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

Article abstract of DOI:10.1002/rcm.4760

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 [¹³C₂₀]-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. Copyright

Full text of DOI:10.1002/rcm.4760

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rcm.4760
Analysis of epoxyeicosatrienoic acids by chiral liquid
chromatography/electron capture atmospheric pressure
13
chemical ionization mass spectrometry using [ C]-analog
internal standards
1
2
1
Clementina Mesaros , Seon Hwa Lee and Ian A. Blair *
1
Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6160, USA
2
Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 13 July 2010; Revised 22 August 2010; Accepted 29 August 2010
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
1
3
closely eluting isomers. We report the preparation of [ C ]-EET analog internal standards and the
0
2
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.
Copyright # 2010 John Wiley & Sons, Ltd.
1
0,11
P450s are membrane-bound hemoproteins that are involved
in the metabolism of drugs, carcinogens, steroids, and
either by cytosolic epoxide hydrolases
to dihydroxyei-
cosatrienoic acids or by glutathione S-transferases to
12
glutathione adducts. The regioselectivity and enantioselec-
1
,2
arachidonic acid (AA). Details of the individual genes,
sequences, and basic catalytic mechanisms are now well
tivity of EET formation is P450 isoform specific and is
thought to involve primarily P450s from the 2 family in
3
established. P450s can catalyze three different types of AA
13–15
16–19
oxidation. First, they can induce bis-allylic oxidation of AA
humans (2C8, 2C19, 2D6, and 2J2).
Endogenous EETs
to produce 7-, 10-, and 13-hydroxyeicosatetraenoic acids
are normally found esterified at the sn-2 position of cellular
(
HETEs) or lipoxygenase-like products such as 11-, 12-, and
glycerophospholipids but can be readily released by
4
19,20
19,21,22
1
5-HETEs. Second, P450s primarily of the 4 family can
basic hydrolysis.
and anti-inflammatory activity.
The EETs have potent vasodilator
2
3–26
perform conventional hydroxylation reactions on the v-
In addition, they can
5
27–31
terminus of AA to produce 16-, 17-, 18-, 19-, and 20-HETEs.
prevent apoptosis
and, depending on their chirality
Third, they can epoxidize AA at each cis-olefin to produce
four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, 14,15-
EET) each of which can be formed as an enantiomeric pair
and regiochemistry, they can also inhibit the platelet
1
3,32
aggregation.
Determination of the enantioselectivity that occurs in the
formation of regioisomeric EETs is important in order to
establish their potential biological activities and to assess
which P450 isoforms are involved in their formation.
There are a number of reports describing the enantiomeric
separation of the EET regioisomers using chiral columns
6
–8
(
Fig. 1). The 5,6-EET regioisomer is rapidly converted into
the corresponding lactone, due to the proximity of the
9
terminal carboxylic group and the 5,6-epoxide. However,
the other EETs are relatively stable until they are metabolized
2
0,33,34
*
Correspondence to: I. A. Blair, Center for Cancer Pharmacology,
University of Pennsylvania School of Medicine, 854 BRB II/III,
21 Curie Boulevard, Philadelphia, PA 19104-6160, USA.
E-mail: ianblair@mail.med.upenn.edu
with normal- or reversed-phase chromatography.
However, it has been extremely difficult to determine the
enantioselectivity of EET formation in cell and tissue samples
4
Copyright # 2010 John Wiley & Sons, Ltd.

3
238 C. Mesaros, S. H. Lee and I. A. Blair
Figure 1. Biosynthesis of epoxyeicosatrienoic acids (EETs) by cytochrome
P450 isoforms.
because of the problems in analyzing trace amounts of these
Cambridge Isotope Laboratories, Andover, MA, USA).
Diisopropylethylamine (DIPEA), 2,3,4,5,6-pentafluorobenzyl
bromide (PFB-Br), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
nicotinamide adenine dinucleotide phosphate-oxidase
(NADPH), ethylenediaminetetraacetic acid (EDTA), 3-chlor-
operoxybenzoic acid (MCPBA), sodium thiosulfate, aceto-
nitrile, methylene chloride and diethyl ether were purchased
from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade
hexane, isopropanol and ethanol were obtained from Fisher
Scientific Co. (Fair Lawn, NJ, USA). ACS-grade ethanol was
obtained from Pharmco (Brookfield, CT, USA). Gases were
supplied by BOC Gases (Lebanon, NJ, USA). The Chiralpak
AD-H column was obtained from Chiral Technologies (West
Chester, PA, USA). Microsomes of recombinant human or
rat P450 expressed in insect cells (hP4501A1, hP4501B1,
hP4502D6, hP4502C19 and rP4501A1, rP4501B1) were
purchased from Gentest (Woburn, MA, USA). Mouse
2
0,33,35
potent biologically active substances.
