Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP‐HPLC method to serve as activity marker

In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r² = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10^?2 μm . Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (K_m, V_max and K_i) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Development and validation of a high performance liquid chromatography quantification method of levo‐tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study

Levo ‐tetrahydropalmatine (l‐ THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l‐ THP and its desmethyl metabolites l‐ corydalmine (l‐ CD) and l‐ corypalmine (l‐ CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid–liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed‐phase Symmetry® C₁₈ column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile–methanol–10 mm ammonium phosphate (pH 3) (10:30:60, v /v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1–10,000 ng/mL. The intra‐ and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l‐ THP in rats. Taken together, the developed method can be applied for bioanalysis of l‐ THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.     

Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma

Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis HLB cartridges. Chromatographic separation was achieved by isocratic elution with water-methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4-8 microg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4-80 microg/mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy did not exceed 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 microg/mL for all compounds. This method seems to be a useful tool for clinical research and therapeutic drug monitoring of eslicarbazepine acetate and its metabolites S-licarbazepine, R-licarbazepine and oxcarbazepine.     

Development and validation of a HPLC-MS/MS method for the determination of venlafaxine enantiomers and application to a pharmacokinetic study in healthy Chinese volunteers

An HPLC‐MS/MS method has been developed and validated for the determination of venlafaxine enantiomers in human plasma and applied to a pharmacokinetic study in healthy Chinese volunteers. The method was carried out on a vancomycin chiral column (5 µm, 250 × 4.6 mm) maintained at 25°C. The mobile phase was methanol–water containing 30 mmol/L ammonium acetate, pH 3.3 adjusted with aqueous ammonia (8:92, v/v) at the flow rate 1.0 mL/min. A tandem mass spectrometer with an electrospray interface was operated in the multiple reaction monitoring mode to detect the selected ions pair at m/z 278.0 → 120.8 for venlafaxine enantiomers and m/z 294.8 → 266.7 for estazolanm (internal standard). The method was linear in the concentration range of 0.28–423.0 ng/mL. The lower limit of quantification was 0.28 ng/mL. The intra‐and inter‐day relative standard deviations were less than 9.7%. The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine enantiomers in 18 healthy volnteers. Validation parameters such as the specificity, linearity, precision, accuracy and stability were evaluated, giving results within the acceptable range. Pharmacokinetic parameters of the venlafaxine enantiomers were measured in the 18 healthy Chinese volunteers who received a single regimen with venlafaxine hydrochloride capsules. The results show that AUC_(0–∞), C_max and t_1/2 between S‐venlafaxine and R‐venlafaxine are significantly different (p < 0.05). Copyright © 2010 John Wiley & Sons, Ltd.     

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7‐hydroxymethotrexate (7‐OH‐MTX) and 2,4‐diamino‐N^10‐methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7‐OH‐MTX, DAMPA and 2,4‐diaminopteroic acid (DAPA) as an internal standard were separated on a reversed‐phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7‐OH‐MTX and DAMPA from plasma at 100 pmol/mL were 81.5 ± 5.4, 82.5 ± 5.3 and 56.2 ± 7.0%, respectively. The limits of quantification for MTX, 7‐OH‐MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within‐ and between‐day variations for MTX, 7‐OH‐MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a novel ion‐pairing HPLC‐FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood

Hydroxychloroquine (HCQ) is an old antimalarial drug that has proven to be a safe and effective treatment for systemic lupus erythematosus (SLE) and other autoimmune diseases. Since hematic concentration of HCQ is closely related to the therapeutic response, monitoring the levels of the drug and its metabolites in the blood of HCQ‐treated patients helps the clinician in the evaluation of partial or complete unresponsiveness to treatment. We developed and validated a novel ion‐pairing HPLC‐FL method for the simultaneous dosage of HCQ, and its major metabolites desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, after extraction from whole blood. This methodological approach was used for the analysis of real samples obtained from patients affected by SLE and undergoing HCQ treatment. The same samples were also analyzed using a previously validated LC/MS/MS method and data obtained with the two approaches were in substantial agreement with each other. Results presented in this work indicate that this approach can be successfully used to monitor the level of HCQ and its metabolites in the blood of various categories of patients (i.e. low and high responders, or those not adhering to the therapy). Comparison of HPLC‐FL and LC/MS/MS data confirmed the efficacy of the proposed method for routine clinical analyses.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a stereoselective HPLC method for the determination of the in vitro transport of nateglinide enantiomers in rat intestine

