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Please find below a short description of research undertaken by Yashaswini Srinivas into methods of detection of DMAA, undertaken at Sheffield Hallam University.
DMAA has been in the news recently as it was implicated in the death of a runner who collapsed and died during the London Marathon 2012. My thoughts on DMAA and it’s properties are in my blog here.
ANALYTICAL METHOD DEVELOPMENT FOR DETECTION OF 4-METHYL-2-HEXANAMINE IN BIOLOGICAL SAMPLES
RESEARCHER - Yashaswini Bangalore Srinivas
RESEARCH UNIT - Sheffield Hallam University, Sheffield, United Kingdom
Date of Research - June-July, 2012
SUMMARY OF THE RESEARCH PROJECT:
4-methyl-2-hexanamine/DMAA, structural congener of 2-aminoheptane is a banned stimulant listed under WADA list of banned substances and whose use is specifically prohibited ‘in-competition’. Claimed to be of herbal origin, obtained from the oil of Pelargonium graveolens, it is said to satisfy the definition of a ‘stimulant’ by agonistically binding at α1 adrenoceptors in the liver leading to glycogenolysis. Since, the physiological effect of glycogenolysis is to release large amount of energy instantaneously, consumption of substances containing DMAA just before the sporting event confers unfair advantages to the athlete possibly leading to enhanced sporting performance.
Worldwide, WADA accredited laboratories employ the sensitive, selective analytical techniques LC-MS and GC-MS to detect the presence of doping agents in an athlete’s urine sample. In this research project, the foremost aim was to validate LC-MS & GC-MS techniques and assess reproducibility of the protocol as followed by Perrenoud et al. (2009) for the available analytical conditions. Calibration experiments were performed on both LC-MS and GC-MS instruments.
The research hypothesis was ‘the two analytical techniques can achieve separation and detect both analyte and internal standard with calibration standard solutions to check the linearity range of the instrument’.
The LC-MS technique failed to achieve resolved peaks for internal (2-aminoheptane) and reference standards (DMAA) in the calibration standard solutions dissolved in the mobile phase. Since both DMAA and internal standards have the same mass (MW=115), and being primary amine compounds can accept a proton from the solvent and attain ion of m/z=116, without separation the interpretation is ambiguous.
Therefore, the focus of the research then shifted towards GC-MS wherein calibration was carried out employing two different internal standards- tuaminoheptane and 1-hexylamine.
Using the internal standard tuaminoheptane gave was the same issue as encountered by Perrenoud et al. (2009). It could not be concluded whether the single peak observed in the TIC represented reference/ internal standard. On the other hand, 1-hexylamine which differs from tuaminoheptane by a methyl unit proved be a slightly better internal standard in that different ion peaks formed as a result of reaction between derivatising agent with internal and reference standards.
In summary, it became clear by the end of this research project that GC-MS could be adopted as a quantification tool for examining the drug under investigation.
Not mentionned here are the alternative derivatising reagents used to try and effect separation using the GC-MS. The separation was achieved initially using cyclohexanone, as published by Perrenoud et al (Journal of Chromatography B, 877 (2009) 3767–3770), then using an alternative.
The internal standard tuaminoheptane was used by Perrenoud whilst in the above project the alternative 1-hexylamine was used with initially promising results.
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