Determination of Amphetamine, Methamphetamine, and Hydroxyamphetamine Derivatives in Urine by Gas Chromatography-Mass Spectrometry and Its Relation to CYP2D6 Phenotype of Drug Users

Abstract

Amphetamine, a CYP2D6 substrate, is widely used by truck drivers, and the extent to which different people metabolize the drug has only been determined in an isolated or reduced number of samples. A gas chromatography-mass spectrometry method is implemented to simultaneously determine amphetamine, methamphetamine, and hydroxyamphetamine in the urine of drug users. This method is a useful contribution to a well-established field. The main improvements are the use of liquid-liquid extraction, the trapping of the amphetamines as their hydrochloride salt, as a solution to the volatility of these analytes, and its application to assess the CYP2D6 metabolic phenotype of amphetamine users, which is innovative. Calibration curves ranged from 125 to 1000 ng/mL and had an r(2) greater than 0.99. The validation data (precision, accuracy, and recovery) shows the reproducibility and selectiveness of the method. The method is applied to determine the metabolic ratio (MR) in 121 urine specimens of federal highway drivers who underwent random mandatory roadside testing for drugs. The statistical analysis of the MR shows the presence of three different groups, which according to the established groups for CYP2D6 and the amount of the drug metabolized, are classified into extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). The biological consequences of these differences in amphetamine metabolism, such as impaired driving, a risk to develop Parkinson's disease, or an addiction, need to be further studied.

Cytochrome P450-2D6 extensive metabolizers are more vulnerable to methamphetamine-associated neurocognitive impairment: Preliminary findings

“…Thus, extrapolation of neurotoxicity findings involving the P450 system from animals to humans must be done cautiously. While no studies, to our knowledge, have investigated links between amphetamine metabolite concentrations and neurotoxicity in humans, it has been demonstrated that EM have greater urinary excretion of the hydroxy metabolite, followed by IM, and then by PM (Miranda, Sordo, Salazar, Contreras, Bautista, & Rojas Garcia, 2007). …”

Solid-phase extraction followed by dispersive liquid-liquid microextraction for the sensitive determination of ecstasy compounds and amphetamines in biological samples

“…Amphetamines and related compounds have usually been extracted from urine samples by liquid-liquid extraction (LLE) [17][18][19], supercritical fluid extraction (SFE) [20], solid-phase extraction (SPE) [21,22], and solid-phase microextraction (SPME) [23][24][25]. However, there are several disadvantages of conventional extraction procedures such as LLE and SPE.…”

Mixed-Hemimicelle Solid Phase Extraction Followed by Dispersive Liquid-Liquid Microextraction of Amphetamines from Biological Samples

“…Therefore, several methods, such as gas chromatography (GC), 3,4 high performance liquid chromatography (HPLC), 5 GC-mass spectrometry (MS), [6][7][8][9][10][11][12] HPLC-MS, 13,14 capillary electrophoresis (CE), 15 and CE-MS, 16 along with sample preparation methods have been developed for the determination of amphetamine and related compounds at low concentration in biological matrices. In order to eliminate interferences from the biological matrices, liquid-liquid extraction (LLE), [17][18][19] supercritical fluid extraction (SFE), 20 solid-phase extraction (SPE), 21,22 and solid-phase microextraction (SPME) [23][24][25] have been proposed. However, there are several disadvantages for conventional extraction procedures such as LLE and SPE.…”