Analysis of amphetamine, metamphetamine and MDMA ("ecstasy") in human plasma and urine by means of liquid chromatography with fluorimetric detection

Introduction: Amphetamine (alpha-methylphenylethylamine) and its analogues such as methamphetamine (N,alpha-dimethylphenylethylamine) and MDMA (N,alpha-dimethyl-3,4-methylenedioxyphenylethylamine) are currently among the most well-known abuse drugs. They are used especially by young people as stimulants during the week-end (e.g. in discos), and are thus often referred to as "recreational drugs". Their mechanism of action is complex, but surely involves the release of catecholamines and serotonin from synaptic vesicles. Aim of this study is the development of a reliable analytical method for the simultaneous determination of amphetamine, methamphetamine and MDMA, and its application to the analysis of these compounds in human biological fluids, such as plasma and urine. Materials and Methods: Since the analytes show native fluorescence, liquid chromatography (HPLC) with fluorimetric detection was chosen for the purpose of separating and quantitating them. Separation was achieved on a C8 reversed-phase column using a phosphate buffer/acetonitrile (88:12) mixture (apparent pH = 2.8) as the mobile phase, flowing at 1 mL/min. Quinine was chosen as the Internal Standard (IS). Detection wavelengths were: lambda(exc) = 210 nm; lambda(em) = 300 nm for the analytes; lambda(exc) = 340 nm; lambda(em) = 420 nm for the IS. The sample pre-treatment procedure was carried out by means of solid-phase extraction (SPE) on hydrophilic-lipophilic balance cartridges. The cartridges were loaded with 250 µL of plasma or urine; the analytes were then eluted, dried and redissolved with 250 µL of mobile phase. Results: Under the described leading conditions, all analytes are baseline separated and detected within 13 minutes. Good linearity was obtained over different concentration ranges, according to the drug and the matrix. The sample pre-treatment procedure gave good extraction yields for all analytes, with mean recovery values ranging from 85 to 95% in plasma. Furthermore, the samples are devoid of interference from the biological matrices, thus good purification has been achieved. Conclusion: The method thus developed seems to be suitable for the determination of amphetamine, methamphetamine and MDMA in human plasma and urine