Simultaneous determination of major components of Huangqi–Honghua extract in rat plasma using LC–MS/MS and application to a pharmacokinetic study

A sensitive and reliable LC–MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi–Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin‐7‐O‐β‐d ‐glucoside (CAG), calycosin, calycosin‐3′‐O‐glucuronide (C‐3′‐G) and calycosin‐3′‐O‐sulfate (C‐3′‐S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C₁₈ column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra‐ and inter‐assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from ?8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S in rat after an oral administration of Huangqi–Honghua extract.     

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

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

Simultaneous determination of evodiamine and its four metabolites in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography tandem mass spectrometry method was validated for simultaneous quantification of evodiamine and its metabolites 10‐hydroxyevodiamine (M1), 18‐hydroxyevodiamine (M2), 10‐hydroxyevodiamine‐glucuronide (M3) and 18‐hydroxy‐ evodiamine‐glucuronide (M4) in rat plasma for the first time. The analytes were extracted with acetonitrile and separated on a C₁₈ column within 3 min. The detection was achieved in positive selected reaction monitoring mode with precursor‐to‐product transitions at m/z 304.1 → 161.1 for evodiamine, m/z 320.1 → 134.1 for M1, m/z 320.1 → 150.1 for M2, m/z 496.2 → 134.1 for M3, m/z 496.2 → 171.1 for M4 and m/z 349.2 → 305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients >0.9991. The lower limits of quantification for evodiamine, M1, M2, M3 and M4 were 0.1, 0.1, 0.1, 0.25 and 0.25 ng mL⁻¹, respectively. Extraction recoveries and matrix effects of the analytes were within the ranges of 84.51–97.21 and 90.13–103.30%, respectively. The accuracy (relative error) ranged from −8.14 to 7.23% while the intra‐ and inter‐day precisions (relative standard deviation) were < 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study will be helpful in understanding the in vivo disposition of evodiamine.     

A sensitive LC–MS/MS method for simultaneous quantification of geniposide and its active metabolite genipin in rat plasma and its application to a pharmacokinetic study

Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH₃COO]⁻ m/z 447.3 → 225.3 for GE and [M − H]⁻ m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE.     

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.     

Simultaneous determination of major type‐B trichothecenes and the de‐epoxy metabolite of deoxynivalenol in chicken tissues by HPLC–MS/MS

In this study, a specific and sensitive LC–MS/MS method for the simultaneous analysis of type‐B trichothecenes (deoxynivalenol, 3‐acetyldeoxynivalenol, and 15‐acetyldeoxynivalenol) and the de‐epoxy metabolite of deoxynivalenol (de‐epoxy‐deoxynivalenol) in chicken muscle, liver, kidney, and fat tissues was developed and validated. The method involved an extraction step using ethyl acetate, followed by the evaporation of the supernatant, which was further purified by an Oasis HLB SPE cartridge (Waters, Milford, MA, USA). Chromatographic separation was performed on a C₁₈ column by detection with MS in multiple‐reaction monitoring mode and using a gradient elution program with 0.1% formic acid in water and methanol. The correlation coefficients (r) for each calibration curve were >0.99 within the experimental concentration range. The extraction recoveries ranged from 73.7 to 106.4%, with intraday and interday RSD < 11.6% at three levels of concentrations of 2, 10, and 100 μg/kg. The decision limits and the detection capabilities of the analytes in the chicken tissues ranged from 0.16 to 0.92 and 0.68 to 2.07 μg/kg, respectively. The results demonstrated the applicability of this sensitive procedure to the determination of trichothecenes in chicken tissue samples.     

A sensitive LC–MS/MS method for simultaneous determination of cabozantinib and its metabolite cabozantinib N‐oxide in rat plasma and its application in a pharmacokinetic study

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N‐oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC–MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C₁₈ column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500–5000 ng/mL for CBZ and 0.525–2100 ng/mL for CBN. The extraction recoveries were constant and the intra‐ and inter‐batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague–Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose‐proportional increases in exposure.     

Development of a simple HPLC–MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide

A simple high‐performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4‐trifluoro‐methylaniline oxanilic acid (4‐TMOA) in human plasma and urine. Merely 50 μL plasma and 20 μL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C₁₈ column was selected to achieve rapid separation for TER and 4‐TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4‐TMOA (m/z 231.9 → 160.0), internal standard teriflunomide‐d4 (m/z 273.0 → 164.0) and 2‐amino‐4‐trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10–5000 ng/mL in plasma as well as 10–10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.     

Simultaneous determination of tandospirone and its active metabolite, 1‐[2‐pyrimidyl]‐piperazine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1‐[2‐pyrimidyl]‐piperazine (1‐PP) in Sprague–Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C₁₈ column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000–500.0 ng/mL for TDS and 10.00–500.0 ng/mL for 1‐PP. Total time for each chromatograph was 3.0 min. The intra‐day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter‐day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1‐PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1‐PP in plasma necessary for the pharmacokinetic investigation.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.     

