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(unpublished data – Rodrigues et al., manuscript accepted by The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.