Development and comparison of reversed-phase ultra high-performance liquid chromatography (RP-UHPLC) and hydrophilic interaction liquid chromatography (HILIC) approaches to the analysis of regioisomeric fluorofentanyl derivatives and related compounds

This study describes the development and comparison of low, intermediate and high pH gradient RP-UHPLC-MS/MS with that of gradient HILIC-MS/MS analysis for a range of fluorofentanyl derivatives including four families of ortho- , meta- and para -regioisomers. High pH RP-UHPLC-MS/MS using an ammonium hydroxide and methanol gradient on a high pH stable SuperC18 column at low temperature was demonstrated to be the most successful chromatographic mode for separating 26 analytes including: regioisomeric fluorofentanyls ( n = 10); fentanyl analogues (n = 10), despropionyl precursors ( n = 4) and two commonly encountered related substances (heroin and xylazine). Low and intermediate pH RP-UHPLC failed to afford separation of many of the fluorofentanyl regioisomers on stationary phases possessing complementary selectivity with either acetonitrile or methanol over a wide temperature range. HILIC on a bare silica column using an acetonitrile and ammonium acetate / acetic acid gradient provided good separation of fluorofentanyl regiosiomers except for the despropionyl series. High pH gradient RP-UHPLC was demonstrated to provide orthogonal chromatographic selectivity to that of HILIC in the gradient analysis of 18 fentanyl and related substances. Seven isobaric fluorofentanyl structural isomers could be readily discriminated from the unique fragmentation ions obtained using positive electrospray ionization MS/MS. The optimum high pH RP-UHPLC chromatographic conditions for the separation of the fluorofentanyls was equally successful for the rapid separation of a wide range of fentanyl regio- and structural isomers. • High pH gradient RP-UHPLC afforded resolution of fluorofentanyl regioisomers. • Gradient HILIC provided resolution of N -acylated fluorofentanyl regioisomers. • RP-UHPLC and HILIC showed orthogonal selectivity of fentanyl and related substances. • Discrimination of isobaric fluorofentanyls achieved by positive electrospray MS/MS. • Chromatography and MS/MS ions facilitated separation of isobaric fluorofentanyls.