2.3. Hypoglycin A Assay

HGA in serum was performed according to a previously described protocol [17]. Quantification of HGA was carried out using a TRAQ^® kit for amino acid analysis of physiological fluids (Sciex, Farmingham, MA, USA). Samples’ HGA was derivatized using an isotopic tag (mass m/z 121), while a second labeling reagent (mass m/z 113) allowed absolute quantification. Derivatized samples were introduced into a TQ5500 tandem mass spectrometer (Sciex, Farmingham, MA, USA) using a Prominence AR HPLC system (Shimadzu, Kyoto, Japan). This method presented a lower limit of quantification of 0.090 μmol/L [17].

To improve method sensitivity on milk’s sample, milk’s HGA was assessed using a slightly modified protocol. Briefly, we increased the initial sample volume (40 > 80 µL) and the injection volume into the analytical system (10 > 25 µL).