Optimization of the chromatographic conditions for the determination of hormones by gas chromatography with mass spectrometry detection Original Research Article

A gas chromatographic method with mass spectrometry detection (GC/MS) has been developed for the analysis of eight hormones: diethylstilbestrol, hexestrol, dienestrol, nor-testosterone, methyl-testosterone, 17-α-estradiol, 17-β-estradiol and 17-α-ethynylestradiol. As the diastereoisomers, α and β-estradiol, coelute and both have the same mass fragments, the experimental design methodology has been applied for obtaining their chromatographic separation. In this paper, the temperature programme of the column with two ramps has been optimized to improve not only the resolution between the peaks of both isomers but also the quality of the peaks (sharp and symmetric peaks). Firstly, a 16-experiment screening design was carried out to determine with a reduced number of experiments which factors affect both the resolution and the peak width. The results concluded that the final temperature of the first ramp mainly affects the resolution whereas the final temperature of the second ramp influences both the resolution and the peak width. A two-factor Doehlert design was subsequently performed to fit a second-order model and jointly optimize the resolution and the peak width through a global desirability function. Thus, the final temperatures of the first and the second ramp are 260 and 300 °C, respectively. The method developed has been validated according to the European Decision 2002/657/EC, including the estimation of the capability of detection (CCβ, X0 = 0, for banned substances) with evaluation of the probability of false positive, α, and of false negative, β.