Post-metabolism impurity profiling of carfentanil, remifentanil, sufentanil, and benzylfentanyl

Carfentanil, remifentanil, and sufentanil are potent fentanyl analogues that are regularly mixed with illicit drugs causing many overdose deaths. Chemical impurity profiling of these drugs is a well-established technique for linking evidence found at a crime scene to other seized samples. The current study aims to expand the application of impurity profiling to metabolized samples to find synthesis specific markers. This is particularly relevant when the drug has been consumed, and no intact material is present at a crime scene. Carfentanil, remifentanil, and sufentanil were synthesized according to the Ugi or 7-step method and benzylfentanyl was produced using the Siegfried method. After in-vitro metabolism with human liver microsomes, the samples were analyzed by gas chromatography-mass spectrometry (GC–MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS). Characteristic markers were found by applying a match criterion approach and principal component analysis (PCA). The precursors 4-ANBP, aniline, and N-phenylacetamide and several metabolites were identified in post-metabolism samples, indicating that specific synthesis information is retained after in-vitro metabolism. The detected levels were in line with concentrations reported in case work. In addition, LDA was applied to maximize discrimination between synthesis methods and to establish likelihood ratios (LRs). Calibrated LR values were in the range of 0.083 to 16 with very low false positive and false negative error rates. In conclusion, the presented work demonstrates the possibility of combining chemical profiling and retrospective biomarker analysis to obtain information about the synthesis method, which could be useful for forensic reconstructions and attribution investigations.