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For the quantification of resveratrol (RV) in the Wistar rat skin, an ultra-performance liquid chromatography-mass spectrometric (UPLC–MS/MS) method was developed and validated on ACQUITY UPLC BEH C18 column (1.7 μm). The mobile phase ratio of methanol (A) and 2% formic acid (B) (ratio 10: 90% v/v, 80: 20 v/v) at isocratic elution with flow rate 0.3 mL/min, and run time 3 min was used for analysis. In addition, the use of multiple reaction monitoring (MRM)/ES+ mode to detect the analytes and to track parents to daughter ion transition of 229.17 > 107.04 m/z (time scan 3 min, retention time 1.48) for RV and curcumin as an internal standard shows 369.16 > 176.93m/z (scan time is 2.80 min, retention time is 1.11), respectively. Linearity was observed in the range of 2.5 to 2,000 ng/mL (R2 = 0.987). Precision and accuracy on rat skin were within the acceptability range (RE%: ±15; RSD%: ±15). Moreover, it showed a good percentage recovery and found within acceptance limit 90–110%. Lower limit of detection and quantitation for the method observed to be 2.5 and 20 ng.mL−1, respectively. Method application indicated successful determination of dermatokinetics parameters of RV from lipospheres gel and suspension in the rats. [ABSTRACT FROM AUTHOR]

In this work, a rapid, simple, sensitive and specific LC-MS/MS method was developed and validated for the quantification of S-Allylmercaptocysteine (SAMC) in plasma. After a simple sample procedure by one step protein precipitation with acetontrile, the samples were separated on Gemini-NX C18 column (2.1mm i.d. 150 mm, 3 μm, Phenomenex). The mobile phase was composed of water-acetonitrile (20:80, v/v) at an isocratic flow rate of 0.3 mL/min. The developed method was validated based on the International Conference on Harmonization (ICH) guidelines. The results show that the method had satisfactory specificity, precision and accuracy in a linear range of 50-3,000 ng/mL for SAMC. The precision conformed to the acceptance criteria, and the lower limit of quantification was 50 ng/mL for the analyte. The plasma samples stored for 10 days or after two freeze-thaw cycles (-80°C) were stable. This method was successfully applied to a pharmacokinetics study of SAMC in rats. It was found that SAMC metabolized very quickly in rats and its plasma half-life was less than 5 min. [ABSTRACT FROM AUTHOR]

Currently, patients with co-infection with HIV and tuberculosis are treated with more than one drug. PA-824 a new chemical entity and a member of a class of compounds known as nitroimidazo-oxazines, has significant antituberculosis activity and a unique mechanism of action. Darunavir (Prezista™) is a new protease inhibitor of HIV-1. A simple, sensitive and rapid LC-MSMS method has been developed and validated for simultaneous determination of PA-824 and darunavir. Chromatographic separation was achieved on Agilent Eclipse plus C18 column (100mm x 2.1 mm, 3.5 µm) using gradient elution of acetonitrile--water (90:10, v/v) with fast gradient elution at a flow rate of 0.6 mL/min and run time of 4.5 min. The mass spectrometer was run in positive electrospray ionization mode using multiple reaction monitoring to monitor the mass transitions. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification, recovery and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. The method had been successfully applied to a pharmacokinetic study of fixed dose administration of PA-824, darunavir and their combination in rats. The results indicated that when co-administration of darunavir could decrease the amount of PA-824 in vivo and extend the elimination half-life. [ABSTRACT FROM AUTHOR]