Typically, this
has required initial preparative chiral high-performance
liquid chromatography (HPLC) separations followed by
the analysis of each of the individual isomers using stable
isotope dilution gas chromatography/electron capture
3
4,36
negative chemical ionization mass spectrometry (MS).
There is a diverse array of chiral HPLC stationary phases
available that are derived from silica matrices coated with
carbamates or benzyl esters. Some of them are suitable for
the enantiomeric separation of one regioisomer but not for
others. Chiralcel OJ was found to resolve all regioisomers
into enantiomeric pairs, but different mobile phase flow
rates were required for optimal separation of the various
3
7
regioisomers. Previous studies have reported the analysis
3
8
of EETs by LC/MS but the methods did not employ
3
7
internal standards for the individual enantiomers and long
TM
chromatographic run times were required for the chiral
epithelial hepatoma Hepa-1c1c7 cells (CRL-2026 ) were
3
5,39
separations.
Over the last decade, we have developed
from the American Type Culture Collection (ATCC,
Manassas, VA, USA). a-Minimum essential medium
(MEM), fetal bovine serum (FBS), penicillin and streptomy-
cin were from Invitrogen (Carlsbad, CA, USA).
highly specific and sensitive methodology for the analysis
of eicosanoids based on the use of stable isotope dilution
normal-phase chiral liquid chromatography/electron cap-
ture atmospheric pressure chemical ionization mass spec-
4
0–43
13
Synthesis of [ C ] EETs
trometry (LC/ECAPCI-MS).
This methodology has
20
1
3
now been applied to determine the enantioselectivity of
AA-mediated EETs formation by different P450 isoforms and
to quantify AA-mediated chiral EETs formation in the mouse
epithelial hepatoma Hepa cell line.
[ C]-AA (2 mg, 6.1 mmol) was epoxidized by adding a
solution of 8 equivalents of MCPBA (8.6 mg, 50 mmol) in
1 mL of methylene chloride. The reaction mixture was stirred
4
4
for 2 h at room temperature. Excess reagent was quenched
with aqueous sodium thiosulfate (10% v/v, 1 mL). The
EETs were extracted three times with 4 mL of diethyl ether.
The combined organic phases were washed with aqueous
sodium hydrogen carbonate (10% v/v), water and dried over
anhydrous sodium sulfate. The filtrate was evaporated to
dryness under a nitrogen stream. After re-suspension in
100 mL of hexanes, the reaction mixture was filtered through
a small flash silica gel column under atmospheric pressure
and 4 mL of hexane was added to the column to elute all the
EXPERIMENTAL
Materials
(
ꢀ)-8,9-Epoxy-(5Z,11Z,14Z)-eicosatrienoic acid [(ꢀ)8,9-EET],
(
ꢀ)-11,12-epoxy-(5Z,8Z,14Z)-eicosatrienoic acid [(ꢀ)11,12-
EET], and (ꢀ)-14,15-epoxy-(5Z,8Z,11Z)-eicosatrienoic acid
[
(
(ꢀ)14,15-EET] were purchased from Biomol International
Plymouth Meeting, PA, USA). Arachidonic acid (AA) was
1
3
purchased from Cayman Chemical Co. (Ann Arbor, MI,
regioisomeric EETs from the silica gel. The purified [ C20]-
EETs were diluted in acetonitrile, divided into 1 mL aliquot
stock solutions and stored at ꢁ808C until ready to use.
1
3
13
USA). [ C]-Labeled AA for the [ C]-labeled EETs synthesis
was purchased from Spectra Stable Isotopes (currently
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

Chiral LC/MS analysis of epoxyeicosatrienoic acids 3239
1
3
Preparation of EET-PFBs
A racemic mixture of each EET regioisomer (50 ng, 0.156
[ C20]-internal standard (Fig. 2). Amounts of EET enantio-
mers were calculated by interpolation from the relevant
calibration curve. Quality control (QC) samples were
prepared by spiking a 3 mL buffer solution with a certain
amount of authentic standard (0.02, 0.04, 0.2 and 0.8 ng of
each stereoisomer, 3ꢂ) and internal standards (10 ng of
nmol) in methylene chloride (100 mL) was treated with
1
00 mL of DIPEA in methylene chloride (1:19, v/v) followed
by 100 mL of PFB-Br in methylene chloride (1:9, v/v) and
the solution was shaken at room temperature for 30 min.
The solution was evaporated to dryness under a nitrogen
stream at room temperature, and re-dissolved in 500 mL of
hexane/ethanol (97:3, v/v) for analysis.
1
3
[ C20]-EETs). The QC samples underwent the same sample
preparation and analytical procedures as the study samples.
