QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

The present studies describe the systematic quality by design (QbD)‐oriented development and validation of a simple, rapid, sensitive and cost‐effective reversed‐phase HPLC bioanalytical method for nevirapine in rat plasma. Chromatographic separation was carried out on a C₁₈ column using isocratic 68:9:23% v/v elution of methanol, acetonitrile and water (pH 3, adjusted by orthophosphoric acid) at a flow rate of 1.0 mL/min using UV detection at 230 nm. A Box–Behnken design was applied for chromatographic method optimization taking mobile phase ratio, pH and flow rate as the critical method parameters (CMPs) from screening studies. Peak area, retention time, theoretical plates and peak tailing were measured as the critical analytical attributes (CAAs). Further, the bioanalytical liquid–liquid extraction process was optimized using an optimal design by selecting extraction time, centrifugation speed and temperature as the CMPs for percentage recovery of nevirapine as the CAA. The search for an optimum chromatographic solution was conducted through numerical desirability function. Validation studies performed as per the US Food and Drug Administration requirements revealed results within the acceptance limit. In a nutshell, the studies successfully demonstrate the utility of analytical QbD approach for the rational development of a bioanalytical method with enhanced chromatographic separation and recovery of nevirapine in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

QbD-driven development and validation of an efficient bioanalytical UPLC method for estimation of olmesartan medoxomil

The present studies describe quality by design-based development of bioanalytical ultra performance liquid chromatography method of olmesartan medoxomil. Initially, method objectives were defined and critical analytical attributes (CAAs) earmarked. Method optimization was conducted using a central composite design for optimizing mobile phase ratio and injection volume as the critical method parameters (CMPs) identified from risk assessment and factor screening studies, and evaluated for their influence on peak area, theoretical plates, and asymmetry factor as CAAs. Chromatographic separation was achieved using acetonitrile:water solvent system containing 0.1% orthophosphoric acid (54:46, v/v) as the mobile phase with UV detection at 243 nm. Further optimization of bioanalytical extraction process was accomplished using a Box–Behnken design selecting extraction time, centrifugation speed, and centrifugation time as the CMPs identified from failure mode and effect analysis, and evaluated for percent recovery, peak asymmetry, and theoretical plate count as the CAAs. Establishment of calibration curve indicated linearity between concentration range of 100 and 800 ng mL^?1, excellent accuracy and precision with limit of detection and limit of quantification as 6.2 and 19.0 ng mL^?1, respectively. Drug stability studies indicated mean percent recovery ranging between 92.4 and 97.3% under various stress conditions.     

Development and Validation of RP-UHPLC Method for Quantification of Gliclazide in Bulk and Pharmaceutical Dosage Form Using Quality-by-Design (QbD) Approach: A Shifting Paradigm

The current research endeavours quality-by-design (QbD)-aided chromatographic techniques for the quantification of gliclazide (GLZ) in bulk and pharmaceutical dosage forms. Analytical QbD was initiated by assigning both an analytical target profile (ATP) and critical analytical attributes (CAAs). Furthermore, risk evaluation studies, along with factor screening studies, helped identify critical method parameters (CMPs). Optimisation was carried out using a 3² full factorial design by utilising the identified CMP, that is, flow rate (X1) and pH of buffer (X2) at three different levels along with evaluation of the selected CAA, that is, the retention time (Y1) and the peak area (Y2). In addition, the influence of sole and interactive CMPs on CAAs was checked using the data obtained statistically and with response surface plots. The confirmation of significance (P?<?0.05) of the method parameters was determined using analysis of variance (ANOVA). Chromatographic separation was achieved using a stainless-steel C8 column (25 cm?×?4 mm) in isocratic elution mode using phosphate buffer (pH 3.4) and HPLC-grade acetonitrile (50:50 v/v) as the eluent. The flow rate was adjusted to 1 mL min^?1 and the eluent was detected at 230 nm. The validated method, alongside subsequent stress degradation studies conducted according to the ICH guidelines, further favours it as a highly efficient method for the analysis of regular drugs as well as their degraded products. The method proposed above provided a successful demonstration of the QbD-based approach in developing an extremely sensitive and dependable technique for estimating the GLZ for routine analysis and pre-clinical applications.