1,6-O,O-Diacetylbritannilactone is a natural sesquiterpene lactone isolated from Inula britannica that has displayed cytotoxic effects against several human cancer cell lines. In this study, a selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for determination of 1,6-O,O-diacetylbritannilactone in rat plasma. Chromatographic separation was achieved on an Agilent C18 column (4.6mm × 75 mm, 3.5 μm) with methanol and water (80:20, v/v) as the mobile phase. An ESI source was applied and operated in positive ion mode; selected-reaction monitoring was used for quantification using target fragment ions m/z 373.2→312.9 for 1,6-O,O-diacetylbritannilactone and m/z 331.2→144.1 for the IS. Calibration plots were linear in the range of 1.5-1350 ng/mL for 1,6-O,O-diacetylbritannilactone in rat plasma. Intra- and inter-day precisions were <8.5%, and the accuracy ranged from -2.7 to 12.8%. The LC-MS-MS method was successfully applied in a pharmacokinetic study of 1,6-O,O-diacetylbritannilactone in rats. [ABSTRACT FROM AUTHOR]

A sensitive and reliable high-performance liquid chromatography with tandem mass spectrometry was developed and validated for the quantification of swertisin in rat plasma. The samples were prepared with methanol by protein precipitation. Swertisin was separated by using an Agilent Poroshell 120 EC-C18 column (4.6mm × 50 mm, 2.7 µm) with the mobile phase consisted of acetonitrile and water containing 0.1% formic acid running at a flow rate of 0.3 mL/min for 5 min. The analytes were detected with the multiple reaction monitoring in the negative electrospray ionization source for quantitative response of swertisin [M-H]- (445.1→281.7) and puerarin (internal standard) [M-H]- (415.1×295.0). Precision (relative standard deviation, RSD%) of the inter-day and the intra-day was <9.89% and 11.34% while accuracy (relative error, RE%) ranged from -6.01% to 4.92% and -3.97% to 4.39%, respectively. Interestingly, the expression of the γ-aminobutyric acid (GABA) receptor subunits (GABAAα1, GABAAα5 and GABAAμ1) mRNAs was enhanced in the rat hippocampal neurons treated with swertisin as analyzed by real-time polymerase chain reaction. Moreover, the method was successfully applied to determine the pharmacokinetic of swertisin in rats after tail vein injection with 4.0 mg/kg of the compound. Our results provide useful pharmacokinetic information for swertisin in vivo and suggest that the sedative function of this compound may be through inducing the expression of the GABAA receptor subunits. [ABSTRACT FROM AUTHOR]

A rapid and sensitive ultra-fast liquid chromatography tandem mass spectrometry method, followed by simple protein precipitation and solid-phase extraction, has been developed and validated for the quantitative determination of four azo dyes (Para red, Solvent yellow 2, Solvent red 1 and Sudan red 7B) in rat plasma using D5-Sudan I as the internal standard. The optimal separation was accomplished on an Agilent Eclipse Plus C18 column (100 ? 2.1 mm, 1.8 µm) with gradient elution using the mobile phase including acetonitrile and water (containing 0.1% formic acid). The flow rate was 0.45 mL/min. The detection was conducted by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring. The calibration curves showed good linearity, with correlation coefficients >0.998 for all of the analytes within the concentration range. The lower limits of quantification (LLOQs) of Para red, Solvent yellow 2, Solvent red 1 and Sudan red 7B in rat plasma were 1.0, 0.1, 0.1 and 0.1 µg/L, respectively. The intra- and interday relative standard deviations were ≤9.6 and ≥12.4%, respectively, and the accuracy was in the range of -5.8 to -9.5%. The average recoveries were between 81.49 and 118.65%, and the matrix effects were satisfactory in the biological matrices. The fully validated method has been successfully applied in measuring levels of the four azo dyes in rat plasma following oral administration of 20.0 mg/kg of analytes in rats, which was suitable for the pharmacokinetic studies of the azo dyes. [ABSTRACT FROM AUTHOR]

