TY - JOUR
T1 - Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome p450 metabolites of arachidonic acid
AU - Nithipatikom, Kasem
AU - Grall, Andrew J.
AU - Holmes, Blythe B.
AU - Harder, David R.
AU - Falck, J R
AU - Campbell, William B.
N1 - Funding Information:
The authors thank the A. O. Smith Foundation for their generosity in providing funds for the purchase of the LC-MS system. We thank Ms. Marilyn A. Isbell, Sarah B. Hittner, Jayashree Narayanan, and Dr. Kathryn M. Gauthier for their technical assistance in this study and Ms. Mary A. Christian for her secretarial assistance. These studies were supported by funds from NIH Grant HL-51055 (W.B.C.), VA Merit Review 3440-02P and NIH NS-32321-05 (D.R.H.), NIH DK-38226 and the Robert A. Welch Foundation (J.R.F.), and the NIH Postdoctoral Training Grant (A.J.G.).
PY - 2001/12/15
Y1 - 2001/12/15
N2 - 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.
AB - 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.
KW - 20-hydroxyeicosatetraenoic acid
KW - Arachidonic acid
KW - Astrocytes
KW - Cytochrome P450
KW - Dihydroxyeicosatrienoic acids
KW - Endothelial cells
KW - Endothelial-derived hyperpolarizing factor (EDHF)
KW - Epoxyeicosatrienoic acids
KW - LC-MS
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U2 - 10.1006/abio.2001.5395
DO - 10.1006/abio.2001.5395
M3 - Article
C2 - 11700990
AN - SCOPUS:0035892637
SN - 0003-2697
VL - 298
SP - 327
EP - 336
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -