Development and validation of highly selective and robust method for simultaneous estimation of pioglitazone, hydroxypioglitazone and metformin in human plasma by LC-MS/MS: application to a pharmacokinetic study.

A simple, selective and robust reverse phase high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous quantitation of pioglitazone (PIO), pioglitazone metabolite M-IV - hydroxypioglitazone (OH-PIO) and metformin (MET) in human plasma using deuterated internal standards (IS) is developed and fully validated as per industrial practices. After acetonitrile-induced protein precipitation of the plasma samples; PIO, OH-PIO, MET and IS were chromatographed on reverse phase column and analyzed in the multiple reaction monitoring in positive ion mode. The ion transitions were monitored at m/z 357.2→134.2 for PIO, 373.0→150.1 for OH-PIO, 130.2→71.0 for MET, 361.1→134.2 for PIO-IS and 136.1→77.1 for MET-IS. The total chromatographic run time was 4.0min. A linear response function (r>0.998) was established for the range of concentrations 15-2500ng/mL, 10-1500ng/mL and 25-3000ng/mL for PIO, OH-PIO and MET respectively in human plasma. The intra and inter-day precision and accuracy values have met the set acceptance criteria. The method is simple, selective, robust economic and has been applied successfully to more than 2000 plasma samples as part of pharmacokinetic 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-(13) C8 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.

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.

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.

Validation and clinical application of an LC-ESI-MS/MS method for simultaneous determination of tolmetin and MED5, the metabolites of amtolmetin guacil in human plasma.

A highly sensitive, rapid assay method has been developed and validated for the simultaneous estimation of tolmetin (TMT) and MED5 in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. A simple solid-phase extraction process was used to extract TMT and MED5 along with mycophenolic acid (internal standard, IS) from human plasma. Chromatographic separation was achieved with 0.2% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.50 mL/min on an X-Terra RP(18) column with a total run time of 2.5 min. The MS/MS ion transitions monitored were 258.1 → 119.0 for TMT, 315.1 → 119.0 for MED5 and 321.2 → 207.0 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 20 ng/mL and the linearity was observed from 20 to 2000 ng/mL, for both the anlaytes. The intra-day and inter-day precisions were in the range 3.27-4.50 and 5.32-8.18%, respectively for TMT and 4.27-5.68 and 5.32-8.85%, respectively for MED5. This novel method has been applied to a clinical pharmacokinetic study.

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 C(18) 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.

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.

LC and LC-MS/MS study of forced decomposition behavior of anastrozole and establishment of validated stability-indicating analytical method for impurities estimation in low dose anastrozole tablets.

Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. Separation of anastrozole from its potential impurities, degradation products and five anastrozole related compounds as main impurities were achieved on Inertsil ODS-3V, 250 mm x 4.6 mm i.d, 5 microm analytical column using reversed phase high performance liquid chromatography (RP-HPLC). The elution of impurities employed time dependent gradient programmed mobile phase consisting of water as mobile phase-A and acetonitrile as mobile phase-B at column flow rates of 1 ml/min and at 215 nm UV detection. The same method was also extended to LC-MS/MS studies which were carried out to identify the degradation product. The method developed was established to have sufficient intermediate precision as similar separation was achieved on another instrument handled by a different operator. The LOQ for anastrozole related compound-A (RC-A), related compound-B (RC-B), related compound-C (RC-C), related compound-D (RC-D), related compound-E (RC-E) and anastrozole were 0.05, 0.03, 0.03, 0.06, 0.06 and 0.06 microg ml(-1) respectively. The linearity of the proposed method for all the above related compounds was investigated in the range of LOQ to 0.600 microg ml(-1) respectively. The specificity was established through peak purity testing using a photo-diode array detector. Method was validated according to ICH guidelines and statistical analysis of the data proved to be suitable for stability testing at quality control.

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.

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.

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.

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.

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.

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.

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.

Structural identification and characterization of impurities in ceftizoxime sodium.

Ceftizoxime sodium is a parenteral beta-lactamic antibacterial drug. In the synthesis of ceftizoxime sodium, eight process related impurities were detected in HPLC analysis. Pure impurities obtained by both synthesis and preparative HPLC were co-injected with ceftizoxime sample to confirm the retention times in HPLC. The impurities were characterized as, (6R,7R)-7-amino-3-cephem-4-carboxylic acid (impurity I); (6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-3-cephem-1-oxo-4-carboxylic acid (impurity II); (4RS,6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino) acetamido]-3,4-dihydro-3-cephem-4-carboxylic acid (impurity III); (6R,7R)-7-[(E)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-3-cephem-4-carboxylic acid (impurity IV); (6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetamido]-3-cephem-N-(3-cephem-4-carboxy-7-yl)-4-carboxamide (impurity V); (6R,7R)-7-[(Z)-2-[[(Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetylamino]thiazol-4-yl]-2-methoxyiminoacetamido]-3-cephem-4-carboxylic acid (impurity VI); 2-mercaptobenzothiazole (impurity VII) and 2-mercapto benzothiazolyl [(Z)-2-(2-amino-4-thiazolyl)-2-methoxyimino] acetate (impurity VIII). Structural elucidation of all impurities by spectral data ((1)H NMR, (13)C NMR, MS and IR) has been discussed.