 330 nm and 484 nm, respectively. The results showed that the recoveries of formaldehyde and acetaldehyde spiked in the samples were 81.64%-106.78%, and the relative standard deviations (RSDs) were 2.02%-5.53% ( n =5). The limits of detection of formaldehyde and acetaldehyde were 19.2 μ g/kg and 20.7 μ g/kg, respectively. The limits of quantification of formaldehyde and acetaldehyde were 63.9 μ g/kg and 69.1 μ g/kg, respectively. The method is simple, sensitive and reproducible. It provides a basic approach for the determination of trace formaldehyde and acetaldehyde.

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.

Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection.

PubMed

Vermeij, T A C; Edelbroek, P M

2004-10-25

A rapid, simple and robust method is presented for the simultaneous determination of the gamma-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by high-performance liquid chromatography (HPLC). Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant are precolumn derivatized with o-phtaldialdehyde (OPA) and 3-mercaptopropionic acid. Separation is achieved on a Alltima 3C18 column using isocratic elution; the drugs are monitored using fluorescence detection. Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2% for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8 mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB. No interferences were found from commonly coadministered antiepileptic drugs (AEDs) and from endogenous amino acids. Experimental design in combination with statistical evaluation (ANOVA) was used to study the robustness of chromatography and sample preparation. The method is very suitable for routine therapeutic drug monitoring and for pharmacokinetic studies.

An alternative derivatization reaction to the determination of doramectin in bovine milk using spectrofluorimetry

NASA Astrophysics Data System (ADS)

Maia, Priscila M. S.; de F. Rezende, Flavia B.; Netto, Annibal D. Pereira; de C. Marques, Flávia F.

The doramectin (DOR), which belongs to the avermectins group (AVM), has a high antiparasitic activity and so it has been widely used in food-producing animals. The DOR shows low fluorescence quantum efficiency and as a consequence, chemical derivatization reactions are necessary to produce derivatives with improved luminescent properties before its determination by fluorimetry. As the presence of this compound in food represents a risk to human health, an easy, clean and low cost derivatization reaction, which is alternative to those usually employed and that enables its spectrofluorimetric determination in milk samples, was developed. Ethanolic solutions of DOR, containing sodium hydroxide at a final concentration of 0.25 mol L-1, after 60 min of heating at 50 °C, produced fluorescent signals 1000 times higher than the original ethanolic solution. Using these optimized conditions, a linear response range that extended from 50.00 to 1000 μg L-1, with a value of (R2) equal to 0.9970, was obtained. Average recovery of DOR was 92.5 ± 1.5% (n = 3) in bovine milk fortified samples submitted to a liquid-liquid extraction at low temperature and pre concentration process, indicating the usefulness and effectiveness of the proposed method. The proposed spectrofluorimetric method is an alternative to high-performance liquid chromatography (HPLC) based methods, allowing rapid and simple detection of doramectin in milk samples.

Sensitive high-performance liquid chromatographic method with fluorometric detection for the simultaneous determination of gabapentin and vigabatrin in serum and urine.

PubMed

Wad, N; Krämer, G

1998-01-23

Serum concentrations of the antiepileptic drug gabapentin (GBP) are usually determined by high-performance liquid chromatography (HPLC) using UV photometric detection after pre-column derivatization with 2,4,6-trinitrobenzenesulphonic acid. Vigabatrin levels in serum are determined by HPLC using fluorescence detection. Like vigabatrin (VGB), gabapentin has also a primary amine group that easily reacts with o-phthaldialdehyde reagent and produces a fluorescing substance. By the use of fluorometric detection, GBP can be determined more simply, sensitively and simultaneously with VGB. The day-to-day coefficient of variation for the determination of GBP in a pooled serum was 4.0% (n=17; serum concentration, 13.8 micromol/l) and forVGB was 3.1% (n=21; serum concentration, 26.4 micromol/l). The lower limit of detection is 0.5 micromol/l for both drugs and the method is linear up to 500 micromol/l for GBP and 1300 micromol/l for VGB.

On-line MSPD-SPE-HPLC/FLD analysis of polycyclic aromatic hydrocarbons in bovine tissues.

PubMed

Gutiérrez-Valencia, Tania M; García de Llasera, Martha P

2017-05-15

A fast method was optimized and validated for simultaneous trace determination of four polycyclic aromatic hydrocarbons: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in bovine tissues. The determination was performed by matrix solid-phase dispersion (MSPD) coupled on-line to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection (FLD). The sample was dispersed on C 18 silica sorbent and then the on-line MSPD-SPE-HPLC/FLD method was applied. Several parameters were optimized: cleaning and elution sequences applied to the MSPD cartridge, the flow rate and dilution of extract used for SPE loading. The on-line method was validated over a concentration range of 0.1-0.6ngg -1 obtaining good linearity (r⩾0.998) and precision (RSD)⩽10%. Recovery ranged from 96 to 99% and the limits of detection were 0.012ngg -1 . This methodology was applied to liver samples from unhealthy animals. The results demonstrate that MSDP-SPE-HPLC/FLD method provides reliable, sensitive, accurate and fast data to the food control. Copyright © 2016 Elsevier Ltd. All rights reserved.

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%.

Determination of low molecular weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

NASA Technical Reports Server (NTRS)

Fahey, Robert C.; Newton, Gerald L.

1988-01-01

Methods are described for the preparation and high-performance liquid chromatography (HPLC) analysis of monobromobimane derivatives of low molecular weight thiols in extracts of biological samples. Typical problems encountered in the development and application of these methods are discussed. Analysis of mung bean extract is used as an example.

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

High-performance liquid chromatographic characterization of some medical plant extracts used in cosmetic formulas.

PubMed

Schulz, H; Albroscheit, G

1988-06-17

Rapid and reliable methods are presented for the characterization of biologically active and/or characteristic constituents in aqueous extracts of Hamamelis virginiana, Matricaria chamomilla, Achillea millefolium, Thymus vulgaris, Althaea officinalis and Cinchonia spp. Prior to high-performance liquid chromatographic (HPLC) separation a clean-up step was performed using a solid-phase extraction system. The purified extracts were analysed by HPLC coupled with a diode-array detector and a fluorescence detector. In some instances, previously unreported components of the aqueous plant extracts were found.

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

Liquid chromatography determination of 10-hydroxycamptothecin in human serum by a column-switching system containing a pre-column with restricted access media and its application to a clinical pharmacokinetic study.

PubMed

Ma, Jun; Jia, Zheng-Ping; Zhang, Qiang; Fan, Jun-Jie; Jiang, Ning-Xi; Wang, Rong; Xie, Hua; Wang, Juan

2003-10-25

A simple, rapid, sensitive column-switching HPLC method is described for the analysis of the 10-hydroxycamptothecin (HCPT) in human serum. A pre-column containing restricted access media (RAM) is used for the sample clean-up and trace enrichment and is combined with a C18 column for the final separation. The analytical time is 8 min. The HCPT is monitored with fluorescence detector, excitation and emission wavelengths being 385 and 539 nm, respectively. There is a linear response range of 1-1000 ng/ml with correlation coefficient of 0.998 while the limit of quantification is 0.1 ng/ml. The intra-day and inter-day variations are less than 5%. This analytic procedure has been applied to a pharmacokinetic study of HCPT in clinical patients and the pharmacokinetic parameters of one-compartment model are calculated.

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.

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.

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags

PubMed Central

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-01-01

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1]). PMID:27222848

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.

PubMed

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-06-01

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).

Simultaneous determination of methocarbamol and aspirin by RP-HPLC using fluorescence detection with time programming: its application to pharmaceutical dosage form.

PubMed

El-Din, M Sharaf; Eid, M; Zeid, A M

2013-01-01

A new simple, rapid and sensitive reversed-phase liquid chromatographic method was developed and validated for the simultaneous determination of methocarbamol (MET) and aspirin (ASP) in their combined dosage form. The separation of these compounds was achieved within 6.0 min on a CLC Shim-pack C8 column (250 × 4.6 mm, 5 µm particle size) using isocratic mobile phase consisting of acetonitrile and 0.02 M dihydrogenphosphate buffer (30:70, v/v) at pH = 5.0. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence detection at 277/313 nm for MET and 298/410 nm for ASP using real-time programming. The selectivity, linearity of calibration, accuracy, inter- and intra-day precision and recovery were examined as parts of the method validation. The concentration-response relationship was linear over concentration ranges of 0.02-0.20 and 0.02-0.40 µg/mL for MET and ASP, respectively, with a limit of detection of 6 and 32 ng/mL for MET and ASP, respectively. The proposed method was successfully applied for the analysis of both MET and ASP in prepared tablets with average recoveries of 99.88 ± 0.65% for MET and 100.44 ± 0.78% for ASP. The results were favourably compared to those obtained by a reference method. Copyright © 2012 John Wiley & Sons, Ltd.

Online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection.

PubMed

Li, Dapeng; Wang, Ting; Guo, Yujie; Hu, Yuanjia; Yu, Boyang; Qi, Jin

2015-12-18

Nitric oxide (NO) is an important cellular signaling molecule with extensive physiological and pathophysiological effects. NO scavengers have the potential to treat inflammation, septic shock and other related diseases, and numerous examples have been chemically synthesized or isolated from natural products. The chemical diversity of natural products, however, means that a huge effort is necessary to efficiently screen and identify bioactive compounds, especially NO scavengers. In this article, we propose an effective analytical method to screen for NO scavengers in three natural products using an online system that couples high performance liquid chromatography with tandem diode array and fluorescence detection (HPLC-DAD-FLD). Eighteen compounds from radix of Scutellaria baicalensis Georgi and green tea displayed significant NO scavenging activity whereas components of Pueraria lobata (Willd.) Ohwi had no discernable activity. The structures of the active compounds were elucidated using Agilent Accurate-Mass Q-TOF LC/MS system. Preliminary analysis of structure-activity relationships indicated that, in flavonoids, a 2,3-double bond and a 3-H atom or a 3-OH group are essential for activity. In tannins, poly-hydroxyl groups are important for NO scavenging activity. Method validation indicated that the newly developed method is both reliable and repeatable. The online method that we present provides a simple, rapid and effective way to identify and characterize NO scavengers present in natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

Simplified RP-HPLC method for multi-residue analysis of abamectin, emamectin benzoate and ivermectin in rice.

PubMed

Xie, Xianchuan; Gong, Shu; Wang, Xiaorong; Wu, Yinxing; Zhao, Li

2011-01-01

A rapid, reliable and sensitive reverse-phase high-performance liquid chromatography method with fluorescence detection (RP-FLD-HPLC) was developed and validated for simultaneous analysis of the abamectin (ABA), emamectin (EMA) benzoate and ivermectin (IVM) residues in rice. After extraction with acetonitrile/water (2 : 1) with sonication, the avermectin (AVMs) residues were directly derivatised by N-methylimidazole (N-NMIM) and trifluoroacetic anhydride (TFAA) and then analysed on RP-FLD-HPLC. A good linear relationship (r(2 )> 0.99) was obtained for three AVMs ranging from 0.01 to 5 microg ml(-1), i.e. 0.01-5.0 microg g(-1) in rice matrix. The limit of detection (LOD) and the limit of quantification (LOQ) were between 0.001 and 0.002 microg g(-1) and between 0.004 and 0.006 microg g(-1), respectively. Recoveries were from 81.9% to 105.4% and precision less than 12.4%. The proposed method was successfully applied to routine analysis of the AVMs residues in rice.

Development and validation of a high-performance liquid chromatography method for determination of lisinopril in human plasma by magnetic solid-phase extraction and pre-column derivatization.

PubMed

Rastkari, Noushin; Ahmadkhaniha, Reza

2018-03-01

A sensitive, reliable and simple HPLC method was developed for the determination of lisinopril in human plasma. The method consists of extraction and clean-up steps based on magnetic solid-phase extraction and pre-column derivatization with a fluorescent reagent. The mobile phase consisted of a mixture of methanol-sodium dihydrogen phosphate (pH 3.0; 0.005 m; 75:25, v/v). The flow rate was set at 0.7 mL/min. Fluorescence detection was performed at 470nm excitation and 530nm emission wavelengths. Total chromatography run time was 5 min. The average extraction recovery of lisinopril and fluvoxamine (internal standard) was ≥82.8%. The limits of detection and quantification were determined as 1 and 3 ng/mL respectively. The method exhibited a linear calibration line over the concentration range of 3-1000 ng/mL with coefficient of determination (r 2 ) of ≥0.98. The within-run and between-run precisions were satisfactory with values of CV of 1.8-12.8% (accuracy from 99.2 to 94.7%) and 2.4-13.7% (accuracy from 99.5 to 92.2%), respectively. These developments led to considerable improvement in method sensitivity and reliability. The method was validated according to the US Food and Drug Administration guidelines. Therefore, it can be considered as a suitable method for determination of lisinopril in plasma samples. Copyright © 2017 John Wiley & Sons, Ltd.

Single Lab Validation of a LC/UV/FLD/MS Method for Simultaneous Determination of Water-soluble Vitamins in Multi-Vitamin Dietary Supplements

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to develop a Single-Lab Validated Method using high-performance liquid chromatography (HPLC) with different detectors (diode array detector - DAD, fluorescence detector - FLD, and mass spectrometer - MS) for determination of seven B-complex vitamins (B1 - thiamin, B2 – ...

Quantitation of fumonisin B1 and B2 in feed using FMOC pre-column derivatization with HPLC and fluorescence detection.

PubMed

Smith, Lori L; Francis, Kyle A; Johnson, Joseph T; Gaskill, Cynthia L

2017-11-01

Pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was determined to be effective for quantitation of fumonisins B 1 and B 2 in feed. Liquid-solid extraction, clean-up using immunoaffinity solid phase extraction chromatography, and FMOC-derivatization preceded analysis by reverse phase HPLC with fluorescence. Instrument response was unchanged in the presence of matrix, indicating no need to use matrix-matched calibrants. Furthermore, high method recoveries indicated calibrants do not need to undergo clean-up to account for analyte loss. Established method features include linear instrument response from 0.04-2.5µg/mL and stable derivatized calibrants over 7days. Fortified cornmeal method recoveries from 0.1-30.0μg/g were determined for FB 1 (75.1%-109%) and FB 2 (96.0%-115.2%). Inter-assay precision ranged from 1.0%-16.7%. Method accuracy was further confirmed using certified reference material. Inter-laboratory comparison with naturally-contaminated field corn demonstrated equivalent results with conventional derivatization. These results indicate FMOC derivatization is a suitable alternative for fumonisins B 1 and B 2 quantitation in corn-based feeds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selective detection of Co2+ by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and biological activities

NASA Astrophysics Data System (ADS)

Mahajan, Prasad G.; Dige, Nilam C.; Desai, Netaji K.; Patil, Shivajirao R.; Kondalkar, Vijay V.; Hong, Seong-Karp; Lee, Ki Hwan

2018-06-01

Nowadays scientist over the world are engaging to put forth improved methods to detect metal ion in an aqueous medium based on fluorescence studies. A simple, selective and sensitive method was proposed for detection of Co2+ ion using fluorescent organic nanoparticles. We synthesized a fluorescent small molecule viz. 4,4‧-{benzene-1,4-diylbis-[(Z)methylylidenenitrilo]}dibenzoic acid (BMBA) to explore its suitability as sensor for Co2+ ion and biocompatibility in form of nanoparticles. Fluorescence nanoparticles (BMBANPs) prepared by simple reprecipitation method. Aggregation induced enhanced emission of BMBANPs exhibits the narrower particle size of 68 nm and sphere shape morphology. The selective fluorescence quenching was observed by addition of Co2+ and does not affected by presence of other coexisting ion solutions. The photo-physical properties, viz. UV-absorption, fluorescence emission, and lifetime measurements are in support of ligand-metal interaction followed by static fluorescence quenching phenomenon in emission of BMBANPs. Finally, we develop a simple analytical method for selective and sensitive determination of Co2+ ion in environmental samples. The cell culture E. coli, Bacillus sps., and M. tuberculosis H37RV strain in the vicinity of BMBANPs indicates virtuous anti-bacterial and anti-tuberculosis activity which is of additional novel application shown by prepared nanoparticles.

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…

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.

Plasma L-ergothioneine measurement by high-performance liquid chromatography and capillary electrophoresis after a pre-column derivatization with 5-iodoacetamidofluorescein (5-IAF) and fluorescence detection.

PubMed

Sotgia, Salvatore; Pisanu, Elisabetta; Pintus, Gianfranco; Erre, Gian Luca; Pinna, Gerard Aime; Deiana, Luca; Carru, Ciriaco; Zinellu, Angelo

2013-01-01

Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%).

Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection

PubMed Central

Sotgia, Salvatore; Pisanu, Elisabetta; Pintus, Gianfranco; Erre, Gian Luca; Pinna, Gerard Aime; Deiana, Luca; Carru, Ciriaco; Zinellu, Angelo

2013-01-01

Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%). PMID:23922985

HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6.

PubMed

Arakawa, K; Kawai, Y; Ito, Y; Nakamura, K; Chujo, T; Nishimura, J; Kitazawa, H; Saito, T

2010-04-01

The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results. Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues. The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Occurrence of ivermectin in bovine milk from the Brazilian retail market.

PubMed

Lobato, V; Rath, S; Reyes, F G R

2006-07-01

High-performance liquid chromatography (HPLC) with fluorescence detection was used for the quantification of ivermectin residues in bovine milk intended for human consumption. After liquid-liquid extraction of ivermectin and purification of the extract, the compound was derivatized with 1-methylimidazol in N,N-dimethyl formamide to form a fluorescent derivative, which was separated by HPLC, using reversed-phase C18, with methanol : water (96 : 4 v/v) mobile phase at a flow rate 0.7 ml min-1. The excitation and emission wavelengths of the fluorescence detector were adjusted at 360 and 470 nm, respectively. The linearity of the method was in the range 10-100 ng ivermectin ml-1. Based on a sample of 5.0 ml, the limit of detection and the limit of quantification for ivermectin in milk were 0.6 and 2 ng ml-1, respectively. The recovery rate varied from 76.4 to 87.2%, with an average of 77.9 +/- 3.2%, at four fortification levels. The inter-day precision of the method was 13% (n = 5). Of 168 samples analysed, 17.8% contained ivermectin above the limit of quantification. Nevertheless, none of the samples contained ivermectin above the maximum residue limit (10 ng ml-1) established by the Brazilian Ministry of Agriculture.

Rapid purification method for vitamin A-derived aging pigments A2E and iso-A2E using cation exchange resin.

PubMed

Jee, Eun Hye; Kim, So Ra; Jang, Young Pyo

2012-08-17

A2E, known to be involved in the pathogenesis of age-related macular degeneration (AMD), is one of the major compounds that accumulate as fluorescent pigments in retinal pigment epithelial (RPE) cells with age and in some retinal disorders. While the biomimetic synthesis of A2E and its cis-isomer, iso-A2E is as simple as 'one-pot' reaction, the purification of these amphiphillic compounds has been a bottleneck for the mass production of these pathophysiologically important eye pigments. In order to provide a new method of rapid purification of A2E and iso-A2E, we employed a cation exchange resin for the separation of these pigments from crude reaction mixture. The reaction mixture was loaded on a weak acid resin and was eluted with 80% methanol with sodium hydroxide (pH 12), 100% methanol, and 100% methanol with 0.1% trifluoroacetic acid (TFA) in sequence. A2E and isoA2E were eluted only with 100% methanol solution containing TFA. Most of unreacted starting materials and intermediates were removed with 80% methanol containing sodium hydroxide. The new method can be used as a relatively simple and economic way to purify A2E and iso-A2E compared to conventional HPLC technique. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC—Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma

PubMed Central

El-Zaher, Asmaa A.; Mahrouse, Marianne A.

2013-01-01

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min−1. A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20–200 ng mL−1. The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference. PMID:23700362

Electrochemical vapor generation of selenium species after online photolysis and reduction by UV-irradiation under nano TiO2 photocatalysis and its application to selenium speciation by HPLC coupled with atomic fluorescence spectrometry.

PubMed

Liang, Jing; Wang, Qiuquan; Huang, Benli

2005-01-01

An online UV photolysis and UV/TiO2 photocatalysis reduction device (UV-UV/TiO2 PCRD) and an electrochemical vapor generation (ECVG) cell have been used for the first time as an interface between high-performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS) for selenium speciation. The newly designed ECVG cell of approximately 115 microL dead volume consists of a carbon fiber cathode and a platinum loop anode; the atomic hydrogen generated on the cathode was used to reduce selenium to vapor species for AFS determination. The noise was greatly reduced compared with that obtained by use of the UV-UV/TiO2 PCRD-KBH4-acid interface. The detection limits obtained for seleno-DL: -cystine (SeCys), selenite (Se(IV)), seleno-DL: -methionine (SeMet), and selenate (Se(VI)) were 2.1, 2.9, 4.3, and 3.5 ng mL(-1), respectively. The proposed method was successfully applied to the speciation of selenium in water-soluble extracts of garlic shoots cultured with different selenium species. The results obtained suggested that UV-UV/TiO2 PCRD-ECVG should be an effective interface between HPLC and AFS for the speciation of elements amenable to vapor generation, and is superior to methods involving KBH4.

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.

