An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of a novel anticancer c‐Met inhibitor LS‐177 in rat plasma and tissues with a validated UPLC‐MS/MS method: application to pharmacokinetics and tissue distribution study

LS‐177 is a novel small‐molecule kinase inhibitor employed to interrupt the c‐Met signaling pathway. A rapid and sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for determination of LS‐177 in rat plasma and tissues. The biosamples were extracted by liquid–liquid extraction with methyl tert‐butyl ether and separated on a C₁₈ column (50 × 4.6 mm, 2.6 µm) using a gradient elution mobile phase consisting of acetonitrile–0.1% formic acid water. Under the optimal conditions, the selectivity of the method was satisfactory with no endogenous interference. The intraday and interday precisions (relative standard deviation) were <10.5% and the accuracy (relative error) was from ?12.5 to 12.5% at all quality control levels. Excellent recovery and negligible matrix effects were observed. Stability studies showed that LS‐177 was stable during the preparation and analytical processes. The UPLC‐MS/MS method was successfully applied to pharmacokinetic and tissue distribution studies. The results indicated that there was no significant drug accumulation after multiple‐dose oral administration of LS‐177. The tissue distribution study exhibited significant higher uptakes of LS‐177 in stomach, intestine, lung and liver among all of the tissues. The results in pharmacokinetics and tissue distribution may provide a meaningful basis for clinical application. Copyright © 2014 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.     

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.     

A validated LC‐MS/MS method for rapid determination of brazilin in rat plasma and its application to a pharmacokinetic study

Brazilin is a major homoisoflavonoid component isolated from the dried heartwood of traditional Chinese medicine Caesalpinia sappan L., which is a natural red pigment used for histological staining. Herein a sensitive, specific and rapid analytical LC‐MS/MS method was established and validated for brazilin in rat plasma. After a simple step of protein precipitation using acetonitrile, plasma samples were analyzed using an LC‐MS/MS system. Brazilin and the IS (protosappanin B) were separated on a Diamonsil C₁₈ analytical column (150 × 4.6 mm, 5 µm) using a mixture of water and 10 mm ammonium acetate in methanol (20:80, v/v) as mobile phase at a flow rate of 0.6 mL/min. The method was sensitive with a lower limit of quantitation of 10.0 ng/mL, with good linearity (r² ≥ 0.99) over the linear range 10.0–5000 ng/mL. All the validation data, such as accuracy and precision, matrix effect, extraction recovery and stability tests were within the required limits. The assay method was successfully applied to evaluate the pharmacokinetics parameters of brazilin after an oral dose of 100 mg/kg brazilin in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of a UPLC‐MS/MS method for the analysis of alosetron in human plasma to support a bioequivalence study in healthy males and females

A simple, rapid and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for the determination of alosetron (ALO) in human plasma. The assay method involved solid‐phase extraction of ALO and ALO 13C‐d3 as internal standard (IS) on a LichroSep DVB‐HL (30 mg, 1 cm³) cartridge. The chromatography was performed on an Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 µm) column using acetonitrile and 2.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (80:20, v/v) as the mobile phase in an isocratic mode. For quantitative analysis, the multiple reaction monitoring transitions studied were m/z 295.1/201.0 for ALO and m/z 299.1/205.1 for IS in the positive ionization mode. The method was validated over a concentration range of 0.01–10.0 ng/mL for ALO. Post‐column infusion experiment showed no positive or negative peaks in the elution range of the analyte and IS after injection of extracted blank plasma. The extent of ion‐suppression/enhancement, expressed as IS‐normalized matrix factor, varied from 0.96 to 1.04. The assay recovery was within 97–103% for ALO and IS. The method was successfully applied to support a bioequivalence study of 1.0 mg alosetron tablets in 28 healthy Indian male and female subjects. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of swertianolin in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C₁₈ column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra‐day and inter‐day precision was less than 6.8%. The accuracy was in the range of ?13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 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.     

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.     

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.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Determination of aripiprazole in rat plasma and brain using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Aripiprazole is an important antipsychotic drug. A simple, sensitive and rapid ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC‐ESI‐MS/MS) method was developed and validated for the simultaneous quantification of this compound in rat plasma and brain homogenate. The analyte was extracted from rat plasma and brain homogenate using a weak cation exchange mixed‐mode resin‐based solid phase extraction. The compound was separated on an Agilent Eclipse Plus C₁₈ (2.1 × 50 mm, 1.8 µm) column using a mobile phase of (A) 0.1% formic acid aqueous and (B) acetonitrile with gradient elution. The analyte was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, recoveries and stability were determined. The LOQ was 0.5 ng/mL for aripiprazole in plasma and 1.5 ng/g in brain tissue. The MS response was linear over the concentration range 0.5–100 ng/mL for aripiprazole in plasma and 1.5–300 ng/g in brain tissue. The precision and accuracy for intra‐day and inter‐day were better than 14%. The relative and absolute recoveries were above 72% and the matrix effects were low. This validated method was successfully used to quantify the rat plasma and brain tissue concentrations of the analyte following chronic treatment with aripiprazole. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid HPLC‐ESI‐MS/MS method for determination of β‐asarone,a potential anti‐epileptic agent,in plasma after oral administration of Acorus calamus extract to rats

β‐Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography–tandem mass spectrometric method (HPLC‐MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre‐treated using a simple solid‐phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C₁₈ column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q‐Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79–4892.50 ng/mL (r² ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC‐MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats. Copyright © 2012 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.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study

A new, rapid, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous quantification of tenofovir and lamivudine in human plasma using abacavir as an internal standard. An API‐4000 LC‐MS/MS with electrospray ionization was operated in multiple‐reaction monitoring mode for the analysis. The analytes were extracted from plasma by solid‐phase extraction technique using an Oasis HLB cartridge. The reconstituted samples were chromatographed on a Chromolith ROD speed C₁₈ column using a mixture of 0.1% formic acid in water and acetonitrile (90:10 v/v) at a flow‐rate of 1 mL/min. The method was validated as per the FDA guidelines. The calibration curves were found to be linear in the range of 5–600 ng/mL for tenofovir and 25– 4000 ng/mL for lamivudine. The intra‐ and inter‐day precision and accuracy results were well within the acceptable limits. A run time of 2.8 min consumed for each sample made it possible to analyze more samples per day. The proposed assay method was found to be applicable to a pharmacokinetic study in human male volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioanalytical method development,validation and quantification of dorsomorphin in rat plasma by LC‐MS/MS

The present investigation describes the development and validation of a sensitive liquid chromatography–mass spectrometry/mass spectrometry (LC‐MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC‐MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C₁₈ column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r² ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter‐day and intra‐day precision and accuracy were found to be within 85–115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Bioanalytical LC‐MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers

A LC‐MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge‐coupled solid‐phase extraction and reverse‐phase chromatographic separation was performed on an Ascentis C₁₈ column. Turbo‐spray negative‐ion mode multiple‐reaction monitoring was selected for mass pair detection at m/z 338.3 → 78.0 and m/z 407.3 → 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half‐life matching for test and reference drug was achieved with 73.43 ± 9.68 and 73.06 ± 14.03 h, respectively, and intra‐subject coefficient of variation achieved within 11% for AUCs and C_max evaluated by non‐compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright © 2009 John Wiley & Sons, Ltd.