Quantitative determination of periplocymarin in rat plasma and tissue by LC‐MS/MS: application to pharmacokinetic and tissue distribution study

A simple, rapid and sensitive liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method for the determination of periplocymarin in biological samples was developed and successfully applied to the pharmacokinetic and tissue distribution study of periplocymarin after oral administration of periplocin. Biological samples were processed with ethyl acetate by liquid–liquid extraction, and diazepam was used as the internal standard. Periplocymarin was analyzed on a C₁₈ column with isocratic eluted mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min (73:27, v/v). Detection was performed on a triple‐quadrupole tandem mass spectrometer using positive‐ion mode electrospray ionization in the selected reaction monitoring mode. The MS/MS ion transitions monitored were m/z 535.3→355.1 and 285.1→193.0 for periplocymarin and diazepam, respectively. Good linearity was observed over the concentration ranges. The lower limit of quantification was 0.5 ng/mL in plasma and tested tissues. The intra‐and inter‐day precisions (relative standard deviation) were <10.2 and 10.5%, respectively, and accuracies (relative error) were between ?6.8 and 8.9%. Recoveries in plasma and tissue were >90%. The validated method was successfully applied to the pharmacokinetic and tissue distribution studies of periplocymarin in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study

In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacokinetic study in nude mice. Using testosterone as an internal standard, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) approach after one‐step precipitation with acetonitrile was validated and used to determine the concentrations of dexamethasone in nude mice plasma. The method utilized a simple isocratic reverse phase separation over a Dionex C₁₈ column with a mobile phase composed of acetonitrile–water (40:60, v/v). The analyte was detected by a triple quadrupole tandem mass spectrometer via electrospray and multiple reaction monitoring was employed to select both dexamethasone at m/z 393.0/147.1 and testosterone at m/z 289.5/97.3 in the positive ion mode. The calibration curves were linear (r >0.99) ranging from 2.5 to 500 ng/mL with a lower limit of quantitation of 2.5 ng/mL. The relative standard deviation ranged from 1.69 to 9.22% while the relative error ranged from ?1.92 to ?8.46%. This method was successfully applied to a preclinical pharmacokinetic study of dexamethasone and its pharmacokinetics was characterized by a two‐compartment model with first‐order absorption in female nude mice. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of LC‐MS/MS method for simultaneous determination of sofosbuvir and daclatasvir in human Plasma: Application to pharmacokinetic study

A simple and highly sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) bioanalytical method was developed and fully validated for the first time for the simultaneous determination of newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid–liquid extraction technique with methyl tert‐butyl ether. The chromatographic separation was carried out using ZorbaxSB‐C₁₈ column (4.6 × 50 mm,5 μm) and 5 mm ammonium formate buffer (pH 3.5)–acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min⁻¹. The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring mode. Validation was applied according to US Food and Drug Administration guidelines for bio‐analytical methodswith respect to linearity, precision, accuracy, selectivity, carry‐over, stability and dilution integrity. Linearity was obtained over concentration ranges of 0.3–3000 and 3–3000 ng mL⁻¹ for SOF and DAC, respectively, by applying a weighted least‐squares linear regression method (1/x²). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies with excellent assay ruggedness and reproducibility.     

Determination of pinocembrin in human plasma by solid‐phase extraction and LC/MS/MS: application to pharmacokinetic studies

A sensitive, fast and specific method for the quantitation of pinocembrin in human plasma based on high‐performance liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed and validated. Clonazepam was used as the internal standard (IS). After solid‐phase extraction of 500 μL plasma, pinocembrin and the IS were separated on a Luna C₈ column using the mobile phase composed of acetonitrile–0.3 mm ammonium acetate solution (65:35, v/v) at a flow rate of 0.25 mL/min in isocratic mode. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source in negative mode by AB SCIEX Qtrap 5500. The assay was linear from 1 to 400 ng/mL, with within‐ and between‐run accuracy (relative error) from ?1.82 to 0.54%, and within‐ and between‐run precision (CV) below 5.25%. The recovery was above 88% for the analyte at 1, 50 and 300 ng/mL. This analytical method was successful for the determination of pinocembrin in human plasma and applied to a pharmacokinetic study of pinocembrin injection in healthy volunteers after intravenous drip administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative bioanalysis of bavachalcone in rat plasma by LC–MS/MS and its application in a pharmacokinetics study

This study aims to develop and validate a simple and sensitive liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for investigating the pharmacokinetic characteristics of bavachalcone. Liquid–liquid extraction was used to prepare plasma sample. Chromatographic separation of bavachalcone and IS was achieved using a Venusil ASB C₁₈ (2.1 × 50 mm, 5 μm) column with a mobile phase of methanol (A)–water (B) (70:30, v /v). The detection and quantification of analytes was performed in selected‐reaction monitoring mode using precursor → product ion combinations of m/z 323.1 → 203.2 for bavachalcone, and m/z 373.0 → 179.0 for IS. Linear calibration plots were achieved in the range of 1–1000 ng/mL for bavachalcone (r ² > 0.99) in rat plasma. The recovery of bavachalcone ranged from 84.1 to 87.0%. The method was precise, accurate and reliable. It was fully validated and successfully applied to pharmacokinetic study of bavachalcone.     

