Determination of the urinary cortisol production rate using (1,2,3,4-13C)cortisol. Isotope dilution analyses at very small enrichments.

Abstract

An isotope dilution mass spectrometric method to determine the urinary cortisol production rate (CPR) in babies and children is described. The method uses stable isotopically labelled (1,2,3,4-13C)cortisol. The tracer is intravenously administered to the patient and urine is collected for the following three days. Following extraction, enzymic hydrolysis, purification and isolation by high-performance liquid chromatography (HPLC) the urinary cortisol metabolites tetrahydrocortisone, tetrahydrocortisol, alpha- and beta-cortolone are separately oxidized the common product, 11-oxo-aetiocholanolone. The methyl oxime tert-butyldimethylsilyl ether derivative (MO TBDMS) was analysed by gas chromatography mass spectrometry. Quantification of the isotope enrichment was carried out by unlabelled, and at m/z 348 for labelled 11-oxo-aetiocholanolone. HPLC isolation of the metabolites together with the oxidation step allowed very small isotope enrichments, sometimes down to 0.1% (1:1000), to be reliably measured against a linear calibration graph containing 0 to 1% (13C4) enrichments. The standards for the calibration graph were synthesized from mixtures of labelled (13C4) cortisol and natural cortisol, and the calibration graph was prepared each time samples were measured. The long term instrumental precision of the isotope dilution analyses was 0.91% for a derivatized sample containing a (13C4) enrichment of 0.5% (measured on six different days over seven months). The coefficient of variation of the complete procedure for the four cortisol metabolites was between 1.17 and 2.14%. The clinical applicability of the method is demonstrated by presenting the results of a CPR determination in a patient.