Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in human plasma by liquid chromatographic-mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 microL of supernatant was mixed with 100 microL of 10 mM ammonium formate (pH = 4.0), and a 50 microL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) >/= 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples.     

Determination of rosuvastatin in rat plasma by HPLC: Validation and its application to pharmacokinetic studies

A specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method was developed for the estimation of rosuvastatin (RST), a novel, synthetic and potent HMG-CoA inhibitor in rat plasma. The assay procedure involved simple liquid-liquid extraction of RST and internal standard (IS, ketoprofen) from a small plasma volume directly into acetonitrile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). Mobile phase consisting of 0.05 m formic acid and acetonitrile (55:45, v/v) was used at a flow rate of 1.0 mL/min for the effective separation of RST and IS. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 240 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of RST and IS were 8.6 and 12.5 min, respectively. The standard curve for RST was linear (r2 > 0.999) in the concentration range 0.02-10 microg/mL. Absolute recoveries of RST and IS were 85-110 and >100%, respectively, from rat plasma. The lower limit of quantification (LLOQ) of RST was 0.02 microg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.02, 0.06, 1.6 and 8.0 microg/mL, were in the range 7.24-12.43% relative standard deviation (RSD) and 2.28-10.23% RSD, respectively. Accuracy in the measurement of QC samples was in the range 93.05-112.17% of the spiked nominal values. Both analyte and IS were stable in the battery of stability studies, viz. benchtop, autosampler and freeze-thaw cycles. RST was found to be stable for a period of 30 days on storage at -80 degrees C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.     

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

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of doxofylline (DFL) with 300 microL human serum using imipramine as the internal standard (IS). The API-3,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved direct precipitation of DFL and IS from human serum with acetonitrile. The resolution of peaks was achieved with formic acid (pH 2.5): acetonitrile (10:90, v/v) on an Amazon C(18) column. The total chromatographic run time was 3.0 min and the elution of DFL and IS occurred at approximately 1.46 and 2.15 min, respectively. The MS/MS ion transitions monitored were 267.5 --> 181.1 for DFL and 281.1 --> 86.2 for IS. The method was proved to be accurate and precise at linearity range of 1.00-5,000 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 1.00 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 DFL tablet.     

Highly sensitive LC‐MS/MS method for determination of galantamine in rat plasma: application to pharmacokinetic studies in rats

A rapid and highly sensitive assay method has been developed and validated for the estimation of galantamine (GLM) in rat plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of GLM and phenacetin (internal standard, IS) from rat plasma using acetonitrile. Chromatographic separation was achieved with 0.2% formic acid:acetonitrile (50:50, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 2.5 min. The MS/MS ion transitions monitored were 288.10 → 213.10 for GLM and 180.10 → 110.10 for IS. Method validation was performed as per United States Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.12 ng/mL and linearity was observed from 0.12 to 525 ng/mL. The intra‐ and inter‐day precision were in the ranges of 4.73–11.7 and 5.83–8.64%, respectively. This novel method has been applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for quantitation of indomethacin in rat plasma and its application to a pharmacokinetic study in rats

A highly sensitive and rapid bioanalytical method has been developed and validated for the estimation of indomethacin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of indomethacin and phenacetin (internal standard, IS) from rat plasma with acetonitrile. Chromatographic separation was achieved with 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 3.0 min. The MS/MS ion transitions monitored were 357.7 → 139.1 for indomethacin and 180.20 → 110.10 for IS. Method validation and pharmacokinetic study plasma analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.51 ng/mL and the linearity was observed from 0.51 to 25.5 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.00–10.2 and 5.88–9.80%, respectively. This novel method has been applied to an oral pharmacokinetic study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Rapid and specific approach for direct measurement of glimepiride in human plasma by LC-ESI-MS-MS employing automated 96 well format: application to a bioequivalence study

A rapid liquid chromatographic method with electrospray ionization tandem mass spectrometric (LC-MS-MS) detection is developed and validated for quantification of glimepiride in heparinized human plasma. Plasma samples, without a drying and reconstitution step, are extracted by solid-phase extraction (SPE) and eluted with 0.9 mL of acetonitrile-methanol (1:1, v/v) containing 0.05% formic acid. The analyte and glimepiride d8 (internal standard, IS) are chromatographed on a C(18) column; the mobile phase is acetonitrile-2 mm ammonium formate (88:12, v/v), with the pH adjusted to 3.5 with formic acid, at a flow rate of 0.5 mL/min. The retention times of glimepiride and the IS are 0.93 min, and the runtime is 1.6 min per sample. Selected reaction monitoring of MH(+) at m/z 491.20 and 499.26 result in stable fragment ions with m/z 351.80 and 359.96 for glimepiride and the IS, respectively. The response was a linear function of the concentration in the range of 2.0-650.0 ng/mL, with r ≥ 0.9994. The recovery of glimepiride and the IS ranged from 81.91 to 83.36%. The assay has excellent characteristics and has been successfully used for the analysis of glimepiride in healthy human subjects in a bioequivalence study. It was well suited to clinical studies of the drug involving large numbers of samples.     

