Quantification of Epoxyeicosatrienoic acids Enantiomers: The development of reliable and practical liquid chromatography mass Spectrometry assay

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2024-10, Vol.1247, p.124346, Article 124346
• Main Authors: A. Isse, Fadumo, Helal, Sara, El-Sherbeni, Ahmed A., Brocks, Dion R, El-Kadi, Ayman O.S.
• Format: Article
• Language: English
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - analysis; 8,11,14-Eicosatrienoic Acid - chemistry; 8,11,14-Eicosatrienoic Acid - metabolism; Animals; Arachidonic acid; Assay; Chromatography, Liquid - methods; EET; Enantiomer; Humans; LC-MS/MS; Limit of Detection; Linear Models; Liquid Chromatography-Mass Spectrometry; Microsome matrix; Microsomes, Liver - chemistry; Microsomes, Liver - metabolism; Reproducibility of Results; Stereoisomerism; Tandem Mass Spectrometry - methods; Underived
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2024.124346

•Validated assay for arachidonic acid (AA)-underived chiral epoxy-metabolites.•A reliable and reproducible chiral analysis for epoxy-metabolites of AA.•Enantioselective analysis of epoxy-metabolites formed by human liver microsomes.•The assay showed acceptable linearity, precision, and accuracy. Epoxyeicosatrienoic acids (EETs) are increasingly recognized as key metabolites in the arachidonic acid (AA) metabolic pathway. EETs are epoxy derivatives of AA with two chiral centers formed by cytochrome P450 (CYP) enzymes. EETs have reported biological activities as racemates; however, knowledge on specific optical isomers of EET is lacking. A main reason is the absence of practical assay to quantify EETs isomers associated with specific pathological conditions and enzymes. The reported underivatized chiral LC-MS/MS assays utilize different mobile phases and flow rates or required long run times to achieve separation of EET stereoisomers. Others incorporated a derivatization step before the separation of EETs in their assays. Therefore, the objective of this study was to develop and validate a stereoselective assay for the simultaneous quantitation of underivatized EET enantiomers using Liquid Chromatography Mass Spectrometry (LC-MS/MS) with an optimum baseline separation using binary mobile phase and gradient elution. Herein, we report the development and validation of an LC-MS/MS assay, and its application to quantify the formation of EET enantiomers mediated by human liver microsomes. Assay linearity extends over 10–600 ng/mL with r2 > 0.99 for all EETs enantiomers. The inter-run accuracy was within ± 15 %, and precision was ≤ 15 %, and