Analysis of Infant Formula for Steroid Hormones by Gas Chromatography-Tandem Mass Spectrometry Using Microwave-Assisted Extraction and Gel Permeation Chromatography Clean Up

A new method was developed for effective separation and simultaneous determination of estrogens, gestagens, and androgens, including estrone, 17 beta-estradiol, testosterone, progesterone, and estriol, in infant formula. The samples were enzymatically digested with beta-glucuronidase/arylsulfatase prior to microwave-assisted extraction. After the extract was cleaned up by gel permeation chromatography the hormones were derivatived with 50 mu L BSTFA containing 1 % TMCS. The derivatived hormones were measured with GC-MS/MS using electron impact ionization source in the positive multi-reaction monitoring mode. The calibration curves showed good linearity with correlation coefficient (r) of >0.999. The limit of quantification of five hormones ranged from 0.094 to 0.265 mu g kg(-1), which is below the minimum required performance limits established by the European Community. The intra- and inter-day precision (as RSD) for six determinations of five analytes at 40 mu g kg(-1) spiked level was in range of 3.4-5.4 % and 3.5-6.8 %, respectively. The recovery of five analytes was obtained to be 84.5-104 %, with relative standard deviations (RSD, n=6) of 1.7-5.5 %. This method has been successfully used for the qualitatively and quantitatively determination of estrone, 17 beta-estradiol, testosterone, progesterone, and estriol in infant formula.