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 (E1), 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 clean-up 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 5.0min was achieved on a BEH C18 XP column. The use of 0.1mM ammonium fluoride (NH