Wheelan P, Murphy R C
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Anal Biochem. 1997 Jan 1;244(1):110-5. doi: 10.1006/abio.1996.9885.
A reverse-phase-HPLC/tandem mass spectrometric method (LC/MS/MS) was developed for the quantitation of leukotriene B4 (LTB4), the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), as well as the omega-oxidation metabolites of LTB4, 20-hydroxy-LTB4, and 20-carboxy-LTB4. Electrospray-generated carboxylate anions were collisionally activated and decomposed to specific and abundant product ions and multiple reaction monitoring was used to analyze LTB4 (m/z 335-->195), 20-hydroxy-LTB4 (m/z 351-->195), 20-carboxy-LTB4 (m/z 365-->195), and 5-HETE (m/z 319-->115). A linear correlation was observed in comparison of results obtained from quantitation by LC/MS/MS with quantitation by gas chromatography/MS of the pentafluorobenzyl ester/trimethylsilyl ether derivative of LTB4 produced during opsonized zymosan stimulation of human neutrophils. Detection limits at the low picogram level were obtained for all metabolites. This method was applied to the quantitation of 5-lipoxygenase products produced from zymosan-stimulated human neutrophils in the presence of ethanol. At physiologically relevant concentrations of ethanol, production of all 5-lipoxygenase products generated by stimulated neutrophils was markedly attenuated.
建立了一种反相高效液相色谱/串联质谱法（LC/MS/MS），用于定量测定白三烯B4（LTB4）、5-脂氧合酶产物5-羟基二十碳四烯酸（5-HETE）以及LTB4的ω-氧化代谢产物20-羟基-LTB4和20-羧基-LTB4。电喷雾产生的羧酸根阴离子经碰撞激活并分解为特定且丰富的产物离子，采用多反应监测来分析LTB4（m/z 335→195）、20-羟基-LTB4（m/z 351→195）、20-羧基-LTB4（m/z 365→195）和5-HETE（m/z 319→115）。在比较LC/MS/MS定量结果与气相色谱/MS定量结果时观察到线性相关性，气相色谱/MS定量的是在调理酵母聚糖刺激人中性粒细胞过程中产生的LTB4的五氟苄基酯/三甲基硅醚衍生物。所有代谢产物的检测限均达到低皮克水平。该方法用于定量测定在乙醇存在下酵母聚糖刺激的人中性粒细胞产生的5-脂氧合酶产物。在生理相关浓度的乙醇条件下，刺激的中性粒细胞产生的所有5-脂氧合酶产物的生成均明显减弱。
