scholarly journals Development and Validation of an HPLC Method for Simultaneous Quantification of Clopidogrel Bisulfate, Its Carboxylic Acid Metabolite, and Atorvastatin in Human Plasma: Application to a Pharmacokinetic Study

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Octavian Croitoru ◽  
Adela-Maria Spiridon ◽  
Ionela Belu ◽  
Adina Turcu-Ştiolică ◽  
Johny Neamţu
Keyword(s):  
Carboxylic Acid ◽  
Internal Standard ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Acid Metabolite ◽  
Buffer Solution ◽  
Solution Ph ◽  
Limit Of Quantification ◽  
Simultaneous Quantification

A simple, sensitive, and specific reversed phase liquid chromatographic method was developed and validated for simultaneous quantification of clopidogrel, its carboxylic acid metabolite, and atorvastatin in human serum. Plasma samples were deproteinized with acetonitrile and ibuprofen was chosen as internal standard. Chromatographic separation was performed on an BDS HypersilC18column (250 × 4.6 mm; 5 μm) via gradient elution with mobile phase consisting of 10 mM phosphoric acid (sodium) buffer solution (pH = 2.6 adjusted with 85% orthophosphoric acid) : acetonitrile : methanol with flow rate of 1 mL·min−1. Detection was achieved with PDA detector at 220 nm. The method was validated in terms of linearity, sensitivity, precision, accuracy, limit of quantification, and stability tests. Calibration curves of the analytes were found to be linear in the range of 0.008–2 μg·mL−1for clopidogrel, 0.01–4 μg·mL−1for its carboxylic acid metabolite, and 0.005–2.5 μg·mL−1for atorvastatin. The results of accuracy (as recovery) with ibuprofen as internal standard were in the range of 96–98% for clopidogrel, 94–98% for its carboxylic acid metabolite, and 90–99% for atorvastatin, respectively.

OPTIMISATION AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF RIFAMPICIN, ISONIAZID, PYRAZINAMIDE, AND ETHAMBUTOL HYDROCHLORIDE IN ANTI-TUBERCULOSIS 4-FDC TABLET

2015 ◽  
Vol 77 (1) ◽  
Author(s):  
Sholihul Khoiri ◽  
Sudibyo Martono ◽  
Abdul Rohman
Keyword(s):  
High Performance ◽  
Phosphate Buffer Solution ◽  
Gradient Elution ◽  
Hplc Method ◽  
Fixed Dose ◽  
Buffer Solution ◽  
Solution Ph ◽  
Combination Tablet ◽  
Simultaneous Quantification ◽  
Dose Combination

High-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous quantification of four components, namely rifampicin (RIF), isoniazid (INH), pyrazinamide (PYR), and ethambutol hydrochloride (ETM), contained in anti-tuberculosis drugs in fixed dose combination tablet (4-FDC). In order to increase the sensitivity of EMB, the pre-column derivatization technique with phenethyl isocyanate (PEIC) was carried out. The separation was accomplished using Waters Symmetry C8 (250× 4.6 mm i.d.; 5 μm) at 30oC. The mobile phase used was a mixture of acetonitrile and 20 mM phosphate buffer solution (pH 6.8) containing triethylamine and delivered at 1.5 mL/minute using gradient elution. TheUV detector was set at 210 nm. The method was validated in terms of selectivity, linearity, accuracy, precision, detection limit, quantification limit, and robustness according to International Conference on Harmanization (ICH). The optimized method is succcesfully used for quantitative analysis of RIF, INH, PYR and ETM in 4-FDC tablets. The level of these drugs in 4-FDC tablets were in accordance to that specified in Indonesian pharmacopeia.

scholarly journals Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure

1998 ◽  
Vol 44 (7) ◽  
pp. 1481-1488 ◽  
Author(s):  
Maria Shipkova ◽  
Paul Dieter Niedmann ◽  
Victor William Armstrong ◽  
Ekkehard Schütz ◽  
Eberhard Wieland ◽  
...  
Keyword(s):  
Mycophenolic Acid ◽  
High Performance ◽  
Internal Standard ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Free Concentration ◽  
Elution Chromatography ◽  
Glucuronide Metabolite

Abstract We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r >0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95–106%;recovery of MPAG was 96–106%. The imprecision (CV) for MPA (0.2–25 mg/L) was <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10–250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <5.9% (215 nm) between days. For quantification of MPAf, 100 μL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18–210 μg/L, the within-day CVs were <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).

