scholarly journals Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4196
Author(s):  
Faisal Nuhu ◽  
Andrew Gordon ◽  
Roger Sturmey ◽  
Anne-Marie Seymour ◽  
Sunil Bhandari
Keyword(s):  
Oxidative Stress ◽  
High Performance ◽  
Limit Of Detection ◽  
Kidney Diseases ◽  
Biological Tissues ◽  
Clinical Samples ◽  
Range Limit ◽  
Limit Of Quantification ◽  
Reliable Determination

Background: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. Methods: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. Results: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM–4 mM and 0.2 µM–0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. Conclusion: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

scholarly journals Stability-Indicating Assay for the Determination of Pentobarbital Sodium in Liquid Formulations

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Myriam Ajemni ◽  
Issa-Bella Balde ◽  
Sofiane Kabiche ◽  
Sandra Carret ◽  
Jean-Eudes Fontan ◽  
...  
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Drug Stability ◽  
Reversed Phase ◽  
Hplc Method ◽  
Limit Of Quantification ◽  
Reliable Determination ◽  
Pentobarbital Sodium ◽  
Stability Indicating

A stability-indicating assay by reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of pentobarbital sodium in oral formulations: a drug used for infant sedation in computed tomography (CT) or magnetic resonance imaging (MRI) scan. The chromatographic separation was achieved on a reversed-phase C18 column, using isocratic elution and a detector set at 214 nm. The optimized mobile phase consisted of a 0.01 M potassium buffer pH 3 and methanol (40 : 60, v/v). The flow rate was 1.0 mL/min and the run time of analysis was 5 min. The linearity of the method was demonstrated in the range of 5 to 250 μg/mL pentobarbital sodium solution (r2= 0.999). The limit of detection and limit of quantification were 2.10 and 3.97 μg/mL, respectively. The intraday and interday precisions were less than 2.1%. Accuracy of the method ranged from 99.2 to 101.3%. Stability studies indicate that the drug is stable to sunlight and in aqueous solution. Accelerated pentobarbital sodium breakdown by strong alkaline, acidic, or oxidative stress produced noninterfering peaks. This method allows accurate and reliable determination of pentobarbital sodium for drug stability assay in pharmaceutical studies.

scholarly journals Simultaneous determination of phytoestrogens in dietary supplements by high-performance liquid chromatography

2021 ◽  
Vol 4 (3) ◽  
pp. 24-35
Author(s):  
Thanh An Vu Thi ◽  
Thanh Hoa Mac Thi ◽  
Khanh Cao Cong ◽  
◽  
◽  
...  
Keyword(s):  
Dietary Supplements ◽  
Mobile Phase ◽  
High Performance ◽  
Limit Of Detection ◽  
Ultrasonic Method ◽  
Gradient Elution ◽  
Analysis Procedure ◽  
Range Limit ◽  
Limit Of Quantification

The bio-active phytoestrogen compounds (puerarin, daidzin, glycitin, genistin, miroestrol, daidzein, glycitein, genistein) in dietary supplements were extracted by ultrasonic method with methanol solvent in 40oC. The analysis procedure was carried out on HPLC Alliance e2695 (Waters) system, with RP-C18 Reliant (25 mm × 4.6 mm; 5µm) column, at 30oC. Phytoestrogens were separated by using gradient elution with a mobile phase consisting 0.1% (v/v) phosphoric aqueous solution and methanol in 45 minutes. The method was validated by determining its specificity, linear range, limit of detection, limit of quantification, repeatability and accuracy. This method was applied succesfully to determine content of Phytoestrogens in commercial dietary supplement products.

scholarly journals DETERMINATION OF 10-GINGEROL IN INDIAN GINGER BY VALIDATED HPTLC METHOD OF SAMPLES COLLECTED ACROSS SUBCONTINENT OF INDIA

2016 ◽  
Vol 8 (12) ◽  
pp. 190
Author(s):  
Kamran Ashraf ◽  
Syed Adnan Ali Shah ◽  
Mohd Mujeeb
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Limit Of Detection ◽  
Limit Of Quantification ◽  
Chromatography Method ◽  
Aluminum Plates ◽  
Layer Chromatography ◽  
Hptlc Method

