An Improved Technique for Extraction, Identification, and Quantification of Leukotrienes

Abstract

An improved method for extraction, separation and quantification of leukotrienes is described. Critical steps include extraction in thoroughly cleaned Sep-Pak C₁₈ mini columns, elution with 90 percent methanol in water, addition of 0.25 percent Na₄EDTA to the solvent system of methanol/water 60/40, pH 7.4, injection of the samples in 1 ml of the same system after lowering the pH to 3.0 with glacial acetic acid and utilization of a Z-module containing a 5 μm C₁₈ reverse phase cartridge. Recovery of the leukotrienes was 94 pm 8% mean pm SD for LTC₄, 89 pm 5% for LTD₄, 89 pm 3% for LTB₄ and 84 pm 6% for LTE₄. We had no problems with precipitation of Na₄EDTA and occlusion of the HPLC fittings. The method is simple, reproducible and easily adaptable to any research laboratory.