Development of a method for the quantification of estrone and estradiol in serum by LC-MS/MS

Estrogens are the main female sex hormones and play a key role both in physiological processes and in some pathological conditions. They are present in both sexes, but their serum levels are much higher in fertile women. In pre-menopause the most abundant estrogen is estradiol (E2), while in post-menopause estrone (E1) prevails. In addition to intervening in the development of primary and secondary sexual functions, the beneficial effects of estrogens are reflected on various organs and tissues. However, elevated estrogen levels in women have been associated with breast cancer and ovarian cancer, while in men, an imbalance between estrogens and androgens appears to be associated with the development of prostate cancer. Their dosage is therefore necessary but the immunoassays currently used in clinical laboratories result inaccurate because of their cross-reactivity and are not sufficiently sensitive to detect concentrations <10 pg/ml typical of menopausal and male serum samples. For this purpose LCMS/MS methods have been developed for their quantitative determination and are actually indicated as the best choice for diagnostic purposes . Unfortunately the low levels of serum estrogens and difficulties in the ionization of these molecules force to introduce time and cost expensive sample purification and concentration steps that are unsuitable for a routine method. To overcome this problem we set up a method that involves a simplified sample preparation through a single LLE with hexane /Ethyl acetate 75/25, supported by an on line 2D chromatography consisting of a trapping/purification step on a C18 luna 5 m 20x2mm, and a separation step on a 3 m 50x2mm PFP analytical column. The mobile phases consist of NH4F 0.2mM in water, and methanol [4]. The analysis was performed with a mass spectrometer AB Sciex 6500 QTRAP with an ESI source, operating in negative ion mode; E2-d3 and E1-d4 are used as ISTD and two transitions for each analyte are acquired and starting serum volume is 300 l. Method validation showed an excellent sensitivity ( E1: LOD 1.8 pg / mL and LOQ 5.3 pg / mL; E2 LOD 1.2 pg / mL and LOQ is 3.7 pg / mL) and a very good precision and accuracy (Intra-assay% CV is 7% and inter-assay% CV = 2% and accuracy% 95% at 83 pg/mL for E2). Matrix effect% and recovery% calculated at100pg / ml for E2 are 46.2% and 80.9% respectively. Statistical comparison with the Elecsys Estradiol III immunoassay method produced by Roche Diagnostics showed good overall correlation (R2= 0.97) however becoming decisively less evident (R2= 0,61) in the low concentration range 5-25 pg/ml were the immunoassay is less sensitive and precise. The method performances and the easy and fast sample preparation allow its introduction in the routine of a clinical laboratory.