Development and validation of a UPLC‐MS/MS method for the determination of cucurbitacin B in rat plasma and application to a pharmacokinetic study

Cucurbitacin B (CuB), one of the most abundant forms of cucurbitacins, is a promising natural anticancer drug candidate. Although the anticancer activity of CuB has been well demonstrated, information regarding the pharmacokinetics is limited. A rapid, selective and sensitive UPLC‐MS/MS for CuB was developed and validated using hemslecin A (HeA) as internal standard (IS). Plasma samples were pre‐treated by liquid–liquid extraction with dichloromethane. Separation was achieved on a reversed‐phase C₁₈ column (50 × 4.6 mm, 5 µm) at 35°C using isocratic elution with water–methanol (25:75, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with positive electrospray ionization mode. The calibration curve was linear (r > 0.995) in a concentration range of 0.3–100 ng/mL with a limit of quantification of 0.3 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by percentage relative error and relative standard deviation, respectively, which were both lower than the limit of 15%. This assay was successfully applied to a pharmacokinetic study of CuB in Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of pethidine in human plasma by LC–MS/MS

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of pethidine in human plasma was developed and validated over the concentration range of 4–2000 ng/mL. After addition of ketamine as internal standard, liquid–liquid extraction was used to produce a protein‐free extract. Chromatographic separation was achieved on a 100 × 2.1 mm, 5 µm particle, Allure^TM PFP propyl column, with 45:40:15 (v/v/v) acetonitrile–methanol–water containing 0.2% formic acid as mobile phase. The MS data acquisition was accomplished by multiple reactions monitoring mode with positive electrospray ionization interface. The lower limit of quantification was 4 ng/mL; for inter‐day and intra‐day tests, the precision (RSD) for the entire validation was less than 7%, and the accuracy was within 95.9–106.5%. The method is sensitive and simple, and was successfully applied to analysis of samples of clinical intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a rapid UPLC-MS/MS method for quantification of saxagliptin in rat plasma and application to pharmacokinetic study

A novel, simple and rapid ultraperformance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) assay was established for quantification of saxagliptin in rat plasma. Plasma samples were processed by liquid–liquid extraction with ethyl acetate and chromatographed on a C₁₈ column (2.1 × 50 mm i.d., 1.7 µm). The mobile phase consisted of methanol and 0.1% formic acid (40:60, v/v). Multiple reaction monitoring transitions were performed for detection in positive‐ion mode with an electrospray ionization source. The calibration curve was linear over the concentration range of 0.5–100 ng/mL (R² > 0.99). All accuracy values were between 90.62 and 105.60% relative error and the intra‐ and inter‐day precisions were less than 9.66% relative standard deviation. Extraction recovery was more than 81.01% and the matrix effect ranged from 90.27 to 109.15%. After validation, the method was applied to a pharmacokinetic study where healthy rats were orally given 0.5 mg/kg saxagliptin. Copyright © 2012 John Wiley & Sons, Ltd.     

LC–MS/MS method for simultaneous determination of ramelteon and its metabolite M‐II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M‐II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS‐2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol–0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500–30.0 ng/mL for ramelteon and 1.00–250 ng/mL for M‐II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (C_max), the time to the C_max and the elimination half‐life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M‐II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.     

Bioanalytical method development and validation of novel antithrombotic agent S002-333 by LC-MS/MS and its application to pharmacokinetic studies

A rapid, sensitive and simple liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method using an electrospray ionization (ECI) source for the quantification of novel anti‐thrombotic agent S002‐333 [2‐(4‐methoxy‐benzenesulfonyl)‐2,3,4,9‐tetrahydro‐1H‐β‐carboxylic acid amide] in rabbit plasma was developed and validated. The extraction from plasma was carried out by simple protein precipitation extraction method. The chromatographic separation was performed on an Ultramex Cyno, (150 × 4.6 mm, 5 µm) with a guard column, using acetonitrile–water (75:25,v/v) with flow rate of 0.6 mL/min as the mobile phase. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 386.4/215.4 for S002‐333 and m/z 393.4/171for the internal standard dexamethasone, using positive ion mode. The MS/MS response was linear over the concentration range from 1.56 to 200 ng/mL, with a lower limit of detection of 0.78 ng/mL. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations and showed no significant matrix effect. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics

A rapid and sensitive high‐performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid–liquid extraction. Chromatographic separation was achieved on a C₁₈ column (2.1 × 30 mm, 3.5 μm) using acetonitrile–water (90:10, v /v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r ² = 0.9998). The intra‐ and inter‐day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC–MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability.     

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.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. 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.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.