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.

Keywords: Methylenecyclopropylacetyl-carnitine; Methylenecyclopropylacetyl-glycine; Methylenecyclopropylformyl-glycine; Sycamore maple (Acer pseudoplatanus).

Fig. 1

Chemical structures of the toxins and their metabolites investigated in this study

Fig. 2

Extraction of HGA, MCPrG and their metabolites from raw milk

Fig. 3

Overlaid MRM extracted ion chromatograms of HGA, MCPrG, MCPA-glycine, MCPF-glycine, and MCPA-carnitine in spiked cow’s raw milk

Fig. 4

Overlaid MRM extracted ion chromatograms of HGA, MCPrG, MCPA-glycine, MCPF-glycine, and MCPA-carnitine in spiked cow’s urine

Fig. 5

Long-term stability of HGA (upper panel) and MCPrG (lower panel) in spiked milk samples stored at − 20 °C. QC samples at 5 and 50 µg/L have been routinely analyzed. The data are presented as the % ratio ± standard deviation of the calculated concentrations of the stored QC samples as compared to those obtained with freshly prepared ones (reference value at day 0)