A validated LC–MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study

A highly sensitive LC–MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid–liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25–500 ng/ml for SV (r² ≥ 0.9923) and 0.24–481.23 ng/ml for SVA (r² ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.     

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

A simple, sensitive and specific LC–MS/MS method for simultaneous determination of simvastatin (SV), lovastatin (LV) and niacin (NIA) in human plasma was developed and validated on API‐4000 in positive ion mode. Nevirapine was used as internal standard (IS). The assay procedure involved a simple one‐step liquid–liquid extraction of SV, LV, NIA and the IS from plasma into ethyl acetate. Separation of SV, LV, NIA and the IS was achieved on an Alltima C₁₈ column with a mobile phase consisting of 5 mm ammonium acetate (pH 4.5) and acetonitrile (20:80, v/v) pumped at a flow rate of 1 mL/min. Nominal retention times obtained for SV, LV, NIA and IS were 2.12, 1.67, 0.50 and 0.65 min, respectively. The lower limits of quantification (LLOQ) for SV, LV and NIA were 0.10, 0.10 and 25.2 ng/mL, respectively. The response function was established for the range of concentrations 0.10–101 ng/mL for SV and LV, and 25.2–5020 ng/mL for NIA, with a coefficient of correlation of >0.99 for all the compounds. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The proposed method was found to be applicable to clinical studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitation of simvastatin and its beta-hydroxy acid in human plasma by liquid-liquid cartridge extraction and liquid chromatography/tandem mass spectrometry

A sensitive and reliable procedure for the simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite (SVA) in human plasma was developed and validated. The analytes were extracted simultaneously from 0.5 ml aliquots of human plasma samples by methyl tert-butyl ether (MTBE) via Chem Elut cartridge extraction [also called liquid-solid extraction (LSE) or liquid-liquid cartridge extraction (LLCE)], separated through a Kromasil C(18) column (50 x 2 mm i.d. 5 microm) and detected by tandem mass spectrometry with a turbo ionspray interface. Stable isotope-labeled SV and SVA, (13)CD(3)-SV and (13)CD(3)-SVA, were used as internal standards. SV and SVA were detected in positive and negative ion modes, respectively, via within-run polarity switching. The use of Chem Elut cartridges not only provided a simple and efficient means of plasma sample extraction but also successfully reduced the interconversion between SV and SVA to an undetectable (for lactonization of SVA) or negligible (<0.07%, for hydrolysis of SV) level. The method showed excellent reproducibility, with intra- and inter-assay precisions <4.5% (RSD), and intra- and inter-assay accuracy between 94% and 107% of nominal values, for both analytes. The extraction recoveries were 78% and 87% on average for SV and SVA, respectively. The analyte was found to be stable in plasma through three freeze (-70 degrees C)-thaw (4 degrees C) cycles and for at least 3 h under bench-top storage condition in an ice-bath (4 degrees C), and also in the reconstitution solution at 4 degrees C for at least 24 h. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1) for both analytes, and has proved to be very reliable for the analysis of clinical samples.     

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.     

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.     

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.     

Highly sensitive method for the determination of omeprazole in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of omeprazole (OPZ) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves alkalinization of plasma followed by simple liquid-liquid extraction of OPZ and lansoprazole (internal standard, IS) from human plasma with acetonitrile. Chromatographic separation was achieved with 0.01 M ammonium acetate:acetonitrile (40:60, v/v) at a flow rate of 0.25 mL/min on an Inertsil ODS 3 column with a total run time 2.5 min. The MS/MS ion transitions monitored were 346.1 --> 198.1 for OPZ and 370.1 --> 252.1 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity was observed from 0.05 to 10.0 ng/mL. The intra-day and inter-day precisions were in the ranges 2.09-8.56 and 5.29-8.19%, respectively. This novel method has been applied to a pharmacokinetic study of OPZ in humans.     

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.     

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.     

Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for simultaneous quantification of olanzapine, clozapine, ziprasidone, haloperidol, risperidone, and its active metabolite 9-hydroxyrisperidone, in rat plasma using midazolam as internal standard (IS). The analytes were extracted from rat plasma using a single step liquid-liquid extraction technique. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 0.1 ng/mL and correlation coefficient (R(2)) values for the linear range of 0.1-100 ng/mL were 0.997 or greater for all the analytes. The intra-day and inter-day precision and accuracy were better than 8.05%. The relative and absolute recovery was above 77% and matrix effects were low for all the analytes except for ziprasidone. This validated method has been successfully used to quantify the plasma concentration of the analytes after chronic treatment with antipsychotic drugs.     

UPLC‐MS/MS assay for the simultaneous quantification of carvedilol and its active metabolite 4′‐hydroxyphenyl carvedilol in human plasma to support a bioequivalence study in healthy volunteers

An ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the simultaneous determination of carvedilol and its pharmacologically active metabolite 4′‐hydroxyphenyl carvedilol in human plasma using their deuterated internal standards (IS). Samples were prepared by solid‐phase extraction using 100 μL human plasma. Chromatographic separation of analytes was achieved on UPLC C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile‐4.0 mm ammonium formate, pH 3.0 adjusted with 0.1% formic acid (78:22, v/v) as the mobile phase. The multiple reaction monitoring transitions for both the analytes and IS were monitored in the positive electrospray ionization mode. The method was validated over a concentration range of 0.05–50 ng/mL for carvedilol and 0.01‐10 ng/mL for 4′‐hydroxyphenyl carvedilol. Intra‐ and inter‐batch precision (% CV) and accuracy for the analytes varied from 0.74 to 3.88 and 96.4 to 103.3% respectively. Matrix effect was assessed by post‐column analyte infusion and by calculation of precision values (coefficient of variation) in the measurement of the slope of calibration curves from eight plasma batches. The assay recovery was within 94–99% for both the analytes and IS. The method was successfully applied to support a bioequivalence study of 12.5 mg carvedilol tablets in 34 healthy subjects. Copyright © 2013 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.     

Determination of apomorphine in canine plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a pharmacokinetic study

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were < 5.9% RSD and < 7.5% bias for apomorphine. Extraction recoveries were > 80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.     

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.     

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

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

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.     

Simultaneous determination of cucurbitacin IIa and cucurbitacin IIb of Hemsleya amabilis by HPLC–MS/MS and their pharmacokinetic study in normal and indomethacin‐induced rats

A selective and sensitive HPLC–MS/MS method was developed for the simultaneous determination of cucurbitacin IIa (cuIIa) and cucurbitacin IIb (cuIIb), the major bioactive cucurbitacins of Hemsleya amabilis, in rat plasma using euphadienol as internal standard (IS). After liquid–liquid extraction with dichloromethane, separation was achieved on a Syncronis HPLC C₁₈ column (150 mm × 4.6 mm, 5 μm) using an isocratic mobile phase system consisting of acetonitrile–water (85:15, v/v) at a flow rate of 0.6 mL/min with a split ratio of 1:2. Detection was performed on a TSQ Quantum Ultra mass spectrometer equipped with an positive‐ion electrospray ionization source. The lower limits of quantification (LLOQs) were 0.25 and 0.15 ng/mL for cuIIa and cuIIb, respectively. The intra‐ and inter‐day precision was <11.5% for the LLOQs and each quality control level of the analytes, and accuracy was between ?9.1 and 7.6%. The extraction recoveries of the analytes and IS from rat plasma were all >87.1%. The method was fully validated and applied to compare the pharmacokinetic profiles of the two cucurbitacins in rat plasma after oral administration of H. amabilis extract between normal and indomethacin‐induced rats. Copyright © 2016 John Wiley & Sons, Ltd.     

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

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

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.