Study of profile analysis on urinary free steroids using high performance liquid chromatography with spectrophotometric scanning by photodiode array--application of Girard reagent T for sample preparation

Abstract

High performance liquid chromatography (HPLC) has been useful for profile analysis of steroids. However, the conventional extraction of urinary free steroids using urine specimens has some disadvantages because of lots of interfering substances simultaneously extracted from the urine. These substances were usually detected on the chromatogram at the range of relatively short retention time within which some urinary free steroids were heavily contaminated. Therefore it seemed unsuitable for profile analysis of urinary free steroids by HPLC. In this study, we developed a relatively simple and reproducible method for removing the interfering substances by Girard reagent T. In addition, the purity of each extracted free steroids were confirmed by Photodiode Array continuous scanning system, together with 3-dimensional chromatogram as well as contour map analyzed by the attached computer. The extraction procedure was as follows: (1) 10% volume of 24-h specimens of urine included 1 microgram internal standard was charged to Sep-pack C18 cartridge. The cartridge was eluted with 20 micromilligram ethyl acetate and the eluate was evaporated. (2) 10 mg Girard reagent T dissolved in 0.5 micromilligram acetic acid and 0.5 micromilligram ethanol was added to the residue, then left at room temperature for 2 hours. During this time, ketosteroids formed by the action of Girard reagent T turned to be water-soluble hydrazone complex. (3) After the addition of 10 micromilligram cold water, it was adjusted to pH 8 with NaOH and NaHCO3, then washed with 5 volumes of ethyl acetate (non-ketotic fraction). (4) The lower layer were hydrolyzed by adding 0.5 micromilligram concentrated hydrogen chloride and left for an hour at room temperature, then the liberated steroids were extracted with ethyl acetate (ketotic fraction). (5) Ethyl acetate extract was evaporated and redissolved in the mobile phase, then injected to HPLC. To determine the effect of Girard's separation non-ketotic fraction was also applied to HPLC. The results were as follows: (1) 3-dimensional chromatogram and contour map confirmed the effectiveness of our Girard's separation. The chromatogram of ketotic fraction showed the clear separation of the peaks of 4-ene-3-one steroids in ultraviolet absorbance at 246 nm and each steroid peaks were identical to those of the reference steroids. (2) Urinary non-specific substances moving into non-ketotic fraction was complete and satisfactory.(ABSTRACT TRUNCATED AT 400 WORDS)