Doran Gregory S, Smith Alistair K, Rothwell Jim T, Edwards Scott H
Charles Sturt University, School of Agricultural and Wine Sciences, Wagga Wagga 2678, NSW, Australia; Graham Centre for Agricultural Innovation.
Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga 2678, NSW, Australia; Graham Centre for Agricultural Innovation.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Feb 15;1076:84-90. doi: 10.1016/j.jchromb.2018.01.018. Epub 2018 Jan 31.
The anaesthetic lidocaine is metabolised quickly to produce a series of metabolites, including several hydroxylated metabolites, which are further metabolised by addition of a glucuronic acid moiety. Analysis of these glucuronide metabolites in urine is performed indirectly by cleaving the glucuronic acid group using β-glucuronidase. However, direct analysis of intact glucuronide conjugates is a more straightforward approach as it negates the need for long hydrolysis incubations, and minimises the oxidation of sensitive hydrolysis products, while also distinguishing between the two forms of hydroxylated metabolites. A method was developed to identify three intact glucuronides of lidocaine in sheep urine using LC-MS/MS, which was further confirmed by the synthesis of glucuronide derivatives of 3OH-MEGX and 4OH-LIDO. Direct analysis of urine allowed the detection of the glucuronide metabolites of hydroxylidocaine (OH-LIDO), hydroxyl-monoethylglycinexylidide (OH-MEGX), and hydroxy-2,6-xylidine (OH-XYL). Analysis of urine before and after β-glucuronidase digestion showed that the efficiency of hydrolysis of these glucuronide metabolites may be underestimated in some studies. Analysis of urine in the current study from three different sheep with similar glucuronide metabolite concentrations resulted in different hydrolysis efficiencies, which may have been a result of different levels of substrate binding by matrix components, preventing enzyme cleavage. The use of direct analysis of intact glucuronides has the benefit of being less influenced by these matrix effects, while also allowing analysis of unstable metabolites like 4OH-XYL, which rapidly oxidises after hydrolysis. Additionally, direct analysis is less expensive and less time consuming, while providing more information about the status of hydroxylated metabolites in urine.
麻醉剂利多卡因迅速代谢产生一系列代谢产物，包括几种羟基化代谢产物，这些产物通过添加葡糖醛酸部分进一步代谢。尿液中这些葡糖醛酸代谢产物的分析是通过使用β-葡糖醛酸酶裂解葡糖醛酸基团间接进行的。然而，直接分析完整的葡糖醛酸缀合物是一种更直接的方法，因为它无需长时间的水解孵育，将敏感水解产物的氧化降至最低，同时还能区分两种羟基化代谢产物的形式。开发了一种方法，使用液相色谱-串联质谱法（LC-MS/MS）鉴定绵羊尿液中利多卡因的三种完整葡糖醛酸，3OH-MEGX和4OH-LIDO的葡糖醛酸衍生物的合成进一步证实了这一点。对尿液的直接分析能够检测到羟基利多卡因（OH-LIDO）、羟基单乙基甘氨酰二甲苯胺（OH-MEGX）和羟基-2,6-二甲苯胺（OH-XYL）的葡糖醛酸代谢产物。β-葡糖醛酸酶消化前后尿液的分析表明，在一些研究中，这些葡糖醛酸代谢产物的水解效率可能被低估。在本研究中，对三种葡糖醛酸代谢产物浓度相似的不同绵羊的尿液进行分析，得到了不同的水解效率，这可能是由于基质成分对底物的结合水平不同，阻止了酶的裂解。使用直接分析完整葡糖醛酸的方法的好处是受这些基质效应的影响较小，同时还能分析像4OH-XYL这样水解后迅速氧化的不稳定代谢产物。此外，直接分析成本更低、耗时更少，同时能提供更多关于尿液中羟基化代谢产物状态的信息。