A rapid and sensitive bioassay based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) has been developed and validated to measure arbutin in rat plasma. Sample preparation of plasma after the addition of indapamide as internal standard (IS) involved solid-phase extraction (SPE) on C18 cartridges. Reversed-phase chromatography using acetonitrile and 0.5% formic acid solution (pH 2.56) was used for separation in a run time of 4.0 min. The analytes were detected in the negative ion mode using selective reaction monitoring (SRM) of the transitions at m/z 271.2→107.8 for arbutin and 364.3→189.0 for indapamide. The method has the following performance characteristics: a reliable response range of 7.5-5250.0 ng/mL with correlation coefficients (r) of >0.995. The lower limit of quantitation (LLOQ) was 7.5 ng/mL. The intra- and inter-day precision and accuracy of the quality control (QC) samples at low, medium and high concentration levels showed ≤8.79% relative standard deviation (RSD) and −1.15 to 1.49% relative error (RE). The method was successfully applied to a pharmacokinetic study to measure arbutin in rats after extracts of Vaccinium vitis-idaea was orally administered. [ABSTRACT FROM AUTHOR]

A rapid and specific LC-MS-MS method has been developed for simultaneous analysis of quercetin and naringenin in rat plasma. The method was applied to the pharmacokinetics studies of quercetin and naringenin after oral administration of Pollen Typhae extract. The samples were prepared by the protein precipitation method. The analysis was carried out on an ACQUITY UPLC™BEH C18 column with gradient elution using mobile phase, which included acetonitrile and water (containing 0.1% formic acid). The flow rate was 0.4 mL/min. All analytes including internal standard (IS) were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for quercetin ranging from 0.5 to 100 ng/mL and naringenin ranging from 5 to 1000 ng/mL. The intra- and interday precisions (RSD) were less than 10.78 and 11.20%. The extraction recovery of the analytes was acceptable. Stability studies showed that quercetin and naringenin were stable in the preparation and analytical process. The validated method was successfully used to determine the concentration-time profiles of quercetin and naringenin. [ABSTRACT FROM AUTHOR]

A rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was developed and validated for the determination and pharmacokinetic investigation of apremilast in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run timewas 3.0 min, and the elution of apremilast was at 1.27 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring mode using the respective transitions m/z 461.3→257.1 for apremilast and m/z 237.2→194.2 for carbamazepine (internal standard). The calibration curve was linear over the range of 0.1-100 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The mean recovery of apremilast in plasmawas in the range of 83.2-87.5%. Both intraday and interday precision were <9.6%. This method was successfully applied in the pharmacokinetic study after oral administration of 6.0 mg/kg apremilast in rats. [ABSTRACT FROM AUTHOR]

N-Acetylcysteine (NAC) is the N-acetyl derivative of the amino acid L-cysteine and is extensively used as a medicine to treat a variety of diseases. High-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method has been developed for the quantitative assessment of N-acetyl-L-cysteine. The method was further applied to study the distribution of the intraperitoneal injected drug into different tissues and plasma of Wistar rats, including liver, kidney, heart, lungs and spleen. The drug was having highest concentration in plasma and liver followed by kidney, lungs, heart and spleen. Method validation studies suggested being linear in the range of 1-15 µg mL-1 for liver, kidney, heart, lungs and spleen and 1-120 µg mL-1 for the plasma. The limit of detection and limit of quantitation were found to be 0.20 and 0.66 ug mL-1, respectively. The recovery studies suggested that in all the cases, the obtained recovery was in the range of 98.51-101.88%. Our analyses provide a validated UPLC-MS method for the determination of NAC and its successful application for the analysis in plasma and tissues obtained from Wistar rats. [ABSTRACT FROM AUTHOR]

