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A more recent version of this article appeared on April 1, 2008

Papers In Press, published online ahead of print January 9, 2008
J. Lipid Res., doi:10.1194/jlr.D700030-JLR200
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Submitted on September 10, 2007
Revised on November 7, 2007
Accepted on January 8, 2008

An improved procedure for brain prostanoid analysis using liquid-liquid extraction combined with LC/MS/MS and requirement for brain fixation using head-focused microwave irradiation

Mikhail Y. Golovko and Eric J. Murphy

Pharmacology, Physiology, and Therapeutics, University of North Dakota, Grand Forks, ND 58202-9037

Corresponding Author: emurphy{at}medicine.nodak.edu

LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC/MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80oC was reduced 2- to 4- fold in 4 weeks, however PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was significantly elevated when mice were euthanized by decapitation and the brains rapidly flash frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass, under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.


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S. E. Farias, M. Basselin, L. Chang, K. A. Heidenreich, S. I. Rapoport, and R. C. Murphy
Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain
J. Lipid Res., September 1, 2008; 49(9): 1990 - 2000.
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