Mishra Rupesh K, Robert-Peillard Fabien, Ravier Sylvain, Coulomb Bruno, Boudenne Jean-Luc
Aix Marseille Université, Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie de l'Environnement (FRE 3416), 13331 Marseille, France.
Aix Marseille Université, Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie de l'Environnement (FRE 3416), 13331 Marseille, France.
Anal Biochem. 2015 Feb 1;470:71-7. doi: 10.1016/j.ab.2014.10.013. Epub 2014 Oct 31.
An analytical chemical method has been developed for determination of β-hydroxymyristic acid (β-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the β-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ). A high-performance liquid chromatographic separation of the β-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of β-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5μM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25M) at 60°C for 2h. After hydrolysis, free acid was detected as its NBD-PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for β-HMA in dialysis water.
已开发出一种分析化学方法，用于测定透析水中脂多糖（LPS/内毒素）的一种成分β-羟基肉豆蔻酸（β-HMA）。在我们的研究中，β-HMA成分被用作透析水中内毒素存在的化学标志物，因为它存在于分子亚基（脂质A）中且具有毒性。它是该亚基中含量最丰富的饱和脂肪酸。所开发的方法基于用4-硝基-7-哌嗪基-2,1,3-苯并恶二唑（NBD-PZ）进行荧光衍生化。使用十八烷基硅胶柱在梯度洗脱条件下实现了β-HMA衍生物的高效液相色谱分离。测试了β-HMA的宽动态范围，并构建了校准曲线，其准确度为90%，变异性小于10%。获得的检测限和定量限分别为2μM和5μM。所开发的方法应用于通过在60°C下用0.25M NaOH对LPS进行碱性水解来检测透析水中的内毒素。水解后，使用高效液相色谱/质谱（HPLC/MS）将游离酸检测为其NBD-PZ衍生物。透析水中β-HMA的回收率良好，范围为98%至105%。
