Ultra‐performance liquid chromatography–tandem mass spectrometric assay for the simultaneous determination of brucine,strychnine and brucine N‐oxide in rat plasma: application to a pharmacokinetic study

A rapid, simple and sensitive UHPLC‐MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N‐oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP₁₈ UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N‐oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1–500 ng/mL for brucine and strychnine and 0.2?50 ng/mL for brucine N‐oxide. The intra‐ and inter‐day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N‐oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a simple,sensitive and accurate LC‐MS/MS method for the determination of guanfacine,a selective α2A‐adrenergicreceptor agonist,in plasma and its application to a pharmacokinetic study

A simple, practical, accurate and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C₁₈ chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1–20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra‐ and inter‐day precisions were <10.8% relative standard deviation with an accuracy of 92.9–108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended‐release tablets. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation and pharmacokinetics of 1,4‐diamino‐2,3‐dicyano‐1,4‐bis (2‐aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography‐tandem mass spectrometry

A simple, robust, and rapid LC‐MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 μL) were deproteinized using 200 μL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC‐C₁₈ column (4.6 × 50 mm, 2.7 μm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20–5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra‐ and interday, was less than 10.1% with an accuracy of 90.7–99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3‐h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.     

Validated LC‐MS/MS method for the simultaneous determination of hyperoside and 2′′–O‐galloylhyperin in rat plasma: application to a pharmacokinetic study in rats

An LC‐MS/MS method was developed for the first time to simultaneously determine hyperoside and 2′′–O‐galloylhyperin, two major components in Pyrola calliantha extract, in rat plasma. Following extraction by one‐step protein precipitation with methanol, the analytes were separated on a Venusil MP‐C₁₈ column within 2 min, using methanol–water–formic acid (50:50:0.1, v/v/v) as the mobile phase at a flow rate of 0.4 mL/min. Detection was performed on electrospray negative ionization mass spectrometry by multiple‐reaction monitoring of the transitions of 2′′–O‐galloylhyperin at m/z 615.1 → 301.0, of hyperoside at m/z 463.1 → 300.1, and of internal standard at m/z 415.1 → 295.1. The limits of quantification were 2 ng/mL for both hyperoside and 2′′–O‐galloylhyperin. The precisions were <13.1%, and the accuracies were between ?9.1 and 5.5% for both compounds. The method was successfully applied in pharmacokinetic studies following intravenous administration of the total flavonoids of P. calliantha extract in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU‐48 in rat plasma and its application to a pharmacokinetic study

A specific, sensitive and stable high‐performance liquid chromatographic–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantitative determination of methyl 3‐amino‐6‐methoxythieno [2,3‐b]quinoline‐2‐carboxylate (PU‐48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU‐48 was achieved with a reversed‐phase C₁₈ column (100 × 2.1 mm, 3 μm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple‐quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU‐48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass‐to‐charge ratio (m/z) 289.1 → 229.2 for PU‐48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU‐48 was linear over the concentration range of 0.1–1000 ng/mL (r² > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU‐48 in rats.     

Simultaneous determination of ferulic acid,paeoniflorin, and albiflorin in rat plasma by ultra‐high performance liquid chromatography with tandem mass spectrometry: Application to a pharmacokinetic study of Danggui‐Shaoyao‐San

A rapid, selective, and sensitive ultra‐high performance liquid chromatography‐tandem mass spectrometry method was developed for simultaneous determination of ferulic acid, paeoniflorin, and albiflorin, the major active constituents of Danggui‐Shaoyao‐San, in rat plasma using geniposide as the internal standard. The plasma samples were processed by protein precipitation with acetonitrile, and then separated on a Shim‐Pack XR‐ODS C₁₈ column (75 mm × 3.0 mm, 2.2 μm) using gradient elution program with a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.4 mL/min. The detection was achieved on a 3200 QTRAP mass spectrometer equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reaction monitoring mode by monitoring the fragmentation of m/z 192.9→134.0 for ferulic acid, m/z 525.0→120.9 for paeoniflorin, m/z 525.2→121.0 for albiflorin, and m/z 433.1→225.1 for the internal standard, respectively. The calibration curve was linear in the range of 5–2500 ng/mL for all the three analytes (r ≥ 0.9972) with the lower limit of quantitation of 5 ng/mL. The intraday and interday precisions were below 12.1% for all the analytes in terms of relative standard deviation, and the accuracy was within ±11.5% in terms of relative error. The extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of ferulic acid, paeoniflorin, and albiflorin after oral administration of Danggui‐Shaoyao‐San to rats.     

Quantitative determination of saroglitazar,a predominantly PPAR alpha agonist,in human plasma by a LC–MS/MS method utilizing electrospray ionization in a positive mode

A sensitive LC–MS/MS method was developed and validated for quantitation of saroglitazar using turboion spray interface with positive ion mode. A liquid–liquid extraction, with a mixture of dichloromethane and diethyl ether, was employed for the extraction of saroglitazar and glimepiride (IS) from human plasma. The chromatographic separation was achieved using an ACE‐5, C₁₈ (4.6 × 100 mm) column with a gradient mobile phase comprising acetonitrile and ammonium acetate buffer with trifluoracetic acid in purified water. Both analytes were separated within 10 min with retention times of 4.52 and 2.57 min for saroglitazar and IS, respectively. Saroglitazar quantitation was achieved by the summation of two MRM transition pairs (m/z 440.2 to m/z 366.0 and m/z 440.2 to m/z 183.1), while that of IS was achieved using transition pair m/z 491.3 to m/z 352.0. The calibration standards of saroglitazar showed linearity from 0.2 to 500 ng/mL, with a lower limit of quantitation of 0.2 ng/mL. The biases for inter‐ and intra‐batch assays were ?7.51–1.15% and ?11.21 to ?3.25%, respectively, while the corresponding precisions were 5.04–8.06% and 1.53–7.68%, respectively. The developed method was used to monitor the plasma concentrations of saroglitazar in clinical samples.     

Simultaneous determination of idelalisib and its metabolite GS‐563117 in dog plasma by LC–MS/MS: Application to a pharmacokinetic study

The purpose of this study was to develop and validate an LC–MS/MS method for simultaneous determination of idelalisib and GS‐563117 in dog plasma. The analytes were extracted using ethyl acetate and then separated on a Waters Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, i. d., 1.7 μm) using 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.3 mL/min in gradient elution mode. The analytes were quantified using selected reaction monitoring with precursor‐to‐product transitions at m/z 416.2 → 176.1, m/z 432.2 → 192.1 and m/z 421.2 → 176.1 for idelalisib, GS‐563117 and [²H₅]‐idelalisib (internal standard). The assay showed good linearity (r > 0.9992) over the tested concentration range of 0.1–600 ng/mL for idelalisib and 0.1–300 ng/mL for GS‐563117. The intra‐ and inter‐day RSD values for idelalisib and GS‐563117 were <8.84 and 12.41%, respectively. The intra‐ and inter‐day RE values were within the range of ?7.21–8.52%, and ?6.44–14.23%, respectively. The extraction recovery was found to be >84.59% and no matrix effects were observed. The validated LC–MS/MS method has been successfully applied for the simultaneous determination of idelalisib and GS‐563117 in a pharmacokinetic study in dogs. Our results suggested that idelalisib was rapidly metabolized into its metabolite GS‐563117 in dog and the in vivo exposure of GS‐563117 was 17.59% of that of idelalisib.     

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

A simple and sensitive analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood–brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC^TM BEH C₁₈ column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol–water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0–83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra‐ and inter‐day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1–9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood–brain barrier permeability. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantitation of rosuvastatin and ezetimibe in human plasma by LC–MS/MS: Pharmacokinetic study of fixed‐dose formulation and separate tablets

