Determination of a novel paclitaxel derivative (NPD‐103) in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry

A sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS‐MS) method for quantification of a newly developed anticancer agent NPD‐103 has been established. An aliquot of human plasma sample (200 µL) was spiked with ¹³C‐labeled paclitaxel (internal standard) and extracted with 1.3 mL of tert‐butyl methyl ether. NPD‐103 was quantitated on a C₁₈ column with methanol–0.1% formic acid (75:25, v/v) as mobile phase using UPLC‐MS‐MS operating in positive electrospray ionization mode with a total run time of 3.0 min. For NPD‐103 at the concentrations of 1.0, 5.0 and 10.0 µg/mL in human plasma, the absolute extraction recoveries were 95.58, 102.43 and 97.77%, respectively. The linear quantification range of the method was 0.1–20.0 µg/mL in human plasma with linear correlation coefficients greater than 0.999. The intra‐ and inter‐day accuracy for NPD‐103 at 1.0, 5.0 and 10.0 µg/mL levels in human plasma fell into the ranges of 95.29–100.00% and 91.04–94.21%, and the intra‐ and inter‐day precisions were in the ranges of 8.96–11.79% and 7.25–10.63%, respectively. This assay is applied to determination of half‐life of NPD‐103 in human plasma. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of pinaverium bromide in human plasma: application to a pharmacokinetic study in healthy volunteers

A sensitive and specific method using liquid chromatography–electrospray tandem mass spectrometry (LC‐MS/MS) for the determination of pinaverium bromide in human plasma was developed and validated. Pinaverium bromide and an internal standard (paclitaxel) were isolated from plasma samples by precipitating plasma, and determined by LC‐MS/MS in multiple‐reaction monitoring mode. The main metabolite of pinaverium bromide and endogenous substances in plasma did not show any interference. The calibration curve was linear over the plasma concentration range of 10.0–10000.0 pg/mL with a correlation coefficient of 0.9979. The relative standard derivations intra‐ and inter‐day at 30.0, 300.0 and 8000.0 pg/mL in plasma were less than 15%. The absolute recoveries of pinaverium bromide and the internal standard were 99.7–111.7 and 106.2%, respectively. The lower limit of quantitation was 10 pg/mL. The analytical method was successfully applied to study the pharmacokinetics of pinaverium bromide tablets in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of penciclovir in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the determination of penciclovir in human plasma. The method involved simple, one‐step SPE procedure coupled with a C₁₈, 75 × 4.mm, 3µm column with a flow‐rate of 0.5 mL/min, and acyclovir was used as the internal standard. The Quattro Micro mass spectrometry was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. Using 250 µL plasma, the methods were validated over the concentration range 52.555–6626.181 ng/mL, with a lower limit of quantification of 52.55 ng/mL. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a clinical pharmacokinetic study in human volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) was developed. This assay represents the first LC‐MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3‐atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/mL and 10 nm for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3–900 ng/mL and 10 nm to 10 µm for human plasma and cellular samples, respectively (r² > 0.999). The intra‐ and inter‐day assay accuracy and precision were evaluated using quality control samples at three different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect and recovery were also successfully demonstrated. The present assay is superior to previously published LC‐MS and LC‐MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of amitriptyline and its metabolite in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study

A method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) in combination with solid‐phase extraction for sample pretreatment has been developed for the simultaneous analysis of amitriptyline and its main metabolite in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective SPE procedure using hydrophilic–lipophilic balance cartridges. The assay involves a simple solid‐phase extraction (SPE) procedure of 0.2 mL of human plasma and analysis was performed on a triple‐quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode via electrospray ionization (ESI). The standard calibration curve was linear over the ranges 0.370–95.539 ng/mL for amitriptyline and 0.365–94.374 ng/mL for nortriptyline, expressed by the linear correlation coefficient r², which was better than 0.995 for both. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recovery was 85.3, 88.4 and 80.7% for amitriptyline, nortriptyline and doxepin respectively. Total run time was 1.2 min only for each sample, which makes it possible to analyze more than 400 samples per day. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2010 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.     