A simple stereoselective high performance liquid chromatographic method was developed for the determination of the in vitro transport of the enantiomers of nateglinide (N-(trans-4-isopropylcyclohexyl-carbonyl)-phenylalanine) in the rat intestine using a Chiralcel OJ-RH column (150 x 4.0 mm, 5 microm). The effects of the mobile phase composition, pH, the flow rate, and the temperature on the chromatographic separation were investigated. The enantioseparation was achieved at 33 degrees C using a mobile phase containing 100 mM potassium dihydrogen phosphate, pH 2.5, and ACN (32:68 v/v) delivered at a flow rate of 1 mL/min. The analytes were monitored at 210 nm and linearity (r >0.99) was obtained for a concentration range of 0.5-50 microg/mL. The LOD and LOQ were 0.2 and 0.5 microg/mL for the R-enantiomer and 0.2 and 0.8 microg/mL for the S-enantiomer, respectively. Both, the intra- and interday accuracy and precision of the calibration curves were determined. The method was successfully applied to estimate the in vitro passage of the enantiomers and the racemate of nateglinide in duodenum, jejunum, and ileum of rats. Generally, higher concentrations of nateglinide and the S-enantiomer were observed when the racemate was administered compared to administration of the individual enantiomers of nateglinide.     

An HPLC‐UV method for the simultaneous quantification of curcumin and its metabolites in plasma and lung tissue: Potential for preclinical applications

Curcumin, derived from turmeric, has been extensively investigated for its broad spectrum of biological activities. Previously reported HPLC‐UV methods have focussed on analysis of the parent compound. Here, a sensitive HPLC‐UV method was developed and partially validated, then used for the simultaneous determination of curcumin and its glucuronide and sulfate metabolites in plasma and lung tissue from mice. The assay was applied to an in vivo pharmacokinetic study comparing formulated curcumin (Meriva™) with standard curcumin. Plasma levels of glucuronide and sulfate metabolites were 5‐ and 2‐fold higher after Meriva™ administration compared with standard curcumin. In lung tissue, free curcumin was 4‐fold higher following Meriva™ administration vs standard curcumin. This assay represents a rapid, cheap method for simultaneous detection of curcumin and its major metabolites that has applicability in pre‐clinical settings.     

Development of a simple and rapid HPLC‐MS/MS method for quantification of streptomycin in mice and its application to plasma pharmacokinetic studies

Streptomycin was the first discovered aminoglycoside antibiotic. It has been widely applied in veterinary medicine for the prevention and treatment of bacterial infection. However, the current detection methods are not satisfactory in terms of sensitivity and sample process, which makes them unsuitable for a pharmacokinetic study. A high‐performance liquid chromatography–mass spectrometric method employing positive electrospray ionization was developed and validated for the determination of streptomycin concentration in mice plasma. A simple protein precipitation method was utilized to extract streptomycin as well as the internal standard (kanamycin) from mouse plasma. This assay method was validated in terms of specificity, sensitivity, precision, accuracy and recovery. This method was applied to a pharmacokinetic study in mice following intramuscular administration of 200 mg/kg streptomycin. The lower limit of quantification of the developed assay method for streptomycin was 10 ng/mL. The intra‐day and inter‐day precision was evaluated with the coefficient of variations <14.3%, whereas the mean accuracy ranged from 87.0 to 105.0%. The samples were stable under the experimental conditions. The present method provides a robust, fast and sensitive analytical approach for the quantification of streptomycin in mouse plasma and has been successfully applied to a pharmacokinetic study in mice.     

Development of solid-phase extraction method and its application for determination of hydrochlorothiazide in human plasma using HPLC

A high-performance liquid chromatographic method was developed, validated and applied for the determination of hydrochlorothiazide in human plasma. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of hydrochlorothiazide and internal standard were investigated. The method involves solid-phase extraction on RP-select B cartridges followed by isocratic reversed-phase chromatography on a Hibar Lichrospher 100 RP-8 column with UV detection at 230 nm. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. Limit of quantification was 10 ng mL(-1). The method has been implemented to monitor hydrochlorothiazide levels in patient samples.     

A simple and sensitive HPLC-UV method for the determination of ponicidin in rat plasma and its application in a pharmacokinetic study

A simple, rapid, selective and sensitive HPLC‐UV method has been developed and validated for the determination of ponicidin in rat plasma. The analyte was extracted from rat plasma by liquid–liquid extraction with ethyl acetate as the extraction solvent. The LC separation was performed on a Zorbax Eclipse XDB C₁₈ analytical column (150 × 4.6 mm i.d., 5 µm) with an isocratic mobile phase consisting of methanol–water–phosphoric acid (45:55:0.01, v/v/v) at a flow rate of 1.0 mL/min. There was a good linearity over the range of 0.1–25 µg/mL (r = 0.9995) with a weighted (1/C²) least square method. The lower limit of quantification was proved to be 0.1 µg/mL. The accuracy was within ±10.0% in terms of relative error and the intra‐ and inter‐day precisions were less than 9.1% in terms of relative standard deviation. After validation, the method was successfully applied to characterize the pharmacokinetics of ponicidin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a reversed-phase liquid chromatographic method for separation and simultaneous determination of COX-2 inhibitors in pharmaceuticals and its application to biological fluids