scholarly journals Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

2008 ◽  
Vol 54 (5) ◽  
pp. 901-906 ◽  
Author(s):  
Jun Lu ◽  
Elizabeth L Frank
Keyword(s):  
Whole Blood ◽  
Clinical Laboratory ◽  
Vitamin B1 ◽  
Current Method ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Phosphate Esters ◽  
Healthy Population ◽  
Limit Of Quantification

Abstract Background: Thiamine (vitamin B1) deficiency is associated with severe diseases such as beriberi and Wernicke encephalopathy. Although most Americans have sufficient dietary intake, thiamine deficiency is observed in the alcohol-dependent and elderly populations. Measurement of thiamine concentration in whole blood provides an assessment of vitamin B1 status in at-risk individuals. Method: We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase. Results: The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP). The means (ranges) for an apparently healthy population were 114 (70–179) nmol/L for TDP and 125 (75–194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min. Conclusion: We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.

scholarly journals A New Validated Stability indicating RP-HPLC Method for Simultaneous Quantification of Impurities of Fluticasone Propionate and Salmeterol Xenafoate in Metered Dose Inhalation Aerosol

2021 ◽  
Vol 33 (4) ◽  
pp. 867-872
Author(s):  
Surya Prakash Mamillapalli ◽  
Shirisha Koyya ◽  
B. Venkata Subbaiah ◽  
N. Annapurna
Keyword(s):  
Fluticasone Propionate ◽  
Drug Product ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Forced Degradation ◽  
Stability Indicating ◽  
Simultaneous Quantification ◽  
Metered Dose ◽  
Dose Inhalation

A simple, specific, precise, accurate and stability indicating reversed phase HPLC method for simultaneous quantification of total 12 impurities of fluticasone propionate and salmeterol xenafoate in metered dose inhalation aerosol has been developed in the present work. Chromatographic separation between impurities of both compounds were achieved on Altima C18 250 × 4.6 mm, 5 μ column using a step-gradient elution at a flow rate of 1.4 mL/min, 0.1% v/v orthophosphoric acid as buffer and acetonitrile as mobile phase constituents. Forced degradation studies for drug product were performed and revealed that Salmeterol is acid sensitive (about 21.3%), degrades to IMP-D and fluticasone is alkali sensitive (about 7.6%) and degrades to IMP-A. All degradant and process related impurities of both compounds were monitored at 214 nm and spectral purity along with % mass balance is assessed using PDA detector, which proved stability indicating capability of the method. The developed method is fully validated as per current ICH guidelines, where precision is achieved at % RSD of < 5, Correlation of < 0.999 for linearity, LOD-LOQ at < 0.02% and < 0.05%, along with satisfactory system suitability results under robustness conditions.

scholarly journals A Rapid and Sensitive HPLC Method for Quantitation of Paclitaxel in Biological Samples using Liquid-Liquid Extraction and UV Detection: Application to Pharmacokinetics and Tissues Distribution Study of Paclitaxel Loaded Targeted Polymeric Micelles in.....

2015 ◽  
Vol 18 (5) ◽  
pp. 647 ◽  
Author(s):  
Mahboubeh Rezazadeh ◽  
Jaber Emami ◽  
Abolfazl Mostafavi ◽  
Mahboubeh Rostami ◽  
Farshid Hassanzadeh ◽  
...  
Keyword(s):  
Polymeric Micelles ◽  
Internal Standard ◽  
Reversed Phase ◽  
Hplc Method ◽  
Acetate Buffer Solution ◽  
Tissue Homogenate ◽  
Buffer Solution ◽  
Column Temperature ◽  
Distribution Study ◽  
Tissue Homogenates

A simple, rapid, and sensitive reversed-phase HPLC method was developed and validated for determination of paclitaxel (PTX) in plasma, various organs and tumor tissues of tumor-bearing mice. Tissue specimens of liver, kidneys, spleen, lungs, heart and tumor were separately homogenized in normal saline. Plasma or tissue homogenate (250 µl) containing PTX and internal standard (diazepam) were extracted by diethyl ether (6 ml). The separation was achieved on a µ-Bondapak C18 HPLC column using sodium acetate buffer solution (0.01 M)/acetonitrile (58/42 v/v) at pH 5 ± 0.1 and flow rate of 1.9 mL/min. The effluent was monitored at 227 nm and column temperature was adjusted at 58ºC. The internal standard and PTX were eluted at 4.2 and 5.2 min, respectively and no interfering peaks were observed.  Calibration curves were linear over the concentration range of 0.25-10 µg/ml of PTX in plasma and 0.3-20 µg/ml PTX in tissue homogenates with acceptable precision and accuracy (<15%).  The mean recoveries of the drug after plasma extraction was 87.4% ± 3.6 while those of tissue homogenates ranged from 62.1± 4.5 to 75.5± 3.2 depending on the type of tissues studied.  PTX was stable in samples with no evidence of degradation during 3 freeze–thaw cycles and 3 months storage at −70 °C.  The developed HPLC method was applied to quantify PTX in the mouse plasma and tissues after intravenous administration of 10 mg equivalent PTX/Kg dose of PTX-loaded tocopherol succinate-chitosan-polyethylene glycol-folate (TS-CS-PEG-FA) micelles formulation or Anzatax® (Cremophor® EL- based formulation of PTX) to female Balb/c mice. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

