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.     

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.     

High‐throughput salting‐out‐assisted liquid–liquid extraction for the simultaneous determination of atorvastatin,ortho‐hydroxyatorvastatin,and para‐hydroxyatorvastatin in human plasma using ultrafast liquid chromatography with tandem mass spectrometry

A high‐throughput, specific, and rapid liquid chromatography with tandem mass spectrometry method was established and validated for the simultaneous determination of atorvastatin and its two major metabolites, ortho‐hydroxyatorvastatin and para‐hydroxyatorvastatin, in human plasma. A simple salting‐out‐assisted liquid–liquid extraction using acetonitrile and a mass‐spectrometry‐friendly salt, ammonium acetate, was employed to extract the analytes from human plasma. A recovery of more than 81% for all analytes was achieved in 1 min extraction time. Chromatographic separation was performed on a Kinetex XB C₁₈ column utilizing a gradient elution starting with a 60% of water solution (1% formic acid), followed by increasing percentages of acetonitrile. Analytes were detected on a tandem mass spectrometer equipped with an electrospray ionization source that was operated in the positive mode, using the transitions of m/z 559.3 → m/z 440.2 for atorvastatin, and m/z 575.3 → m/z 440.2 for both ortho‐ and para‐hydroxyatorvastatin. Deuterium‐labeled compounds were used as the internal standards. The method was validated over the concentration ranges of 0.0200–15.0 ng/mL for atorvastatin and ortho‐hydroxyatorvastatin, and 0.0100–2.00 ng/mL for para‐hydroxyatorvastatin with acceptable accuracy and precision. It was then successfully applied in a bioequivalence study of atorvastatin.     

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2‐carboxyethyl)phosphine as a reducing agent by LC–MS/MS

A simple, robust, and rapid LC–MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2‐carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide‐bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel‐d₄ as an internal standard (IS). The mixtures were separated on a C₁₈ RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI‐MS. The detector was operated in selected reaction‐monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05–20 and 0.5–200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra‐ and interday, was less than 8.26% with an accuracy of 87.6–106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.     

Simultaneous quantification of imatinib and its main metabolite N‐demethyl‐imatinib in human plasma by liquid chromatography–tandem mass spectrometry and its application to therapeutic drug monitoring in patients with gastrointestinal stromal tumor

The aim of this study was to improve and validate a more stable and less time‐consuming method based on liquid chromatography and tandem mass spectrometry (LC‐ MS/MS) for the quantitative measurement of imatinib and its metabolite N‐ demethyl‐imatinib (NDI) in human plasma. Separation of analytes was performed on a Waters XTerra RP₁₈ column (50 × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of methanol–acetonitrile–water (65:20:15, v /v/v) with 0.05% formic acid at a flow‐rate of 0.2 mL/min. The Quattro MicroTM triple quadruple mass spectrometer was operated in the multiple‐reaction‐monitoring mode via positive electrospray ionization interface using the transitions m /z 494.0 → 394.0 for imatinib, m /z 479.6 → 394.0 for NDI and m /z 488.2 → 394.0 for IS. The method was linear over 0.01–10 μg/mL for imatinib and NDI. The intra‐ and inter‐day precisions were all <15% in terms of relative standard deviation, and the accuracy was within ±15% in terms of relative error for both imatinib and NDI. The lower limit of quantification was identifiable and reproducible at 10 ng/mL. The method was sensitive, specific and less time‐consuming and it was successfully applied in gastrointestinal stromal tumor patients treated with imatinib.     

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.     

Simultaneous determination of evobrutinib and its metabolite evobrutinib‐diol in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography hyphenated with electrospray ionization tandem mass spectrometric method (LC–ESI–MS/MS) was developed and validated for simultaneous determination of evobrutinib and evobrutinib‐diol in dog plasma. The plasma sample was processed using acetonitrile and chromatographic separation was carried out on a Waters Acquity BEH C₁₈ column (50 × 2.1 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile, with an optimized gradient elution at a flow rate of 0.4 mL/min. Detection was accomplished in selective reaction monitoring mode via electrospray ionization interface operated in positive ion mode. The precursor‐to‐product transitions for quantification were m/z 430.2 → 98.1 for evobrutinib, m/z 464.2 → 98.1 for evobrutinib‐diol and m/z 441.2 → 138.1 for ibrutinib (internal standard). The developed assay was linear over the tested concentration ranges with correlation coefficient >0.995. The LLOQ was 0.1 ng/mL for both analytes. The inter‐ and intra‐day precisions were <9.65% and the accuracy ranged from ?3.94 to 6.37%. The extraction recovery was >85.41% and no significant matrix effect was observed. The developed assay was successfully applied to the pharmacokinetic study of evobrutinib and evobrutinib‐diol in dogs after oral administration of evobrutinib at a single dose of 5 mg/kg.     

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.     

