Journal of Chromatography A, 1214 (2008) 134–142 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Application of comprehensive two-dimensional gas chromatography to sterols analysis Blagoj S. Mitrevski a , J. Thomas Brenna b , Y. Zhang b , Philip J. Marriott a,∗ a Australian Centre for Research on Separation Science, School of Applied Sciences, RMIT University, G.P.O. Box 2476V, Melbourne 3001, Australia b Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA article info Article history: Received 19 September 2008 Received in revised form 8 October 2008 Accepted 14 October 2008 Available online 6 November 2008 Keywords: Comprehensive two-dimensional gas chromatography GC×GC Modulator temperature TM Doping control Steriods Flame ionisation detector Time-of-flight mass spectrometry Minimum acceptable match (MAM) abstract The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for sterol analysis was investigated by separation and identification of endogenous sterols in standards, and spiked in human urine. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of sterols was compared on two complementary column sets. Whilst the BPX5/BPX50 column set offers better overall separation, BPX50/BPX5 provides better peak shape and sensitivity. Comparison of the identification power of GC×GC–TOFMS against both the NIST05 MS library and a laboratory created (in-house) TOFMS library was carried out on a free sterols extract of urine, derivatised and spiked at the World Anti-Doping Agency (WADA) limit of 2 ng mL -1 . The average match quality for 19 analysed sterols on the BPX50/BPX5 column set was 950/1000 when searched against the in-house library; only four were identified against the NIST05 library, at a match threshold of 800. The match quality of GC×GC–TOFMS spectra was superior to that for analysis using 1D GC–TOFMS for sterols spiked in urine at 10 ng mL -1 . An r 2 > 0.997 was obtained for the concentration range between 0.25 ng mL -1 and 10 ng mL -1 for three selected sterols. The lowest limit of detection (LOD) was obtained for estrone (0.1 ng mL -1 ) and the highest LOD was for 5-androstan-3,11-diol-17-one, epitestosterone and cholesteryl butyrate (1 ng mL -1 ), using a match threshold of at least 800 and signal-to-noise ratio of at least 10. TOFMS coupled to GC×GC enabled satisfactory identification of sterols in urine at their LOD. A minimum acceptable match (MAM) criterion for urinary sterols using 2D retention times and TOF mass spectra is introduced. This study shows that GC×GC–TOFMS yields high specificity for steroids derived from urine, with detection limits appropriate for use in doping control. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The analysis of sterols is challenged by their usually low concen- tration in samples, and complex matrices in which they are found. It requires extraction/pre-concentration, followed by a separation technique, normally chromatographic, coupled to a sensitive and selective detector, such as mass spectrometry (MS). Complexity of samples, as well as low level analytes, and the need for precise results demands best possible identification and quantification. Sterols are important for a number of reasons. The occurrence of estrogenic sterols in surface water is environmentally important. Endocrine-disrupting chemicals (EDC) can interfere with the func- tion of the endocrine systems in humans and animals, and estrogen sterols are one of the most obvious and important of these. Con- centrations may be low (ng L -1 ), but can be sufficient to interfere ∗ Corresponding author. Tel.: +61 3 99252632; fax: +61 3 99253747. E-mail address: philip.marriott@rmit.edu.au (P.J. Marriott). with the normal reproduction and development of wildlife [1]. Nat- urally produced estrogens such as estrone and 17-estradiol, are mainly released in water by inefficient removal from waste-water treatment plants (WWTPs), where they are found in humans and livestock excreta [2,3]. Many sterols indicated as estrogens, gestagens and androgens (EGAs) are forbidden in products of animal origin due to the pos- sible toxicological effect on consumer’s health [4,5]. The minimum required performance limit (MRPL) of the methods for analysis of these banned hormonal growth promoters is as little as 2 gL -1 , as reported in the Italian Monitoring Plan [6]. Plasma or urine levels of some sterols can be used as a marker of biosynthesis deficiency [7]. The desire for maximum athletic performance in sporting are- nas, in addition to social recognition and financial rewards, leads some competitors to use performance enhancing drugs. Among the most important are anabolic steroids similar in structure and activ- ity to the male hormone testosterone. They are used by competitors to improve muscle mass and to accelerate recovery times after 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.045