Liquid chromatographic determination of urinary 6β-hydroxycortisol to assess cytochrome p-450 3A activity in HIV positive pregnant women

Assessing the activity of CYP3A4 is important for predicting the pharmacokinetic behavior of protease inhibitors in HIV positive patients, especially in pregnant women. The endogenous hormonal ratio of 6β- hydroxycortisol (6β-OHF) to cortisol (F) in the urine is an index for metabolic enzyme activity of cytochrome p-450 (CYP) 3A4. Because the ratio is a unique way to assess the enzyme activity without using any exogenous probes for this isozyme, it is practical for use in pregnant women. In this paper, we describe a method using high performance liquid chromatography (HPLC) for 6β-OHF in urine from pregnant women to estimate the ratio of 6β-OHF/F. Urinary 6β-OHF was measured by using C18-cartridge solid phase extraction and isocratic HPLC. Aliquots (1 ml) of urine samples spiked with internal standard, 6β-hydroxyprednisolone (6β-OHPSL), were alkalinized with NaOH, then applied to C18-cartridges, which were washed with water and hexane and eluted with ethyl acetate. After the effluents were dried and reconstituted in 10% acetonitrile, the samples were analyzed by HPLC using an isocratic mobile phase (acetic acid/acetonitrile/50 mM potassium dihydrogenphosphate; 0.2/9/90.8; v/v) and ultraviolet detection at 245 nm. The recoveries of 6β- OHF from C18 cartridges were 93.2 and 93.9% when the authentic 6β-OHF was added to the urine sample at the concentration of 50 and 300 ng/ml, respectively. Intra- and inter-day variations estimated at concentrations of 113-674 ng/ml were 2.9-5.6 and 4.9-8.1%, respectively. The method was applied to morning urine samples collected from HIV-positive pregnant women managed with protease inhibitor containing anti-retroviral regimens. (C) Elsevier Science B.V.