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

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

Rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantification of ambroxol in human plasma

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.     

A rapid LC-MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine 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 ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of pramipexole 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 pramipexole (PPX) with 500 microL human plasma using memantine as an internal standard (IS). The API-4000 was operated under multiple-reaction monitoring mode (MRM) using the electrospray ionization technique. Solid-phase extraction was used to extract PPX and IS from human plasma. The resolution of peaks was achieved with 0.01 m ammonium acetate buffer (pH 4.4):acetonitrile (30:70, v/v) on a Discovery CN column. The total chromatographic run time was 3.0 min and the elution of PPX and IS occurred at approximately 2.32 and 2.52, respectively. The MS/MS ion transitions monitored were 212.10 --> 153.10 for PPX and 180.20 --> 107.30 for IS. The method was proved to be accurate and precise at linearity range of 20-3540 pg/mL with a correlation coefficient (r) of > or =0.999. The intra- and inter-day precision and accuracy values 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 0.25 mg PPX tablet.     

Simultaneous quantification of atorvastatin and active metabolites in human plasma by liquid chromatography-tandem mass spectrometry using rosuvastatin as internal standard

A simple, sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of atorvastatin and its active metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin in human plasma using rosuvastatin as internal standard (IS). Following simple liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 559/440 for atorvastatin, m/z 575/466 for ortho-hydroxyatorvastatin, m/z 575/440 for para-hydroxyatorvastatin and m/z 482/258 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for atorvastatin and its two metabolites in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin and the IS from spiked plasma samples were 54.2 +/- 3.2, 50.1 +/- 3.8, 65.2 +/- 3.6 and 71.7 +/- 2.7%, respectively. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid liquid chromatography-tandem mass spectrometry method for quantification of ziprasidone in human plasma

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ziprasidone (ZIP) in human plasma was developed. ZIP and N-methyl ziprasidone as internal standard (IS) were extracted from alkalinized plasma using tert- butyl methyl ether. Separation was performed isocratically on a C8 column with 90% acetonitrile containing 2 mmol/L ammonium acetate as a mobile phase with a total run time of 2.5 min. MS/MS transitions of m/z 413 --> 194 and m/z 427 --> 177 of the analyte and internal standard were used for quantification. Confirmatory ions of m/z 413 --> 177 and m/z 427 --> 180 were collected as well. The calibration curve based on peak-area ratio was linear up to at least 200 ng/mL with a detection limit of 0.1 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 5%. The method was successfully applied to the analysis of ZIP in spiked human plasma.     

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

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

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.     

Effect of collision-activated dissociation gas and collision energy on the fragmentation of dipyridamole and its rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric determination in human plasma

The effect of nitrogen as the collision-activated dissociation (CAD) gas on the fragmentation of dipyridamole was investigated in the range of 10-90 eV collision energy. The results support the collision model reported elsewhere, that the degree of ion fragmentation increases with the increasing mass of the collision gas. A simple, sensitive and high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of dipyridamole, a platelet aggregation inhibitor in human plasma, using granisetron as internal standard (IS). The method involved liquid-liquid extraction of the analyte and IS from 0.5 mL human plasma with diethyl ether. The chromatographic separation was achieved under isocratic conditions and the ion transitions for dipyridamole (m/z 505.40 --> 429.60) and the IS (m/z 313.10 --> 138.20) were monitored on a triple quadrupole mass spectrometer, operating in positive ion multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.1-4499.1 ng/mL for dipyridamole. The method was rugged and rapid with a total run time of 1.2 min. It was successfully applied to a pivotal bioequivalence study in 67 healthy human subjects after oral administration of a 75 mg extended release formulation under fasting condition.     

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.     

Development and validation of a highly sensitive LC-MS/MS method for quantitation of dexlansoprazole in human plasma: application to a human pharmacokinetic study

A highly sensitive, specific and simple LC-MS/MS method was developed for the simultaneous estimation of dexlansoprazole (DEX) with 50 μL of human plasma using omeprazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under multiple reaction-monitoring mode using electrospray ionization. A simple liquid-liquid extraction process was used to extract DEX and IS from human plasma. The total run time was 2.00 min and the elution of DEX and IS occurred at 1.20 min. This was achieved with a mobile phase consisting of 0.2% ammonia-acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X-terra RP 18 (50 × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 2 ng/mL for DEX. A linear response function was established for the range of concentrations 2.00-2500.0 ng/mL (r > 0.998) for DEX. The intra- and inter-day precision values for DEX met the acceptance criteria as per FDA guidelines. DEX was stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

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 plasma topiramate concentration using LC-MS/MS for pharmacokinetic and bioequivalence studies in healthy Korean volunteers

A rapid, simple and validated liquid chromatography coupled to tandem mass spectrometric method (LC-MS/MS) for topiramate analysis in human plasma has been applied to pharmacokinetic and bioequivalence studies in 24 healthy male Korean volunteers. The procedure involves a simple liquid extraction of topiramate and prednisone (internal standard) with acetonitrile and separation by HPLC equipped with a Capcell Pak C18 column using acetonitrile-0.1% triethylamine (80:20, v/v) as a mobile phase. Detection was carried out on an API 2000 MS system by multiple reactions monitoring mode. The ionization was optimized using ESI(-) and selectivity was achieved by MS/MS analysis, m/z 338.0 --> 77.5 and m/z 357.1 --> 327.2 for topiramate and prednisone, respectively. The method had a total run time of 2.5 min and showed good linearity over a working range of 20-5000 ng/mL in human plasma with a lower limit of quantification of 20 ng/mL. No metabolic compounds were found to interfere with the analysis. The inter-day and intra-day accuracy were in the ranges of 99.24-116.63 and 93.45-108.68%, respectively, and inter-day and intra-day precisions were below 6.24 and 5.25%, respectively. This method was successfully applied for pharmacokinetic and bioequivalence studies by analysis of blood samples taken up to 96 h after an oral administration of 100 mg of topiramate in 24 healthy Korean volunteers.     

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.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). 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 NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

A rapid liquid chromatography-tandem mass spectrometry method for quantification of a biologically active molecule camboginol in the extract of Garcinia cambogia

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of camboginol in the extract of fruit rinds of Garcinia cambogia has been developed. Separation was achieved isocratically on an RP C(18) column using a solvent system consisting of a mixture of acetonitrile-water (9:1) and methanol-acetic acid (99.5:0.5) in the ratio of 30:70 as mobile phase at a flow rate of 0.4 mL/min. A multiple reaction monitoring (MRM) method was developed for quantification of camboginol in the fruit rinds extract of G. cambogia using MRM transitions of m/z 601.4 --> m/z 176.7 and m/z 601.4 --> m/z 448.9, respectively. The calibration curve based on peak area against concentration was linear up to 50 ng/mL with a detection limit of 0.5 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 6%. The method was successfully applied for quantification of camboginol in different Garcinia extracts.     

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.