scholarly journals Development and Validation of a Novel RP-HPLC Analytical Method for Sitagliptin Determination in Human Plasma

2021 ◽  
pp. 92-101
Author(s):  
Sagar Pamu ◽  
Sazal Patyar ◽  
Lakshmi Thakkalapally
Keyword(s):  
Human Plasma ◽  
Analytical Method ◽  
Internal Standard ◽  
Accurate Method ◽  
Pharmacokinetic Studies ◽  
Chromatographic Separations ◽  
Rp Hplc ◽  
Analytical Estimation ◽  
The Stability ◽  
Stock Solutions

Background: Different bio-analytic methods have been developed for determining drug concentration in plasma, but methods for sitagliptin determination are still very rare. In this study, RP-HPLC based method has been developed for assessing sitagliptin concentration in plasma. Aim: To develop and validate RP-HPLC based analytical method for estimating sitagliptin in human plasma for pharmacokinetic applications. Methods: In the present study, the mobile phase composed of acetonitrile: 0.5% triethanolamine (20:80) with pH 6.5 has been utilized. Samples of plasma containing sitagliptin and internal standard (IS)-rosiglitazone were extracted with dichloromethane:diethyl ether (4:6; v/v) at pH 7.4. The rate of flow was 1 ml/min. The retention time was about 5,232 and 6,903 minutes respectively for sitagliptin and rosiglitazone. Results: At concentrations of 100-3200 ng/ml in plasma, calibration curves of sitagliptin were linear. The inter- and intra-day precision and accuracy ranged in between 93.56-98.56% and 1.09-4.55% respectively. For specificity, the study findings showed no co-eluting peaks occurring with IS drug (rosiglitazone) and confirmed that no percentage of interferences at analyte (sitagliptin) retention in presence of rosiglitazone. The sitagliptin recovery was 96.442%. Chromatographic separations were performed on HI Qsil C-18 HS column (250mm x 4.6mm x 5μm). The stability of stock solutions of sitagliptin and IS at room temperature was 98.06% and 100.79% respectively while under refrigerated conditions stability was 98.19% and 96.59% respectively. Freeze-thaw stability for sitagliptin was performed with low & high QC and shown as 98.26% for and 97.45% respectively. The limits of detection and quantification were 8.592 ng/ml and 28.641 ng/ml respectively. Conclusion: A simple, sensitive and accurate method was developed for bio analytical estimation of sitagliptin in human plasma using liquid-liquid extraction technique. The validation results of linearity, accuracy, precision, stability, selectivity, ruggedness, LOD and LOQ were good under acceptable limit and can be applied for pharmacokinetic studies.

scholarly journals DEVELOPMENT AND VALIDATION OF A SIMPLE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–UV METHOD FOR THE DETERMINATION OF VINORELBINE - A CHEMOTHERAPEUTIC DRUG IN SPIKED HUMAN PLASMA

2019 ◽  
Vol 12 (1) ◽  
pp. 369
Author(s):  
Useni Reddy Mallu ◽  
Venkateswara Rao Anna ◽  
Bikshal Babu Kasimala
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Studies ◽  
Chemotherapeutic Drug ◽  
Spiked Human Plasma ◽  
Rp Hplc

Objective: Vinorelbine (VNRB) is a chemotherapeutic drug used to treat non-small cell lung cancer and breast cancer. Literature survey reveals that there are no reverse-phase high-performance liquid chromatography (RP-HPLC) methods reported for the estimation of VNRB in spiked human plasma. Hence, the present work aimed to develop a simple and efficient RP-HPLC method for the estimation of VNRB in human plasma.Methods: Specimen preparation for the measurement of VNRB was performed through liquid-liquid extraction using methanol as extracting solvent and reconstructed with mobile phase. Paclitaxel (PCTX) was used as internal standard. HPLC method was optimized and validated as per the US FDA bioanalytical guidelines. VNRB and internal standard were separated on Kromasil® C18 (250×4.6 mm; id 5 μ) using acetate buffer (pH=5.9) and methanol in the ratio of 85:15 (v/v) at 1 ml/min flow rate. Eluted compounds were recorded using UV detector at 235 nm.Results: The retention time of PCTX and internal standard was found to be 4.3 and 9.0 min, respectively. The analytical measuring ranges were found to be 5–750 ng/ml (r2>0.9998). The method was found to be simple, accurate, precise, and stable and there is no interference of plasma matric components.Conclusion: The described HPLC method allows for the measurement of total and free PCTX in both plasma and cord blood and can utilize for the estimation of drug in pharmacokinetic studies.

