Synthesis of 13C-labeled drugs of abuse as internal standards for reducing differences in ion suppressing/alteration effects in LC/MS-MS quantification

Abstract

Stable isotope labelled internal standards (SIL ISs) are often used when legal and illegal drugs are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ideally an IS behaves identically as its corresponding analyte during sample preparation and LC-MS/MS analysis. However, isotope effects (IEs) may cause differences in the behaviour between the SIL IS and its analyte. In reversed phase (RP) LC-MS/MS separations deuterium (2H) labelled ISs often elutes slightly earlier than their corresponding analytes, something that reduces the ISs possibility to correct for ion suppression/enhancement effects. There is a greater difference in physico-chemical properties between hydrogen isotopes than between isotopes of other elements. Therefore, ISs labelled with 13C, 15N and/or 18O are expected to behave more similarly to their analytes than 2H labelled ISs. The availability of high quality 13C-SIL IS will improve accurate quantification of narcotics and drugs in biological samples. Thus, the synthesis of some of the most important misused drugs and metabolites was undertaken to detect and control misuse and monitor administration in clinical settings. Ten [13C6]-labelled phenethylamine derivatives, namely: amphetamine, methamphetamine, 3,4- methylenedioxyamphetamine, 3,4-methylenedioxy-methamphetamine, 3,4- methylenedioxy-N-ethylamphetamine, 4-methoxyamphetamine, 4- methoxymethamphetamine, 3,5-dimethoxyphenethylamine 4-bromo-2,5- dimethoxyphenethyl-amine and 2,5-dimethoxy-4-iodo-phenethylamine, was synthesized. [13C6]-Phenol proved to be an excellent starting material for making 13C-labelled narcotic substances, and a developed Stille type coupling enabled an efficient synthesis of the 3,4- methylenedioxy and 4-methoxy derivatives. (-)-Δ9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in the prescribed drug Dronabinol™. Reference samples of the native drug [13C4]-Δ9- tetrahydrocannabinol and the most important metabolite [13C4]-11-nor-9-carboxy-Δ9- tetrahydrocannabinol were developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-Δ9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)- apoverbenone. The illegal use of opiates and (-)-cocaine is a challenge World-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the native drugs and their metabolites are needed. Thus, the synthesis of [13C4]-heroin, [13C5]-heroin, [13C3]-6- acetylmorphine, [13C2]-codeine, [13C6]-(-)-cocaine, [13C6]-(-)-benzoylecgonine, [13C6]-(-)- norcocaine, and [13C6]-(-)-cocaethylene was undertaken to provide such reference materials. The 13C-labelled opiates and cocaine derivatives were made from the corresponding natural products. The synthetic work in this thesis has focused on identifying 13C atom efficient routes towards the SIL IS. This has been accomplished by finding atom efficient routes, shortening existing methods or starting by chemical manipulation of the natural products. Thus, to provide internal standards for more accurate quantification and for identification purpose the final SIL ISs were tested for their efficiency. 13C-SIL ISs proved in some cases to be superior over existing reference materials based on 2H.