A background subtraction approach for determination of endogenous cortisol and 6β-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women

Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6 beta-hydroxycortisol/cortisol ratio (6 beta-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6 beta-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6 ss-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquidliquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC (R) BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1-1.000 ng/mL and 2-1.000 ng/mL for C and 6 beta-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6 beta-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6 beta-OHF:C ranged from 2.57 to 51.69, with a mean +/- standard deviation (SD) of 15.12 +/- 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6 beta-OHF:C ranging from 3.48 to 44.54 with a mean +/- SD of 14.37 +/- 5.73 (n = 7). Even though the 6 beta-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability.