Simultaneous quantification of hyperin,reynoutrin and guaijaverin in mice plasma by LC‐MS/MS: application to a pharmacokinetic study

A specific and sensitive LC‐MS/MS assay was developed to simultaneously quantify three structurally similar flavonoid glycosides – hyperin, reynoutrin and guaijaverin – in mouse plasma. Biosamples were prepared by solid‐phase extraction. Isocratic chromatographic separation was performed on an AichromBond‐AQ C₁₈ column (250 × 2.1 mm, 5 μm) with methanol–acetonitrile–water–formic acid (20:25:55:0.1) as the mobile phase. Detection of hyperin, reynoutrin, guaijaverin and internal standard [luteolin‐7‐O‐β‐d ‐apiofuranosyl‐(1 → 6)‐β‐d ‐glucopyranoside] was achieved by ESI‐MS/MS in the negative ion mode using m/z 463 → m/z 300, m/z 433 → m/z 300, m/z 433 → m/z 300 and m/z 579 → m/z 285 transitions, respectively. Linear concentration ranges of calibration curves were 4.0–800.0 ng/mL for hyperin and reynoutrin and 8.0–1600.0 ng/mL for guaijaverin when 100 μL of plasma was analyzed. We used this validated method to study the pharmacokinetics of hyperin, reynoutrin and guaijaverin in mice following oral and intravenous administration. All three quercetin‐3‐O‐glycosides showed poor oral absorption in mice, and the absolute bioavailability of hyperin after oral administration of 100 mg/kg was 1.2%. Pretreatment with verapamil increased the peak concentration and area under the concentration–time curve of hyperin, which were significantly higher than the control values. The half‐life of hyperin with verapamil was significantly prolonged compared with that of the control. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

LC‐MS/MS methods for the determination of edaravone and/or taurine in rat plasma and its application to a pharmacokinetic study

Three liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid–methanol, isocratic 0.1% formic acid–methanol (90:10) and 0.02% formic acid–methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H]⁺ 175.1 → 133.0 and [M + H]⁺ 189.2 → 147.0 for edaravone and its IS, m/z [M ? H]^? 124.1 → 80.0 and [M ? H]^? 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co‐administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration–time curve, mean residence time, half‐life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. 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.     

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 application of a LC–MS/MS assay for the simultaneous quantification of edaravone and taurine in beagle plasma

An LC–MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 μm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor‐to‐product transitions of m/z [M+H]⁺ 175.1 → 133.0 (edaravone), m/z [M+H]⁺ 189.1 → 147.0 (3‐methyl‐1‐p‐tolyl‐5‐pyrazolone, internal standard, IS), m/z [M–H]^? 124.1→80.0 (taurine), and m/z [M–H]^? 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 μg/mL for edaravone and 0.66 and 2 μg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r ＞ 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs.     

Development of an LC/MS/MS method in order to determine arctigenin in rat plasma: its application to a pharmacokinetic study

In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2–500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and ?80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half‐life and area under the concentration–time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. 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.     

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.     

Determination of asperosaponin VI in dog plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pilot pharmacokinetic study

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the determination of asperosaponin VI in beagle dog plasma using glycyrrhizic acid as the internal standard (IS). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on a Hedera ODS‐2 column using mobile phase of methanol–10 mm ammonium acetate buffer solution containing 0.05% acetic acid (71:29, v/v) at a flow rate of 0.38 mL/min. Asperosaponin VI and the IS were eluted at 2.8 and 1.9 min, respectively, ionized in negative ion mode, and then detected by multiple reaction monitoring. The detection used the transitions of the deprotonated molecules at m/z 927.5 → 603.4 for asperosaponin VI and m/z 821.4 → 645.4 for glycyrrhizic acid (IS). The assay was linear over the concentration range of 0.15–700 ng/mL and was successfully applied to a pilot pharmacokinetic study in beagle dogs. Copyright © 2011 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.     

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.     

A high performance liquid chromatography-tandem mass spectrometric method for the determination of mefenamic acid in human plasma: application to pharmacokinetic study

In the present study, the development and validation of an LC‐MS/MS method for quantifying mefenamic acid in human plasma is described. The method involves liquid–liquid extraction using diclofenac as an internal standard (IS). Chromatographic separation was achieved on a Thermo Hypurity C₁₈, 50 × 4.6 mm, 5 µm column with a mobile phase consisting of 2 m m ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) at a flow‐rate of 0.75 mL/min and the total run time was 1.75 min. Analyte was introduced to the LC‐MS/MS using an atmospheric pressure ionization source. Both the drug and IS were detected in negative‐ion mode using multiple reaction monitoring m/z 240.0 → 196.3 and m/z 294.0 → 250.2, respectively, with a dwell time of 200 ms for each of the transitions. The standard curve was linear from 20 to 6000 ng/mL. This assay allows quantification of mefenamic acid at a concentration as low as 20 ng/mL in human plasma. The observed mean recovery was 73% for the drug. The applicability of this method for pharmacokinetic studies has been established after successful application during a 12‐subject bioavailibity study. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of isoorientin levels in rat plasma after oral administration of Vaccinum bracteatum Thunb. methanol extract by high‐performance liquid chromatography‐tandem mass spectrometry

A simple, sensitive and rapid liquid chromatography tandem mass spectrometry method (LC‐MS/MS) was developed and validated for the determination of plasma isoorientin levels in rats. After simple protein precipitation using methanol, chromatographic analysis was performed using a Synergi 4μ polar‐RP 80A column (150 × 2.0 mm, 4μm) under isocratic conditions and a mobile phase consisting of 0.1% formic acid in water and methanol (80:20, v/v) at a flow rate of 0.2 mL/min. In positive electrospray ionization mode, the protonated precursor and product ion transitions of isoorientin (m/z 449.0 → 299.1) and of puerarin (the internal standard; m/z 417.1 → 297.1) were acquired by multiple reaction monitoring. Calibration curves obtained for plasma showed good linearity over the concentration range 1–1000 ng/mL. The lower limit of quantification was 1 ng/mL. Intra‐ and inter‐day precisions were within 8.8% relative standard deviation. Accuracies ranged from 92.1 and 109.7%. The isoorientin stability in rat plasma under typical handling/storage conditions also found to be acceptable. The developed method was applied successfully to a pharmacokinetic study of isoorientin orally administered as the methanol extract of Vaccinium bracteatum Thunb. or administered as pure isoorientin.     

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.     

An LC–MS/MS method for quantification of HR011303, a novel highly selective urate transporter 1 inhibitor in beagle dogs and the application to a pharmacokinetic study

HR011303 is a novel and highly selective urate transporter 1 (URAT1) inhibitor. In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of HR011303 in beagle dog plasma. Plasma samples were pretreated with protein‐precipitation extraction by acetonitrile and added with a trifluoromethyl substituted analog of HR011303 as internal standard. The chromatographic separation was performed on a Shiseido C₁₈ column (100 × 4.6 mm, i.d., 5 μm) by mobile phases consisting of 5 mm ammonium–formic acid (100:0.1) and acetonitrile–formic acid (100:0.1) solutions in gradient elution. The MS detection was conducted in electrospray positive ionization with multiple reactions monitoring at m/z 338 → 240 for HR011303 and m/z 328 → 230 for the internal standard using 25 eV argon gas collision induced dissociation. The established LC–MS/MS method showed good selectivity, sensitivity, precision and accuracy. The plasma pharmacokinetics of HR011303 in beagle dogs following both oral and intravenous administration were then successfully evaluated using this LC–MS/MS method.     

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.     

Liquid chromatographic/mass spectrometry assay of bromotetrandrine in rat plasma and its application to pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric method (LC‐MS/MS) for the determination of bromotetrandrine in rat plasma has been developed and applied to pharmacokinetic study in Sprague–Dawley (SD) rats after a single oral administration. Sample preparation involves a liquid–liquid extraction with n‐hexane–dichlormethane (65:35, containing 1% 2‐propanol isopropyl alcohol, v/v). Bromotetrandrine and brodimoprim (internal standard, IS) were well separated by LC with a Dikma C₁₈ column using methanol–ammonium formate aqueous solution (20 mm ) containing 0.5% formic acid (60:40, v/v) as mobile phase. Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 703.0 → 461.0 and m/z 339.0 → 281.0 for bromotetrandrine and IS, respectively. The present method exhibited good linearity over the concentration range of 20–5000 ng/mL for bromotetrandrine in rat plasma with a lower limit of quantification of 20 ng/mL. The intra‐ and inter‐day precisions were 2.8–7.5% and 3.2–8.1%, and the intra‐ and inter‐day accuracy ranged from ?4.8 to 8.2% and ?5.6 to 6.2%, respectively. The method was successfully applied to a pharmacokinetic study after a single oral administration to SD rats with bromotetrandrine of 50 mg/kg. Copyright © 2009 John Wiley & Sons, Ltd.