scholarly journals DEVELOPMENT AND VALIDATION OF A SIMPLE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–UV METHOD FOR THE DETERMINATION OF VINORELBINE - A CHEMOTHERAPEUTIC DRUG IN SPIKED HUMAN PLASMA

2019 ◽  
Vol 12 (1) ◽  
pp. 369 ◽  
Author(s):  
Useni Reddy Mallu ◽  
Venkateswara Rao Anna ◽  
Bikshal Babu Kasimala
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Studies ◽  
Chemotherapeutic Drug ◽  
Spiked Human Plasma ◽  
Rp Hplc

Objective: Vinorelbine (VNRB) is a chemotherapeutic drug used to treat non-small cell lung cancer and breast cancer. Literature survey reveals that there are no reverse-phase high-performance liquid chromatography (RP-HPLC) methods reported for the estimation of VNRB in spiked human plasma. Hence, the present work aimed to develop a simple and efficient RP-HPLC method for the estimation of VNRB in human plasma.Methods: Specimen preparation for the measurement of VNRB was performed through liquid-liquid extraction using methanol as extracting solvent and reconstructed with mobile phase. Paclitaxel (PCTX) was used as internal standard. HPLC method was optimized and validated as per the US FDA bioanalytical guidelines. VNRB and internal standard were separated on Kromasil® C18 (250×4.6 mm; id 5 μ) using acetate buffer (pH=5.9) and methanol in the ratio of 85:15 (v/v) at 1 ml/min flow rate. Eluted compounds were recorded using UV detector at 235 nm.Results: The retention time of PCTX and internal standard was found to be 4.3 and 9.0 min, respectively. The analytical measuring ranges were found to be 5–750 ng/ml (r2>0.9998). The method was found to be simple, accurate, precise, and stable and there is no interference of plasma matric components.Conclusion: The described HPLC method allows for the measurement of total and free PCTX in both plasma and cord blood and can utilize for the estimation of drug in pharmacokinetic studies.

scholarly journals DEVELOPMENT AND VALIDATION OF BIOANALYTICAL HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF CILNIDIPINE AND NEBIVOLOL IN HUMAN PLASMA

2017 ◽  
Vol 9 (10) ◽  
pp. 253
Author(s):  
Aruna G. ◽  
Bharathi K ◽  
Kvsrg Prasad
Keyword(s):  
Human Plasma ◽  
Analytical Method ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Parameters ◽  
Simultaneous Estimation ◽  
Time Data ◽  
Bioanalytical Method Validation ◽  
Limit Of Quantitation ◽  
The Mean

Objective: To develop and validate a modified isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of cilnidipine and nebivolol in human plasma to be used for pharmacokinetic studies.Methods: The drug was extracted from plasma samples by direct protein precipitation technique using acetonitrile. Amlodipine was used as internal standard (IS). Samples were analyzed on BDS C18 column (250 x 4.6 mm, 5 µm), applying ortho phosphoric acid (0.1%): Acetonitrile, at a ratio of 45:55 v/v in isocratic mode as a mobile phase at a flow rate of 1 ml/min to attain adequate resolution. Separations were performed at room temperature and monitored at a wavelength of 260 nm after injection of 50μl samples into the HPLC system. The analytical method was validated according to FDA bioanalytical method validation guidance. The method was applied for pharmacokinetic study of cilnidipine and nebivolol tablets-10 mg and 5 mg were administered as a single dose to 6 healthy male rabbits under fasting condition. Twelve blood samples were withdrawn from each rabbit over 24 h periods. From the plasma concentration-time data of each individual, the pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were calculated.Results: A peak area was obtained for cilnidipine and nebivolol at 3.943 and 4.719 min retention time respectively. Linearity was established at a concentration range of 0.20-20 μg/ml (r2=0.999, n=8) for cilnidipine and 0.02-2 μg/ml (r2=0.999, n=8) for nebivolol. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 0.2μg/ml for cilnidipine and 0.02 μg/ml for nebivolol. The coefficients of variation (%cv) of the intra-day and inter-day precision of cilnidipine at 600, 1000 and 1600ng/ml levels were found to be 6.90%, 6.19%, 5.22%; and 7.74%, 6.54%, 5.77%, respectively, which are lower than the accepted criteria limits (15-20 %). The mean recovery (%) cilnidipine at 600, 1000, and 1600ng/ml was found to be 101.03%, 99.27% and 104.87%, and for nebivolol 60, 100, and 160 ng/ml was found to be 106.13%, 107.03% and 98.06% respectively. Stability at different conditions and in autosampler was also established. The mean pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were 6 ng/ml, 2 hr, 96.76 mg. hr/ml, 63.45 mg. hr/ml for cilnidipine and 5.8ng/ml, 2hr, 74.78 mg. hr/ml, 100.25 mg. hr/ml for nebivolol respectively.Conclusion: The present analytical method was found to be specific, sensitive, accurate and precise for quantification of cilnidipine and nebivolol in human plasma. It can be successively applied for pharmacokinetics, bioavailability and bioequivalence studies.