Development and validation of an LC-MS/MS-ESI method for the determination of lorglumide, a CCK-1 antagonist in mouse plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method was developed and validated for the estimation of lorglumide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in positive‐ion mode. The assay procedure involves extraction of lorglumide and phenacetin (internal standard, IS) from mouse plasma with simple protein precipitation. Chromatographic separation was achieved using an isocratic mobile (0.2% formic acid solution–acetonitrile, 20:80, v/v) at a flow‐rate of 0.5 mL/min on an Atlantis dC₁₈ column maintained at 40 °C with a total run time of 4.0 min. The MS/MS ion transitions monitored were 459.2 → 158.4 for lorglumide and 180.1 → 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 0.42 ng/mL and the linearity range extended from 0.42 to 500 ng/mL. The intra‐ and inter‐day precisions were in the ranges of 1.47–10.9 and 3.56–7.53, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of lipoic acid in rat plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a pharamcokinetic study

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization using multiple ions for quantitation of torcetrapib in hamster and dog plasma

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of torcetrapib (TTB) with 100 microL hamster/dog plasma using DRL-16126 as an internal standard (IS). The API-4000 Q Trap LC-MS/MS was operated under multiple-reaction monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of TTB and IS from plasma with acetonitrile, which yielded consistent recoveries of 65.73 and 94.01% for TTB and 79.68 and 90.70% for IS in hamster and dog plasma, respectively. The total chromatographic run time was 3.0 min and the elution of TTB and IS occurred at approximately 2.25 and 2.20 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate:acetonitrile (15:85, v/v) at a flow rate of 0.40 mL/min on an Inertsil ODS-3 column. The method was proved to be accurate and precise at linearity range of 1.00-200 ng/mL with a correlation coefficient (r) of > or = 0.993. The method was rugged with 1.00 ng/mL as the lower limit of quantitation. TTB was stable in the battery of stability studies. The application of the assay to preclinical pharmacokinetic studies confirmed the utility of the assay to derive hamster/dog pharmacokinetic parameters.     

Quantitative determination of DRF-1042 in human plasma by HPLC: validation and application in clinical pharmacokinetics

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.     

Eco-Friendly,Simple, Fast,and Sensitive UPLC-MS/MS Method for Determination of Pexidartinib in Plasma and Its Application to Metabolic Stability

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C₁₈ column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.     

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of nobiletin in rat plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of nobiletin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of nobiletin and citalopram (internal standard, IS) from rat plasma with liquid–liquid extraction. Chromatographic separation wa s achieved using an isocratic mobile phase (0.2% formic acid–acetonitrile, 20:80, v/v) at a flow rate of 0.6 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1 °C) with a total run time of 2.0 min. The MS/MS ion transitions monitored were 403.2 → 373.0 for nobiletin and 325.2 → 109.0 for IS. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range extended from 0.05 to 51.98 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.96–14.3 and 6.21–12.1, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of bisoprolol in human plasma

An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned up by protein precipitation with acetonitrile, reconstituted in mobile phase and separated by reversed-phase high-performance liquid chromatography (HPLC) using methanol:10 mm ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on an LC-MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5-100 ng/mL with a limit of quantitation (LOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 and 9.95%, respectively, while the accuracy was in the range 89.4-113%. This method has been utilized in a pharmacokinetic study, where healthy volunteers were treated with an oral dose of 5 mg bisoprolol.     

A Simple HPLC Method for the Determination of Trazodone Human Serum

Abstract

A simple HPLC method with minimal sample preparation and good reproducibility for the determination of trazodone in serum is described. Basified serum samples were extracted using ethyl acetate containing diazepam as the internal standard (IS). Chromatography was performed on a cyanopropylsilane column with 15 μL sample injection. The mobile phase consisted of 0.02 M ammonium phosphate, pH 7.5 : acetonitrile (70:30 v/v). The eluent was monitored at 220 nm. The serum standard curve was linear from 10.0 to 8000.0 ng/mL serum. The overall within-run quality control CV was 6.3% for five concentrations (20.0, 40.0, 100.0, 250.0 and 1000.0 ng/mL) and the overall recovery from serum was 85.4%. This method has been applied to the analysis of human serum samples.     

Development and validation of a highly sensitive LC‐MS/MS‐ESI method for the determination of bicalutamide in mouse plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of bicalutamide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves extraction of bicalutamide and tolbutamide (internal standard, IS) from mouse plasma with a simple protein precipitation method. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid:acetonitrile, 35:65, v/v) at a flow rate of 0.5 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were m/z 428.9 → 254.7 for bicalutamide and m/z 269.0 → 169.6 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 1.04 ng/mL and the linearity range extended from 1.04 to 1877 ng/mL. The intra‐ and inter‐day precisions were in the ranges of 0.49–4.68 and 2.62–4.15, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. 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 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.