Dried blood spots analysis of 6β-hydroxycortisol and cortisol using liquid chromatography/tandem mass spectrometry for calculating 6β-hydroxycortisol to cortisol ratio.

Dried blood spot (DBS) sampling is a minimally invasive method used to collect blood samples of any population for personalized medicine. We aimed to develop a sensitive and reliable analytical method for measuring 6β-hydroxycortisol (6β-OHF) and cortisol concentrations in DBS by liquid chromatography/tandem mass spectrometry so as to utilize DBS as a less invasive blood sampling method for calculating the ratio of 6β-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 μl for 6β-OHF and 1.01 pg/50 μl for cortisol. The 6β-OHF and cortisol in DBS were stable for 28 days at room temperature. The intraday and interday accuracy and precision of the method was <12%. Additionally, the 6β-OHF and cortisol in DBS were measured before, during, and after 3 days of clarithromycin administration to two of the subjects. Then, their concentration was compared in the plasma and whole blood collected simultaneously. The concentrations of 6β-OHF and cortisol in four DBS ranged from 0.007 to 0.079 ng/50 μl and from 1.15 to 6.66 ng/50 μl, respectively. The 6β-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6β-OHF/cortisol ratio in DBS also showed a strong correlation with that in whole blood (r = 0.9694) and plasma (r = 0.9383). This method provides high accuracy and precision for measuring 6β-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6β-OHF/cortisol ratio. The 6β-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting.