Simultaneous Determination Method of Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids by LC-MS/MS System

Abstract

Epoxyeicosatrienoic acids (EETs) are produced primarily by CYPs from arachidonic acid (AA) and then further metabolized to the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs play important roles in physiological processes such as regulating vasodilation and inflammation. Thus, the drug inhibition of CYP-mediated AA metabolism could reduce production of EETs, potentially resulting in adverse cardiovascular events. The aim of this study was to develop a simple method to simultaneously determine the concentrations of both EETs and DHETs using a conventional LC-MS/MS system to evaluate drug-endogenous substance interactions, including eicosanoids. Eight eicosanoids (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) were detected with their corresponding deuterium-labeled eicosanoids as internal standards. The samples were purified by solid-phase extraction columns. Liquid chromatographic separation was achieved on a C18 column. DHETs and EETs were eluted at 4–7 and 18–26 min, respectively. The weighted (1/y²) calibration curves were linear over a range of 5–2000 nmol/L for EETs and 2–2000 nmol/L for DHETs. In quality control (QC) samples, the recoveries of eicosanoids were 95.2–118%. The intra-day precisions were within 6% in all three QC samples, and the inter-day precisions were <16.7% at 50 nmol/L, <8.6% at 200 nmol/L, and <9.8% at 1000 nmol/L. We have applied this method for the determination of the eicosanoid levels in samples from incubation studies of AA by using human recombinant CYP enzyme (rCYP), and confirmed that the method has sensitivity sufficient for assessment of rCYP incubation study.

Arachidonic acid (AA) is metabolized to various eicosanoids via several pathways.¹⁾ These eicosanoids play important roles in physiological processes, including the regulation of inflammation.^2–5) Three major metabolic pathways for conversion of AA to eicosanoids are recognized, including routes that incorporate cyclooxygenase (COX), lipoxygenase (LOX), and CYP.⁶⁾ The CYP pathway, which is mediated primarily by CYP2C9, CYP2C8, and CYP2J2, produces epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs).^7,8) EETs exist as one of 4 regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), depending on the location of the epoxidized residue. Each EET is substantially metabolized to its corresponding diol, a dihydroxyeicosatrienoic acid (DHET), by soluble epoxide hydrolase (sEH).^1,9–12) The EETs have been shown to play important roles on cardiovascular homeostasis.^13–15) Thus, the drug inhibition of CYP-mediated AA metabolism via could reduce production of EETs, potentially resulting adverse cardiovascular events.

Traditional analytical methods such as HPLC or capillary electrophoresis with UV or fluorescence detection,^16,17) and GC-MS¹⁸⁾ lack sufficient sensitivity to detect the low levels of AA metabolites typically found in physiological matrices. Several assay methods for eicosanoids employing LC/MS have been reported.^19–21) A method using a tandem MS (LC-MS/MS) system^22–26) was recently developed to enhance sensitivity. However, the method described by Bylund et al.²²⁾ employed an ion trap MS system which generally shows a smaller dynamic range than a triple-quadrupole MS/MS system, and other methods use one or no internal standard for determination of eicosanoid concentrations.^23–26) Edpuganti and Mehvar have recently developed a method sensitive enough to measure free concentrations of AA and its P450 metabolites using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) instrumentation.²⁷⁾ However, the authors used only one or two internal standards to measure the concentration of eicosanoid groups. The use of deuterium-labeled internal standards for each of the eicosanoid compounds is expected to minimize determination and variation errors.

The present report describes a simple method for the detection of EETs and DHETs by conventional LC-MS/MS using the corresponding deuterium-labeled eicosanoids as internal standards (except for the commercially unavailable 5,6-DHET-d₁₁) to evaluate the effect of an administered drug on AA metabolism. We further demonstrated that our technique was applicable to incubation studies of AA exposed to recombinant human CYP (rCYP) enzymes.

