Identification of arachidonate epoxides/diols by capillary chromatography-mass spectrometry.
The identification of epoxide regioisomers of arachidonic acid (EETs) as methyl esters is difficult because they coelute during gas chromatography and possess similar mass spectra. In the present study, EETs and their hydrolysis products, dihydroxyeicosatrienoic acids (DHETs), were analyzed as pentafluorobenzyl ester derivatives and their properties were compared to other esters. The four EET regioisomers were not resolved by gas chromatography as pentafluorobenzyl, trimethylsilyl, t-butyldimethylsilyl, or methyl esters. However, after being hydrolyzed to DHETs, three of the four regioisomers were resolved as (bis)-t-butyldimethylsilyl ether, pentafluorobenzyl esters. The fourth regioisomer (5,6-DHET) was resolved after being converted to a delta-lactone. Thus, the EETs could be resolved by capillary gas chromatography once converted to DHETs. Pentafluorobenzyl esters of both EETs and DHETs (15-40 ng) provided diagnostic spectra when analyzed by electron ionization mass spectrometry. The mass spectral interpretations that indicated epoxide and diol positions were validated using synthesized EET/DHET [17,17,18,18-d4, 5,6,8,9,11,12,14,15 d8] standards. Lesser amounts of DHETs (5-150 fg) also indicated molecular weights when analyzed in the negative-ion chemical-ionization mode. In summary, EETs in nanogram quantities were identified as pentafluorobenzyl esters using electron ionization mass spectrometry. EETs in femtogram-to-picogram quantities were also identified after conversion to DHETs and analysis by gas chromatography-mass spectrometry in the negative ion-chemical ionization mode.
VanRollins
M
M
Department of Internal Medicine, University of Iowa, Iowa City 52242, USA.
Knapp
H R
HR
D016718:Arachidonic Acid / Q000737:chemistry*; D004537:Eicosanoic Acids / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry*; D004951:Esterification; D005464:Fluorobenzenes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006900:Hydroxylation; D015394:Molecular Structure; D015203:Reproducibility of Results; D014297:Trimethylsilyl Compounds / Q000737:chemistry
D004537:Eicosanoic Acids; D004852:Epoxy Compounds; D005464:Fluorobenzenes; D014297:Trimethylsilyl Compounds; D016718:Arachidonic Acid