Fluorometric Liquid-Chromatographic Determination of Serum Cortisol

We describe a sensitive and specific liquid-chromato-graphic assay for serum cortisol, exploiting acid-induced fluorescence. Each analysis requires only 50 zL of serum, and chromatography is complete in 7 mm. Analytical re-covery of cortisol added to serum ranged from 90 to 105% and between-run precision (CV) from 6 to 8%. The lower limit of detection for cortisol is 10 g/L, and linearity ex-tends to 1600 tgIL. Numerous steroids and drugs tested didnot interfere. The technique of sulfuric acid-induced fluorescence of steroids was first applied to the measurement of plasma cor-ticosteroids by Sweat in 1954 (1). In 1962, Mattingly (2) modified the technique and developed a more practical method for the measurement of plasma cortisol. Subsequent modifications, to develop simpler but more specific assays, were reviewed by Rubin (3). Although cortisol is the principal adrenal corticosteroid found in plasma, other steroids, drugs, and several normal blood constituents can interfere with fluorometric methods (1, 4-6). Liquid chromatography, however, can provide the necessary specificity needed for the determination of serum cortisol. Recently, Reardon et al. (7) and Kabra et al. (8) have de-scribed specific liquid-chromatographic methods; however, these methods involve spectrophotometric detection, a technique generally less sensitive than fluorometric detection. Fluorometric liquid-chromatographic methods have been described by Kawasaki et al. (9) and Goehl et al. (10), but these methods involve derivatization with dansylhydrazine and thus require additional time. We describe a simple, specific, and sensitive fluorometric liquid-chromatographic method for serum cortisol in which corticosterone, 11-deoxycortisol, and numerous other steroids and drugs do not interfere. Only 50 1zL of serum is required foranalysis,making themethod suitable for pediatric use. Materials and Method Equipment We used a Model 601 liquid-chromatograph witha Model 023 recorder and a Model 650-1OLC fluorescence spectro