Simultaneous analysis of 17alpha-estradiol and 17beta-estradiol in bovine serum by liquid chromatography-tandem mass spectrometry.

A new LC–MS/MS method for the separation, identification and quantification of residues of 17α-estradiol (17α-E2) and 17β-estradiol (17β-E2) in bovine serum is reported. Deuterium-labelled 17β-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M−H]−, were generated at m/z 271 and 274 for 17α/17β-E2 and 17β-E2-d3, respectively. The decision limits obtained (CCα, i.e., critical concentration alpha) were 0.06ng/mL and 0.03ng/mL, respectively for 17α-E2 and 17β-E2. Detection capability (CCβ, i.e., critical concentration beta) values were 0.08ng/mL and 0.04ng/mL, respectively, for 17α-E2 and 17β-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.