Examining Alkaloid Profiles in Milk with LC-MS

A recent study conducted by The Technology Transfer Center of the Edmund Mach Foundation (San Michele all'Adige, Italy) and the University of Udine (Udine, Italy) investigated alkaloid (alk) transfer from alpine pastures to milk, with a particular focusing particularly on identification of profiles in milk samples from 16 cows grazing in two distinct alpine regions. This study aimed to evaluate the influence of different grazing environments on the alk content in the milk, to explore the potential toxicity risks, and to examine the use of these profiles as identifying the geographical origin and product authenticity of the milk. Liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) was used in the analysis of both herbage and milk samples, and 41 alkaloids with pure standards were identified, and putatively identifying another 116. A paper based on this study was published in the Journal of Food Science (1).

A varied group of naturally occurring chemical compounds yielding major pharmacological properties, alks have garnered significant attention in recent years due to their presence in a variety of food products, including dairy products. The transfer of alks into dairy products raises concerns regarding the safety of food and potential health implications that might result (1). The investigation of alks spotlight specific toxic varieties, notably pyrrolizidines (Pyzs), tropanes (Trps), or quinolizidines (Qnzs) (2-4); the presence of these varieties in milk is mainly derived from contaminated animal feed products that have been contaminated in some way. When ingested, these alks are metabolized in the body when ingested to create toxic metabolites, which may damage liver cells and ultimately lead to cirrhosis, liver failure, or even death in extreme cases (5,6).

For this study, a group of 16 lactating cows was chosen from a herd of 110 Italian Simmental dairy cows grazing in two different pastures (with eight selected from each pasture). The vegetation selected by each cow was manually sampled by using a hand-plucking technique which copied animal intake (7). Profile evaluation of alks was performed on 48 vegetation samples (N = 48), on the unpasteurized milk samples (48 individual ones and 12 masses), and on the mixed concentrate used for the cattle’s feed supplement (1).

Through chromatographic analysis, the researchers determined that a wide, complex range of alks are synthesized by alpine plants and become part of the cow's intake. The research provided valuable evidence of the varied alk compositions in different pasture herbs despite the difficulty resulting from the limited availability of pure commercial standards. While the confirmed alk content in the milk samples was found to be significantly lower than the potential found in plants from mountain pastures, the researchers believed that the results were still intriguing (1).

Despite limitations, such as a small sample size and short study duration, the research points to the potential of using alks as markers for milk origin, offering a tool for fraud prevention and product transparency. The authors of the study state that further investigation is needed to confirm these findings (1).

References

(1) Nardin, T.; Piasentier, E.; Romanzin, A.; Martinelli, F.; Larcher, R. Alkaloid Profile of Italian Alpine Milk. J. Food Sci. 2025, 90 (2), e70027. DOI: 10.1111/1750-3841.70027

(2) Engel, A. M.; Klevenhusen, F.; Moenning, J. L.; Numata, J.; Fischer-Tenhagen, C.; Sachse, B.; Schäfer, B.; Fry, H.; Kappenstein, O.; Pieper, R. Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows. J. Agric. Food Chem. 2022, 70 (37), 11749-11758. DOI: 10.1021/acs.jafc.2c02517

(3) Klein, L. M.; Gabler, A. M.; Rychlik, M.; Gottschalk, C.; Kaltner F. A Sensitive LC-MS/MS Method for Isomer Separation and Quantitative Determination of 51 Pyrrolizidine Alkaloids and Two Tropane Alkaloids in Cow's Milk. Anal. Bioanal, Chem. 2022, 414 (28), 8107-8124. DOI: 10.1007/s00216-022-04344-5

(4) Lamp, J.; Knappstein, K.; Walte, H. G.; Krause, T.; Steinberg, P.; Schwake-Anduschus, C. Transfer of Tropane Alkaloids (Atropine and Scopolamine) into the Milk of Subclinically Exposed Dairy Cows. Food Control 2021, 126, 108056. DOI: 10.1016/j.foodcont.2021.108056

(5) Kohnen-Johannsen K. L.; Kayser O. Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production. Molecules 2019, 24 (4), 796. DOI: 10.3390/molecules24040796

(6) Wang, Z.; Han, H.; Wang, C.; Zheng, Q.; Chen, H.; Zhang, X.; Hou R. Hepatotoxicity of Pyrrolizidine Alkaloid Compound Intermedine: Comparison with Other Pyrrolizidine Alkaloids and Its Toxicological Mechanism. Toxins (Basel) 2021, 13 (12), 849. DOI: DOI: 10.3390/toxins13120849

(7) Berry, N. R.; Jewell, P. L.; Sutter, F.; Edwards, P. J.; Kreuzer, M. Selection, Intake and Excretion of Nutrients by Scottish Highland Suckler Beef Cows and Calves, and Brown Swiss Dairy Cows in Contrasting Alpine Grazing Systems. J. Agric. Sci. 2002, 139 (4), 437-453. DOI: 10.1017/S002185960200271X