Serially coupled columns enhance rapid separation and predictive interaction understanding of 93 fentanyl analogs

The simplicity of synthesis methods has facilitated the illegal manufacture of various fentanyl analogs, leading to numerous fatal overdoses worldwide, particularly in North America. Fentanyl analogs with similar structures are difficult to distinguish due to their fragmentation patterns, making separation using chromatography essential. Additionally, because fentanyl analogs are lethal even in trace amounts, they are easily smuggled, and commonly used fentanyl test strips often fail to detect them due to their low sensitivity. Therefore, the urgent need for analytical methods that can simultaneously identify multiple analogs and swiftly detect them at low concentrations.

In this study, liquid chromatography-tandem mass spectrometry was conducted to screen 93 types of fentanyl analogs among the illegal fentanyl substances. The phenyl-hexyl columns enhance fentanyl analog separation through strong π-π interactions. The serially coupled column system increased the separation efficiency and mitigated peak distortion, particularly those of polar fentanyl analogs. The selectivity varied significantly, depending on the interactions with the combined columns. The phenyl-hexyl column's superior ability to predict fentanyl analog interactions based on molecular structure was confirmed by retention factor analysis. The resolution of fentanyl isomers increased significantly when methanol was used instead of acetonitrile as an organic modifier in the mobile phase. The approach was validated by determining the limits of detection and quantification, specificity, detection capability, recovery, and relative ion intensity.

The fentanyl analogs, including 23 sets of isomeric and isobaric compounds, were analyzed via separation using a phenyl-hexyl column serially coupled with a cyano column. The serially coupled column system increased the separation efficiency and mitigated peak distortion, particularly those of polar fentanyl analogs. The proposed strategy can be adopted in exploring methods of effectively separating mixtures with diverse properties, aiding the prevention of drug abuse and bolstering public health and safety efforts.