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. [ABSTRACT FROM AUTHOR]