Research article Received: 8 October 2014,
Revised: 2 December 2014,
Accepted: 8 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3426
Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study Vijay Kumar S, Vinay Dhiman, Kalpesh Kumar Giri, Kuldeep Sharma, Mohd Zainuddin and Ramesh Mullangi* ABSTRACT: 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. Keywords: tofacitinib; HPLC; method validation; rat plasma; pharmacokinetics
Introduction Tofacitinib (Fig. 1, CAS no: 477600-75-2) is a novel, potent and selective JAK1/JAK3 (Janus kinase) inhibitor in cells and shows dose-dependent reduction in joint inflammation in mouse and rat arthritis models (Milici et al., 2008; Meyer et al., 2010; Ghoreschi et al., 2011; LaBranche et al., 2012). In November 2012, the US Food and Drug Administration approved tofacitinib for the treatment of moderate to severe rheumatoid arthritis. It is recommended (5 mg twice a day) with or without methotrexate for moderate to severe active rheumatoid arthritis adult patients, who have an adequate response or are intolerant to diseasemodifying antirheumatic drugs. To date there is no HPLC method reported for quantification of tofacitinib in rat plasma. In order to support the pharmacokinetic studies in small animals a method is needed that requires a small volume of plasma for quantification. In this paper, we report the development and validation of a simple, specific, sensitive and reproducible HPLC method for quantitation of tofacitinib in rat plasma (100 μL). The method was successfully applied to quantitate levels of tofacitinib in a rat pharmacokinetic study.
Experimental Chemicals and reagents Tofacitinib was procured from Cayman Chemicals (Ann Arbor, MI, USA). Itraconazole was purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from Rankem, Ranbaxy Fine Chemicals Limited (New Delhi, India). Ammonium acetate
Biomed. Chromatogr. 2015
was purchased from Sigma Aldrich (St Louis, MO, USA). Blank rat plasma . (with Na2EDTA as an anticoagulant) was procured from Jubilant Biosys Animal House, Bangalore, India. All other chemicals/reagents were of research grade and used without further purification.
HPLC operating conditions The HPLC system consisted of Waters 2695 Alliance system (Waters, Milford, USA) equipped with performance plus inline degasser along with an autosampler and ultraviolet (UV) detector set at 287 nm for tofacitinib and IS. The chromatographic separation of tofacitinib and IS in processed samples was achieved on a Symmetry Shield RP18 column (250 × 4.6 mm, 5 μm; Waters Corporation, Milford, MA, USA), which was maintained at 40 ± 1 °C. The binary mobile phase system consisting of reservoir A (0.01 m ammonium acetate in Milli Q water, pH 5.0 adjusted with acetic acid) and reservoir B (acetonitrile) was run on a gradient program (0–4.9 min, 95% A and 5% B; 5.0–5.4 min, 5% A and 95% B; and 5.5–9.0 min, 95% A and 5% B). A flow rate of 1.0 mL/min was used throughout the analytical run.
Preparation of stock and standard solutions Tofacitinib and IS were accurately weighed into volumetric flasks using an analytical microbalance. They were then diluted with methanol. Tofacitinib stock solutions for spiking the calibration standards and
* Correspondence to: Ramesh Mullangi, Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India. Email: [email protected] Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India
Copyright © 2015 John Wiley & Sons, Ltd.
Vijay Kumar S et al. calibration curve had to have a correlation coefficient (r) of 0.99 or better. The acceptance criteria for each back-calculated standard concentration were ±15% deviation from the nominal value except at LLOQ, which was set at ±20% (US DHHS, FDA and CDER 2001).
Figure 1. Structural representation of tofacitinib. quality control (QC) samples were prepared from separate weighings at 215 μg/mL. The IS stock solution of 200 μg/mL was prepared in methanol. The stock solutions of tofacitinib and IS were stored at 4 °C, and were found to be stable for 1 month (data not shown). They were successively diluted with methanol–water (50:50, v/v) to prepare working solutions to prepare a calibration curve. Another set of working stock solutions of tofacitinib were made in methanol–water (50:50, v/v) (from different stock) for preparation of QC samples. Appropriate dilutions of tofacitinib stock solution were made in methanol–water (50:50, v/v) to produce working stock solutions for calibration curve and QC spiking. A working IS solution (2000 ng/mL) was prepared in methanol–water (50:50, v/v). Calibration samples were prepared by spiking 90 μL of blank rat plasma with the appropriate working solution of tofacitinib (10 μL) on the day of analysis. Samples for the determination of precision and accuracy were prepared by spiking blank rat plasma in bulk with tofacitinib at appropriate concentrations (182, 546, 2312 and 3975 ng/mL) and 100 μL aliquots were distributed into different tubes. All the samples were stored together at 80 ± 10 °C until analysis.
