Abstract

The detection of the five low level anabolic agents 19-Norandrosterone, 3’-OH-Stanozolol, Clenbuterol, 17β-methyl-5β-androst-1-ene-3α,17α-diol (EMD) and 17α-methyl-5β-androst-3α,17β-diol (MethylT metabolite) by LC/MS/MS will require the investigation of matrix effects. Matrix effects are the Achilles heel of electrospray ionisation (ESI) quantification (Taylor, 2005). Ionisation suppression or enhancement can markedly affect quantitative results in ESI. In extreme cases the suppression can be sufficient to prevent detection of an analyte. If ESI is to be used to detect low level analytes then a means of ensuring that the analytes will be detected at required levels in all samples needs to be proven. There are several proposed methods which can be used for the calculation of recoveries. Two will be examined, the addition of internal deuterated standards for each analyte added prior to extraction or post column infusion of internal standards. Three of the low level anabolic agents were already available commercially in deuterated form but EMD and MethylT metabolite needed to be synthesised by the reference standards group of NMI (Australia). The post column infusion of a standard has been used by several investigators to examine matrix affects but it has also been suggested as a complete replacement for a spiked internal standard (Cheng and Tsai, 2008). The selected internal standard is added post column as a continual infusion into the HPLC column eluent. This can be used to correct for matrix effects by using either a single standard of similar structure to the group of analytes of interest, in this case progesterone was used, or a mixture of deuterated internal standards associated with the compounds of interest (deuterated mixture of 5 anabolic agents for this study). Aus dem Text (geändert)