Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a rapid LC‐MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring

A selective, rapid, and sensitive liquid chromatography–tandem mass spectrometry(LC‐MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one‐step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed‐phase YMC‐ODS‐C₁₈ column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0–60.0 ng/mL. Intra‐ and inter‐batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions. Copyright © 2015 John Wiley & Sons, Ltd.     

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

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

An improved LC‐MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study

An improved LC‐MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide‐d3 was used as internal standard (IS) and liquid–liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP‐column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 → m/z 188.9 and m/z 367.0 → m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25–50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration–time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

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

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

A validated HPLC‐MS/MS method for the quantification of caderofloxacin in human plasma and its application to a clinical pharmacokinetic study

A simple, selective and rapid HPLC‐MS/MS method was developed and validated for the determination of caderofloxacin in human plasma. Sparfloxacin was used as the internal standard (IS). After precipitation with methanol and dilution with the mobile phase, the samples were injected into the HPLC‐MS/MS system. The chromatographic separation was performed on a Zorbax XDB Eclipse C₁₈ column (150 × 4.6 mm, 5 µm) with a mobile phase of ammonium acetate buffer (20 mm, pH 3.0)–methanol, 45:55 (v/v). The MS/MS analysis was done in positive mode. The multiple reaction monitoring transitions monitored were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The calibration curve was linear over the range of 50.0–8000 ng/mL with an aliquot of 100 μL plasma. The precision of the assay was 2.0–9.4 and 6.6–11.5% for the intra‐ and inter‐run variability, respectively. The intra‐ and inter‐run accuracy (relative error) was 4.4–10.0 and ?1.2–4.0%. The total run time was 3.5 min. The assay was fully validated in accordance with the US Food and Drug Administration guidance. It was successfully applied to a pharmacokinetic study of caderofloxacin in healthy Chinese volunteers. Copyright © 2015 John Wiley & Sons, Ltd.     

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

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

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

In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2–500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and ?80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half‐life and area under the concentration–time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

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 sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a sensitive and high‐throughput LC‐MS/MS method for the simultaneous determination of esomeprazole and naproxen in human plasma

A sensitive and high‐throughput LC‐MS/MS method has been developed and validated for the combined determination of esomeprazole and naproxen in human plasma with ibuprofen as internal standard. Solid‐phase extraction was used to extract both analytes and internal standard from human plasma. Chromatographic separation was achieved in 4.0 min on XBridge C₁₈ column using acetonitrile–25 mM ammonium formate (70:30, v/v) as mobile phase. Mass detection was achieved by ESI/MS/MS in negative ion mode, monitoring at m/z 344.19 → 194.12, 229.12 → 169.05 and 205.13 → 161.07 for esomeprazole, naproxen and IS, respectively. The calibration curves were linear from 3.00 to 700.02 ng/mL for esomeprazole and 0.50 to 150.08 ng/mL for naproxen. The intra‐ and inter‐batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. The assay is suitable for measuring accurate esomeprazole and naproxen plasma concentrations in human bioequivalence study following combined administration. Copyright © 2013 John Wiley & Sons, Ltd.     

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

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

An improved LC–MS/MS method for determination of docetaxel and its application to population pharmacokinetic study in Chinese cancer patients

Because of its unpredictable side effects and efficacy, the anticancer drug docetaxel (DTX) requires improved characterisation of its pharmacokinetic profiles through population pharmacokinetic studies. A sensitive and rugged LC–MS/MS method for the detection of DTX in human plasma was developed and optimised using paclitaxel as an internal standard (IS). The plasma samples underwent rapid extraction using hybrid solid-phase extraction-protein precipitation. The analyte and IS were separated with an isocratic system on a Zorbax Eclipse Plus C₁₈ column using water containing 0.05% acetic acid along with 20 μM of sodium acetate and methanol (30/70, v/v) as the mobile phase. Quantification was performed using a triple quadrupole mass spectrometer through multiple reaction monitoring in positive mode, using the m/z 830.3 → 548.8 and m/z 876.3 → 307.7 transitions for DTX and paclitaxel, respectively. The range of the calibration curve was 1–500 ng/mL for DTX, and the linear correlation coefficient was >0.99. The accuracies ranged from −4.6 to 4.2%, and the precision was no higher than 7.0% for the analytes. No significant matrix effect was observed. Both DTX and the IS showed considerable recovery. This method was finally applied to the establishment of a population pharmacokinetic model to optimise the clinical use of DTX.     

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 tapentadol and its carbamate prodrug in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study

A prodrug of tapentadol, namely tapentadol carbamate (WWJ01), was synthesized to improve the bioavailability of tapentadol owing to its extensive first‐pass metabolism. In this study, a highly rapid and sensitive UPLC‐MS/MS method was developed and validated for the simultaneous determination of tapentadol and WWJ01 in rat plasma with fluconazole as an internal standard. The analytes and internal standard were treated by methanol and then separated on a Phenomenex Kinetex® XB‐C₁₈ (2.1 × 50 mm × 2.6 μm) column at a flow rate of 0.3 mL/min. The mobile phase comprised methanol and water with a gradient elution. The mass transition ion‐pairs were m/z 222.2 → 107.0, m/z 293.2 → 71.9 and m/z 307.1 → 220.0 for tapentadol, WWJ01 and IS, respectively. Excellent linearity was observed over the concentration range of 2–1250 ng/mL (r = 0.995) with a lower limit of quantification of 2 ng/mL for both tapentadol and WWJ01. The intra‐ and inter‐day accuracy and precision for all quality control samples were within ±15%. The validated method was accurate, rapid and reproducible, and was successfully applied to a pharmacokinetic study of tapentadol and WWJ01.