Sample preparation A liquid–liquid extraction method was followed for extraction of tofacitinib from rat plasma. To an aliquot of 100 μL plasma sample, 25 μL of 20% ammonia solution and IS solution (20 μL of 2000 ng/mL) were added and mixed for 15 s on a cyclomixer (Remi Instruments, Mumbai, India). After the addition of 1.5 mL of tert-butyl methyl ether, the mixture was vortexed for 3 min, followed by centrifugation for 10 min at 14,000 rpm on a Centrifuge 5430R (Eppendorff, Germany) at 4 °C. The organic layer (1.3 mL) was separated and evaporated to dryness at 50 °C using a gentle stream of nitrogen (Turbovap®, Zymark®, Kopkinton, MA, USA). The residue was reconstituted in 200 μL of the 80% acetonitrile in Milli Q water and 50 μL was injected onto the HPLC system.
Validation procedures A full validation according to the US Food and Drug Administration guidelines (US DHHS et al., 2001) was performed for the assay in rat plasma. Specificity and selectivity. The specificity of the method was evaluated by analyzing rat plasma samples from at least six different lots to investigate the potential interferences at the LC peak region for analytes and IS. The acceptance criterion for experiment was that at least four out of six lots should have response less than 5 times the lower limit of quantitation (LLOQ) level response in the same matrix. Recovery. The efficiency of tofacitinib and IS extraction from rat plasma was determined by comparing the responses of the analyte extracted from replicate QC samples (n = 6) with the response of analyte from neat standards at equivalent concentrations by liquid–liquid extraction process. Recoveries of tofacitinib was determined at QC low, QC medium and QC high concentrations, viz. 546, 2312 and 3975 ng/mL, whereas the recovery of the IS was determined at a single concentration of 4000 ng/mL. Calibration curve. The eight-point calibration curve (182, 364, 728, 1325, 2385, 3710, 4240 and 5035 ng/mL) was constructed by plotting the peak area ratio of tofacitinib–IS against the nominal concentration of calibration standards in blank rat plasma. Following the evaluation of different weighting factors, the results were fitted to linear regression 2 analysis with the use of a 1/x (x = concentration) weighting factor. The
wileyonlinelibrary.com/journal/bmc
Precision and accuracy. The intra-assay precision and accuracy were estimated by analyzing six replicates containing tofacitinib at four different QC levels, that is, 182, 546, 2312 and 3975 ng/mL. The inter-assay precision was determined by analyzing the four levels of QC samples on three different runs. The criteria for acceptability of the data included accuracy within 85–115% from the nominal values and a precision of within ±15% relative standard deviation (RSD) except for the LLOQ, which should be within 80–120% for accuracy and
Revised: 2 December 2014,
Accepted: 8 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3426
Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study Vijay Kumar S, Vinay Dhiman, Kalpesh Kumar Giri, Kuldeep Sharma, Mohd Zainuddin and Ramesh Mullangi* ABSTRACT: 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. Keywords: tofacitinib; HPLC; method validation; rat plasma; pharmacokinetics
Introduction Tofacitinib (Fig. 1, CAS no: 477600-75-2) is a novel, potent and selective JAK1/JAK3 (Janus kinase) inhibitor in cells and shows dose-dependent reduction in joint inflammation in mouse and rat arthritis models (Milici et al., 2008; Meyer et al., 2010; Ghoreschi et al., 2011; LaBranche et al., 2012). In November 2012, the US Food and Drug Administration approved tofacitinib for the treatment of moderate to severe rheumatoid arthritis. It is recommended (5 mg twice a day) with or without methotrexate for moderate to severe active rheumatoid arthritis adult patients, who have an adequate response or are intolerant to diseasemodifying antirheumatic drugs. To date there is no HPLC method reported for quantification of tofacitinib in rat plasma. In order to support the pharmacokinetic studies in small animals a method is needed that requires a small volume of plasma for quantification. In this paper, we report the development and validation of a simple, specific, sensitive and reproducible HPLC method for quantitation of tofacitinib in rat plasma (100 μL). The method was successfully applied to quantitate levels of tofacitinib in a rat pharmacokinetic study.
Experimental Chemicals and reagents Tofacitinib was procured from Cayman Chemicals (Ann Arbor, MI, USA). Itraconazole was purchased from Sigma-Aldrich (St Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from Rankem, Ranbaxy Fine Chemicals Limited (New Delhi, India). Ammonium acetate
Biomed. Chromatogr. 2015
was purchased from Sigma Aldrich (St Louis, MO, USA). Blank rat plasma . (with Na2EDTA as an anticoagulant) was procured from Jubilant Biosys Animal House, Bangalore, India. All other chemicals/reagents were of research grade and used without further purification.
