Simultaneous determination of lamivudine,stavudine and nevirapine in human plasma by LC–MS/MS and its application to pharmacokinetic study in clinic

A new high‐throughput LC–MS/MS method for the simultaneous determination of lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma is presented, with zidovudine as an internal standard. The analytes were extracted from plasma by protein precipitation and only 150 μL plasma was needed. Chromatographic separation was achieved on a Shiseido C₈ column (150 × 2.0 mm, 5 μm) with a total run time of 6 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was developed and validated over the concentration range of 25–5000 ng/mL for 3TC and NVP and 20–4000 ng/mL for d4T. The method was validated in terms of intra‐ and inter‐day precision (≤8.6%), accuracy (within ± 8.4%), linearity and specificity. The method has been successfully applied to the pharmacokinetic study of a combination treatment of 300 mg lamivudine, 30 mg stavudine and 200 mg nevirapine in 22 healthy male volunteers under fasting conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative analysis of enasidenib in dried blood spots of mouse blood using an increased‐sensitivity LC–MS/MS method: Application to a pharmacokinetic study

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guidelines for the estimation of enasidenib on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The method employs liquid extraction of enasidenib from DBS disks of mouse whole blood followed by chromatographic separation using 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 1.0 mL/min on an Atlantis dC₁₈ column with a total run time of 2.0 min. The MS/MS ion transitions monitored were m/z 474.0 → 267.1 for enasidenib and m/z 309.2 → 251.3 for the internal standard (warfarin). The assay was linear in the range of 1.01 – 3044 ng/mL. The within‐run and between‐run precisions were in the range of 3.18 – 9.06 and 4.66 – 8.69%, respectively. Stability studies showed that enasidenib was stable on DBS cards for 1 month. This novel method has been applied to analyze the DBS samples of enasidenib obtained from a pharmacokinetic study in mice.     

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 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.     

A rapid and sensitive UHPLC–MS/MS method for the determination of celosin A in rat plasma with application to pharmacokinetic study

Celosin A (CA), a natural compound isolated from Celosia argentea L., has been shown significant hepatoprotective effect on AHNP‐induced liver injury. This study described a rapid and sensitive ultra‐high‐pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) assay for determination of CA in rat plasma. Methanol‐mediated precipitation was used for sample pretreatment. Chromatographic separation was achieved on a T3 column with gradient elution using water and acetonitrile as mobile phase. Determination was obtained using an electrospray ionization source in negative selected reaction monitoring mode at the transitions of m/z 793.3 → m/z 661.2 and m/z 955.6 → m/z 793.2 for CA and IS, respectively. The assay was linear over the concentration range 0.25–2500 ng/mL (r > 0.995) with a lowest limit of quantification (LLOQ) of 0.25 ng/mL. The intra‐ and inter‐day precisions (RSD) were 1.65–9.84 and 2.46–13.49%, respectively, while accuracy (RR) ranged from 96.21 to 99.45%, respectively. The recovery ranged from 95.09 to 102.22% and the matrix effect from 98.29 to 100.13%. The analyte was stable under the tested storage conditions. The method has been successfully applied to a preclinical pharmacokinetic study in rats after a single intravenous (2 mg/kg) or oral (50 mg/kg) administration. The oral bioavailability of CA was ~1.94%; in addition, there was no difference between male and female rats. This is the first time of the use of an UHPLC–MS/MS method for determination of CA concentration in rat plasma and for evaluation of its pharmacokinetic behavior.     

Development and validation of an LC-MS-MS method for determination of methyl kulonate in rat plasma

A sensitive, rapid and specific LC‐MS‐MS method was established and validated for determination of methyl kulonate, a major bioactive constituent isolated from Meliae Cortex, in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed using a Capcell Pak C₁₈ column (100 × 4.6 mm, 5 µm) with the mobile phase comprising a mixture of methanol, 10 m m ammonium formate and formic acid (95:5:0.1, v/v/v). Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive atmospheric ionization at m/z 467 → 311 for methyl kulonate, and m/z 469 → 451 for dubione B (internal standard), respectively. A good linear response was observed over the concentration range 1.00–500 ng/mL with the lower limit of quantification 1.00 ng/mL in rat plasma. The method also afforded satisfactory results base on sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐time stability. The validated method was successfully applied to determine the pharmacokinetic properties of methyl kulonate in rats after oral administration at dose of 100 mg/kg. This pharmacokinetic study of methyl kulonate is reported here for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