Liquid chromatographic/mass spectrometry assay of bromotetrandrine in rat plasma and its application to pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric method (LC‐MS/MS) for the determination of bromotetrandrine in rat plasma has been developed and applied to pharmacokinetic study in Sprague–Dawley (SD) rats after a single oral administration. Sample preparation involves a liquid–liquid extraction with n‐hexane–dichlormethane (65:35, containing 1% 2‐propanol isopropyl alcohol, v/v). Bromotetrandrine and brodimoprim (internal standard, IS) were well separated by LC with a Dikma C₁₈ column using methanol–ammonium formate aqueous solution (20 mm ) containing 0.5% formic acid (60:40, v/v) as mobile phase. Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 703.0 → 461.0 and m/z 339.0 → 281.0 for bromotetrandrine and IS, respectively. The present method exhibited good linearity over the concentration range of 20–5000 ng/mL for bromotetrandrine in rat plasma with a lower limit of quantification of 20 ng/mL. The intra‐ and inter‐day precisions were 2.8–7.5% and 3.2–8.1%, and the intra‐ and inter‐day accuracy ranged from ?4.8 to 8.2% and ?5.6 to 6.2%, respectively. The method was successfully applied to a pharmacokinetic study after a single oral administration to SD rats with bromotetrandrine of 50 mg/kg. Copyright © 2009 John Wiley & Sons, Ltd.     

Solid‐phase extraction and analysis of paroxetine in human plasma by ultra performance liquid chromatography–electrospray ionization mass spectrometry

A rapid, sensitive and rugged solid‐phase extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed for determination of paroxetine in human plasma. The procedure for sample preparation includes simple SPE extraction procedure coupled with Hypersil Gold C₁₈ column (100 mm ? 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow‐rate of 0.350 mL/min and fluoxetine was used as the internal standard. The analysis was performed on a triple‐quadrupole tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization. Using 500 μL plasma, the methods were validated over the concentration range 0.050–16.710 ng/mL for paroxetine, with a lower limit of quantification of 0.050 ng/mL. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recovery was 69.2 and 74.4% for paroxetine and fluoxetine respectively. Total run time was only 1.9 min. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated LC‐MS/MS assay for the quantitative determination of vardenafil in human plasma and its application to a pharmacokinetic study

A sensitive high‐performance liquid chromatography–tandem mass spectrometric (HPLC‐MS/MS) assay has been developed for the quantitative analysis of vardenafil in human plasma. Vardenafil and the internal standard, alprazolam, were extracted from 0.2 mL aliquots of alkalinized plasma by a single solvent extraction into hexane : dichloromethane. Reversed‐phase chromatographic separation was affected by gradient elution with mobile phases consisting of 10 mM ammonium formate pH 7.0 (solvent A) and methanol (100%, solvent B), delivered at a flow rate of 0.4 mL/min. The analytes were detected by using an electrospray ion source on a 4000 QTrap triple quadrupole mass spectrometer operating in positive ionization mode. The mass transitions were m/z 489.3 → 312.2 for vardenafil and m/z 309.2 → 281.0 for alprazolam. The assay was linear over the concentration range of 0.2–100 ng/mL, with correlation coefficients ≥0.995. The intra‐ and inter‐day precision was less than 5.4% in terms of relative standard deviation and the accuracy was within 12.7% in terms of relative error. The lower limit of quantitation was set at 0.2 ng/mL. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of plasma samples obtained following the oral administration of vardenafil to healthy male volunteers in a pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC‐ ESI‐ MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin‐d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid–liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin‐d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis‐RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025–753.217 ng/mL for acitretin and 0.394–289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra‐day and inter‐day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid and sensitive LC-MS/MS method for quantification of lamotrigine in human plasma: application to a human pharmacokinetic study

A highly sensitive, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lamotrigine (LAM) with 100 μL of human plasma using flucanozole as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using electrospray ionization. A simple liquid–liquid extraction process was used to extract LAM and IS from human plasma. The total run time was 2.0 min and the elution of LAM and IS occurred at 1.25 and 1.45 min; this was achieved with a mobile phase consisting of 0.1% formic acid–methanol (20:40:40, v/v) at a flow rate of 0.50 mL/min on a Discovery CN (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for LAM. A linear response function was established for the range of concentrations 0.1–1500 ng/mL (r > 0.998) for LAM. The intra‐ and inter‐day precision values for LAM met the acceptance as per Food and Drug Administration guidelines. LAM was stable in the set of stability studies, viz. bench‐top, autosampler and freeze–thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans. Copyright © 2011 John Wiley & Sons, Ltd.     

