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  • Title: Quantitation of leukotriene B4 in human serum by negative ion gas chromatography-mass spectrometry.
    Author: Hughes H, Mitchell JR, Gaskell SJ.
    Journal: Anal Biochem; 1989 Jun; 179(2):304-8. PubMed ID: 2549806.
    Abstract:
    Leukotriene B4 (LTB4) is a potent chemotactic agent formed via the 5-lipoxygenase pathway from arachidonic acid. To understand the role LTB4 plays in several pathological processes it is essential that endogenous concentrations of LTB4 be accurately quantitated. We have developed a method based on electron capture negative ion mass spectrometry for the analysis of LTB4 in serum at low picogram per milliliter concentrations. Blood is collected into the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to suppress ex vivo formation. Serum is isolated, equilibrated with the internal standard [2H4]LTB4, and extracted using octadecyl-silica (C-18) cartridges. After conversion of the carboxylic acids to their pentafluorobenzyl esters the extract is purified by straight-phase HPLC. Gas chromatographic-mass spectrometric analysis is accomplished on the tert-butyldimethylsilyl ether derivatives using dual-selected ion monitoring of m/z 431 and 435. These ions correspond to loss of tert-butyldimethylsilanol from the (M-PFB)- ion of endogenous and [2H4]LTB4, respectively. The concentration of LTB4 in human serum samples was 10.0 +/- 4.0 pg/ml (n = 5). The assay exhibited satisfactory precision, with an intraassay coefficient of variation of 17% and a high degree of accuracy. The concentration of LTB4 in serum collected with (NDGA) was less than 10% of that observed in blood collected without the lipoxygenase inhibitor. Ex vivo formation can therefore be a major obstacle in assessing circulating levels of LTB4.
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