J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D700030-JLR200 on January 9, 2008

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Journal of Lipid Research, Vol. 49, 893-902, April 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

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

Mikhail Y. Golovko* and Eric J. Murphy1,*,{dagger}

* Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, Grand Forks, ND 58202-9037
{dagger} Department of Chemistry, University of North Dakota, Grand Forks, ND 58202-9037

Published, JLR Papers in Press, January 9, 2008.

1 To whom correspondence should be addressed. e-mail: emurphy{at}medicine.nodak.edu

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows 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 –80°C 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 increased significantly 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.

Supplementary key words prostaglandin • isoprostane • high-performance liquid chromatography-tandem mass spectrometry

Abbreviations: BHT, butylated hydroxytoluene; LC-MS/MS, high-performance liquid chromatography with tandem mass spectrometry detection; PG, prostanoid; PGD2, 9{alpha},15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid; PGD2-d4, 9{alpha},15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic-3,3,4,4-2H4 acid; PGE2, 9-oxo-11{alpha},15S-dihydroxyprosta-5Z,13E-dien-1-oic acid; 11β-PGE2, 9-oxo-11β,15S-dihydroxyprosta-5Z,13E-dien-1-oic acid; 8-isoPGE2, 9-oxo-11{alpha},15R-dihydroxyprosta-5Z,13E-dien-1-oic acid; PGE2-d4, 9-oxo-11{alpha},15S-dihydroxyprosta-5Z,13E-dien-1-oic-3,3,4,4-2H4 acid; PGF2{alpha}, 9{alpha},11{alpha},15S-trihydroxyprosta-5Z,13E-dien-1-oic acid; 11β-PGF2{alpha}, 9{alpha},11β,15S-trihydroxyprosta-5Z,13E-dien-1-oic acid; 8-isoPGF2{alpha}, 9{alpha},11{alpha},15S-trihydroxy-(8β)-prosta-5Z,13E-dien-1-oic acid; PGF2{alpha}-d4, 9{alpha},11{alpha},15S-trihydroxyprosta-5Z,13E-dien-1-oic-3,3,4,4-2H4 acid; 6-oxo-PGF1{alpha}, 6-oxo-9{alpha},11{alpha},15S-trihydroxy-prosta-13E-en-1-oic acid; 6-oxo-PGF1{alpha}-d4, 6-oxo-9{alpha},11{alpha},15S-trihydroxy-prosta-13E-en-1-oic-3,3,4,4-2H4 acid; TXB2, 9{alpha},11,15S-trihydroxythromba-5Z,13E-dien-1-oic acid; TXB2-d4, 9{alpha},11,15S-trihydroxythromba-5Z,13E-dien-1-oic-3,3,4,4-2H4 acid


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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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