van de Kerkhof, D. H., van Ooijen, R. D., de Boer, D., Fokkens, R. H., Nibbering, N. M., Zwikker, J. W., Thijssen, J. H., & Maes, R. A. (2002). Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in doping analysis. Journal of Chromatography A, 954, 199-206.

"Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltri-fluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography-mass spectrometry (GC-MS) in steroid analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix, and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation".

Implication. The chemicals used in drug analysis do interact with the substances in urine and increase their presence. When in particularly small amounts, the increases still will not surpass anti-doping standards. However, in the unusual situation where naturally occurring substances are of high levels, the interaction could produce false-positive results.

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