Determination of cortisol in human plasma by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, I.

A definitive method is described for the determination of cortisol in human plasma. The method is based on the principle of isotope dilution-mass spectrometry. The analytical procedure comprises the following steps: (1) Addition of [4-14C]cortisol to the plasma sample; (2) extraction of the 14C-labelled and of the non-labelled cortisol by dichloromethane; (3) purification of the cortisol fraction by column chromatography on Sephadex LH-20; (4) formation of the trimethylsilyl ether of cortisol methoxime; (5) selected ion monitoring at m/z-values 605, 607, 636 and 638 during gas liquid chromatography. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. The precision of the method was evaluated by calculation of the coefficient of variation from day to day; this varied from 0.7-2.3% in the range of 256-845 nmol/l. The lower limit of detection (ratio of signal to noise 3:1) is 200 pg cortisol per sample.