A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) 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 S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Validation of a chiral LC‐MS/MS‐ESI method for the simultaneous quantification of darolutamide diastereomers in mouse plasma and its application to a stereoselective pharmacokinetic study in mice

A simple, selective and reliable LC‐MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 μL mouse plasma using warfarin as an internal standard (IS) as per regulatory guidelines. Plasma samples were extracted by liquid–liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mm ammonium acetate–absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done in multiple reaction monitoring mode following the transitions m/z 397 → 202 and 307 → 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100–2400 ng/mL for each diastereomer. The intra‐ and inter‐day precisions were in the ranges of 1.78–4.20 and 4.34–14.6, and 3.63–4.74 and 4.78–5.15 for diastereomer‐1 and diastereomer‐2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer‐1 and diastereomer‐2, respectively. The terminal half‐life was found to be ~0.50 h for both the diastereomers. The AUC_(0–t) was found to be 18,961 ng*h/mL for diastereomer‐1 and 1340 ng*h/mL diastereomer‐2.     

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.     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 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 HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a sensitive LC‐MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection

A sensitive LC‐MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two‐step liquid–liquid extraction. Chromatographic separation was achieved on a C₁₈ analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid–acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r² ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044–1.190 ng/mL in rat serum. Intra‐ and inter‐day precisions were <14.2%. The accuracy expressed as recovery was within the range of 85.1–112.8%. The extraction recoveries were >75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection. Copyright © 2014 John Wiley & Sons, Ltd.     

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. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

A highly sensitive LC-MS/MS method for the determination of S-raclopride in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and rapid assay method has been developed and validated for the estimation of S‐(−)‐raclopride (S‐RCP) 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 technique for extraction of S‐RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C₁₈ column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S‐RCP and 180.1 → 110.1 for IS. Method validation and pre‐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 range was extended from 0.05 to 152 ng/mL in rat plasma. The intra‐day and inter‐day precisions were 0.23–10.5 and 3.74–7.29%, respectively. This novel method was applied to a pharmacokinetic study of S‐RCP in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a rapid LC‐MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring

A selective, rapid, and sensitive liquid chromatography–tandem mass spectrometry(LC‐MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one‐step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed‐phase YMC‐ODS‐C₁₈ column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0–60.0 ng/mL. Intra‐ and inter‐batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions. Copyright © 2015 John Wiley & Sons, Ltd.     

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 C₁₈ 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. Copyright © 2010 John Wiley & Sons, Ltd.     

A UPLC–MS/MS method for comparative pharmacokinetics study of morusin and morin in normal and diabetic rats

The aim of this study was to establish and validate a rapid, selective and reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for simultaneous quantitations of morin and morusin, and to investigate their pharmacokinetics difference between normal and diabetic rats after oral administration. Plasma samples were pretreated via protein precipitation with acetonitrile. Genkwanin was used as internal standard (IS). Analytes and IS were separated on a Thermo Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 μm) using gradient elution. The mobile phase consisted of acetonitrile and 0.1% formic acid in water at a flow rate of 0.5 mL/min. Mass spectrometry detection was carried out by means of negative electrospray ionization source and multipe‐reaction monitoring mode. The transitions of m/z 300.9 → 151.2 for morin, m/z 419.2 → 297.1 for morusin and m/z 283.1 → 268.2 for IS were chosen for quantification. Calibration curves were linear in the range of 1.01–504.2 ng/mL (r² ≥ 0.99) for morin and 1.02–522.3 ng/mL (r² ≥ 0.99) for morusin. The lower limit of quantification was 1.02 ng/mL for morin and 1.05 ng/mL for morusin. The extraction recovery was >85.1% for each analyte. No obvious matrix effect was observed under the present UPLC–MS/MS conditions during all of the bioanalysis. The stability study demonstrated that morin and morusin remained stable during the whole analytical procedure. The method was successfully applied to support the pharmacokinetic comparisons of morin and morusin between normal and diabetic rats.     

Development and validation of a robust LC‐MS‐MS with atmospheric pressure chemical ionization for quantitation of carbazochrome sodium sulfonate in human plasma: application to a pharmacokinetic study

A highly selective and sensitive liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC‐APCI‐MS‐MS) was developed and validated for the quantitation and pharmacokinetic study of carbazochrome sodium sulfonate in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and amiloride hydrochloride was employed as an internal standard. The analytes were separated on a Hypersil ODS‐2 column by a multiple‐step linear gradient elution with a mobile phase consisting of 0.2% formic acid solution and methanol pumped at a flow rate of 1.0 mL/min. The determination was optimized and carried out with positive atmospheric pressure chemical ionization by selective reaction monitoring of the ion of m/z 148, the protonated thermodegraded fragment of the free acidic form of carbazochrome sodium sulfonate selected as the parent, and the ion of m/z 107 as the optimum collision induced dissociation (CID) product. The method was fully validated over a concentration range of 0.5–50 ng/mL, with the lower limit of quantitation of 0.5 ng/mL. The application of the LC‐MS‐MS method was demonstrated for the specific and quantitative analysis of carbazochrome sodium sulfonate in human plasma from a pharmacokinetic study in 24 healthy male Chinese volunteers after a single oral administration of 90 mg carbazochrome sodium sulfonate capsules. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation of a sensitive LC/MS/MS method for the determination of telaprevir and its R‐isomer in human plasma

The purpose of this study was to validate a reversed‐phase high‐performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R‐diastereomer (VRT‐127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir‐R was performed on a Waters XBridge^TM BEH Shield C₁₈, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir‐d11). An internal standard for the telaprevir‐R (telaprevir‐R‐d11) was also prepared by incubating telaprevir‐d11 in basic solution, which facilitated isomer inter‐conversion. The detection and quantitation of telaprevir, telaprevir‐R, telaprevir‐IS and telaprevir‐R‐IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and application of an analytical method for curdione quantification in pregnant sprague–dawley rats by LC‐MS/MS

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague–Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C₁₈ MGIII column. A gradient elution profile with 0.5% formic acid (A)–0.5% formic acid–acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5–500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5–101.1%. The intra‐day and inter‐day precisions were between 2.35 and 9.08%. This LC‐MS/MS method provides a simple and sensitive means for determining the plasma concentration. Copyright © 2015 John Wiley & Sons, Ltd.     

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. Copyright © 2013 John Wiley & Sons, Ltd.     

An improved LC‐MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study

An improved LC‐MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide‐d3 was used as internal standard (IS) and liquid–liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP‐column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 → m/z 188.9 and m/z 367.0 → m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25–50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration–time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption. Copyright © 2014 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.