An isocratic reversed-phase high-performance liquid chromatographic method has been developed for separation and simultaneous determination of COX-2 inhibitors, viz., celecoxib, rofecoxib, valdecoxib, nimesulide and nabumetone, using 4-chloro-2-nitroaniline as internal standard. Good chromatographic separation was achieved using a reversed-phase Inertsil C(18) column with mobile phase consisting of methanol and 0.05% aqueous glacial acetic acid (68:32 v/v) using photodiode array (PDA) detector at 230 nm. It was validated with respect to accuracy, precision, linearity, limit of detection and quantification. The linearity range was found to be 1.0--20 microg/mL and the percentage recoveries were between 97.55 and 100.14. The method is suitable not only for the estimation of active ingredients in pharmaceutical dosage forms but also in vitro estimations in human plasma. It is simple, rapid, selective and capable of detecting and determining COX-2 inhibitors with a detection limit of 0.127--1.040 microg/mL simultaneously.     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

A rapid and simple high-performance liquid chromatography method for the determination of human plasma levofloxacin concentration and its application to bioequivalence studies

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of levofloxacin in human plasma is described. Neutralized with phosphate buffer (pH 7.0), the sample (0.1 mL) was extracted with dichlormethane (1 mL). After voltex-mixing and centrifuged at 3000g for 6 min at 4 degrees C, the upper aqueous layer was aspirated using a micro vacuum pump and the organic layer was directly transferred to a clean test tube without pipetting. The organic solvent was evaporated and the residues were reconstituted with the mobile phase. Levofloxacin and terazosin (internal standard, IS) were chromatographically separated on a C(18) column with a mobile phase containing phosphate buffer (pH 3.0, 10 mm), acetonitrile and triethylamine (76:24:0.076, v/v/v) at a flow rate of 1 mL/min. The analytes were detected using fluorescence detection at an excitation and emission wavelength of 295 and 440 nm, respectively. The linear range of the calibration curves was 0.0521-5.213 microg/mL for levofloxacin with a lower limit of quantitation (0.0521 microg/mL). The retention times of levofloxacin and terazosin were 2.5 and 3.1 min, respectively. Within- and between-run precision was less than 12 and 11%, respectively. Accuracy ranged from -6.3 to 4.5%. The recovery ranged from 86 to 89% at the concentrations of 0.0521, 0.5213 and 5.213 microg/mL. The present HPLC-FLD method is sensitive, efficient and reliable. The method described herein has been successfully used for the pharmacokinetic and bioequivalence studies of a levofloxacin formulation product after oral administration to healthy Chinese volunteers.     

Sensitive method for the determination of paricalcitol by liquid chromatography and mass spectrometry and its application to a clinical pharmacokinetic study

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for the quantification of paricalcitol (PAR) in human plasma (500 μL) using paricalcitol‐d₆ (PAR‐d₆) as an internal standard (IS) as per regulatory guidelines. A liquid–liquid extraction method was used to extract the analyte and IS from human plasma. Chromatography was achieved on Zorbax SB C₁₈ column using an isocratic mobile phase in a gradient flow. The total chromatographic run time was 6.0 min and the elution of PAR and PAR‐d₆ occurred at ~2.6 min. A linear response function was established for the range of concentrations 10–500 pg/mL in human plasma. The intra‐ and inter‐day accuracy and precision values for PAR met the acceptance criteria. The validated assay was applied to quantitate PAR concentrations in human plasma following oral administration of 4 µg capsules to humans. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction

An HPLC method was developed for the simultaneous determination of seven water-soluble vitamins, viz. thiamine, riboflavin, nicotinic acid, nicotinamide, pyridoxine, cyanocobalamin, and folic acid, in multivitamin pharmaceutical formulations and biological fluids (blood serum and urine). Separation was achieved at ambient temperature on a Phenomenex Luna C18 (150 x 4.6 mm) analytical column. Gradient elution was performed starting at a 99:1 A:B v/v composition, where A: 0.05 M CH3COONH4/CH3OH (99/1) and B: H2O/CH3OH (50/50), at a flow rate of 0.8 mL/min. After a 4-min isocratic elution the composition was changed to 100% of B in 18 min and elution continued isocratically for 8 min. Detection was performed with a photodiode array detector at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples. Detection limits were in the range of 1.6-3.4 ng, per 20-microL injection, while linearity held up to 25 ng/microL. Accuracy, intra-day repeatability (n = 6), and inter-day precision (n = 7) were found to be satisfactory. Theobromine (2 ng/microL) was used as internal standard. Sample preparation of biological fluids was performed by SPE on Supelclean LC-18 cartridges with methanol-water 85/15 v/v as eluent. Extraction recoveries from biological matrices ranged from 84.6% to 103.0%.     

Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 μm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.