Accurate Biological Testing for Amphetamine and Methamphetamine Abuse Using Chiral HPLC and MS Detection

Introduction

Methamphetamine and amphetamine, powerful CNS stimulants that have a variety of ethical uses, have side effects of increased confidence, sociability, and energy that have resulted in their extensive abuse as recreational psychoactive drugs. This extends to their abuse in sports because of the additional properties of increased mental alertness and suppression of fatigue.¹ Negative side effects include hypertension and tachycardia. In the usually uncontrolled situations of drug abuse, they exhibit similar psychoactive effects, although amphetamine is generally less potent than methamphetamine.

Illicit use of these drugs continues to be high. Public Health England recently reported that the number of people stating their main injecting drug to be amphetamines and amphetamine-type substances (such as mephedrone) nearly tripled between 2002 and 2012.² Additionally, unlike the less-volatile amphetamine sulphate, methamphetamine hydrochloride (sometimes known as “ice” or “crystal meth”) can be smoked, giving highs that are similar to crack cocaine, although significantly longer lasting. It has been reported that routine co-monitoring of methamphetamine and amphetamine is not normally carried out, so there is little information on which is more widely used. It was noted however, that amphetamine is more widely available than methamphetamine in Europe, whereas the reverse is true for North America and Asia.³

Chirality

Amphetamine and methamphetamine are chiral molecules (Figure 1). In both cases the D-enantiomer has greater biological activity than the L-enantiomer. Although methamphetamine is a controlled substance (Class A in Europe and Schedule II in the US, both as individual enantiomers and as the racemate), the L-isomer is in fact used in several over-the-counter medicines (e.g. Vicks^® inhaler in North America) and so can alter the true level arising from abuse of the drug. The analysis is further complicated by the fact that L-methamphetamine is also a metabolite of certain therapeutic drugs, such as selegiline, a treatment for early-onset Parkinson’s disease, depression, and dementia. Amphetamine quantification is more complex, again, simply because it is also the main metabolite of methamphetamine. The traditionally used method for drug analysis, immunoassay, cannot distinguish between the enantiomers and so can easily give incomplete, inconclusive results.

Figure 1. Structures of D- and L-Amphetamine and Methamphetamine

When methamphetamine is sold illegally, it is usually as the D-enantiomer or as the racemate. When ingested, a dose may vary from several tens to several hundreds of milligrams, depending on the purity and the isomeric composition, and will metabolize to amphetamine and 4-hydroxymethamphetamine.³ Amphetamine metabolizes mainly to 1-phenyl-2-propanone, with smaller amounts of 4-hydroxyamphetamine. However, since up to 54% of methamphetamine is excreted unchanged and 10-23% as amphetamine following oral ingestion, it is usually the parent drug that is monitored.¹ Culpability for the illicit use of amphetamine and methamphetamine rests on the ability to distinguish the contribution to the measured abused product from possible alternative sources of the L-enantiomer. Therefore, the separation of the enantiomers is a more accurate approach for testing. A chiral method can also be used to indicate the synthetic route that was used by the illegal source, which can be useful as part of a criminal investigation. Indeed, detection of just the D-methamphetamine isomer has been found in product prepared through either ephedrine or pseudoephedrine.⁴

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