Sample multiplexing for increasing throughput for quantification of estrogens in serum by LC-MS/MS.

Abstract

Estrogens are involved in many physiological processes in vivo. The accurate and rapid quantification of estrogens is required for the diagnosis and prognosis of estrogen-related diseases. To achieve high-volume assays, we developed and validated a sample-multiplexing liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of serum estrogens including estrone (E1), estradiol (E2), and estriol (E3). A total of 100 μL serum samples were extracted using ethyl acetate. After derivatization with either dansyl chloride or pyridine-3-sulfonyl chloride, derivatized samples were combined. Then we performed the second liquid-liquid extraction using hexane to purify the mixture. Finally, the reconstitution solutions were injected into LC-MS/MS. In addition, the proposed LC-MS/MS method was validated according to FDA and CLSI guidelines. Within a single run (7min), this sample-multiplexing LC-MS/MS method could simultaneously analyze E1, E2, and E3 in 2 serum samples. Meanwhile, the method demonstrated satisfactory analytical characteristics including accuracy (87.7-110.3%), linearity (2-1000pg/mL, R2 > 0.99), precision (intra-assay CV, 1.7-8.7%; inter-assay CV, 1.9-9.4%), and negligible interference and carry-over effect as well as acceptable matrix effect. In conclusion, this sample-multiplexing LC-MS/MS method has achieved a doubled-throughput assay for simultaneous quantification of E1, E2, and E3 without compromising analytical characteristics.