High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

Background: There are considerable demands to accurately measure estradiol (E2) at low concentrations (<20 pg/mL) in postmenopausal women, men, pediatric patients, and patients receiving breast cancer treatment. Most current high-sensitivity LC-MS/MS E2 methods require large sample volumes and involve complex sample preparations with dansyl chloride derivatization. Our study aims to develop a high-sensitivity, underivatized method using micro LC-MS/MS to reliably measure E2 concentrations below 5 pg/mL by the use of low sample volume. Methods: A total of 290 μL of sample was mixed with internal standard (IS), E2-d4, and extracted with a mixture of hexane/ethyl acetate (90/10) (v/v). After extraction, sample was separated by Eksigent Ekspert™ micro LC 200 system with a flow rate of 35 μL/min in a total run time of 3.5 min and detected by SCIEX QTRAP 6500 mass spectrometer in a negative mode using transitions: 271/145 (quantifier) and 271/143 (qualifier). In this method, it was crucial to use HPLC columns with stability at a pH >10. Results: The validation study demonstrated broad linear ranges (3.0-820.0 pg/mL) with r 2 > 0.999. Total precision was below 15% at all QC levels, and limit of quantification (LOQ) was 3.0 pg/mL. Our method showed good correlation with E2 RIA (r 2 = 0.96, bias = -1.0 pg/mL) and modest correlation with E2 Roche Cobas automated immunoassay (r 2 = 0.86, bias = 6.0 pg/mL). Conclusions: In conclusion, we developed and validated a routinely applicable micro LC-MS/MS method without derivatization for E2 in blood samples with an LOQ of 3.0 pg/mL.