YuYu Pharma (South Korea)

Amphetamine (AP) and amphetamine-type stimulants, methamphetamine (MA) and N,N-dimethylamphetamine (DMA), are known as central nervous system stimulants, and their abuse throughout the world has recently increased. Since it is difficult to physically distinguish among AP, MA and DMA, analysts may not be aware of what abusers have administered. In this study, following the detection of specific metabolites of AP, MA and DMA as biomarkers in abuser urines, a rapid and sensitive method was developed for the identification and classification of AP-type stimulants abusers. After the simple filtration of the urine samples, the samples were directly analyzed using a liquid chromatography/tandem mass spectrometry system with selected reaction monitoring (SRM)-triggered quantitation-enhanced data-dependent MS/MS (QED-MS/MS) for the simultaneous qualitative and quantitative analysis of p-hydroxy AP, p-hydroxy MA, p-hydroxy DMA, AP, MA, DMA and DMA N-oxide. The determination of p-hydroxy AP, p-hydroxy MA, AP, MA, DMA and DMA N-oxide was accurate and reproducible, with the limits of quantitation of 5 ng/mL in urine. When applied to the urine samples of suspected AP-type stimulants abusers, the abused drugs were precisely identified between MA and DMA abusers.

% RSD Accuracy (%)TMA (ng/mL)25 26.4 ± 1.3 5.2 105.7 26.4 ± 0.9 3.7 105.8100 99.3 ± 3.0 3.0 99.3 106.9 ± 3.9 3.9 106.9500 500.1 ± 26.5 5.3 100.0 509.0 ± 12.8 2.6 101.8TMANO (µg/mL)1 0.9 ± 0.1 10.6 89.1 0.9 ± 0.1 9.2 91.75 4.6 ± 0.4 7.7 92.5 4.5 ± 0.5 9.5 89.625 25.6 ± 1.4 5.6 102.6 24.3 ± 1.3 5.1 97.1

N,N-dimethylamphetamine (DMA) is a methamphetamine analogue known to be a weaker central nervous system stimulant than methamphetamine. Although a major metabolite of DMA is known to be DMA N-oxide (DMANO), which may be catalysed by flavin-containing monooxygenase (FMO), the specific enzyme(s) involved in this biotransformation has not been identified. In this study, the specific enzyme(s) involved with DMA N-oxidation was characterized by several assays. When DMA was incubated with different human recombinant drug-metabolizing enzymes, including FMOs and cytochrome P450s (CYPs), the formation of DMANO by FMO1 was the most predominant. The Michaelis–Menten kinetic constants for DMA N-oxidation by FMO1 were: Km of 44.5 μM, Vmax of 7.59 nmol min−1 mg−1 protein, and intrinsic clearance of 171 μl min−1 mg−1 protein, which was about twelve-fold higher than that by FMO3. Imipramine, an FMO1-specific inhibitor, selectively inhibited DMA N-oxidation. The resulting data showed that DMA N-oxidation is mainly mediated by FMO1.