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Simple, robust, rapid, reproducible method development of ZSTK474 for routine analysis.Full validated LCMS/MS Method.Successfully applied to pharmacokinetics study.Mice pharmacokinetics is first time reported.ZSTK474, a promising novel anticancer molecule derived from s-triazine, found to have antitumor activities against different cancer cell lines. However, neither LCMS method nor pharmacokinetics of ZSTK474 has been reported till now. A sensitive, simple, short and specific liquid chromatography tandem mass spectrometry (LCMS/MS) method was developed for the quantification of ZSTK474 in mouse plasma accordance with the US Food and Drug Administration guidelines. Extraction of drug molecule was carried out using protein precipitation. Chromatographic analyte separation was achieved on Atlantis dC18 (4.6 × 50 mm, 3 μm). Composition of isocratic mobile phase consists of 90% acetonitrile and 0.2% formic acid, at 0.7 mL/min flow rate, having short 2.5 min run time. Method development was validated and found to be linear over a dynamic range between 1.9–1000 ng/mL; having a correlation coefficient (r 2) ≥ 0.9978. The analyte was found to be stable under short and long term storage conditions. LCMS/MS method developed was validated and found to be selective, reproducible, precise and accurate to quantify ZSTK474 in plasma samples, and first time successfully applied to pharmacokinetic studies. Pharmacokinetic data showed fast absorption attaining Cmax at 0.25 h and half life (t1/2) 5.18 h after oral administration of ZSTK474 at 20 mg/kg in mouse.