草甘膦是农业中使用最多的除草剂。它的主要代谢产物是AMPA(氨甲基膦酸),但N-乙酰-AMPA和N-乙酰草甘膦也是感兴趣的代谢产物。对于风险评估,提出了一个通用的残基定义,即草甘膦的总和,AMPA,N-乙酰草甘膦和N-乙酰-AMPA,表示为草甘膦。一种确认脂肪中草甘膦的方法,肝脏和肾脏,以及AMPA和N-乙酰草甘膦在所有基质中的确认方法,仍然失踪。在本文中,我们提出了一种定量测定草甘膦残留及其代谢物AMPA的方法,脂肪组织中N-乙酰-AMPA和N-乙酰-草甘膦的液相色谱-质谱(LC-MS/MS)分析,肝脏,鸡蛋,没有衍生的牛奶和蜂蜜。测试了不同的色谱柱,Hypercarb柱提供最好的结果。用在乙腈中的1.2%甲酸和0.5%甲酸的酸化水的流动相洗脱分析物。还通过改变溶剂萃取混合物(水,甲醇和混合物Φ(甲醇,水)=1:1,每个添加1%甲酸(v/v)),在固相萃取(SPE)(聚合阳离子(PCX)和阴离子(PAX))中使用不同的吸附剂,并通过修改萃取程序使用分散固相萃取(dSPE)(C18和PSA)。最后,用1%甲酸水溶液(v/v)从样品中提取分析物。牛奶和脂肪组织通过添加二氯甲烷进行纯化,而肝脏和鸡蛋样品用混合阳离子交换吸附剂进行SPE纯化,并用截止过滤器进行超滤。根据SANTE/11312/2021指南:线性,量化的极限,确定所有矩阵的精度和准确度。定量限(LOQs)范围为0.025至0.2mgkg-1。Precision,表示为相对标准偏差,<20%,虽然准确性,表示为分析回收率,范围从70%到120%。在方法验证期间,所有分析物的测量不确定度估计为<50%.对于所有分析物,实现了根据SANTE文件的良好验证参数。因此,该方法对于极性农药的常规监测具有足够的可靠性和灵敏度。认可方法在常规分析中的应用将提供有助于重新评估动物源性食品风险评估研究的数据。
Glyphosate is the most used herbicide in agriculture. Its major metabolite is
AMPA (aminomethylphosphonic acid), but N-acetyl-
AMPA and N-acetylglyphosate are also metabolites of interest. For risk assessment, a general residue definition was proposed as the sum of glyphosate, AMPA, N-acetyl-glyphosate and N-acetyl-
AMPA, expressed as glyphosate. A confirmatory method for glyphosate in fat, liver and kidneys, as well as a confirmatory method for
AMPA and N-acetyl-glyphosate in all matrices, are still missing. In this paper, we present a method for the quantitative determination of glyphosate residues and its metabolites
AMPA, N-acetyl-
AMPA and N-acetyl-glyphosate by liquid chromatography-mass spectrometry (LC-MS/MS) in adipose tissue, liver, eggs, milk and honey without derivatization. Different chromatographic columns were tested, with the Hypercarb column providing the best results. The analytes were eluted with mobile phases of acidified water with 1.2% formic acid and 0.5% formic acid in acetonitrile. Sample purification procedures were also optimized by varying the solvent extraction mixtures (water, methanol and mixture ψ (methanol, water) = 1:1, each with the addition of 1% formic acid (v/v)), using different sorbents in solid phase extraction (SPE) (polymeric cationic (PCX) and anionic (PAX)) and using dispersive solid phase extraction (dSPE) (C18 and PSA) by modifying the extraction procedures. Finally, the analytes were extracted from the samples with 1% formic acid in water (v/v). Milk and adipose tissue were purified by the addition of dichloromethane, while liver and egg samples were purified by SPE with a mixed cation exchange sorbent and ultrafiltration with cut-off filters. The proposed analytical procedures were validated according to SANTE/11312/2021 guidelines: linearity, limits of quantification, precision and accuracy were determined for all matrices. The limits of quantification (LOQs) ranged from 0.025 to 0.2 mg kg-1. Precision, expressed as relative standard deviation, was <20%, while accuracy, expressed as analytical recovery, ranged from 70% to 120%. During method validation, the measurement uncertainty was estimated to be <50% for all analytes. Good validation parameters according to the SANTE document were achieved for all analytes. Therefore, the method can be considered reliable and sensitive enough for routine monitoring of polar pesticides. The application of the accredited method in routine analysis will provide data that are useful for the re-evaluation of risk assessment studies in foods of animal origin.