Automated on-line SPE LC–MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine: Use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC–MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC–MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7–9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2–10 pm urine collection period.