Derivatização de anfetaminas: quais são os desafios na análise de sangue post mortem por GC-MS?

Abstract

Amphetamines are a class of synthetic compounds that act as central nervous system stimulants and their clinical use is restricted to the treatment of obesity, narcolepsy and attention deficit hyperactivity disorder (ADHD). They are represented by substances such as methamphetamine, ecstasy (MDMA) and 3,4-Methylenedioxyamphetamine (MDA). Recreational use promotes euphoria and well-being, but it can cause seizures, hallucinations, severe intoxication and death. In this study, two derivatization reagents were evaluated, pentafluoropropionic anhydride (PFPA) and trifluoroacetic anhydride (TFAA), to stablish which would allow the detection of amphetamine, methamphetamine, MDMA, MDA, 3,4-methylenedioxy-N-ethylamphetamine (MDEA) and fenproporex. TFAA enabled the detection of all the analytes and was therefore chosen for optimization. For the other derivatization conditions, a central composite design was conducted varying the volume of the derivatizing agent (40 to 60 µL), reaction time (20 to 40 min) and temperature (50 to 70ºC). MDA, MDMA and MDEA were only detected when the reaction occurred at a minimum of 60ºC and all the analytes had the best analytical response at 50 µL of TFAA with the reaction being heated at 60ºC for 30 minutes and these were the conditions defined as optimal and used in the subsequent analyses. For the sample preparation, the liquid-liquid partitioning at low-temperature extraction was performed with methyl tert-butyl ether (MTBE) as the extracting solvent. However, a large amount of interferents were extracted, which hampered derivatization and the presence of the characteristic peaks of the analytes. For this reason, it was required an extraction approach that would remove the interferents more effectively, which was only achieved with solid phase extraction. After determining the limit of detection of 100 ng.mL-1 for methamphetamine, 200 ng.mL-1 for MDMA, MDA and femproporex and 250 ng.mL-1 for amphetamine, two cases were evaluated: in the first, amphetamine was detected because it was a metabolite of Vyvanse® (which the victim used continuously) and in the second, MDMA was identified in a suspected overdose. Thus, with the elucidation of real cases, the present method of derivatization with TFAA of amphetamines in post mortem blood for analysis by GC-MS was developed and successfully applied to real samples.