AA incubation with supersomes
LC
To supersomes containing 50 pmol of the relevant P450 in
0.25 mL incubation buffer (0.05 mM Tris-HCl, pH 7.4,
containing 1 mM EDTA) and 1 mM NADPH was added
5 mM AA in ethanol (95% (v/v)). The mixtures were
incubated at 378C for 30 min. All incubations were performed
in duplicate. The reactions were terminated by placing the
samples on ice and adding 1 mL of ice-cold ethanol. The
Normal-phase chiral chromatography for LC/MS exper-
iments was performed using a Waters Alliance 2690 HPLC
system (Waters Corp., Milford, MA, USA). Gradient elution
was performed in the linear mode. A Chiralpak AD-H
column (250 ꢂ 4.6 mm i.d., 5 mm; Daicel Chemical Industries,
Ltd., Tokyo, Japan) was employed at a flow rate of 1 mL/min.
Solvent A was hexanes and solvent B was 2-propanol/
hexanes (6:4, v/v). Isocratic elution was used with 1.5% B for
1
3
internal standard mixture (10 mL stock solution of [ C20]-
EET) was added to all samples and they were centrifuged
at 10 000 rpm for 10 min. The supernatants were moved to
larger glass tubes and lipids were extracted with diethyl
ether (3 mL ꢂ 2) and the combined organic layers were
evaporated to dryness under nitrogen. Analysis of the PFB
derivatives (prepared as described above for the standards)
by LC/ECAPCI-MRM-MS analysis was conducted on a
1
2 min and then a linear gradient to 100% A for 10 min
for washing the column. The separation was performed
at 308C. Always, after PFB derivatization, the samples were
re-suspended in 100 mL of hexanes/2-propanol (95/5, v/v)
and were maintained at 48C in the autosampler tray;
injections of 20 mL were made.
2
0 mL aliquot of this solution. Concentrations of EETs from
ECAPCI-MS
the supersomes were calculated by interpolation from the
calculated regression lines.
Mass spectrometry was conducted on a Thermo Finnigan
TSQ Quantum Ultra AM mass spectrometer (Thermo
Finnigan, San Jose, CA, USA) equipped with an APCI
source in the EC negative ion mode. The TSQ Quantum
operating conditions were as follows: vaporizer temperature,
Culture of epithelial mouse Hepa-1c1c7 cells
Hepa1c1c7 cells were cultured in a-MEM with 10% FBS
and 100 U/mL penicillin/streptomycin. Cells grew to 90%
6
4
508C; heated capillary temperature, 2508C; corona discharge
confluence, approximately 1 ꢂ 10 cells per plate, at 378C and
needle, set at 30 mA. The sheath gas (nitrogen) and auxiliary
gas (nitrogen) pressures were 25 psi and 5 (arbitrary units),
respectively. Collision-induced dissociation (CID) was
performed using argon as the collision gas at 1.5 mTorr in
the second (rf-only) quadrupole. For full-scan and multiple
reaction monitoring (MRM) analyses, unit resolution was
maintained for both precursor and product ions. MS analysis
was conducted by infusing a solution in hexanes of 10 ng/mL
of each compound as its PFB derivative infused at 5 mL/min.
The following MRM transitions were monitored: (ꢀ)8,9-EET-
PFB, m/z 319 ! 155 (collision energy 12 eV), (ꢀ)11,12-EET-
PFB, m/z 319 ! 208 (collision energy 14 eV), (ꢀ)14,15-EET-
PFB, m/z 319 ! 219 (collision energy 12 eV). The internal
2
5% CO and were passaged every 4 days at a 1:3 dilution.
Incubations of Hepa cells with AA
When the cells were approximately 90% confluent, the media
was removed and the cells washed with phosphate-buffered
saline (PBS). Fresh FBS-free media was added and the cells
were treated with vehicle alone (dimethyl sulfoxide (DMSO),
ethanol) or 5 nM TCDD in DMSO (95% (v/v)) and 10 mM AA
in ethanol (95% (v/v)). All treatments were performed in
duplicate. After completion of the treatment, the media was
removed, and the cells were scraped into polypropylene
Eppendorf tubes. They were centrifuged at 10 000 rpm for
10 min. The supernatants were removed, 1 mL of fresh PBS
was added to the cell pellet, and the cells were re-suspended
1
3
standards were: (ꢀ)[ C ]-8,9-EET-PFB m/z 339 ! 163
2
0
1
3
6
(
collision energy 12 eV), (ꢀ)[ C20]-11,12-EET-PFB m/z
by pipetting for 10 s. The cell suspension (typically 10 cells)
1
3
3
39 ! 220 (collision energy 14 eV), (ꢀ)[ C20]-14,15-EET-PFB
was transferred to a clean glass tube containing chloroform
m/z 339 ! 233 (collision energy 12 eV).
and methanol (2:1 v/v, 5 mL). The lipids were extracted
4
5
by Folch extraction. Briefly: the samples were shaken for
15 min and centrifuged at 3000 g for 10 min. The supernatant
from each tube was transferred to a new tube and washed
with 1 mL of 0.9% NaCl solution. After vortex mixing and
centrifugation to separate the two phases, the upper phase
was removed. The steps for washing with 0.9% NaCl
solution and separation were repeated and the combined
lower phases were evaporated to dryness under nitrogen.
