Evaluation of Deuterium-Labeled Internal Standard for the Measurement of Venetoclax by HPLC-ESI -Tandem Mass Spectrometry

Objective: Venetoclax is a selective, potent, first-in-class BCL-2 inhibitor that restores apoptosis in cancer cells and has demonstrated clinical efficacy in a variety of haematological malignancies. There is no reported evidence for its measurement in human plasma. Method: A simple, sensitive and specific high-throughput HPLC-ESI-Tandem Mass Spectrometric method was developed for the estimation of Venetoclax (VX) in human plasma using Venetoclax-D8 (VXD8) as an internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 75 x 4.6 mm, 3.5 mm, 80 Å column with an isocratic mobile phase composed of Methanol and 5mM Ammonium acetate in the ratio of (70:30 v/v), at a flow-rate of 0.6 mL/min. The proton adducts of VX and VXD8 were detected at m/z 868.12 → 321.54 and 876.9 → 329.7 respectively, using multiple reaction monitoring (MRM) positive mode respectively. The Liquid-Liquid extraction method was used to extract the analyte and IS. Results: The method was successfully validated over linearity concentration range of 10.0-10000.0 pg/mL with the correlation coefficient (r2) ≥ 0.9997. This method demonstrated intra and inter-day precision within 5.7 to 7.7 and 5.95 to 8.5 and % Accuracy within 96.3 to 98.7 and 98 to 100.4 %. Venetoclax was found to be stable throughout freeze-thawing cycles, bench top, postoperative stability studies. Conclusion: The method was suitable and conveniently applicable to pharmacokinetic and bioavailability studies for estimation of Venetoclax in biological matrices by HPLC-MS/MS.