Green autofluorescence, a double edged monitoring tool for bacterial growth and activity in micro-plates

NASA Astrophysics Data System (ADS)

Mihalcescu, Irina; Van-Melle Gateau, Mathilde; Chelli, Bernard; Pinel, Corinne; Ravanat, Jean-Luc

2015-12-01

The intrinsic green autofluorescence of an Escherichia coli culture has long been overlooked and empirically corrected in green fluorescent protein (GFP) reporter experiments. We show here, by using complementary methods of fluorescence analysis and HPLC, that this autofluorescence, principally arise from the secreted flavins in the external media. The cells secrete roughly 10 times more than what they keep inside. We show next that the secreted flavin fluorescence can be used as a complementary method in measuring the cell concentration particularly when the classical method, based on optical density measure, starts to fail. We also demonstrate that the same external flavins limit the dynamical range of GFP quantification and can lead to a false interpretation of lower global dynamic range of expression than what really happens. In the end we evaluate different autofluorescence correction methods to extract the real GFP signal.

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.

Solid-phase microextraction of benzimidazole fungicides in environmental liquid samples and HPLC-fluorescence determination.

PubMed

López Monzón, A; Vega Moreno, D; Torres Padrón, M E; Sosa Ferrera, Z; Santana Rodríguez, J J

2007-03-01

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) with fluorescence detection was optimized for extraction and determination of four benzimidazole fungicides (benomyl, carbendazim, thiabendazole, and fuberidazole) in water. We studied extraction and desorption conditions, for example fiber type, extraction time, ionic strength, extraction temperature, and desorption time to achieve the maximum efficiency in the extraction. Results indicate that SPME using a Carboxen-polydimethylsiloxane 75 microm (CAR-PDMS) fiber is suitable for extraction of these types of compound. Final analysis of benzimidazole fungicides was performed by HPLC with fluorescence detection. Recoveries ranged from 80.6 to 119.6 with RSDs below 9% and limits of detection between 0.03 and 1.30 ng mL-1 for the different analytes. The optimized procedure was applied successfully to the determination of benzimidazole fungicides mixtures in environmental water samples (sea, sewage, and ground water).

Analytical determination of flavonoids aimed to analysis of natural samples and active packaging applications.

PubMed

Castro-López, María del Mar; López-Vilariño, José Manuel; González-Rodríguez, María Victoria

2014-05-01

Several HPLC and UHPLC developed methods were compared to analyse the natural antioxidants catechins and quercetin used in active packaging and functional foods. Photodiode array detector coupled with a fluorescence detector and compared with LTQ-Orbitrap-MS was used. UHPLC was investigated as quick alternative without compromising the separation, analysis time shortened up to 6-fold. The feasibility of the four developed methods was compared. Linearity up to 0.9995, low detection limits (between 0.02 and 0.7 for HPLC-PDA, 2 to 7-fold lower for HPLC- LTQ-Orbitrap-MS and from 0.2 to 2mgL(-)(1) for UHPLC-PDA) and good precision parameters (RSD lower than 0.06%) were obtained. All methods were successfully applied to natural samples. LTQ-Orbitrap-MS allowed to identify other analytes of interest too. Good feasibility of the methods was also concluded from the analysis of catechin and quercetin release from new active packaging materials based on polypropylene added with catechins and green tea. Copyright © 2013 Elsevier Ltd. All rights reserved.

[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.

Rapid analysis of aflatoxins B1, B2, and ochratoxin A in rice samples using dispersive liquid-liquid microextraction combined with HPLC.

PubMed

Lai, Xian-Wen; Sun, Dai-Li; Ruan, Chun-Qiang; Zhang, He; Liu, Cheng-Lan

2014-01-01

A novel, simple, and rapid method is presented for the analysis of aflatoxin B1, aflatoxin B2, and ochratoxin A in rice samples by dispersive liquid-liquid microextraction combined with LC and fluorescence detection. After extraction of the rice samples with a mixture of acetonitrile/water/acetic acid, mycotoxins were rapidly partitioned into a small volume of organic solvent (chloroform) by dispersive liquid-liquid microextraction. The three mycotoxins were simultaneously determined by LC with fluorescence detection after precolumn derivatization for aflatoxin B1 and B2. Parameters affecting both extraction and dispersive liquid-liquid microextraction procedures, including the extraction solvent, the type and volume of extractant, the volume of dispersive solvent, the addition of salt, the pH and the extraction time, were optimized. The optimized protocol provided an enrichment factor of approximately 1.25 and with detection of limits (0.06-0.5 μg/kg) below the maximum levels imposed by current regulations for aflatoxins and ochratoxin A. The mean recovery of three mycotoxins ranged from 82.9-112%, with a RSD less than 7.9% in all cases. The method was successfully applied to measure mycotoxins in commercial rice samples collected from local supermarkets in China. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of Selected Amino Acids in Serum of Patients with Liver Disease.

PubMed

Kanďár, Roman; Drábková, Petra; Toiflová, Tereza; Čegan, Alexander

2016-01-01

The determination of amino acids can be a reliable approach for extended diagnosis of liver diseases. This is because liver disease can be a cause of impaired amino acid metabolism. Therefore, a method for the determination of serum amino acids, applicable for clinical purposes, is necessary. The aim of this study was to find differences in the levels of selected amino acids between patients with liver disease and a control group. Samples of peripheral venous blood were obtained from a group of patients with liver disease (n = 131, 59 women at an average age of 60 years and 72 men at an average age of 52 years) and a control group (n = 105, 47 women at an average age of 62 years and 58 men at an average age of 58 years). Before the separation, the amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde. For the separation, reverse phase column was used. The effluent was monitored with a fluorescence detector. There were significant differences in the concentrations of some amino acids between the patients and the control group, but also between women and men. Correlations between some amino acids and markers of liver blood tests and lipid metabolism were observed. A simple, relatively rapid and selective HPLC method with fluorescence detection for the determination of selected amino acids in serum has been developed.

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.

Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

PubMed

Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

2015-05-01

Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.

Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

PubMed

Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

2014-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.

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.

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

PubMed Central

Wilkinson, T C; Wilton, D C

1986-01-01

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

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.

Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods

PubMed Central

Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.

2015-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

Determination of N-glycans by high performance liquid chromatography using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the glycosylamine labeling reagent.

PubMed

Wu, Yike; Sha, Qiuyue; Du, Juan; Wang, Chang; Zhang, Liang; Liu, Bi-Feng; Lin, Yawei; Liu, Xin

2018-02-02

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging.

PubMed

Paul, Albert Jesuran; Bickel, Fabian; Röhm, Martina; Hospach, Lisa; Halder, Bettina; Rettich, Nina; Handrick, René; Herold, Eva Maria; Kiefer, Hans; Hesse, Friedemann

2017-07-01

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000 μm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10 μmol L -1 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

Separation and characterization of polycyclic aromatic hydrocarbons and alkylphenols in coal derived solvents

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

Hurtubise, R.J.; Allen, T.W.; Hussain, A.

1981-03-29

Dry-column chromatography with an aluminum oxide stationary phase and a n-hexane-ether (19:1) mobile phase was used to separate polycyclic aromatic hydrocarbons (PAH) by ring size. Prior to the dry-column chromatography step, the coal derived solvents were added to an acid treated silica gel column and eluted with chloroform. This step removed pyridine-type nitrogen heterocycles. After separation of the individual ring fractions, the fractions were further separated by either thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). If TLC was used, then after separation fluorescence profiles of each PAH ring fraction distributed on 30%-acetylated cellulose chromatoplates were obtained withmore » a spectrodensitometer. Measurement of fluorescence peak heights gave an approximate measure of the amount of the 3-, 4-, 5-, and 6- ring PAH. For HPLC separation, the 3- and 4- ring PAH fractions obtained from the dry-column chromatography step were separated with a ..mu..-Bondapak C/sub 18/ column and methanol:water (65:35) mobile phase. The HPLC separated PAH were characterized by chromatographic correlation factors and corrected fluorescence excitation spectra. Alkylphenols were identified in coal recycle solvent sample following separation by HPLC.« less

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

[Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS].

PubMed

Tabata, Setsuko; Iida, Kenji; Kimura, Keisuke; Iwasaki, Yumiko; Nakazato, Mitsuo; Kamata, Kunihiro; Hirokado, Masako

2008-04-01

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).

Optimal Conditions for the Asymmetric Polymerase Chain Reaction for Detecting Food Pathogenic Bacteria Using a Personal SPR Sensor.

PubMed

Nagai, Haruka; Tomioka, Kanji; Okumura, Shiro

2018-06-26

We have been developing quick and simple system for detecting food-poisoning bacteria using a combination of an asymmetric PCR and a portable surface plasmon resonance (SPR) sensor. The system would be suitable for point-of-care detection of food-poisoning bacteria in the field of food industry. In this study, we established a novel method for quantifying the amplified forward (F) and reverse (R) chains of Staphylococcus aureus separately by high-performance liquid chromatography (HPLC). The concentration of single-stranded DNA amplicon excessively amplified, which is crucial for the system, could be calculated as the difference between those of the F- and R-chains. For the R-chain, a correction based on the F-chain concentration in the sample was used to obtain a more accurate value, because the determination of the R-chain concentration was affected by that of the coexisting F-chain. The concentration values were also determined by fluorescence imaging for electrophoresis gels of amplicons with FITC- or Cy5-conjugated primers, and they were in good agreement with the values by the HPLC. The measured concentration of the single-strand F-chain correlated well with the value of the SPR response against the probe that was a complementary sequence of the F-chain, immobilized on the sensor chip of the SPR sensor.

In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R. Br. ex roemer and schultes through callus culture under abiotic stress conditions.

PubMed

Ali Ahmed, Abdul Bakrudeen; Rao, Adhikarla Suryanarayana; Rao, Mandali Venkateswara

2009-01-01

Plant secondary metabolites have enormous potential for research and new drug development. Many secondary metabolites have a complex and unique structure and their production is often enhanced by biotic and abiotic stress conditions. Gymnemic acid (C(43)H(68)O(14)), a pentacyclic triterpenoid isolated from the leaves of Gymnema sylvestre, exhibits potent inhibitory effect on diabetes. The gymnemic acid content is determined by chromatographic methods: Camag HPTLC system equipped with a sample applicator Linomat IV and TLC scanner and integration software CAT 4.0. In HPLC C(18) (ODS) reverse phase column; water 486 UV detector; mobile phase, water/methanol (35:65, HPLC grade) + 0.1% acetic acid are used. Sample (20 microL) is applied with a flow rate of 1 mL/min and read at 230 nm with UV detector. The production of gymnemic acid is significantly higher in callus treated with 2,4-dichloro phenoxy acetic acid (2,4-D) and kinetin (KN). The blue light increases gymnemic acid accumulation upto 4.4-fold as compared with fluorescent light treatment and out of which 2.8 is found in leaves. Gymnemic acid is isolated from callus, grown under stress conditions followed by preparative TLC, simple and reproducible character based on HPTLC and high performance liquid chromatography.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

PubMed Central

Li, Yinghong; Yang, Yang; Guan, Xiangming

2012-01-01

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

A base-modified PNA-graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats

NASA Astrophysics Data System (ADS)

Sabale, Pramod M.; George, Jerrin Thomas; Srivatsan, Seergazhi G.

2014-08-01

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures. Electronic supplementary information (ESI) available. Figures, tables, experimental procedures and NMR spectra. See DOI: 10.1039/c4nr00878b

Development of a facile and sensitive HPLC-FLD method via fluorescence labeling for triterpenic acid bioavailability investigation.

PubMed

You, Jinmao; Wu, Di; Zhao, Mei; Li, Guoliang; Gong, Peiwei; Wu, Yueyue; Guo, Yu; Chen, Guang; Zhao, Xianen; Sun, Zhiwei; Xia, Lian; Wu, Yongning

2017-06-01

Triterpenic acids are widely distributed in many fruits and are known for their medicinal benefits. The study of bioavailability has been an important task for a better understanding of the triterpenic acids. Although many methods based on fluorescence labeling for triterpenic acid determination have been established, these reported methods needed anhydrous conditions, which are not suitable for the convenient study of triterpenic acid bioavailability. Inspired by that, a versatile method, which overcomes the difficulty of the reported methods, has been first developed in this study. The novel method using 2-[12-benzo[b]acridin-5- (12H)-yl]-acetohydrazide (BAAH) as the fluorescence labeling reagent coupled with high-performance liquid chromatography with fluorescence detection was first developed for the study of triterpenic acid bioavailability. Furthermore, the labeling conditions have been optimized in order to achieve the best fluorescence labeling yield. Under the optimal conditions, the quantitative linear range of analytes was 2-1000 ng mL -1 , and the correlation coefficients were >0.9998. The detection limits for all triterpenic acid derivatives were achieved within the range of 0.28-0.29 ng mL -1 . The proposed method was successfully applied to the study of triterpenic acid bioavailability with excellent applicability and good reproducibility. Copyright © 2016 John Wiley & Sons, Ltd.

Application of solid-phase microextraction in the investigation of protein binding of pharmaceuticals.

PubMed

Theodoridis, Georgios

2006-01-18

Protein-drug interactions of seven common pharmaceuticals were studied using solid-phase microextraction (SPME). SPME can be used in such investigations on the condition that no analyte depletion occurs. In multi-compartment systems (e.g. a proteinaceous matrix) only the free portion of the analyte is able to partition into the SPME fiber. In addition if no sample depletion occurs, the bound drug-free drug equilibria are not disturbed. In the present study seven pharmaceuticals (quinine, quinidine, naproxen, ciprofloxacin, haloperidol, paclitaxel and nortriptyline) were assayed by SPME. For quantitative purposes SPME was validated first in the absence of proteins. Calibration curves were constructed for each drug by HPLC-fluorescence and HPLC-UV analysis. SPME was combined to HPLC off-line, desorption occurring in HPLC inserts filled with 200 microL methanol. Binding of each drug to human serum albumin was studied independently. Experimental results were in agreement with literature data and ultrafiltration experiments, indicating the feasibility of the method for such bioanalytical purposes.

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

Rapid screening of transferrin-binders in the flowers of Bauhinia blakeana Dunn by on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-transferrin-fluorescence detection system.

PubMed

Liu, Meixian; Dong, Jing; Lin, Zongtao; Niu, Yanyan; Zhang, Xiaotian; Jiang, Haixiu; Guo, Ning; Li, Wei; Wang, Hong; Chen, Shizhong

2016-06-10

Transferrin (Transferrin, TRF, TF) has drawn increasing attention in cancer therapy due to its potential applications in drug delivery. TF receptor, highly expressed in tumor cells, recognizes and transports Fe(3+)-TF into cells to release iron into cytoplasm. Thus, discovering TF-binding compounds has become an active research area and is of great importance for target therapy. In this study, an on-line analysis method was established for screening TF-binding compounds from the flowers of Bauhinia blakeana Dunn using a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-transferrin-fluorescence detector (HPLC-DAD-MS(n)-TF-FLD) method. As a result, 33 of 80 identified or tentatively characterized compounds in the sample were TF-binding active. Twenty-five flavonol glycosides and eight phenolic acids were identified as TF-binders. Twelve of these active compounds together with six standard compounds were used to study the dose-response effects and structure-activity relationships of flavonoids and phenolic acids. The method was validated by vitexin with a good linearity in the range of concentrations used in the study. The limit of detection for vitexin was 0.1596 nmol. Our study indicated that the established method is simple, rapid and sensitive for screening TF-binding active compounds in the extract of Bauhinia blakeana Dunn, and therefore is important for discovering potential anti-cancer ingredients from complex samples for TF related drug delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

Separation and determination of citrinin in corn using HPLC fluorescence detection assisted by molecularly imprinted solid phase extraction clean-up

USDA-ARS?s Scientific Manuscript database

A liquid chromatography based method to detect citrinin in corn was developed using molecularly imprinted solid phase extraction (MISPE) sample clean-up. Molecularly imprinted polymers were synthesized using 1,4-dihydroxy-2-naphthoic acid as the template and an amine functional monomer. Density func...

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.

Glycation of human lens proteins: preferential glycation of alpha A subunits.

PubMed

Swamy, M S; Abraham, A; Abraham, E C

1992-03-01

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16-85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg-1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg-1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of alpha-crystallin, while the increases in beta and gamma were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10-50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Topiramate: A Review of Analytical Approaches for the Drug Substance, Its Impurities and Pharmaceutical Formulations.

PubMed

Pinto, Eduardo Costa; Dolzan, Maressa Danielli; Cabral, Lucio Mendes; Armstrong, Daniel W; de Sousa, Valéria Pereira

2016-02-01

An important step during the development of high-performance liquid chromatography (HPLC) methods for quantitative analysis of drugs is choosing the appropriate detector. High sensitivity, reproducibility, stability, wide linear range, compatibility with gradient elution, non-destructive detection of the analyte and response unaffected by changes in the temperature/flow are some of the ideal characteristics of a universal HPLC detector. Topiramate is an anticonvulsant drug mainly used for the treatment of different types of seizures and prophylactic treatment of migraine. Different analytical approaches to quantify topiramate by HPLC have been described because of the lack of chromophoric moieties on its structure, such as derivatization with fluorescent moieties and UV-absorbing moieties, conductivity detection, evaporative light scattering detection, refractive index detection, chemiluminescent nitrogen detection and MS detection. Some methods for the determination of topiramate by capillary electrophoresis and gas chromatography have also been published. This systematic review provides a description of the main analytical methods presented in the literature to analyze topiramate in the drug substance and in pharmaceutical formulations. Each of these methods is briefly discussed, especially considering the detector used with HPLC. In addition, this article presents a review of the data available regarding topiramate stability, degradation products and impurities. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products.

PubMed

Hurst, William J; Stanley, Bruce; Glinski, Jan A; Davey, Matthew; Payne, Mark J; Stuart, David A

2009-10-15

This report describes the characterization of a series of commercially available procyanidin standards ranging from dimers DP = 2 to decamers DP = 10 for the determination of procyanidins from cocoa and chocolate. Using a combination of HPLC with fluorescence detection and MALDI-TOF mass spectrometry, the purity of each standard was determined and these data were used to determine relative response factors. These response factors were compared with other response factors obtained from published methods. Data comparing the procyanidin analysis of a commercially available US dark chocolate calculated using each of the calibration methods indicates divergent results and demonstrate that previous methods may significantly underreport the procyanidins in cocoa-containing products. These results have far reaching implications because the previous calibration methods have been used to develop data for a variety of scientific reports, including food databases and clinical studies.

An optical spot test for the detection of dopamine in human urine using stabilized in air lipid films.

PubMed

Nikolelis, Dimitrios P; Drivelos, Dimitrios A; Simantiraki, Maria G; Koinis, Spyros

2004-04-15

The present technique describes a simple, sensitive spot test for the rapid one-shot detection of dopamine in human urine using lipid films with incorporated resorcin[4]arene receptor that are synthesized by a chemical reaction with a methacrylate polymer on a glass fiber filter. The lipid films without the receptor provided fluorescence under a UV lamp. The use of the receptor in these films quenched this fluorescence, and the color became similar to that of the filters without the lipid films. A drop of dopamine or urine containing this stimulant provided a "switching on" of the fluorescence, which allows the rapid detection of this stimulant in human urine at 10(-8) M concentrations. The novelty of the present work is that it opens new routes in the field of biosensing, i.e., development of sensitive, rapid, and simple methods for detecting species based on the fluorescence of the lipid membranes on a polymer film, and provides a spot test technique for the rapid detection of dopamine. The effect of potent interferences including a wide range of compounds usually found in human urine (i.e., ascorbic aid, glucose, leucine, glycine, tartrate, citrate, bicarbonate, and caffeine) was examined using an aqueous buffered solution that contained the potent interference and dopamine at two lower concentration levels (i.e., 3 x 10(-8)-10(-8) M). The effect of proteins and lipids was also investigated at these two lower dopamine concentration levels in aqueous buffered solution. The results showed no interferences from all these constituents at concentrations usually found in human urine samples; for example, albumin up to 3.22 g/L concentration levels did not provide any interference (i.e., no fluorescence). A drop of urine containing this stimulant provided similar results, i.e., a "switching on" of the fluorescence that allows a technique for the rapid detection of this stimulant in human urine at 10(-8) M concentrations. The technique is not based on a calibration graph but is a semiquantitative method for the detection of dopamine in real samples of urine that can be complimentary to HPLC methods. The difference in color between the samples containing dopamine at concentration levels of 10(-8)-10(-7) M can be easily distinguished by naked eye and a digital camera. An increase of dopamine concentration from 10(-8) to 10(-7) M makes the color more blue whereas the color of the filters remains purple in the blank test (i.e., addition of a urine sample without dopamine or dopamine at concentration levels of 10(-9) M to the filters that contain the lipid membranes with incorporated receptor). The reproducibility of the method was checked in approximately 100 samples, and all of them were found to provide similar results. Note that it was also found that the colors remain stable in the samples containing dopamine for periods of more than two months.

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.

Simultaneous detection of six urinary pteridines and creatinine by high-performance liquid chromatography-tandem mass spectrometry for clinical breast cancer detection.

PubMed

Burton, Casey; Shi, Honglan; Ma, Yinfa

2013-11-19

Recent preliminary studies have implicated urinary pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary pteridines: 6-biopterin, 6-hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the pteridines is between 0.025 and 0.5 μg/L, and for creatinine, it is 0.15 μg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary pteridines as breast cancer biomarkers.

Greek PDO saffron authentication studies using species specific molecular markers.

