Quantification of 17β-estradiol residues in bovine serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization†
A method for the quantification of the natural hormone 17β-estradiol (17β-E2) in bovine serum by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) was developed. Ethinylestradiol (EE2) was used as internal standard. Analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a polymeric reversed-phase (PLRP-S) LC column. They were ionized in a heated nebulizer (HN) interface operating in the negative ion mode, where only the intact deprotonated molecules, [M – H]–, were generated at m/z 271 and 295 for 17β-E2 and EE2, respectively. These served as precursor ions for collision-induced dissociation (CID) and diagnostic product ions were identified for the unambiguous hormone confirmation by selected reaction monitoring (SRM) LC-APCI-MS-MS. The method was validated on bovine serum and the limit of quantification (LOQ) was 30 pg ml–1 for 17β-E2. The inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) derived from the analyses of validation samples at three concentrations ranged from 1.76 to 3.76 and from –4.18 to –2.01%, respectively. This method is currently being successfully applied to measure the bovine serum concentration of 17β-E2 in order to discriminate between the physiological concentrations of 17β-E2 and the hormone levels resulting from illegal administration.