GC–MS/MS method for the quantification of α‐cedrene in rat plasma and its pharmacokinetic application

α‐Cedrene is a pharmacologically active ingredient isolated from the essential oil of cedar. A selective and sensitive GC–MS/MS method was developed for the quantification of α‐cedrene in rat plasma for the first time. α‐Cedrene was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were determined in selective reaction monitoring mode using MS/MS: m/z 204.3→119.0 for α‐cedrene and m/z 146.0→111.0 for 1,4‐dichlorobenzene (internal standard). The standard curve was linear (r² ≥ 0.995) over the concentration ranges of 5–800 ng/mL. The lower limit of quantification was 5 ng/mL using 50 μL of rat plasma. The coefficient of variation and relative error for intra‐ and interassays at four quality control levels were 3.1–13.9% and ?4.0–2.6%, respectively. The stability of processing (freeze–thaw, long‐term storage at ?80°C, and short‐term storage at room temperature) and chromatography (reinjection) was shown to be of insignificant effect. The present method was applied successfully to the pharmacokinetic study of α‐cedrene after its intravenous (10 mg/kg) and oral (25 mg/kg) administration in male Sprague‐Dawley rats.     

Quantification of methyllycaconitine,selective α7 nicotinic receptor antagonist,in rodent plasma and brain tissue by liquid chromatography tandem mass spectrometry – application to neuropharmacokinetics of methyllycaconitine in rats

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of methyllycaconitine (MLA) in rat plasma and brain tissue. Following acetonitrile protein precipitation, the analyte was separated using a gradient mobile phase on a reversed‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 683–216 for MLA and m/z 260–116 for the internal standard. The assay exhibited a linear dynamic range of 0.5–250 ng/mL for MLA in rat plasma and brain tissue. The lower limit of quantification was 0.5 ng/mL. Acceptable precision (<12%) and accuracy (100 ± 6%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify MLA concentrations in a rodent pharmacokinetic and brain penetration study. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1–500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C_max; 616 ± 73 ng/mL) was achieved at 0.5 h (T_max) and area under curve (AUC_0–24) was 1841 ± 308 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials. Copyright © 2011 John Wiley & Sons, Ltd.     

Stereoselective determination of ginsenosides Rg3 and Rh2 epimers in rat plasma by LC‐MS/MS: Application to a pharmacokinetic study

We developed and validated an accurate and sensitive LC–MS/MS method for the simultaneous quantitation of ginsenoside Rg3 and Rh2 epimers (R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2) in rat plasma. Analytes were extracted from 0.1 mL aliquots of rat plasma by liquid–liquid extraction, using 2 mL of ethyl acetate. In this assay, dioscin (500 ng/mL) was used as an internal standard. Chromatographic separation was conducted using an Acclaim RSLC C₁₈ column (150 × 2.1 mm, 2.2 μm) at 40°C, with a gradient mobile phase consisting of 0.1% formic acid in distilled water and in acetonitrile, a flow rate of 0.35 mL/min, and a total run time of 20 min. Detection and quantification were performed using a mass spectrometer in selected reaction‐monitoring mode with negative electrospray ionization at m/z 783.4 → 161.1 for R‐Rg3 and S‐Rg3, m/z 621.3 → 161.1 for R‐Rh2 and S‐Rh2, and m/z 867.2 → 761.5 for the internal standard. For R‐Rg3 and S‐Rg3, the lower limit of quantification was 5 ng/mL, with a linear range up to 500 ng/mL; for R‐Rh2 and S‐Rh2, the lower limit of quantification was 150 ng/mL, with a linear range up to 6000 ng/mL. The coefficient of variation for assay precision was less than 10.5%, with an accuracy of 86.4–112%. No relevant cross‐talk or matrix effect was observed. The method was successfully applied to a pharmacokinetic study after oral administration of 400 mg/kg and 2000 mg/kg of BST204, a fermented ginseng extract, to rats. We found that the S epimers exhibited significantly higher plasma concentrations and area under curve values for both Rg3 and Rh2. This is the first report on the separation and simultaneous quantification of R‐Rg3, S‐Rg3, R‐Rh2, and S‐Rh2 in rat plasma by LC‐MS/MS. The method should be useful in the clinical use of ginseng or its derivatives.     

