A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive LC‐MS/MS method for determination of galantamine in rat plasma: application to pharmacokinetic studies in rats

A rapid and highly sensitive assay method has been developed and validated for the estimation of galantamine (GLM) in rat plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of GLM and phenacetin (internal standard, IS) from rat plasma using acetonitrile. Chromatographic separation was achieved with 0.2% formic acid:acetonitrile (50:50, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 2.5 min. The MS/MS ion transitions monitored were 288.10 → 213.10 for GLM and 180.10 → 110.10 for IS. Method validation was performed as per United States Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.12 ng/mL and linearity was observed from 0.12 to 525 ng/mL. The intra‐ and inter‐day precision were in the ranges of 4.73–11.7 and 5.83–8.64%, respectively. This novel method has been applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS‐ESI method for the determination of bicalutamide in mouse plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of bicalutamide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves extraction of bicalutamide and tolbutamide (internal standard, IS) from mouse plasma with a simple protein precipitation method. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid:acetonitrile, 35:65, v/v) at a flow rate of 0.5 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were m/z 428.9 → 254.7 for bicalutamide and m/z 269.0 → 169.6 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 1.04 ng/mL and the linearity range extended from 1.04 to 1877 ng/mL. The intra‐ and inter‐day precisions were in the ranges of 0.49–4.68 and 2.62–4.15, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of sitagliptin and simvastatin in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and validated for simultaneous quantification of sitagliptin and simvastatin in human plasma. Carbamazepine was used as an internal standard (IS). The analytes and IS were extracted from the human plasma by liquid–liquid extraction technique. The reconstituted samples were chromatographed on an Alltima HP C₁₈ column using an isocratic solvent mixture [acetonitrile–5 mm ammonium acetate (pH 4.5), 85:15 (v/v)] at a flow rate of 1.0 mL/min. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.10–501 and 0.05–105 ng/mL for sitagliptin and simvastatin, respectively. The results of the intra‐ and inter‐day precision and accuracy studies were well within the acceptable limits. Both the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay was successfully applied to a pharmacokinetic study in human volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Highly sensitive method for the determination of ropinirole with a lower limit of quantitation of 3.45 pg/mL in human plasma by LC‐ESI‐MS/MS: application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of ropinirole (RPR) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. A solid‐phase process was used to extract RPR and citalopram (internal standard, IS) from human plasma. Chromatographic separation was operated with 0.2% ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Hypurity C₁₈ column with a total run time of 3.2 min. The MS/MS ion transitions monitored were 261.2 → 114.2 for RPR and 325.1 → 209.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 3.45 pg/mL and the linearity was observed from 3.45 to 1200 pg/mL. The intra‐day and inter‐day precisions were in the range of 4.71–7.98 and 6.56–8.31%, respectively. This novel method has been applied to a pharmacokinetic study of RPR in humans. Copyright © 2008 John Wiley & Sons, Ltd.     

Simultaneous determination of atorvastatin,amlodipine, ramipril and benazepril in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

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 C₁₈ 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. Copyright © 2010 John Wiley & Sons, Ltd.     

A highly sensitive LC-MS/MS method for the determination of S-raclopride in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and rapid assay method has been developed and validated for the estimation of S‐(−)‐raclopride (S‐RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid–liquid extraction technique for extraction of S‐RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C₁₈ column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S‐RCP and 180.1 → 110.1 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra‐day and inter‐day precisions were 0.23–10.5 and 3.74–7.29%, respectively. This novel method was applied to a pharmacokinetic study of S‐RCP in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation and clinical application of an LC‐ESI‐MS/MS method for simultaneous determination of tolmetin and MED5, the metabolites of amtolmetin guacil in human plasma

