Accurate Quantitation of Metabolites in Human Urine

Our R&D team presented a technical poster at ASMS 2017. The study and quantitation of metabolites can give insights to health state and human diseases. This poster focuses on cortisol metabolism as an example. Cortisol is quantitatively the major glucocorticoid product of the adrenal cortex. The deficiency of ad-renal steroid excretion is found in Addison’s disease leading to hypotension, and on the other hand the over-production is found in Cushing’s syndrome leading to hypertension (3).


Several metabolites are known to be glucuronidated and sulfated in the human body. Monitoring the aglycones with the gluruco-nidated and sulfated metabolites creates six additional masses to monitor on tandem mass spectrometry, which can limit the total number of metabolites being monitored. In addition, the conjugated standards are very expensive and often not available. Herein, we report the use of purified β-glucuronidase (IMCSzyme®) and recombinant arylsulfatase that effectively cleave glucuronides and sulfates at neutral pH with no detectable CYP activity or esterase activity. The initial studies explored urinary corticosteroid metabolites on tandem mass spectrometry before and after cleaving glucuronides and sulfates with the purified enzymes. Among corticosteroids, glucuronidated allo-tetrahydrocortisol (aTHF) has been reported to be recalcitrant substrate for enzymatic decon-jugation. We demonstrate rapid hydrolysis within 10 minutes, while the reported 4 hour incubation using crude snail enzyme is in-adequate for recovering the higher levels of aTHF glucuronide (1, 2).


Download poster presentation. 


  1. Raffaelli, A., Saba, A., Vignali, E., Marcocci, C., and Salvadori, P. (2006) Direct determination of the ratio of tetrahydrocortisol allo-tetrahydrocortisol to tetrahy-drocortisone in urine by LC–MS–MS. Journal of Chromatography B, 830, 278-285.
  2. Cuzzola, A., Mazzini, F., and Petri, A. (2014) A comprehensive study for the validation of a LC–MS/MS method for the determination of free and total forms of uri-nary cortisol and its metabolites. Journal of Pharmaceutical and Biomedical Analysis, 94, 203-209.
  3. Turpeinen, U., and Hämäläinen, E. (2013) Determination of cortisol in serum, saliva and urine. Best Practice & Research Clinical Endocrinology & Metabolism, 27, 795-801.