In this study, the procedure for analyzing amphetamines and synthetic cathinones (also known as "bath salts") 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 ÁL 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.
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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 1: Structure of amphetamine.
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.
Figure 2: Structure of cathinone.
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.
Amphetamine is deactivated during metabolism in the human system, 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 amphetamines requires derivatization using compounds such as heptafluorobutyric anhydride (HFAA) or
pentafluoropropionic anhydride (PFAA) (14,15). Because amphetamine exists as a d–l 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.