Simultaneous determination of multicomponent of acetylkitasamycin and kitasamycin by LC–MS/MS in swine plasma and its application in a pharmacokinetic study

A simple and reliable LC–MS/MS method was established for simultaneous determination of 12 components from acetylkitasamycin and kitasamycin in swine plasma. The analytes were separated on a Shim‐pack VP‐ODS column with a 25 min gradient elution using 5 mmol/L ammonium acetate and acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. Identification and quantification were accomplished by electrospray ionization) in positive mode using multiple reaction monitoring. The limits of quantitation of acetylkitasamycin A₁A₃, A₁₃ and kitasamycin A₃, A₁₃ were 3 μg/L, and that of the other eight components was 5 μg/L. The mean recoveries of kitasamycin and acetylkitasamycin ranged from 85.3 to 103.5%. The developed method was successfully applied to a pharmacokinetic study in swine after intravenous (i.v.) and oral (p.o.) administration of acetylkitasamycin. The result showed that the plasma concentrations of acetylkitsamycin components were much higher than that of kitasamycin in swine after i.v. and p.o., in which acetylkitsamycin A₄A₅ was the highest component at each time point.     

Simultaneous determination of 20(S)‐protopanaxadiol and its three metabolites in rat plasma by LC–MS/MS: application to their pharmacokinetic studies

The aim of this study was to develop an LC–MS/MS method for simultaneous determination of 20(S) protopanaxadiol (PPD) and its three metabolites, PPD‐glucuronide (M1), (20S,24S)‐epoxy‐dammarane‐3,12,25‐triol (M2) and (20S,24R)‐epoxydammarane‐3,12,25‐triol (M3), in rat plasma. Precipitation with acetonitrile was employed for sample preparation and chromatographic separations were achieved on a C₁₈ column. The sample was detected using triple quadrupole tandem mass spectrometer with selected reaction monitoring mode. The monitored precursor‐to‐product ion transitions were m/z 459.4 → 375.3 for PPD, m/z 635.4 → 113.0 for M1, m/z 477.4 → 441.4 for M2 and M3 and m/z 475.4 → 391.3 for IS. The developed assay was validated according to the guidelines of the US Food and Drug Administration. The calibration curves showed good linearity over the tested concentration ranges (r > 0.9993), with the LLOQ being 1 ng/mL for all analytes. The intra‐ and inter‐day precisions (RSD) were < 9.51% while the accuracy (RE) ranged from −8.91 to 12.84%. The extraction recovery was >80% and no obvious matrix effect was detected. The analytes were stable in rat plasma with the RE ranging from −12.34 to 9.77%. The validated assay has been successfully applied to the pharmacokinetic study of PPD as well as its metabolites in rat plasma. According to the pharmacokinetic parameters, the in vivo exposures of M1, M2 and M3 were 11.91, 47.95 and 22.62% of that of PPD, respectively.     

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.     

An LC–MS/MS method for quantification of demethylzeylasteral,a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study

In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC–MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Simultaneous determination of wogonin,oroxylin a,schisandrin, paeoniflorin and emodin in rat serum by HPLC–MS/MS and application to pharmacokinetic studies

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti‐inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high‐performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). The separation was performed on a Symmetry C₁₈ column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple‐reaction monitoring with electrospray ionization source in positive–negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu‐gan‐kan‐kang‐yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.     

A reliable LC–MS/MS method for the quantification of five bioactive saponins of crude and processed Bupleurum scorzonerifolium in rat plasma and its application to a pharmacokinetic study

A simple and reliable liquid chromatography–mass spectrometry (LC–MS) method was developed for simultaneous determination of saikosaponin A, saikosaponin B₁, saikosaponin C, saikosaponin D and saikosaponin F in rat plasma using glycyrrhetinic acid as an internal standard (IS). The separation was operated on a Waters BEH C₁₈ column. The mobile phases of gradient elution consisted of acetonitrile (A) and 0.1% aqueous acetic acid (B). The mass spectrometric detection was accomplished in multiple reaction monitoring mode. The five saponins displayed good linearity (r² > 0.9996). The lower limits of quantitation of saikosaponin A, saikosaponin B₁, saikosaponin C, saikosaponin D and saikosaponin F were determined to be 2.9, 2.3, 3.5, 2.9 and 3.1 ng/mL, respectively. Moreover, the intra‐ and inter‐day precisions of the five saponins showed an RSD within 2.96%, whereas the accuracy (RE) ranged from ?2.28 to 2.78%. Finally, the developed method was fully validated and applied to a comparative pharmacokinetic study of the five bioactive saponins in rats following oral administration of crude and vinegar‐processed Bupleurum scorzonerifolium.     

LC–MS/MS method for simultaneous determination of ramelteon and its metabolite M‐II in human plasma: Application to a clinical pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M‐II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS‐2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol–0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500–30.0 ng/mL for ramelteon and 1.00–250 ng/mL for M‐II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (C_max), the time to the C_max and the elimination half‐life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M‐II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.