A simple, high‐throughput and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous estimation of rosuvastatin and free ezetimibe. Liquid–liquid extraction was carried out using methyl‐tert butyl ether after prior acidification from 300 μL human plasma. The recovery for both the analytes and their deuterated internal standards (ISs) ranged from 95.7 to 99.8%. Rosuvastatin and ezetimibe were separated on Symmetry C₁₈ column using acetonitrile and ammonium formate buffer, pH 3.5 (30:70, v/v) as the mobile phase. The analytes were well resolved with a resolution factor of 3.8. Detection and quantitation were performed under multiple reaction monitoring using ESI(+) for rosuvastatin (m/z 482.0 → 258.1) and ESI(−) for ezetimibe (m/z 407.9 → 271.1). A linear response function was established in the concentration ranges of 0.05–50.0 ng/mL and 0.01–10.0 ng/mL for rosuvastatin and ezetimibe, respectively, with correlation coefficient, r² ≥ 0.9991. The IS‐normalized matrix factors for the analytes ranged from 0.963 to 1.023. The developed method was successfully used to compare the pharmacokinetics of a fixed‐dose combination tablet of rosuvastatin‐ezetimibe and co‐administered rosuvastatin and ezetimibe as separate tablets to 24 healthy subjects. The reliability of the assay was also assessed by reanalysis of 115 subject samples.     

Ultra‐performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand,in rat plasma and its application to in vivo pharmacokinetics

Methamphetamine abuse continues as a major problem in the USA owing to its powerful psychological addictive properties. AZ66, 3‐[4‐(4‐cyclohexylpiperazine‐1‐yl)pentyl]‐6‐fluorobenzo[d]thiazole‐2(3H)‐one, an optimized sigma receptor ligand, is a promising therapeutic agent against methamphetamine. To study the in vivo pharmacokinetics of this novel sigma receptor ligand in rats, a sensitive ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed in rat plasma and validated. The developed method requires a small volume of plasma (100 μL) and a simple liquid–liquid extraction. The chromatographic separations were achieved in 3.3 min using an Acquity UPLC BEH Shield RP₁₈ column. The mass spectrophotometric detection was carried out using a Waters Micromass Quattro MicroTM triple‐quadrupole system. Multiple reaction monitoring was used for the quantitation with transitions m/z 406 → m/z 181 for AZ66 and m/z 448 → m/z 285 for aripiprazole. The method was validated over a concentration range of 1–3500 ng/mL and the lower limit of quantitation was determined to be 1 ng/mL. Validation of the assay demonstrated that the developed UPLC/MS/MS method was sensitive, accurate and selective for the determination of AZ66 in rat plasma. The present method has been successfully applied to an i.v. pharmacokinetic study in Sprague–Dawley rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of silodosin and its active glucuronide metabolite KMD‐3213G in human plasma by LC–MS/MS for a bioequivalence study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is described for the simultaneous determination of silodosin (SLD) and its active metabolite silodosin β‐d ‐glucuronide (KMD‐3213G) in human plasma. Liquid–liquid extraction of plasma samples was carried out with ethyl acetate and methyl tert‐butyl ether solvent mixture using deuterated analogs as internal standards. The extraction recoveries of SLD and KMD‐3213G were in the ranges 90.8–93.4 and 87.6–89.9%, respectively. The extracts were analyzed on a Symmetry C₁₈ (50 × 4.6 mm, 5 μm) column under gradient conditions using 10 mm ammonium formate in water and methanol–acetonitrile (40:60, v/v), within 6.0 min. For MS/MS measurements, ionization of the analytes was carried out in the positive ionization mode and the transitions monitored were m/z 496.1 → 261.2 for SLD and m/z 670.2 → 494.1 for KMD‐3213G. The method showed good linearity, accuracy, precision and stability in the range 0.10–80.0 ng/mL for SLD and KMD‐3213G. The IS‐normalized matrix factors obtained were highly consistent, ranging from 0.962 to 1.023 for both analytes. The method was used to support a bioequivalence study of SLD and its metabolite in healthy volunteers after oral administration of 8 mg silodosin capsules.     

A validated LC–MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay

An accurate and sensitive LC–MS/MS method for determining thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 μL) by liquid–liquid extraction with ethyl acetate and then separated on a BETASIL C₁₈ column (4.6 × 150 mm, 5 μm) with mobile phase composed of methanol–water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor‐to‐product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5‐hydroxy thalidomide, m/z 273.2 → 146.1 for 5′‐hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0–2000.0 ng/mL for thalidomide, 0.2–50.0 ng/mL for 5‐hydroxy thalidomide and 1.0–200.0 ng/mL for 5′‐hydroxy thalidomide. The method was validated with respect to linear, within‐ and between‐batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.     

