Autor:innen:
R. Zeidler (Leipzig, DE)
R. Biemann (Leipzig, DE)
U. Ceglarek (Leipzig, DE)
J. Kratzsch (Leipzig, DE)
B. Isermann (Leipzig, DE)
A. Gaudl (Leipzig, DE)
AIMS 11-Oxygenated androgens (11-OAs) are discussed as potential biomarkers for adrenal androgen excess such as congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS). However, quantification of 11 OAs by LC-MS/MS still relies on extensive sample preparation including liquid-liquid extraction, derivatization and partial long runtimes, which is unsuitable for high-throughput analysis under routine laboratory settings. Aim of this study was to validate a method to quantify 11-OAs using a clinical routine LC-MS/MS method for steroid hormone profiling with minimal sample preparation.
METHODS A DIN ISO 15189 accredited LC-MS/MS method for quantitation of 7 serum steroids in daily routine use was extended by 11-ketoandrostendione (11-KA), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11-OHT), 11β-hydroxytestosterone (11-OHT) and validated. In brief, online solid phase extraction was combined with reverse phase liquid chromatography using methanol/H2O/0.2 mM NH4F as mobile phase. Detection was conducted using a Sciex QTrap 6500plus in positive ionization and multiple reaction mode. Calibrators and controls were produced inhouse by spiking certified Chromsystems® calibration and control material for steroid hormone analysis covering expected endogenous concentration ranges. Method validation included thorough examination of reproducibility, recovery, linearity, sensitivity and specificity according to FDA guidelines. Possible effects of freeze/thaw cycles were addressed. For clinical verification, 13 CAH patients and healthy controls were compared.
RESULTS Chromatographic separation was achieved within a total run time of 6.6 min to simultaneously quantify 4 11-OAs as well as 7 other steroid hormones from the original method. Lower limits of quantification were well below endogenous ranges (63-320 pmol/l), recoveries ranged from 85% to 117% with CVs ≤ 15%. The concentration of the analytes were comparable to published values of healthy individuals and showed high stability across five freeze/thaw cycles (CV ≤ 3.1%). Clinical verification revealed, as expected, an increased concentration of 11-OAs and decreased ratios of androstendione or testosterone to 11-OAs in CAH patients.
CONCLUSION We present a robust high-throughput method with high sensitivity for simultaneous quantification of four 11-OAs and seven routine steroid hormones using minimal sample preparation. By multiplex-design, ratios of clinically relevant steroids to 11 OAs are instantly accessible, allowing the use of 11-OAs for diagnosis and therapy monitoring in androgen excess-related disorders. In current studies, the method is used for determination of reference intervals (from birth to 80 years) as well as to investigate the relationship between 11-OAs with obesity, metabolic syndrome, and puberty.