A rapid and sensitive determination of aucubin in rat plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

A sensitive, accurate, rapid and robust LC‐MS‐MS method for the quantification of aucubin, a major bioactive constituent of Aucuba japonica, Eucommia ulmoides and Plantago asiatica, was established and validated in rat plasma. Plasma samples were simply precipitated by adding methanol and the supernatant was chromatographed by a Diamonsil® C₁₈(2) column with the mobile phase comprising a mixture of 10 mm ammonium acetate in methanol and that in water with the ratio of 50:50 (v/v). Quantification of aucubin was performed by mass spectrometry in the multiple‐reaction monitoring mode with positive atmospheric ionization at m/z 364 → 149 for aucubin, and m/z 380 → 165 for catalpol (IS), respectively. The retention time was 2.47 and 2.44 min for aucubin and the IS, respectively. The calibration curve (10.0–30,000 ng/mL) was linear (r² > 0.99) and the lower limit of quantification was 10.0 ng/mL in the rat plasma sample. The method showed satisfactory results such as sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐term stability. This simple LC‐MS method was successfully applied in a pharmacokinetic study carried out in Sprague–Dawley rats after oral administration of aucubin at a single dose of 50 mg/kg. Herein the pharmacokinetic study of aucubin is reported for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

A sensitive and selective LC‐MS/MS method for the quantitative determination of segetalin A from the plasma of rats

A sensitive and selective LC‐MS/MS method was developed and validated for the determination and pharmacokinetic investigation of segetalin A in rat plasma. Sample preparation was accomplished through a simple SPE procedure for the removal and preconcentration of the analyte and IS. Plasma samples were separated by HPLC on a Symmetry C₁₈ column using a mobile phase consisting of methanol and 0.1% formic acid in water (70:30, v/v) with isocratic elution. The quantification was performed using multiple reaction monitoring with the transitions m/z 610.3 → 511.2 for segetalin A and m/z 779.4 → 751.4 for IS, respectively. The calibration curve was linear over the range of 8.0–4000 ng/mL with a limit of quantitation (LOQ) of 8.0 ng/mL. This method was applied in a pharmacokinetic study of segetalin A in rats. For intravenous (i.v.) administration, the plasma concentrations of segetalin A decreased quickly (t_1/2z, 1.31 ± 0.341 h). For oral administration, the plasma concentrations of segetalin A increased to a peak value at 1.50 ± 0.577 h, followed by a gradual decrease to the LOQ in 12 h. The mean AUC values after i.v. and oral administration were 553 ± 105 and 1482 ± 110 ng h/mL, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of rhynchophylline and hirsutine in rat plasma by UPLC‐MS/MS after oral administration of Uncaria rhynchophylla extract

An ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to concurrently determine rhynchophylline and hirsutine in rat plasma. The sample preparation of rat plasma was achieved by alkalization and liquid–liquid extraction. The mass transition of precursor ion → product ion pairs were monitored at m/z 385.2 → 160.0 for rhynchophylline, m/z 369.3 → 144.0 for hirsutine and m/z 414.0 → 220.0 for noscapine (internal standard). This method revealed linear relationships from 2.5 to 50 ng/mL (r² > 0.997) for rhynchophylline and from 2.5 to 50 ng/mL (r² > 0.998) for hirsutine. The limit of quantification values for rhynchophylline and hirsutine in rat plasma were both 2.5 ng/mL. Intra‐day and inter‐day precisions were within 10.6% and 12.5%, respectively, for rhynchophylline and hirsutine, and the accuracy (bias) was <10%. Liquid–liquid extraction of rat plasma samples resulted in insignificant matrix effect, and the extraction recoveries were >83.6% for rhynchophylline, 73.4% for hirsutine and 90.7% for the internal standard. This method was applied successfully to a pharmacokinetic study of rhynchophylline and hirsutine in rats after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of metformin and glipizide in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A selective, sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one‐step protein precipitation of 200 μL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol–10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10–656 ng/mL for metformin and 2.55–408 ng/mL for glipizide. The relative standard deviation of intra‐day and inter‐day precision was below 10% and the relative error of accuracy was between ?7.0 and 4.6%. The presented HPLC‐MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration. Copyright © 2012 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.     