Hydrolysis of the esterified lipids was performed after
dissolving the dry residue in 500 mL of 80% methanolic
Preparation of standards and QC solutions
For calibration curves of the EETs, the three racemic
stereoisomers of the authentic EETs (0, 0.002, 0.004, 0.1,
1
3
0
.2, 0.4, 0.8, 1.6 ng of each) together with 10 ng of each [ C ]-
20
EET were spiked into 3 mL buffer solution. The standard
solutions underwent the same sample preparation and
analytical procedures as the samples. Calibration curves
were calculated with a linear regression analysis of the peak
area ratios of authentic standard against the appropriate
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

3
240 C. Mesaros, S. H. Lee and I. A. Blair
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.
NaOH for 30 min at 608C under nitrogen. After completion of
the hydrolysis, samples were transferred to larger glass tubes
and acidified to pH 4 with 2 N HCl. Then 10 ng of the internal
standard mixture was added and the lipids were extracted
with diethyl ether (3 mL ꢂ 2). The combined organic layers
were evaporated to dryness under nitrogen. Analysis of
the PFB derivatives (prepared as described above for the
standards) by LC/ECAPCI-MRM-MS analysis was con-
ducted on a 20 mL aliquot of this 100 mL solution. Concen-
trations of EETs from the cells were calculated by
interpolation from the calculated regressions lines.
RESULTS
MS analysis of EETs
Each PFB derivative of the three EET regioisomers was
analyzed under ECAPCI conditions. All of the EETs
exhibited an intense precursor ion at m/z 319 corresponding
to the loss of the PFB moiety in the source of the mass
4
0
ꢁ
spectrometer. The product spectra of the [M–PFB] ion for
each of the regioisomers were almost identical with those
reported by Bernstrom et al. from CID of the fast atom
4
6
bombardment-generated carboxylate anions and Naka-
mura et al. from CID of the same carboxylate anions
generated by electrospray ionization in a tandem quadrupole
4
7
mass spectrometer. The EET-PFB derivatives had common
Data analysis
All data analysis was performed using Xcalibur software
product ions formed by loss of water at m/z 301, at m/z 275
(-COO) and at m/z 257 (-(H O þ COO)). The MS and MS/MS
2
(
version 2.0 SR2; Thermo Analytical) from raw mass
spectra for the R,S isomer and S,R isomers were identical.
spectral data. Calibration curves were plotted using a linear
regression with weighting index of 1/x. Concentrations of
EETs in validation samples were determined from the
calibration line, and used to calculate the accuracy and
precision of the method within the study.
The most intense product ion from the CID and MS/MS
ꢁ
analysis of 8,9-EET-PFB [M–PFB] was observed at m/z 155
(Fig. 3(A)). This corresponded to the breaking of the C8ꢁC9
bond and C9ꢁO. CID and MS/MS analysis of 11,12-EET-PFB
ꢁ
[M–PFB] showed a characteristic odd-electron ion at m/z
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

Chiral LC/MS analysis of epoxyeicosatrienoic acids 3241
Figure 4. LC/MRM-MS chromatograms of EET-PFB stan-
1
dards and corresponding [ C]-labeled internal standards.
3
showed peaks in the channel for 11,12-EET. For quantifi-
1
3
cation of the EETs the [ C ]-EET mixture was added as
2
0
the internal standard (Fig. 4). The following specific MRM
ꢁ
transitions were monitored for the EETs: m/z 319 [M–PFB]
ꢁ
to m/z 155 for 8,9-EET, m/z 319 [M–PFB] to m/z 208 for 11,12-
ꢁ
EET and m/z 319 [M–PFB] to m/z 219 for 14,15-EET. For the
internal standards the following transitions were monitored:
ꢁ
13
m/z 339 [M–PFB] to m/z 163 for [ C20]-8,9-EET, m/z 339
ꢁ
M–PFB] to m/z 220 for [ C ]-11,12-EET and m/z 339
20
13
[
[
ꢁ
13
M–PFB] to m/z 233 for [ C ]-14,15-EET. The chiral
2
0
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.
separation of the EET-PFB derivatives was performed on
the Chiralpak AD-H column, an amylose tris(3,5-dimethyl-
phenyl carbamate) chiral stationary phase. The separation of
the EETs enantiomers required a non-polar phase. The best
separation was archived with 0.9% 2-propanol in hexanes.
