Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.     

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.     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Determination of bergenin in human plasma after oral administration by HPLC-MS/MS method and its pharmacokinetic study

A highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and applied to the determination of bergenin concentration in human plasma. Bergenin and the internal standard (IS) thiamphenicol in plasma were extracted with ethyl acetate, separated on a C(18 )reversed-phase column, eluted with mobile phase of acetonitrile-water, ionized by negative ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor --> product ions of m/z 327.1 --> 192 for bergenin and 354 --> 185.1 for the IS, respectively. The linear range of the calibration curve for bergenin was 0.25-60 ng mL(-1), with the lowest limit of quantification of 0.25 ng mL(-1), and the intra/inter-day relative standard deviation (RSD) was less than 10%. The method is suitable for the determination of low bergenin concentration in human plasma after therapeutic oral doses, and has been first and successfully used for its pharmacokinetic studies in healthy Chinese volunteers.     

Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry

A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.     

UPLC–MS/MS assay for simultaneous determination of four compounds in rat plasma: application to pharmacokinetic study after oral administration of Caulis Spatholobi extract

An ultra‐performance liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of protocatechuic acid, catechin, gallocatechin and formononetin in rat plasma, with genkwanin as the internal standard in this study. Plasma samples were prepared by liquid–liquid extraction with ethyl acetate. The four components were separated on an Agilent Zorbax Eclipse Plus C₁₈ column (2.1 × 50 mm, 1.8 μm) with the mobile phase consisting of water containing 0.05% formic acid and methanol (35:65, v/v), and detected by negative ion electrospray ionization in the selected reaction monitoring mode. The method was linear for all analytes over the investigated ranges, with all correlation coefficients >0.99. The validated lower limit of quantification was 0.5 ng/mL for protocatechuic acid, catechin, and gallocatechin and 0.8 ng/mL for formononetin. The intra‐ and inter‐day precisions (RSD, %) were <13.1%, and accuracy (RE, %) ranged from ?13.8 to 9.9%. The mean absolute extraction recoveries of the analytes and internal standard from rat plasma were all >80.7%. The validated method was successfully applied for the first time to investigate the pharmacokinetics of four chemical ingredients after oral administration of Caulis Spatholobi Extract in rats. Copyright © 2016 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 ipriflavone in human plasma by LC-MS and its application in a pharmacokinetic study

A sensitive liquid chromatography-mass spectrometric method was developed for the quantification of ipriflavone in human plasma. The method utilized liquid-liquid extraction of plasma with ethyl acetate. A gradient elution was performed on a Hedera ODS-2 column (150×2.1 mm i.d., 5 μm), using a mobile phase consisting of 0.1% formic acid solution and methanol at a flow rate of 0.5 mL/min. The single quadrupole mass spectrometer was operated in selected-ion monitoring mode via positive electrospray ionization interface detecting m/z 239.1 and 285.1 for ipriflavone and diazepam (the internal standard), respectively. To improve the selectivity and sensitivity, the fragment ion m/z 239.1, which was produced by in-source collision-induced dissociation, was chosen as the quantitative ion for ipriflavone. The method was fully validated and applied to a pharmacokinetic study of ipriflavone. After oral administration of a single 200 mg ipriflavone tablet, the C(max,) AUC(0-72 h) , t(1/2) and T(max) were 6.3±6.3 ng/mL, 80.0±69.1 μg h/L, 23.0±8.6 h and 3.4±2.1 h, respectively.     

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.     

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.     

The simultaneous UPLC–MS/MS determination of emerging drug combination; candesartan and chlorthalidone in human plasma and its application

A novel, precise, sensitive and accurate ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the simultaneous determination of a novel drug combination, candesartan (CAN) and chlorthalidone (CHL), in human plasma. Chromatographic separation was achieved on Waters Acquity UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm). Mobile phase consisting of 1 mm ammonium acetate in water–acetonitrile (20:80 v /v) was used. The total chromatographic runtime was 1.9 min with retention times for CAN and CHL at 0.7 and 1.1 min respectively. Ionization and detection of analytes and internal standards was performed on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and negative ionization mode. Quantitation was done to monitor protonated precursor → product ion transition of m /z 439.2 → 309.0 for CAN, 337.0 → 189.8 for CHL and 443.2 → 312.1 for candesartan D4 and 341.0 → 189.8 for chlorthalidone D4. The method was validated over a wide dynamic concentration range of 2.0–540.0 ng/mL for candesartan and 1.0–180.0 ng/mL for chlorthalidone. The validated method was successfully applied for the assay of CAN and CHL in healthy volunteers.     

