Targeted and untargeted alkaloids characterisation of pasture herbs in eastern Italian Alps using high resolution mass spectrometry

Alkaloids, widely present in herbs, have attracted the attention of phytochemists for over 150 years due to their potential value as medicinal agents or their toxic principles. In this work we developed a method for targeted and untargeted alkaloid investigation that automatically combines SPE-on line sample purification with UHPLC/ESI/High Resolution Mass Spectrometry (Q-Orbitap) detection. Thirty-five alkaloids were quantified in comparison with analytical standards, 48 were putatively identified and confirmed using the chromatographic retention time and the fragmentation profile obtained analysing the extracts of plants already well-documented in the literature, and further 200 using an in-house database built from literature information on exact mass and isotopic pattern. Besides, the alkaloid profile of 67 single herbage plants, characteristic of the local alpine flora, and that of 48 herbal mix samples representative of the exact daily intake of 8 cows grazing in three consecutive days on two distinct alpine pastures in north-eastern Italy, was described. Moreover, also the corresponding 48 milks produced by those cows were analysed. Among the quantified alkaloids, the most abundant were Lycopsamine and Gramine (42 and 31% of pasture samples, and 52 and 44% of milks, respectively; contents from 0.4 to 80 µg kg-1 in herb, and 0.04-0.4 µg L-1 in milk). The untargeted analysis found over 120 different alkaloids in herbal mixes and roughly 40 in milks.