Searching for potential markers for monitoring the presence of opiates in urine exposed to oxidising adulterants

Publication Type:
Thesis
Issue Date:
2014
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Urine is a long accepted biological matrix used for the detection of prescription and illicit drug use in the population. In today’s society, there is still a social stigma attached to individuals that have been found to be using contraband drugs. Being labelled a “drug addict” or a “drug cheat” in sports can potentially be detrimental to a person’s reputation. As such, it is not surprising to learn that they are motivated to discover and utilise new and ingenious ways of circumventing routine drug testing protocol. A very effective method for doing so is to purposefully tamper a urine specimen to invalidate the results of a drug test. Currently, urine samples deemed to be tampered are not analysed further for drugs of abuse as the presence of the target analytes may be significantly deteriorated or even undetectable using routine testing methods. One pathway for the mechanism of action of commercially available urine adulterants is through oxidation. The research carried out in this project has shown that following exposure of six opiates (6-MAM, morphine, codeine, codeine-6-glucuronide, morphine-3-glucuronide and morphine-6-glucuronide) to various oxidising adulterants (nitrite, PCC and hypochlorite), stable reaction products were identified in urine. The structures of 12 reaction products were elucidated using high resolution mass spectrometry and nuclear magnetic resonance spectroscopy, where possible. The reaction products were characterised to be: 2-nitro-MAM, 2-nitro-morphine, 2-nitro-M6G, codeinone, 14-hydroxycodeinone, 6-Omethylcodeine, 8-hydroxy-7,8-dihydrocodeinone, a lactone derivative of C6G, morphinone-3-glucuronide, 7,14-dihydroxy-6-MAM, a 7,8-di-keto analogue of 6-MAM and a 7,8-di-keto analogue of morphine. In all cases, the original opiate abundances were found to be diminished or undetectable. However, the reaction products were found to be stable for at least seven days using LC-MS. Reaction mechanisms for the formation of the 2-nitro analogues and codeinone were also proposed. The formation of the 2-nitro analogues was hypothesised to follow an electrophilic substitution reaction. The production of codeinone was suggested to be initiated by the chromium (VI) complex found in PCC. It was discovered that both nitrite and PCC caused a decrease in the response of the CEDIA 6-AM and opiate assays, respectively. In addition, the morphine/codeine ratios (used during confirmation testing) were found to be affected by the presence of PCC, due to the loss of both native and internal standard species. The exposure of the opiates to hypochlorite in water resulted in the detection of several potential reaction products. However, it is disadvantageous that they appear to be relatively unstable, only forming under narrow hypochlorite concentration ranges. Due to these reasons, further investigation was not pursued. Finally, an in-house quantitative NMR procedure for the certification of reaction product material was demonstrated using 2-nitro-MAM and 2-nitro-morphine following their syntheses and isolation. This method can be used as a quick alternative to certifying material through commercial institutions when there are constraints with time and funding. Overall, the research carried out in this project has laid the groundwork for future work concerning the use of the reaction products as markers for monitoring the presence of opiates in adulterated urine. Due to its relative stability, ease of formation and detection, the identified reaction products show potential for their incorporation into drug testing programs as a way of monitoring opiate positive urine specimens adulterated with nitrite.
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