Micro determination of cortisol and cortisone in umbilical cord blood by chemiluminescent high-performance liquid chromatography

Abstract

A simple, sensitive and specific chemiluminescent high-performance liquid chromatography method, based on the luminol reaction, for determination of serum cortisol and cortisone, was established. In infants, placental 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD2) activity may affect adrenal function early after birth. The cortisol-cortisone ratio of serum concentrations in umbilical cord blood is an indicator of placental 11beta-HSD2 activity. The optimum conditions for the luminol reaction were determined to be 1.5 mM luminol, 0.6 M sodium hydroxide, 0.15 mm potassium hexacyanoferrate(III) and 200 mM potassium hexacyanoferrate (II). The calibration curves for cortisol and cortisone exhibited good linearity. The correlation coefficients of the calibration curves were 0.996. The intra- and inter-day precisions were in the ranges: cortisol 7.0-12.2 and 4.4-9.2%, cortisone 5.3-7.0 and 6.2-9.9%. The recoveries of these steroids were in the ranges: cortisol 97-105%, cortisone 94-102%. The limits of detection were as follows: cortisol, 0.17 microg/dl; cortisone 0.15 microg/dl. This assay could be successfully applied to determination of the cortisol-cortiosone ratio of serum concentrations in umbilical cord bloods.