Salting-out Assisted Liquid-Liquid Extraction for the rapid and simple simultaneous analysis of pyrrolizidine alkaloids and related N-oxides in honey and pollen

The application of salting out assisted liquid-liquid extraction for the rapid and simple simultaneous determination of pyrrolizidine alkaloids and related N-oxide in honey and pollen was evaluated. The determination of PAs and PANOs was obtained by ultra high performance liquid chromatography coupled with tandem mass spectrometry. The critical parameters (pH of the aqueous solution, extraction solvent, type and amount of salting-out agents) affecting SALLE performance were carefully studied and optimized using a chemometric approach. Under the optimal experimental conditions, the developed procedure showed excellent extraction efficiency (82–104 %) and recoveries (82–113 %) for all target analytes, except for Intermedine N-oxide, at three concentration levels (0.25, 2.5 and 25 μg kg−1 and 2.5, 25 and 250 μg kg-1 for honey and pollen samples, respectively), and satisfactory intra-day (<10) and inter-day (<13) precisions. The proposed method showed negligible matrix effect for both studied matrices and method quantification limits at very low levels (0.1−0.2 and 1−2 μg kg-1 for honey and pollen, respectively). The method was successfully applied to the analysis of honey (n = 71) and pollen (n = 6) samples. The 97 % and the 67 % of analyzed honey and pollen samples, respectively, turned out to be contaminated by at least one PA. Echimidine, echimidine N-oxide, intermedine and intermedine N-oxide significantly contributed to the contamination of the samples.