A novel and sensitive UPLC–MS/MS method to determine mequitazine in rat plasma and urine: Validation and its application to pharmacokinetic studies

The aim of this study was to develop an analytical method to determine mequitazine in rat plasma and urine. Mequitazine was separated by UPLC–MS/MS equipped with a Kinetex core–shell C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.3 mL/min. Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring positive ion mode. Mass transitions were m/z 323.3 → 83.1 for mequitazine and 281.3 → 86.3 for imipramine as internal standard. Liquid–liquid extraction with ethyl acetate and protein precipitation with methanol were used for sample extraction. Chromatograms showed that the method had high resolution, sensitivity and selectivity without interference from plasma constituents. Calibration curves for mequitazine in rat plasma and urine were 0.02–200 ng/mL, showing excellent linearity with correlation coefficients (r²) >0.99. Both intra‐ and inter‐day precisions (CV%) were within 4.08% for rat plasma and urine. The accuracies were 99.58–102.03%. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of mequitazine after oral and intravenous administration to rats.     

Determination of quinapril and quinaprilat in human plasma by ultraperformance liquid chromatography–electrospray ionization mass spectrometry

A novel, specific and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma. The method involves a simple, one‐step extraction procedure coupled with an Acquity UPLC? BEH C₁₈ column (100 × 2.1 mm, i.d., 1.7 µm) with isocratic elution at a flow‐rate of 0.2 mL/min and lisinopril as the internal standard. Detection was performed on a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. Using 250 µL plasma, the methods were validated over the concentration range 5.010–500.374 ng/mL for quinapril and 10.012–1000 ng/mL for quinaprilat, with a lower limit of quantification of 5.010 ng/mL for quinapril and 10.012 ng/mL for quinaprilat. The intra‐ and inter‐day precision and accuracy were within 10.0%. The recovery was 85.8, 62.6 and 61.3% for quinapril, quinaprilat and lisinopril, respectively. Total run time was 3.0 min only. Copyright © 2008 John Wiley & Sons, Ltd.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

Liquid chromatography–tandem mass spectrometry method for simultaneous quantification of urapidil and aripiprazole in human plasma and its application to human pharmacokinetic study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of urapidil and aripiprazole in human plasma. A simple liquid–liquid extraction with ethyl acetate was used for the sample preparation. Chromatographic separation was achieved on a Phenomenex C₁₈ (4.6 × 50 mm, 5 µm) column with 0.1% formic acid–acetonitrile (10:90, v/v) as the mobile phase with flow rate of 0.6 mL/min. The quantitation of the target compounds was determined in a positive ion multiple reaction monitoring mode. Calibration plots were linear over the range of 2.0–2503.95 ng/mL for urapidil and 1.0–500.19 ng/mL for aripiprazole. The lower limit of quantitation for urapidil and aripiprazole was 2.0 and 1.0 ng/mL, respectively. Mean recovery was in the range of 69.94–75.62% for both analytes and internal standards. Intra‐day and inter‐day precisions of the assay at three concentrations were 2.56–5.89% with accuracy of 92.31–97.83% for urapidil, and 3.14–6.84% with accuracy of 91.38–94.42% for aripiprazole. The method was successfully applied to human pharmacokinetic study of urapidil and aripiprazole in healthy human male volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative analysis of ezetimibe in human plasma by gas chromatography‐mass spectrometry

A new, specific and sensitive GC‐MS method with electron impact ionization technique was developed for quantitative analysis of ezetimibe (EZE) in human plasma. Prior to GC analysis, EZE was derivatized with N‐methyl‐N‐trimethylsilyl‐trifluoroacetamide (MSTFA), which is a trimethyl silylating reagent. The derivatization reaction was optimized and parameters such as catalyst, derivatization time, temperature, solvent and the volume of silylating reagent were investigated. Trimethylsilyl ether derivative of EZE was determined in selected ion monitoring (SIM, mass‐to‐charge ratio (m/z): 326) mode. The method was validated with respect to LOD and LOQ, precision, accuracy, linearity, specificity, stability, and recovery. The LOQ and LOD were found as 15 and 10 ng/mL, respectively. The linearity of the method ranged from 15 to 250 ng/mL. The correlation coefficient of the calibration curve was 0.9977 ± 0.0004 (± S.E.M.). The intra‐ and inter‐day precisions (RSD) were less than 6% and accuracies (bias) for intra‐ and inter‐day accuracy were found between –4.04 and 9.71% at four different concentration levels (15, 40, 100, 250 ng/mL). The proposed method was successfully applied to real human plasma samples for determination of total EZE.     

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.