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

Abstract

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 2α , PGE 2 , PGD 2 , PGJ 2 ,14,11,8,5,14,11,8,9-EET, and 5,6-EET in rat brain tissues. Deuterium labeled PGF 2α -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.

An improved LC–MS/MS method for the quantification of prostaglandins E2 and D2 production in biological fluids

“…To overcome the limitations of GC-MS and immunoassays for prostaglandin measurements, LC-MS [15][16][17] and LC-MS-MS [18][19][20][21][22][23][24][25][26][27] based methods have evolved as powerful tools for measuring prostaglandins in biological samples because of their high sensitivity, high selectivity and simplicity of sample preparation. Although five of these LC-MS or LC-MS-MS methods have measured PGD 2 [15][16][17][18][19], none have controlled for the inherent chemical instability of PGD 2 which is critical for determining accurate levels and for comparison with the values of other, more stable eicosanoids.…”

“…Although five of these LC-MS or LC-MS-MS methods have measured PGD 2 [15][16][17][18][19], none have controlled for the inherent chemical instability of PGD 2 which is critical for determining accurate levels and for comparison with the values of other, more stable eicosanoids. Our method addresses this important issue by incorporation of both a d 4 -PGD 2 and d 4 -PGE 2 internal standards in the reaction mixture in order to accurately quantify the relative amounts of PGE 2 and PGD 2 in the same sample.…”

Simultaneous Determination Method of Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids by LC-MS/MS System

“…We speculate that these peaks corresponded to HETEs, [19][20][21][22][23] which are also metabolites of AA, and/or impurities contained in standard solution of AA. EETs and DHETs were detected at much higher levels than the respective LLOQs in the samples incubated for 30 min with rCYP2C9 (Fig.…”

“…Several assay methods for eicosanoids employing LC/ MS have been reported. [19][20][21] A method using a tandem MS (LC-MS/MS) system [22][23][24][25][26] was recently developed to enhance sensitivity. However, the method described by Bylund et al 22) employed an ion trap MS system which generally shows a smaller dynamic range than a triple-quadrupole MS/MS system, and other methods use one or no internal standard for determination of eicosanoid concentrations.…”

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

“…To extract all analytes of interest with one common sample preparation approach, different extraction procedures ( 29,31,(36)(37)(38)(39). Therefore, we tested sample preparation using C18 RP SPE and polymeric RP SPE.…”