Simultaneous quantification of leukotrienes and hydroxyeicosatetraenoic acids in cell culture medium using liquid chromatography/tandem mass spectrometry.

Abstract

Leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) are important bioactive lipid mediators that participate in various pathophysiological processes. To advance understanding of the mechanisms that regulate these mediators in physiological and pathological processes, an analytical method using liquid chromatography/tandem mass spectrometry for the simultaneous quantification of LTB4, LTC4, LTD4, LTE4, 5-HETE, 8-HETE, 12-HETE and 15-HETE in cell culture media was developed. A Supel™-Select HLB solid-phase extraction cartridge was used for sample preparation. The compounds were separated on a C18 column using gradient elution with acetonitrile-water-formic acid (20:80:0.1, v/v/v) and acetonitrile-formic acid (100:0.1, v/v). The calibration curves of LTB4, LTD4, LTE4 and HETEs were linear in the range of 0.025-10 ng/mL, and the calibration curve of LTC4 was linear in the range of 0.25-10 ng/mL. Validation assessment showed that the method was highly reliable with good accuracy and precision. The stability of LTs and HETEs was also investigated. Using the developed method, we measured LTs and HETEs in the culture supernatant of the human mast cell line HMC-1. The present method could facilitate investigations of the mechanisms that regulate the production, release and signaling of LTs and HETEs.