Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay
Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay
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DOI：
10.1194/jlr.d059725
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发表时间：
2015-07-01
影响因子：
6.5
通讯作者：
Lagrost, Laurent
中科院分区：
文献类型：
--
作者：
de Barros, Jean-Paul Pais;Gautier, Thomas;Lagrost, Laurent
血浆脂多糖(LPS)的定量可以用来证明革兰氏阴性细菌感染。本工作用有机溶剂从血浆样品中提取内毒素衍生的3-羟基肉豆蔻酸盐，用反相高效液相色谱分离，并用MS/MS进行定量，该质量法与凝胶生物测定法(LAL)相结合以监测生物样品中内毒素活性的中和。该方法是一种可靠、实用、准确、灵敏的内毒素定量检测方法。LAL和LC/MS/MS分析的结合为血浆脂蛋白和磷脂转运蛋白具有中和LPS活性的内在能力提供了新的证据。在一组全身性炎症反应综合征患者中，有记录的感染但血浆LAL试验阴性的患者，用高效液相色谱/质谱仪/质谱法测定了显著含量的内毒素。血浆内毒素浓度最高的患者病情更严重。HPLC/MS/MS是定量检测样品中的内毒素、评价内毒素中和效果、评价体内内毒素促炎作用的一种相关方法。
Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.