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LC‐MS measurement of the epoxyeicosatrienoic acid mimetic EET‐8‐ZE‐D in rat serum
Author(s) -
Shaw Sean,
Shrestha Binaya,
Nithripatikom Kasem,
Imig John
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.1019.13
Subject(s) - chemistry , acetic acid , chromatography , methanol , acetonitrile , derivatization , extraction (chemistry) , chloroform , carboxylic acid , high performance liquid chromatography , in vivo , organic chemistry , microbiology and biotechnology , biology
Epoxyeicosatrienoic acids (EET) are created by CYP450 epoxygenases and possess a wide variety of cardiovascular protective actions. Since their presence is transitory the synthesis of EET mimetics with longer lifetimes offers a potential route for cardiovascular drug development. While one such mimetic, an esterified EET analogue (EET‐8‐ZE), had no effect in SHR rats, further modification by amidation of the carboxylic group with aspartic acid (EET‐8‐ZE‐D) has been shown to reduce blood pressure in SHR rats (injected at 3mg/day; at Day 2 the MAPδ was ‐8±1.3 mmHg). From there we have developed an LC‐MS method for the detection of EET‐8‐ZE‐D in rat serum. A 200μl sample of rat serum was diluted with water to a total of 1ml then spiked with both EET‐8‐ZE‐D and deuterated‐EET standards; after adding acetic acid to a 20% concentration it underwent a chloroform/methanol liquid‐phase extraction. The organic phase was collected dried down under nitrogen, briefly resuspended in methanol then dried and suspended in 30ul acetonitrile. After reverse‐phase separation on an HPLC column (acetonitrile + 0.1% acetic acid against water + 0.1% acetic acid) the samples were run on an Agilent 1100 triple quadrupole MS, providing sufficient quantitative sensitivity to isolate 300pg. These data demonstrate that the EET‐8ZE‐D mimetic has in vivo cardiovascular activity and that EET‐8ZE‐D can be accurately measured in biological fluids.