High-Sensitivity Micro LC-MS/MS Assay Developed for Serum Estradiol

Automated immunoassays are the most widely used techniques for E2 measurements in clinical laboratories; however, they suffer from poor accuracy and precision at low physiological E2 concentrations. A high-sensitivity, underivatized method has been developed using micro liquid chromatography–mass spectrometry (LC-MS/MS) to reliably measure E2 concentrations below 5 pg/mL by the use of low sample volume.

Scientists at the University of Chicago (IL, USA) used samples for method comparison from leftover samples retrieved from the clinical chemistry laboratory. 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 an Eksigent Ekspert micro LC 200 system with a flow rate of 35 μL/min in a total run time of 3.5 minutes and detected by a QTRAP 6500 mass spectrometer (Sciex, Framingham, MA, USA) 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.

E2 measurements by LC-MS/MS were compared with the Cobas 8000 E2 II automated immunoassay (Roche Diagnostics, Basel, Switzerland) using 42 plasma samples collected in lithium heparin tubes and with a laboratory-developed indirect radioimmunoassay using 38 serum samples collected in plain serum tubes. The LC-MS/MS method showed good correlation with E2 RIA and modest correlation with E2 Roche Cobas automated immunoassay. The validation study demonstrated broad linear ranges 3.0 to 820 pg/mL. Total precision was below 15% at all quality control (QC) levels, and limit of quantification (LOQ) was 3.0 pg/mL. The study was published in the July 2016 issue of the Journal of Applied Laboratory Medicine.

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University of Chicago
Sciex
Roche Diagnostics