High-performance liquid chromatographic determination of cortolic and cortolonic acids as pyrenyl ester derivatives

A new procedure is described for the detection of the acidic metabolites of cortisol (cortoic acids) as the pyrenylmethyl-21-oic esters. The derivatizing reagent, diazomethylpyrene, was prepared by an improved procedure. The reagent was used at room temperature, required no catalyst, and was not restricted by stoichiometric requirements. The steroid esters were separated by reversed-phase high-performance liquid chromatography and analyzed simultaneously by their ultraviolet absorbance and fluorescence characteristics. Identities of the products were confirmed using the photodiode array detector to determine spectral profiles, absorbance maxima, and absorbance ratios. Further confirmation of identity of the cortoic acid esters used mass spectrometry under normal and collision-activated dissociation conditions. With the method described, a linear spectral response was obtained between 8 and 1680 fmol. Application of the technique to the analysis of steroid acids in human urine indicated the presence of cortoic acids.