Development and validation of a sensitive and selective UHPLC–MS/MS method for quantitation of an investigational anti-malarial compound, 2-tert-butylprimaquine (NP-96) in rat plasma, and its application in a preclinical pharmacokinetic study

An ultra-high performance liquid chromatographic tandem mass spectroscopy (UHPLC–MS/MS) method was developed and validated for the quantification of an investigational anti-malarial entity, 2-tert-butylprimaquine (NP-96), in rat plasma. Simple protein precipitation by acetonitrile was used for the sample preparation. Effective separation of NP-96, internal standard (IS) and matrix components were achieved on an UHPLC column (Hypersil Gold C18, 50 mm × 2.1 mm, 1.9 μm) using a mobile phase composed of acetonitrile and 20 mM ammonium acetate, which was pumped in a gradient mode at a flow rate of 450 μl/min. Selective reaction monitoring (SRM) was utilized for quantitation of the molecules. To increase sensitivity of the method, two ions of m/z 299 and m/z 231 were selected for NP-96, while IS was monitored for an ion of m/z 489. The method was validated according to FDA guideline on bioanalytical method validation and showed good compliance. The intra-day and inter-day precision expressed as R.S.D. was lower than 15% at all the tested quality control levels, including upper and lower limits of quantification. The calibration range was 2.5–500 ng/ml. Total runtime for the method was 5 min, which was suitable to produce high-throughput results for pharmacokinetic evaluation.