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

Abstract

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.

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.

• Authors: U Turpeinen, H Markkanen, T Sane, E Hämäläinen
• Journal: Scandinavian journal of clinical and laboratory investigation (Scand J Clin Lab Invest) Vol. 66 Issue 2 Pg. 147-59 ( 2006) ISSN: 0036-5513 [Print] England
• PMID: 16537248 (Publication Type: Evaluation Study, Journal Article)

Chemical References

• Tetrahydrocortisone
• Tetrahydrocortisol

Topics

• Chromatography, Liquid (methods)
• Humans
• Mass Spectrometry (methods)
• Mineralocorticoid Excess Syndrome, Apparent (diagnosis)
• Tetrahydrocortisol (urine)
• Tetrahydrocortisone (urine)