Acotiamide is a new prokinetic drug that is used to treat functional dyspepsia (FD). A sensitive and specific LC-MS-MS method has been developed and validated for the analysis of acotiamide in rat plasma. The assay involved a simple protein precipitation (PPT) step with methanol-acetonitrile (50:50, v/v) and a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid. The analytes were chromatographed on a reversephase Agilent Zorbax XDB C18 column (2.1 mm× 50 mm, 3.5 μm) with a flowrate of 0.50 mL/min. The analytes were monitored by tandem-mass spectrometry with positive electrospray ionization. The precursor-product transitions (m/z) in the positive ion mode were 451.4→271.3 and 386.2→122.2 for acotiamide and buspirone (internal standard, IS), respectively. The assay was shown to be linear over the range of 0.10-200 ng/mL, with a lower limit of quantification of 0.10 ng/mL. The method was shown to be reproducible and reliable with the inter- and intra-batch accuracy and precision were within ±15%. The assay has been successfully used for pharmacokinetic evaluation of acotiamide after intravenous and oral administration of 10 mg/kg acotiamide in rats. The oral absolute bioavailability (F) of acotiamide in rats was estimated to be 38.4 ± 13.5% with an elimination half-life (t1/2) value of 9.11 ± 0.40 h. [ABSTRACT FROM AUTHOR]

This studywas designed to develop a sensitive, simple and rapid method for the quantitation of morroniside in rat urine using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) and to investigate the excretion of morroniside in rat urine. The mobile phase consisted of water-acetonitrile (gradient elution) at a flow rate of 0.4 mL/min. Detection was performed using positive-ion electrospray ionization in multiple reaction monitoring (MRM) modes. And the detection of morroniside in rat urine by the LC-MS-MS was accurate and precise from 1.0 to 2,500 ng/mL (a correlation coefficient of 0.9953). The recoveries and matrix effects were all in line with the biological sample measurement requirements. The intraday accuracy was 88.68-105.78% with precision of 6.50-11.19% and the interday accuracy was 95.77-102.43% with precision of 7.08-10.40%. Excretion data of morroniside in rat urine indicated that 21.43? (i.g.) and 100.35% (i.v.) of the dose administered was excreted as unconverted form, respectively. And the maximal excretion ratewas 27.57 and 482.42 µg/h after oral and intravenous administration, respectively. These results indicated that the developed method has satisfactory sensitivity, accuracy and precision for the quantification of morroniside in rat urine. [ABSTRACT FROM AUTHOR]

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous quantitation of metformin (MTF), metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma using paracetamol as an internal standard (IS), respectively. The sample preparation involved a proteinprecipitation method with methanol after the addition of IS. The separation was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 μm) at a flowrate of 1.0 mL/min, using methanol-water containing 0.1% formic acid (39:61, v/v) as mobile phase, and total run time was 8.5 min. MS-MS detection was accomplished in multiple reaction monitoring mode with positive electrospray ionization. The monitored transitions were m/z 130.1→60.2 for MTF, m/z 268.2→116.1 for MET, m/z 284.2→ 116.1 for HMT, m/z 254.2→116.1 for DMT and m/z 152.3→110.1 for IS. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 19.53-40,000 ng/mL for MTF, 3.42-7,000 ng/mL for MET, 2.05-4,200 ng/mL for HMT and 1.95-4,000 ng/mL for DMT, respectively. The analytical method was successfully applied to drug interaction study of MTF and MET after oral administration of MTF and MET. Results suggested that the coadministration of MTF and MET results in a significant drug interaction in rat. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia is a common occurrence in many neurodegenerative diseases, including tauopathies. We developed and validated a simple and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of homocysteine (Hcy) in rat plasma. Hcy was analyzed using ultra-performance liquid chromatography on a C8 column with detection by positive ESI tandem mass spectrometry. For optimal retention and separation, we used ion-pair reagent-heptafluorobutyric acid. The method utilizes heavy labeled internal standard and does not require any derivatization or extraction step. The procedure was validated in compliance with the European Medicines Agency guideline. The limit of detection was 0.15 µmol/L and the limit of quantification was 0.5 µmol/L. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was in the range of 93-98%. The inter-day precision (n = 5 days), expressed as % relative standard deviation, was in the range 3-8%. Using this method, we analyzed plasma samples from two transgenic lines of the rat model for tauopathies. [ABSTRACT FROM AUTHOR]

