Simultaneous separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

A method for separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, was developed using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry. The collision energy, chromatographic column, and mobile phase were optimized. All compounds were efficiently flushed out of a universal C18 column with a soft gradient consisting of solvent A (2 mM ammonium formate and 0.1% formic acid in water) and solvent B (2 mM ammonium formate and 0.1% formic acid in methanol) in only 20 min, achieving excellent resolution. Detection and analysis were carried out simultaneously in the positive ion mode using the full scan and data-dependent tandem mass spectrometry modes with a normalized collision energy of 40. The method was validated in terms of limit of detection, limit of quantification, linearity, accuracy, and precision. For all fentanyl-related substances, the limit of detection (0.5 ng/mL) and limit of quantification (1 ng/mL) were adequate for screening and quantification in daily drug control. Calibration curves for all compounds were established in the range of 1-500 ng/mL. The intra- and interday precision (RSD%) were within 0.4-2.3 and 0.7-2.7%, respectively. The accuracy ranged from 99 to 106%. The method was applied to analyze seized drug samples.