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Abstract: Background: Salivary steroid measurements have the potential to provide a non-invasive assessment of serum free steroid concentrations. Salivary testing has been proposed as a possible substitute for serum testing partly because of easy sample collection. Methods: We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous measurement of testosterone and androstendione in saliva. We spiked 100μL of saliva with isotope-labeled internal standards and performed extraction with diethyl ether. After evaporation both steroids were analyzed by LC–MS/MS operating in the positive mode electrospray ionization (ESI) after separation on a reversed-phase column. Results: The calibration curves for analysis of testosterone and androstendione exhibited consistent linearity and reproducibility in the range of 20–1000pmol/L. The limit of quantitation (LOQ) was 10pmol/L for both steroids. The mean recovery of the analytes added to saliva ranged from 97 to 102%. Between-run CVs were 3.6–4.0% for testosterone and 4.8–8.0% for androstendione. The upper limit (the 97.5 percentile) of testosterone and androstendione reference ranges for males was 356pmol/L and 344pmol/L and that for females 39pmol/L and 349pmol/L, respectively. Conclusions: This LC–MS/MS method has the required lower limit of quantification for analysis of salivary concentrations of testosterone and androstendione. The assay is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer with required sensitivity. [Copyright &y& Elsevier]

Abstract: Background: Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is warranted. Methods: Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d8-17-OHP, d7-androstenedione, and d4-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Results: Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9–9.9% to 3.6–10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%. Conclusions: The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families. [Copyright &y& Elsevier]