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

By LabMedica International staff writers
Posted on 10 Aug 2016
There are considerable demands to accurately measure estradiol (E2) at low concentrations of less than 20 pg/mL in postmenopausal women, men, pediatric patients, and patients receiving breast cancer treatment.

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.

Image: The Eksigent Ekspert micro liquid chromatography (LC) 200 system (Photo courtesy of Sciex).

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.

Related Links:
University of Chicago
Sciex
Roche Diagnostics

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