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 11beta-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.


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.
Helsinki University Central Hospital, Laboratory, Finland. ursula.turpeinen@hus.fi
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U
Turpeinen
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H
Markkanen
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T
Sane
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Hämäläinen
D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D043204:Mineralocorticoid Excess Syndrome, Apparent / Q000175:diagnosis*; D013760:Tetrahydrocortisol / Q000652:urine*; D013761:Tetrahydrocortisone / Q000652:urine*
D013761:Tetrahydrocortisone; D013760:Tetrahydrocortisol