MATERIALS AND METHODS

Materials

AA, EETs, DHETs, and their deuterium-labeled analogues commercially available were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Chemical structures of the eicosanoids are shown in Fig. 1. Human CYP2C9*1 expressed with P450 reductase and cytochrome b₅ Supersomes™ (rCYP2C9), insect cell control Supersomes™ (control microsomes), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system solutions A and B were purchased from BD Gentest (Franklin Lakes, NJ, U.S.A.). Other chemicals and solvents were of special or HPLC grade.

Fig. 1. Structures of Unlabeled and Deuterated Eicosanoids

Sample Preparation

The AA stock solution was formulated at 20 mmol/L in ethanol. All stock solutions of eicosanoids and their deuterium-labeled versions were formulated at 40 µmol/L in ethanol, except for 5,6-EET, which was formulated in acetonitrile. All solutions were stored frozen at −80°C before use. Mixed solutions with the deuterium-labeled materials were evaporated and then reconstituted in ethanol to 4 µmol/L (8,9-DHET-d₁₁ only) or 8 µmol/L (all others).

Solid-Phase Extraction

Each stock solution of eicosanoid was mixed to yield final concentrations of 2–2000 nmol/L. After evaporation, the residue was reconstituted with 186 µL of Tris–HCl buffer (100 mmol/L, pH 7.5), 12 µL of water, 2 µL of ethanol, and 50 µL acetonitrile. An aliquot (50 µL) of the mixed solution of the deuterium-labeled material in acetonitrile then was added to the sample, which was further diluted by addition of 700 µL of water. The sample was extracted using an Oasis HLB cartridge (1 cc Vac Cartridge, 30 mg Sorbent, Waters Corp., Milford, MA, U.S.A.). Eicosanoids were eluted with 2 mL of ethyl acetate. After evaporating the solvent, the residue was reconstituted in 50 µL of 50% acetonitrile, and an aliquot (40 µL) was injected into the LC-MS/MS.

LC-MS/MS Conditions

HPLC was performed using an Agilent 1200 series machine (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with binary pump, in-line degasser, column oven, and thermostatically controlled autosampler. A triple quadrupole mass spectrometer API3200 QTRAP LC/MS/MS System (AB Sciex, Framingham, MA, U.S.A.) was coupled to the HPLC system. Eicosanoids were separated using an Ascentis Express C18 (2.1 mm×100 mm) column with 2.7-µm particle size (Sigma-Aldrich, St. Louis, MO, U.S.A.) at 50°C. The mobile phase consisted of 0.1% formic acid in 50% acetonitrile. The flow rate was 0.3 mL/min.

Calibration and Validation

Standard curves were constructed by analyte/IS ratios of LC-MS/MS peak areas of each sample against analyte concentration using weighted (1/y²) linear regression analysis and were analyzed over 3–5 separate experiments. Quantitation was performed using each deuterated internal standard, with the exception of 5,6-DHET; 8,9-DHET-d₁₁ was used instead.

Accuracy and precision were evaluated in quality control (QC) samples at three levels (50, 200, 1000 nmol/L) for each eicosanoid. Accuracy was determined as the calculated concentration for each QC sample based on the standard curve equation. Recovery was calculated as the (calculated concentration/theoretical concentration)×100. Precision was determined by calculating the relative standard deviation (CV%) for all injections of QC samples. Intra-day precision was determined by comparing among 7 separate injections of the QC samples prepared for the accuracy analysis. To obtain inter-day precision, the QC samples were analyzed on 5–7 consecutive days. According to the procedures of the United States Pharmacopeia Convention, the limit of detection (LOD) and the lower limit of quantitation (LLOQ) of the signal to noise ratio (S/N) were set at 3 and 10, respectively.

Data Analysis

Data were acquired by Analyst^® ver. 1.4.2 software (AB Sciex). The standard curves also were generated using Analyst 1.4.2.

Incubation Study Using Recombinant Enzymes

We have applied our method for the determination of eicosanoid levels in samples obtained from incubation studies in which AA was exposed to rCYP2C9 or control microsomes for 30 min.

The pre-incubation mixture consisted of rCYP2C9 (10 µL at 1000 pmol P450/mL), NADPH regeneration system solutions A and B (12 µL), Tris–HCl buffer (176 µL at 50 mmol/L). The mixture was pre-incubated for 3 min at 37°C. The reaction then was initiated by adding 2 µL of AA solution (2 mmol/L) and incubating for 30 min at 37°C. For the control reaction, 10 µL of control microsomes were added instead of the rCYP2C9. The reaction was stopped by adding 50 µL of ice-cold acetonitrile. The sample was subjected to solid-phase extraction accordingly, and then analyzed using our LC-MS/MS method for the determination of the eicosanoid levels.

RESULTS

Optimization of Multiple Reaction Monitoring (MRM) Conditions

The m/z values of parent and daughter ions of eicosanoids assayed in this study and the eicosanoid determination parameters used to obtain the optimal MRM condition are shown in Table 1.

Table 1. Optimal Determination Parameters of Eicosanoids

Eicosanoids or I.S.	Parent to daughter ion	Dwell time (ms)	DP (V)	EP (V)	CEP (V)	CE (eV)	CXP (V)
14,15-EET	m/z 319.2→219.2	232.0	−40.0	−8.0	−23.47	−22.0	−6.0
11,12-EET	m/z 319.2→167.0	232.0	−35.0	−6.5	−22.00	−20.0	0.0
8,9-EET	m/z 319.2→167.0	232.0	−35.0	−8.0	−28.00	−22.0	−6.0
5,6-EET	m/z 319.2→191.2	232.0	−40.0	−4.5	−20.00	−16.0	0.0
14,15-DHET	m/z 337.1→207.2	117.0	−40.0	−9.5	−30.00	−24.0	−4.0
11,12-DHET	m/z 337.2→167.0	117.0	−45.0	−8.0	−32.00	−26.0	0.0
8,9-DHET	m/z 337.2→127.0	117.0	−45.0	−10.0	−36.00	−34.0	0.0
5,6-DHET	m/z 337.2→145.0	117.0	−50.0	−12.0	−30.00	−24.0	0.0
14,15-EET-d11	m/z 330.2→219.2	232.0	−40.0	−8.0	−23.87	−22.0	−6.0
11,12-EET-d11	m/z 330.2→167.0	232.0	−40.0	−8.0	−23.87	−22.0	−6.0
8,9-EET-d11	m/z 330.2→167.0	232.0	−40.0	−8.0	−23.87	−22.0	−6.0
5,6-EET-d11	m/z 330.2→202.2	232.0	−40.0	−8.0	−23.87	−22.0	−6.0
14,15-DHET-d11	m/z 348.1→207.2	117.0	−40.0	−8.0	−24.54	−22.0	−6.0
11,12-DHET-d11	m/z 348.2→167.0	117.0	−40.0	−8.0	−24.54	−22.0	−6.0
8,9-DHET-d11	m/z 348.2→127.0	117.0	−40.0	−8.0	−24.54	−22.0	−6.0

EET: Epoxyeicosatrienoic acid, DHET: Dihydroxyeicosatrienoic acid, DP: Declustering potential, EP: Entrance potential, CEP: Collision cell exit potential, CE: Collision energy; CXP: Collision cell exit potential.

Typical MRM chromatograms of eicosanoids are shown in Fig. 2A (unlabeled eicosanoids) and Fig. 2B (deuterated eicosanoids). DHETs and EETs eluted at retention times of 4–7 and 18–26 min, respectively.

Fig. 2. Typical MRM Chromatograms of (A) Eicosanoid Standards (100 nmol/L Each) and (B) Their Deuterated Substances (200 nmol/L for 8,9-DHET-d₁₁ and 400 nmol/L for All Others)

Validation of the Method

Table 2 shows the linearity of determination of eicosanoid levels by this method. The calibration curves were linear over a range of 5–2000 nmol/L for EETs and 2–2000 nmol/L for DHETs. The correlation coefficients were 0.972–0.999. The LOD and LLOQ were 1.5 and 5 nmol/L for EETs, and 0.6 and 2 nmol/L for DHETs, respectively. Table 3 shows the recoveries, and intra- and inter-day precisions of eicosanoids in the QC samples. The recoveries of eicosanoids were 95.2–118%. The intra-day precisions were within 6% in all three QC samples, and the inter-day precisions were <16.7%, <8.6%, and <9.8% at 50, 200, and 1000 nmol/L, respectively.

