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.     

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 a high‐throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of oseltamivir phosphate and its oseltamivir carboxylate metabolite in human plasma for pharmacokinetic studies

A rapid, sensitive and rugged solid‐phase extraction ultraperformance liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for determination of oseltamivir phosphate (OP) and oseltamivir carboxylate (OC) in human plasma. The procedure for sample preparation includes a simple SPE extraction procedure coupled with a Chromatopack C₁₈ column (50 × 3.0 mm, i.d., 3.0 µm) with isocratic elution at a flow‐rate of 0.600 mL /min and acyclovir was used as the internal standard. The analysis was performed on a triple‐quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. Using 500 µL plasma, the methods were validated over the concentration ranges 0.92–745.98 and 5.22–497.49 ng/mL for OP and OC, with a lower limit of quantification of 0.92and 5.22 ng/mL. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.1%. The recovery was 68.72, 70.66 and 71.59% for OP, OC and IS, respectively. Total run time was only 1.0 min. The method was highly reproducible with excellent chromatography properties. Copyright © 2010 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.     

Development and validation of UPLC tandem mass spectrometry assay for separation of a phase II metabolite of ramipril using actual study samples and its application to a bioequivalence study

In this paper, we present a validated UPLC‐MS/MS assay for determination of ramipril and ramiprilat from human plasma samples. The assay is capable of isolating phase II metabolites (acylglucornides) of ramipril from in vivo study samples which is otherwise not possible using conventional HPLC conditions. Both analytes were extracted from human plasma using solid‐phase extraction technique. Chromatographic separation of analytes and their respective internal standards was carried out using an Acquity UPLC BEH C₁₈ (2.1 × 100 mm), 1.7 µm column followed by mass spectrometric detection using an Waters Quattro Premier XE. The method was validated over the range 0.35–70.0 ng/mL for ramipril and 1.0–40.0 ng/mL for ramiprilat. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Determination of Ginsenoside Rg2 in Rat Plasma by High‐Performance Liquid Chromatography–Mass Spectrometry after Solid‐Phase Extraction

Abstract

A sensitive liquid chromatography‐mass spectrometric (LC/MS) method for the quantification of ginsenoside Rg2 (Rg2) in rat plasma was developed after solid‐phase extraction (SPE). Chromatographic separation was achieved on a reversed‐phase Kromasil C₁₈ column with the mobile phase of acetonitrile‐ammonium chloride (500 µM/L) and step gradient elution resulted in a total run time of about 9 min. The analytes were detected using electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range (5–2500 ng/mL) (r=0.9999). Limit of quantification (LOQ) was 5 ng/mL and the limit of detection (LOD) was 2 ng/mL using 100 µL plasma sample. Average recoveries ranged from 72.43–84.73% in plasma at the concentrations of 20, 200, and 2000 ng/mL. Intra‐ and interday coefficients of variation for the assay were 4.93–10.87% and 4.06–7.84%, respectively. The method was successfully applied to the analysis of ginsenoside Rg2 in rat plasma. The applicability of this assay was examined in a preliminary pharmacokinetic study of ginsenoside Rg2 in rats.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

An LC-MS/MS method for simultaneous determination of vincristine and verapamil in rat plasma after oral administration of a dual agent formulation

A rapid and sensitive method for simultaneous determination of vincristine and verapamil in rat plasma was first developed and validated, using high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) in multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). The method, which required a small sample volume (25 µL) of plasma, was linear in the concentration range of 0.5–500 ng/mL for vincristine and 0.1–100.0 ng/mL for verapamil. Finally, the method was successfully employed in a pharmacokinetic study of vincristine and verapamil in rats after an oral administration of a dual‐agent formulation containing vincristine and verapamil. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study

A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of a sensitive LC/MS/MS method for the determination of telaprevir and its R‐isomer in human plasma

The purpose of this study was to validate a reversed‐phase high‐performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R‐diastereomer (VRT‐127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir‐R was performed on a Waters XBridge^TM BEH Shield C₁₈, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir‐d11). An internal standard for the telaprevir‐R (telaprevir‐R‐d11) was also prepared by incubating telaprevir‐d11 in basic solution, which facilitated isomer inter‐conversion. The detection and quantitation of telaprevir, telaprevir‐R, telaprevir‐IS and telaprevir‐R‐IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of Cefalothin and Cefazolin in Human Plasma,Urine and Peritoneal Dialysate by UHPLC‐MS/MS: application to a pilot pharmacokinetic study in humans

An ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1–500 µg/mL. Unbound concentrations are measured from ultra‐filtered plasma acquired using Centrifree^® devices and are suitable for the concentration range of 0.1–500 µg/mL for cefazolin and 1–500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1–20 mg/mL for cefazolin and 0.2–20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2–100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter‐assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis‐associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid LC‐MS/MS determination and pharmacokinetic application of linarin in rat plasma

