Systematic evaluation of derivatization strategies and GC column polarity for reliable GC–MS separation of amphetamine and phenethylamine in putrefied postmortem samples

PURPOSE: The reliable identification of amphetamine in degraded biological samples is often complicated by the presence of putrefactive amines, such as phenethylamine, which exhibit similar physicochemical properties and can interfere with chromatographic separation. This study aimed to systematically evaluate the conditions of gas chromatography-mass spectrometry (GC-MS), including derivatization methods and analytical column selection, to achieve effective separation and identification of amphetamine in the presence of phenethylamine. METHODS: Various derivatization approaches, including acylation, alkoxycarbonylation, silylation, and pentafluorobenzylation, were examined in combination with non-polar, slightly polar, and semi-polar GC columns. The chromatographic behavior of amphetamine and phenethylamine, including the retention time, peak shape, and formation of mono- or di-substituted derivatives, was systematically assessed to optimize separation and mass spectral identification. RESULTS: Alkoxycarbonylation, silylation, and alkylation produced overlapping peaks, mixed derivatives, or unreacted compounds, thereby limiting their reliable identification. Acyl derivatization allowed for effective separation using non-polar and semi-polar columns, with the non-polar column minimizing peak tailing that is commonly caused by high phenethylamine concentrations. Differences in the elution order were observed between column types, and semi-polar columns provided satisfactory chromatographic resolution and high-quality mass spectra, consistent with spectral databases. CONCLUSIONS: Successful GC-MS identification of amphetamine in putrefied samples requires careful selection of the derivatization chemistry and GC column polarity. Acyl derivatization combined with an appropriate column choice enables reliable separation from phenethylamine, improves spectral quality, and enhances the evidential value of forensic analysis.