scholarly journals Validation of developed method by RP-HPLC for simultaneous estimation of famotidine and ibuprofen in human plasma and studying the stability of the drugs in plasma

2018 ◽  
Vol 12 (1) ◽  
pp. 34-48
Author(s):  
K. S Ashutosh ◽  
D. Manidipa ◽  
R. J. V. L. N. Seshagiri ◽  
S. D. Gowri
Keyword(s):  
Human Plasma ◽  
Mobile Phase ◽  
Simultaneous Estimation ◽  
Hplc Separation ◽  
Reverse Phase ◽  
Rp Hplc ◽  
The Stability ◽  
Sodium Dihydrogen ◽  
Isocratic Mode

The RP-HPLC separation was carried out by reverse phase chromatography on a Symmetry C18 (4.6 x 150 mm, 3.5 μm, make: XTerra) with a mobile phase composed of sodium dihydrogen ortho phosphate [pH 2.5] and acetonitrile in the ratio of 30:70 v/v in an isocratic mode at a flow rate of 1.2 mL/min. The run time was maintained for 8.0 min. The detection was monitored at 236 nm. The accuracy was calculated in human plasma and the % recovery was found 99.80 - 99.85 for famotidine and 99.56 -99.85.5 for ibuprofen and reproducibility was found to be satisfactory. The calibration curve for famotidine in human plasma was linear over 3.32 to 6.65 μg/mL and 100- 200 μg/mL for ibuprofen in human plasma respectively. The inter-day and intra-day precision in human plasma was found within limits. The proposed method has adequate sensitivity, reproducibility, and specificity for the determination of famotidine and ibuprofen in plasma. The LLOQ obtained by the proposed method in human plasma were 1.24 and 5.0 μg/mL for famotidine and ibuprofen respectively. The proposed method is simple, fast, accurate, and precise for the quantification of famotidine and ibuprofen in plasma as per the ICH guidelines.Kathmandu University Journal of Science, Engineering and TechnologyVol. 12, No. I, June, 2016, Page: 34-48

scholarly journals Validation of developed method by RP-HPLC for estimation of Prasugrelin human plasma and studying the stability of the drugs in plasma

2018 ◽  
Vol 13 (1) ◽  
pp. 65-75
Author(s):  
Kumar S. Ashutosh ◽  
Debnath Manidipa ◽  
Rao J.V.L.N. Seshagiri ◽  
Sankar D. Gowri
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Method Development ◽  
Potassium Dihydrogen Phosphate ◽  
Array Detector ◽  
Dihydrogen Phosphate ◽  
Limit Of Quantification ◽  
Rp Hplc ◽  
Relative Standard ◽  
The Stability

This paper is concern with a reverse phase high performance liquid chromatography (RP-HPLC) bio-analytical method development and validation for Prasugrel in human plasma using photo diode array detector (PDA detector). The HPLC separation was carried out in an isocratic mode on an X-Terra C18 column (4.6 x 150 mm; 5 μm) with a mobile phase consisting of potassium dihydrogen phosphate [pH 3.0] and acetonitrile in the ratio of 30:70 v/v at a flow rate of 1.0 mL/min. The run time was maintained for 5 mins and the detection was monitored at 210 nm. The percentage recovery was found 99.61-100.06 in human plasma. This reveals that the method is quite accurate. The linearity was found 15-40 μg/mL in human plasma. The inter-day and intra-day precision in plasma was found within the limits. The lower limit of quantification (LLOQ) obtained by the proposed method was 0.05 μg/mL. The percentage relative standard deviation (%RSD) obtained for the drug spiked in plasma for stability studies were less than 2 %.Kathmandu University Journal of Science, Engineering and TechnologyVol. 13, No. 1, 2017, Page: 65-75