PubMed

Bosmali, I; Ordoudi, S A; Tsimidou, M Z; Madesis, P

2017-10-01

Saffron, the spice produced from the red stigmas of the flower of Crocus sativus L. is a frequent target of fraud and mislabeling practices that cannot be fully traced using the ISO 3632 trade standard specifications and test methods. A molecular approach is proposed herein as a promising branding strategy for the authentication of highly esteemed saffron brands such as the Greek Protected Designation of Origin (PDO) "Krokos Kozanis". Specific ISSR (inter-simple sequence repeat) markers were used to assess for the first time, the within species variability of several populations of C. sativus L. from the cultivation area of "Krokos Kozanis" as well as the potential differences with the band pattern produced by other Crocus species. Then, species-specific markers were developed taking advantage of an advanced molecular technique such as the HRM analysis coupled with universal DNA barcoding regions (trnL) (Bar-HRM) and applied to saffron admixtures with some of the most common plant adulterants (Calendula officinalis, Carthamus tinctorius, Gardenia jasminoides, Zea mays and Curcuma longa). The sensitivity of the procedure was tested for turmeric as a case study whereas HPLC-fluorescence determination of secondary metabolites was also employed for comparison. The overall results indicated that the Bar-HRM approach is quite effective in terms of specificity and sensitivity. Its effectiveness regarding the detection of turmeric was comparable to that of a conventional HPLC method (0.5% vs 1.0%, w/w). Yet, the proposed DNA-based method is much faster, cost-effective and can be used even by non-geneticists, in any laboratory having access to an HRM-capable real-time PCR instrumentation. It can be, thus, regarded as a strong analytical tool in saffron authentication studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

A rhodamine-based fluorescent probe for colorimetric and fluorescence lighting-up determination of toxic thiophenols in environmental water and living cells.

PubMed

Wu, Juanjuan; Ye, Zhuo; Wu, Feng; Wang, Hongying; Zeng, Lintao; Bao, Guang-Ming

2018-05-01

Thiophenols are a class of highly toxic environmental pollutant, hence it is very necessary to monitor thiophenols in environment and living cells with an efficient and reliable method. Herein, a novel fluorescent probe for thiophenols has been developed, which exhibited a colorimetric and fluorescence turn-on dual response towards thiophenols with good selectivity and fast response. The sensing mechanism for thiophenols was attributed to nucleophilic substitution reaction, which was confirmed by HPLC. The probe exhibited good recovery (from 90% to 107%) and low limit of detection for thiophenols (37nM) in industrial wastewater. Moreover, the probe has been successfully employed to visualize thiophenol in living cells. Therefore, the fluorescent probe has good capability for monitoring thiophenols in environmental samples and biological systems. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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.

A facile fluorescent "turn-off" method for sensing paraquat based on pyranine-paraquat interaction

NASA Astrophysics Data System (ADS)

Zhao, Zuzhi; Zhang, Fengwei; Zhang, Zipin

2018-06-01

Development of a technically simple yet effective method for paraquat (PQ) detection is of great importance due to its high clinical and environmental relevance. In this study, we developed a pyranine-based fluorescent "turn-off" method for PQ sensing based on pyranine-PQ interaction. We investigated the dependence of analytical performance of this method on the experimental conditions, such as the ion strength, medium pH, and so on. Under the optimized conditions, the method is sensitive and selective, and could be used for PQ detection in real-world sample. This study essentially provides a readily accessible fluorescent system for PQ sensing which is cheap, robust, and technically simple, and it is envisaged to find more interesting clinical and environmental applications.

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.

An HPLC method for the determination of selected amino acids in human embryo culture medium.

PubMed

Drábková, Petra; Andrlová, Lenka; Kanďár, Roman

2017-02-01

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP 18e or Ascentis Express C 18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos. Copyright © 2016 John Wiley & Sons, Ltd.

Development of an analytical method for antimony speciation in vegetables by HPLC-hydride generation-atomic fluorescence spectrometry.

PubMed

Olivares, David; Bravo, Manuel; Feldmann, Jorg; Raab, Andrea; Neaman, Alexander; Quiroz, Waldo

2012-01-01

A new method for antimony speciation in terrestrial edible vegetables (spinach, onions, and carrots) was developed using HPLC with hydride generation-atomic fluorescence spectrometry. Mechanical agitation and ultrasound were tested as extraction techniques. Different extraction reagents were evaluated and optimal conditions were determined using experimental design methodology, where EDTA (10 mmol/L, pH 2.5) was selected because this chelate solution produced the highest extraction yield and exhibited the best compatibility with the mobile phase. The results demonstrated that EDTA prevents oxidation of Sb(III) to Sb(V) and maintains the stability of antimony species during the entire analytical process. The LOD and precision (RSD values obtained) for Sb(V), Sb(III), and trimethyl Sb(V) were 0.08, 0.07, and 0.9 microg/L and 5.0, 5.2, and 4.7%, respectively, for a 100 microL sample volume. The application of this method to real samples allowed extraction of 50% of total antimony content from spinach, while antimony extracted from carrots and onion samples ranged between 50 and 60 and 54 and 70%, respectively. Only Sb(V) was detected in three roots (onion and spinach) that represented 60-70% of the total antimony in the extracts.

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.

PubMed

Chen, Tianfeng; Wong, Yum-Shing; Zheng, Wenjie

2006-11-01

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.

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.

Determination of sodium benzoate in food products by fluorescence polarization immunoassay.

PubMed

Ren, Linlin; Meng, Meng; Wang, Peng; Xu, Zhihuan; Eremin, Sergei A; Zhao, Junhong; Yin, Yongmei; Xi, Rimo

2014-04-01

A rapid and sensitive fluorescence polarization immunoassay (FPIA), based on a polyclonal antibody, has been developed for the detection of sodium benzoate in spiked samples. The immunogen and fluorescein-labeled analyte conjugate were successfully synthesized, and the tracer was purified by TLC. Under the optimal assay conditions, the FPIA shows a detection range of 0.3-20.0 μg mL(-1) for sodium benzoate with a detection limit of 0.26 μg mL(-1) in the borate buffer. In addition, the IC₅₀ value was 2.48 μg mL(-1), and the cross-reactivity of the antibodies with ten structurally and functionally related analogs were detected respectively. Four kinds of food samples (energy drink, candy, ice sucker, RIO(TM) cocktail) were selected to evaluate the application of FPIA in real systems. The recoveries were 96.68-106.55% in energy drink; 95.78-100.80% in candy, 86.97-102.70% in ice sucker, and 103.58-109.87% in benzoate contained sample RIO(TM) cocktail, and coefficients of variation of this method were all lower than 11.25%. Comparing with the detection results of HPLC, the developed FPIA has comparative performance in the real sample determination. The results suggest that the FPIA developed in this study is a rapid, convenient and simple method, which is suitable to be used as a screening tool for homogeneous detection of sodium benzoate in food products. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Dual-wavelength excitation to reduce background fluorescence for fluorescence spectroscopic quantitation of erythrocyte zinc protoporphyrin-IX and protoporphyrin-IX from whole blood and oral mucosa

NASA Astrophysics Data System (ADS)

Hennig, Georg; Vogeser, Michael; Holdt, Lesca M.; Homann, Christian; Großmann, Michael; Stepp, Herbert; Gruber, Christian; Erdogan, Ilknur; Hasmüller, Stephan; Hasbargen, Uwe; Brittenham, Gary M.

2014-02-01

Erythrocyte zinc protoporphyrin-IX (ZnPP) and protoporphyrin-IX (PPIX) accumulate in a variety of disorders that restrict or disrupt the biosynthesis of heme, including iron deficiency and various porphyrias. We describe a reagent-free spectroscopic method based on dual-wavelength excitation that can measure simultaneously both ZnPP and PPIX fluorescence from unwashed whole blood while virtually eliminating background fluorescence. We further aim to quantify ZnPP and PPIX non-invasively from the intact oral mucosa using dual-wavelength excitation to reduce the strong tissue background fluorescence while retaining the faint porphyrin fluorescence signal originating from erythrocytes. Fluorescence spectroscopic measurements were made on 35 diluted EDTA blood samples using a custom front-face fluorometer. The difference spectrum between fluorescence at 425 nm and 407 nm excitation effectively eliminated background autofluorescence while retaining the characteristic porphyrin peaks. These peaks were evaluated quantitatively and the results compared to a reference HPLC-kit method. A modified instrument using a single 1000 μm fiber for light delivery and detection was used to record fluorescence spectra from oral mucosa. For blood measurements, the ZnPP and PPIX fluorescence intensities from the difference spectra correlated well with the reference method (ZnPP: Spearman's rho rs = 0.943, p < 0.0001; PPIX: rs = 0.959, p < 0.0001). In difference spectra from oral mucosa, background fluorescence was reduced significantly, while porphyrin signals remained observable. The dual-wavelength excitation method evaluates quantitatively the ZnPP/heme and PPIX/heme ratios from unwashed whole blood, simplifying clinical laboratory measurements. The difference technique reduces the background fluorescence from measurements on oral mucosa, allowing for future non-invasive quantitation of erythrocyte ZnPP and PPIX.

[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.

Rapid Estimation of Astaxanthin and the Carotenoid-to-Chlorophyll Ratio in the Green Microalga Chromochloris zofingiensis Using Flow Cytometry.

PubMed

Chen, Junhui; Wei, Dong; Pohnert, Georg

2017-07-19

The green microalga Chromochloris zofingiensis can accumulate significant amounts of valuable carotenoids, mainly natural astaxanthin, a product with applications in functional food, cosmetics, nutraceuticals, and with potential therapeutic value in cardiovascular and neurological diseases. To optimize the production of astaxanthin, it is essential to monitor the content of astaxanthin in algal cells during cultivation. The widely used HPLC (high-performance liquid chromatography) method for quantitative astaxanthin determination is time-consuming and laborious. In the present work, we present a method using flow cytometry (FCM) for in vivo determination of the astaxanthin content and the carotenoid-to-chlorophyll ratio (Car/Chl) in mixotrophic C. zofingiensis . The method is based on the assessment of fluorescent characteristics of cellular pigments. The mean fluorescence intensity (MFI) of living cells was determined by FCM to monitor pigment formation based on the correlation between MFI detected in particular channels (FL1: 533 ± 15 nm; FL2: 585 ± 20 nm; FL3: >670 nm) and pigment content in algal cells. Through correlation and regression analysis, a linear relationship was observed between MFI in FL2 (band-pass filter, emission at 585 nm in FCM) and astaxanthin content (in HPLC) and applied for predicting astaxanthin content. With similar procedures, the relationships between MFI in different channels and Car/Chl ratio in mixotrophic C. zofingiensis were also determined. Car/Chl ratios could be estimated by the ratios of MFI (FL1/FL3, FL2/FL3). FCM is thus a highly efficient and feasible method for rapid estimation of astaxanthin content in the green microalga C. zofingiensis . The rapid FCM method is complementary to the current HPLC method, especially for rapid evaluation and prediction of astaxanthin formation as it is required during the high-throughput culture in the laboratory and mass cultivation in industry.

Rapid Estimation of Astaxanthin and the Carotenoid-to-Chlorophyll Ratio in the Green Microalga Chromochloris zofingiensis Using Flow Cytometry

PubMed Central

Chen, Junhui; Pohnert, Georg

2017-01-01

The green microalga Chromochloris zofingiensis can accumulate significant amounts of valuable carotenoids, mainly natural astaxanthin, a product with applications in functional food, cosmetics, nutraceuticals, and with potential therapeutic value in cardiovascular and neurological diseases. To optimize the production of astaxanthin, it is essential to monitor the content of astaxanthin in algal cells during cultivation. The widely used HPLC (high-performance liquid chromatography) method for quantitative astaxanthin determination is time-consuming and laborious. In the present work, we present a method using flow cytometry (FCM) for in vivo determination of the astaxanthin content and the carotenoid-to-chlorophyll ratio (Car/Chl) in mixotrophic C. zofingiensis. The method is based on the assessment of fluorescent characteristics of cellular pigments. The mean fluorescence intensity (MFI) of living cells was determined by FCM to monitor pigment formation based on the correlation between MFI detected in particular channels (FL1: 533 ± 15 nm; FL2: 585 ± 20 nm; FL3: >670 nm) and pigment content in algal cells. Through correlation and regression analysis, a linear relationship was observed between MFI in FL2 (band-pass filter, emission at 585 nm in FCM) and astaxanthin content (in HPLC) and applied for predicting astaxanthin content. With similar procedures, the relationships between MFI in different channels and Car/Chl ratio in mixotrophic C. zofingiensis were also determined. Car/Chl ratios could be estimated by the ratios of MFI (FL1/FL3, FL2/FL3). FCM is thus a highly efficient and feasible method for rapid estimation of astaxanthin content in the green microalga C. zofingiensis. The rapid FCM method is complementary to the current HPLC method, especially for rapid evaluation and prediction of astaxanthin formation as it is required during the high-throughput culture in the laboratory and mass cultivation in industry. PMID:28753934

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.

Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD-FLD) in human urine samples.

PubMed

Fernandez-Torres, R; Consentino, M Olías; Lopez, M A Bello; Mochon, M Callejon

2010-05-15

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N(4)-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N(4)-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N(4)-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex Gemini C(18) (150mm x 4.6mm I.D., particle size 5microm) analytical column with LiChroCART LiChrospher C(18) (4mm x 4mm, particle size 5microm) guard column. Analyzed drugs were determined within 34min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut Plexa columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.

Metallothionein quantification in clams by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization.

PubMed

Alhama, José; Romero-Ruiz, Antonio; López-Barea, Juan

2006-02-24

In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.

A quantum dot-based immunoassay for screening of tylosin and tilmicosin in edible animal tissues.

PubMed

Le, Tao; Zhu, Liqian; Yang, Xian

2015-01-01

A rapid, indirect competitive fluorescence-linked immunosorbent assay (ic-FLISA) based on quantum dots (QDs) as the fluorescent marker was developed for the detection of tylosin and tilmicosin in edible animal tissues. The end point fluorescent detection system was carried out using QDs conjugated with goat anti-mouse secondary antibody. The limits of detection (LODs) for the determination of tylosin and tilmicosin were 0.02 and 0.04 μg kg(-1), respectively. This detection method was used to analyse spiked samples and the recoveries ranged from 83.5% to 98.7% for tylosin and from 81.8% to 98.2% for tilmicosin. In real porcine tissue sample analysis, the results of ic-FLISA were similar to those obtained from an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to an HPLC method indicating its potential for tylosin and tilmicosin screening in edible animal tissues.

Utility of eosin Y as a complexing reagent for the determination of citalopram hydrobromide in commercial dosage forms by fluorescence spectrophotometry.

PubMed

Azmi, Syed Najmul Hejaz; Al-Fazari, Ahlam; Al-Badaei, Munira; Al-Mahrazi, Ruqiya

2015-12-01

An accurate, selective and sensitive spectrofluorimetric method was developed for the determination of citalopram hydrobromide in commercial dosage forms. The method was based on the formation of a fluorescent ion-pair complex between citalopram hydrobromide and eosin Y in the presence of a disodium hydrogen phosphate/citric acid buffer solution of pH 3.4 that was extractable in dichloromethane. The extracted complex showed fluorescence intensity at λem = 554 nm after excitation at 259 nm. The calibration curve was linear over at concentrations of 2.0-26.0 µg/mL. Under optimized experimental conditions, the proposed method was validated as per ICH guidelines. The effect of common excipients used as additives was tested and the tolerance limit calculated. The limit of detection for the proposed method was 0.121 μg/mL. The proposed method was successfully applied to the determination of citalopram hydrobromide in commercial dosage forms. The results were compared with the reference RP-HPLC method. Copyright © 2015 John Wiley & Sons, Ltd.

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

Nanofluidic Lab-On-Chip Technology for DNA Identification

DTIC Science & Technology

2013-09-30

samples Fluorescently labeled (FAM tag) DNA oligomers (10, 20, and 50 bases long) were purchased with standard desalting and additional HPLC purification...A.2 DNA samples: DNA oligomers (10, 20, 50 nt long) were purchased with standard desalting and additional HPLC purification for the 50 base

[Determination of four phenolic endocrine disruptors in environmental water samples by high performance liquid chromatography-fluorescence detection using dispersive liquid-liquid microextraction coupled with derivatization].

PubMed

Wang, Xiaoyan; Qi, Weimei; Zhao, Xian'en; Lü, Tao; Wang, Xiya; Zheng, Longfang; Yan, Yehao; You, Jinmao

2014-06-01

To achieve accurate, fast and sensitive detection of phenolic endocrine disruptors in small volume of environmental water samples, a method of dispersive liquid-liquid microextraction (DLLME) coupled with fluorescent derivatization was developed for the determination of bisphenol A, nonylphenol, octylphenol and 4-tert-octylphenol in environmental water samples by high performance liquid chromatography-fluorescence detection (HPLC-FLD). The DLLME and derivatization conditions were investigated, and the optimized DLLME conditions for small volume of environmental water samples (pH 4.0) at room temperature were as follows: 70 microL chloroform as extraction solvent, 400 microL acetonitrile as dispersing solvent, vortex mixing for 3 min, and then high-speed centrifugation for 2 min. Using 2-[2-(7H-dibenzo [a, g] carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC-Cl) as precolumn derivatization reagent, the stable derivatives of the four phenolic endocrine disruptors were obtained in pH 10.5 Na2CO3-NaHCO3 buffer/acetonitrile at 50 degrees C for 3 min, and then separated within 10 min by HPLC-FLD. The limits of detection (LODs) were in the range of 0.9-1.6 ng/L, and the limits of quantification (LOQs) were in the range of 3.8-7.1 ng/L. This method had perfect linearity, precision and recovery results, and showed obvious advantages and practicality comparing to the previously reported methods. It is a convenient and validated method for the routine analysis of phenolic endocrine disruptors in waste water of paper mill, lake water, domestic wastewater, tap water, etc.

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.

Synthesis and binding studies of Alzheimer ligands on solid support.

PubMed

Rzepecki, Petra; Geib, Nina; Peifer, Manuel; Biesemeier, Frank; Schrader, Thomas

2007-05-11

Aminopyrazole derivatives constitute the first class of nonpeptidic rationally designed beta-sheet ligands. Here we describe a double solid-phase protocol for both synthesis and affinity testing. The presented solid-phase synthesis of four types of hybrid compounds relies on the Fmoc strategy and circumvents subsequent HPLC purification by precipitating the final product from organic solution in pure form. Hexa- and octapeptide pendants with internal di- and tetrapeptide bridges are now amenable in high yields to combinatorial synthesis of compound libraries for high-throughput screening purposes. Solid-phase peptide synthesis (SPPS) on an acid-resistant PAM allows us, after PMB deprotection, to subject the free aminopyrazole binding sites in an immobilized state to on-bead assays with fluorescence-labeled peptides. From the fluorescence emission intensity decrease, individual binding constants can be calculated via reference curves by simple application of the law of mass action. Gratifyingly, host/guest complexation can be monitored quantitatively even for those ligands, which are almost insoluble in water.

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.

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions.

PubMed

Satínský, Dalibor; Havlíková, Lucie; Solich, Petr

2013-08-01

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min(-1) and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5% triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min(-1) and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL(-1).

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

Statistical image segmentation for the detection of skin lesion borders in UV fluorescence excitation

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Padilla-Martinez, Juan Pablo; Franco, Walfre

2016-04-01

The skin contains several fluorescent molecules or fluorophores that serve as markers of structure, function and composition. UV fluorescence excitation photography is a simple and effective way to image specific intrinsic fluorophores, such as the one ascribed to tryptophan which emits at a wavelength of 345 nm upon excitation at 295 nm, and is a marker of cellular proliferation. Earlier, we built a clinical UV photography system to image cellular proliferation. In some samples, the naturally low intensity of the fluorescence can make it difficult to separate the fluorescence of cells in higher proliferation states from background fluorescence and other imaging artifacts -- like electronic noise. In this work, we describe a statistical image segmentation method to separate the fluorescence of interest. Statistical image segmentation is based on image averaging, background subtraction and pixel statistics. This method allows to better quantify the intensity and surface distributions of fluorescence, which in turn simplify the detection of borders. Using this method we delineated the borders of highly-proliferative skin conditions and diseases, in particular, allergic contact dermatitis, psoriatic lesions and basal cell carcinoma. Segmented images clearly define lesion borders. UV fluorescence excitation photography along with statistical image segmentation may serve as a quick and simple diagnostic tool for clinicians.

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.

Simple interface of high-performance liquid chromatography-atomic fluorescence spectrometry hyphenated system for speciation of mercury based on photo-induced chemical vapour generation with formic acid in mobile phase as reaction reagent.

PubMed

Yin, Yongguang; Liu, Jingfu; He, Bin; Shi, Jianbo; Jiang, Guibin

2008-02-15

Photo-induced chemical vapour generation (CVG) with formic acid in mobile phase as reaction reagent was developed as interface to on-line couple HPLC with atomic fluorescence spectrometry for the separation and determination of inorganic mercury, methylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg). In the developed procedure, formic acid in mobile phase was used to decompose organomercuries and reduce Hg(2+) to mercury cold vapour under UV irradiation. Therefore, no post-column reagent was used and the flow injection system in traditional procedure is omitted. A number of operating parameters including pH of mobile phase, concentration of formate, flow rate of mobile phase, length of PTFE reaction coil, flow rate of carrier gas and Na(2)S(2)O(3) in sample matrix were optimized. The limits of detection at the optimized conditions were 0.085, 0.033, 0.029 and 0.038 microg L(-1) for inorganic mercury, MeHg, EtHg and PhHg, respectively. The developed method was validated by determination of certified reference material DORM-2 and was further applied in analyses of seafood samples from Yantai port, China. The UV-CVG with formic acid simplifies the instrumentation and reduces the analytical cost significantly.

