Determination of free tetrahydrocortisol and tetrahydrocortisone ratio in urine by liquid chromatography-tandem mass spectrometry

"Objective. Measurement of urinary free tetrahydrocortisol and tetrahydrocortisone ratio (allo-THF+THF)/ THE is clinically important in the diagnosis of hypertension caused by congenital absence of 11 beta-hydroxysteroid dehydrogenase type 2 ( apparent mineralocorticoid excess, AME) or inhibition of the enzyme after licorice ingestion. Although gas chromatography-mass spectrometry (GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. The purpose of this study was to demonstrate that detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. Material and methods. The analysis utilizes 1 mL urine. The samples were extracted with solid-phase extraction (SPE) using ethyl acetate as eluent. The extract was evaporated to dryness, and allo-tetrahydrocortisol (allo-THF), THF and THE concentrations were analyzed by LC-MS/MS operating in the negative mode after separation on a reversed-phase column. The calibration curves exhibited consistent linearity and reproducibility in the range of 7.5 - 120 nmol/L. Interassay CVs were 7.0 - 10 % at mean ratios of (allo-THF+ THF)/ THE of 0.54 - 1.9. The detection limit of the analytes was 0.4 - 0.8 nmol/L (signal-to-noise ratio=3). The mean recovery of the three analytes ranged from 88 to 95 %. The regression equation for the free ratio using the LC-MS/MS ( x) method and the total ratio using the GC-MS (y) method was: y=0.30x+ 0.91 (r=0.61; n=25). Conclusions. The sensitivity and specificity of the LC-MS/MS method offer an advantage over GC-MS by eliminating derivatization. The high costs of equipment are balanced by higher through-put, owing also to shorter chromatographic run times."