Liquid Chromatographic–Electrospray Ionization–Mass Spectrometric Analysis of Cytochrome P450 Metabolites of Arachidonic Acid
| Autor: | Blythe B. Holmes, John R. Falck, David R. Harder, Andrew J. Grall, William B. Campbell, Kasem Nithipatikom |
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| Rok vydání: | 2001 |
| Předmět: |
Male
Spectrometry Mass Electrospray Ionization Electrospray Electrospray ionization Biophysics Kidney Mass spectrometry Biochemistry Rats Sprague-Dawley Acetic acid chemistry.chemical_compound Dogs Cytochrome P-450 Enzyme System Liquid chromatography–mass spectrometry Microsomes Animals Selected ion monitoring Molecular Biology Arachidonic Acid Chromatography Myocardium Cell Biology Reference Standards 20-Hydroxyeicosatetraenoic acid Rats chemistry Astrocytes cardiovascular system Cattle lipids (amino acids peptides and proteins) Arachidonic acid Endothelium Vascular Chromatography Liquid |
| Zdroj: | Analytical Biochemistry. 298:327-336 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2001.5395 |
| Popis: | Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples. |
| Databáze: | OpenAIRE |
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