Analyzing Normetabolites of the Fentanyls by Gas Chromatography/Electron Capture Detection

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Abstract
A selective and sensitive gas-liquid chromatographic (GC) method has been developed for analyzing the normetabolites of fentanyl and 3-methylfentanyl in urine. The method employs differential pH extraction of 1 mL samples, extractive acylation with pentafluoropropionic anhydride (PFPA), GC separation on a fused-silica capillary column (DB-1701), and detection by electron capture detector (ECD) or mass spectroscopy (MS). Limit of sensitivity for this method is 2 ng/mL for norfenianyl (NF) and nor-3-methylfentanyl (N-3-MF) using a 1-mL urine sample and a 2-µL injection from a final volume of 20 µL. Within-run precision, expressed as the coefficient of variation (CV), was 14% and 5% for 4 ng/mL and 16 ng/mL of NF and 9% and 4% for the same concentrations of N-3-MF. Between-run precision was 30% and 12% for NF and 11% and 10% for N-3-MF, at 4 ng/mL and 16 ng/mL, respectively. Metabolites are stable in urine for at least one month at room temperature (25° or −20°C. PFP-derivatives of the metabolites were confirmed by high-resolution MS in the electron-impact mode. Three characteristic ions for each metabolite were identified—m/z 392 (molecular ion), m/z 336 (loss of propionyl), and m/z 244 (loss of propionanilide) for N-3-MF-PFP and m/z 378 (molecular ion), m/z 322 (lose of propionyl), and m/z 230 (loss of propionanilide) for NF-PFP, suitable for use in GC/MS with selected ion monitoring as a complimentary confirming technique. This method was validated by analyzing urine samples from individuals suspected of using fentanyl or 3-methylfentanyl. Concentrations of the parent drugs, as determined by RIA, were approximately 1 ng/mL, while concentrations of the normetabolites, as determined by PFPA derivatization and GC/ECD, were generally tenfold higher. Thus, this GC/ECD method for the normetabolites of the fentanyls, when coupled with the RIA screening technique, may be used in urine testing to detect abuse of both the licit and illicit fentanyls.

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