Simultaneous determination of novel ketolide antibiotic nafithromycin and its major metabolite in human plasma using liquid chromatography tandem mass spectrometry

Bioanalysis ◽  
2019 ◽  
Vol 11 (19) ◽  
pp. 1767-1776
Author(s):  
Kiran R Patil ◽  
Ravindra D Yeole ◽  
Marcel de Zwart ◽  
Peter Pruim
Keyword(s):  
Phase I ◽  
Human Plasma ◽  
Internal Standard ◽  
Pharmacokinetic Studies ◽  
Simultaneous Quantification ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Us Fda ◽  
Standard Protein

Aim: A sensitive method to quantify nafithromycin and its N-desmethyl metabolite in human plasma was necessary for Phase I pharmacokinetic studies. Methodology: A precise and accurate LC–MS/MS bioanalytical method has been developed and validated for the simultaneous quantification of nafithromycin (NFT, WCK 4873) and N-desmethyl metabolite (M1, WCK 4978) in human plasma. Clarithromycin was used as an internal standard. Protein precipitation technique was used as sample preparation approach. The calibration curve was linear (r ≥ 0.99) over the concentration range of 10–5000 ng/ml for NFT and M1. Method was validated as per US FDA guideline. Conclusion: The proposed method was successfully applied for determination of plasma levels of the NFT and M1 during Phase I clinical studies.

Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt

2019 ◽  
Vol 57 (7) ◽  
pp. 636-643 ◽  
Author(s):  
Aya A Youssef ◽  
N Magdy ◽  
Lobna A Hussein ◽  
A M El-Kosasy
Keyword(s):  
Human Plasma ◽  
Method Validation ◽  
Treatment Protocol ◽  
Internal Standard ◽  
Gradient Elution ◽  
Hplc Method ◽  
Array Detector ◽  
Therapeutic Drug ◽  
Bioanalytical Method Validation ◽  
Rp Hplc

Abstract Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid–liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 μm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5–80, 0.1–40 and 0.5–80 μg/mL) with average recoveries (100.64–108.28%, 98.48–105.91% and 97.68–101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients’ follow-up especially in Egypt and other developing countries fighting HCV.

scholarly journals DEVELOPMENT AND VALIDATION OF LC-MS/MS METHOD FOR ESTIMATION OF UROCARB IN HUMAN PLASMA

2019 ◽  
pp. 125-130
Author(s):  
IRYNA DRAPAK ◽  
BORYS ZIMENKOVSKY ◽  
LINA PEREKHODA ◽  
SERGIY KOVALENKO ◽  
LILIYA LOGOYDA
Keyword(s):  
Formic Acid ◽  
Human Plasma ◽  
Internal Standard ◽  
Sample Volume ◽  
Pharmacokinetic Studies ◽  
Coefficients Of Variation ◽  
Highly Sensitive ◽  
Regulatory Guidelines ◽  
Initial Content ◽  
Development And Validation

Objective: The present study was aimed to develop a rapid, specific and sensitive method based on LC-MS/MS method was developed for the determination of urocarb using etomidate as an internal standard. Methods: Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile-water–formic acid, 5: 95: 0.1 v/v), eluent B (acetonitrile–formic acid, 100: 0.1 v/v)). The initial content of the eluent B of 8%, which increases linearly to 1.0 min to 100%, is maintained up to 1.5 min and returned to the original 8% to 1.51 min. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 4 μl. Results: The total chromatographic run time was 2.0 min and the elution of urocarb and IS (etomidate) occurred at ~1.53 and 1.67 min, respectively. A linear response function was established at 1-100 ng/ml for urocarb and etomidate in human plasma. The % mean recovery for urocarb in LQC, MQC and HQC was 104.1 %, 100.0 % and 97.4 %. The lowest concentration with the RSD<20% was taken as LLOQ and was found to be 1.03 ng/ml for urocarb. The within-run coefficients of variation ranged between 0.271 % and 0.478 % for urocarb. The within-run percentages of nominal concentrations ranged between 99.12 % and 100.21 % for urocarb. The between-run coefficients of variation ranged between 0.388 % and 0.601 % for urocarb. The between-run percentages of nominal concentrations ranged between 98.78 % and 101.11 % for urocarb. Conclusion: A highly sensitive, specific, reproducible, rapid and high-throughput LC-MS/MS assay was developed and validated to quantify urocarb in human plasma as per the regulatory guidelines. Due to the sensitivity of the developed method, it can be applied to the monitoring of plasma levels in the analysis of drug in preclinical and clinical pharmacokinetic studies. All the parameters and results were found within the acceptance limit as given in the validation protocol.

