Highly sensitive assay for the determination of therapeutic peptide desmopressin in human plasma by UPLC-MS/MS.

An analytical method based on ultra-performance liquid chromatography with positive ion electrospray ionization (ESI) coupled with tandem mass spectrometry detection (UPLC-MS/MS) was developed and validated for the determination of therapeutic peptide desmopressin in human plasma. A desmopressin stable labeled isotope (desmopressin d8) was used as an internal standard. Analyte and the internal standard were extracted from 200 µL of human plasma via solid-phase extraction technique using Oasis WCX cartridges. The chromatographic separation was achieved on an Aquity UPLC HSS T3 column by using a gradient mixture of methanol and 1 mM ammonium formate buffer as the mobile phase. The calibration curve obtained was linear (r2 ≥0.99) over the concentration range of 1.01-200 pg/mL. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. The proposed method was successfully applied to pharmacokinetic studies in humans.

LC-MS/MS assay for the determination of lurasidone and its active metabolite, ID-14283 in human plasma and its application to a clinical pharmacokinetic study.

The authors proposed a sensitive, selective and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay procedure for the quantification of lurasidone and its active metabolite, i.e. ID-14283 in human plasma simultaneously using corresponding isotope labeled compounds as internal standards as per regulatory guidelines. After liquid-liquid extraction with tert-butyl methyl ether, the analytes were chromatographed on a C18 column using an optimized mobile phase composed of 5 mm ammonium acetate (pH 5.0) and acetonitrile (15:85, v/v) and delivered at a flow rate of 1.00 mL/min. The assay exhibits excellent linearity in the concentration ranges of 0.25-100 and 0.10-14.1 ng/mL for lurasidone and ID-14283, respectively. The precision and accuracy results over five concentration levels in four different batches were well within the acceptance limits. Lurasidone and ID-14283 were found to be stable in battery of stability studies. The method was rapid with the chromatographic run time 2.5 min, which made it possible to analyze 300 samples in a single day. Additionally, this method was successfully used to estimate the in vivo plasma concentrations of lurasidone and ID-14283 obtained from a pharmacokinetic study in south Indian male subjects and the results were authenticated by conducting incurred samples reanalysis. Copyright © 2015 John Wiley & Sons, Ltd.

Liquid chromatography-tandem mass spectrometric assay for eltrombopag in 50µL of human plasma: a pharmacokinetic study.

Eltrombopag is a thrombopoietin receptor agonist, used in the treatment of thrombocytopenia. This paper describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay method for the determination of eltrombopag in human plasma samples using eltrombopag 13C4 as internal standard (IS). Analyte and the IS were extracted from 50µL of human plasma using protein precipitation technique with no drying, evaporation and reconstitution steps. The chromatographic separation was achieved on a C18 column by using a mixture of 10mM ammonium formate (pH3) and acetonitrile (10:90, v/v) as the mobile phase at a flow rate of 1.0mL/min. The linearity of the method was established in the concentration range 50.0-10007ng/mL with r(2)≥0.99. The intra-day and inter-day precision and accuracy results in four validation batches across five concentration levels were well within the acceptance limits. The proposed method was found to be applicable to pharmacokinetic studies.

Bioanalysis of tolvaptan, a novel AVP-V2 receptor antagonist in human plasma by a novel LC-ESI-MS/MS method: a pharmacokinetic application in healthy South Indian male subjects.

A simple, rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) assay method is proposed for the determination of tolvaptan in human plasma samples using tolvaptan d7 as internal standard (IS). Analyte and the IS were extracted from 100 µL of human plasma via simple liquid-liquid extraction. The chromatographic separation was achieved on a C18 column using a mixture of methanol and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r(2) ≥ 0.99) over the concentration range of 0.05-501 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The intra-day and inter-day precision (coefficient of variation) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. A run time of 2.0 min for each sample made it possible to analyze more samples in a short time, thus increasing the productivity. The proposed method was successfully applied to a pharmacokinetic study of 15 mg and 60 mg tolvaptan tablet formulation in healthy South Indian male subjects under fasting condition.

A sensitive LC-MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application.

An improved, simple and highly sensitive LC-MS/MS method has been developed and validated for quantification of febuxostat with 100 µL human plasma using febuxostat-d7 as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid-liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C18 column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1-6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra- and inter-day precisions (%RSD) were within 1.29-9.19 and 2.85-7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans.

Liquid chromatography-tandem mass spectrometric assay for aliskiren, a novel renin inhibitor in micro-volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects.

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 µL of human plasma via liquid-liquid extraction using tert-butyl methyl ether. The chromatographic separation was achieved on a C18 column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r(2) ≥ 0.99) over the concentration range of 0.10-1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies.

Liquid chromatography-tandem mass spectrometric assay for the determination of tetrabenazine and its active metabolites in human plasma: a pharmacokinetic study.

