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

Simultaneous determination of 6β-hydroxycortisol and cortisol in human urine and plasma by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity

“…The ratio of 6␤-OHF/F in the period of 20:00-23:00 was significantly higher than other collection intervals (P < 0.05). The circadian variation in the ratio of 6␤-OHF/F observed in our lab is in general agreement with that observed by Ohno et al [16] and Barrett et al [17]. We find out a parallel diurnal rhythm in the urinary excretion of 6␤-OHF and F, with the lowest excretion rates observed in the period of 16:00-23:00 for both 6␤-OHF and F, in agreement with the results obtained by Lee [18] and Barrett et al [17].…”

Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS, and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity

“…The limit of detection was 1 pg on column for both cortisol (S/N = 8) and 6␤-OHC (S/N = 6), which corresponds to a concentration of 0.01 ng/mL. This is more sensitive by more than a factor of 10 than the LC-MS/MS method with the Spark on-line SPE sample cleanup [10] that showed S/N = 10 for 0.2 ng/mL cortisol and S/N = 42 for 1 ng/mL 6␤-OHC.…”

“…The most sensitive measurement was obtained in the negative ion mode by monitoring the transition of the adduct of cortisol or 6␤-OHC with the formic acid anion (m/z 407 or m/z 423) in the first quadrupole and the fragments m/z 331 for cortisol or m/z 347 for 6␤-OHC [10], corresponding to the loss of CH 2 O from the molecular ion after breakage between carbon atoms 20 and 21 of the steroid moiety [15]. The amount of urine used, injection volume and washing time over the trap column were optimized to give the best signal-to-noise ratio (S/N).…”

“…More recently, an on-line solid phase extraction (SPE) LC-MS/MS method [10] was reported. It is based on a special on-line SPE equipment (Prospect-2 TM , Spark) that is not routinely available.…”

Principles and Applications of LC-MS/MS for the Quantitative Bioanalysis of Analytes in Various Biological Samples

“…Method development for determining drug or metabolite concentrations from urine samples has been simplified with the implementation of on-line SPE. A sensitive method using on-line SPE and LC-MS/MS system for quantification of urinary cortisol metabolite and cortisol was developed and human urine sample were injected directly onto an on-line SPE apparatus followed by HPLC separation and LC-MS/MS detection (Barrett et al, 2005).…”