Simultaneous determination of three estrogens in human saliva without derivatization or liquid-liquid extraction for routine testing via miniaturized solid phase extraction with LC-MS/MS detection

Accurate quantitation of estrogens (i.e, estrone (El), estradiol (E2) and estriol (E3)) is valuable for clinical assessment of human health and disease. Alterations in estrogen levels have been implicated in numerous pathological conditions. However, inadequacies in sensitivity and specificity, cumbersome sample preparation and invasive specimen collection hamper the usability of available methods for clinical applications. Herein, a simple, rapid, highly sensitive and specific LC-MS/MS method was developed and validated for the simultaneous determination of three estrogens in human saliva providing a non-invasive alternative to conventional, blood samples. For the first time, a 96-well hydrophilic-lipophilic-balanced (HLB) microplate was employed for cleanup and enrichment of estrogens in a single extraction without the requirements of derivatization, evaporation, liquid-liquid extraction and online extraction. A rapid LC chromatographic separation with a turnaround time of 5.0 min was achieved on a BEH C18 XP column. The use of 0.1 mM ammonium fluoride (NH4F) as LC additive, and integration of summated and scheduled multiple reaction monitoring (MRM) transitions substantially improved the sensitivity to 1 pg/mL, allowing the accurate quantitation of trace levels of three estrogens in one run. The assay was fully validated with good performance for extraction efficiency (67.0-85.6%), matrix effect (89.6-100.2%), linearity (from 1.0 pg/mL up to 1000 pg/mL), accuracy (98.9-112.4%) and precision (<= 7.4%). Additionally, the assay was unaffected by 34 structurally-similar, potentially interfering substances tested at high clinical concentrations. The applicability of the assay was demonstrated by assessing the reference intervals of authentic saliva samples from healthy adult males, pre- and post-menopausal females. The easy sample preparation, fast LC and multi-analyte MS/MS detection utilizing noninvasive saliva as a specimen delivers a simple, practical, sensitive and accurate tool suitable for the high throughput measurement of El, E2 and E3 in clinical laboratories.