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A critical appraisal of the analytical methods for the qualitative and quantitative analysis of amphetamine

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Amphetamine and related derivatives are powerful stimulants of the central nervous system and are widely abused to create hallucination and euphoria, and also as doping agents. The psycho-stimulant activity of amphetamine and amphetamine-derived designer drugs depends on the type of enantiomer (5 times higher for S-(+) enantiomes). The target analytes in biological samples are the parent compounds rather then products of metabolism, although for some designer drugs, such as MDMA and MDEA, the target analyte is also the N-dealkyl metabolite MDA. They are most commonly detected in urine and/or serum samples using gas chromatography-mass spectrometry (GC/MS). Other analytical strategies include high-performance liquid chromatography (HPLC), and liquid chromatography- tandem mass spectrometry (LC-MS/MS). Capillary gas chromatography/Fourier transformed infrared spectroscopy has also been employed to identify amphetamines. Several clean-up and extraction procedures, such as liquid-liquid extraction, solid-phase extraction or solid-phase microextraction, are required prior to the injection of samples into the GC column. Although amphetamine is typically consumed in the form of water-soluble salt, a free form of the drug is a volatile oil. This characteristic has been utilized to detect amphetamine in biological fluids using atmospheric pressure ionization method (Chen et al. 2009). Finally, immunoassays are usually used to detect amphetamines in laboratory screening and workplace point-of-care devices. For any method it is of paramount importance to test limit of detection (LOD), limit of quantification (LOQ), as well as linearity.

Immunoassays in detection of amphetamines

Immunoassays involve the use of immunoglobins (antibodies), and include ELISA (Enzyme-Linked Immunosorbent Assay) and RIA (radio-immunoassay), or EMIT (enzyme-multiplied immunoassay technique). EMIT is based on competition for an antibody binding between drug in the sample and drug labelled with an enzyme. A non-competitive version of EMIT has also been developed which utilizes an excess of drug-specific antibody. RIA is in principal similar to EMIT, but uses a radioisotope, usually radioactive iodine (I125) which emits gamma radiation. Other modern assays, such as Fluorescence Polarization Immunoassay (FPIA) and CEDIA (cloned enzyme donor immunoassay), are also applicable for detection of amphetamines and may be advantageous for clinical toxicology. In FPIA, the fluorescently labelled drug competes with unlabelled drug in tested sample for binding sites on the antibody. The assay is performed as a single solution reaction, and uses the principle of rotation of molecules in solution. Different fluorescence polarisation properties allow distinguishing between free and antibody-bound antigen fluorescence. Overall, FPIA provides an inverse dose-response curve with lower levels of analyte resulting in a higher signal. FPIA has also been used to detect drugs of abuse, including amphetamine, in hair. Briefly, hair was decontaminated in ethanol, incubated with 3M sodium hydroxide, and the aliquots were subsequently neutralized and analysed with FPIA-based ADx kit from Abbott (Kintz et al. 1992). CEDIA is based on the use of two polypeptides, constituting 95% (enzyme acceptor) and 5% (enzyme donor) of E.coli beta-galactosidase. Recombination of these polypeptides regenerates the enzymatic activity. In an assay a combination of drug-specific antibody, drug-labelled enzyme donor peptide (ligand conjugate) and sample analyte create a competitive binding environment. Higher amount of analyte in a sample leads to the better regeneration of beta-galactosidase enzymatic activity. The advantages of immunoassays include low cost and rapid turnaround time. However, GC/MC analysis is usually required for confirmation and verification of the results of an immunological assay.

Gas chromatography/Fourier transform infrared spectroscopy (GC/FT-IR)

GC/MS can sometimes lead to false positive identifications in drug testing labs. GC/FT-IR reportedly provides absolute identification through infrared fingerprinting with routine detection in the ppb range. GC-FT-IR has recently become an efficient and powerful analytical approach for the indentification of amphetamine analogs. Similarly to GC/MS it involves derivatization, e.g. HFBA derivatization results in more defined and sharper chromatographic peaks. Praisler and co-authors have recently developed a computer-aided procedure automating the identification of amphetamine derivatives eluted from a gas chromatograph coupled to a Fourier transforminfrared spectrophotometer (Praisler et al. 2000). Thus, it is currently possible to identify a compound within seconds using soft independent modeling of class analogy (SIMCA) providing tests that are of high specificity, highly selective, extremely fast and user friendly.


Immunoassays can be used as method of choice for screening purposes and as point-of-care detection devices. However, the accurate quantification and validation of the results should be performed using a more sensitive technology. Among these, LC-MS/MS appears as a sensitive and rapid approach. In addition, a recently developed APC/CI-MS technology can be applied to detect amphetamines quickly and with a good sensitivity. The infrared fingerprinting offers probably the highest identification accuracy, which is strengthen further by the recent developments of knowledge bases defining the reference Fourier transform infrared spectroscopic (FTIR) spectral patterns.

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