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

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.

Download Full-text

LC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF CARDIAZOL IN HUMAN PLASMA

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2019v11i4.33482 ◽

2019 ◽

pp. 380-385

Author(s):

IRYNA DRAPAK ◽

BORYS ZIMENKOVSKY ◽

LINA PEREKHODA ◽

SERGIY KOVALENKO ◽

Liliya Logoyda

Keyword(s):

Formic Acid ◽

Human Plasma ◽

Method Development ◽

Accurate Method ◽

Sample Volume ◽

Ich Guidelines ◽

Coefficients Of Variation ◽

Method Development And Validation ◽

Development And Validation

Objective: The main purpose of this study was to develop a simple, precise, rapid and accurate method for the quantification of cardiazol in human plasma. 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 300 μl. Results: The total chromatographic run time was 2.5 min and the elution of cardiazol and IS (difenoconazole) occurred at ~2.15 and 1.98 min, respectively. A linear response function was established at 1-100 ng/ml for cardiazol and difenoconazole in human plasma. The % mean recovery for cardiazol in LQC, MQC and HQC was 102.8 %, 100.3 % and 95.9 %. The lowest concentration with the RSD<20% was taken as LLOQ and was found to be 1.10 ng/ml for cardiazol. The % accuracy of LLOQ samples prepared with the different biological matrix lots was found 109.7 %, which were found within the range of 80.00-120.00 % for the seven different plasma lots. % CV for LLOQ samples was observed as 11.9 %, which are within 20.00% of the acceptance criteria. The within-run coefficients of variation ranged between 0.311 % and 0.601 % for cardiazol. The within-run percentages of nominal concentrations ranged between 99.80 % and 100.41 % for cardiazol. The between-run coefficients of variation ranged between 0.332 % and 0.615 % for cardiazol. The between-run percentages of nominal concentrations ranged between 98.18 % and 101.21 % for cardiazol. Conclusion: A rapid method was developed for simultaneous determination of cardiazol in human plasma. The method was strictly validated according to the ICH guidelines. Acquired results demonstrate that the proposed strategy can be effortlessly and advantageously applied for routine examination of cardiazol in human plasma.

Download Full-text

A HPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF BISOPROLOL AND ENALAPRIL IN THE PRESENT OF ENALAPRILAT IN HUMAN PLASMA

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2018v10i2.23195 ◽

2018 ◽

Vol 10 (2) ◽

pp. 31 ◽

Cited By ~ 8

Author(s):

Liliya Logoyda ◽

Dmytro Korobko ◽

Oleksandra Oleshchuk ◽

Taras Proniv ◽

Mariya Dmutriv

Keyword(s):

Formic Acid ◽

Simultaneous Determination ◽

Human Plasma ◽

Method Development ◽

Sample Volume ◽

Ich Guidelines ◽

Method Development And Validation ◽

Initial Content ◽

Development And Validation

Objective: A highly specific, sensitive and rapid HPLC-MS/MS method has been developed and validated for the simultaneous quantification of bisoprolol and enalapril in the present of enalaprilat in human plasma.Methods: Analytes were extracted from plasma using a protein precipitation extraction method. 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 is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: The total chromatographic run time was 2.0 min and the elution of bisoprolol, enalapril, enalaprilat and IS (verapamil) occurred at ~1.01, 1.03, 0.96 and 1.09 min, respectively. A linear response function was established at 0.5-50 ng/ml for bisoprolol fumarate, 2-200 ng/ml for enalapril maleate, 1-100 ng/ml for enalaprilat dehydrate in human plasma. The intraday and interday accuracy and precisions were in the range of 0.311 %-0.647 % and 0.364 %-0.572 % for bisoprolol, 0.321 %-0.747 % and 0.390 %-0.673 % for enalapril, 0.221 %-0.547 % and 0.264 %-0.773 % for enalaprilat, respectively.Conclusion: A new rapid method was developed for simultaneous determination of bisoprolol and enalapril in the present of enalaprilat in human plasma. The method was strictly validated according to the ICH guidelines. The information thus obtained from the study can be used for the full pharmacokinetic profiling in individuals.