A highly sensitive, rapid assay method has been developed and validated for the simultaneous estimation of tolmetin (TMT) and MED5 in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. A simple solid‐phase extraction process was used to extract TMT and MED5 along with mycophenolic acid (internal standard, IS) from human plasma. Chromatographic separation was achieved with 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.50 mL/min on an X‐Terra RP₁₈ column with a total run time of 2.5 min. The MS/MS ion transitions monitored were 258.1 → 119.0 for TMT, 315.1 → 119.0 for MED5 and 321.2 → 207.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 20 ng/mL and the linearity was observed from 20 to 2000 ng/mL, for both the anlaytes. The intra‐day and inter‐day precisions were in the range 3.27–4.50 and 5.32–8.18%, respectively for TMT and 4.27–5.68 and 5.32–8.85%, respectively for MED5. This novel method has been applied to a clinical pharmacokinetic study. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for quantitation of indomethacin in rat plasma and its application to a pharmacokinetic study in rats

A highly sensitive and rapid bioanalytical method has been developed and validated for the estimation of indomethacin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of indomethacin and phenacetin (internal standard, IS) from rat plasma with acetonitrile. Chromatographic separation was achieved with 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 3.0 min. The MS/MS ion transitions monitored were 357.7 → 139.1 for indomethacin and 180.20 → 110.10 for IS. Method validation and pharmacokinetic study plasma analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.51 ng/mL and the linearity was observed from 0.51 to 25.5 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.00–10.2 and 5.88–9.80%, respectively. This novel method has been applied to an oral pharmacokinetic study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC‐MS/MS‐ESI method for the determination of ivabradine in human plasma: application to a pharmacokinetic study

A sensitive, rapid assay method for estimating ivabradine in human plasma has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The procedure involved extraction of ivabradine and the internal standard (IS) from human plasma by solid‐phase extraction. Chromatographic separation was achieved using an isocratic mobile phase (0.1% formic acid–methanol, 60:40, v/v) at a flow rate of 1.0 mL/min on an Aglient Eclipse XDB C₈ column (150 × 4.6 mm, 5 µm; maintained at 35°C) with a total run time of 4.5 min. Detection was achieved using an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 3200 triple‐quadrupole mass spectrometer. The MS/MS ion transitions monitored were 469–177 for ivabradine and 453–177 for IS. Method validation was performed according to Food and Drug Administration guidelines, and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 0.1–200 ng/mL. The lower limit of quantitation achieved was 0.1 ng/mL. Intra‐ and inter‐day precisions were in the range of 1.23–14.17% and 5.26‐8.96%, respectively. Finally, the method was successfully used in a pharmacokinetic study that measured ivabradine levels in healthy volunteers after a single 5 mg oral dose of ivabradine. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐ESI‐MS/MS determination of 4‐methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs

A simple, specific, sensitive and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of 4‐methylpyrazole in dog plasma using N‐methylnicotinamide‐d₄ as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4‐methylpyrazole and the IS was performed on a monolithic (Chromolith RP_18e) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4‐methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96–4955 ng/mL. The intra‐ and inter‐day accuracy and precision were in the ranges 1.81–12.9 and 3.80–11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs.     

Validated LC‐ESI‐MS/MS method for simultaneous quantitation of felodipine and metoprolol in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and specific LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of felodipine (FDP) and metoprolol (MPL) in rat plasma (50 μL) using phenacetin as an internal standard (IS) as per the FDA guidelines. Liquid–liquid extraction method was used to extract the analytes and IS from rat plasma. The chromatographic resolution of FDP, MPL and IS was achieved with a mobile phase consisting of 0.2% formic acid in water–acetonitrile (25:75, v/v) with a time program flow gradient on a C₁₈ column. The total chromatographic run time was 4.0 min and the elution of FDP, MPL and IS occurred at 1.05, 2.59 and 1.65 min, respectively. A linear response function was established for the range of concentrations 0.59–1148 and 0.53–991 ng/mL for FDP and MPL, respectively, in rat plasma. The intra‐ and inter‐day accuracy and precision values for FDP and MPL met the acceptance as per FDA guidelines. FDP and MPL were stable in battery of stability studies viz., bench‐top, auto‐sampler and freeze–thaw cycles. The validated assay was applied to a pharmacokinetic study in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.