Table 2. Performance Values of Calibration Curves of Eicosanoids

Eicosanoids	Linear range (nmol/L)	Line equation (weighting by 1/y2)	Correlation coefficient
14,15-EET	5–2000	y=0.00561x−0.0155	r=0.972–0.997
11,12-EET	5–2000	y=0.00843x−0.0350	r=0.990–0.999
8,9-EET	5–2000	y=0.0106x+0.0386	r=0.990–0.999
5,6-EET	5–2000	y=0.0215x−0.139	r=0.979–0.994
14,15-DHET	2–2000	y=0.00579x+0.149	r=0.994–0.998
11,12-DHET	2–2000	y=0.0122x+0.398	r=0.991–0.995
8,9-DHET	2–2000	y=0.0281x+1.033	r=0.982–0.994
5,6-DHET	2–2000	y=0.0184x+0.144	r=0.987–0.991

EET: Epoxyeicosatrienoic acid; DHET: Dihydroxyeicosatrienoic acid.

Table 3. Recoveries and Intra- and Inter-Day Precisions of Eicosanoids in Quality Control Samples

Eicosanoids	Concentration (nmol/L)	Recovery (%) (n=3–5)	Intra-day precision CV% (n=7)	Inter-day precision CV% (n=3–5)
14,15-EET	50	99.4	3.9	8.1
	200	96.9	2.6	8.6
	1000	107.7	2.1	6.5
11,12-EET	50	99.2	2.2	7.2
	200	97.3	1.8	3.9
	1000	107.6	1.2	3.3
8,9-EET	50	99.7	4.8	5.9
	200	95.2	3.0	4.8
	1000	105.3	3.0	7.6
5,6-EET	50	100.9	3.3	6.5
	200	98.6	2.9	6.9
	1000	105.8	2.9	6.0
14,15-DHET	50	114.2	4.1	6.5
	200	111.2	2.7	4.0
	1000	97.7	1.4	2.4
11,12-DHET	50	118.0	2.6	5.4
	200	110.6	2.9	4.1
	1000	96.4	2.1	2.9
8,9-DHET	50	118.3	3.2	5.2
	200	114.7	2.5	2.5
	1000	98.1	3.1	2.6
5,6-DHET	50	111.4	5.1	16.7
	200	115.7	1.6	5.0
	1000	103.6	2.6	9.8

EET: Epoxyeicosatrienoic acid; DHET: Dihydroxyeicosatrienoic acid.

Incubation Study

Typical chromatograms of the samples from an incubation study using rCYP2C9 or control microsomes are shown in Fig. 3. There were no interfering peaks in either of the blank samples (data not shown).

Fig. 3. Typical MRM Chromatograms of Samples from Incubation Studies Using rCYP2C9 (A) or Control Microsomes (B)

Calculated concentrations (values in nmol/L): (A) 14,15-EET 1400; 11,12-EET 661; 8,9-EET 237; 5,6-EET 33.6; 14,15-DHET 40.4; 11,12-DHET 49.8; 8,9-DHET 53.5; and 5,6-DHET ND. (B) 14,15-EET 8.5; 11,12-EET 5.8; 8,9-EET 7.2; 5,6-EET 14.0; 14,15-DHET 2.9; 11,12-DHET 2.3; 8,9-DHET ND; and 5,6-DHET ND. ND: Not detected.

