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Sci. Pharm. 2016, 84(2), 347-359; doi:10.3797/scipharm.1510-08

Simultaneous Quantification of Baricitinib and Methotrexate in Rat Plasma by LC-MS/MS: Application to a Pharmacokinetic Study

1
Incozen Therapeutics Pvt. Ltd, Alexandria Knowledge Park, Shamirpet, Hyderabad, 500078, India
2
CRD, PRIST University, Vallam, Thanjavur 613403. India, India
3
Dept of Pharmaceutics, Annai JKK Sampoorani Ammal College of Pharmacy, Namakkal - 638 183, India
*
Author to whom correspondence should be addressed.
Received: 17 October 2015 / Accepted: 20 December 2015 / Published: 20 December 2015
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Abstract

Efficacy assessments using a combination of baricitinib and methotrexate necessitate the development of an analytical method for the determination of both drugs in plasma with precision. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of baricitinib and methotrexate in rat plasma. Extraction of baricitinib, methotrexate, and tolbutamide (internal standard; IS) from 50 µL of rat plasma was carried out by protein precipitation with methanol. Chromatographic separation of the analytes was performed on the YMC pack ODS AM (150 mm × 4.6 mm, 5 µm) column under gradient conditions with methanol: 2.0 mM ammonium acetate buffer as the mobile phases at a flow rate of 1 mL/min. The precursor ion and product ion transition for both analytes and IS were monitored on a triple quadrupole mass spectrometer, operated with selective reaction monitoring in positive ionization mode. The method was validated over a concentration range of 0.5-250.00 ng/mL for baricitinib and methotrexate. Mean extraction recoveries for baricitinib, methotrexate, and IS of 86.8%, 89.4%, and 91.8% were consistent across low, medium, and high QC levels, respectively. Precision and accuracy at low, medium, and high quality control levels were less than 15% across the analytes. Benchtop, wet, freeze-thaw, and long-term stability were evaluated for both of the analytes. The analytical method was applied to support the pharmacokinetic study of simultaneous estimation of baricitinib and methotrexate in Wistar rats. Assay reproducibility was demonstrated by reanalysis of 18 incurred samples.
Keywords: Baricitinib; Methotrexate; Plasma; LC-MS/MS; Bioanalytical Baricitinib; Methotrexate; Plasma; LC-MS/MS; Bioanalytical
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

VEERARAGHAVAN, S.; THAPPALI, S.R.S.; VISWANADHA, S.; VAKKALANKA, S.; RANGASWAMY, M. Simultaneous Quantification of Baricitinib and Methotrexate in Rat Plasma by LC-MS/MS: Application to a Pharmacokinetic Study. Sci. Pharm. 2016, 84, 347-359.

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