Addition of more polar (ethanol, methanol) or less polar
(butanol) solvents to hexanes did not separate all the
regioisomers into their corresponding enantiomers in the
same chromatographic run. The retention times were 7.9 min
for 8(S),9(R)-EET-PFB and 8.4 min for 8(R),9(S)-EET-PFB;
9.0 min for 11(S),12(R)-EET-PFB and 9.6 min for 11(R),12(S)-
EET-PFB; 8.9 min for 14(R),15(S)-EET-PFB and 9.6 min for
2
08, corresponding to the cleavage of the C12ꢁC13 bond
(
Fig. 3(B)). The product ions at m/z 167 and 179 corresponded
to the breaking of the C10ꢁC11 bond with an additional
proton and the breaking of C11ꢁC12 and C11ꢁO, respect-
ꢁ
ively. CID and MS/MS analysis of 14,15-EET-PFB [M–PFB]
revealed a product ion at m/z 219 resulting from the cleavage
of the C14–C15 bond and C15ꢁO (Fig. 3(C)) and a product
ion at m/z 175 corresponding to the loss of water from the m/z
1
3
14(S),15(R)-EET-PFB. The [ C20]-EETs had the exact same
retention times as their corresponding unlabeled EETs
(Fig. 4).
2
19 ion. The mechanisms of formation of all the EET-derived
product ions were assumed to be similar to those described
4
8
by Murphy et al.
The authentic EET standards were commercially available
as a racemic mixture. LC/MS analysis of 14,15-EET-PFB
revealed that interference from MRM transitions m/z 319 to
155 (8,9-EET-PFB) and m/z 319 to 208 (11,12-EET-PFB) was
<0.5% (0.5% and 0.3%, respectively; Fig. 5). Thus, although
m/z 155 and 208 were minor product ions present in the
MS/MS spectrum of 14,15-EET-PFB, they did not cause
significant interference in the LC/MRM-MS analysis of 8,9-
Analysis of EET-PFB derivatives by chiral
phase LC/ECAPCI-MRM-MS
Specific product ions were selected for each EET-PFB
derivative. For 11,12-EET, the product ion at m/z 208 had
to be picked over the more intense ion at m/z 167 because
8
,9-EET also exhibited a ion fragment at m/z 167, which
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

3
242 C. Mesaros, S. H. Lee and I. A. Blair
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
4
2
and 11-12-EET enantiomers. It is known that different polar
modifiers of the mobile phase will change the elution order.
Due to lack of the pure enantiomers of each EET, the elution
order was established by comparison of the most abundant
enantiomer formed by different supersome isoforms with
the enantiomer reported in the literature. For P4502C8, an
1
3
their corresponding C-labeled internal standards for estab-
lishing the order of elution of the each enantiomer.
(ꢀ)8,9-EET-PFB, (ꢀ)11,12-EET-PFB and (ꢀ)14,15-EET-PFB
were 800, 100 and 100 fg on-column, respectively. Calibration
curves were prepared in the range from 1 to 40 ng/mL
(Fig. 2). Samples were stored in hexanes/2-propanol (95:5, v/v).
4
9
enzyme with major epoxygenase activity, it was reported
that the 11(R),12(S)-EET was produced with 82% enantio-
meric excess (ee). In our experiments, we found the later peak
eluting in the 11,12-EET channel to be the most abundant
2
Calibration curves for 8(S),9(R)-EET (y ¼ 0.408ꢂ – 0.0001; r ¼
2
(
data not shown), so the first eluting peak was from
0.996), 8(R),9(S)-EET (y ¼ 0.4863ꢂ – 0.0002; r ¼ 1), 11(S),12(R)-
2
1
1(S),12(R)-EET and the later eluting peak arose from
1(R),12(S)-EET (Fig. 4). This assignment was confirmed
EET (y ¼ 0.2283ꢂ – 0.0004; r ¼ 0.999), 11(R),12(S)-EET (y ¼
2
1
0.2247ꢂ – 0.0002; r ¼ 0.999), 14(R),15(S)-EET (y ¼ 0.2143ꢂ –
2
by the order of elution of the products of P4502C9-mediated
0.0014; r ¼ 0.998) and 14(S),9(R)-EET (y ¼ 0.2227ꢂ – 0.0002;
2
AA metabolism. The reported ee for 11(S),12(R)-EET was
r ¼ 0.998) were fitted to a linear regression with a 1/x
6
9% and we obtained almost exactly the same value for
weighting.
P4502C9-mediated AA metabolism to 11,12-EET (data not
shown). The order of elution of the 14,15-EET-PFB deriva-
tives was established by a similar rationale; the first eluting
peak was from 14(R),15(S)-EET and the second one was from
Assay validation
Validations were performed on five replicate samples at the
lower limit of quantification (LLOQ) of 0.02 ng, lower quality
control (LQC) of 0.04 ng, middle quality control (MQC) of
0.2 ng, and high quality control (HQC) of 0.8 ng. Precision
was between 2.0% and 14.7% and accuracy was between
91.8% and 107.8% for each individual enantiomer (Table 1).
