RP-HPLC法测定白细胞中LTB_4和5-HETE的含量

Determination of LTB_4 and 5-HETE in Leukocytes by RP-HPLC

目的:建立了5-脂氧酶抑制剂筛选模型的RP-HPLC测定方法。方法:采用Brava BDSC_(18)柱(250mm×4.6mm,5μm),流动相为甲醇-0.02%乙酸水溶液,流速为0.8 mL·min^(-1),5-LOX的主要产物LTB_4和5-HETE的检测波长分别为275nm和235nm。结果:LTB_4和5-HETE分别在2.0～50.0μg·mL^(-1)(r=0.9999)和5.0～125.0μg·mL^(-1)(r=1.0000)范围内呈良好的线性关系;回收率分别为100.0%和97.7%(RSD分别为1.1%和1.5%);日内、日间精密度的RSD分别为0.7%,1.1%和0.3%,1.3%。结论:该法分离效果和重复性均良好,且结果准确可靠,可用于LTB_4和5-HETE的检测,为筛选新的具有抗炎活性的中药成分奠定了基础。

This study aimed to develop an efficient RP-HPLC method for determination of 5-1ipoxygenase （5-LOX） inhibitory activity. The content of LTB4 and 5-HETE, the main products of the 5-LOX metabolism pathway of arachidonic acid, was determined by the RP-HPLC method. The RP-HPLC was performed on a Brava BDS C18 column （250mm×4.6mm, 5μm） using a mobile phase consisting of 0.02% acetic acid solution-methanol at 30 ℃ with the flow rate of 0.8mL·min^-1 and detection wavelength at 235nm and 275nm. As a result, the calibration curve was hnear in the range of 2.0-50.0 μg·mL^-1 （r = 0.9999） for determination of LTB4, and 5.0-125.0μg·mL^-1 （r=1.0000） for determination of 5-HETE. The average recoveries of LTB4 and 5-HETE were 100.0% （RSD=1.1%） and 97.7% （RSD=1.5%）, respectively. The intra-day and inter-day RSDs were 0.7%, 1.1% and 0.3%, 1.3%. Being sensitive, accurate and reliable with good reproducibility, this method is available for the screening of new anti-inflammatory active components of Chinese medicine.