A validated liquid chromatography–mass spectrometry method for the determination of leukotrienes B 4 and B 5 produced by stimulated human polymorphonuclear leukocytes

Abstract

A validated high-performance liquid chromatography (HPLC)–mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B 4 and B 5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization–single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M − H] − and m/z 335.2 for prostaglandin B 2/LTB 5 and LTB 4, respectively. The calibration curves for LTB 4 and LTB 5 were linear over the ranges 165–990 and 0.825–13.2 ng/ml, respectively. The lower limit of quantification for LTB 5 was 0.66 ng/ml. The mean absolute recoveries for LTB 4 and LTB 5 were 81 ± 4.8% and 82 ± 5.9%, respectively. The method is precise with mean interday CVs for LTB 4 and LTB 5 within 7.1–10.7, and 3.8–9.4%, respectively, and accurate (range of interday deviations for LTB 4 and LTB 5 were −7.8 to 1, and −5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B 4 and B 5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.