Determination of rosamultin in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A specific and reliable LC–MS/MS method for the determination of rosamultin in rat plasma was validated. Plasma samples were prepared with protein precipitation method, and chromatographic separation was performed on a Thermo C₁₈ analytical column (4.6 mm × 50 mm, 3.0 μm). The mass spectrometry (MS) analysis was conducted in positive SRM mode for the transitions of m/z 673.2 → 511.1 for rosamultin and m/z 601.1 → 330.9 for IS. The method validation was conducted over the calibration range of 1.0–500 ng/mL with the precision ≤11.03% and accuracy within ±14.64%. The assay was applied to the pharmacokinetic study after oral administration of rosamultin at a dose of 20 mg/kg in rats.     

An LC‐MS/MS method for determination of 3,6′‐disinapoylsucrose in rat plasma and its application to a pharmacokinetic study

3,6′‐Disinapoylsucrose (DSS), a major active component of traditional Chinese medicine Yuan‐Zhi (the roots of Polygala tenuifolia), has significant effects for neuroprotection and improving learning memory. In order to explore the pharmacokinetic properties of DSS so as to further understand its in vivo activities, a sensitive LC‐MS/MS method was developed for determination of DSS in rat plasma and applied to a pharmacokinetic study in the present study. After treatment by protein precipitation, the plasma sample was separated on a C₁₈ HPLC column and analyzed by a mass spectrometry under positive electrospray ionization. Multiple‐reaction monitoring was employed to measure the ion transition at m/z 777.4 → 409.2 for DSS and m/z 557.2 → 309.1 for forsythin as internal standard. The method was linear over the studied concentration range of 0.5–1000.0 ng/mL. The precision and accuracy ranged from 1.4 to 18.4%, and from ?3.7 to ?9.5%, respectively, for within‐day and between‐day assay. Extraction recovery was higher than 86.6%. The limits of detection and quantification were 0.3 and 0.5 ng/mL, respectively. The present method was successfully applied to a pharmacokinetic study. DSS was found to have poor oral absorption with only about 0.5% bioavailability. Copyright © 2009 John Wiley & Sons, Ltd.     

An LC–MS/MS method for quantitation of cyanidin‐3‐O‐glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin‐induced diabetic rats

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to determine cyanidin‐3‐O‐glucoside (Cy‐3G) in normal and streptozotocin‐induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ (50 × 4.6 mm, 5 μm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple‐quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy‐3G and m/z 463.0 → 300.1 for quercetin‐3‐O‐glucoside (internal standard). The calibration curve was linear over the range 3.00–2700 ng/mL (r² ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra‐ and inter‐day precision was <14.5% and mean accuracy was from −11.5 to 13.6%. Stability testing showed that Cy‐3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy‐3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy‐3G after oral administration in rats.     

Liquid chromatography tandem mass spectrometry method for the quantification of sarpogrelate, a selective 5-HT(₂A) receptor antagonist, in plasma: application to a pre-clinical pharmacokinetic study

A simple LC‐MS/MS method was developed and validated for the estimation of sarpogrelate in 50 µL of rat plasma. The analyte and internal standard (IS) were extracted from rat plasma by acetonitrile precipitation and they were separated on a reversed‐phase C₈ column with gradient program. The MS acquisition was performed with multiple reaction monitoring mode using m/z 430.2 to m/z 135.0 for analyte and m/z 448.2 to m/z 285.3 for IS. The calibration curves were linear over the range of 1–1000 ng/mL with the correlation coefficient greater than 0.999. With dilution integrity up to 20‐fold, the upper limit of quantification was extendable up to 15,000 ng/mL. The method was successfully applied to the analysis of rat plasma samples after single dose oral administration of sarpogrelate at 5 mg/kg to rats for the determination of its pharmacokinetics. Following oral administration the maximum mean concentration in plasma (C_max, 11514 ng/mL) was achieved at 0.25 h (T_max) and the area under curve (AUC_0–24) was 11051 ± 3315 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.9 ± 1.1 h and 490 ± 171 mL/h/kg, respectively. We believe that development of a method in rodent plasma would facilitate the ease of adaptability of sarpogrelate in human plasma. Copyright © 2010 John Wiley & Sons, Ltd.