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α-dihydrotetrabenazine and ß-dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 µL aliquots of human plasma via solid-phase extraction using C18 solid-phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C18 column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API-4000 LC-MS/MS in multiple reaction-monitoring mode was used for detection. The calibration curves obtained were linear (r(2) ≥ 0.99) over the concentration range of 0.01-5.03 ng/mL for tetrabenazine and 0.50-100 ng/mL for α-dihydrotetrabenazine and ß-dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.

A rapid and sensitive liquid chromatography-tandem mass spectrometric assay for cycloserine in 50µL of human plasma: Its pharmacokinetic application.

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of cycloserine in human plasma using carbamazepine as internal standard (IS). Analyte and the IS were extracted from the 50µL of human plasma via protein precipitation using acetonitrile. The chromatographic separation was achieved on a C(18) column by using a mixture of acetonitrile-0.5% formic acid buffer (60:40, v/v) as the mobile phase at a flow rate of 0.8mL/min. The calibration curve obtained was linear (r(2)≥0.99) over the concentration range of 50-15,000ng/mL. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5min for each sample made it possible to analyze more number of samples in short time, thus increasing the productivity. The proposed method was found to be applicable to pharmacokinetic studies.

Simultaneous determination of atorvastatin, metformin and glimepiride 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 fully validated for the simultaneous quantification of atorvastatin, metformin and glimepiride in human plasma. Carbamazepine was used as internal standard (IS). The analytes were extracted from 200 µL aliquots of human plasma via protein precipitation using acetonitrile. The reconstituted samples were chromatographed on a Alltima HP C18 column by using a 60:40 (v/v) mixture of acetonitrile and 10 mM ammonium acetate (pH 3.0) as the mobile phase at a flow rate of 1.1 mL/min. The calibration curves obtained were linear (r2 ≥0.99) over the concentration range of 0.50-150.03 ng/mL for atorvastatin, 12.14-1207.50 ng/mL for metformin and 4.98-494.29 ng/mL for glimepiride. The API-4000 LC-MS/MS in multiple reaction monitoring (MRM) mode was used for detection. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. All 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 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers.

A rapid and sensitive liquid chromatography-tandem mass spectrometric assay for duloxetine in human plasma: Its pharmacokinetic application.

This paper describes a simple, rapid and sensitive liquid chromatography-tandem mass spectrometry assay for the determination of duloxetine in human plasma. A duloxetine stable labeled isotope (duloxetine d5) was used as an internal standard. Analyte and the internal standard were extracted from 100 µL of human plasma via solid phase extraction technique using Oasis HLB cartridges. The chromatographic separation was achieved on a C18 column by using a mixture of acetonitrile-5 mM ammonium acetate buffer (83:17, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r2≥0.99) over the concentration range of 0.05-101 ng/mL. Multiple-reaction monitoring mode (MRM) was used for quantification of ion transitions at m/z 298.3/154.1 and 303.3/159.1 for the drug and the internal standard, respectively. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.

Simultaneous determination of carisoprodol and aspirin in human plasma using liquid chromatography-tandem mass spectrometry in polarity switch mode: application to a human pharmacokinetic study.

A simple, sensitive and rapid LC-MS/MS-ESI method has been developed and validated for simultaneous quantification of the carisoprodol and aspirin in human plasma. Carisoprodol was detected in positive ion mode, whereas aspirin was detected in negative ion mode. Carbamazepine and furosemide were used as internal standards (IS) for quantification of carisoprodol and aspirin, respectively. The extraction procedure involves a liquid-liquid extraction method with ter-butyl methyl ether. Chromatographic separation was achieved on a Zorbax XDB-Phenyl (4.6 × 75 mm, 3.5 µm) column using an isocratic mobile phase (5 mm ammonium acetate:methanol, 20:80, v/v) at a flow rate of 0.8 mL/min with a total run time of 2.2 min. A detailed method validation was performed as per the FDA guidelines. The standard curves found to be linear in the range of 25.5-4900 and 15.3-3000 ng/mL for carisoprodol and aspirin, respectively. The results met the acceptance criteria. Carisoprodol and aspirin were found to be stable in various stability studies. The validated method was successfully applied to a pharmacokinetic study following co-administration of carisoprodol (250 mg) and aspirin (75 mg) tablets by oral route to human volunteers.

Liquid chromatography-tandem mass spectrometric assay for the non-nucleoside reverse transcriptase inhibitor rilpivirine in human plasma.

An analytical method based on liquid chromatographic-tandem mass spectrometry (LC-MS/MS) was developed for the determination of the non-nucleoside reverse transcriptase inhibitor rilpivirine in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from human plasma by liquid-liquid extraction. The reconstituted samples were chromatographed on a C(18) column using a mixture of acetonitrile and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The linearity was confirmed in the concentration range 0.51-200 ng/mL in human plasma. Multiple reaction monitoring mode was used for quantification of ion transitions at m/z 367.2/195.1 and 267.1/226.1 for the drug and the internal standard, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Extraction recoveries of drug from plasma were >69.5%. A run time of 2.50 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed method is simple, rapid and sensitive for the determination of rilpivirine concentrations in real-time plasma samples obtained from pharmacokinetic studies.