1
4(S),15(R)-EET (Fig. 4). Previous studies of supersome-
mediated 8,9-EET formation do not provide data for
unequivocal identification of the elution order for the
3
7
individual EET-PFB enantiomers. However, Wei et al.
reported that on the Chiralcel OJ column, 8(R),9(S)-EET and
(S),9(R)-EET were separated by 7 min. Each enantiomer
was collected after Chiralcel OJ separation, mixed with the
8
Analysis of EET enantiomers in supersomes
treated with AA
1
3
appropriate [ C20]-internal standards and analyzed after
PFB derivatization using the AD-H column. This made
it possible to assign the first eluting enantiomer as 8(S),9(R)-
EET and the later eluting enantiomer as 8(R),9(S)-EET
Recombinant supersomes containing appropriate P450s
were treated with 5 mM AA for 30 min after adding the
NADPH. The control experiments were the exact same
amounts minus the NADPH. There was a striking difference
between hP4502C19 and hP4502D6 (Fig. 7, Table 2).
14(R),15(S)-EET was formed in 96% ee in hP4502C19,
whereas 14(S),15(R)-EET was formed in 96% ee in
hP4502D6. hP4502C19 and 2D6 were also very enantiose-
lective for 8(R),9(S)-EET formation (Figs. 7(A) and 7(B),
Table 2). However, hP4502C19-mediated formation of 11,12-
EET was almost racemic (Fig. 7(A)), whereas hP4502D6 was
highly enantioselective for the formation of 11(R),12(S)-EET
(
Fig. 6). Therefore, the order of elution of the EET PFB-
derivatives on the AD-H column was the opposite of that
3
7
reported for the underivatized EETs on the OJ column.
Sensitivity and linearity
To determine the limit of detection (LOD), a serial dilution
of each racemic EET was prepared (0.02 to 80 ng/mL). The
LOD determined at a S/N (signal/noise) ratio of 3:1 for
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

Chiral LC/MS analysis of epoxyeicosatrienoic acids 3243
Table 1. Precision and accuracy of EETs analyses (n ¼ 5)
LLOQ
LQC
(ng)
MQC
(ng)
HQC
(ng)
(
ng)
Analyte
Parameter
0.02
0.04
0.2
0.8
8
8
1
1
1
1
(R),9(S)-EET
Mean (ng)
0.02
10.3
99.3
0.02
14.8
98.2
0.021
7.8
103.5
0.018
8.8
91.8
0.021
7.2
0.04
11.3
100.5
0.04
12.1
100.4
0.038
9.3
95.9
0.04
6.6
99.7
0.04
9.6
0.203
3.3
101.6
0.205
7.3
102.7
0.208
7.6
103.9
0.213
6.4
106.3
0.220
4.1
110.2
0.198
2.4
0.86
5.6
100.7
0.839
8.9
104.8
0.863
3.5
107.8
0.848
2.7
106.1
0.860
3.0
107.5
0.790
6.8
Precision (%)
Accuracy (%)
Mean (ng)
Precision (%)
Accuracy (%)
Mean (ng)
Precision (%)
Accuracy (%)
Mean (ng)
Precision (%)
Accuracy (%)
Mean (ng)
Precision (%)
Accuracy (%)
Mean (ng)
Precision (%)
Accuracy (%)
(S),9(R)-EET
1(R),12(S)-EET
1(S),12(R)-EET
4(R),15(S)-EET
4(S),15(R)-EET
107.2
0.02
14.7
101.7
101.2
0.042
2.0
105.6
99.1
98.8
(
Fig. 7(B)). hP4501A1 and rP4501A1 gave a remarkably
P4502C19 but preferentially formed 14(S),15(R)-EET in a
similar pattern of EET metabolites (Fig. 8). Enantioselective
formation of 8(R),9(S)-EET (Fig. 8) was similar to hP4502D6
and 2C19 (Fig. 7), whereas enantioselective formation of
manner analogous to hP4502D6 (Table 2).