Analysis and pharmacokinetic study of polyphyllin H in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC‐MS/MS

A highly sensitive, rapid assay method has been developed and validated for the analysis of polyphyllin H 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 polyphyllin H and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8μm) 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 2.5 min. The MS/MS ion transitions monitored were 870.46 → 869.6 for polyphyllin H and 947.12 → 969.60 for IS. Linear responses were obtained for polyphyllin H ranging from 1 to 50 ng/mL. The intra‐and inter‐day precisions (RSDs) were <1.77 and 3.39% and the extraction recovery ranged from 91.89 to 93.33% with RSD <2.68%. Stability studies showed that polyphyllin H was stable in the preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of polyphyllin H. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of zafirlukast, a selective leukotriene antagonist in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of zafirlukast (ZFK) with 500 microL human plasma using valdecoxib as an internal standard (IS). The API-4,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of ZFK and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with 10 mm ammonium acetate (pH 6.4):acetonitrile (20:80, v/v) on a Hypersil BDS C(18) column. The total chromatographic run time was 2.0 min and the elution of ZFK and IS occurred at approximately 1.11 and 1.58 min, respectively. The MS/MS ion transitions monitored were 574.2 --> 462.1 for ZFK and 313.3 --> 118.1 for IS. The method was proved to be accurate and precise at a linearity range of 0.15-600 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 0.15 ng/mL as lower limit of quantitation. 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 pharmacokinetic study in human volunteers following oral administration of 20 mg ZFK tablet.     

A simple,rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K₂EDTA plasma using levonorgestrel d₆ as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid–liquid extraction. Chromatographic separation was achieved on a Zorbax XDB‐Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile–5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003–200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra‐ and inter‐day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of a rapid and selective method for the simultaneous determination of carebastine and pseudoephedrine in human plasma by liquid chromatography–electrospray mass spectrometry for bioequivalence study in Korean subjects

We describe a simple, rapid and sensitive high‐performance liquid chromatography–electrospray ionization tandem mass spectrometric method that was developed for the simultaneous determination of carebastine and pseudoephedrine in human plasma using cisapride as an internal standard. Acquisition was performed in multiple‐reaction monitoring mode by monitoring the transitions: m/z 500.43 > 167.09 for carebastine and m/z 166.04 > 147.88 for pseudoephedrine. The devised method involves a simple single‐step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a C₁₈ reversed‐phase chromatographic column at 0.2 mL/min by isocratic elution with 10 mM ammonium formate buffer–acetonitrile (30:70, v/v; adjusted to pH 3.3 with formic acid). The devised method was validated over 0.5–100 ng/mL of carebastine and 5–1000 ng/mL of pseudoephedrine with acceptable accuracy and precision, and was successfully applied to a bioequivalence study involving a single oral dose (10 mg of ebastine plus 120 mg of pseudoephedrine complex) to healthy Korean volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive method for the determination of abiraterone in rat and human plasma by LC-MS/MS-ESI: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of abiraterone (ART) in rat and human plasma with liquid chromatography coupled to tandem mass spectrometry and electrospray ionization in the positive-ion mode. The assay procedure involves extraction of ART and phenacetin (internal standard, IS) from rat and human plasma with a simple protein precipitation extraction process. Chromatographic separation was achieved using an isocratic mobile (10 mm ammonium acetate:acetonitrile, 10:90, v/v) at a flow-rate of 0.70 mL/min on an Atlantis dC(18) column maintained at 40 °C with a total run time of 3.5 min. The MS/MS ion transitions monitored were 350.3 → 156.0 for ART and 180.2 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.20 ng/mL and the linearity range extended from 0.20 to 201 ng/mL. The intra- and inter-day precisions were in the ranges 2.39-10.4 and 4.84-9.53% in rat plasma and 3.82-10.8 and 6.97-8.94% in human plasma.     

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–Electrospray Ionization–Mass Spectrometric Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Pharmacokinetic Study

Abstract

A rapid, convenient, and sensitive liquid chromatography–electrospray ionization–mass spectrometry method was developed and validated for the quantification of hydrochlorothiazide in human plasma. The samples were first spiked with the internal standard, and the analyte was then extracted with ethyl acetate. The chromatographic separation was achieved on a C₁₈ column by using water–acetonitrile (68:32, v/v) as mobile phase. The method was linear within the range of 2.5–200 ng/ml. The lower limit of quantification was 1.0 ng/ml. Finally, the validated method was successfully applied for the evaluation of the pharmacokinetic profiles of hydrochlorothiazide in healthy male Chinese volunteers.     

Improved liquid chromatographic tandem mass spectrometric determination and pharmacokinetic study of glimepiride in human plasma

An improved liquid chromatographic tandem mass spectrometric method for the determination of glimepiride in human plasma has been developed and fully validated. The article describes in detail the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using liquid--liquid extraction with a mixture of 1-chlorobutane-isopropanol-ethyl acetate (88:2:10, v/v/v). The chromatographic separation was performed on a reversed-phase Hypersil ODScolumn (250 x 4.6 mm i.d.; 5 microm particle size) using a mobile phase consisting of formic acid 0.05 M-acetonitrile (28:72, v/v), pumped at a flow rate of 0.3 ml min(-1) heated to 25 degrees C. The analytes were detected using an API 3000 triple quadrupole mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. Tandem mass spectrometric detection enabled the quantitation of glimepiride down to 0.50 ng mL(-1). Calibration graphs were linear (r better than 0.998, n=1), in concentration range 0.50--1000 ng mL(-1), and the intra- and inter- day RSD values were less than 10.37 and 11.55% for glimepiride. The method was successfully applied to a kinetic study in order to assess the main pharmacokinetic parameters of glimepiride.