Download Full-text

Development and validation of bexarote by bioanalytical methods using liquid chromatography-tandem mass spectroscopy (LC-MS/MS)

Future Journal of Pharmaceutical Sciences ◽

10.1186/s43094-020-00155-6 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

K. Nagaraju ◽

Y. A. Chowdary ◽

M. V. Basaveswara Rao

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Human Plasma ◽

Internal Standard ◽

Pharmacokinetic Studies ◽

Tandem Mass ◽

System Suitability ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectroscopy ◽

Development And Validation

Abstract Background The aim of this study was to develop and validate accurate and precise UPLC method with tandem mass spectrometry (Waters) for the determination of bexarotene in human plasma using bexarotene D4 as internal standard (IS). Results The retention time of bexarotene was 2.75 ± 0.30 min. The method was validated with respect to system suitability, linearity, accuracy, precision, matrix effect, auto sampler carryover test, and recovery. Linearity was found to be 1.04 to 351.93 μg/mL. LOQQC, LQC, INTQC, MQC, and HQC were found to be 1.0550, 2.7800, 25.2700, 131.61, and 263.23 respectively. The mean percentage recovery was found to be 95.72% Conclusion The bioanalytical method, a selective and sensitive liquid chromatography-mass spectrometry method to quantitate bexarotene in K2EDTA human plasma over the concentration range 1.0440 to 351.9320 ng/mL, was successfully validated. This method is suitable for sample analysis to support bioequivalence/bioavailability and/or pharmacokinetic studies involving formulations of bexarotene.

Download Full-text

Simultaneous Determination of Candesartan and Hydrochlorothiazide in Human Plasma by HPLC-MS/MS

Drug development & registration ◽

10.33380/2305-2066-2021-10-4-177-189 ◽

2021 ◽

Vol 10 (4) ◽

pp. 177-189

Author(s):

P. K. Karnakova ◽

T. N. Komarov ◽

O. A. Archakova ◽

D. S. Shchelgacheva ◽

A. A. Aleshina ◽

...

Keyword(s):

Formic Acid ◽

Simultaneous Determination ◽

Human Plasma ◽

Internal Standard ◽

Human Blood Plasma ◽

Pharmacokinetic Studies ◽

Mixed Solution ◽

Ionization Source ◽

Analytical Range

Introduction. Combined drugs have the greatest efficacy and safety in arterial hypertension treatment. The combination of candesartan and hydrochlorothiazide (AT1-receptor antagonist and a thiazide diuretic, respectively) provides high efficiency of antihypertensive combination therapy, therefore it is widely used in medical practice. Developing a method for simultaneous determination of candesartan and hydrochlorithiazide in human blood plasma is necessary for performing the analytical part of pharmacokinetic studies and bioequivalence studies of multicomponent drugs.Aim. The aim of this study is to develop a method for quantitative determination of candesartan and hydrochlorothiazide in human plasma by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) for further bioequivalence studies.Materials and methods. Determination of candesartan and hydrochlorothiazide in human plasma by HPLC-MS/MS. The samples were processed by acetonitrile protein precipitation. Internal standard: mixed solution of valsartan and indapamide. Mobile phase: 0.1 % formic acid solution in water (eluent A), 0.1 % formic acid in acetonitrile (eluent B). Column: Phenomenex Luna Phenyl-Hexyl, 50x4.6 mm, 5 μm. Analytical range: 2.00– 300.00 ng/mL for candesartan, 2.00–200.00 ng/mL for hydrochlorothiazide in human plasma. Ionization source: electrospray ionization. Detection conditions: 441.10 → 192.00 m/z, 441.10 → 263.15 m/z (candesartan), 295.85 → 269.00 m/z (hydrochlorothiazide), 436.00 → 207.05 m/z (valsartan), 363.85 → 132.10, 363.85 → 189.00 m/z (indapamide).Results and discussion. This method was validated by selectivity, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability. The developed method meets the requirements for conducting bioequivalence studies of medicinal products within the framework of the Eurasian Economic Union.Conclusion. The analytical range was 2.00–300.00 ng/mL for candesartan, 2.00–200.00 ng/mL for hydrochlorothiazide in human plasma. The method was applied in BE study of the combination of candesartan and hydrochlorothiazide.