<p><strong>Objective: </strong>A simple, sensitive, precise, and accurate stability indicating HPTLC (high-performance thin-layer chromatography) method for analysis of 10-gingerol in ginger has been developed and validated as perICH guidelines.</p><p><strong>Methods: </strong>The separation was achieved on TLC (thin layer chromatography) aluminum plates pre-coated with silica gel 60F<sub>254</sub> using n-hexane: ethyl acetate 55:45 (%, v/v) as a mobile phase. Densitometric analysis was performed at 569 nm.</p><p><strong>Results: </strong>This system was found to have a compact spot of 10-gingerol at <em>R</em><sub>F</sub> value of 0.57±0.03. For the proposed procedure, linearity (<em>r</em><sup>2</sup> = 0.998±0.02), limit of detection (18ng/spot), limit of quantification (42 ng/spot), recovery (ranging from 98.35%–100.68%), were found to be satisfactory.</p><p><strong>Conclusion: </strong>Statistical analysis reveals that the content of 10-gingerol in different geographical region varied significantly. The highest and lowest concentration of 10-gingerol in ginger was found to be present in a sample of Patna, Lucknow and Surat respectively which inferred that the variety of ginger found in Patna, Lucknow are much superior to other regions of India.</p>

Determination of Potential Genotoxic Impurity, 5-Amino-2-Chloropyridine, in Active Pharmaceutical Ingredient Using the HPLC-UV System

2020 ◽  
Author(s):  
Murat Soyseven ◽  
Rüstem Keçili ◽  
Hassan Y Aboul-Enein ◽  
Göksel Arli
Keyword(s):  
Analytical Method ◽  
High Performance ◽  
Orthophosphoric Acid ◽  
Limit Of Detection ◽  
Detection System ◽  
Active Pharmaceutical Ingredient ◽  
Limit Of Quantification ◽  
Column Temperature ◽  
Water Ph

Abstract A novel analytical method, based on high-performance liquid chromatography with a UV (HPLC-UV) detection system for the sensitive detection of a genotoxic impurity (GTI) 5-amino-2-chloropyridine (5A2Cl) in a model active pharmaceutical ingredient (API) tenoxicam (TNX), has been developed and validated. The HPLC-UV method was used for the determination of GTI 5A2Cl in API TNX. The compounds were separated using a mobile phase composed of water (pH 3 adjusted with orthophosphoric acid): MeOH, (50:50: v/v) on a C18 column (150 × 4.6 mm i.d., 2.7 μm) at a flow rate of 0.7 mL min−1. Detection was carried out in the 254 nm wavelength. Column temperature was maintained at 40°C during the analyses and 10 μL volume was injected into the HPLC-UV system. The method was validated in the range of 1–40 μg mL−1. The obtained calibration curves for the GTI compound was found linear with equation, y = 40766x − 1125,6 (R2 = 0.999). The developed analytical method toward the target compounds was accurate, and the achieved limit of detection and limit of quantification values for the target compound 5A2Cl were 0.015 and 0.048 μg mL−1, respectively. The recovery values were calculated and found to be between 98.80 and 100.03%. The developed RP-HPLC-UV analytical method in this research is accurate, precise, rapid, simple and appropriate for the sensitive analysis of target GTI 5A2Cl in model API TNX.

Determination of tigecycline in turkey plasma by LC-MS/MS: validation and application in a pharmacokinetic study

2017 ◽  
Vol 20 (2) ◽  
pp. 241-249 ◽  
Author(s):  
A. Jasiecka-Mikołajczyk ◽  
J.J. Jaroszewski
Keyword(s):  
Pharmacokinetic Study ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Reversed Phase ◽  
Selected Reaction Monitoring ◽  
Limit Of Quantification ◽  
Complicated Skin ◽  
Intravenous And Oral Administration

Abstract Tigecycline (TIG), a novel glycylcycline antibiotic, plays an important role in the management of complicated skin and intra-abdominal infections. The available data lack any description of a method for determination of TIG in avian plasma. In our study, a selective, accurate and reversed-phase high performance liquid chromatography-tandem mass spectrometry method was developed for the determination of TIG in turkey plasma. Sample preparation was based on protein precipitation and liquid-liquid extraction using 1,2-dichloroethane. Chromatographic separation of TIG and minocycline (internal standard, IS) was achieved on an Atlantis T3 column (150 mm × 3.0 mm, 3.0 μm) using gradient elution. The selected reaction monitoring transitions were performed at 293.60 m/z → 257.10 m/z for TIG and 458.00 m/z → 441.20 m/z for IS. The developed method was validated in terms of specificity, selectivity, linearity, lowest limit of quantification, limit of detection, precision, accuracy, matrix effect, carry-over effect, extraction recovery and stability. All parameters of the method submitted to validation met the acceptance criteria. The assay was linear over the concentration range of 0.01-100 μg/ml. This validated method was successfully applied to a TIG pharmacokinetic study in turkey after intravenous and oral administration at a dose of 10 mg/kg at various time-points.

