Structural modifications of certain pyrrolizidine alkaloids during sample extraction and its impact on analytical results

Pyrrolizidine alkaloids (PAs), a group of plant toxins often contaminating food or feed, are typically extracted from samples using liquid extraction. The crude extracts are then often purified using solid-phase extraction (SPE) cartridges before being analysed by LC-MS/MS. During the development of analytical methods based on strong cation exchange SPE, certain structurally related PAs showed unexpectedly low or significantly increased recoveries, suggesting transformation reactions may be at play. To investigate this hypothesis, sample preparations were conducted using PA-free milk as food matrix, water or organic solvents, into which PA reference standards were spiked before or after critical steps of the protocol. The results revealed a significant decrease in acetylated PA N -oxides to their corresponding deacetylated compounds, as well as the formation of epoxydic PAs from PA compounds containing chlorine and hydroxyl groups in the α position. Evaporation of the alkaline SPE eluates, combined with the use of the protic solvent methanol in cases of deacetylation, was responsible for these phenomena. An alkaline ester hydrolysis mechanism was hypothesised for the deacetylation, while an internal S _N 2 reaction, similar to the chlorohydrin reaction, was suggested for the formation of epoxy PA compounds. Consequently, using different sample preparation methods may inadvertently bias the determined PA patterns.