Simultaneous determination of tapentadol and its carbamate prodrug in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study

A prodrug of tapentadol, namely tapentadol carbamate (WWJ01), was synthesized to improve the bioavailability of tapentadol owing to its extensive first‐pass metabolism. In this study, a highly rapid and sensitive UPLC‐MS/MS method was developed and validated for the simultaneous determination of tapentadol and WWJ01 in rat plasma with fluconazole as an internal standard. The analytes and internal standard were treated by methanol and then separated on a Phenomenex Kinetex® XB‐C₁₈ (2.1 × 50 mm × 2.6 μm) column at a flow rate of 0.3 mL/min. The mobile phase comprised methanol and water with a gradient elution. The mass transition ion‐pairs were m/z 222.2 → 107.0, m/z 293.2 → 71.9 and m/z 307.1 → 220.0 for tapentadol, WWJ01 and IS, respectively. Excellent linearity was observed over the concentration range of 2–1250 ng/mL (r = 0.995) with a lower limit of quantification of 2 ng/mL for both tapentadol and WWJ01. The intra‐ and inter‐day accuracy and precision for all quality control samples were within ±15%. The validated method was accurate, rapid and reproducible, and was successfully applied to a pharmacokinetic study of tapentadol and WWJ01.     

Determination of sunitinib and its active metabolite,N‐desethyl sunitinib in mouse plasma and tissues by UPLC‐MS/MS: assay development and application to pharmacokinetic and tissue distribution studies

A simple, sensitive and specific method using ultraperformance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) was developed to determine sunitinib and N‐desethyl sunitinib in mouse plasma and tissues. The analytes were separated by an isocratic mobile phase consisting of acetonitrile and buffer solution (water with 0.1% formic acid and 5 m m ammonium acetate; 40: 60, v/v) running at a flow rate of 0.35 mL/min for 2 min. Quantification was performed using a mass spectrometer by multiple reaction monitoring in positive electrospray ionization mode. The transition was monitored at m/z 399 → 283, m/z 371 → 283 and m/z 327 → 270 for sunitinib, N‐desethyl sunitinib and internal standard, respectively. Calibration curves were linear over concentration ranges of 2–500, 0.5–50 and 1–250 ng/mL for plasma, heart and other biosamples. The method was successfully applied to animal experiments. The pharmacokinetic study indicated that sunitinib was eliminated quickly in mice with a half‐life of 1.2 h; tissue distribution data showed more sunitinib and its metabolite in liver, spleen and lung, which provided reference for further study. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC‐MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under multiple‐reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C₁₈ 100 × 2.1 mm column with a mobile phase of water–methanol–0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple‐reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20–10,000 µg/L (r > 0.998) for bivalirudin. The intra‐ and inter‐day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench‐top, freeze–thaw cycles and long‐term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

A quantitative determination of fluorochloridone in rat plasma by UPLC‐MS/MS method: application to a pharmacokinetic study

A precise, high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC‐MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r² > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra‐ and inter‐day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a sensitive and high‐throughput LC‐MS/MS method for the simultaneous determination of esomeprazole and naproxen in human plasma

A sensitive and high‐throughput LC‐MS/MS method has been developed and validated for the combined determination of esomeprazole and naproxen in human plasma with ibuprofen as internal standard. Solid‐phase extraction was used to extract both analytes and internal standard from human plasma. Chromatographic separation was achieved in 4.0 min on XBridge C₁₈ column using acetonitrile–25 mM ammonium formate (70:30, v/v) as mobile phase. Mass detection was achieved by ESI/MS/MS in negative ion mode, monitoring at m/z 344.19 → 194.12, 229.12 → 169.05 and 205.13 → 161.07 for esomeprazole, naproxen and IS, respectively. The calibration curves were linear from 3.00 to 700.02 ng/mL for esomeprazole and 0.50 to 150.08 ng/mL for naproxen. The intra‐ and inter‐batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. The assay is suitable for measuring accurate esomeprazole and naproxen plasma concentrations in human bioequivalence study following combined administration. Copyright © 2013 John Wiley & Sons, Ltd.