scholarly journals A Validated RP-HPLC Method for the Determination of Butamirate Citrate and Benzoic Acid in Syrup, Based on an Experimental Design Assessment of Robustness

Separations ◽  
2021 ◽  
Vol 8 (10) ◽  
pp. 163
Author(s):  
Antonios-Dionysios G. Neofotistos ◽  
Kostas Gkountanas ◽  
Haris Boutsikaris ◽  
Yannis Dotsikas
Keyword(s):  
Experimental Design ◽  
Benzoic Acid ◽  
Reversed Phase ◽  
Hplc Method ◽  
Array Detector ◽  
Buffer Solution ◽  
Solution Ph ◽  
Design Assessment ◽  
Rp Hplc

A reversed-phase high-pressure liquid chromatography (RP-HPLC) method was developed and subsequently validated for the simultaneous determination of butamirate citrate (BC) and benzoic acid (BA) in cough syrup. The separation was performed employing a cyanopropyl column with a mobile phase consisting of 50%/50% v/v MeOH/NaH2PO4 * H2O 50 mM aqueous solution pH = 3.0. The quantitation was achieved with a diode array detector (DAD) at 210 nm. The method demonstrated a congenitally satisfactory separation, yet the acquired peaks were asymmetrical. This effect was eliminated by using 1% triethylamine in the buffer solution as a silanol blocker. In addition, the method was found to unequivocally assess the target analytes in the sample matrix and fulfilled the required specifications in relevance to specificity, linearity, accuracy, precision and stability of both the standard solutions and of the sample solutions. Lastly, an experimental design was designed in order to assess the robustness of the proposed assay. To this purpose, a graphical and a statistical approach were utilized and compared to identify the factors that should be strictly controlled during each execution of the method.

scholarly journals Stability Indicating Method for Simultaneous RP HPLC Determination of Camylofin Dihydrochloride and Nimesulide in Pharmaceutical Preparations

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Rajeev Kumar R. Singh ◽  
Manapragada V. Rathnam ◽  
Sangeeta J. Singh ◽  
Raju V. K. Vegesna
Keyword(s):  
Internal Standard ◽  
Pharmaceutical Preparations ◽  
Reversed Phase ◽  
Control Analysis ◽  
Solution Stability ◽  
Buffer Solution ◽  
Solution Ph ◽  
Column Temperature ◽  
Stability Indicating

A simple, fast, and precise reversed phase high-performance liquid chromatographic method has been developed for the simultaneous determination of camylofin dihydrochloride and nimesulide using caffeine as an internal standard. The stability indicating capability of the method was proved by subjecting the drugs to stress conditions as per ICH-recommended test conditions. Separation was achieved using Varian Chromspher 5 C18 column (250 mm × 4.6 mm, 5 μm) as stationary phase with a mobile phase comprising of buffer solution pH 5.0 : methanol (600 : 400, v/v) at a flow rate of 1.0 mL min−1, column temperature of 30∘C and UV detection at 220 nm. The retention time of caffeine, camylofin dihydrochloride, and nimesulide was about 5.0 min, 6.1 min, and 12.7 min, respectively. The proposed method was validated for linearity, accuracy, precision, sensitivity, robustness and solution stability. Linearity, accuracy, and precision were found to be acceptable over the ranges of 250–750 μg mL−1 for Nimesulide and 125–375 μg mL−1 for camylofin dihydrochloride. The test solution was found to be stable for 72 h. It can be conveniently adopted for routine quality control analysis.

scholarly journals Development and Validation of an LC Method for Simultaneous Determination of Amiloride Hydrochloride and Hydrochlorothiazide in Human Urine

2009 ◽  
Vol 92 (3) ◽  
pp. 813-819 ◽  
Author(s):  
Abdalla A Elshanawane ◽  
Samia M Mostafa ◽  
Mohamed S Elgawish
Keyword(s):  
Human Urine ◽  
Internal Standard ◽  
Reversed Phase ◽  
Hplc Method ◽  
Solution Ph ◽  
Photometric Detection ◽  
Coefficients Of Variation ◽  
Development And Validation ◽  
Amiloride Hydrochloride

