Kishore Kumar Hotha

Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: application to a bioequivalence study.

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 microL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C(18) (50 mm x 4.6 mm, 5 microm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r>0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

Simultaneous estimation of four proton pump inhibitors—lansoprazole, omeprazole, pantoprazole and rabeprazole: development of a novel generic HPLC-UV method and its application to clinical pharmacokinetic study

A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of four proton-pump inhibitors (PPI), lansoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ), with 500 microL human plasma using zonisamide as an internal standard (IS). The sample preparation involved simple liquid-liquid extraction of LPZ, OPZ, PPZ and RPZ and IS from human plasma with ethyl acetate. The baseline separation of all the peaks was achieved with 0.1% triethylamine (pH 6.0):acetonitrile (72:28, v/v) at a flow rate of 1 mL/min on a Zorbax C(8) column. The total chromatographic run time was 11.0 min and the simultaneous elution of IS, OPZ, RPZ, PPZ and LPZ occurred at approximately 2.42, 4.45, 5.02 and 9.37 min, respectively. The method was proved to be accurate and precise at linearity range of 20.61-1999.79 ng/mL with a correlation coefficient (r) of >or=0.999. The limit of quantitation for each of the PPI studied was 20.61 ng/mL. 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.

LC–MS/MS method for simultaneous estimation of candesartan and hydrochlorothiazide in human plasma and its use in clinical pharmacokinetics

BACKGROUND ATACAND HCT(®) (candesartan cilexetil-hydrochlorothiazide [CAN-HCTZ]) combines an angiotensin II receptor (type AT1) antagonist and a diuretic. Quantification of CAN and HCTZ in biological matrices has traditionally been difficult - developing a single method with the desired sensitivity has been the issue. RESULTS A high-throughput bioanalytical method for the analysis of CAN and HCTZ in human plasma using liquid-liquid extraction and LC coupled to negative ion mode MS/MS has been developed and validated according to US FDA guidelines. The method uses 100 µl plasma and covers the calibration range 1-160 ng/ml for CAN and 2-160 ng/ml for HCTZ for routine pharmacokinetic studies in humans. The intra- and inter-day precision values for CAN and HCTZ met the acceptance criteria. CAN and HCTZ were stable in a battery of stability studies (benchtop, autosampler and long-term). CONCLUSION The advantages of the described technique included a single method with a shorter run time (2.5 min), simple extraction technique, LLOQ of 1 ng/ml for CAN and 2 ng/ml for HCTZ and lower sample volume (0.10 ml), which overcomes drawbacks of two single methods for each analyte, such as higher analysis time, LOQ and sample volume, as in previously published methods. The developed assay was applied to an oral pharmacokinetic study in humans.

Quantification of montelukast, a selective cysteinyl leukotriene receptor (CysLT1) antagonist in human plasma by liquid chromatography–mass spectrometry: validation and its application to a human pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of montelukast (MTK) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive-ion mode. Liquid-liquid extraction was used to extract MTK and amlodipine (internal standard, IS) from human plasma. Chromatographic separation was achieved with 10 mM ammonium acetate (pH 6.4): acetonitrile (15:85, v/v) at a flow rate of 0.50 mL/min on a Discovery HS C(18) column with a total run time of 3.5 min. The MS/MS ion transitions monitored were 586.10 --> 422.10 for MTK and 409.20 --> 238.30 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 0.25 ng/mL and linearity was observed from 0.25 to 800 ng/mL. The intra-day and inter-day precisions were 5.97-8.33 and 7.09-10.13%, respectively. This novel method has been applied to a pharmacokinetic study of MTK in humans.

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.

Simultaneous estimation of amlodipine and atenolol in human plasma: a sensitive LC–MS/MS method validation and its application to a clinical PK study

BACKGROUND A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of amlodipine (AMD) and atenolol (ATL) in human plasma (200 µl) using AMD-d4 and ATL-d7, respectively, as an internal standard (IS) as per the regulatory guidelines. RESULTS The SPE method was used to extract the analytes and IS from human plasma. The chromatographic resolution of AMD, ATL and corresponding IS was achieved using an isocratic flow on a C18 column. The total chromatographic run time was 3 min. A linear response function was established for the range of concentrations 50-8000 pg/ml and 10-800 ng/ml for AMD and ATL, respectively, in human plasma. CONCLUSION The intra- and inter-day accuracy and precision values for AMD and ATL met the acceptance as per regulatory guidelines. The validated assay was applied to a fixed-dose combination of AMD and ATL (Adopin-AT(®)) PK study in humans.

Determination of the quaternary ammonium compound trospium in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A highly sensitive, specific and evaporation free SPE extraction, LC-MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M(+)] cations, m/z 392-164 for trospium and m/z 400-172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05-10 ng/mL (r>0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.

Highly sensitive method for the determination of ropinirole with a lower limit of quantitation of 3.45 pg/mL in human plasma by LC-ESI-MS/MS: application to a clinical pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of ropinirole (RPR) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. A solid-phase process was used to extract RPR and citalopram (internal standard, IS) from human plasma. Chromatographic separation was operated with 0.2% ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Hypurity C(18) column with a total run time of 3.2 min. The MS/MS ion transitions monitored were 261.2 --> 114.2 for RPR and 325.1 --> 209.0 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 3.45 pg/mL and the linearity was observed from 3.45 to 1200 pg/mL. The intra-day and inter-day precisions were in the range of 4.71-7.98 and 6.56-8.31%, respectively. This novel method has been applied to a pharmacokinetic study of RPR 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.