Development and Validation of an UHPLC-MS/MS Method for the Determination of 32 Pyrrolizidine Alkaloids in Chinese Wild Honey

Background Studies on pyrrolizidine alkaloid (PA) contamination in honey produced in China are scarce. Previously reported HPLC-MS/MS methods for the determination of PAs in honey often suffer from insufficient separation and uncertainties in PA isomers. Objective To develop and validate an Ultra-HPLC (UHPLC)-MS/MS method for baseline separation of PA isomers towards precise determination of 32 PAs in Chinese wild honey. Methods PAs were extracted from honey samples and separated on an ACQUITY BEH C-18 (2.1 mm x 100 mm, 1.7 mu m) column with (A) 0.1% formic acid aqueous solution containing 5 mM ammonium acetate and (B) methanol as mobile phase. The column temperature was maintained at 30 degrees C, and flow rate was 0.3 mL/min. Detection was performed by tandem mass spectrometry. The total run time was reduced to 18 min. Results Thirty-one of 32 PAs were baseline separated efficiently within 18 min. The LOD and LOQ were 0.06-0.25 mu g/kg and 0.22-0.82 mu g/kg, respectively, except for that of clivorine, for which LOD and LOQ were 2.03 and 6.78 mu g/kg, respectively. The average recoveries ranged between 66.3 and 95.1% and the average RSDs were 3.2 to 8%. The established method was used to analyze PAs in 22 types of Chinese wild honey, and the predominant PAs found in these honey samples were intermedine and lycopsamine. Conclusion A high-throughput method for the determination of isomeric PAs in honey was developed and validated. Five of the 22 types of Chinese wild honey were contaminated with PAs concentrations of 2.2-207.0 mu g/kg.