Authors:Camila C. Pires, Moacir Kaiser, Lauren D. Grünspan, Fabiano Barreto, Adrine Innocente, Simone Gnoatto, João V. Laureano, Bibiana V. Araujo, Teresa Dalla Costa, and Leandro Tasso
An accurate and reliable LC—MS/MS assay was firstly developed and validated for quantitative determination of a new antimalarial prototype drug, 3β-hydroxyurs-12-en-28-oic acid (LAFIS 01), in rat plasma. Dexamethasone was employed as internal standard. Simple protein precipitation by acetonitrile for the sample preparation was used. Effective separation was achieved with Phenomenex Luna C18 (50 × 2 mm, 5 μm) column. The mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.1% acetic acid, delivered by gradient elution. The column temperature was maintained at 40 °C. The LAFIS 01 was monitored by electrospray ionization interface, operating in the negative mode (ESI−) in multiple reactions monitoring (MRM), checking the transitions 455 > 455 for LAFIS 01 and 451 > 361 for the IS. Once LAFIS 01 demonstrated low fragmentation by collision-induced dissociation (CID) nonpresenting abundant high-intensity fragments to meet the desired concentration levels quantification, only pseudomolecular ion was monitored. The flow rate was 500 μL min−1. The lower limit of quantitation achieved was 10 ng mL−1 and linearity was observed from 10 to 500 ng mL−1. The relative standard deviation (RSD) values of the intra- and inter-assay precisions of the method were below 8.42 and 7.94%, respectively. The accuracy ranged from 92.05 to 102.94%. The extraction recovery of LAFIS 01 and IS was up to 90%. The method showed linearity, precision, accuracy, sensitivity, and stability required to quantify LAFIS 01 in preclinical pharmacokinetic study.