Determination of 17beta-estradiol in bovine plasma: development of a highly sensitive technique by ion trap gas chromatography-tandem mass spectrometry using negative chemical ionization

A novel approach to the determination of 17beta-estradiol in bovine plasma is presented. The observed enhanced sensitivity is gained by the application of tandem mass spectrometry (MS) fragmentation to a stable, well characterized negative ion produced by chemical ionization (methane as reagent gas). A specific derivatizing reactant is employed (pentafluorobenzyl bromide), combined with bis-trimethylsilyltrifluoroacetamide, to favor the formation of a diagnostic precursor negative ion. Plasma samples are purified through a C18 solid phase extraction column and derivatized before gas chromatography-MS analysis. The accuracy and the precision of the method, tested over a set of spiked samples, were satisfactory. The limit of detection was found to be 5 pg ml(-1) and the limit of quantification was fixed at 20 pg ml(-1). The fragmentation pattern is fully explained and the method is applicable for the official analysis of bovine plasma for the detection of 17beta-estradiol according to the European criteria 256/93 and to the draft SANCO/1805/2000 rev. 3. The quantification of incurred positive samples was performed according to the proposed procedure and compared with the results obtained by standardized radio immuno assay; the estimated concentrations were significantly similar.