Validation of a gas chromatography–mass spectrometry method for the determination of pg/ml levels of 17β-estradiol and 17β-trenbolone in bovine serum

A method for the quantitation of pg/ml levels of 17β-estradiol and 17β-trenbolone in bovine serum by gas chromatography/electron-capture mass spectrometry has been developed and validated. Using the area ratios of the integrated molecular-ion peaks of the analytes to their corresponding deuterated internal standards, [2,4,16,16-2H4] 17β-estradiol (17β-estradiol-d4) and [16,16-2H2] 17β-trenbolone (17β-trenbolone-d2), and non-weighted linear regression, two calibration curves per analyte; 5–50 and 50–500 pg/ml for 17β-estradiol in sera, and 25–250 and 250–2500 pg/ml for 17β-trenbolone in sera, respectively, were constructed. Splitless injection of 200 fg 17β-estradiol and 1000 fg 17β-trenbolone could be detected and quantified. Tested batches of control bovine sera did not exhibit interference for 17β-trenbolone, and showed expected background presence of endogenous 17β-estradiol. Intra-day residual errors did not exceed 20%, and regression correlations were greater than 0.99. Intra-day precision data was similar to inter-day precision data. Using this method, 16 samples can be processed within one working day.