Abstract
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.
Highlights
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
To analyze epoxyeicosatrienoic acid (EET) by gas chromatography-mass spectrometry (GC-MS), the four regioisomers must first be isolated by reversed-phase and normal-phase high performance liquid chromatography (HPLC) because they are poorly resolved by capillary GC, at least as methyl or pentafluorobenzyl esters [5, 6]
Ng amounts of' EETs can be used to establish the molecular weights when pentafluorobenzyl esters are analyzed by GC-MS in the negative ion-chemical ionization (NICI) mode [6]
Summary
EET and D H E T derivatives were examined using two Hewlett-Packard quadrupole systems: models 5970B (MSD) and 5989A (Engine). Samples were dissolved in 1.0 p1 isooctane or tridecane and injected into a wall-coated (0.25 pm film of dimethylpolysiloxane; DB-1, J&W), fused-silica column [0.25 mm (i.d.) x 28 m]. The oven temperature was ramped 70°C/min from 90°C (isooctane) or 225OC (tridecane) to a final constant temperature. A Hewlett-Packard 5989A, interfaced to a 5980 GC (Series 11) with on-column (“duckbill”) injector and programmable flow rates, was used for analysis of pentafluorobenzyl ester derivatives. A 15-m column with helium flowing at 90 cm/sec was used; 1.0 min after injection, the oven temperature was ramped from 90°C at 30°C/min to 280OC. A 60-m column with helium flowing at 24 cm/sec was used; 1.0 min after injection, the oven temperature was ramped from 23OOC at 70°C/min to 300OC. Signal-to-noise measurements (root mean square) were performed using the Hewlett-Packard software
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