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.

Supersonic molecular beam-hyperthermal surface ionisation coupled with time-of-flight mass spectrometry applied to trace level detection of polynuclear aromatic hydrocarbons in drinking water for reduced sample preparation and analysis time.

PubMed

Davis, S C; Makarov, A A; Hughes, J D

1999-01-01

Analysis of sub-ppb levels of polynuclear aromatic hydrocarbons (PAHs) in drinking water by high performance liquid chromatography (HPLC) fluorescence detection typically requires large water samples and lengthy extraction procedures. The detection itself, although selective, does not give compound identity confirmation. Benchtop gas chromatography/mass spectrometry (GC/MS) systems operating in the more sensitive selected ion monitoring (SIM) acquisition mode discard spectral information and, when operating in scanning mode, are less sensitive and scan too slowly. The selectivity of hyperthermal surface ionisation (HSI), the high column flow rate capacity of the supersonic molecular beam (SMB) GC/MS interface, and the high acquisition rate of time-of-flight (TOF) mass analysis, are combined here to facilitate a rapid, specific and sensitive technique for the analysis of trace levels of PAHs in water. This work reports the advantages gained by using the GC/HSI-TOF system over the HPLC fluorescence method, and discusses in some detail the nature of the instrumentation used.

Identification of Furosemide Photodegradation Products in Water-Acetonitrile Mixture.

PubMed

Katsura, Shinji; Yamada, Nobuo; Nakashima, Atsushi; Shiraishi, Sumihiro; Furuishi, Takayuki; Ueda, Haruhisa

2015-01-01

The aim of this study was to identify the chemical structure of the photodegradation products of furosemide in a water-acetonitrile mixture (1 : 1). Furosemide solution was irradiated with a D65 fluorescent lamp and the products were isolated by preparative HPLC. The fractions were evaporated to dryness in vacuo. The purity of the photodegradation products was measured by HPLC. The purity of products 1, 3, and 4 was greater than 90%, whereas that of product 2 was 13%, therefore, photodegradation product 2 was unstable. We identified photodegradation products 1 and 3 as 4-chloro-5-sulfamoylanthranilic acid and 4-hydroxy-N-furfuryl-5-sulfamoylanthranilic acid, respectively, by LC/MS and NMR. Additionally, we assumed that photodegradation product 4 was methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-(methyleneamino)-5-sulfamoylbenzoate by LC/MS and NMR. This showed that furosemide underwent hydrolysis and substitution, and reacted with the acetonitrile under the light of a D65 fluorescent lamp. We were furthermore able to determine the elution times of the photodegradation products of furosemide by applying the Japanese Pharmacopoeia chromatographic method for related substances to the isolated products.

Direct observation of redox reactions in Candida parapsilosis ATCC 7330 by Confocal microscopic studies.

PubMed

Venkataraman, Sowmyalakshmi; Narayan, Shoba; Chadha, Anju

2016-10-14

Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells. This alcohol, itself non-fluorescent, gets oxidized to produce a fluorescent ketone, 1-(6-methoxynaphthalen-2-yl)ethanone. Kinetic studies showed that the reaction occurs spontaneously and the products get released out of the cells in less time [5 mins]. The biotransformation was validated using HPLC.

Use of image analysis to estimate anthocyanin and UV-excited fluorescent phenolic compound levels in strawberry fruit

PubMed Central

Yoshioka, Yosuke; Nakayama, Masayoshi; Noguchi, Yuji; Horie, Hideki

2013-01-01

Strawberry is rich in anthocyanins, which are responsible for the red color, and contains several colorless phenolic compounds. Among the colorless phenolic compounds, some, such as hydroxycinammic acid derivatives, emit blue-green fluorescence when excited with ultraviolet (UV) light. Here, we investigated the effectiveness of image analyses for estimating the levels of anthocyanins and UV-excited fluorescent phenolic compounds in fruit. The fruit skin and cut surface of 12 cultivars were photographed under visible and UV light conditions; colors were evaluated based on the color components of images. The levels of anthocyanins and UV-excited fluorescent compounds in each fruit were also evaluated by spectrophotometric and high performance liquid chromatography (HPLC) analyses, respectively and relationships between these levels and the image data were investigated. Red depth of the fruits differed greatly among the cultivars and anthocyanin content was well estimated based on the color values of the cut surface images. Strong UV-excited fluorescence was observed on the cut surfaces of several cultivars, and the grayscale values of the UV-excited fluorescence images were markedly correlated with the levels of those fluorescent compounds as evaluated by HPLC analysis. These results indicate that image analyses can select promising genotypes rich in anthocyanins and fluorescent phenolic compounds. PMID:23853516

[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.

Development of achiral and chiral 2D HPLC methods for analysis of albendazole metabolites in microsomal fractions using multivariate analysis for the in vitro metabolism.

PubMed

Belaz, Kátia Roberta A; Pereira-Filho, Edenir Rodrigues; Oliveira, Regina V

2013-08-01

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid resolution liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one.

PubMed

Zhao, Ying-Yong; Qin, Xiang-Yang; Cheng, Xian-Long; Liu, Xue-Ying; Lin, Rui-Chao; Zhang, Yongmin; Li, Xiao-Ye; Sun, Xiao-Li; Sun, Wen-Ji

2010-08-24

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) from many medicinal plants has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic and immunosuppressive activity. Metabolism and pharmacokinetic studies on rat were conducted for ergone. Rapid resolution liquid chromatography with atmospheric pressure chemical ionization tandem multi-stage mass spectrometry (RRLC-APCI-MS(n)) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) methods were applied for the identification and quantification of ergone and its metabolite from rat plasma, faeces and urine. A metabolite was identified by RRLC-DAD-APCI-MS(n): 22,23-epoxy-ergosta-4,6,8(14)-triaen-3-one (epoxyergone). The concentrations of the analyte with its metabolites were determined by HPLC-FLD at excitation wavelength of 370 nm and emission wavelength of 485 nm. The samples were deproteinized with methanol after addition of camptothecin as internal standard (IS). The analysis was performed on a Diamonsil C18 column (150 mm x 4.6 mm x 5 microm) with a mobile phase gradient consisting of methanol and water at a flow rate of 1 mL min(-1). The assay was linear over the concentration range of 42-1500, 36-7500 and 42-1500 ng mL(-1) for plasma, faecal homogenate and urine respectively. The absolute recoveries were found to be 97.0+/-1.2%, 98.1+/-0.7% and 96.6+/-1.8% for plasma, faecal homogenate and urine respectively. The intra-day and inter-day relative standard deviations (RSD) were less than 10%. The previous HPLC-MS/MS method is not affordable for most laboratories because of the specialty requirement and high equipment cost. However, the HPLC-FLD method is economic and operating simply for quantitative determination of ergone and its metabolite in rat plasma, faeces and urine. In addition, liquid chromatography coupled with ion trap multi-stage mass spectrometry is becoming a useful technique for ergone metabolite identification. 2010 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.

HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their Leuco Metabolites in Catfish Muscle

USDA-ARS?s Scientific Manuscript database

Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in catfish muscle were individually determined by HPLC using visible and fluorescence detectors. This detection scheme obviated a PbO2 column that converts leuco forms to chromatic forms for visible detection, thus el...

Analytical method for the fast time-domain reconstruction of fluorescent inclusions in vitro and in vivo.

PubMed

Han, Sung-Ho; Farshchi-Heydari, Salman; Hall, David J

2010-01-20

A novel time-domain optical method to reconstruct the relative concentration, lifetime, and depth of a fluorescent inclusion is described. We establish an analytical method for the estimations of these parameters for a localized fluorescent object directly from the simple evaluations of continuous wave intensity, exponential decay, and temporal position of the maximum of the fluorescence temporal point-spread function. Since the more complex full inversion process is not involved, this method permits a robust and fast processing in exploring the properties of a fluorescent inclusion. This method is confirmed by in vitro and in vivo experiments. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Insights into accelerated liposomal release of topotecan in plasma monitored by a non-invasive fluorescence spectroscopic method

PubMed Central

Fugit, Kyle D.; Jyoti, Amar; Upreti, Meenakshi; Anderson, Bradley D.

2014-01-01

A non-invasive fluorescence method was developed to monitor liposomal release kinetics of the anticancer agent topotecan (TPT) in physiological fluids and subsequently used to explore the cause of accelerated release in plasma. Analyses of fluorescence excitation spectra confirmed that unencapsulated TPT exhibits a red shift in its spectrum as pH is increased. This property was used to monitor TPT release from actively loaded liposomal formulations having a low intravesicular pH. Mathematical release models were developed to extract reliable rate constants for TPT release in aqueous solutions monitored by fluorescence and release kinetics obtained by HPLC. Using the fluorescence method, accelerated TPT release was observed in plasma as previously reported in the literature. Simulations to estimate the intravesicular pH were conducted to demonstrate that accelerated release correlated with alterations in the low intravesicular pH. This was attributed to the presence of ammonia in plasma samples rather than proteins and other plasma components generally believed to alter release kinetics in physiological samples. These findings shed light on the critical role that ammonia may play in contributing to the preclinical/clinical variability and performance seen with actively-loaded liposomal formulations of TPT and other weakly-basic anticancer agents. PMID:25456833

Steviol quantification at the picomole level by high-performance liquid chromatography.

PubMed

Minne, Veerle J Y; Compernolle, Frans; Toppet, Suzanne; Geuns, Jan M C

2004-05-05

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.

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.

Detection of aflatoxin M1 in powdered milk and sweetened condensed milk products in several cities in Java with HPLC-fluorescence method

NASA Astrophysics Data System (ADS)

Wijaya, H.; Wardayanie, N. I.; Widjajanti, R.; Silitonga, R. F.

2018-01-01

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) produced by lactating animals due to consuming AFB1-contaminated feed. AFM1 can be found in dairy products because it is resistant to heat during processing. This study aimed to detect AFM1 in powdered milk and sweetened condensed milk sold in several cities in Java. The amount of powdered milk sample was 20, while the amount of sweetened condensed milk sample was 16. AFM1 detection in powdered milk and sweetened condensed milk was conducted by HPLC-fluorescence method. The results showed that the concentration of AFM1 in powdered milk ranged from undetectable to 0.549 μg/kg and the highest data (55%) was distributed in concentration range of >0.05 μg/kg - 0.2 μg/kg. On the other hand, AFM1 levels in sweetened condensed milk ranged from undetectable to 0.056 μg/kg and 43.75% data was distributed in concentration range of >0.025 μg/kg - 0.05 μg/kg. All powdered milk and sweetened condensed milk samples have met the maximum level of AFM1 according to Indonesian regulation.

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.

Isolation and Characterization of Precise Dye/Dendrimer Ratios

PubMed Central

Dougherty, Casey A.; Furgal, Joseph C.; van Dongen, Mallory A.; Goodson, Theodore; Banaszak Holl, Mark M.; Manono, Janet; DiMaggio, Stassi

2014-01-01

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne `click' ligand, separation into sample containing precisely defined `click' ligand/particle ratios using reverse-phase high performance liquid chromatography (rp-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using rp-HPLC. These materials were characterized using 1H and 19F NMR, rp-HPLC, UV-Vis and fluorescence spectroscopy, lifetime measurements, and MALDI. PMID:24604830

[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

Determination of curcumin in biologically active supplements and food spices using a mesofluidic platform with fluorescence detection.

PubMed

Petrova, Anastasiia; Dar'in, Dmitriy; Ivanov, Aleхander; Moskvin, Leonid; Ishimatsu, Ryoichi; Nakano, Koji; Imato, Toshihiko; Bulatov, Andrey

2016-10-01

A mesofluidic platform (MP) with fluorescence detection based on a stepwise injection analysis (SWIA) was used for the determination of curcumin in biologically active supplements and food spices. The main units of the MP are a mixing chamber (MC) and an optical channel with a quartz capillary inside. The MC provides rapid and complete mixing solutions by gas bubbling. The proposed method is based on the new rapid and sensitive reaction of curcumin with a fluorescence reagent - 4-(2,3,3-trimethyl-3H-indolium-1-yl)butane-1-sulfonate (TIBS). The fluorescence intensity of TIBS is greatly quenched in the presence of curcumin in an alkaline medium. The linear range was from 1 to 10µM of curcumin, and the limit of detection, calculated as 3σ of a blank test (n=5), was found to be 0.3µM. The sample throughput was 24h(-1). The proposed method was successfully applied for the determination of curcumin in biologically active supplements and samples of food spices. The obtained data were in good agreement with those measured by a HPLC-UV method. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

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

Moester, Martiene J.C.; Schoeman, Monique A.E.; Oudshoorn, Ineke B.

2014-01-03

Highlights: •We validate a simple and fast method of quantification of in vitro mineralization. •Fluorescently labeled agents can detect calcium deposits in the mineralized matrix of cell cultures. •Fluorescent signals of the probes correlated with Alizarin Red S staining. -- Abstract: Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and ismore » therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.« less

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma.

PubMed

Zaffaroni, M; Mucignat, C; Pecere, T; Zagotto, G; Frapolli, R; D'Incalci, M; Zucchetti, M

2003-10-25

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.

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.

Coral Pigments: Quantification Using HPLC and Detection by Remote Sensing

NASA Technical Reports Server (NTRS)

Cottone, Mary C.

1995-01-01

Widespread coral bleaching (loss of pigments of symbiotic dinoflagellates), and the corresponding decline in coral reef health worldwide, mandates the monitoring of coral pigmentation. Samples of the corals Porites compressa and P. lobata were collected from a healthy reef at Puako, Hawaii, and chlorophyll (chl) a, peridinin, and Beta-carotene (Beta-car) were quantified using reverse-phase high performance liquid chromatography (HPLC). Detailed procedures are presented for the extraction of the coral pigments in 90% acetone, and the separation, identification, and quantification of the major zooxanthellar pigments using spectrophotometry and a modification of the HPLC system described by Mantoura and Llewellyn (1983). Beta-apo-8-carotenal was found to be inadequate as in internal standard, due to coelution with chl b and/or chl a allomer in the sample extracts. Improvements are suggested, which may result in better resolution of the major pigments and greater accuracy in quantification. Average concentrations of peridinin, chl a, and Beta-car in corals on the reef were 5.01, 8.59, and 0.29, micro-grams/cm(exp 2), respectively. Average concentrations of peridinin and Beta-car did not differ significantly between the two coral species sampled; however, the mean chl a concentration in P. compressa specimens (7.81 ,micro-grams/cm(exp 2) was significantly lower than that in P. lobata specimens (9.96 11g/cm2). Chl a concentrations determined spectrophotometrically were significantly higher than those generated through HPLC, suggesting that spectrophotometry overestimates chl a concentrations. The average ratio of chl a-to-peridinin concentrations was 1.90, with a large (53%) coefficient of variation and a significant difference between the two species sampled. Additional data are needed before conclusions can be drawn regarding average pigment concentrations in healthy corals and the consistency of the chl a/peridinin ratio. The HPLC pigment concentration values contribute to the limited database of pigment concentrations in healthy corals, from which quantitative definitions of 'healthy' vs. 'bleached' coral may emerge. They also serve as ground-truth, corresponding to fluorescence data collected from the reef at Puako using airborne remote sensing of laser induced fluorescence. Fluorescence spectra from several overflights using the NASA AOL (airborne oceanographic lidar) system show consistent chlorphyll fluorescence peaks around 685 nm, as well as consistence peaks in the 400-600 nm range which may emanate from granules in the coral tissue. These data, along with results from previous studies of coral fluorescence, suggest that remote sensing of laser-induced fluorescence may become a rapid and efficient means of monitoring coral pigmentation and coral reef bleaching.

Determination of 3,6-dinitrobenzo[e]pyrene in surface soil and airborne particles by high-performance liquid chromatography with fluorescence detection.

PubMed

Hasei, Tomohiro; Watanabe, Tetsushi; Hirayama, Teruhisa

2006-11-24

We developed a sensitive analytical method and an efficient clean-up method to quantify 3,6-dinitrobenzo[e]pyrene (3,6-DNBeP) in surface soil and airborne particles. After purification using a silica gel column and two reversed-phase columns, 3,6-DNBeP was reduced to 3,6-diaminobenzo[e]pyrene by a catalyst column and analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. 3,6-DNBeP was detected in all of the soil samples and airborne particles examined. The concentration of 3,6-DNBeP in surface soil and airborne particles was determined in the ranges of 347-5007 pg/g of soil and 137-1238 fg/m3, respectively.

Proline derivatives in fruits of bergamot (Citrus bergamia Risso et Poit): presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine.

PubMed

Servillo, Luigi; Giovane, Alfonso; Balestrieri, Maria Luisa; Cautela, Domenico; Castaldo, Domenico

2011-01-12

The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported.

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.

Improved conditions for the application of solid phase microextraction prior to HPLC-FLD analysis of anatoxin-a.

PubMed

Rellán, Sandra; Gago-Martínez, Ana

2007-10-01

Solid phase microextraction coupled with high performance liquid chromatography with fluorescence detection has been optimized and evaluated for a simple, rapid, and selective analysis of anatoxin-a. Four kinds of fiber (100 microm polydimethylsiloxane, 60 microm polydimethylsiloxane/divinylbenzene, 50 microm Carbowax/templated resin-100, and 85 microm polyacrylate) were evaluated for an efficient extraction of the toxin. Parameters relating to the desorption step, such as desorption mode, solvent composition, time for both static and dynamic desorption, as well as carryover, have been studied and optimized. The derivatization process was investigated using NBD-F as derivatizing reagent. Anatoxin-a derivative was formed when the anatoxin-a-loaded fiber was inserted in a vial containing 5 microL of NBD-F. Variables affecting extraction such us ionic strength, temperature, and time have been also optimized. The results obtained showed linearity in the range of 10-2000 ng and a limit of detection of 0.29 ng/mL in river water. The presented method has been applied to different environmental samples.

Molecularly Imprinted Polymer of Colocynthin, An Effective Tool for Quality Control of Citrullus colocynthis Extracts.

PubMed

Farid, Ramezani; Mahnaz, Khanavi; Ardekani Mohammad Reza, Shams; Börje, Sellergren; Mahdieh, Eftekhari; Hossein, Rastegar; Maryam, Shekarchi

2017-01-01

Different parts of Colocynth, Citrullus colocynthis (L.) Schrad., are used in traditional phytotherapy and homeopathy. In our new approach, a molecularly imprinted polymer was synthesized to absorb colocynthin, the major plant marker, and its capability was evaluated using HPLC-UV. A new method was considered to achieve optimal conditions. FT-infrared, N2 adsorption porosimetry, fluorescent and scanning electron microscopy and thermo gravimetric profile of the polymers were studied. The imprinted polymer was applied as molecularly imprinted solid phase extraction sorbent to enrich colocynthin from colocynth oil extract, a traditional medicine dosage form. The imprinted polymer showed high capacity and affinity toward colocynthin. Physical assessments demonstrated no major differences between imprinted and nonimprinted polymers. The imprinted polymer was able to absorb colocynthin more efficiently than non-imprinted and control simple solvent extraction from the real sample. In conclusion, this polymer is capable of being applied as a promising adsorbent for analysis of colocynth traditional medicine products. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Teaching about photosynthesis with simple equipment: analysis of light-induced changes in fluorescence and reflectance of plant leaves.

PubMed

Björn, Lars Olof; Li, Shaoshan

2013-10-01

Solar energy absorbed by plants results in either reflection or absorption. The latter results in photosynthesis, fluorescence, or heat. Measurements of fluorescence changes have been used for monitoring processes associated with photosynthesis. A simple method to follow changes in leaf fluorescence and leaf reflectance associated with nonphotochemical quenching and light acclimation of leaves is described. The main equipment needed consists of a green-light emitting laser pointer, a digital camera, and a personal computer equipped with the camera acquisition software and the programs ImageJ and Excel. Otherwise, only commonly available cheap materials are required.

Investigating rhodamine B-labeled peptoids: scopes and limitations of its applications.

PubMed

Birtalan, Esther; Rudat, Birgit; Kölmel, Dominik K; Fritz, Daniel; Vollrath, Sidonie B L; Schepers, Ute; Bräse, Stefan

2011-01-01

The fluorophore rhodamine B is often used in biological assays. It is inexpensive, robust under a variety of reaction conditions, can be covalently linked to bioactive molecules, and has suitable spectral properties in terms of absorption and fluorescence wavelength. Nonetheless, there are some drawbacks: it can readily form a spirolactam compound, which is nonfluorescent, and therefore may not be the dye of choice for all fluorescence microscopy applications. Herein this spirolactam formation was observed by purifying such a labeled peptoid with high performance liquid chromatography (HPLC) and monitored in detail by making a series of analytical HPLC runs over time. Additionally, a small library of eight peptoids with rhodamine B as label was synthesized. Analysis of the absorption properties of these molecules demonstrated that the problem of fluorescence loss can be overcome by coupling secondary amines with rhodamine B.

Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes

PubMed Central

Zhang, Xiaoyun; Li, Yulin; Wang, Haiying; Gu, Xiangyu; Zheng, Xiangfeng; Wang, Yun; Diao, Junwei; Peng, Yaping; Zhang, Hongyin

2016-01-01

Ochratoxin A (OTA) contamination has been established as a world-wide problem. In this study, the strains with the ability of OTA production were screened by analyzing the green fluorescence of the isolates colonies from the grapes in Zhenjiang with 365 nm UV light and confirmed by HPLC with fluorescent detection (HPLC-FLD). The results showed that seven isolates acquired the characteristic of the fluorescence, of which only five showed the ability of OTA production as confirmed by HPLC-FLD analysis. The five OTA-producing strains were identified based on comparative sequence analysis of three conserved genes (ITS, BenA and RPB2) of the strains, and they are Talaromyces rugulosus (O1 and Q3), Penicillium commune (V5-1), Penicillium rubens (MQ-5) and Aspergillus aculeatus (MB1-1). There are two Penicillium species of the five OTA-producing strains and our study is the first to report that P. rubens, T. rugulosus and A. aculeatus can produce OTA. This work would contribute to comprehensively understanding the fungi with an OTA-producing ability in grapes before harvest and then take effective measures to prevent OTA production. PMID:27845758

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

Polypyrrole nanowire as an excellent solid phase microextraction fiber for bisphenol A analysis in food samples followed by ion mobility spectrometry.

PubMed

Kamalabadi, Mahdie; Mohammadi, Abdorreza; Alizadeh, Naader

2016-08-15

A polypyrrole nanowire coated fiber was prepared and used in head-space solid phase microextraction coupled with ion mobility spectrometry (HS-SPME-IMS) to the analysis of bisphenol A (BPA) in canned food samples, for the first time. This fiber was synthesized by electrochemical oxidation of the monomer in aqueous solution. The fiber characterization by scanning electron microscopy (SEM) revealed that the new fiber exhibited two-dimensional structures with a nanowire morphology. The effects of important extraction parameters on the efficiency of HS-SPME were investigated and optimized. Under the optimum conditions, the linearity of 10-150ngg(-1) and limit of detection (based on S/N=3) of 1ngg(-1) were obtained in BPA analysis. The repeatability (n=5) expressed as the relative standard deviation (RSD%) was 5.8%. At the end, the proposed method was successfully applied to determine BPA in various canned food samples (peas, corns, beans). Relative recoveries were obtained 93-96%. Method validation was conducted by comparing our results with those obtained through HPLC with fluorescence detection (FLD). Compatible results indicate that the proposed method can be successfully used in BPA analysis. This method is simple and cheaper than chromatographic methods, with no need of extra organic solvent consumption and derivatization prior to sample introduction. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection.

PubMed

Inoue, K; Kato, K; Yoshimura, Y; Makino, T; Nakazawa, H

2000-11-10

A simple and sensitive method using high-performance liquid chromatography with multi-electrode electrochemical detection (HPLC-ED) including a coulometric array of four electrochemical sensors has been developed for the determination of bisphenol A in water and human serum. For good separation and detection of bisphenol A, a CAPCELL PAK UG 120 C18 reversed-phase column and a mobile phase consisting of 0.3% phosphoric acid-acetonitrile (60:40) were used. The detection limit obtained by the HPLC-ED method was 0.01 ng/ml (0.5 pg), which was more than 3000-times higher than the detection limit obtained by the ultraviolet (UV) method, and more than 200-times higher than the detection limit obtained by the fluorescence (FL) method. Bisphenol A in water and serum samples was pretreated by solid-phase extraction (SPE) after removing possible contamination derived from a plastic SPE cartridges and water used for the pretreatment. A trace amount (ND approximately 0.013 ng/ml) of bisphenol A was detected from the parts of cartridges (filtration column, sorbent bed and frits) by extraction with methanol, and it was completely removed by washing with at least 15 ml of methanol in the operation process. The concentrations of bisphenol A in tap water and Milli-Q-purified water were found to be 0.01 and 0.02 ng/ml, respectively. For that reason, bisphenol A-free water was made to trap bisphenol A in water using an Empore disk. In every pretreatment, SPE methods using bisphenol A-free water and washing with 15 ml of methanol were done in water and serum samples. The yields obtained from the recovery tests using water to which 0.5 or 0.05 ng/ml of bisphenol A was added were 83.8 to 98.2%, and the RSDs were 3.4 to 6.1%, respectively. The yields obtained from the recovery tests by OASIS HLB using serum to which 1.0 ng/ml or 0.1 ng/ml of bisphenol A was added were 79.0% and 87.3%, and the RSDs were 5.1% and 13.5%, respectively. The limits of quantification in water and serum sample were 0.01 ng/ml and 0.05 ng/ml, respectively. The method was applied to the determination of bisphenol A in healthy human serum sample, and the obtained detection was 0.32 ng/ml. From these results, the HPLC-ED method should be the most useful in the determination of bisphenol A at low concentration levels in water and biological samples.

Guanidinium ionic liquid-based surfactants as low cytotoxic extractants: Analytical performance in an in-situ dispersive liquid-liquid microextraction method for determining personal care products.

PubMed

Pacheco-Fernández, Idaira; Pino, Verónica; Ayala, Juan H; Afonso, Ana M

2018-07-20

The IL-based surfactant octylguanidinium chloride (C 8 Gu-Cl) was designed and synthetized with the purpose of obtaining a less harmful surfactant: containing guanidinium as core cation and a relatively short alkyl chain. Its interfacial and aggregation behavior was evaluated through conductivity and fluorescence measurements, presenting a critical micelle concentration value of 42.5 and 44.6mmolL -1 , respectively. Cytotoxicity studies were carried out with C 8 Gu-Cl and other IL-based and conventional surfactants, specifically the analogue 1-octyl-3-methylimidazolium chloride (C 8 MIm-Cl), and other imidazolium- (C 16 MIm-Br) and pyridinium- (C 16 Py-Cl) based surfactants, together with the conventional cationic CTAB and the conventional anionic SDS. From these studies, C 8 Gu-Cl was the only one to achieve the classification of low cytotoxicity. An in situ dispersive liquid-liquid microextraction (DLLME) method based on transforming the water-soluble C 8 Gu-Cl IL-based surfactant into a water-insoluble IL microdroplet via a simple metathesis reaction was then selected as the extraction/preconcentration method for a group of 6 personal care products (PCPs) present in cosmetic samples. The method was carried out in combination with high-performance liquid chromatography (HPLC) and diode array detection (DAD). The method was properly optimized, requiring the use of only 30μL of C 8 Gu-Cl for 10mL of aqueous sample with a NaCl content of 8% (w/v) to adjust the ionic strength and pH value of 5. The metathesis reaction required the addition of the anion exchange reagent (bis[(trifluoromethyl)sulfonyl]imide - 1:1 molar ratio), followed by vortex and centrifugation, and dilution of the final microdroplet up to 60μL with acetonitrile before the injection in the HPLC-DAD system. The optimum in situ DLLME-HPLC-DAD method takes ∼10min for the extraction step and ∼22min for the chromatographic separation, with analytical features of low detection limits: down to 0.4μgL -1 ; high reproducibility: with RSD values lower than 10% (intra-day) and 16% (inter-day) for a spiked level of 15μgL -1 ; and an average enrichment factor of 89. The requirement of low volumes (30μL) of a low cytotoxic IL-based surfactant allows the method to be considered less harmful than other common analytical microextraction approaches. Copyright © 2017 Elsevier B.V. All rights reserved.

A synthesis of fluorescent starch based on carbon nanoparticles for fingerprints detection

NASA Astrophysics Data System (ADS)

Li, Hongren; Guo, Xingjia; Liu, Jun; Li, Feng

2016-10-01

A pyrolysis method for synthesizing carbon nanoparticles (CNPs) were developed by using malic acid and ammonium oxalate as raw materials. The incorporation of a minor amount of carbon nanoparticles into starch powder imparts remarkable color-tunability. Based on this phenomenon, an environment friendly fluorescent starch powder for detecting latent fingerprints in non-porous surfaces was prepared. The fingerprints on different non-porous surfaces developed with this powder showed very good fluorescent images under ultraviolet excitation. The method using fluorescent starch powder as fluorescent marks is simple, rapid and green. Experimental results illustrated the effectiveness of proposed methods, enabling its practical applications in forensic sciences.

A sensitive and rapid determination of ranitidine in human plasma by HPLC with fluorescence detection and its application for a pharmacokinetic study.

PubMed

Ulu, Sevgi Tatar; Tuncel, Muzaffer

2012-04-01

A novel precolumn derivatization reversed-phase high-performance liquid chromatography method with fluorescence detection is described for the determination of ranitidine in human plasma. The method was based on the reaction of ranitidine with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole forming yellow colored fluorescent product. The separation was achieved on a C(18) column using methanol-water (60:40, v/v) mobile phase. Fluorescence detection was used at the excitation and emission of 458 and 521 nm, respectively. Lisinopril was utilized as an internal standard. The flow rate was 1.2 mL/min. Ranitidine and lisinopril appeared at 3.24 and 2.25 min, respectively. The method was validated for system suitability, precision, accuracy, linearity, limit of detection, limit of quantification, recovery and robustness. Intra- and inter-day precisions of the assays were in the range of 0.01-0.44%. The assay was linear over the concentration range of 50-2000 ng/mL. The mean recovery was determined to be 96.40 ± 0.02%. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of ranitidine. © The Author [2012]. Published by Oxford University Press. All rights reserved.

Separation methods applicable to urinary creatine and creatinine.

PubMed

Smith-Palmer, Truis

2002-12-05

Urinary creatinine has been analyzed for many years as an indicator of glomerular filtration rate. More recently, interest in studying the uptake of creatine as a result of creatine supplementation, a practice increasingly common among bodybuilders and athletes, has lead to a need to measure urinary creatine concentrations. Creatine levels are of the same order of magnitude as creatinine levels when subjects have recently ingested creatine, while somewhat elevated urinary creatine concentrations in non-supplementing subjects can be an indication of a degenerative disease of the muscle. Urinary creatine and creatinine can be analyzed by HPLC using a variety of columns. Detection methods include absorption, fluorescence after post-column derivatization, and mass spectrometry, and some methods have been automated. Capillary zone electrophoresis and micellar electrokinetic capillary chromatography have also been used to analyze urinary creatine and creatinine. Creatine and creatinine have also been analyzed in serum and tissue using HPLC and CE, and many of these separations could also be applicable to urinary analysis.

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.

Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone.

PubMed

Galeotti, Fabio; Volpi, Nicola

2011-09-01

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.

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.

Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection.

PubMed

Selvaggini, Roberto; Servili, Maurizio; Urbani, Stefania; Esposto, Sonia; Taticchi, Agnese; Montedoro, GianFrancesco

2006-04-19

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.

Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging†

PubMed Central

Digman, Michelle A.; Gratton, Enrico; Storti, Barbara; Beltram, Fabio

2013-01-01

A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy. PMID:22517076

[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

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour - Determination by high-performance thin-layer chromatography-fluorescence detection.

PubMed

Oellig, Claudia

2017-07-21

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography-fluorescence detection (HPTLC-FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC-FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26μg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitation of polycyclic aromatic hydrocarbons (PAH4) in cocoa and chocolate samples by an HPLC-FD method.

PubMed

Raters, Marion; Matissek, Reinhard

2014-11-05

As a consequence of the PAH4 (sum of four different polycyclic aromatic hydrocarbons, named benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene) maximum levels permitted in cocoa beans and derived products as of 2013, an high-performance liquid chromatography with fluorescence detection method (HPLC-FD) was developed and adapted to the complex cocoa butter matrix to enable a simultaneous determination of PAH4. The resulting analysis method was subsequently successfully validated. This method meets the requirements of Regulation (EU) No. 836/2011 regarding analysis methods criteria for determining PAH4 and is hence most suitable for monitoring the observance of the maximum levels applicable under Regulation (EU) No. 835/2011. Within the scope of this work, a total of 218 samples of raw cocoa, cocoa masses, and cocoa butter from several sample years (1999-2012), of various origins and treatments, as well as cocoa and chocolate products were analyzed for the occurrence of PAH4. In summary, it is noted that the current PAH contamination level of cocoa products can be deemed very slight overall.

Multiresidue Screening Method for Detection of Benzimidazoles and their Metabolites in Liver and Muscle by High-Performance Liquid Chromatography: Method Development and Validation According to Commission Decision 2002/657/EC

PubMed Central

Prearo, Marino; Stella, Paola; Ostorero, Federica; Abete, Maria Cesarina

2014-01-01

The use of veterinary drugs may cause the presence of residues in food of animal origin if appropriate withdrawal periods are not respected. A high-performance liquid chromatography (HPLC) method has been developed for the simultaneous detection of 11 benzimidazole residues, including metabolites – albendazole, albendazole sulphoxide, albendazole sulphone, fenbendazole, fenbendazole sulphoxide (oxfendazole), fenbendazole sulphone, flubendazole, mebendazole, oxibendazole, thiabendazole, 5-hydroxythiabendazole – in bovine, ovine, equine, swine, rabbit and poultry liver and in bovine, swine and fish muscle. After extraction with a dicloromethane/acetonitrile solution (35/65 v/v) containing 5% ammonium hydroxide, the solvent was evaporated to dryness, the residue was dissolved in HCl 0.1 M, defatted with hexane, purified on a strong cation exchange solid-phase extraction cartridge and analysed in HPLC with diode array and fluorescence detectors. The method was validated as screening qualitative method evaluating, according to Commission Decision 2002/657/EC criteria, specificity, CCβ and β error at cut off level of 25 μg/kg and ruggedness. PMID:27800310

[Study on fluorescence labeling and determination of polypeptide (PC2~PC6) by high performance liquid chromatography].

PubMed

Li, Jing-xi; Gao, Li-jie; Cao, Wei; Zheng, Li; Chen, Jun-hui; Xu, Xiu-li; Wang, Xiao-ru

2014-12-01

This study was based on the thiol groups (-SH) of PC2~PC6, which could be reacted with the Monobromobimane (mBBr), in order to get polypeptide derivatives with fluorescent signal. A new method was developed for measuring the Polypeptides by high performance liquid chromatography with fluorescence detector, then the chromatographic conditions of HPLC was optimized; meawhile the reaction proportion of PCs and mBBr was identified by Trap-MS. The results showed that, the reaction proportion of PCs and mBBr was 1:1, the polypeptide derivatives had good stability; the five compounds separation was better, and the peak time focused on the 16.6~22.0 min; the linear correlation coefficient of PC2, PC3, PC4, PC5 and PC6 was >0.9991, and the limits of quantification were 0.3, 0.05, 0.3, 0.5 and 0.8 mg · L(-1) respectively, the recovery rate was 83.0%-102.0%; the method was reproducible, RSD<2%, this method for measuring the peptide compounds was rapid and accurate.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

PubMed

Kramer, S; Blaschke, G

2001-02-10

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

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

Simple and Sensitive Molecularly Imprinted Polymer - Mn-Doped ZnS Quantum Dots Based Fluorescence Probe for Cocaine and Metabolites Determination in Urine.

PubMed

Chantada-Vázquez, María Pilar; Sánchez-González, Juan; Peña-Vázquez, Elena; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2016-03-01

A new molecularly imprinted polymer (MIP)-based fluorescent artificial receptor has been prepared by anchoring a selective MIP for cocaine (COC) on the surface of polyethylene glycol (PEG) modified Mn-doped ZnS quantum dots (QDs). The prepared material combines the high selectivity attributed to MIPs and the sensitive fluorescent property of the Mn-doped ZnS QDs. Simple and low cost methods have therefore been optimized for assessing cocaine abuse in urine by monitoring the fluorescence quenching when the template (COC) and also metabolites from COC [benzoylecgonine (BZE) and ecgonine methyl ester (EME)] are present. Fluorescence quenching was not observed when performing experiments with other drugs of abuse (and their metabolites) or when using nonimprinted polymer (NIP)-coated QDs. Under optimized operating conditions (1.5 mL of 200 mg L(-1) MIP-coated QDs solution, pH 5.5, and 15 min before fluorescence scanning) two analytical methods were developed/validated. One of the procedures (direct method) consisted of urine sample 1:20 dilution before fluorescence measurements. The method has been found to be fast, precise, and accurate, but the standard addition technique for performing the analysis was required because of the existence of matrix effect. The second procedure performed a solid phase extraction (SPE) first, avoiding matrix effect and allowing external calibration. The limits of detection of the methods were 0.076 mg L(-1) (direct method) and 0.0042 mg L(-1) (SPE based method), which are lower than the cutoff values for confirmative conclusions regarding cocaine abuse.

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.

Screening and determination of polycyclic aromatic hydrocarbons in seafoods using QuEChERS-based extraction and high-performance liquid chromatography with fluorescence detection.

PubMed

Gratz, Samuel R; Ciolino, Laura A; Mohrhaus, Angela S; Gamble, Bryan M; Gracie, Jill M; Jackson, David S; Roetting, John P; McCauley, Heather A; Heitkemper, Douglas T; Fricke, Fred L; Krol, Walter J; Arsenault, Terri L; White, Jason C; Flottmeyer, Michele M; Johnson, Yoko S

2011-01-01

A rapid, sensitive, and accurate method for the screening and determination of polycyclic aromatic hydrocarbons (PAHs) in edible seafood is described. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction and HPLC with fluorescence detection (FLD). The method was developed and validated in response to the massive Deepwater Horizon oil spill in the Gulf of Mexico. Rapid and highly sensitive PAH screening methods are critical tools needed for oil spill response; they help to assess when seafood is safe for harvesting and consumption. Sample preparation involves SPE of edible seafood portions with acetonitrile, followed by the addition of salts to induce water partitioning. After centrifugation, a portion of the acetonitrile layer is filtered prior to analysis via HPLC-FLD. The chromatographic method uses a polymeric C18 stationary phase designed for PAH analysis with gradient elution, and it resolves 15 U.S. Environmental Protection Agency priority parent PAHs in fewer than 20 min. The procedure was validated in three laboratories for the parent PAHs using spike recovery experiments at PAH fortification levels ranging from 25 to 10 000 microg/kg in oysters, shrimp, crab, and finfish, with recoveries ranging from 78 to 99%. Additional validation was conducted for a series of alkylated homologs of naphthalene, dibenzothiophene, and phenanthrene, with recoveries ranging from 87 to 128%. Method accuracy was further assessed based on analysis of National Institute of Standards and Technology Standard Reference Material 1974b. The method provides method detection limits in the sub to low ppb (microg/kg) range, and practical LOQs in the low ppb (microg/kg) range for most of the PAH compounds studied.

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.

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling.

PubMed

Beland, Frederick A; Churchwell, Mona I; Hewer, Alan; Phillips, David H; Gamboa da Costa, Gonçalo; Marques, M Matilde

2004-07-23

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

PubMed Central

Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

2015-01-01

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma

PubMed Central

Gounder, Murugesan K.; Lin, Hongxia; Stein, Mark; Goodin, Susan; Bertino, Joseph R.; Kong, Ah-Ng Tony; DiPaola, Robert S.

2015-01-01

2-deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with the glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate/boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45min. The analytes were separated on a YMC ODS C18 reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425nm. The 2-DG calibration curves were linear over the range of 0.63 to 300μg/mL with the limit of detection of 0.5μg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8% and the accuracy ranged from 86.8% to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors. PMID:21932382

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.

[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.

A Zn-porphyrin complex contributes to bright red color in Parma ham.

PubMed

Wakamatsu, J; Nishimura, T; Hattori, A

2004-05-01

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Simple and robust diagnosis of early, small and AFP-negative primary hepatic carcinomas: an integrative approach of serum fluorescence and conventional blood tests.

PubMed

Wang, Ting; Zhang, Kun-He; Hu, Piao-Ping; Huang, Zeng-Yong; Zhang, Pan; Wan, Qin-Si; Huang, De-Qiang; Lv, Nong-Hua

2016-09-27

The diagnosis of early, small and alpha-fetoprotein (AFP)-negative primary hepatic carcinomas (PHCs) remains a significant challenge. We developed a simple and robust approach to noninvasively detect these PHCs. A rapid, high-throughput and single-tube method was firstly developed to measure serum autofluorescence and cell-free DNA (cfDNA)-related fluorescence using a real-time PCR system, and both types of serum fluorescence were measured and routine laboratory data were collected in 1229 subjects, including 353 PHC patients, 331 liver cirrhosis (LC) patients, 213 chronic hepatitis (CH) patients and 332 normal controls (NC). The results showed that fluorescence indicators of PHC differed from those of NC, CH and LC to various extents, and all of them were not associated with age, gender, or AFP level. The logistic regression models established with the fluorescence indicators alone and combined with AFP, hepatic function tests and blood cell analyses were valuable for distinguishing early, small, AFP-negative and all PHC from LC, CH, NC and all non-PHC, with areas under the receiver operating characteristic curves 0.857-0.993 and diagnostic accuracies 80.2-97.7%. Conclusively, serum autofluorescence and cfDNA-related fluorescence are able to be rapidly and simultaneously measured by our simple method and valuable for diagnosing early, small and AFP-negative PHCs, especially integrating with AFP and conventional blood tests.

