INRA, UMRH1213 Herbivores, F-63122 Saint-Genès-Champanelle, France.
J Chromatogr A. 2012 Sep 21;1256:169-76. doi: 10.1016/j.chroma.2012.07.094. Epub 2012 Aug 4.
Microbial protein synthesis and nitrogen balance status in ruminants can be evaluated by the presence of metabolites in urine. This work aims to develop and validate a simple and sensitive method for simultaneous determination of nine markers of nitrogen status in ruminant's urine using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS system, operated in multiple reaction monitoring (MRM) in positive mode, was used for determining selected purine (allantoin, uric acid, xanthine, and hypoxanthine) and pyrimidine derivatives (β-aminobutyric acid and β-alanine), which are used to estimate rumen microbial protein synthesis - the main source of protein for ruminants. Creatinine, creatine and urea, three other metabolites involved in nitrogen metabolism were also measured by this method. The procedure was based on a simple dilution of urine samples in acetonitrile, followed by LC-ESI-MS/MS analysis. Chromatographic separation was tested with three different columns. A zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) column provided an optimal separation for all metabolites. Precision of the method was typically below 10%, and accuracy was above 90% with the exceptions of allantoin and urea at pH 6 and 3, and β-alanine at pH 3. Metabolites were stable after 3 months of storage at -20°C, except for xanthine, hypoxanthine and β-aminobutyric acid that lost up to 48, 50 and 39% of initial concentration after only 1month of storage in acidified urine. This LC-ESI-MS/MS method is more specific, unequivocally detecting target metabolites at lower detection limits than methods using UV detection. The method was suitable for the determination of all metabolites tested. The developed method was subsequently used to compare total and spot urine sampling obtained from dairy cows fed diets with contrasting levels of protein.
利用尿液中的代谢物可以评估反刍动物的微生物蛋白质合成和氮平衡状态。本研究旨在开发并验证一种简单、灵敏的方法，采用亲水相互作用液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）同时测定反刍动物尿液中 9 种氮状态标志物。该 LC-ESI-MS/MS 系统在正模式下采用多重反应监测（MRM）进行操作，用于测定所选嘌呤（尿囊素、尿酸、黄嘌呤和次黄嘌呤）和嘧啶衍生物（β-氨基丁酸和β-丙氨酸），这些物质可用于估计瘤胃微生物蛋白质合成-反刍动物的主要蛋白质来源。肌酐、肌酸和尿素这三种其他参与氮代谢的代谢物也可通过该方法进行测量。该方法的步骤基于尿液样品在乙腈中的简单稀释，然后进行 LC-ESI-MS/MS 分析。通过三种不同的色谱柱对色谱分离进行了测试。两性离子亲水作用液相色谱（ZIC-HILIC）柱为所有代谢物提供了最佳的分离效果。该方法的精密度通常低于 10%，准确度均高于 90%，除了 pH 值为 6 和 3 时的尿囊素和尿素，以及 pH 值为 3 时的β-丙氨酸。代谢物在-20°C 下储存 3 个月后稳定，除了黄嘌呤、次黄嘌呤和β-氨基丁酸在酸化尿液中仅储存 1 个月后分别损失了初始浓度的 48%、50%和 39%。与使用紫外检测的方法相比，这种 LC-ESI-MS/MS 方法具有更高的特异性，能够在更低的检测限下明确地检测到目标代谢物。该方法适用于所有测试代谢物的测定。随后，该方法被用于比较采食不同蛋白水平日粮的奶牛的总尿液和点尿液采样。