A sensitive, selective and robust liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the rapid determination of linarin in rat plasma. Separation of the analyte and warfarin as internal standard (IS) from 100 μL rat plasma was carried out by simple protein precipitation treatment. Chromatographic separation of the analyte was performed on a Diamonsil® C₁₈ column (150 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of methanol–0.5% formic acid (80:20, v/v). The flow rate was 0.6 mL/min and the total run time was not more than 4.0 min. The method was validated over a wide dynamic concentration range of 1.00–1000 ng/mL for linarin. The precision and accuracy values for linarin met the acceptance criteria according to US Food and Drug Administration guidelines. Linarin was stable in the stability studies including a long‐term test (?80°C for 43 days), a short‐term test (ambient for 2 h and autosampler for 8 h) and three freeze–thaw cycles (?80–25°C). The developed assay method was applied to the pharmacokinetic study in rats after a single intramuscular administration of 713 µg/kg linarin. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasma was carried out by solid‐phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C₁₈ (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate–methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1–350 ng/mL for BUP and 0.1–600 ng/mL for HBUP. The matrix effect was assessed by post‐column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions. Copyright © 2011 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.     

Simultaneous quantification of olanzapine and its metabolite N‐desmethylolanzapine in human plasma by liquid chromatography tandem mass spectrometry for therapeutic drug monitoring

A simple, sensitive, and selective liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of olanzapine (OLZ) and its metabolite N‐desmethylolanzapine (DMO) in human plasma for therapeutic drug monitoring. Sample preparation was performed by one‐step protein precipitation with methanol. The analytes were chromatographed on a reversed‐phase YMC‐ODS‐AQ C₁₈ Column (2.0 × 100 mm,3 µm) by a gradient program at a flow rate of 0.30 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in positive ion mode. The method was validated for selectivity, linearity, accuracy, precision, matrix effect, recovery and stability. The calibration curve was linear over the concentration range 0.2–120 ng/mL for OLZ and 0.5–50 ng/mL for DMO. Intra‐ and interday precisions for OLZ and DMO were <11.29%, and the accuracy ranged from 95.23 to 113.16%. The developed method was subsequently applied to therapeutic drug monitoring for psychiatric patients receiving therapy of OLZ tablets. The method seems to be suitable for therapeutic drug monitoring of patients undergoing therapy with OLZ and might contribute to prediction of the risk of adverse reactions. Copyright © 2014 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

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

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra‐and inter‐day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of hyperoside. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of an antitumor agent (copen) in rat plasma by liquid chromatography–electrospray ionization tandem mass spectrometry and its application in a preclinical pharmacokinetic study

Copen is a derivative obtained from the structural modification of osthole, which inhibits tumoral proliferation in many tumor cell lines. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was established for the quantification of copen in rat plasma. After a simple sample preparation procedure by one‐step protein precipitation with methanol, copen and bicalutamide (internal standard, IS) were chromatographed on a Zorbax SB‐C₁₈ (4.6×100 mm, 1.8 µm) column with a mobile phase consisting of methanol–5 mm ammonium formate water with 0.1% formic acid (80:20, v/v). MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration range of 51.58–20630 ng/mL, with a limit of quantitation (LOQ) of 51.58 ng/mL. The intra‐ and inter‐day precisions (relative standard deviation) were ≤3.21 and ≤11.3%, respectively, with accuracy (%) in the range of 94.66–102.1%. The method was fully validated in a study of the pharmacokinetics of copen (25 mg/kg) after intragastric administration in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Study the pharmacokinetics of AV-45 in rat plasma and metabolism in liver microsomes by ultra-performance liquid chromatography with mass spectrometry

An ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC‐MS/MS) method was developed and validated to determine AV‐45 in rat plasma. After the addition of the internal standard benzophenone, plasma samples were pretreated by protein precipitation. Chromatographic separation was achieved on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 µm) by gradient elution at a flow rate of 0.4 mL/min. Detection of analytes and internal standard (IS) was done by tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring mode. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect and stability study. The calibration curve showed good linearity over the concentration range 2.00–1000 ng/mL for AV‐45. Intra‐ and inter‐day precisions were less than 7.6%, and accuracy ranged from 100.6 to 107.8%. There was no matrix effect. The validated method was successfully applied to a pharmacokinetic study of AV‐45 in rats. Additionally, the metabolism of AV‐45 in rat liver microsomes was also studied by ultra‐performance liquid chromatography combined with time‐of‐flight mass spectrometry (UPLC/TOF‐MS). With the help of chromatographic behavior and accurate mass measurements, the metabolites were characterized. Copyright © 2011 John Wiley & Sons, Ltd.