Quantification of EETs from AA-treated Hepa
cells
1
4(R),15(S)-EET (Fig. 8) and racemic 11,12-EET (Fig. 8)
was similar to hP4502C19 (Fig. 7(A)) and contrasted with
Hepa cells were treated with 10 mM AA before or after pre-
treatment with 5 nM TCDD. The total amount of EETs
(esterified and free) was determined by LC/ECAPCI-MRM-
MS. Enantioselective formation of 8(S),9(R)-EET, 11(S),12(R)-
the enantioselective formation of 14(S),15(R)-EET and
1
1(R),12(S)-EET by hP4502D6 (Fig. 7(B)). rP4501B1 formed
(R),9(S)-EET and racemic 11,12-EET in a similar manner to
8
Figure 7. Enantioselective biosynthesis of EETs by P450 family 2 isoforms: (A) hP4502C19 and (B)
hP4502D6.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

3
244 C. Mesaros, S. H. Lee and I. A. Blair
Table 2. Enantiomeric composition of each EETregioisomer formed through metabolism of AA by recombinant P450-containing
supersomes
8(S),9(R)-EET
8(R),9(S)-EET
11(S),12(R)-EET
11(R),12(S)-EET
14(R),15(S)-EET
14(S),15(R)-EET
(%)
(%)
(%)
(%)
(%)
(%)
hP4501A1
hP4501B1
rP4501A1
rP4501B1
hP4502C19
hP4502D6
2
4
2
2
2
0
98
96
98
98
98
13
47
49
50
40
1
87
53
51
50
60
99
90
92
90
25
96
4
10
8
10
75
4
100
96
6
EET, and 14(R),15(S)-EET was observed (Fig. 9). From the
0.53 ꢀ 0.71 ng and 0.26 ꢀ 0.01 ng/10 cells (Fig. 10(A)). The
amounts of 11(S),12(R)-EET and 11(R),12(S)-EET were 0.25 ꢀ
chromatograms of the Hepa cells (Fig. 9) one can appreciate the
13
6
usefulness of the [ C20]-internal standards. In the channel
corresponding to 8,9-EET (m/z 319 to 155) (Fig. 9(A)), there
were two intense peaks, that could have been easily mistaken
for both enantiomers of the 8,9-EET, if the corresponding
0.09 ng and 0.2 ꢀ 0.05 ng/10 cells (Fig. 10(B)). The amounts
of 14(R),15(S)-EET and 14(S),15(R)-EET were 1.25 ꢀ 0.09 ng
6
and 0.2 ꢀ 0.02 ng/10 cells (Fig. 10(C)). After 4 h treatment,
the amounts of 8(S),9(R)-EET and 8(R),9(S)-EET increased to
1
3
6
[
C
20]-internal standards had not shown that only one of
0.95 ꢀ 0.08 ng and 0.45 ꢀ 0.09 ng/10 cells. The amounts of
the intense peaks was indeed the 8(S),9(R)-EET. The other
enantiomer appeared as a much smaller peak, corresponding
to the enantioselective formation of 8(R),9(S)-EET. The
11(S),12(R)-EET and 11(R),12(S)-EET were 0.5 ꢀ 0.06 ng and
6
0.4 ꢀ 0.06 ng/10 cells. The amounts of 14(R),15(S)-EET and
6
14(S),15(R)-EET were 2.5 ꢀ 0.14 ng and 0.4 ꢀ 0.1 ng/10 cells.
1
3
[
C
20]-internal standards proved even more useful for the
TCDD pre-treatment of the Hepa cells also resulted in
enantioselective formation of the EETs. The 1 h treatment
with AA gave: 8(S),9(R)-EET and 8(R),9(S)-EET 1.5 ꢀ 0.17 ng
correct assignment of 11,12-EET enantiomers (Fig. 9(A)). In
the channel m/z 319 to 208 there were three peaks, and the
most intense one did not correspond to any of the peaks of
the internal standard, so the two lower intensity peaks were
assigned as 11(S),12(R)-EET and 11(R),12(S)-EET, respect-
6
and 0.3 ꢀ 0.04 ng/10 cells. The amounts of 11(S),12(R)-EET
6
and 11(R),12(S)-EET were 0.9 ꢀ 0.05 ng and 0.2 ꢀ 0.03 ng/10
cells. The amounts of 14(R),15(S)-EET and 14(S),15(R)-EET
1
3
6
ively. In the case of the 14,15-EET enantiomers, the [ C ]-
were 3.2 ꢀ 0.07 ng and 0.5 ꢀ 0.3 ng/10 cells. The 4 h
2
0
internal standards showed the enantiomeric formation of
4(R),15(S)-EET (Fig. 9(A)). In Fig. 9(B), the enantiomer
treatment with AA gave: 8(S),9(R)-EET and 8(R),9(S)-EET
6
1
1.5 ꢀ 0.17 ng and 0.3 ꢀ 0.04 ng/10 cells. The amounts of
assignments were easier since increased amounts were
formed after TCDD induction, but using the labeled internal
standards, it was possible to unequivocally quantify the
11(S),12(R)-EET and 11(R),12(S)-EET were 1 ꢀ 0.02 ng and
6
0.4 ꢀ 0.06 ng/10 cells. The amounts of 14(R),15(S)-EET
and 14(S),15(R)-EET were 4.9 ꢀ 0.02 ng and 0.6 ꢀ 0.08 ng/
6
1
4(R),15(S)-EET. When the cells were treated with 10 mM AA
10 cells. Therefore, the predominant isomer with or without
for 1 h the amounts of 8(S),9(R)-EET and 8(R),9(S)-EET were
TCDD induction was 14(R),15(S)-EET (Fig. 10(C)). The
Figure 8. Enantioselective biosynthesis of EETs by P450 family 1 isoforms:(A) hP4501A1 and (B)
rP4501A1.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

Chiral LC/MS analysis of epoxyeicosatrienoic acids 3245
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 mM AA without TCDD induction and (B) 1 h
treatment with 10 mM AA after 6 h TCDD (5 nM) induction. Abbreviations: IS, internal standard; IS1,
1
3
13
13
[
C ]-8(S),9(R)-EET-PFB; IS2, [ C ]-8(R),9(S)-EET-PFB; IS3, [ C ]-11(S)129(R)-EET-PFB;
20 20 20
1
3
13
IS4, [ C ]-11(R),12(S)-EET-PFB; IS5, [ C ]-14(R),15(S)-EET-PFB; IS6, [ C ]-14(S),15(R)-
13
2
0
20
20
EET-PFB.
