Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry

Presented here is a stable isotope dilution technique for determining corti sol production rate (CPR), The method involves extraction and derivatizatio n of cortisol isoforms from serum (0.5 ml), separation of derivatives by ga s chromatography, and detection by using negative ion chemical ionization m ass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol prod uction rate with the use of small amounts of pooled serum, even in the pres ence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d(0), m/z 782) and deuterated cortisol (d(3), m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) stan dards: ddd, ratios ranging from 1 to 8%; 2) controls: d(3)-cortisol added t o serum with known cortisol concentration; 3) subjects: 24-h pooled serum s amples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d(3)-cortisol at 2-4% of the ir estimated CPR. Recovery after the solid phase extraction and derivatizat ion process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d(3)/d(0) in standards and serum controls were h ighly correlated (r(standard)(2) = 0.99; r(control)(2) = 0.99) over a wide range of d(3)-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m(2)/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/ m(2)/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotop ic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production. (C) 1999 Published by Elsevier Science Inc. All rights reserved.