Simultaneous determination of atorvastatin and niacin in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

A rapid, simple, sensitive and selective LC‐MS/MS method has been developed and validated for quantification of the atorvastatin (AT) and niacin (NA) in 250 μL human plasma. The analytical procedure involves a liquid–liquid extraction method using nevirapine as an internal standard (IS). The chromatographic separation was achieved on a Hypurity Advance (4.6 × 50 mm, 5 µm) column using a mobile phase consisting of 0.1% formic acid buffer–acetonitrile (20:80, v/v) at flow rate of 0.8 mL/min. The API‐4000 LC‐MS/MS was operated in the multiple‐reaction monitoring mode using electrospray ionization. The total run time of analysis was 3 min and elution of AT, NA and IS occurred at 1.06, 1.84 and 0.92 min, respectively. A detailed validation of the method was performed as per the US Food and Drug Administration guidelines and the standard curves found to be linear in the range of 0.10–30.0 ng/mL for AT and 20.2–6026 ng/mL for NA, with a coefficient of correlation of ≥0.99 for both the compounds. AT and NA were found to be stable in a battery of stability studies, viz. bench‐top, autosampler, re‐injection, wet‐extract and repeated freeze–thaw cycles. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of sitagliptin and simvastatin in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and validated for simultaneous quantification of sitagliptin and simvastatin in human plasma. Carbamazepine was used as an internal standard (IS). The analytes and IS were extracted from the human plasma by liquid–liquid extraction technique. The reconstituted samples were chromatographed on an Alltima HP C₁₈ column using an isocratic solvent mixture [acetonitrile–5 mm ammonium acetate (pH 4.5), 85:15 (v/v)] at a flow rate of 1.0 mL/min. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.10–501 and 0.05–105 ng/mL for sitagliptin and simvastatin, respectively. The results of the intra‐ and inter‐day precision and accuracy studies were well within the acceptable limits. Both the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay was successfully applied to a pharmacokinetic study in human volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Simultaneous quantitation of nicorandil and its denitrated metabolite in plasma by LC-MS/MS: application for a pharmacokinetic study

A liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantitation of nicorandil and its denitrated metabolite, N-(2-hydroxyethyl)-nicotinamide, in rat plasma. After a liquid-liquid extraction step, chromatographic separation was performed on a ShinPack C(18) column with an isocratic mobile phase composed of methanol and 2 mM aqueous ammonium acetate containing 0.03% (v/v) formic acid (33:67 v/v). Procainamide was used as an internal standard (IS). Selected reaction monitoring was performed using the transitions m/z 212 → m/z 135, m/z 166 → m/z 106 and m/z 236 → m/z 163 to quantify nicorandil, its denitrated metabolite and IS, respectively. Calibration curves were constructed over the range of 5-15,000 ng.ml(-1) for both nicorandil and its metabolite. The mean relative standard deviation (RSD%) values for the intra-run precision were 5.4% and 7.3% and for the inter-run precision were 8.5% and 7.3% for nicorandil and its metabolite, respectively. The mean accuracy values were 100% and 95% for nicorandil and its metabolite, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after per os administration of nicorandil in rats.     

Simultaneous determination of atorvastatin,amlodipine, ramipril and benazepril in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A rapid, simple, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple‐reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C₁₈ column by pumping 0.1% formic acid–acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26–210 ng/mL for ATO; 0.05–20.5 ng/mL for AML; 0.25–208 ng/mL for RAM and 0.74–607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra‐day and inter‐day precision and accuracy results were well with in the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Simultaneous determination of simvastatin and simvastatin acid in human plasma by LC-MS/MS without polarity switch: application to a bioequivalence study

A simple, specific and sensitive LC-MS/MS assay for simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite, simvastatin acid (SVA) in human plasma was developed using a statin analog as internal standard (IS). The method was validated over a dynamic linear range of 0.20-100.00 ng/mL for SV and 0.10-50.00 ng/mL for SVA with correlation coefficient r > or = 0.9987 and 0.9989, respectively. The analytes and IS were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction using methyl tert-butyl ether and separated through an Aquasil C18 column (100 mm x 2.1 mm, 5 microm). Detection of analytes and IS was done by MS/MS with a turbo ion spray interface operating in positive ion and selective reaction monitoring acquisition mode. The total chromatographic run time was 3.0 min. Flash freezing of the aqueous phase was an added advantage during liquid-liquid extraction, which considerably reduced time and labour. The method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect. The method was successfully used for bioequivalence study of 40 mg SV tablet formulation in 12 human subjects under fasting condition.     

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

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

Validated LC-MS/MS assay for the quantitative determination of clematichinenoside AR in rat plasma and its application to a pharmacokinetic study

This study aimed to develop and validate a liquid chromatography tandem mass spectrometry method for measuring clematichinenoside AR in rat plasma. Clematichinenoside AR was extracted by solid‐phase extraction and chromatographed on an XTerra MS C₈ column. Pulchinenoside B4 was used as the internal standard. Elution was achieved using an isocratic mobile phase of acetonitrile with 0.1% acetic acid (21:79, v/v) at a flow‐rate of 0.2 mL/min. The detection was performed by multiple reaction monitoring mode via a negative electrospray ionization interface. Standard curves were linear, ranging from 2.5 to 500 ng/mL. The intra‐ and inter‐day precision values were <14.0% and the accuracy was within ±13%. Extraction recovery ranged from 93.2 to 93.9%. This proposed method was successfully applied to a pharmacokinetic study on clematichinenoside AR in rats after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 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.     

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.     

Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of rhein in human plasma: application to a pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of rhein with 100 microL human plasma using celecoxib as an internal standard (IS). The API-4,000 Q-Trap LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of rhein and IS from human plasma with acetonitrile, which yielded consistent recoveries of 36.01 and 65.85% for rhein and IS, respectively. The total chromatographic run time was 5.0 min and the elution of rhein and IS occurred at approximately 1.60 and 3.96 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate (pH 6.0):acetonitrile:methanol (30:58:12, v/v) on an Inertsil ODS-3 column. The method was proved to be accurate and precise at a linearity range of 0.005-5.00 microg/mL with a correlation coefficient (r) of >or=0.995. The lower limit of quantitation was 0.005 microg/mL. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. Rhein was found to be stable in the battery of stability studies. The application of the assay to pre-clinical pharmacokinetic studies confirmed the utility of the assay to derive pharmacokinetic parameters.     

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.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

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

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

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.