     

The application of qNMR for the determination of rosuvastatin in tablet form

In this study, quantative nuclear magnetic resonance (qNMR) method was used to determine the content of rosuvastatin in tablet. Linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were determined in validation study of rosuvastatin. Furthermore, validation study of rosuvastatin was performed with high performance liquid chromatography (HPLC). Uncertainties of qNMR and HPLC methods were determined using per EURACHEM/CITAC Guide CG 4 (3th edition), quantifying uncertainty in analytical measurement. qNMR and HPLC methods were linear in the ranges of 0.10 - 5.00 mg/mL and 0.001 - 0.0995 mg/mL, respectively and these lineraties indicate very good linearity performance with regression coefficients (R2 value) above > 0.99. Moreover, LOD and LOQ values using qNMR method were observed as 0.25 mg/mL and 0.80 mg/mL, respectively. These values using HPLC method were found as 0.00051 µg/mL and 0.001695 µg/mL, respectively. The strengths and weaknesses of qNMR method and HPLC method were determined with spectral emphasis on the role of identical reference standards in qualitive and quantitative analyses. It was found that qNMR method is simple, efficient, reliable, and accurate method. Moreover, qNMR method is an easy, practical, and useful method for the validation and optimization of rosuvastatin in the tablet.     

Integrated quality by design (QbD) and design of experiments (DoE) approach for UFLC determination of telaprevir in rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of telaprevir in Wistar albino rat serum. Principles of quality by design (QbD) were implemented for enhancing the bioanalytical liquid–liquid extraction of telaprevir from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the critical analytical attribute, i.e., %recovery of telaprevir. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:borate buffer of pH 9 (90:10 v/v) flowing at 1.2?mL/min. Photodiode array detection was performed at 270?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 25–10,000?ng/mL. Limit of detection for the developed method was 10?ng/mL. Further, design of experiments (DoE) used for inter-day accuracy and precision study suggested superior method reliability. This integrated QbD- and DoE-based approach ensured the development of a validated and reliable analytical method for optimum bioanalysis of telaprevir in biological matrix.     

Complementary mobile-phase optimisation for resolution enhancement in high-performance liquid chromatography

An optimisation methodology in high-performance liquid chromatography (HPLC) is presented for the selection of two or more mobile phases having an optimal complementary resolution. The complementary mobile phases (CMPs) are selected in such a way that each one resolves optimally only some compounds in the mixture, while the remainder, resolved by the other mobile phase(s), can overlap among them. The methodology is based on the computation of a peak purity measurement for each solute, using an asymmetrical peak model for peak simulation. Two global resolution criteria (product of elementary resolutions and worst elementary resolution) and two methods for solving the problem (a systematic examination of all possible solute arrangements, and the use of genetic algorithms to expedite the calculation time) were used to find the optimal CMPs. The CMP optimisation methodology was applied to the resolution of a mixture of 10 diuretics and beta-blockers, which could not be resolved using a single mobile phase; virtual baseline resolution was achieved, however, with two CMPs.     

Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate

The current work entails development of rapid, sensitive, and inexpensive high-performance liquid chromatographic method of quercetin dihydrate using the quality by design approach. Quality target method profile was defined and critical analytical attributes (CAAs) were earmarked. Chromatographic separation was accomplished on a C₁₈ column using acetonitrile and ammonium acetate buffer (35:65) %v/v (containing 0.1% acetic acid, pH 3.5) as mobile phase at 0.7?mL/min flow rate with UV detector at 237?nm. Screening studies using fractional factorial design revealed that organic modifier, injection volume, column temperature, and buffer strength have significant influence on method CAAs, namely, peak area, retention time, and peak tailing. The critical method parameters were systematically optimized using Box–Behnken design. Response surface mapping was used along with numerical optimization and desirability function for identifying the optimal chromatographic conditions. Linearity was observed in the drug concentration ranging between 2 and 50?µg/mL. Accuracy analysis revealed mean % recovery between 93.6 and 96.2%, while precision study revealed mean % recovery between 93.7 and 96.5%. Limits of detection and quantification of the developed method were found to be 12.1 and 36.6?ng/mL. Overall, the studies construed successful development of chromatographic method of quercetin with enhanced method performance.     