DISCUSSION

We have developed a simultaneous determination method for eight AA metabolites using LC-MS/MS. Although Shinde et al. have separated EETs and DHETs at comparable retention times, their method used only one internal standard (20-HETE-d₆) for determination of eicosanoids and excluded an information for 5,6-EET.²⁵⁾ To minimize determination and variation errors, we used the deuterated eicosanoids as internal standards for each unlabeled eicosanoid, with the exception of 5,6-DHET-d₁₁ (which is not available commercially). The deuterated materials are adequate internal standards for determination by the MS(MS) system, because the physico-chemical properties of the deuterated materials are identical to those of the unlabeled materials, with the exception of molecular weights.²⁸⁾ Furthermore, we detected 5,6-EET with its deuterated material in this method.

Blewett et al. used a similar gradient mobile phase system (a mixture of acetonitrile and water containing 0.1% formic acid) for the assaying of 23 eicosanoids.¹⁹⁾ Their methodology was time-consuming, with DHETs, EETs, and AA eluting at approximately 29, 45, and 52 min, respectively. Our method focused on the CYP pathway of AA metabolism to investigate drug-endogenous interactions, and we sought to determine EETs and DHETs in a shorter time. Therefore, in the present study, the constitution of acetonitrile and water was set to 1 : 1 at the start of the determination. Using our method, DHETs and EETs eluted at retention times within 30 min.

Recently, Edpuganti and Mehvar have developed the method to permit separation of AA and its P450 metabolites within 2.5 min using an UHPLC-MS/MS system,²⁷⁾ and applied the technique to determine the levels of free eicosanoids in rat livers. Those researchers used only one or two internal standards to measure EETs and DHETs. In contrast, the present study used a conventional LC-MS/MS system to determine eicosanoid levels using the seven internal standards, permitting determination without overlapping interference peaks.

Our method was designed to facilitate incubation studies of AA exposed to rCYP enzymes. Therefore, we used a solid-phase extraction step to purify the samples. The extraction rates of eicosanoids by solid-phase extraction were 75–101% and CV% values were less than 10%, except for that for 5,6-EET (13.4%, data not shown).

In the present study, use of the deuterated eicosanoids as internal standards provided reduced intra- and inter-day CV%s for almost all samples (Table 3), especially EETs, compared to values obtained by previous assay methods.^25,26,29) However, the inter-day CV% of 50 nmol/L 5,6-DHET (for which deuterium-labeled material was not commercially available) was 16.7%. Some researchers have reported that DHETs also have biological effects, albeit with much lower activities than EETs.^5,30,31) Since 5,6-EET is a poor substrate for sEH,³²⁾ the amount of 5,6-DHET formed in the incubation study with AA is expected to be negligible.

In our experiments, we observed peaks that eluted after DHETs and before EETs. We speculate that these peaks corresponded to HETEs,^19–23) which are also metabolites of AA, and/or impurities contained in standard solution of AA. EETs and DHETs were detected at much higher levels than the respective LLOQs in the samples incubated for 30 min with rCYP2C9 (Fig. 3A), while none of the EETs and DHETs were detected in blank rCYP2C9 reactions (data not shown). These results demonstrate sensitivity sufficient to measure EETs and DHETs generated in samples derived from studies in which AA was incubated in the presence of rCYP enzymes. Although the amounts of endogenous EETs and DHETs contained in the control microsomes were negligible, EETs also were detected at levels close to the LLOQ in the samples incubated with control microsomes (Fig. 3B). These results suggest that non-specific AA metabolism (e.g., by extraneous proteins co-purifying with recombinant enzymes) may occur with rCYP2C9 derived from bacurovirus. Thus, such non-specific effects should be considered in the analysis of incubation experiments using rCYPs; the inclusion of control microsomes is expected to address these limitations.

In conclusion, we have developed a simple LC-MS/MS method for simultaneous detection of EETs and DHETs. This technique is applicable for the determination of the levels of eicosanoids in samples derived from incubation of AA with rCYP enzymes. This method is expected to be useful in inhibition studies designed to investigate drug-endogenous substance interactions.

Acknowledgment

This work was supported by JSPS KAKENHI Grant No. 22590141.

Conflict of Interest

The authors declare no conflict of interest.

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