Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using [13C]-analog internal standards

Abstract

The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [(13)C(20)]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line.

Figure 1

Biosynthesis of epoxyeicosatrienoic acids (EETs) by cytochrome P450 isoforms.

Figure 2

Calibration curves: (A) 8(S),9(R)-EET-PFB; (B) 8(R),9(S)-EET-PFB; (C) 11(S),12(R)-EET-PFB; (D) 11(R),12(S)-EET-PFB; (E) 14(R),15(S)-EET-PFB; and (F) 14(S),15(R)-EET-PFB.

Figure 3

Product ion spectra of EETs: (A) CID of 8,9-EET-PFB after PFB loss in the source; (B) CID of 11,12-EET-PFB after PFB loss in the source; and (C) CID of 14,15-EET-PFB after PFB loss in the ion source of the mass spectrometer.

Figure 4

LC/MRM-MS chromatograms of EET-PFB standards and corresponding [¹³C]-labeled internal standards.

Figure 5

LC/MRM-MS chromatograms of 14,15-EET-PFB standard injected alone. Intensities in the MRM channels for 8,9-EET-PFB and 11,12-EET-PFB were <0.5%.

Figure 6

LC/MRM-MS chromatograms of synthetic pure 8(R),9(S)-EET-PFB and 8(S),9(R)-EET-PFB standards and their corresponding ¹³C-labeled internal standards for establishing the order of elution of the each enantiomer.

Figure 7

Enantioselective biosynthesis of EETs by P450 family 2 isoforms: (A) hP4502C19 and (B) hP4502D6.

Figure 8

Enantioselective biosynthesis of EETs by P450 family 1 isoforms:(A) hP4501A1 and (B) rP4501A1.

Figure 9

LC/MRM-MS chromatograms of EET-PFB in Hepa cells and their corresponding 13C-labeled internal standards: (A) 1 h treatment with 10 μM AA without TCDD induction and (B) 1 h treatment with 10 μM AA after 6 h TCDD (5 nM) induction. Abbreviations: IS, internal standard; IS1, [¹³C₂₀]-8(S),9(R)-EET-PFB; IS2, [¹³C₂₀]-8(R),9(S)-EET-PFB; IS3, [¹³C₂₀]-11(S)129(R)-EET-PFB; IS4, [¹³C₂₀]-11(R),12(S)-EET-PFB; IS5, [¹³C₂₀]-14(R),15(S)-EET-PFB; IS6, [¹³C₂₀]-14(S),15(R)-EET-PFB.

Figure 10

EET formation in Hepa cells treated with 10 μM of arachidonic acid with or without 5 nM TCDD induction.