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 C(18) 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 (r(2) ≥ 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.

Simultaneous Determination of Atorvastatin and Aspirin in Human Plasma by LC-MS/MS: Its Pharmacokinetic Application.

A simple, rapid, and sensitive liquid chromatography tandem mass spectro-metric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin and aspirin in human plasma using a polarity switch. Proguanil and furosemide were used as the internal standards for the quantification of atorvastatin and aspirin, respectively. The analytes were extracted from human plasma by the liquid-liquid extraction technique using methyl tert-butyl ether. The reconstituted samples were chromatographed on a Zorbax XDB Phenyl column by using a mixture of 0.2% acetic acid buffer, methanol, and acetonitrile (20:16:64, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Prior to detection, atorvastatin and aspirin were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The ions were monitored at the positive m/z 559.2→440.0 transition for atorvastatin and the negative m/z 179.0→136.6 transition for aspirin. The calibration curve obtained was linear (r(2) ≥ 0.99) over the concentration range of 0.20-151 ng/mL for atorvastatin and 15.0-3000 ng/mL for aspirin. The method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 3.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The proposed method was found to be applicable to clinical studies.

A rapid and sensitive liquid chromatography-tandem mass spectrometric assay for moexipril, an angiotensin-converting enzyme inhibitor in human plasma.

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of angiotensin-converting enzyme inhibitor, moexipril, in human plasma. Benazepril was used as an internal standard (IS). Analyte and IS were extracted from the human plasma by liquid-liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on a C18 column by using a mixture of methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.2-204 ng/mL. The multiple reaction-monitoring mode was used for quantification of ion transitions at m/z 499.4/234.2 and 425.2/351.1 for moexipril and IS, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 400 plasma samples per day. The proposed method was found to be applicable to clinical studies.

Liquid chromatography-tandem mass spectrometric assay for the light sensitive calcium channel antagonist lacidipine in human plasma.

A novel, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of calcium channel antagonist lacidipine in human plasma. Carbamazepine was used as an internal standard. Analyte and the internal standard were extracted from human plasma by solid-phase extraction technique. The reconstituted samples were chromatographed on a C(18) column by using a mixture of acetonitrile-ammonium acetate buffer (5 mM) (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r(2)≥0.9990) over the concentration range of 0.05-12.5 ng/mL. The multiple reaction-monitoring mode was used for quantification of ion transitions at m/z 456.2/354.2 and 237.1/194.1 for the drug and the internal standard, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.2 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.

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.

A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study.

A new, rapid, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous quantification of tenofovir and lamivudine in human plasma using abacavir as an internal standard. An API-4000 LC-MS/MS with electrospray ionization was operated in multiple-reaction monitoring mode for the analysis. The analytes were extracted from plasma by solid-phase extraction technique using an Oasis HLB cartridge. The reconstituted samples were chromatographed on a Chromolith ROD speed C(18) column using a mixture of 0.1% formic acid in water and acetonitrile (90:10 v/v) at a flow-rate of 1 mL/min. The method was validated as per the FDA guidelines. The calibration curves were found to be linear in the range of 5-600 ng/mL for tenofovir and 25- 4000 ng/mL for lamivudine. The intra- and inter-day precision and accuracy results were well within the acceptable limits. A run time of 2.8 min consumed for each sample made it possible to analyze more samples per day. The proposed assay method was found to be applicable to a pharmacokinetic study in human male volunteers.

Simultaneous determination of losartan, losartan acid and amlodipine in human plasma by LC-MS/MS and its application to a human pharmacokinetic study.

INTRODUCTION: A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric assay method has been developed and fully validated for simultaneous quantification of losartan and its active metabolite, losartan carboxylic acid, and amlodipine in human plasma. Irbesartan was used as an internal standard. MATERIALS AND METHODS: The analytes were extracted from human plasma samples by solid-phase extraction technique using Oasis HLB cartridges, (Waters Corporation, Mumbai, India). The reconstituted samples were chromatographed on a C18 column by using an 85:15, v/v mixture of methanol and 0.1% v/v formic acid as the mobile phase at a flow rate of 1.0 mL/min. A detailed validation of the method was performed as per the FDA guidelines. RESULTS: The calibration curves obtained were linear (r ≥ 0.99) over the concentration range of 0.5-1000 ng/mL for losartan and for its active metabolite losartan acid and 0.05-10.1 ng/mL for amlodipine. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. CONCLUSIONS: A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.

High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma: Pharmacokinetic application.

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of pravastatin and aspirin in human plasma. Furosemide was used as an internal standard. Analytes and the internal standard were extracted from human plasma by liquid-liquid extraction technique using methyl tertiary butyl ether. The reconstituted samples were chromatographed on a Zorbax SB-C18 column by using a mixture of 5 mM ammonium acetate buffer and acetonitrile (20:80, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 0.50-600.29 ng/mL for pravastatin and 20.07-2012.00 ng/mL for aspirin. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.