Journal
CLINICA CHIMICA ACTA
Volume 409, Issue 1-2, Pages 78-84Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.cca.2009.09.003
Keywords
Testosterone; Dihydrotestosterone; Estradiol; Androgen; Estrogen; LC-MS/MS
Categories
Funding
- Australian Research Council
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Background: Immunoassays are widely used to quantify steroid hormones in biological samples. However, they lack specificity, especially at low levels. This study aimed to develop a sensitive LC-MS/MS method to measure serum androgens and estrogens without derivatization within a single run. Methods: A stable-isotope dilution LC-MS/MS method was established using atmospheric pressure photoionization to quantify testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) from serum. Sample preparation involved liquid-liquid extraction (LLE) with hexane:ethyl acetate (3:2) containing deuterated internal standards. Accuracy was assessed by spiked recovery of serum pools, and imprecision by quality controls. Results: Using 200 mu L serum, limits of quantification were 0.3 pg (1.5 pg/mL) E(1), 0.5 pg (2.5 pg/mL) E(2), 2 pg (10 pg/mL) T and 10 pg (50 pg/mL) DHT. Accuracy (93-110%) and precision (median 4%, all <15%) were determined to be well within acceptable limits for bioanalytical method validation. An analysis time of less than 10 min allowed up to 150 samples (600 analytes) to be processed per day. Conclusions: The method is sufficiently sensitive and precise to accurately quantify serum T levels in females and E(2) in males, and is readily adapted to tissue and non-human samples. (C) 2009 Elsevier B.V. All rights reserved.
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