Download Full-text

A HPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF NIFEDIPINE AND ENALAPRIL IN HUMAN PLASMA

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2018v10i4.24528 ◽

2018 ◽

Vol 10 (4) ◽

pp. 35 ◽

Cited By ~ 7

Author(s):

Liliya Logoyda

Keyword(s):

Formic Acid ◽

Simultaneous Determination ◽

Human Plasma ◽

Method Development ◽

Accurate Method ◽

Sample Volume ◽

Ich Guidelines ◽

Method Development And Validation ◽

Development And Validation

Objective: The main purpose of this study was to develop a simple, precise, rapid and accurate method for the simultaneous quantification of nifedipine and enalapril in human plasma.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 is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: The total chromatographic run time was 2.5 min and the elution of nifedipine, enalapril and IS (verapamil) occurred at ~1.83, 1.57 and 1.61 min, respectively. A linear response function was established at 1-100 ng/ml for nifedipine and 2-200 ng/ml for enalapril maleate in human plasma. The % mean recovery for enalapril in LQC, MQC and HQC was 114.0 %, 112.9 % and 113.2 %, for nifedipine in LQC, MQC and HQC was 104.1 %, 105.0 % and 108.7 % respectively. The lowest concentration with the RSD<20% was taken as LLOQ and was found to be 2.16 ng/ml for enalapril, 1.01 ng/ml for nifedipine. The % accuracy of LLOQ samples prepared with the different biological matrix lots was found 108.2 % for enalapril and 100.5 % for nifedipine, which were found within the range of 80.00-120.00 % for the seven different plasma lots. % CV for LLOQ samples was observed as 3.2 % and 7.4 % respectively, which are within 20.00% of the acceptance criteria.Conclusion: A rapid method was developed for simultaneous determination of nifedipine and enalapril in human plasma. The method was strictly validated according to the ICH guidelines. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for routine examination of nifedipine and enalapril in human plasma.

Download Full-text

Method Development and Validation of Anabasine and Nornicotine in Human Plasma by LC-MS/MS

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i1731301 ◽

2021 ◽

pp. 7-17

Author(s):

P. Rajagopaludu ◽

N. Saritha ◽

N. Devanna ◽

M. Srinivas

Keyword(s):

Human Plasma ◽

Method Development ◽

Internal Standard ◽

Charge Ratio ◽

Coefficients Of Variation ◽

Calculated Curve ◽

Average Recovery Rate ◽

Method Development And Validation ◽

Precision And Accuracy ◽

Development And Validation

Aim: For the Method development and validation of Anabasine and Nornicotine in human plasma LC-MS/MS technique is used. Methodology: Operated in positive electron spray ionization which removes the hardness in ions. Metoprolol is used as an internal standard. Mass analyzers are used to determine the mass to charge ratio. Liquid chromatography separates mixture components in the basis of differences in affinity for stationary and mobile phase. It removes undesired impurities. It increases reproducibility, sensitivity, robustness, detection of low-level proteins. C18 Column (Phenomenex Luna C18, 5 μm, 50*4.6mm ID) is used to for high resolution and peak area. Calibration curve is constructed with the help of linear regression. During the entire process no instability is found. Results: m/z values of Anabasine are 162.84(Q1), 134.68(Q3) and for Nornicotine are 148.90(Q1), 79.82(Q3). Back calculated curve is calculated at different concentrations from 1-1000ng/ml and their accuracy range from 97-105% for Anabasine and 93-108% for Nornicotine. In Anabasine and Nornicotine the precision and accuracy are calculated by Intra-batch and inter-batch. In the process of multi-step preparation, consider n=6 for intra-batch and n=18 for inter-batch the values obtained are between the nominal concentrations i.e., 90-110%. For each analyte the coefficients of variation are below nominal criteria <15%. For Anabasine and Nornicotine the average recovery rate is 98.3% and 94.3%.

