A sensitive LC–MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow’s milk and urine and application to farm milk samples from Germany

Abstract

Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow’s milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89–106% and 85–104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites. Graphical Abstract

Detection of Hypoglycin A and MCPrG Metabolites in the Milk and Urine of Pasture Dairy Cows after Intake of Sycamore Seedlings

“…Toxin Analysis in Milk. Milk samples were analyzed according to El-Khatib et al 37 Briefly, 10 mL of milk samples was mixed with 10 mL of 1% formic acid in methanol (v/v). Additionally, 100 μL of formic acid and 1 mL of EDTA solution were added.…”

“…Urine Sampling and Toxin Analysis in Urine. Urine samples were obtained from all 5 cows on the basis of routine veterinary diagnostics upon spontaneous micturition on day 3 at 4.30 pm and afterward analyzed for their levels of HGA, MCPrG, and their respective metabolites by applying the method of El-Khatib et al 37 Briefly, the urinary creatinine concentration was measured at an accredited medical analytics laboratory (Labor 28 GmbH, Berlin, Germany). The urine samples were subsequently diluted with 5% MeOH to a creatinine concentration of 0.1 mg/dL and then analyzed by LC−MS/MS.…”