A rapid, simple, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid-liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C(18) column by pumping 0.1% formic acid-acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26-210 ng/mL for ATO; 0.05-20.5 ng/mL for AML; 0.25-208 ng/mL for RAM and 0.74-607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra-day and inter-day precision and accuracy results were well with in the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.
Introduction:A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric assay method has been developed and fully validated for simultaneous quantification of losartan and its active metabolite, losartan carboxylic acid, and amlodipine in human plasma. Irbesartan was used as an internal standard.Materials and Methods:The analytes were extracted from human plasma samples by solid-phase extraction technique using Oasis HLB cartridges, (Waters Corporation, Mumbai, India). The reconstituted samples were chromatographed on a C18 column by using an 85:15, v/v mixture of methanol and 0.1% v/v formic acid as the mobile phase at a flow rate of 1.0 mL/min. A detailed validation of the method was performed as per the FDA guidelines.Results:The calibration curves obtained were linear (r ≥ 0.99) over the concentration range of 0.5-1000 ng/mL for losartan and for its active metabolite losartan acid and 0.05-10.1 ng/mL for amlodipine. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits.Conclusions:A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.
A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of donepezil and its active metabolite, 6-o-desmethyl donepezil in human plasma. Analytes and the internal standard were extracted from human plasma by liquid-liquid extraction technique using a 30:70 v/v mixture of ethyl acetate and n-hexane. The reconstituted samples were chromatographed on a C(18) column by using a 70:30 v/v mixture of acetonitrile and ammonium formate (5 mm, pH 5.0) as the mobile phase at a flow rate of 0.6 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.09-24.2 ng/mL for donepezil and 0.03-8.13 ng/mL for 6-o-desmethyl donepezil. The results of the intra-day and inter-day precision and accuracy studies were well within the acceptable limits. The proposed method was successfully applied for the estimation of the drug in real time plasma samples for pharmacokinetic studies.
A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of pioglitazone and candesartan in human plasma. Irbesartan was used as an internal standard. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 μm polymeric sorbent. The reconstituted samples were chromatographed on a C18 column by using a 80:20 (v/v) mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min. The calibration curves obtained were linear (r≥0.99) over the concentration range of 15–3000 ng/mL for pioglitazone and 5–608 ng/mL for candesartan. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.7 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.
A simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of angiotensin-converting enzyme inhibitor, moexipril, in human plasma. Benazepril was used as an internal standard (IS). Analyte and IS were extracted from the human plasma by liquid-liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on a C₁₈ column by using a mixture of methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.2-204 ng/mL. The multiple reaction-monitoring mode was used for quantification of ion transitions at m/z 499.4/234.2 and 425.2/351.1 for moexipril and IS, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 400 plasma samples per day. The proposed method was found to be applicable to clinical studies.
Metformin has been shown to repress transcription of the bile salt export pump (BSEP) in human primary hepatocytes. The primary objective of this study was to assess the effect of oral metformin on the human pharmacokinetics (PKs) of two BSEP probe substrates: pravastatin and chenodeoxycholic acid (CDCA; also known as chenodiol). Endogenous bile acid levels were assessed as a secondary measure of metformin impact. An open‐label, randomized, single‐dose, placebo‐controlled, fasted, crossover PK study was conducted in 12 healthy adult volunteers. Metformin (500 mg b.i.d.) or placebo (b.i.d.) was administered orally for 6 days. On day 7, a single dose of the BSEP substrates pravastatin (80 mg) and CDCA (250 mg) were administered orally. Plasma samples were quantified for pravastatin, CDCA, and endogenous bile acids. Compared to placebo, metformin increased pravastatin plasma exposure, did not impact CDCA plasma exposure, and reduced conjugated primary bile acid levels in the blood. These results are consistent with metformin repressing BSEP expression. This differential effect reflects the degree of enterohepatic recirculation of victim substrates.
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