Download Full-text

Liquid Chromatography-Tandem Mass Spectrometry Method for Ticagrelor and its Active Metabolite Determination in Human Plasma: Application to a Pharmacokinetic Study

Current Analytical Chemistry ◽

10.2174/1573411015666190220144904 ◽

2020 ◽

Vol 16 (5) ◽

pp. 602-608

Author(s):

Niloufar Marsousi ◽

Serge Rudaz ◽

Jules A. Desmeules ◽

Youssef Daali

Keyword(s):

Clinical Trial ◽

Formic Acid ◽

Human Plasma ◽

Active Metabolite ◽

Pharmacokinetic Parameters ◽

Pharmacokinetic Studies ◽

Healthy Male ◽

Coronary Syndrome ◽

Healthy Male Volunteers ◽

Male Volunteers

Background: Ticagrelor is a highly recommended new antiplatelet agent for the treatment of patients with acute coronary syndrome at moderate or high ischemic risk. There is a real need for rapid and accurate analytical methods for ticagrelor determination in biological fluids for pharmacokinetic studies. In this study, a sensitive and specific LC-MS method was developed and validated for quantification of ticagrelor and its Active Metabolite (AM) in human plasma over expected clinical concentrations. Methods: Samples were handled by Liquid-Liquid Extraction (LLE). A linear gradient was applied with a mobile phase composed of formic acid 0.1% and acetonitrile with 0.1% of formic acid using a C18 reversed-phase column. MS spectra were obtained by electrospray ionization in negative mode and optimized at 521.4→360.9 m/z, 477.2→361.2 m/z and 528.1→367.9 m/z transitions for ticagrelor, AM and ticagrelor-d7, respectively. Results: This method allowed rapid elution, in less than 4 minutes, and quantification of concentrations as low as 2 ng/mL for ticagrelor and 1 ng/mL for AM using only 100 μL of human plasma. LLE using hexane/ethyl acetate (50/50) was an optimal compromise in terms of extraction recovery and endogenous compounds interference. Trueness values of 87.8% and 89.5% and precisions of 84.1% and 93.8% were obtained for ticagrelor and AM, respectively. Finally, the usefulness of the method was assessed in a clinical trial where a single 180 mg ticagrelor was orally administered to healthy male volunteers. Pharmacokinetic parameters of ticagrelor and its active metabolite were successfully determined. Conclusion: A sensitive and specific quantification LC-MS-MS method was developed and validated for ticagrelor and its active metabolite determination in human plasma. The method was successfully applied to a clinical trial where a single ticagrelor 180 mg dose was orally administered to healthy male volunteers. The described method allows quantification of concentrations as low as 2 ng/mL of ticagrelor and 1 ng/mL of the metabolite using only 100 μL of plasma.

Download Full-text

A Novel Simultaneous Estimation of Sofosbuvir and Velpatasvir in Human Plasma by Liquid Chromatography Tandem-Mass Spectrometry after Protein Precipitation Method

Current Pharmaceutical Analysis ◽

10.2174/1573412914666180910102353 ◽

2019 ◽

Vol 15 (7) ◽

pp. 710-715

Author(s):

S.T. Narenderan ◽

Basuvan Babu ◽

T. Gokul ◽

Subramania Nainar Meyyanathan

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Multiple Reaction Monitoring ◽

Simultaneous Estimation ◽

Pharmacokinetic Studies ◽

Tandem Mass ◽

Mass Spectrometric Method ◽

Highly Sensitive ◽

Tandem Mass Spectrometric

Objective: The aim of the present work is to achieve a novel highly sensitive chromatographic method for the simultaneous determination of hepatitis C agents, sofosbuvir and velpatasvir from human plasma using ritonavir as an internal standard. Methods: Chromatographic separation was achieved using Hypersil C18 column (50mm x 4.6mm, 3μm) with an isocratic elution mode using the mobile phase composition 10 mM ammonium formate buffer (pH 5.0): acetonitrile (20:80 v/v) pumped at a flow rate of 0.5 ml/min. The detection was carried out by tandem mass spectrometry using Multiple Reaction Monitoring (MRM) positive Electrospray Ionization (ESI) with proton adducts at m/z 530.10 > 243.10, 883.40 > 114.0 and 721.25 > 197.0. Results: The method validated as per USFDA guidelines with respect to linearity, accuracy, and precision was found to be acceptable over the concentration range of 0.2–2000 ng/ml and 5-2000 ng/ml for sofosbuvir and velpatasvir respectively and the method was found to be highly sensitive and selective. Conclusion: The developed tandem mass spectrometric method is robust and can be applied for the monitoring of plasma levels of the analyzed drug in preclinical and clinical pharmacokinetic studies.

Download Full-text