Document Detail

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.
MedLine Citation:
PMID:  22909891     Owner:  NLM     Status:  MEDLINE    
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 B(4), 6-trans-leukotriene B(4), 6-trans-12-epi-leukotriene B(4), 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 16.6mM 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 Ca(2+)-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.
Hans Abromeit; Anja M Schaible; Oliver Werz; Gerhard K E Scriba
Publication Detail:
Type:  Evaluation Studies; Journal Article     Date:  2012-08-04
Journal Detail:
Title:  Journal of chromatography. A     Volume:  1267     ISSN:  1873-3778     ISO Abbreviation:  J Chromatogr A     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-16     Completed Date:  2013-04-22     Revised Date:  2013-07-07    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  217-23     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier B.V. All rights reserved.
Department of Pharmaceutical/Medical Chemistry, Friedrich-Schiller-Universität, Jena, Germany.
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MeSH Terms
Arachidonate 5-Lipoxygenase / chemistry*,  metabolism
Cells, Cultured
Chromatography, Micellar Electrokinetic Capillary / instrumentation,  methods*
Cyclodextrins / administration & dosage,  chemistry
Leukocytes / chemistry,  enzymology*
Reg. No./Substance:
0/Cyclodextrins; EC 5-Lipoxygenase

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