Simultaneous determination of three flavonoids and one coumarin by LC–MS/MS: Application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Daphne genkwa extract

A selective and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple‐quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161 → 133 for umbelliferone, m/z 269 → 117 for apigenin, m/z 283 → 268 for genkwanin and m/z 299 → 284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB‐C₁₈ column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421–1421 ng/mL for umbelliferone, 0.845–845 ng/mL for apigenin, 1.025–1025 ng/mL for genkwanin and 0.845–845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.     

Evaluation of the influence of salt processing on pharmacokinetics of psoralen and isopsoralen in Psoralea corylifolia L.

A sensitive, specific and rapid ultra‐high‐pressure liquid chromatography tandem mass spectrometry (UHPLC‐MS/MS) method has been developed to investigate pharmacokinetic properties of psoralen and isopsoralen, two compounds isolated from raw/salt‐processed fruit of Psoralea corylifolia L. UHPLC‐MS/MS was used with positive ion electrospray. The mobile phase was composed of acetonitrile and 0.1% formic acid aqueous solution and a gradient elution program at flow rate of 0.3 mL/min was applied. Multiple reaction monitoring mode was used for the quantification of psoralen, isopsoralen ([M + H]⁺ m/z 187.0 → m/z 131.0) and scoparone (m/z 207.0 → m/z 151.1). Scoparone served as an internal standard. The method was fully validated for its sensitivity, selectivity, stability, matrix effect and extraction recovery. The obtained results showed that salt‐processed Buguzhi significantly promoted the absorption of psoralen and isopsoralen, and increased the bioavailability of these compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma

A sensitive and fast high‐performance liquid chromatography–electrospray ionization–MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C₁₈ column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple‐quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50–5000 ng/mL for levodopa and 3–600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC–MS/MS assay for quantification of bruceine D in rat plasma

A sensitive LC–MS/MS method for the determination of bruceine D in rat plasma was developed. The analyte and IS were separated on a Luna C₁₈ column (2.1 × 50 mm, 1.7 μm) using a mobile phase of acetonitrile and 0.1% formic acid in water (40:60, v/v) at a flow rate of 0.25 mL/min. The selected reaction monitoring mode was chosen to monitor the precursor‐to‐product ion transitions of m/z 409.2 → 373.2 for bruceine D and m/z 469.2 → 229.3 for IS using a negative ESI mode. The method was validated over a concentration range of 0.5–2000 ng/mL for bruceine D. Total chromatography time for each run was 3.5 min. The method was successfully applied to a pharmacokinetic study of bruceine D in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantification of tizoxanide and tizoxanide glucuronide in mouse plasma by liquid chromatography–tandem mass spectrometry

Nitazoxanide (NTZ) is a broad‐spectrum antimicrobial agent. Tizoxanide (T) and tizoxanide glucuronide (TG) are the major circulating metabolites after oral administration of NTZ. A rapid and specific LC–MS/MS method for the simultaneous quantification of T and TG in mouse plasma was developed and validated. A simple acetonitrile‐induced protein precipitation method was employed to extract two analytes and the internal standard glipizide from 50 μL of mouse plasma. The purified samples were resolved using a C₁₈ column with a mobile phase consisting of acetonitrile and 5 mm ammonium formate buffer (containing 0.05% formic acid) following a gradient elution. An API 3000 triple quadrupole mass spectrometer was operated under multiple reaction‐monitoring mode with electrospray ionization. The precursor‐to‐product ion transitions m/z 264 → m/z 217 for T and m/z 440 → m/z 264 for TG were used for quantification. The developed method was linear in the concentration ranges of 1.0–500.0 ng/mL for T and 5.0–1000.0 ng/mL for TG. The intra‐ and inter‐day precision and accuracy of the quality control samples at low, medium and high concentrations exhibited an RSD of <13.2% and the accuracy values ranged from ?9.6 to 9.3%. We used this validated method to study the pharmacokinetics of T and TG in mice following oral administration of NTZ. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of an LC‐MS/MS method for quantification of kadsurenone in rat plasma and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method was developed and validated for the determination of kadsurenone in rat plasma using lysionotin as the internal standard (IS). The analytes were extracted from rat plasma with acetonitrile and separated on a SB‐C₁₈ column (50 × 2.1 mm, i.d.; 1.8 µm) at 30 °C. Elution was achieved with a mobile phase consisting of methanol–water–formic acid (65:35:0.1, v/v/v) at a flow rate of 0.30 mL/min. Detection and quantification for analytes were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 357.1 → 178.1 for kadsurenone, and m/z 345.1 → 315.1 for IS. Calibration curves were linear over a concentration range of 4.88–1464 ng/mL with a lower limit of quantification of 4.88 ng/mL. The intra‐ and inter‐day accuracies and precisions were <8.9%. The LC‐MS/MS assay was successfully applied for oral pharmacokinetic evaluation of kadsurenone using the rat as an animal model. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma: application to a pharmacokinetic study

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid‐phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP_18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow‐gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49–91.0 and 0.40–74.4 ng/mL for MTX and TFB, respectively. The intra‐ and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post‐dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.