second most abundant regioisomer was 8,9-EET (Fig. 10(A))
with the 8(S),9(R)-EET enantiomer being formed preferen-
tially. The amounts of 11,12-EETs (Fig. 10(B)) were almost
eight times lower than 14,15-EET (Fig. 10(C)). TCDD alone
did not induce significant amounts of EET formation
TCDD is a polycyclic aromatic hydrocarbon that binds to
the aryl hydrocarbon receptor (AhR), translocates into the
5
1,52
nucleus, and up-regulates P4501A1 and 1B1 expression.
Therefore, TCDD pre-treatment of the Hepa cells resulted in
formation of almost twice the amount of EETs when
compared with non-induced cells at both 1 h and 4 h
(
Fig. 10).
(
Fig. 10). Furthermore, the enantioselectivity of 14,15-EET
formation was preserved after TCDD induction (Fig. 10(C)),
which provided additional confirmation that rodent
P4501A1 and 1B1 were responsible for its formation.
Surprisingly, 8(S),9(R)-EET was the major AA-derived 8,9-
EET in both the non-induced and TCDD-induced Hepa cells
(Fig. 10(A)). None of the P450s that were tested produced
significant quantities of this enantiomer (Table 2), which has
been shown previously to be a major metabolite of the rat
DISCUSSION
There was a striking difference in the enantioselectivity of
1
1
4,15-EET formation between P450 2C19 and P450 2D6.
4(R),15(S)-EET was formed with a high ee by P4502C19,
whereas 14(S),15(R)-EET was the predominant enantiomer
formed from P4502D6 (Fig. 7). As expected, hP4501A1 and
rP4501A1 had similar enantioselectivity, converting AA
primarily into the 14(R),15(S)-EET (Fig. 8). P450-mediated
metabolism of AA by mouse Hepa cells also resulted in the
formation of the EETs with high regioselectivity for
5
3
cortex. This suggests that there is another P450 in the mouse
Hepa cell line, which is responsible for the formation of
8(S),9(R)-EET. Interestingly, the 8(S),9(R)-EET enantiomer
has potent vasoactive properties and undergoes COX-
mediated metabolism to a potent mitogen for mesangial
1
4(R),15(S)-EET (Figs. 9 and 10). Hepa cells constitutively
5
0
express CYP1A1 and CYP1B1, and so a predominance of
the 14(R),15(S)-EET would have been predicted from the
supersome studies (Fig. 8, Table 2). Up-regulation of these
P450s would also be expected to increase the amounts of
EETs that are formed from AA.
5
4
cells. The low abundance of the 8(R),9(S)-EET in the TCDD-
induced cells at 1 h and 4 h (Fig. 10(A)), a significant product
of both rP4501A1 and 1B1 (Fig. 8, Table 2), suggests that
1
1
preferential hydrolysis of this EET enantiomer could have
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3237–3247
DOI: 10.1002/rcm

3
246 C. Mesaros, S. H. Lee and I. A. Blair
Figure 10. EET formation in Hepa cells treated with 10 mM of arachidonic acid with or without 5 nM
TCDD induction.
occurred as a result of TCDD treatment. Recent studies have
suggested that TCDD-mediated up-regulation of antioxidant
genes such as epoxide hydrolase could occur through an
P450s and to quantify the individual enantiomers in AA-
treated cultured cell lines.
5
5
interaction between the Ahr and Nrf-2.
1,12-EET, a minor product of AA metabolism of P4501A1
1
Acknowledgements
and 1B1 in the supersomes (Fig. 8, Table 2), was also the least
abundant product in the Hepa cell incubations (Fig. 10(B)).
The expected racemic 11,12-EET was observed in the non-
induced cells, whereas TCDD induction caused an apparent
selective induction of 11(S),12(R)-EET formation (Fig. 10(B)).
However, this could have been due to selective hydrolysis of
the 11(R),12(S)-EET isomer as suggested above for 8(R),9(S)-
EET. Taken together, these data suggest that P4501A1- and
We gratefully acknowledge the support of grants
UO1ES016004 and P30ES0135080 from the National Insti-
tutes of Health.
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