A simple and sensitive liquid chromatography-tandem mass spectrometric method was developed and validated to measure aurantio-obtusin content in rat plasma using rhein as internal standard (IS). The analytes were extracted from plasma by a simple liquid–liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on a Zorbax XDB-C18 column (50 mm × 4.6 mm, 2.1 µm particle size) using the mobile phase consisting of methanol–water (70 : 30, v/v). The detection was achieved by negative ion electrospray ionization in selected reaction monitoring mode, monitoring the transitions m/z 329.1 → 299.2 and m/z 283.1 → 183.0 for aurantio-obtusin and IS, respectively. The assay was linear over a concentration range of 4–4,000 ng/mL with a lower limit of quantification of 4.00 ng/mL for aurantio-obtusin. The intra- and inter-day precision was <6.8%, and the accuracy values were between –1.7 and 7.1%. The validated method was successfully applied to the pharmacokinetics of aurantio-obtusin following intravenous administrations to rats. [ABSTRACT FROM PUBLISHER]

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometry was developed to determine two epimeric furofuran lignans (sesamin and asarinin) simultaneously from Asarum heterotropoides extract in rat plasma. Simple protein precipitation with acetonitrile was performed to extract analytes by using alantolactone as an internal standard. Chromatographic separation was achieved using a DIKMA Diamonsil C18 analytical column (4.6 mm × 150 mm, i.d., 5 µm) by isocratically eluting with a mobile phase consisting of methanol/5 mM ammonium acetate/formic acid (75:25:0.1, v/v/v) at a flow rate of 0.8 mL/min. Tandem mass spectrometric detection with an electrospray ionization interface was performed by multiple reaction monitoring in positive ionization mode. This method was validated according to specificity, sensitivity, linearity, intra- and inter-day precision (<10.7%) and accuracy (<2.3%) and recovery and stability in a concentration range of 25.0–15 000 ng/mL for sesamin and 5.00–3 000 ng/mL for asarinin. This method has been successfully applied in a pharmacokinetic study of A. heterotropoides extract containing sesamin and asarinin after this extract was orally administrated in rats. [ABSTRACT FROM PUBLISHER]

A sensitive and selective liquid chromatography mass spectrometry method was developed for the determination of rhynchophylline in rat plasma. After the addition of estazolam as the internal standard (IS), protein precipitation by acetonitrile was used for sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column (2.1 × 150 mm, 5 µm) with acetonitrile–0.1% formic acid as the mobile phase with gradient elution. The electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification by using target fragment ions m/z 385 for rhynchophylline and m/z 295 for the IS. Calibration plots were linear over the range of 5–500 ng/mL for rhynchophylline in plasma. The lower limit of quantification for rhynchophylline was 5 ng/mL. The mean recovery of rhynchophylline from plasma was in the range of 87.7–92.6%. The coefficients of variation of intra-day and inter-day precision were both less than 11%. This method is sensitive and selective enough to be used in pharmacokinetic research for the determination of rhynchophylline in rat plasma. [ABSTRACT FROM PUBLISHER]

A rapid, sensitive and reliable liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of betulonic acid in rat plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on an Agela MG-C18 analytical column (50 × 2.1 mm, 5 µm) using methanol–water–formic acid (80:20:0.1, v/v/v) as the mobile phase with a constant flow rate of 0.6 mL/min. Mass detection was conducted by electrospray ionization in positive ion multiple reaction monitoring mode. The calibration curves were linear over a concentration range of 3.00–3,000 ng/mL for the analyte. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the estimation of betulonic acid in rat plasma from a preclinical pharmacokinetics study. [ABSTRACT FROM AUTHOR]

