Analytica Chimica Acta 544 (2005) 26–35 Optimization of the chromatographic conditions for the determination of hormones by gas chromatography with mass spectrometry detection Inmaculada Garc´ ıa a , Luis A. Sarabia b , M. Cruz Ortiz a,∗ , J. Manuel Aldama c a Department of Chemistry, Faculty of Sciences, University of Burgos, Pza. Misael Ba˜ nuelos s/n, 09001 Burgos, Spain b Department of Mathematics and Computation, Faculty of Sciences, University of Burgos, Pza. Misael Ba˜ nuelos s/n, 09001 Burgos, Spain c Instituto de Ciencias de la Salud (An´ alisis F´ ısico-Qu´ ımicos), Ctra. Extremadura, Km. 114,1, 45600 Talavera de la Reina, Toledo, Spain Received 7 October 2004; received in revised form 21 March 2005; accepted 8 April 2005 Available online 10 May 2005 Abstract 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--estradiol, 17--estradiol and 17--ethynylestradiol. As the diastereoisomers,  and -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 (CC, X 0 = 0, for banned substances) with evaluation of the probability of false positive, α, and of false negative, β. © 2005 Elsevier B.V. All rights reserved. Keywords: Estrogens; Androgens; -Estradiol; -Estradiol; Screening design; Doehlert design; Desirability; CC 1. Introduction The European Union has forbidden the administration of hormonal growth promotants to farm animals destined for hu- man consumption (Directive 96/22/EC amended by Directive 2003/74/EC). Consequently, the laboratories in charge of de- tecting these residues need to develop techniques allowing not only the analysis in several biological matrices but also the distinction between natural and synthetic hormones. These compounds are often identified and quantified by gas chromatography with mass spectrometry detection (GC/MS) [1,2] because of the low capability of detection pro- ∗ Corresponding author. Fax: +34 947 258 831. E-mail address: mcortiz@ubu.es (M.C. Ortiz). vided by this technique. Derivatization of compounds with polar groups is common in GC in order to decrease their polar- ity, increase the volatility, improve the chromatographic sep- aration or to stabilize thermolabile substances. Trimethylsi- lylation is the most habitual reaction for derivatizating hor- mones and different silylation agents have been used alone [3] or combined with catalysers [1,2,4]. Apart from the increase in the analysis time, some difficulties in derivatization have been observed: hormones have various hydroxyl groups, and consequently, different derivatives might be formed [5]. Be- sides, few intermediate derivatives are not stable over time, which makes the chromatogram more complex and less spe- cific. In this paper, the problem has been solved by employing a midpolarity column DB-17MS (50% phenyl–50% dimethyl arylene siloxane), which allows the analysis of compounds 0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.04.022