Betamethasone assay and analytical validation
BET determination in plasma was performed by an LC-MS/MS system according to a method previously validated by the research group (unpublished data – Rodrigues et al., manuscript accepted by The Brazilian Journal of Pharmaceutical Sciences). Briefly, 500 µL of plasma were added to an internal standard solution (IS; deuterated betamethasone-acetate; 1 µg/mL in methanol) and submitted to the extraction process with 6 mL of diisopropyl ether.
Analytes were separated in a LiChrospher reversed-phase C-8 column with a LiChrospher® 100 RP-8 C-8 guard column, using a mixture of methanol and ammonium formate 0.05 mM (90:10 v/v) as a mobile phase.25,16 The MS/MS detection system was a Quattro Micro LC triple quadrupole (Micromass, Manchester, United Kingdom) equipped with an electrospray interface (ESI) operating in positive ion mode. The method revealed linearity in the range of 2-250 ng/mL plasma for BET. Coefficients of variation and inaccuracy percentage were ≤15%, indicating that the method was precise and accurate. Moreover, the method showed selectivity and did not presented matrix or carry-over effects. Stability tests also presented the coefficient of variation and relative standard errors ≤ 15%. Therefore, the method for BET pharmacokinetics studies had adequate confidence limits, ensuring the results’ reproducibility and repeatability.