Validation and application of an ultra-sensitive liquid chromatography (LC)-tandem mass spectrometry (MS) assay to measure androgens and estrogen in human urine. (#226)
We developed an ultra-sensitive and specific LC tandem MS method to simultaneously determine testosterone (T), estradiol (E2), and dehydroepiandrosterone (DHEA) in extracts of human urine without derivatization. To demonstrate the applicability of this method, we analyzed urine androgens and estrogen changes in adolescents during puberty.
Urine samples (1 mL) underwent enzymatic hydrolysis (β-glucuronidase, overnight, room temperature) followed by extraction with methyl tert-butyl ether. Steroids in reconstituted urine extracts were quantified using the unique LC retention time and monitoring MS mass-to-charge transition using atmospheric pressure photoionization in positive (T, DHEA) or negative (E2) ionization mode. The method was fully validated for linearity, limit of quantification (LOQ), specificity, recovery, accuracy, precision and enzyme hydrolysis efficiency. Matrix effects were evaluated to appraise the potential effects of interfering substances. Concentrations in urine were adjusted to a standard SG of 1.020.
The method is sensitive and specific, and maintains high within-day and between-day accuracy and precision for all analytes within acceptable limits for bioanalytical method validation (Table 1). Matrix effects were minimal with negligible ion suppression or enhancement for all analytes.
Using this method, adolescent males (n=27, 13-16 years old) had lower E2 (1.98±0.22 vs 4.93±0.89 ng/ml, P<0.05) and higher T (26.22±1.95 vs 8.23±1.00 ng/ml, P<0.05) excretion compared to females (n=20, aged 13-16 years old) but the genders did not differ in urine DHEA excretion (22.41±1.49 vs 27.47±4.17 ng/ml, P=0.21).
We successfully developed and validated an ultra-sensitive and specific LC-MS method for valid measurement of endogenous sex steroids (T, E2, DHEA) simultaneously in human urine.