A validated liquid chromatography-mass spectrometry method for the determination of leukotrienes B4 and B5 produced by stimulated human polymorphonuclear leukocytes.
A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, 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 B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 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 B4 and B5 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.
Panchaud
A
A
Unité de Nutrition Clinique, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Avois
L
L
Roulet
M
M
Pilet
M
M
Hug
C
C
Saugy
M
M
Decosterd
L A
LA
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D015118:Eicosapentaenoic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry; D006801:Humans; D007975:Leukotriene B4 / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods / Q000592:standards; D013696:Temperature; D013816:Thermodynamics
D007975:Leukotriene B4; C040212:leukotriene B5; D015118:Eicosapentaenoic Acid