Unique potentiometric detection systems for HPLC determination of some steroids in human urine

Abstract

Unique potentiometric detection systems for HPLC determination of some steroids in human urineIsocratic HPLC with potentiometric detection is used for the determination of some 17-ketosteroids (17-KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17-KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p-chlorophenyl) borate containing PVC-based rubber phase membranes. The neutral 17-KS compounds were derivatized with Girard's reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17-KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion-sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC-based, rubber phase membrane. The three 17-KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time-consuming cleanup and preconcentration step. IntroductionThe 17-ketosteroids (17-KS) are steroids with a ketone functional group at position 17 in their polycyclic carbon skeleton. Typical examples are dehydroepiandrosterone (DHEA), estrone (E 1 ), and androsterone (AD), see Scheme (1) for their structures. DHEA, a representative 17-KS, is synthesized by the adrenal gland and then converted into active hormones, estrogens, and androgens, in the peripheral tissues. Most of the DHEA (over 99%) is present in the blood as the sulfated conjugate (dehydroepiandrosterone sulfate (DHEAS)), which is second to cholesterol in abundance for circulating steroids in humans. In both sexes, serum DHEA levels vary profoundly throughout life. Levels start to increase in children, peakCorrespondence: Professor Grzegorz Bazylak, Department of Pharmaco-Bromatology & Molecular Nutrition, Faculty of Pharmacy, Collegium Medicum, Nicolaus Copernicus University, Jagiellonska 13, PL-85-067 Bydgoszcz, Poland E-mail: gbazylak@cm.umk.pl Fax: +4852-585-3817Abbreviations: AD, androsterone; AD-GP, androsterone derivatized with Girard's reagent P; ADS, androsterone sulfate; BOR_-Comp, TCPB containing graphite/PVC composite electrode used in repeated over several days measurements of sequence of different injected concentration of the analyte; BOR_Comp_1-day, TCPB containing graphite/PVC composite electrode used in consequtive measurements of the same injected concent...