HPLC analysis of para-aminosalicylic acid and its metabolite in plasma, cerebrospinal fluid and brain tissues

PubMed Central

Hong, Lan; Jiang, Wendy; Zheng, Wei; Zeng, Su

2011-01-01

Para-aminosalicylic acid (PAS), an approved drug for treatment of tuberculosis, is a promising therapeutic agent for treatment of manganese (Mn)-induced parkinsonian syndromes. Lack of a quantifying method, however, has hindered the clinical evaluation of its efficacy and thereupon new drug development. This study was aimed at developing a simple and effective method to quantify PAS and its major metabolite, N-acetyl-para-aminosalicylic acid (AcPAS), in plasma, cerebrospinal fluid (CSF) and tissues. Biological samples underwent one-step protein precipitation. The supernatant was fractionated on a reversed-phase C18 column with a gradient mobile system, followed by on-line fluorescence detection. The lower limits of quantification for both PAS and AcPAS were 50 ng/ml of plasma and 17 ng/g of tissues. The intra-day and inter-day precision values did not exceed 5% and 8%, respectively, in all three matrices. The method was used to quantify PAS and AcPAS in rat plasma and brain following a single iv injection of PAS. Data showed a greater amount of PAS than AcPAS in plasma, while a greater amount of AcPAS than PAS was found in brain tissues. The method has been proven to be sensitive, reproducible, and practically useful for laboratory and clinical investigations of PAS in treatment of Mn Parkinsonism. PMID:21159459

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.

Simple fluorescence-based high throughput cell viability assay for filamentous fungi.

PubMed

Chadha, S; Kale, S P

2015-09-01

Filamentous fungi are important model organisms to understand the eukaryotic process and have been frequently exploited in research and industry. These fungi are also causative agents of serious diseases in plants and humans. Disease management strategies include in vitro susceptibility testing of the fungal pathogens to environmental conditions and antifungal agents. Conventional methods used for antifungal susceptibilities are cumbersome, time-consuming and are not suitable for a large-scale analysis. Here, we report a rapid, high throughput microplate-based fluorescence method for investigating the toxicity of antifungal and stress (osmotic, salt and oxidative) agents on Magnaporthe oryzae and compared it with agar dilution method. This bioassay is optimized for the resazurin reduction to fluorescent resorufin by the fungal hyphae. Resazurin bioassay showed inhibitory rates and IC50 values comparable to the agar dilution method and to previously reported IC50 or MICs for M. oryzae and other fungi. The present method can screen range of test agents from different chemical classes with different modes of action for antifungal activities in a simple, sensitive, time and cost effective manner. A simple fluorescence-based high throughput method is developed to test the effects of stress and antifungal agents on viability of filamentous fungus Magnaporthe oryzae. This resazurin fluorescence assay can detect inhibitory effects comparable to those obtained using the growth inhibition assay with added advantages of simplicity, time and cost effectiveness. This high throughput viability assay has a great potential in large-scale screening of the chemical libraries of antifungal agents, for evaluating the effects of environmental conditions and hyphal kinetic studies in mutant and natural populations of filamentous fungi. © 2015 The Society for Applied Microbiology.

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.

PubMed

Bandhuvula, Padmavathi; Fyrst, Henrik; Saba, Julie D

2007-12-01

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.

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.

Development, evaluation and comparison of two independent sampling and analytical methods for ortho-phthalaldehyde vapors and condensation aerosols in air† ‡

PubMed Central

2015-01-01

Two independent sampling and analytical methods for ortho-phthalaldehyde (OPA) in air have been developed, evaluated and compared (1) a reagent-coated solid sorbent HPLC-UV method and (2) an impinger-fluorescence method. In the first method, air sampling is conducted at 1.0 L min−1 with a sampler containing 350 mg of silica gel coated with 1 mg of acidified 2,4-dinitrophenylhydrazine (DNPH). After sampling, excess DNPH in ethyl acetate is added to the sampler prior to storage for 68 hours. The OPA-DNPH derivative is eluted with 4.0 mL of dimethyl sulfoxide (DMSO) for measurement by HPLC with a UV detector set at 3S5 nm. The estimated detection limit is 0.016 µg per sample or 0.067 µg m−3 (0.012 ppb) for a 240 L air sample. Recoveries of vapor spikes at levels of 1.2 to 6.2 µg were 96 to 101%. Recoveries of spikes as mixtures of vapor and condensation aerosols were 97 to 100%. In the second method, air sampling is conducted at 1.0 L mm−1 with a midget impinger containing 10 mL of DMSO solution containing N-acetyl-l-cysteine and ethylenediamine. The fluorescence reading is taken 80 min after the completion of air sampling. Since the time of taking the fluorescence reading is critical, the reading is taken with a portable fluorometer. The estimated detection limit is 0.024 µg per sample or 0.1 µg m−3 (0.018 ppb) for a 240 L air sample. Recoveries of OPA vapor spikes at levels of 1.4 to 5.0 µg per sample were 97 to 105%. Recoveries of spikes as mixtures of vapors and condensation aerosols were 95 to 99%. The collection efficiency for a mixture of vapor and condensation aerosol was 99.4%. The two methods were compared side-by-side in a generation system constructed for producing controlled atmospheres of OPA vapor in air. Average air concentrations of OPA vapor found by both methods agreed within ±10%. PMID:26346658

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform

PubMed Central

Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G.; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C.; Kummel, Andrew C.

2015-01-01

Abstract. A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

Nondestructive application of laser-induced fluorescence spectroscopy for quantitative analyses of phenolic compounds in strawberry fruits (Fragaria x ananassa).

PubMed

Wulf, J S; Rühmann, S; Rego, I; Puhl, I; Treutter, D; Zude, M

2008-05-14

Laser-induced fluorescence spectroscopy (LIFS) was nondestructively applied on strawberries (EX = 337 nm, EM = 400-820 nm) to test the feasibility of quantitatively determining native phenolic compounds in strawberries. Eighteen phenolic compounds were identified in fruit skin by UV and MS spectroscopy and quantitatively determined by use of rp-HPLC for separation and diode-array or chemical reaction detection. Partial least-squares calibration models were built for single phenolic compounds by means of nondestructively recorded fluorescence spectra in the blue-green wavelength range using different data preprocessing methods. The direct orthogonal signal correction resulted in r (2) = 0.99 and rmsep < 8% for p-coumaroyl-glucose, and r (2) = 0.99 and rmsep < 24% for cinnamoyl-glucose. In comparison, the correction of the fluorescence spectral data with simultaneously recorded reflectance spectra did not further improve the calibration models. Results show the potential of LIFS for a rapid and nondestructive assessment of contents of p-coumaroyl-glucose and cinnamoyl-glucose in strawberry fruits.

4-Pyridoxic Acid in the Spent Dialysate: Contribution to Fluorescence and Optical Monitoring.

PubMed

Kalle, Sigrid; Tanner, Risto; Arund, Jürgen; Tomson, Ruth; Luman, Merike; Fridolin, Ivo

2016-01-01

In this work we estimated the contribution of the fluorescence of 4-pyridoxic acid (4-PA) to the total fluorescence of spent dialysate with the aim of evaluating the on-line monitoring of removal of this vitamin B-6 metabolite from the blood of patients with end-stage renal disease (ESRD). Spectrofluorometric analysis of spent dialysate, collected from hemodialysis and hemodiafiltration sessions of 10 patients receiving regularly pyridoxine injections after dialysis treatment, was performed in the range of Ex/Em 220-500 nm. 4-PA in dialysate samples was identified and quantified using HPLC with fluorescent and MS/MS detection. Averaged HPLC chromatogram of spent dialysate had many peaks in the wavelength region of Ex320/Em430 nm where 4-PA was the highest peak with contribution of 42.2±17.0% at the beginning and 47.7±18.0% in the end of the dialysis. High correlation (R = 0.88-0.95) between 4-PA concentration and fluorescence intensity of spent dialysate was found in the region of Ex310-330/Em415-500 nm, respectively. 4-PA elimination from the blood of ESRD patients can be potentially followed using monitoring of the fluorescence of the spent dialysate during dialysis treatments.

Enantioselective determination of metoprolol and its metabolites in human urine high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and tandem mass spectrometry (MS/MS).

PubMed

Baranowska, Irena; Adolf, Weronika; Magiera, Sylwia

2015-11-01

A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025-2.0μg/mL (R(2)>0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50mg rac-metoprolol. Copyright © 2015 Elsevier B.V. All rights reserved.

Spatial distribution of Chloroflexus-like bacteria in the hypersaline artificial microbial mat

NASA Astrophysics Data System (ADS)

Bachar, A.; Polerecky, L.; Vamvakopoulos, K.; de Beer, D.; Jonkers, H. M.

An artificial microbial mat grown in a mesocosm originated from the Hypersaline Lake of La Salada de Chiprana NE Spain was examined with respect to its organism s spatial distribution via high resolution methods A special attention was given to the elucidative Chloroflexus -like bacteria on which spatial distribution data is not available We have characterized this thick 1cm and developed mat for photopigments HPLC and obtained the general pigment distribution pattern Furthermore fiberoptic and photosynthetic microsensor measurements gave inner light attenuations and flux rates of oxygen within the different layers respectively Using fluorescence and spectral imaging we were able to detect characteristic pigmentation in the different layers FISH probes targeting Chloroflexus -like bacteria confirmed the visualization techniques and showed a single hybridized layer below the cyanobacterial layer as did the HPLC fiberoptic microsensor and fluorescence imaging We conclude that Chloroflexus -like bacteria are located below the cyanobacterial layer and above the purple sulfur bacteria and for the firs time we are able to show it by different independent state of the art techniques These approaches can be important for rapid community investigations within a millimeter scale microniches

Assessment of Free Dye in Solutions of Dual-Labeled Antibody Conjugates for In Vivo Molecular Imaging

PubMed Central

Aldrich, Melissa B.; Wang, XueJuan; Hart, Amy; Sampath, Lakshmi; Marshall, Milton V.; Sevick-Muraca, Eva M.

2017-01-01

PURPOSE Recent preclinical and clinical studies show dyes that excite and fluoresce in the near infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic. PROCEDURES Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800CW in (DTPA)n-trastuzumab—(IRDye 800)m conjugate sample preparations in which HPLC assessment of free dye was not possible. RESULTS The SDS-PAGE assay was accurate and valid for free IRDye 800CW amounts between 38 and 4 molar percent of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined. CONCLUSION This method may be applicable to other near infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging. PMID:20458634

Direct-injection HPLC method of measuring micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column.

PubMed

Uranishi, Hiroaki; Nakamura, Mitsuhiro; Nakamura, Hiroki; Ikeda, Yukari; Otsuka, Mayuko; Kato, Zenichiro; Tsuchiya, Teruo

2011-04-15

A direct-injection HPLC-based method has been developed for determining amounts of micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column. The method is easy to perform and requires only 10 μL of a filtered plasma sample. The chromatographic separations were carried out with a gradient mode. The fluorescence detection wavelengths of excitation and emission were set at 273 nm and 464 nm, respectively. Retention times for micafungin and IS were 22.4 and 23.7 min, respectively. Micafungin and FR195743 (IS) peaks were completely separated with little tailing, and no interference was observed. The calibration curve of micafungin showed good linearity in the range of 0.5-20.0 μg/mL (r(2)=1.00). The intra-day accuracy ranged from -4.5 to 5.3%. The inter-day accuracy ranged from -9.8 to 1.5%. The precisions were less than 10%. This method is useful for the determination of micafungin in human plasma. Copyright © 2011 Elsevier B.V. All rights reserved.

HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma and blood cells.

PubMed

Guo, Meihua; Wang, Wenjing; Hai, Xin; Zhou, Jin

2017-10-25

Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia (APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V)) and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and plasma. Blood cells and plasma were digested with mixtures of HNO 3 H 2 O 2 and analyzed by HG-AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein. The supernatant was separated on an anion-exchange column within 6min with isocratic elution using 13mM CH 3 COONa, 3mM NaH 2 PO 4 , 4mM KNO 3 and 0.2mM EDTA-2Na. The methods provided linearity range of 0.2-20ng/mL for total arsenic and 2.0-50ng/mL for four arsenic species. The developed methods for total arsenic and arsenic species determination were precise and accurate. The spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied successfully for the assay of total arsenic and arsenic species in 5 APL patients. The HPLC-HG-AFS may be a good alternative for arsenic species determination in APL patients with its simplicity and low-cost in comparison with HPLC-ICP-MS. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification of the HIV-integrase inhibitor raltegravir (MK-0518) in human plasma by high-performance liquid chromatography with fluorescence detection.

PubMed

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

2008-05-15

A simple and sensitive HLPC method with fluorescence detection was developed for the accurate determination of the first licensed HIV integrase inhibitor raltegravir in human plasma. A 500-microL plasma sample was spiked with delavirdine as internal standard and subjected to liquid-liquid extraction based on a previously described assay i.e. using hexane/methylene chloride (1:1, v/v%) at pH 4.0. HPLC was performed using a Symmetry Shield RP18 column (150 mm x 4.6 mm), a gradient elution of acetonitrile -0.01% (v/v) triethylamine in water adjusted to pH 3.0 at a flow rate of 1 mL/min and a fluorimetric detector set at 299 and 396 nm as excitation and emission wavelengths, respectively. The retention time was 5.0 min for internal standard and 6.4 min for raltegravir. Calibration curves were linear in the range 5-1000 ng/mL and the accuracy of quality control samples in the range 10-750 ng/mL varied from 98.3 to 99.1% and 98.3 to 101.0% of the nominal concentrations for intra-day and day-to-day analysis, respectively with a precision of 6.3% or less. Among the other antiretroviral drugs which can be given in association to HIV-infected patients, none was found to interfere with internal standard or raltegravir. The described assay was developed for the purpose of therapeutic drug of this HIV integrase inhibitor.

Development of a Terbium-Sensitized Fluorescence Method for Analysis of Silibinin.

PubMed

Ershadi, Saba; Jouyban, Abolghasem; Molavi, Ommoleila; Shayanfar, Ali

2017-05-01

Silibinin is a natural flavonoid with potent anticancer properties, as shown in both in vitro and in vivo experiments. Various methods have been used for silibinin analysis. Terbium-sensitized fluorescence methods have been widely used for the determination of drugs in pharmaceutical preparations and biological samples in recent years. The present work is aimed at providing a simple analytical method for the quantitative determination of silibinin in aqueous solutions based on the formation of a fluorescent complex with terbium ion. Terbium concentration, pH, and volume of buffer, the important effective parameters for the determination of silibinin by the proposed method, were optimized using response surface methodology. The fluorescence intensity of silibinin was measured at 545 nm using λex = 334 nm. The developed method was applied for the determination of silibinin in plasma samples after protein precipitation with acetone. Under optimum conditions, the method provided a linear range between 0.10 and 0.50 mg/L, with a coefficient of determination (R2) of 0.997. The LOD and LOQ were 0.034 and 0.112 mg/L, respectively. These results indicate that the developed method is a simple, low-cost, and suitable analytical method for the quantification of silibinin in aqueous solution and plasma samples.

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.

Time-Resolved Fluorescent Immunochromatography of Aflatoxin B1 in Soybean Sauce: A Rapid and Sensitive Quantitative Analysis.

PubMed

Wang, Du; Zhang, Zhaowei; Li, Peiwu; Zhang, Qi; Zhang, Wen

2016-07-14

Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3-10.0 µg·kg(-1), with a limit of detection (LOD) of 0.1 µg·kg(-1) and recoveries of 87.2%-114.3%, within 10 min. The results showed good correlation (R² > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg(-1). The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.

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.

Iron-chelating agents never suppress Fenton reaction but participate in quenching spin-trapped radicals.

PubMed

Li, Linxiang; Abe, Yoshihiro; Kanagawa, Kiyotada; Shoji, Tomoko; Mashino, Tadahiko; Mochizuki, Masataka; Tanaka, Miho; Miyata, Naoki

2007-09-19

Hydroxyl radical formation by Fenton reaction in the presence of an iron-chelating agent such as EDTA was traced by two different assay methods; an electron spin resonance (ESR) spin-trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and high Performance liquid chromatography (HPLC)-fluorescence detection with terephthalic acid (TPA), a fluorescent probe for hydroxyl radicals. From the ESR spin-trapping measurement, it was observed that EDTA seemed to suppress hydroxyl radical formation with the increase of its concentration. On the other hand, hydroxyl radical formation by Fenton reaction was not affected by EDTA monitored by HPLC assay. Similar inconsistent effects of other iron-chelating agents such as nitrylotriacetic acid (NTA), diethylenetriamine penta acetic acid (DTPA), oxalate and citrate were also observed. On the addition of EDTA solution to the reaction mixture 10 min after the Fenton reaction started, when hydroxyl radical formation should have almost ceased but the ESR signal of DMPO-OH radicals could be detected, it was observed that the DMPO-OH* signal disappeared rapidly. With the simultaneous addition of Fe(II) solution and EDTA after the Fenton reaction ceased, the DMPO-OH* signal disappeared more rapidly. The results indicated that these chelating agents should enhance the quenching of [DMPO-OH]* radicals by Fe(II), but they did not suppress Fenton reaction by forming chelates with iron ions.

Determination of catecholamine in human serum by a fluorescent quenching method based on a water-soluble fluorescent conjugated polymer-enzyme hybrid system.

PubMed

Huang, Hui; Gao, Yuan; Shi, Fanping; Wang, Guannan; Shah, Syed Mazhar; Su, Xingguang

2012-03-21

In this paper, a sensitive water-soluble fluorescent conjugated polymer biosensor for catecholamine (dopamine DA, adrenaline AD and norepinephrine NE) was developed. In the presence of horse radish peroxidase (HRP) and H(2)O(2), catecholamine could be oxidized and the oxidation product of catecholamine could quench the photoluminescence (PL) intensity of poly(2,5-bis(3-sulfonatopropoxy)-1,4-phenylethynylenealt-1,4-poly(phenylene ethynylene)) (PPESO(3)). The quenching PL intensity of PPESO(3) (I(0)/I) was proportional to the concentration of DA, AD and NE in the concentration ranges of 5.0 × 10(-7) to 1.4 × 10(-4), 5.0 × 10(-6) to 5.0 × 10(-4), and 5.0 × 10(-6) to 5.0 × 10(-4) mol L(-1), respectively. The detection limit for DA, AD and NE was 1.4 × 10(-7) mol L(-1), 1.0 × 10(-6) and 1.0 × 10(-6) mol L(-1), respectively. The PPESO(3)-enzyme hybrid system based on the fluorescence quenching method was successfully applied for the determination of catecholamine in human serum samples with good accuracy and satisfactory recovery. The results were in good agreement with those provided by the HPLC-MS method.

[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.

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection.

PubMed

Xie, Kaizhou; Jia, Longfei; Yao, Yilin; Xu, Dong; Chen, Shuqing; Xie, Xing; Pei, Yan; Bao, Wenbin; Dai, Guojun; Wang, Jinyu; Liu, Zongping

2011-08-01

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

A simple method to extract DNA from hair shafts using enzymatic laundry powder.

PubMed

Guan, Zheng; Zhou, Yu; Liu, Jinchuan; Jiang, Xiaoling; Li, Sicong; Yang, Shuming; Chen, Ailiang

2013-01-01

A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale.

Simple and cost-effective fluorescent labeling of 5-hydroxymethylcytosine

NASA Astrophysics Data System (ADS)

Shahal, Tamar; Green, Ori; Hananel, Uri; Michaeli, Yael; Shabat, Doron; Ebenstein, Yuval

2016-12-01

The nucleobase 5-hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation. The fluorescence intensity of the labeled sample is directly proportional to its 5-hmC content. We show that this simple and cost-effective technique is suitable for quantification of 5-hmC content in different mouse tissues.

Surface Transient Binding-Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of the Time Window to Seconds.

PubMed

Peng, Sijia; Wang, Wenjuan; Chen, Chunlai

2018-05-10

Fluorescence correlation spectroscopy is a powerful single-molecule tool that is able to capture kinetic processes occurring at the nanosecond time scale. However, the upper limit of its time window is restricted by the dwell time of the molecule of interest in the confocal detection volume, which is usually around submilliseconds for a freely diffusing biomolecule. Here, we present a simple and easy-to-implement method, named surface transient binding-based fluorescence correlation spectroscopy (STB-FCS), which extends the upper limit of the time window to seconds. We further demonstrated that STB-FCS enables capture of both intramolecular and intermolecular kinetic processes whose time scales cross several orders of magnitude.

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

Capillary electrophoresis with laser-induced fluorescence detection: a sensitive method for monitoring extracellular concentrations of amino acids in the periaqueductal grey matter.

PubMed

Bergquist, J; Vona, M J; Stiller, C O; O'Connor, W T; Falkenberg, T; Ekman, R

1996-03-01

The use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the analysis of microdialysate samples from the periaqueductal grey matter (PAG) of freely moving rats is described. By employing 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde (CBQCA) as a derivatization agent, we simultaneously monitored the concentrations of 8 amino acids (arginine, glutamine, valine, gamma-amino-n-butyric acid (GABA), alanine, glycine, glutamate, and aspartate), with nanomolar and subnanomolar detection limits. Two of the amino acids (GABA and glutamate) were analysed in parallel by conventional high-performance liquid chromatography (HPLC) in order to directly compare the two analytical methods. Other CE methods for analysis of microdialysate have been previously described, and this improved method offers greater sensitivity, ease of use, and the possibility to monitor several amino acids simultaneously. By using this technique together with an optimised form of microdialysis technique, the tiny sample consumption and the improved detection limits permit the detection of fast and transient transmitter changes.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

PubMed Central

Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

2015-01-01

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

A new relative referencing method for crop monitoring using chlorophyll fluorescence

NASA Technical Reports Server (NTRS)

Norikane, J.; Goto, E.; Kurata, K.; Takakura, T.

