Selective and sensitive methodology is needed to permit the detection of low levels of potent anabolic steroids and their metabolites in physiological fluids. Electrospray ionization (ESI) is characterized by its high efficiency of ionization (approaching 100 percent) possible in large measure because the ionization process occurs at atmospheric pressure. Thus, for methods striving for low detection limits, it would be desirable to employ the technology of ESI. Unfortunately, many compounds such as free steroids, in particular testosterone and other anabolic steroids, have a low ionization efficiency by ESI, presumably because of the lack of any basic sites in the molecule. The goal of our project has been to prepare derivatives that contain either a fixed charge or a basic site that promote detection and/or ionization and detection by ESI during analysis of extracts by LC-MS. We have demonstrated that the hydrazone derivative and the oxime derivative of keto steroids can be easily and efficiently prepared, and that the resulting derivatized analyte gives a high response to ESI. The chemical reactions can be carried out in the laboratory at room temperature and reach 90% or greater conversion in 15 minutes. Analyses of test samples indicate that methyl testosterone can be detected well into the low picomole regime.
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