Measurements of leukotrienes in human plasma by solid phase extraction and high performance liquid chromatography

Leukotrienes (LTs) are biologically active compounds derived from lipoxygenase catalyzed metabolism of arachidonic acid in mammalian tissues. The present report describes a simple method for extraction and isolation of dihydroxy-LTs; LTB4, LTB5 and the peptido-LTs; LTC4, LTD4 and LTE4 from human plasma, using a pretreatment cartridge which utilizes both hydrophobic and ion-exchange interactions. 5 ml acidified plasma or acetate buffer containing commercially available LT standards were passed through the cartridges under suction, and the absorbed LTs were subsequently eluted in a stepwise manner with acetate buffer containing increasing amounts of methanol. The eluted LTs were analyzed by reversed-phase high performance liquid chromatography (HPLC) on octadecylsilyl (ODS)-silica, using a Waters HPLC unit. Both with plasma and acetate buffer the present methodology resulted in good separation of the LTs with a total run-time of less than 32 min. Recovery of dihydroxy-LTs was approximately 80% (range 73-82%) both when the standards were dissolved in plasma and in acetate buffer. Recovery of the peptido-LTs was, however somewhat lower (47-50%). It should be noted that the present method has the advantage that exposure to chemicals of high toxicity is avoided.