Separation and quantification of novel synthetic regioisomeric fentanyls in clinical extracts by high pH reverse-phase LC-MS/MS

This study compares three different chromatographic methods used for the separation of regioisomeric fluorofentanyl, as described in previous literature, through application to clinical whole blood extracts. A high pH, RP-UHPLC-MS/MS method was selected and a series of opioids ( n = 23), including regioisomeric series of fluorofentanyl, despropionylfluorofentanyl, and fluoroisobutyrylfentanyl ( n = 13), fentanyl and other fentanyl analogues ( n = 8), heroin and xylazine, were successfully validated against UKAS LAB51 and SWGTOX guidelines using this method. The results of the validation demonstrated R 2 greater than 0.99 for all analytes except heroin ( n = 22), accuracy ranging from 96.8 % to 110.1 % and precision between 0.8 % to 13.2 % as percentage coefficient of variation across replicate injections ( n = 9) of three different quality control levels for all analytes except heroin (n = 22). Limits of detection ranged from 0.016 ng/mL to 0.24 ng/mL and limits of quantification ranged from 0.049 ng/mL to 0.729 ng/mL. Clinical whole blood extracts were screened by high-resolution mass spectrometry and triaged for further identification and quantification of regioisomeric compounds where an unknown regioisomeric form of fluorofentanyl was found. Across all sample extracts para -fluorofentanyl was successfully identified, alongside potential interferences including other drugs of abuse (cocaine, methamphetamine, gabapentin) and cutting agents (procaine, lidocaine), and quantified within the range of 0.44 ng/mL to 31.16 ng/mL, with fentanyl, para -despropionylfluorofentanyl, and 4-ANPP detected and quantified in all samples with para -fluorofentanyl. • Comparison of chromatography for quantifying regioisomeric fluorofentanyls. • Partial method validation complying with SWGTOX guidelines. • Screening of extracts by high-resolution mass spectrometry for fluorofentanyl. • Quantification of previously unidentified fluorofentanyl.