A sensitive, simple and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of ginkgolide B (GB) and the prodrug of ginkgolide B (PGB) in rat plasma and brain tissue. Detection was performed on a triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode using the electrospray ionization technique in negative ionization mode. The total run time was only 2.0 min. Good linearity was found between 1–200 ng/mL (r > 0.9993) for plasma samples and 0.5–50 ng/mL (r = 0.9995) for the brain tissue samples. The lower limits of quantification of PGB and GB were 1 ng/mL for plasma and 0.5 ng/mL for brain tissue. Intra-day and inter-day precision was less than 11.67%. Intra-day accuracy was in the range of –2.61–10.67%, inter-day accuracy was in the range of –2.36–8.98%. The mean recovery for PGB and GB was between 82.5 and 97.0%. The validated method was successfully applied to a pharmacokinetic study of PGB and GB in rats after intravenous administration. [ABSTRACT FROM PUBLISHER]

A specific ultra-performance liquid chromatography tandem mass spectrometry method has been described for the simultaneous determination of caffeine, tolbutamide, metoprolol and dapsone in rat plasma, which are the four probe drugs of the four cytochrome P450 (CYP450) isoforms CYP1A2, CYP2C9, CYP2D6 and CYP3A4. The chromatographic separation was achieved using a Waters Acquity UPLC BEH HILIC C18 column (2.1 × 50 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) (15:85, v/v). The triple quadrupole mass spectrometric detection was operated by positive electrospray ionization. Phenacetin was chosen as internal standard. Plasma samples were extracted with dichloromethane–butanol (10:1, v/v). The recoveries ranged from 67.5% to 98.5%. The calibration curves in plasma were linear in the range of 2.5–1,000 ng/mL for caffeine and dapsone, 5–5,000 ng/mL for tolbutamide and 2.5–250 ng/mLfor metoprolol, with correlation coefficient (r2) of 0.9936, 0.9966, 0.9990 and 0.9998, respectively. The method was successfully applied to pharmacokinetic studies of the four probe drugs of the four CYP450 isoforms and used to evaluate the effects of breviscapine on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats. [ABSTRACT FROM AUTHOR]

A rapid, sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed and validated for the quantification of isotoosendanin, an important bioactive component isolated from Meliae cortex. A Capcell PAK C18 column (100 × 4.6 mm) was used for the chromatographic elution using methanol–10 mM ammonium acetate–formic acid (80:20:0.1, v/v/v) as mobile phase at the flow rate of 0.6 mL/min. MS-MS analysis was performed on a triple quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization source in positive ion mode. Extraction of isotoosendanin and genistein (internal standard, IS) from rat plasma was determined by precipitating protein treatment. Quantification was performed by MS in the multiple reaction monitoring mode with positive ionization at m/z 557 → 437 for the analyte and m/z 271 → 215 for IS, respectively. Linear isotoosendanin calibration curves were obtained between 2.0–2,000 ng/mL with a correlation coefficient greater than 0.99. Acceptable precision and accuracy were acquired for concentrations over the standard curve range. Satisfactory results were achieved for sensitivity, specificity, recovery, freeze/thaw and stability. This analytical method was successfully applied to determine the pharmacokinetic parameters of isotoosendanin after an oral administration of 200 mg/kg to rats. [ABSTRACT FROM AUTHOR]

A simple, rapid and sensitive liquid chromatography tandem mass spectrometry method is presented for the simultaneous determination of oleanolic acid, p-coumaric acid, ferulic acid, kaemperol and quercetin in rat plasma. Glycyrrhetinic acid was used as an internal standard, and sample pretreatment consisted of a liquid–liquid extraction. Chromatographic separation was achieved on a Gemini 110A C18 column (50 × 2.0 mm i.d., 5 µm) by gradient elution with a mobile phase consisting of methanol, acetonitrile and 0.01% formic acid in water. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ionization mode. Calibration curves offered linear ranges of two orders of magnitude with r > 0.99. The method was validated in terms of matrix effect, intra-day and inter-day precision, accuracy, linearity, specificity and stability. The relative standard deviation of intra-day and inter-day variations ranged from 2.66 to 14.74% and 1.9 to 14.55%. No substantial endogenous interference from blank plasma was observed. The method has been successfully applied to a pharmacokinetic study of Oldenlandia diffusa extract after oral administration in rats. [ABSTRACT FROM PUBLISHER]