Abstract An HPLC method with photometric detection has been developed for determination of a binary mixture of amiloride hydrochloride and hydrochlorothiazide in human urine using chlorthalidone as the internal standard. Reversed-phase chromatography was performed at room temperature on a cyanopropyl column with the mobile phase consisting of a 10 mM KH2PO4 solution (pH 4.5)methanol (70 + 30, v/v) at a flow rate of 1 mL/min. The detector was set at 214 nm. The total analysis time was 10 min. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability, and re-injection reproducibility. The procedure shows good accuracy, repeatability, and selectivity. Moreover, the method was applied directly to urine that had not undergone prior treatment. The intra- and interday coefficients of variation for all compounds were below 4, and the method was highly accurate, with a relative error for all compounds that was below 8. No interference from endogenous compounds in urine samples was found. The proposed method, which is rapid, simple, and does not require any separation steps, has been successfully applied to the assay of human urine containing amiloride hydrochloride and hydrochlorothiazide.

Precolumn 𝘖-Phthalaldehyde Derivatization and Reversed-Phase Liquid Chromatography of S-Methylmethioninesulfonium in Satsuma Mandarin Juice

1992 ◽  
Vol 75 (1) ◽  
pp. 77-79
Author(s):  
Hidenobu Sumitani ◽  
Sachiko Suekane ◽  
Yasue Sakai ◽  
Kiyoaki Tatsuka
Keyword(s):  
Liquid Chromatography ◽  
Internal Standard ◽  
Reversed Phase ◽  
Acetate Buffer Solution ◽  
Internal Standard Method ◽  
Buffer Solution ◽  
Solution Ph ◽  
Satsuma Mandarin ◽  
Phase Liquid ◽  
Mandarin Juice

Abstract Experiments were conducted to determine the amount of S-methylmethlonlnesulfonium (MMS) in Satsuma mandarin Juice using a precolumn 𝘖-phthalaldehyde (OPA) derivatization and reversedphase liquid chromatography. Favorable analytical conditions that allowed MMS analysis were achieved by using OPA/3-mercaptopropionlc acid (MPA) as a derivatization agent with fluorescence detection (excitation at 330 nm, emission at 450 nm). A C18 reversed-phase column with 5 μm particle size and a length of 250 mm was used. Resolution of MMS/OPA/MPA derivative was accomplished with a linear gradient eluent (30mM sodium acetate buffer solution, pH 7.3, and 70% (v/v) methanol). Quantitative analysis of MMS by the internal standard method using β-alanine gave highly reproducible result with a coefficient of variation less than 3%. Recovery of MMS added to Juice samples was 105%. MMS content in Satsuma mandarin Juice was 28.2μM.

Validation of a simple HPLC method to quantify mycophenolic acid concentrations in human plasma

MedPharmRes ◽  
2021 ◽  
Vol 5 (2) ◽  
pp. 1-6
Author(s):  
Thuan Thi Minh Nguyen ◽  
Truong Huu Tran
Keyword(s):  
Lower Limit ◽  
Mycophenolic Acid ◽  
Solid Phase ◽  
Internal Standard ◽  
Reversed Phase ◽  
Hplc Method ◽  
Solid Organ ◽  
Limit Of Quantification ◽  
Organ Rejection ◽  
Sample Extract

Introduction: Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil and mycophenolate sodium which are widely prescribed to prevent organ rejection after solid organ transplantations. However, MPA induced many side effects on gastrointestinal tract and haematological system. Objectives: The purpose of this study is to establish a high-performance liquid chromatography (HPLC) method to determine the MPA concentration in plasma in order to optimize the treatment efficacy of MPA or apply to bioequivalence studies. MPA and visnadine (as an internal standard) were extracted from plasma samples with methanol by solid phase extraction using Osis HLB 1cc cartridge. 10 µL of sample extract was injected onto LiChroCART®125-4 (C18 reversed-phase column) at 43 °C on a Waters 2695 XE system. The signals were detected by PDA detector (photodiodes array) at 254 nm. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 3) with a flow rate of 1 mL/min. The validation criteria included: selectivity, linearity, accuracy, precision, recovery, lower limit of quantification. Results: Total chromatographic runtime was 15 min. MPA and visnadine were found at 6.45 and 10.79 min, respectively. MPA concentrations were in the linear range from 0.25 to 50 µg/mL. The coefficient of variation (CV) of mean intra-day and inter-day precision levels for MPA was less than 7.5%. The lower limit of quantification was 0.25 µg/mL. No interference was found in the assay. Conclusion: A simple and reliable HPLC method was developed to quantify the MPA concentration in plasma.

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