Yu W, Powell W S
Department of Medicine, Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada.
Anal Biochem. 1995 Apr 10;226(2):241-51. doi: 10.1006/abio.1995.1221.
Arachidonic acid can be converted to a large number of metabolites by various lipoxygenases, cyclooxygenase, and other enzymes. Because of the complex profiles of products formed by many types of cells, high-pressure liquid chromatography (HPLC) has proved to be an invaluable technique for their purification and analysis. In the present study we have developed improved methods for the analysis of complex mixtures of eicosanoids by HPLC using binary gradients containing trifluoroacetic acid (TFA), which allows considerable manipulation of the retention times of the cysteine-containing leukotrienes (LTs) LTC4, LTD4, and LTE4 relative to those of other eicosanoids. With a gradient between 0.003 and 0.005% TFA and a 4.6-mm-i.d. column of Spherisorb ODS-2, cysteinyl-LTs are very well resolved from one another and are separated as a group with retention times longer than those of all other major eicosanoids. These conditions can be used for the analysis of prostaglandin B2 (PGB2), LTB4, monohydroxyeicosatetraenoic acids (HETEs), and cysteinyl-LTs in only 30 min. Slightly longer analysis times must be used for the separation of more polar eicosanoids such as hydroxy metabolites of LTB4 and lipoxins. We have also developed methods for the analysis of eicosanoids using a midbore (3.2 mm i.d.) column containing Spherisorb ODS-2, which improves sensitivity and reduces solvent consumption. In this case higher concentrations (0.04 to 0.05%) of TFA have been used, resulting in retention times for cysteinyl-LTs between those of the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid and the HETEs. This approach permits analysis of PGB2, LTB4, HETEs, and cysteinyl-LTs in only 20 min. Samples which also contain hydroxy-LTB4 and lipoxins can be analyzed in 40 min. The above techniques are highly reproducible and give baselines which are free of interfering peaks.
花生四烯酸可通过多种脂氧合酶、环氧化酶及其他酶转化为大量代谢产物。由于多种细胞形成的产物谱复杂，高压液相色谱法（HPLC）已被证明是用于其纯化和分析的一项极有价值的技术。在本研究中，我们开发了改进方法，通过使用含三氟乙酸（TFA）的二元梯度的HPLC分析类二十烷酸的复杂混合物，这使得含半胱氨酸的白三烯（LTs）LTC4、LTD4和LTE4相对于其他类二十烷酸的保留时间能够得到相当大的调控。在0.003%至0.005% TFA的梯度以及内径4.6毫米的Spherisorb ODS - 2柱条件下，半胱氨酰白三烯彼此间能很好地分离，并且作为一组与所有其他主要类二十烷酸相比，保留时间更长。这些条件可用于仅在30分钟内分析前列腺素B2（PGB2）、LTB4、单羟基二十碳四烯酸（HETEs）和半胱氨酰白三烯。对于分离极性更强的类二十烷酸，如LTB4的羟基代谢产物和脂氧素，则必须使用稍长的分析时间。我们还开发了使用含Spherisorb ODS - 2的中孔径（内径3.2毫米）柱分析类二十烷酸的方法，这提高了灵敏度并减少了溶剂消耗。在这种情况下，使用了更高浓度（0.04%至0.05%）的TFA，导致半胱氨酰白三烯的保留时间介于环氧化酶产物12 - 羟基 - 5,8,10 - 十七碳三烯酸和HETEs之间。这种方法仅需20分钟就能分析PGB2、LTB4、HETEs和半胱氨酰白三烯。同时含有羟基 - LTB4和脂氧素的样品可在40分钟内分析。上述技术具有高度的可重复性，并且给出的基线没有干扰峰。