HPLC operating conditions The HPLC system consisted of Waters 2695 Alliance system (Waters, Milford, USA) equipped with performance plus inline degasser along with an autosampler and ultraviolet (UV) detector set at 287 nm for tofacitinib and IS. The chromatographic separation of tofacitinib and IS in processed samples was achieved on a Symmetry Shield RP18 column (250 × 4.6 mm, 5 μm; Waters Corporation, Milford, MA, USA), which was maintained at 40 ± 1 °C. The binary mobile phase system consisting of reservoir A (0.01 m ammonium acetate in Milli Q water, pH 5.0 adjusted with acetic acid) and reservoir B (acetonitrile) was run on a gradient program (0–4.9 min, 95% A and 5% B; 5.0–5.4 min, 5% A and 95% B; and 5.5–9.0 min, 95% A and 5% B). A flow rate of 1.0 mL/min was used throughout the analytical run.
Preparation of stock and standard solutions Tofacitinib and IS were accurately weighed into volumetric flasks using an analytical microbalance. They were then diluted with methanol. Tofacitinib stock solutions for spiking the calibration standards and
* Correspondence to: Ramesh Mullangi, Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India. Email: [email protected] Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022, India
Copyright © 2015 John Wiley & Sons, Ltd.
Vijay Kumar S et al. calibration curve had to have a correlation coefficient (r) of 0.99 or better. The acceptance criteria for each back-calculated standard concentration were ±15% deviation from the nominal value except at LLOQ, which was set at ±20% (US DHHS, FDA and CDER 2001).
Figure 1. Structural representation of tofacitinib. quality control (QC) samples were prepared from separate weighings at 215 μg/mL. The IS stock solution of 200 μg/mL was prepared in methanol. The stock solutions of tofacitinib and IS were stored at 4 °C, and were found to be stable for 1 month (data not shown). They were successively diluted with methanol–water (50:50, v/v) to prepare working solutions to prepare a calibration curve. Another set of working stock solutions of tofacitinib were made in methanol–water (50:50, v/v) (from different stock) for preparation of QC samples. Appropriate dilutions of tofacitinib stock solution were made in methanol–water (50:50, v/v) to produce working stock solutions for calibration curve and QC spiking. A working IS solution (2000 ng/mL) was prepared in methanol–water (50:50, v/v). Calibration samples were prepared by spiking 90 μL of blank rat plasma with the appropriate working solution of tofacitinib (10 μL) on the day of analysis. Samples for the determination of precision and accuracy were prepared by spiking blank rat plasma in bulk with tofacitinib at appropriate concentrations (182, 546, 2312 and 3975 ng/mL) and 100 μL aliquots were distributed into different tubes. All the samples were stored together at 80 ± 10 °C until analysis.
Sample preparation A liquid–liquid extraction method was followed for extraction of tofacitinib from rat plasma. To an aliquot of 100 μL plasma sample, 25 μL of 20% ammonia solution and IS solution (20 μL of 2000 ng/mL) were added and mixed for 15 s on a cyclomixer (Remi Instruments, Mumbai, India). After the addition of 1.5 mL of tert-butyl methyl ether, the mixture was vortexed for 3 min, followed by centrifugation for 10 min at 14,000 rpm on a Centrifuge 5430R (Eppendorff, Germany) at 4 °C. The organic layer (1.3 mL) was separated and evaporated to dryness at 50 °C using a gentle stream of nitrogen (Turbovap®, Zymark®, Kopkinton, MA, USA). The residue was reconstituted in 200 μL of the 80% acetonitrile in Milli Q water and 50 μL was injected onto the HPLC system.
Validation procedures A full validation according to the US Food and Drug Administration guidelines (US DHHS et al., 2001) was performed for the assay in rat plasma. Specificity and selectivity. The specificity of the method was evaluated by analyzing rat plasma samples from at least six different lots to investigate the potential interferences at the LC peak region for analytes and IS. The acceptance criterion for experiment was that at least four out of six lots should have response less than 5 times the lower limit of quantitation (LLOQ) level response in the same matrix. Recovery. The efficiency of tofacitinib and IS extraction from rat plasma was determined by comparing the responses of the analyte extracted from replicate QC samples (n = 6) with the response of analyte from neat standards at equivalent concentrations by liquid–liquid extraction process. Recoveries of tofacitinib was determined at QC low, QC medium and QC high concentrations, viz. 546, 2312 and 3975 ng/mL, whereas the recovery of the IS was determined at a single concentration of 4000 ng/mL. Calibration curve. The eight-point calibration curve (182, 364, 728, 1325, 2385, 3710, 4240 and 5035 ng/mL) was constructed by plotting the peak area ratio of tofacitinib–IS against the nominal concentration of calibration standards in blank rat plasma. Following the evaluation of different weighting factors, the results were fitted to linear regression 2 analysis with the use of a 1/x (x = concentration) weighting factor. The
wileyonlinelibrary.com/journal/bmc
Precision and accuracy. The intra-assay precision and accuracy were estimated by analyzing six replicates containing tofacitinib at four different QC levels, that is, 182, 546, 2312 and 3975 ng/mL. The inter-assay precision was determined by analyzing the four levels of QC samples on three different runs. The criteria for acceptability of the data included accuracy within 85–115% from the nominal values and a precision of within ±15% relative standard deviation (RSD) except for the LLOQ, which should be within 80–120% for accuracy and