scholarly journals ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF AMLODIPINE IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY/MASS SPECTROMETRY

2018 ◽  
Vol 11 (7) ◽  
pp. 393
Author(s):  
Akiful Haque M ◽  
Shanthi Priya D K ◽  
Dibyalochan Mohanty ◽  
Vasudha Bakshi ◽  
Narender Boggula
Keyword(s):  
Mass Spectrometry ◽  
Human Plasma ◽  
Analytical Method ◽  
High Performance ◽  
Method Development ◽  
Solid Phase ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Economic Method ◽  
Positive Ion

Objective: The objective of the present investigation was to develop a novel, simple, and economic method for the estimation of amlodipine in positive ion mode in human plasma using amlodipine maleate d4 as an internal standard.Methods: The chromatographic separation was performed on Zorbax SB, C18, 50 mm*4.6 mm, and 3.5 mm. The mobile phase was prepared with a mixture of 5 mm ammonium acetate in 0.1% formic acid: High performance liquid chromatographic (HPLC) grade methanol:HPLC grade acetonitrile (40:30:30) that run isocratically at the flow rate of 0.700 ml/min and run time at 2.50 min.Results: The analytical method is valid for the estimation of amlodipine, in human plasma over a range of 0.100 ng/ml–9.990 ng/ml with the detection of amlodipine m/z - 409.10 (parent) and 238.00 (product), and internal standard Amlodipine Maleate d4 m/z - 413.20 (parent), and 238.00 (product) in positive ion mode. The results of carryover test, matrix effect, linearity, precision and accuracy, stabilities, dilution integrity, and run size evaluation test presented in this report are within the acceptance range.Conclusion: A sensitive method for the separation and determination of amlodipine in plasma has been developed based on solid-phase extraction with disposable extraction cartridges in combination with LC and mass spectrophotometers (MS/MS).

scholarly journals Ultra-performance liquid chromatography–tandem mass spectrometric determination of ramipril in human plasma

2020 ◽  
Vol 19 (4) ◽  
pp. 851-857
Author(s):  
Sherif A. Abdel-Gawad
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
Internal Standard ◽  
The United States ◽  
Ultra Performance Liquid Chromatography ◽  
Mass Spectrometric ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Liquid Chromatography Tandem Mass ◽  
Tandem Mass Spectrometric

Purpose: To develop a sensitive and accurate ultra-performance liquid chromatography–tandem mass spectrometric (UPLC-MS) method for quantification of ramipril in human plasma.Methods: Ramipril was extracted from biological fluid using equal volumes of n-hexane and propanol (1:1, v/v), and then chromatographed in a suitable C18 column with methanol: 0.1 % HCOOH (4: 1, v/v) as mobile phase. Atorvastatin was used as an internal standard for the  chromatographic separation and quantification. The method was validated according to the United States Food and Drug Administration guidelines for standard indices.Results: Ramipril was determined in the concentration range 0.05 and 1000 ng/mL the validation procedure exhibited a correlation coefficient of 0.9979 + 0.002 (p = 0.05). The studied drug was quantified with lower ceiling of 0.05 ng/mL, and showed an accuracy of 105.00 %.Conclusion: A sensitive UPLC-MS analytical method has been successfully developed for the quantification of ramipril in human plasma. This method can be applied efficiently for the quantification of ramipril in bioavailability and pharmacokinetic studies. Keywords: Liquid chromatography–tandem mass, Ramipril, Stability, Biological fluids, Plasma

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