Candidate definitive method for the determination of cortisol in human serum

As a substantial part in the development of a reference material in clinical chemistry, a highly accurate and precise isotope dilution mass spectrometric method has been worked out for the determination of cortisol in human serum. The candidate definitive method consists of addition of 4-[14C]cortisol and, following equilibration, solvent extraction of cortisol and its internal standard from the serum matrix. After conversion into methoxime-trimethylsilyl derivatives, the extract is purified by gel chromatography. Measurements are made by combined capillary gas chromatography mass spectrometry. The ratio of peak heights at m/z 605 and 607 for each sample and calibration mixtures is used for quantification. To ensure maximum accuracy each set of calibration mixtures was composed from two stock solutions and closely bracketed the anticipated serum concentration. An analysis of variance, involving comparison of within-run and between-run variability, gave a total coefficient of variation (CV) of 0.38% for a serum pool containing 90.79 ng cortisol ml-1 serum.