A liquid chromatography/mass spectrometric method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites prostaglandins, dihydroxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids in rat brain tissue.
A sensitive, specific, and robust liquid chromatography/mass spectrometric (LC/MS) method was developed and validated that allows simultaneous analysis of arachidonic acid (AA) and its cyclooxygenase, cytochrome P450, and lipoxygenase pathway metabolites prostaglandins (PGs), dihydroxyeicosatrienoic acids (DiHETrEs), hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), including PGF(2alpha), PGE(2), PGD(2), PGJ(2), 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat brain tissues. Deuterium labeled PGF(2alpha)-d(4), PGD(2)-d(4), 15(S)-HETE-d(8), 14,15-EET-d(8), 11,12-EET-d(8), 8,9-EET-d(8), and AA-d(8) were used as internal standards. Solid phase extraction was used for sample preparation. A gradient LC/MS method using a C18 column and electrospray ionization source under negative ion mode was optimized for the best sensitivity and separation within 35 min. The method validation, including LC/MS instrument qualification, specificity, calibration model, accuracy, precision (without brain matrix and with brain matrix), and extraction efficiency were performed. The linear ranges of the calibration curves were 2-1000 pg for PGs, DiHETrEs, HETEs, and EETs, 10-2400 pg for PGE(2) and PGD(2), and 20-2000 ng for AA, respectively.
Yue
Hongfei
H
Temple University, Chemistry Department, Analytical Chemistry, 1901 North 13th Street, Philadelphia, PA 19122, USA.
Jansen
Susan A
SA
Strauss
Kenneth I
KI
Borenstein
Michael R
MR
Barbe
Mary F
MF
Rossi
Luella J
LJ
Murphy
Elise
E
D000818:Animals; D016718:Arachidonic Acid / Q000032:analysis*; D001095:Arachidonic Acids / Q000032:analysis*; D001923:Brain Chemistry / Q000187:drug effects*; D002138:Calibration; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015777:Eicosanoids / Q000032:analysis*; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis*; D008084:Lipoxygenase / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011453:Prostaglandins / Q000032:analysis*; D011786:Quality Control; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results
D001095:Arachidonic Acids; D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; D011453:Prostaglandins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D008084:Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases