UPLC-MS/MS method for determination of endogenous cortisol and 6β-hydroxycortisol in urine using a background subtraction approach: application to a CYP3A phenotyping.

The methodological validation guidelines focus on analyses of exogenous compounds. Methods for the determination of endogenous compounds has insufficient regulation1. Although cortisol and 6β-hydroxycortisol (6β) determination were discussed by many authors, the methodology is frequently not fully described or evaluated. This work describes a simple method for the simultaneous determination of the endogenous compounds cortisol and 6β in urine with the aim of CYP3A phenotyping. Differently from other works, we used a background subtraction approach. This approach allows the use of the same matrix for calibration curve and quality control samples, reducing the variation of matrix effect and recovery between samples and calibrators. This method presented acceptable precision, accuracy, and stability using a C18 UPLC reversed-phase column and a gradient mobile phase constituted by acetonitrile containing 0.1% formic acid, with total time run of 13 minutes. Additionally, the matrix effect evaluated by qualitative and quantitative tests were considered absent.