Quality by Design: Design of Experiments Approach Prior to the Validation of a Stability-Indicating HPLC Method for Montelukast

This paper describes a systematic design of experiments (DoE) approach by applying the principle of quality by design (QbD) to determine the design space for a stability-indicating HPLC method prior to validation. By employing DoE, a simultaneous multivariate approach was carried out for mobile phase pH, flow rate, percentage of organic content and column temperature. A two-level fractional factorial design (2^4?1 + 2 center points = 10 experiments) was employed and statistical analysis of the experimental data uncovered the significant influential chromatographic factors. The experimental data for USP tailing and resolution were analyzed statistically to screen the chromatographic factors. This approach determined the most influential chromatographic factors. During this process, inferences were evaluated from various data tables, for example, analysis of variance, summary of fit, lack of fit, and parameter estimates. The study also explained various plots such as actual vs. predicted plot, Pareto plot, and prediction profiler. The acceptable range of the chromatographic factors was displayed as a Contour plot defining the ‘design space’ of the method. The range of operating conditions that guarantee a satisfactory QbD was deduced to finalize the method prior to validation. The method is simple, rapid, and robust for the determination of montelukast in montelukast sodium oral granules dosage form. The method was validated according to ICH guidelines for accuracy, precision, linearity, range, specificity, ruggedness and robustness (one factor varied at a time). The method has been successfully transferred to the quality control department for quality analysis of manufactured batches and stability samples.     

Quality by design (QbD) based development of a stability indicating HPLC method for drug and impurities

In this paper, an application of Quality by Design (QbD) concepts to the development of a stability indicating HPLC method for a complex pain management drug product containing drug substance, two preservatives, and their degradants is described. The QbD approach consisted of (i) developing a full understanding of the intended purpose, (ii) developing predictive solutions, (iii) designing a meaningful system suitability solution that helps to identify failure modes, and (iv) following design of experiments (DOE) approach. The starting method lacked any resolution among drug degradant and preservative oxidative degradant peaks, and peaks for preservative and another drug degradant. The method optimization was accomplished using Fusion AE? software (S-Matrix Corporation, Eureka, CA) that follows a DOE approach. Column temperature (50 ± 5°C), mobile phase buffer pH (2.9 ± 0.2), initial % acetonitrile (ACN, 2 ± 1%), and initial hold time (2.5, 5, or 10 min) of the HPLC method were simultaneously studied to optimize separation of the unresolved peaks. The optimized HPLC conditions (column temperature of 50°C, buffer pH of 3.1, 3% initial ACN with 2.5 min initial hold) resulted in fully resolved peaks in the two critical pairs. The QbD based method development helped in generating a design space and operating space with knowledge of all method performance characteristics and limitations and successful method robustness within the operating space.     

Quality by Design: Design of Experiments Approach Prior to the Validation of a Stability-Indicating HPLC Method for Montelukast

This paper describes a systematic design of experiments (DoE) approach by applying the principle of quality by design (QbD) to determine the design space for a stability-indicating HPLC method prior to validation. By employing DoE, a simultaneous multivariate approach was carried out for mobile phase pH, flow rate, percentage of organic content and column temperature. A two-level fractional factorial design (2⁴⁻¹ + 2 center points = 10 experiments) was employed and statistical analysis of the experimental data uncovered the significant influential chromatographic factors. The experimental data for USP tailing and resolution were analyzed statistically to screen the chromatographic factors. This approach determined the most influential chromatographic factors. During this process, inferences were evaluated from various data tables, for example, analysis of variance, summary of fit, lack of fit, and parameter estimates. The study also explained various plots such as actual vs. predicted plot, Pareto plot, and prediction profiler. The acceptable range of the chromatographic factors was displayed as a Contour plot defining the ‘design space’ of the method. The range of operating conditions that guarantee a satisfactory QbD was deduced to finalize the method prior to validation. The method is simple, rapid, and robust for the determination of montelukast in montelukast sodium oral granules dosage form. The method was validated according to ICH guidelines for accuracy, precision, linearity, range, specificity, ruggedness and robustness (one factor varied at a time). The method has been successfully transferred to the quality control department for quality analysis of manufactured batches and stability samples.

     

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.     

A new approach for evaluating carryover and its influence on quantitation in high-performance liquid chromatography and tandem mass spectrometry assay

In a high-performance liquid chromatography (HPLC)-based analytical method, carryover denotes one type of systematic error that is derived from a preceding sample and introduced into the next sample. For typical bioanalytical method development, a significant amount of time and resources are spent on reducing carryover for some analytes. In this paper, the statistical characteristics of carryover were analyzed based on the experimental results. The relative carryover (RC), defined as the peak area ratio of a blank sample to the preceding sample, was constant for the analyte and independent of the concentration of the preceding sample. The influence of carryover on the quantitation of the next injected sample or the 'following' sample was proportional to the concentration ratio of two consecutive samples and the relative carryover. Based on these experiments and analyses, the influence of carryover on the quantitation of unknown samples in an HPLC assay can be evaluated by the estimated carryover influence (ECI), which is the product of the relative carryover and the concentration ratio. This new approach provides a quantitative estimation for the influence of carryover on the quantitation of the unknown sample, and removes the limit put on the dynamic range of the assay by the current criterion of carryover. In general, if the relative standard deviation (RSD) of a validated bioanalytical method is less than 10%, the carryover will not have a significant effect on the accuracy of the assay when the estimated carryover influence is less than 5%.     

Robust design space development for HPLC analysis of five chemical components in Panax notoginseng saponins

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous analysis of five chemical components in Panax notoginseng saponins (PNS) using quality by design (QbD). The method was developed in two main phases: screening and optimization. During the screening phase, the most suitable stationary phase, column temperature, and flow rate were identified, while the secondary influential parameters, such as the gradient slope, the initial concentration of acetonitrile, and the initial isocratic hold of the gradient elution system were fine-tuned in the later optimization phase. In this phase, a 17-run experiment was used to examine multifactorial effects of these parameters on the critical resolution, analysis time, and peak symmetry. The Monte Carlo simulation method was successfully used to build the chromatographic design space and the process capability index C_p was introduced to evaluate the robustness of the design space. At last, the verification experiment was performed within the working design space by the accuracy profile methodology and model was found to be accurate. A robust combination of separation conditions predicted in the design space was obtained with the gradient slope of 1.6% · min^?1, the initial concentration of acetonitrile of 20%, and the initial isocratic hold of 20 min, and the total analysis time was no more than 40 min. The results demonstrated that rapid separation of five components of PNS herbal extract could be achieved on the chromatographic column packed with narrow size particles at backpressures available in ordinary high performance liquid chromatography (HPLC) instruments.     

Quantitative determination of rosuvastatin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for determining rosuvastatin in human plasma, a new synthetic hydroxymethylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (IS; cilostazol) were extracted by simple one-step liquid/liquid extraction with ether. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The chromatographic separation was performed on an Atlantis C18 column (2.1 mm x 150 mm, 5.0 microm) with a mobile phase consisting of 0.2% formic acid/methanol (30:70, v/v) at a flow rate of 0.20 mL/min. The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 482 --> 258 and m/z 370 --> 288. The areas of peaks from the analyte and the IS were used for quantification of rosuvastatin. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification (LLOQ) was 0.2 ng/mL and the assay exhibited a linear range of 0.2-50.0 ng/mL and gave a correlation coefficient (r) of 0.9991 or better. Quality control samples (0.4, 8, 25 and 40 ng/mL) in six replicates from three different runs of analysis demonstrated an intra-assay precision (RSD) 7.97-15.94%, an inter-assay precision 3.19-15.27%, and an overall accuracy (relative error) of < 3.7%. The method can be applied to pharmacokinetic or bioequivalence studies of rosuvastatin.     