2003-01-01

The measurement of plant chlorophyll fluorescence has been used for many years as a method to monitor a plant's health status. These types of methods have been mostly relegated to the laboratory. The newly developed Relative Referencing Method allows for the measurement of chlorophyll fluorescence under artificial lighting conditions. The fluorescence signal can be determined by first taking a reference signal measurement, then a second measurement with an additional fluorescence excitation source. The first signal can then be subtracted from the second and the plant's chlorophyll fluorescence due to the second lighting source can be determined. With this simple approach, a photosynthesizing plant can be monitored to detect signs of water stress. Using this approach experiments on tomato plants have shown that it was possible to detect water stress, while the plants were continuously illuminated by fluorescent lamps. This method is a promising tool for the remote monitoring of crops grown in a CELSS-type application. Published by Elsevier Science Ltd on behalf of COSPAR.

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.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

PubMed Central

2016-01-01

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

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.

Simultaneous determination of α-asarone and β-asarone in Acorus tatarinowii using excitation-emission matrix fluorescence coupled with chemometrics methods

NASA Astrophysics Data System (ADS)

Bai, Xue-Mei; Liu, Tie; Liu, De-Long; Wei, Yong-Ju

2018-02-01

A chemometrics-assisted excitation-emission matrix (EEM) fluorescence method was proposed for simultaneous determination of α-asarone and β-asarone in Acorus tatarinowii. Using the strategy of combining EEM data with chemometrics methods, the simultaneous determination of α-asarone and β-asarone in the complex Traditional Chinese medicine system was achieved successfully, even in the presence of unexpected interferents. The physical or chemical separation step was avoided due to the use of ;mathematical separation;. Six second-order calibration methods were used including parallel factor analysis (PARAFAC), alternating trilinear decomposition (ATLD), alternating penalty trilinear decomposition (APTLD), self-weighted alternating trilinear decomposition (SWATLD), the unfolded partial least-squares (U-PLS) and multidimensional partial least-squares (N-PLS) with residual bilinearization (RBL). In addition, HPLC method was developed to further validate the presented strategy. Consequently, for the validation samples, the analytical results obtained by six second-order calibration methods were almost accurate. But for the Acorus tatarinowii samples, the results indicated a slightly better predictive ability of N-PLS/RBL procedure over other methods.

A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent.

PubMed

Bahrami, Gholamreza; Mohammadi, Bahareh

2007-05-01

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid-liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 x 4.6 mm) column. In this method the limit of quantification of 0.01 microg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 microg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from -3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.

HPLC-DAD-FLD Method for Simultaneous Determination of Mycotoxins in Wheat Bran.

PubMed

Irakli, Maria N; Skendi, Adriana; Papageorgiou, Maria D

2017-08-01

Aflatoxins, deoxynivalenol, ochratoxin A and zearalenone are the most important mycotoxins that everyone on its own, in groups or simultaneously contaminate cereals. The external layers of cereal grains (bran) apart from health promoting ingredients are also the most contaminated part with reference to mycotoxin's presence. Therefore, consumption of a high fiber wheat-based diet represent an increased risk to consumer's health. The objective of this study was to develop a simple and reliable high performance liquid chromatography method for the simultaneous determination of these mycotoxins in wheat bran (WB). A double extraction was applied with phosphate buffered saline/methanol and for the clean-up a multi-immunoaffinity column was utilized. The detection was carried out with diode-array and fluorescence detectors linked with a post-column photochemical reactor. After optimization of the chromatographic conditions, all mycotoxins were eluted within ~26 min. Limits of detection for each mycotoxin (0.12-12.58 µg/kg) were below the maximum levels provisioned by European Union regulations. Good linearity was observed for the analytes (r2 ≥ 0.9980). The recovery of analyzed mycotoxins ranged from 70.2 to 105.8%, with a relative standard deviation <12%. The method was successfully applied to quantify mycotoxins in 34 WB samples obtained after pearling of grains that were collected from different regions of Greece. © Crown copyright 2017.

Fluorescence detection of dental calculus

NASA Astrophysics Data System (ADS)

Gonchukov, S.; Biryukova, T.; Sukhinina, A.; Vdovin, Yu

2010-11-01

This work is devoted to the optimization of fluorescence dental calculus diagnostics in optical spectrum. The optimal wavelengths for fluorescence excitation and registration are determined. Two spectral ranges 620 - 645 nm and 340 - 370 nm are the most convenient for supra- and subgingival calculus determination. The simple implementation of differential method free from the necessity of spectrometer using was investigated. Calculus detection reliability in the case of simple implementation is higher than in the case of spectra analysis at optimal wavelengths. The use of modulated excitation light and narrowband detection of informative signal allows us to decrease essentially its diagnostic intensity even in comparison with intensity of the low level laser dental therapy.

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

PubMed

Sotoma, Shingo; Hsieh, Feng-Jen; Chen, Yen-Wei; Tsai, Pei-Chang; Chang, Huan-Cheng

2018-01-23

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

Conjugated polymer nanoparticles-based fluorescent biosensor for ultrasensitive detection of hydroquinone.

PubMed

Liu, Yuan; Wang, Yu-Min; Zhu, Wu-Yang; Zhang, Chong-Hua; Tang, Hao; Jiang, Jian-Hui

2018-07-05

This work describes a simple and sensitive fluorescent method for detection of hydroquinone utilizing conjugated polymer nanoparticles (CPNs). The CPNs serve both as a catalyst to accelerate the conversion of hydroquinone to benzoquinone and a fluorescent probe. In the presence of hydroquinone, the fluorescence of CPNs can be effectively quenched by benzoquinone. The detection limit of hydroquinone was down to 5 nM and excellent selectivity toward possible interferences was obtained. This method was successfully applied for hydroquinone detection in lake water and satisfactory results were achieved. Copyright © 2018 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

Optimization of ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain.

PubMed

Stragierowicz, Joanna; Daragó, Adam; Brzeźnicki, Sławomir; Kilanowicz, Anna

2017-07-26

Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous system for excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishing pathological functions, among others in terms of occupational exposure to toxic substances. We present modification of the HPLC (high-performance liquid chromatography) to the UPLC (ultra-performance liquid chromatography) method for the simultaneous determination of glutamate and γ-aminobutyric acid in a single injection. The isocratic separation of these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column with particle size of 1.7 μm at 35°C using a mobile phase consisting of 0.1 M acetate buffer (pH 6.0) and methanol (60:40, v/v) at a flow rate of 0.3 ml/min. The analytes were detected with the fluorescence detector (FLD) using derivatization with o-phthaldialdehyde (OPA), resulting in excitation at 340 nm and emission at 455 nm. Several validation parameters including linearity (0.999), accuracy (101.1%), intra-day precision (1.52-1.84%), inter-day precision (2.47-3.12%), limit of detection (5-30 ng/ml) and quantification (100 ng/ml) were examined. The developed method was also used for the determination of these neurotransmitters in homogenates of selected rat brain structures. The presented UPLC-FLD is characterized by shorter separation time (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniques such as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods. Med Pr 2017;68(5):583-591. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

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.

Aqueous Peroxyoxalate Chemiluminescence.

DTIC Science & Technology

1982-01-01

fluorescence. It should be noted that the chloromethylstyrene, 45, was actually a mixture of the meta and para isomers as received from the supplier...the pentaetWyleneoxy acetylenic derivative, 65. By the conventional scheme, bis-pentaethyleneoxy-BPEA, 66, was obtained as an oil, even after HPLC ...phenylacetylide or lithium amide. The attempted hydrolysis of 56 using NaOH/DMF at reflux gave a semi-solid product, which was not totally pure even after HPLC

Enumerating viruses by using fluorescence and the nature of the nonviral background fraction.

PubMed

Pollard, Peter C

2012-09-01

Bulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.

[Blood levels of homocysteine by high pressure liquid chromatography and comparison with two other techniques].

PubMed

Ceppa, F; Drouillard, I; Chianea, D; Burnat, P; Perrier, F; Vaillant, C; El Jahiri, Y

1999-01-01

Cardio-vascular diseases are the most common cause of death in industrialized countries. A new marker has emerged among offending risk factors in the past few years: homocysteine. This sulphured amino-acid is an important intermediate in transsulphuration and remethylation reactions of methionine's metabolism. We proposed to evaluate a home made method of determination for this parameter by high performance liquid chromatography (HPLC) and to compare it to fluorescence polarization immunoassay technique (FPIA) and to gaz phase chromatography (CG-SM). This method associated with good sensibility and precision remain much less expensive than FPIA technique.

Application of UHPLC for the determination of free amino acids in different cheese varieties.

PubMed

Mayer, Helmut K; Fiechter, Gregor

2013-10-01

A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ(Ex) 250 nm/λ(Em) 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (>35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200-600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2-deoxyglucose in human plasma.

PubMed

Gounder, Murugesan K; Lin, Hongxia; Stein, Mark; Goodin, Susan; Bertino, Joseph R; Kong, Ah-Ng Tony; DiPaola, Robert S

2012-05-01

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C₁₈ reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors. Copyright © 2011 John Wiley & Sons, Ltd.

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.

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

PubMed

Yamada, Keita; Hirabayashi, Jun; Kakehi, Kazuaki

2013-03-19

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Analysis of abamectin residues in avocados by high-performance liquid chromatography with fluorescence detection.

PubMed

Hernández-Borges, Javier; Ravelo-Pérez, Lidia M; Hernández-Suárez, Estrella M; Carnero, Aurelio; Rodríguez-Delgado, Miguel Angel

2007-09-21

In this work an analytical method for the determination of abamectin residues in avocados is developed using high-performance liquid chromatography (HPLC) with fluorescence (FL) detection. A pre-column derivatization with trifluoroacetic anhydride (TFAA) and N-methylimidazole (NMIM) was carried out. The mobile phase consisted of water, methanol and acetonitrile (5:47.5:47.5 v/v/v) and was pumped at a rate of 1 mL/min (isocratic elution). The fluorescence detector was set at an excitation wavelength of 365 nm and an emission wavelength of 470 nm. Homogenized avocado samples were extracted twice with acetonitrile:water 8:2 (v/v) and cleaned using C(18) solid-phase extraction (SPE) cartridges. Recovery values were in the range 87-98% with RSD values lower than 13%. The limits of detection (LODs) and quantification (LOQs) of the whole method were 0.001 and 0.003 mg/kg, respectively. These values are lower than the maximum residue limit (MRL) established by the European Union (EU) and the Spanish legislation in avocado samples.

A label-free amplified fluorescence DNA detection based on isothermal circular strand-displacement polymerization reaction and graphene oxide.

PubMed

Li, Zhen; Zhu, Wenping; Zhang, Jinwen; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2013-07-07

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

PubMed

Anumula, Kalyan Rao

2012-07-01

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection.

PubMed

Brunnenberg, M; Lindenblatt, H; Gouzoulis-Mayfrank, E; Kovar, K A

1998-11-20

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCI. The maximum plasma concentrations were 235-465 ng/ml (MDE), 67-673 ng/ml (HME) and 7-33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.

New insight in quantitative analysis of vascular permeability during immune reaction (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kalchenko, Vyacheslav; Molodij, Guillaume; Kuznetsov, Yuri; Smolyakov, Yuri; Israeli, David; Meglinski, Igor; Harmelin, Alon

2016-03-01

The use of fluorescence imaging of vascular permeability becomes a golden standard for assessing the inflammation process during experimental immune response in vivo. The use of the optical fluorescence imaging provides a very useful and simple tool to reach this purpose. The motivation comes from the necessity of a robust and simple quantification and data presentation of inflammation based on a vascular permeability. Changes of the fluorescent intensity, as a function of time is a widely accepted method to assess the vascular permeability during inflammation related to the immune response. In the present study we propose to bring a new dimension by applying a more sophisticated approach to the analysis of vascular reaction by using a quantitative analysis based on methods derived from astronomical observations, in particular by using a space-time Fourier filtering analysis followed by a polynomial orthogonal modes decomposition. We demonstrate that temporal evolution of the fluorescent intensity observed at certain pixels correlates quantitatively to the blood flow circulation at normal conditions. The approach allows to determine the regions of permeability and monitor both the fast kinetics related to the contrast material distribution in the circulatory system and slow kinetics associated with extravasation of the contrast material. Thus, we introduce a simple and convenient method for fast quantitative visualization of the leakage related to the inflammatory (immune) reaction in vivo.

A simple one-step method to prepare fluorescent carbon dots and their potential application in non-invasive glioma imaging

NASA Astrophysics Data System (ADS)

Ruan, Shaobo; Qian, Jun; Shen, Shun; Zhu, Jianhua; Jiang, Xinguo; He, Qin; Gao, Huile

2014-08-01

Fluorescent carbon dots (CD) possess impressive potential in bioimaging because of their low photobleaching, absence of optical blinking and good biocompatibility. However, their relatively short excitation/emission wavelengths restrict their application in in vivo imaging. In the present study, a kind of CD was prepared by a simple heat treatment method using glycine as the only precursor. The diameter of CD was lower than 5 nm, and the highest emission wavelength was 500 nm. However, at 600 nm, there was still a relatively strong fluorescent emission, suggesting CD could be used for in vivo imaging. Additionally, several experiments demonstrated that CD possessed good serum stability and low cytotoxicity. In vitro, CD could be taken up into C6 glioma cells in a time- and concentration-dependent manner, with both endosomes and mitochondria involved. In vivo, CD could be used for non-invasive glioma imaging because of its high accumulation in the glioma site of the brain, which was demonstrated by both in vivo imaging and ex vivo tissue imaging. Furthermore, the fluorescent distribution in tissue slices also showed CD distributed in glioma with high intensity, while with a low intensity in normal brain tissue. In conclusion, CD were prepared using a simple method with relatively long excitation and emission wavelengths and could be used for non-invasive glioma imaging.Fluorescent carbon dots (CD) possess impressive potential in bioimaging because of their low photobleaching, absence of optical blinking and good biocompatibility. However, their relatively short excitation/emission wavelengths restrict their application in in vivo imaging. In the present study, a kind of CD was prepared by a simple heat treatment method using glycine as the only precursor. The diameter of CD was lower than 5 nm, and the highest emission wavelength was 500 nm. However, at 600 nm, there was still a relatively strong fluorescent emission, suggesting CD could be used for in vivo imaging. Additionally, several experiments demonstrated that CD possessed good serum stability and low cytotoxicity. In vitro, CD could be taken up into C6 glioma cells in a time- and concentration-dependent manner, with both endosomes and mitochondria involved. In vivo, CD could be used for non-invasive glioma imaging because of its high accumulation in the glioma site of the brain, which was demonstrated by both in vivo imaging and ex vivo tissue imaging. Furthermore, the fluorescent distribution in tissue slices also showed CD distributed in glioma with high intensity, while with a low intensity in normal brain tissue. In conclusion, CD were prepared using a simple method with relatively long excitation and emission wavelengths and could be used for non-invasive glioma imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02657h

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.

NARSTO ICARTT NEAX 2004 DOE G-1 DATA

Atmospheric Science Data Center

2018-04-09

... Probe Chemiluminescence UV Ozone Detector Pulsed Fluorescence HPLC Aerosol Spectrometer Probe Pyranometer Photometer ... Carbon Monixide Hydrogen Peroxide Sulfate Particles Nitrate Particles Crustal Particles Order Data:  ...

[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.

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

CdTe quantum dot as a fluorescence probe for vitamin B12 in dosage form

NASA Astrophysics Data System (ADS)

Vaishnavi, E.; Renganathan, R.

2013-11-01

We here report the CdTe quantum dot (CdTe QDs)-based sensor for probing vitamin B12 derivatives in aqueous solution. In this paper, simple and sensitive fluorescence quenching measurements has been employed. The Stern-Volmer constant (KSV), quenching rate constant (kq) and binding constant (K) were rationalized from fluorescence quenching measurement. Furthermore, the fluorescence resonance energy transfer (FRET) mechanism was discussed. This method was applicable over the concentration ranging from 1 to 14 μg/mL (VB12) with correlation coefficient of 0.993. The limit of detection (LOD) of VB12 was found to be 0.15 μg/mL. Moreover, the present approach opens a simple pathway for developing cost-effective, sensitive and selective QD-based fluorescence sensors/probes for biologically significant VB12 in pharmaceutical sample with mean recoveries in the range of 100-102.1%.

CdTe quantum dot as a fluorescence probe for vitamin B(12) in dosage form.

PubMed

Vaishnavi, E; Renganathan, R

2013-11-01

We here report the CdTe quantum dot (CdTe QDs)-based sensor for probing vitamin B12 derivatives in aqueous solution. In this paper, simple and sensitive fluorescence quenching measurements has been employed. The Stern-Volmer constant (KSV), quenching rate constant (kq) and binding constant (K) were rationalized from fluorescence quenching measurement. Furthermore, the fluorescence resonance energy transfer (FRET) mechanism was discussed. This method was applicable over the concentration ranging from 1 to 14μg/mL (VB12) with correlation coefficient of 0.993. The limit of detection (LOD) of VB12 was found to be 0.15μg/mL. Moreover, the present approach opens a simple pathway for developing cost-effective, sensitive and selective QD-based fluorescence sensors/probes for biologically significant VB12 in pharmaceutical sample with mean recoveries in the range of 100-102.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Noninvasive control of dental calculus removal: qualification of two fluorescence methods

NASA Astrophysics Data System (ADS)

Gonchukov, S.; Sukhinina, A.; Bakhmutov, D.; Biryukova, T.

2013-02-01

The main condition of periodontitis prevention is the full calculus removal from the teeth surface. This procedure should be fulfilled without harming adjacent unaffected tooth tissues. Nevertheless the problem of sensitive and precise estimating of tooth-calculus interface exists and potential risk of hard tissue damage remains. In this work it was shown that fluorescence diagnostics during calculus removal can be successfully used for precise noninvasive detection of calculus-tooth interface. In so doing the simple implementation of this method free from the necessity of spectrometer using can be employed. Such a simple implementation of calculus detection set-up can be aggregated with the devices of calculus removing.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

PubMed

Prince, Linda M

2015-01-01

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

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.

Rapid, simultaneous and interference-free determination of three rhodamine dyes illegally added into chilli samples using excitation-emission matrix fluorescence coupled with second-order calibration method.

PubMed

Chang, Yue-Yue; Wu, Hai-Long; Fang, Huan; Wang, Tong; Liu, Zhi; Ouyang, Yang-Zi; Ding, Yu-Jie; Yu, Ru-Qin

2018-06-15

In this study, a smart and green analytical method based on the second-order calibration algorithm coupled with excitation-emission matrix (EEM) fluorescence was developed for the determination of rhodamine dyes illegally added into chilli samples. The proposed method not only has the advantage of high sensitivity over the traditional fluorescence method but also fully displays the "second-order advantage". Pure signals of analytes were successfully extracted from severely interferential EEMs profiles via using alternating trilinear decomposition (ATLD) algorithm even in the presence of common fluorescence problems such as scattering, peak overlaps and unknown interferences. It is worth noting that the unknown interferents can denote different kinds of backgrounds, not only refer to a constant background. In addition, the method using interpolation method could avoid the information loss of analytes of interest. The use of "mathematical separation" instead of complicated "chemical or physical separation" strategy can be more effective and environmentally friendly. A series of statistical parameters including figures of merit and RSDs of intra- (≤1.9%) and inter-day (≤6.6%) were calculated to validate the accuracy of the proposed method. Furthermore, the authoritative method of HPLC-FLD was adopted to verify the qualitative and quantitative results of the proposed method. Compared with the two methods, it also showed that the ATLD-EEMs method has the advantages of accuracy, rapidness, simplicity and green, which is expected to be developed as an attractive alternative method for simultaneous and interference-free determination of rhodamine dyes illegally added into complex matrices. Copyright © 2018. Published by Elsevier B.V.

Synthesis and characterization of a fluorescent water-soluble paclitaxel prodrug.

PubMed

Sohn, Jeong-Sun; Choi, Eun-Sun; Jo, Byung-Wook; Hess, Michael; Han, Song-Hee

2010-05-01

A fluorescence susceptible water-soluble paclitaxel was synthesized by a condensation reaction between PEGylated paclitaxel (namely, PP7) and 1-pyrene butyric acid (PBA) in order to obtain a better understanding of the mechanism of action of paclitaxel as well as of the environment of the paclitaxel-binding site. The reaction was performed successfully and the resulting paclitaxel was characterized by FT-NMR, analytical-HPLC, UV spectro photometry, and fluorescence spectrometry. The synthesized paclitaxel analogue showed a high susceptibility to fluorescence in both excitation and emission spectra. And we have investigated the time-resolved fluorescence behavior of them in different solvents and at different excitation wavelengths.

Ambient air levels and health risk assessment of benzo(a)pyrene in atmospheric particulate matter samples from low-polluted areas: application of an optimized microwave extraction and HPLC-FL methodology.

PubMed

de la Gala Morales, María; Holgado, Fernando Rueda; Marín, Ma Rosario Palomo; Blázquez, Lorenzo Calvo; Gil, Eduardo Pinilla

2015-04-01

A new methodology involving a simple and fast pretreatment of the samples by microwave-assisted extraction and concentration by N2 stream, followed by HPLC with fluorescence detection, was used for determining the concentration of benzo(a)pyrene (BaP) in atmospheric particulate matter (PM10 fraction). Obtained LOD, 1.0 × 10(-3) ng/m(3), was adequate for the analysis of benzo(a)pyrene in the samples, and BaP recovery from PAH in Fine Dust (PM10-like) certified reference material was nearly quantitative (86%). The validated procedure was applied for analyzing 115 PM10 samples collected at different sampling locations in the low-polluted area of Extremadura (Southwest Spain) during a monitoring campaign carried out in 2011-2012. BaP spatial variations and seasonal variability were investigated as well as the influence of meteorological conditions and different air pollutants concentrations. A normalized protocol for health risk assessment was applied to estimate lifetime cancer risk due to BaP inhalation in the sampling areas, finding that around eight inhabitants per million people may develop lung cancer due to the exposition to BaP in atmospheric particulates emitted by the investigated sources.