scholarly journals Development and method validation for determination of 54 pesticides in Okra by LC-MS/MS analysis

2020 ◽  
Vol 11 (1) ◽  
pp. 985-992
Author(s):  
Hymavati Muppalla ◽  
Kiranmayi Peddi
Keyword(s):  
European Union ◽  
Pesticide Residues ◽  
High Performance ◽  
Limit Of Detection ◽  
Extraction Procedure ◽  
Mass Spectrometry Analysis ◽  
Limit Of Quantification ◽  
Spectrometry Analysis ◽  
Tandem Mass Spectrometry Analysis

The presence of pesticide residues in primary and derived agricultural products raises serious health concerns for consumers across the globe. The aim of the present study was to assess the level of pesticide residues in Okra in India. A multi-residue method for the quantification of fifty-four pesticides in okra is described in this work. The present study employed a modified quick, easy cheap, effective rugged and safe (QuEChERS) extraction procedure followed by UHPLC-MS/MS (Ultra-High-Performance Liquid Chromatography coupled to Tandem Mass Spectrometry) analysis. Validation of the method was according to the guidelines given by European Union SANCO/12571/2013. The levels of validation were 10.0, 50.0 and 100 µg kg-1. The following parameters such as linearity, the limit of detection (LOD) (nearer to 0.005 mg kg-1) and limit of quantification (LOQ) (nearer to 0.01 mg kg-1) were set to be acceptable. The trueness of the method for 54 pesticides in all Okra commodities was between 80-110% with satisfactory repeatability and within-run reproducibility except for the pesticide residues such as Thiamethoxam and Fenamidone. The measurement of uncertainty for each of the pesticide was below 50% and was estimated to be in the range of 5.37% - 10.71%, which meets the criteria established in the SANCO/12571/2013 document (European Union, 2013). This method is concluded to be applicable for the determination of pesticide residues in Okra.

HPTLC Separation of a Hepatoprotective Combination in Pharmaceutical Formulation and Human Plasma

2020 ◽  
Vol 58 (5) ◽  
pp. 411-417
Author(s):  
Maimana A Magdy ◽  
Rehab M Abdelfatah
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Limit Of Detection ◽  
Pharmaceutical Formulation ◽  
Limit Of Quantification ◽  
Ich Guidelines ◽  
Spiked Plasma ◽  
Significant Difference ◽  
Developing System

Abstract A binary mixture of Silymarin (SR) and Vitamin E (VE) acetate, of an antioxidant and a hepatoprotective effect, has been analyzed using a sensitive, selective and economic high performance thin layer chromatographic (HPTLC) method in their pure forms, pharmaceutical formulation and spiked human plasma. SR and VE were separated on 60F254 silica gel plates using hexane:acetone:formic acid (7:3:0.15, v/v/v) as a developing system with UV detection at 215 nm. The method was evaluated for linearity, accuracy, precision, selectivity, limit of detection (LOD) and limit of quantification (LOQ). SR and VE were detected in the linear range of 0.2–2.5 and 0.2–4.5 μg/band, respectively. Method validation was done as per ICH guidelines and acceptable results of accuracy of 99.86 ± 1.190 and 100.22 ± 1.609 for SR and VE, respectively were obtained. The method has been successfully applied for determination of the studied drugs in their pharmaceutical formulation without any interference from excipients, and in spiked plasma samples. Results obtained by the developed HPTLC-densitometric method were statistically compared to those obtained by the reported HPLC methods and no significant difference was found between them.

Simultaneous Determination of Cholecalciferol and 25- Hydroxycholecalceferol in Lipid-based Self-nanoemulsifying formulations and Marketed Product Vi-de 3® by UHPLC-UV

2019 ◽  
Vol 16 (1) ◽  
pp. 100-109
Author(s):  
Ibrahim Aljuffali ◽  
Fahad Almarri ◽  
A. F. M. Motiur Rahman ◽  
Fars Kaed Alanazi ◽  
Musaed Alkholief ◽  
...  
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Simultaneous Estimation ◽  
Limit Of Quantification ◽  
Column Temperature ◽  
Related Substances ◽  
Accuracy And Precision ◽  
Regulatory Guidelines ◽  
25 Hydroxycholecalciferol