Determination of daumone in mouse plasma by HPLC/MS-MS

Daumone, a pheromone secreted by Caenorhabditis elegans, is an essential regulator of chemosensory processes in development and aging. A quantification method using HPLC/MS-MS was developed for the determination of daumone in mouse plasma. After simple protein precipitation with acetonitrile including methaqualone (an internal standard), the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations following a 5-week repeated oral administration of daumone in mice.     

QbD-mediated RP-UPLC method development invoking an FMEA-based risk assessment to estimate nintedanib degradation products and their pathways

QbD is considered an important, fundamental, and integral part of dosage form development. Despite its significance in drug formulations, the knowledge, reference, and guidance for using QbD in analytical science have not been thoroughly documented in the literature. The present study is aimed at bridging the gap between its generated data and the unexplored terrain in formulation science. This study is novel because, for the first time, an exclusive shorter run time UHPLC method for estimating degradation products was developed through the QbD approach, validated, and proved stability indicative. Five degradation impurities were separated and well characterized. Further, the degradation pathway of the anticancer drug nintedanib (NIN) was explored for the first time in the soft gel formulation using tandem quadrupole MS abetted mass identification, and ESI/MS/MS aided structure elucidation was performed. By carefully demonstrating the step-by-step procedure for QbD-based optimization, parameters such as the analytical target profile (ATP) and critical quality attributes (CQAs) were assessed. The risk assessment was performed using failure mode effect analysis (FMEA). Critical method attributes and critical method parameters were identified based on the magnitude of the calculated risk priority number (RPN) value. Designed experiments using 4-factor two-level factorial design monitored three critical quality attributes to arrive at a method operable design space (MODS). The effect of individual method attributes was also analyzed using half-normal and Pareto charts. Control strategies design and RPN values were recalculated based on the DOE output. This RPN value is eventually identified to be significantly smaller and satisfactory within the allowable limit.     

Development of a validated liquid chromatographic method for quantification of sorafenib tosylate in the presence of stress‐induced degradation products and in biological matrix employing analytical quality by design approach

The current research work envisages an analytical quality by design‐enabled development of a simple, rapid, sensitive, specific, robust and cost‐effective stability‐indicating reversed‐phase high‐performance liquid chromatographic method for determining stress‐induced forced‐degradation products of sorafenib tosylate (SFN). An Ishikawa fishbone diagram was constructed to embark upon analytical target profile and critical analytical attributes, i.e. peak area, theoretical plates, retention time and peak tailing. Factor screening using Taguchi orthogonal arrays and quality risk assessment studies carried out using failure mode effect analysis aided the selection of critical method parameters, i.e. mobile phase ratio and flow rate potentially affecting the chosen critical analytical attributes. Systematic optimization using response surface methodology of the chosen critical method parameters was carried out employing a two‐factor–three‐level–13‐run, face‐centered cubic design. A method operable design region was earmarked providing optimum method performance using numerical and graphical optimization. The optimum method employed a mobile phase composition consisting of acetonitrile and water (containing orthophosphoric acid, pH 4.1) at 65:35 v/v at a flow rate of 0.8 mL/min with UV detection at 265 nm using a C₁₈ column. Response surface methodology validation studies confirmed good efficiency and sensitivity of the developed method for analysis of SFN in mobile phase as well as in human plasma matrix. The forced degradation studies were conducted under different recommended stress conditions as per ICH Q1A (R2). Mass spectroscopy studies showed that SFN degrades in strongly acidic, alkaline and oxidative hydrolytic conditions at elevated temperature, while the drug was per se found to be photostable. Oxidative hydrolysis using 30% H₂O₂ showed maximum degradation with products at retention times of 3.35, 3.65, 4.20 and 5.67 min. The absence of any significant change in the retention time of SFN and degradation products, formed under different stress conditions, ratified selectivity and specificity of the systematically developed method.     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study

A selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid‐phase extraction using 100 μL of human plasma. The separation was carried out on a BDS Hypersil C₁₈ (150 × 4.6 mm, 5 μm) column using a mixture of 0.2% formic acid in HPLC‐grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20–20 μg/mL with r² > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.     

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.