[Immuno-affinity chromatographic purification: the study of methods to test citrinin in monascus products by high performance liquid chromatography].

PubMed

Qiu, Wen-qian; Liu, Xiao-xia; Zheng, Kui-cheng; Fu, Wu-sheng

2012-08-01

To establish a method to test citrinin (CIT) in monascus products by immuno-affinity chromatography (IAC)-high performance liquid chromatography (HPLC), and to detect the content of CIT in monascus products in Fujian province. IAC-HPLC was applied to detect the CIT content in monascus products. The conditions to use HPLC were as follows: C(18) reversed-phase chromatographic column, 150.0 mm×4.6mm×3 µm; mobile phase: the volume ratio of acetonitrile and 0.1% phosphoric acid solution at 65:35; isocratic elution; column temperature: 28°C; flow velocity: 0.8 ml/min; fluorescence detector, excitation wavelength (λ(ex)) was 331 nm and emission wavelength (λ(em)) was 500 nm. The standard curved was established by the linear regression of peak area (Y) to CIT content (X, ng/ml). The accuracy and precision of the method would then be verified. And 32 kinds of monascus products were determined and their color values were compared by this method. The standard curve established in this study was Y = 4634.8X-136.42, r = 1.000; whose limits of detection was 20 µg/kg and the limits of qualification was 64 µg/kg. In the range between 200 and 800 µg/kg, the standard recovery rate was 98.9% - 110.0% (n = 3), and the relative standard deviation (RSD) was 0.51% - 1.76%. Out of the 32 samples, CIT was detected from 11 samples of monascus rice, 9 samples of monascus powder and 5 samples of monascus pigments, the content was around 0.212 - 14.500 mg/kg. 4 out of 7 functional monascus samples were detected out CIT, whose content at 0.142 - 0.275 mg/kg. The method to detect CIT in monascus products by IAC-HPLC has been established.

Analysis of Levodopa Content in Commercial Mucuna pruriens Products Using High-Performance Liquid Chromatography with Fluorescence Detection.

PubMed

Soumyanath, Amala; Denne, Tanya; Hiller, Amie; Ramachandran, Shaila; Shinto, Lynne

2018-02-01

Mucuna pruriens (MP) seeds contain levodopa (up to 2% by weight) and have been used in traditional Indian medicine to treat an illness named "Kampavata," now understood to be Parkinson's disease (PD). Studies have shown MP to be beneficial, and even superior, to levodopa alone in treating PD symptoms. Commercial products containing MP are readily available from online and retail sources to patients and physicians. Products often contain extracts of MP seeds, with significantly higher levodopa content than the seeds. However, MP products have limited regulatory controls with respect to quality and content of active ingredient. The aim of this study was to apply a quantitative method to determine levodopa content in readily available MP products that might be used by patients or in research studies. Levodopa present in six commercial MP products was quantified by solvent extraction followed by reversed-phase high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). Certificates of analysis (COA) were obtained, from manufacturers of MP products, to assess the existence and implementation of specifications for levodopa content. HPLC-FD analysis revealed that the levodopa content of the six commercial MP products varied from 6% to 141% of individual label claims. No product contained levodopa within normal pharmacopeial limits of 90%-110% label claim. The maximum daily dose of levodopa delivered by the products varied from 14.4 to 720 mg/day. COAs were inconsistent in specifications for and verification of levodopa content. The commercial products tested varied widely in levodopa content, sometimes deviating widely from the label claim. These deficiencies could impact efficacy and safety of MP products used by PD patients and compromise the results of scientific studies on MP products. The HPLC-FD method described in this study could be utilized by both manufacturers and scientific researchers to verify levodopa content of MP products.

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

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique.

PubMed

Riccò, R; Nizzero, S; Penna, E; Meneghello, A; Cretaio, E; Enrichi, F

2018-01-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. Graphical abstractWe report a simple, cheap, reliable protocol for the synthesis and systematic tuning of ultra-small (< 10 nm) dye-doped luminescent silica nanoparticles.

Simple determination of aflatoxins in rice by ultra-high performance liquid chromatography coupled to chemical post-column derivatization and fluorescence detection.

PubMed

Huertas-Pérez, José Fernando; Arroyo-Manzanares, Natalia; Hitzler, Dominik; Castro-Guerrero, Francisco Germán; Gámiz-Gracia, Laura; García-Campaña, Ana M

2018-04-15

A fast and simple analytical method was developed and characterized for the determination of aflatoxins (B 1 , B 2 , G 1 and G 2 ) in rice. The procedure is based on a simple solid-liquid extraction without further clean-up, and analysis by ultra-high performance liquid chromatography coupled with fluorescence detection. Fluorescence emission of aflatoxins B 1 and G 1 was enhanced by post-column chemical derivatization using pyridinium bromide perbromide. The analytical method was satisfactorily characterized in white and brown rice. Under optimum conditions, external calibration in solvent could be used for quantification purposes and limits of quantification were below the maximum contents established by the European Union regulation for these contaminants/commodity group combination (0.07-0.14 µg/kg for white rice and 0.20-0.28 µg/kg for brown rice). Recovery studies carried out at three different concentration levels (0.5, 2 and 5 µg/kg) showed values in the range of 84.5-105.3%, and RSDs ≤ 5%. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans.

PubMed

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-05-15

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of an Ultrasonication-Assisted Extraction Based HPLC With a Fluorescence Method for Sensitive Determination of Aflatoxins in Highly Acidic Hibiscus sabdariffa

PubMed Central

Liu, Xiaofei; Ying, Guangyao; Sun, Chaonan; Yang, Meihua; Zhang, Lei; Zhang, Shanshan; Xing, Xiaoyan; Li, Qian; Kong, Weijun

2018-01-01

The high acidity and complex components of Hibiscus sabdariffa have provided major challenges for sensitive determination of trace aflatoxins. In this study, sample pretreatment of H. sabdariffa was systematically developed for sensitive high performance liquid chromatography-fluorescence detection (HPLC-FLD) after ultrasonication-assisted extraction, immunoaffinity column (IAC) clean-up and on-line post-column photochemical derivatization (PCD). Aflatoxins B1, B2, G1, G2 were extracted from samples by using methanol/water (70:30, v/v) with the addition of NaCl. The solutions were diluted 1:8 with 0.1 M phosphate buffer (pH 8.0) to negate the issues of high acidity and matrix interferences. The established method was validated with satisfactory linearity (R > 0.999), sensitivity (limits of detection (LODs) and limits of quantitation (LOQs) of 0.15–0.65 and 0.53–2.18 μg/kg, respectively), precision (RSD <11%), stability (RSD of 0.2–3.6%), and accuracy (recovery rates of 86.0–102.3%), which all met the stipulated analytical requirements. Analysis of 28 H. sabdariffa samples indicated that one sample incubated with Aspergillus flavus was positive with aflatoxin B1 (AFB1) at 3.11 μg/kg. The strategy developed in this study also has the potential to reliably extract and sensitively detect more mycotoxins in other complex acidic matrices, such as traditional Chinese medicines, foodstuffs, etc. PMID:29681848

Development of an Ultrasonication-Assisted Extraction Based HPLC With a Fluorescence Method for Sensitive Determination of Aflatoxins in Highly Acidic Hibiscus sabdariffa.

PubMed

Liu, Xiaofei; Ying, Guangyao; Sun, Chaonan; Yang, Meihua; Zhang, Lei; Zhang, Shanshan; Xing, Xiaoyan; Li, Qian; Kong, Weijun

2018-01-01

The high acidity and complex components of Hibiscus sabdariffa have provided major challenges for sensitive determination of trace aflatoxins. In this study, sample pretreatment of H. sabdariffa was systematically developed for sensitive high performance liquid chromatography-fluorescence detection (HPLC-FLD) after ultrasonication-assisted extraction, immunoaffinity column (IAC) clean-up and on-line post-column photochemical derivatization (PCD). Aflatoxins B 1 , B 2 , G 1 , G 2 were extracted from samples by using methanol/water (70:30, v/v ) with the addition of NaCl. The solutions were diluted 1:8 with 0.1 M phosphate buffer (pH 8.0) to negate the issues of high acidity and matrix interferences. The established method was validated with satisfactory linearity ( R > 0.999), sensitivity (limits of detection (LODs) and limits of quantitation (LOQs) of 0.15-0.65 and 0.53-2.18 μg/kg, respectively), precision (RSD <11%), stability (RSD of 0.2-3.6%), and accuracy (recovery rates of 86.0-102.3%), which all met the stipulated analytical requirements. Analysis of 28 H. sabdariffa samples indicated that one sample incubated with Aspergillus flavus was positive with aflatoxin B 1 (AFB 1 ) at 3.11 μg/kg. The strategy developed in this study also has the potential to reliably extract and sensitively detect more mycotoxins in other complex acidic matrices, such as traditional Chinese medicines, foodstuffs, etc.

Estimation of the chemical rank for the three-way data: a principal norm vector orthogonal projection approach.

PubMed

Hong-Ping, Xie; Jian-Hui, Jiang; Guo-Li, Shen; Ru-Qin, Yu

2002-01-01

A new approach for estimating the chemical rank of the three-way array called the principal norm vector orthogonal projection method has been proposed. The method is based on the fact that the chemical rank of the three-way data array is equal to one of the column space of the unfolded matrix along the spectral or chromatographic mode. A vector with maximum Frobenius norm is selected among all the column vectors of the unfolded matrix as the principal norm vector (PNV). A transformation is conducted for the column vectors with an orthogonal projection matrix formulated by PNV. The mathematical rank of the column space of the residual matrix thus obtained should decrease by one. Such orthogonal projection is carried out repeatedly till the contribution of chemical species to the signal data is all deleted. At this time the decrease of the mathematical rank would equal that of the chemical rank, and the remaining residual subspace would entirely be due to the noise contribution. The chemical rank can be estimated easily by using an F-test. The method has been used successfully to the simulated HPLC-DAD type three-way data array and two real excitation-emission fluorescence data sets of amino acid mixtures and dye mixtures. The simulation with added relatively high level noise shows that the method is robust in resisting the heteroscedastic noise. The proposed algorithm is simple and easy to program with quite light computational burden.

Rapid detection of malachite green in fish based on CdTe quantum dots coated with molecularly imprinted silica.

PubMed

Wu, Le; Lin, Zheng-Zhong; Zhong, Hui-Ping; Peng, Ai-Hong; Chen, Xiao-Mei; Huang, Zhi-Yong

2017-08-15

A sensitive fluorescence sensor for the detection of malachite green (MG) was fabricated by grafting molecularly imprinted polymers (MIPs) onto the surface of CdTe quantum dots (QDs). The MIP-coated QDs were synthesized via a reverse microemulsion method using (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as functional monomer and cross-linker, respectively. The optimum molar ratio of MG, functional monomer and cross-linker was 1:3:10. The MIP-coated QDs exhibited uniform spheres with diameter around 49nm and excellent fluorescence emission at λ ex 370nm. A linear relationship with two segments between the relative fluorescence intensities and the MG concentrations ranging from 0.08 to 20μmol·L -1 could be obtained with a detection limit of 12μg·kg -1 . The fluorescent probe was successfully applied to the determination of MG in fish samples with the spiked recoveries ranging from 94.3% to 109.5% which were in accordance with those of the measurement by HPLC-UV. Copyright © 2017 Elsevier Ltd. 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.

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.

Development and certification of a coal fly ash certified reference material for selected polycyclic aromatic hydrocarbons.

PubMed

Cao, X; Xu, X; Cui, W; Xi, Z

2001-08-01

The development and certification of a coal fly ash certified reference material (CRM) for polycyclic aromatic hydrocarbons (PAH) is described; this is the first natural matrix CRM for organic environmental analysis in China. The homogeneity and stability of this material have been tested by HPLC. The concentrations of several PAH were determined by use of two independent, different methods--solvent extraction-HPLC analysis with UV detection coupled with fluorescence detection (FLD) and solvent extraction, isolation with a silica column, and GC analysis with flame ionization detection (FID). Five certified values were determined: phenanthrene 7.1 +/- 2.6 microg g(-1), anthracene 2.0 +/- 0.8 microg g(-1), fluoranthene 7.4 +/- 1.9 microg g(-1), pyrene 7 +/- 2 microg g(-1), and benzo[a]pyrene 1.3 +/- 0.3 microg g(-1). Reference values for several other PAH are also suggested.

A novel application of three phase hollow fiber based liquid phase microextraction (HF-LPME) for the HPLC determination of two endocrine disrupting compounds (EDCs), n-octylphenol and n-nonylphenol, in environmental waters.

PubMed

Villar-Navarro, Mercedes; Ramos-Payán, María; Fernández-Torres, Rut; Callejón-Mochón, Manuel; Bello-López, Miguel Ángel

2013-01-15

This work proposes for the first time the use of a three phase hollow fiber liquid phase microextraction (HF-LPME) procedure for the extraction, and the later HPLC determination using fluorescence detection, of two much known endocrine disrupting compounds (EDCs): n-octylphenol (OP) and n-nonylphenol (NP). The extraction was carried out through a dihexyl ether liquid membrane supported on an Accurel® Q3/2 polypropylene hollow fiber. Optimum pH for donor and acceptor phases and extraction time were established. Enrichment (preconcentration) factors of 50 were obtained that allows detection limits of 0.54 and 0.52 ng mL(-1) for OP and NP, respectively. The method was successfully applied to the determination of these EDCs in environmental water samples, including urban wastewaters. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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 .

Agar/gelatin bilayer gel matrix fabricated by simple thermo-responsive sol-gel transition method.

PubMed

Wang, Yifeng; Dong, Meng; Guo, Mengmeng; Wang, Xia; Zhou, Jing; Lei, Jian; Guo, Chuanhang; Qin, Chaoran

2017-08-01

We present a simple and environmentally-friendly method to generate an agar/gelatin bilayer gel matrix for further biomedical applications. In this method, the thermally responsive sol-gel transitions of agar and gelatin combined with the different transition temperatures are exquisitely employed to fabricate the agar/gelatin bilayer gel matrix and achieve separate loading for various materials (e.g., drugs, fluorescent materials, and nanoparticles). Importantly, the resulting bilayer gel matrix provides two different biopolymer environments (a polysaccharide environment vs a protein environment) with a well-defined border, which allows the loaded materials in different layers to retain their original properties (e.g., magnetism and fluorescence) and reduce mutual interference. In addition, the loaded materials in the bilayer gel matrix exhibit an interesting release behavior under the control of thermal stimuli. Consequently, the resulting agar/gelatin bilayer gel matrix is a promising candidate for biomedical applications in drug delivery, controlled release, fluorescence labeling, and bio-imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of CdSe quantum dots for the direct detection of TNT.

PubMed

Yi, Kui-Yu

2016-02-01

CdSe quantum dots were synthesized through a simple, green organic-phase method. Paraffin was used as the reaction solvent and a reducing agent, oleic acid was the reaction ligand, and oleyl amine was the stabilizer. Based on the phenomenon of TNT quenched oil-soluble CdSe quantum dot fluorescence, a simple, fast, and direct method of TNT detection was established. Under optimum conditions, the degree of fluorescence quenching of oil-soluble CdSe quantum dots had a good linear correlation with TNT concentration in the 1.0×10(-7)-5.0×10(-5) mol/L range, and the correlation coefficient was 0.9990. TNT detection limit was 2.1×10(-8)mol/L. The method was successfully used to determine TNT-explosion dust samples, results were satisfactory. The fluorescence quenching mechanism of oil-soluble CdSe quantum dots by TNT was also discussed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A Simple and Sensitive Method for Auramine O Detection Based on the Binding Interaction with Bovin Serum Albumin.

PubMed

Yan, Jingjing; Huang, Xin; Liu, Shaopu; Yang, Jidong; Yuan, Yusheng; Duan, Ruilin; Zhang, Hui; Hu, Xiaoli

2016-01-01

A simple, rapid and effective method for auramine O (AO) detection was proposed by fluorescence and UV-Vis absorption spectroscopy. In the BR buffer system (pH 7.0), AO had a strong quenching ability to the fluorescence of bovin serum albumin (BSA) by dynamic quenching. In terms of the thermodynamic parameters calculated as ΔH > 0 and ΔS > 0, the resulting binding of BSA and AO was mainly attributed to the hydrophobic interaction forces. The linearity of this method was in the concentration range from 0.16 to 50 μmol L(-1) with a detection limit of 0.05 μmol L(-1). Based on fluorescence resonance energy transfer (FRET), the distance r (1.36 nm) between donor (BSA) and acceptor (AO) was obtained. Furthermore, the effects of foreign substances and ionic strength were evaluated under the optimum reaction conditions. BSA as a selective probe could be applied to the analysis of AO in medicines with satisfactory results.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize.

PubMed

Li, Chenglong; Wen, Kai; Mi, Tiejun; Zhang, Xiya; Zhang, Huiyan; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

2016-05-15

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 μg kg(-1) for DON, 17.8 μg kg(-1) for T-2 toxin and 331.5 μg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

A study of the relationship between the chemical structures and the fluorescence quantum yields of coumarins, quinoxalinones and benzoxazinones for the development of sensitive fluorescent derivatization reagents.

PubMed

Azuma, Kentaro; Suzuki, Sachiko; Uchiyama, Seiichi; Kajiro, Toshi; Santa, Tomofumi; Imai, Kazuhiro

2003-04-01

To develop new fluorescent derivatization reagents, we investigated the relationship between the chemical structures and the fluorescence quantum yields (phi(f)) of coumarins, quinoxalinones and benzoxadinones. Forty-six compounds were synthesized and their fluorescence spectra were measured in n-hexane, ethyl acetate, methanol and water. The energy levels of these compounds were calculated by combination of the semi-empirical AM1 and INDO/S (CI = all) methods. The deltaE(Tn(n,pi*), S1(pi,pi*)) (the energy gap between the Tn(n,pi*) and S1(pi,pi*) states) values were well correlated with the phi(f) values, which enables us to predict the phi(f) values from their chemical structures. Based on this relationship, 3-phenyl-7-N-piperazinoquinoxalin-2(1H)-one (PQ-Pz) and 7-(3-(S)-aminopyrrolidin-1-yl)-3-phenylquinoxalin-2-(1H)-one (PQ-APy) were developed as fluorescent derivatization reagents for carboxylic acids. The derivatives of the carboxylic acids with PQ-Pz and PQ-APy showed large phi(f) values even in polar solvents, suggesting that these reagents are suitable for the microanalysis of biologically important carboxylic acids by reversed phase HPLC.

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.

Glutathione-capped CdTe nanocrystals as probe for the determination of fenbendazole

NASA Astrophysics Data System (ADS)

Li, Qin; Tan, Xuanping; Li, Jin; Pan, Li; Liu, Xiaorong

2015-04-01

Water-soluble glutathione (GSH)-capped CdTe quantum dots (QDs) were synthesized. In pH 7.1 PBS buffer solution, the interaction between GSH-capped CdTe QDs and fenbendazole (FBZ) was investigated by spectroscopic methods, including fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, and resonance Rayleigh scattering (RRS) spectroscopy. In GSH-capped CdTe QDs solution, the addition of FBZ results in the fluorescence quenching and RRS enhancement of GSH-capped CdTe QDs. And the quenching intensity (enhanced RRS intensity) was proportional to the concentration of FBZ in a certain range. Investigation of the interaction mechanism, proved that the fluorescence quenching and RRS enhancement of GSH-capped CdTe QDs by FBZ is the result of electrostatic attraction. Based on the quenching of fluorescence (enhancement of RRS) of GSH-capped CdTe QDs by FBZ, a novel, simple, rapid and specific method for FBZ determination was proposed. The detection limit for FBZ was 42 ng mL-1 (3.4 ng mL-1) and the quantitative determination range was 0-2.8 μg mL-1 with a correlation of 0.9985 (0.9979). The method has been applied to detect FBZ in real simples and with satisfactory results.

Determination of fluoxetine in pharmaceutical and biological samples based on the silver nanoparticle enhanced fluorescence of fluoxetine-terbium complex.

PubMed

Lotfi, Ali; Manzoori, Jamshid L

2016-11-01

In this study, a simple and sensitive spectrofluorimetric method is presented for the determination of fluoxetine based on the enhancing effect of silver nanoparticles (AgNPs) on the terbium-fluoxetine fluorescence emission. The AgNPs were prepared by a simple reduction method and characterized by UV-Vis spectroscopy and transmission electron microscopy. It was indicated that these AgNPs have a remarkable amplifying effect on the terbium-sensitized fluorescence of fluoxetine. The effects of various parameters such as AgNP and Tb 3+ concentration and the pH of the media were investigated. Under obtained optimal conditions, the fluorescence intensity of the terbium-fluoxetine-AgNP system was enhanced linearly by increasing the concentration of fluoxetine in the range of 0.008 to 19 mg/L. The limit of detection (b + 3s) was 8.3 × 10 -4 mg/L. The interference effects of common species found in real samples were also studied. The method had good linearity, recovery, reproducibility and sensitivity, and was satisfactorily applied for the determination of fluoxetine in tablet formulations, human urine and plasma samples. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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.