Background: The purpose of the current study was to develop a selective, precise, fast economical and advanced reverse phase ultra-high-performance liquid chromatography (UHPLC UV) method and validate it for the simultaneous estimation of cholecalciferol and its analogue 25- hydroxycholecalciferol in lipid-based self-nano emulsifying formulation (SNEDDS). Methods: The chromatographic separation was simply performed on a Dionex® UHPLC systems (Ultimate 3000, Thermo scientific) by using HSS C18 (2.1x50 mm, 1.8 µm) analytical column. The elution was carried out isocratically with the mobile phase consisting of acetonitrile and methanol in the ratio of 50:50 %v/v with a flow rate of 0.4 ml/min, followed by the UV detection at 265 nm. The injection volume was 1µl and the column temperature was maintained at 45°C. FDA regulatory guidelines were used to develop and validate the method. Results: The current developed UHPLC-UV method was found to be rapid (run time 2 min), and selective with the high resolution of cholecalciferol and 25-hydroxycholecalciferol (RT=0.530 min & 1.360 min) from different lipid matrices. The method was highly sensitive (Limit of Detection and Lower Limit of Quantification were 0.13 ppm & 0.51ppm, and 0.15 ppm & 0.54 ppm, respectively). The linearity, accuracy and precision were determined as suitable over the concentration range of 0.5-50.0 ppm for both the analytes. Conclusion: The proposed UHPLC-UV method can be used for the determination of cholecalciferol and 25-hydroxycholecalciferol in SNEDDS and marketed Vi-De 3® as pure forms (intact) with no interference of excipients or drug-related substances.

scholarly journals Determination of Formaldehyde by HPLC with Stable Precolumn Derivatization in Egyptian Dairy Products

2018 ◽  
Vol 2018 ◽  
pp. 1-5 ◽  
Author(s):  
Ahmed Salem Sebaei ◽  
Ahmed M. Gomaa ◽  
A. A. El-Zwahry ◽  
E. A. Emara
Keyword(s):  
Dairy Products ◽  
High Performance ◽  
Limit Of Detection ◽  
Precolumn Derivatization ◽  
Limit Of Quantification ◽  
Chromatography Method ◽  
Derivatizing Agent ◽  
Array Detection ◽  
Liquid Chromatography Method

Formaldehyde is one of the most dangerous chemical compounds affecting the human health; exposure to it from food may occur naturally or by intentional addition. In this study a high performance liquid chromatography method for determination of formaldehyde in dairy products was described. The dairy samples were reacted and extracted with a warmed organic solvent in the presence of derivatizing agent 2,4-dinitrophenylhydrazine (DNPH) and formaldehyde; the mixture was centrifuged and followed by diode array detection. The method is validated and gives average recovery of formaldehyde at the three different levels 0.1, 5.0, and 10.0 mg/kg varied between 89% and 96%. The method is linear from the limit of quantification 0.1 mg/kg up to 10 mg/kg levels. This method is intended for formaldehyde analyses in dairy products simply with stable derivatization, minimum residue loss, excellent recovery, and accurate results with a sensitive limit of detection 0.01 mg/kg. 90 dairy samples from milk, cheese, and yogurt were investigated from seven Egyptian governorates and all samples were free from formaldehyde.

scholarly journals HPLC method validation and application for organic acid analysis in wine after solid-phase extraction

2016 ◽  
Vol 35 (2) ◽  
pp. 225 ◽  
Author(s):  
Violeta Ivanova-Petropulos ◽  
Krste Tašev ◽  
Marina Stefova
Keyword(s):  
Solid Phase Extraction ◽  
Organic Acids ◽  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Hplc Method ◽  
Phase Extraction ◽  
Hplc Separation ◽  
Limit Of Quantification

<p>A solid-phase extraction method followed by reverse phase high-performance liquid chromatography (RP-HPLC) was optimized and validated for the quantitative determination of tartaric, malic, shikimic, lactic, citric and succinic acids in wine. Solid-phase extraction was carried out with C18 cartridges and extraction recoveries for all acids ranging from 98.3 to 103% were obtained. HPLC separation was performed with isocratic elution on a LiChrosorb RP-18 column (250 × 4.6 mm I.D., 5 µm) protected with the appropriate guard column. The mobile phase was a 5 mM solution of H<sub>3</sub>PO<sub>4</sub> with pH 2.1 at a flow rate of 1 ml/min. Detection of the organic acids was performed at 210 nm. The developed method was validated by checking its linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery. The method was applied to the analysis of organic acids in Macedonian red and white wines.</p>

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