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Biancotto Giancarlo1; Angeletti Roberto1; Traldi Piero2, Silvestri 1. Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy 2. CNR, Centro di studio sulla Stabilità e Reattività dei composti di 3. R&C Scientific, Altavilla Vicentina, (VI), Italy 4. Thermoquest Italia S.p.a, Rodano (MI), Italy 17ß-Estradiol (17ß-E2) is a natural hormone typically present in plasma of animals at very low concentration (typically lower than 20 pg/ml). Due to its anabolic activity 17ß-E2 has also found illegal diffusion to force the growth in bovine species. In order to prove the illicit abuse of 17ß-E2, it is necessary being able to discriminate between a legal occurring level stated at 40 pg/ml and possible higher concentrations from exogenous origin . Therefore the quantification of 17ß-E2 in a range around 40 pg/ml is a must. In recent years LC-MS/MS provided with triple quadruopole analysers, have been successful chosen for this purpose[1,2], but the high costs of these instruments did not make them available in all the analytical routine laboratory. In the present work, we introduce a novel approach by GC-NCI-MSn Following previous studies proposed by Bagnati and co-workers[3,4], a strategic derivatisation combining pentafluorobenzyl and trimethylsylil moieties on the 17ß-E2 structure, enables to form stable negative ions in chemical ionisation conditions using methane as reagent gas. The subsequent MS and MS fragmentations increase the sensitivity of the method and are the bases for unambiguous identification. The method was applied over real samples of bovine plasma spiked in a range between 30 and 100 pg/ml. Only 4 ml of plasma were used for the analysis consisting of one single SPE (solid phase extraction) purification step in C18 column and subsequent double derivatisation with pentafluorobenzyl bromide (PFBzBr) and Bistrimethylsyliltrifluoro-acetamide (BSTFA) containing of 1% Trimethyl chlorosilane (TMCS). The advantage of the pentafluorobezylic-derivative is represented by the increased stability of the negatively charged 17ß-estradiol-OTMS formed in CI conditions: this fact is responsible for the considerable sensitivity of the method. In MS2 experiments the transition corresponding to m/z 343→253 and 343→145 were monitored, while in MS3 the transitions at m/z 343→253→145+159+237+238 were monitored. The contemporary MS2 and MS3 experiments on each samples gave highly specific structural information and make this methodology suitable
for the determination of 17ß-E2 in plasma permitting to discriminate
between natural levels and higher concentrations due to of illegal
1. Draisci R. et al., the Analyst, 1998, 123, 2605-2609
2. Singh G. et al., Analytical Chemistry, 2000, 72, 3007-3013
3. Bagnati R et al., J-Chromatogr,-Biomed-Appl. May 1990; 527(2): 267-
4. Bagnati R. et al., J-Chromatogr, 1991, 547(1-2): 325-334.


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