Chemometrics-guided development of a cyclodextrin-modified micellar electrokinetic chromatography method with head-column field amplified sample stacking for the analysis of 5-lipoxygenase metabolites

A new sensitive method using α-cyclodextrin-modified micellar electrokinetic chromatography has been developed to separate and quantify arachidonic acid metabolites of the lipoxygenase pathways in human polymorphonuclear leukocytes, i.e. leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, and 15(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid. The electrophoresis system was optimized with regard to the pH, boric acid, SDS and α-cyclodextrin concentration as well as separation voltage and temperature using a three level resolution IV fractional factorial design and a five level circumscribed central composite design. The resulting optimized conditions included 80 mM sodium borate buffer, pH 10.07, containing 16.6 mM sodium dodecyl sulfate, and 15 mM α-cyclodextrin, using a separation voltage of 12.5 kV at 23 °C. Sensitivity was enhanced employing head-column field amplified sample stacking which resulted in limits of quantification between 30 and 50 ng/mL and limits of detection between 10 and 17 ng/mL after solid phase extraction of the lipoxygenase products. The method was validated according to the recommendations of the International Conference on Harmonization and applied to the determination of the lipoxygenase metabolites in polymorphonuclear leukocytes upon stimulation with Ca2+-ionophore A23187 and arachidonic acid. Robustness was confirmed using a three level resolution IV fractional factorial design. The novel method is suitable for the analysis of various arachidonic acid metabolites produced by cells and may be used for evaluation of lipoxygenase inhibitors.

► A new MEKC method allowing the separation of leukotriene diastereomers was developed.
► Chemometrics-guided method development and robustness testing was applied.
► Low LOQs were achieved by using head-column field enhanced sample stacking.
► The application to the analysis of leukotrienes and HETEs in PNML cells is described.