Analysis of Amphetamines and Synthetic Cathinones in Hair Samples Using LC–Tandem Mass Spectrometry and Solid-Phase Extraction

Mar 01, 2014
Volume 32, Issue 3

In this study, the procedure for analyzing amphetamines and synthetic cathinones (also known as bath salts drugs) in hair samples using a mixed-mode solid-phase extraction (SPE) is described. Samples of hair were digested with a dilute solution of base (containing internal standards), neutralized, and diluted with an aqueous phosphate buffer (pH 6). Each sample was applied to a conditioned SPE column, after which the sorbent was rinsed with deionized water, acetic acid, and methanol. After drying, the analytes were eluted and collected from the SPE column with 3 mL of an elution solvent consisting of methylene chloride–isopropanol–ammonium hydroxide. To the eluate, 200 µL of mobile phase was added and the samples were evaporated to the mobile phase for analysis by liquid chromatography–tandem mass spectrometry (LC–MS-MS). Chromatography was performed in gradient mode using a C18 column and a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. The total run time for each analysis was under 5 min.

Figure 1: Structure of amphetamine.
Amphetamine (α-methylphen-ethylamine) (Figure 1) belongs to a class of compounds known as sympathiomimetic amines (1). This class of drugs includes the illicit drugs methamphetamine, methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA), as well as ephedrine and pseudoephedrine, which can be found in over-the-counter medications. Amphetamine is administered as a prescription medication (for example, Adderall [Shire Pharmaceuticals]) for treating medical issues such as narcolepsy, obesity, or hypotension (2), whereas methamphetamine, MDA, and MDMA are considered controlled substances — that is, pharmaceuticals with little or no medical use.

Figure 2: Structure of cathinone.
Synthetic cathinones are derived structurally from the parent compound (Figure 2) and have become noticeable in the scientific literature in recent times because of the fatalities arising from administration (3–6). The drugs are commonly referred to as "bath salts" because they were originally packaged with names such as "Ivory Wave" and marketed as "not for human consumption" or "research chemicals." These drugs are now scheduled in the same way as other controlled substances.

The popularity of amphetamines is because of their euphoria effect and ease of synthesis. Their use or abuse is generally verified by the analysis of biological samples, such as urine, blood, oral fluid, or hair. Of these samples, hair is a biological matrix that has been used as an alternative to urine or blood for drug testing because it allows noninvasive sampling and can document the use of the drugs over a longer period of time than blood or urine (7). In this study, amphetamine in the form of Adderall was determined in the hair of a subject along with several other amphetamines and a range of synthetic cathinones.

In terms of metabolism in the human system, amphetamine is inactivated during this process, undergoing deamination to form phenylacetone, which is converted to benzoic acid and excreted in a conjugated form (2). A small amount of the parent is oxidized to norephedrine, which is also metabolized to the parahydroxylated forms of this compound, all of which is pharmacologically active and are thought to contribute to the effects of the drug (8,9). The therapeutic, toxic, and fatal concentrations of amphetamines in samples such as blood and urine are well documented (2,10), but not so much for the synthetic cathinones because of the recent nature of their abuse. Most of the published studies on hair analysis have been performed in the area of work place drug testing and drugs or driving cases (11,12), not postmortem studies.

Gas chromatography coupled to mass spectrometry (GC–MS) has been reported as a technique for quantifying amphetamines in hair (13). GC–MS analysis of the amphetamines requires derivatization using compounds such as heptafluorobutyric anhydride (HFAA) or pentafluoropropionic anhydride (PFAA) (14,15). Because amphetamine exists as a dl isomeric pair, some laboratories have used chiral modification to separate the isomers in samples such as hair (16). The ratio of the isomeric forms may indicate whether or not the amphetamine has been taken legally. GC–MS analysis of the cathinones used similar fluoroacyl derivatives (3). Liquid chromatography coupled to tandem mass spectrometry (LC–MS-MS) is gaining popularity for analysis in this matrix (17,18). The use of solid-phase extraction (SPE) described in this article uses the LC mobile phase as a keeper solvent for amphetamine, reducing its volatility. In previous methods the addition of methanolic hydrochloric acid or a solvent such as dimethylformamide has been reported, and the solvent was evaporated to dryness (19). The addition of the mobile phase presents the LC–MS-MS with a more amenable analytical solvent. SPE has been reported in the analysis of hair samples previously (20–22), but not using this type of keeper solution format.