Characterization and identification of in vitro metabolites of (-)-epicatechin using ultra-high performance liquid chromatography-mass spectrometry

Purpose: To characterize and identify metabolites of (-)-epicatechin in microsomal fraction of rat hepatocytes (MFRHs). Methods: A single incubation of (-)-epicatechin (1 mL, 50 μg/mL) in MFRH (0.5 mg/mL) was used for the generation of metabolites. Thereafter, the sample was subjected to protein precipitation prior to analysis with ultra-high performance liquid chromatography coupled to linear ion-trap orbitrap mass spectrometry (UHPLC-LTQ-Orbitap MS). Results: Nine metabolites of (-)-epicatechin were characterized on the basis of high resolution mass measurement, MS spectra and literature data. Based on their structures, the major metabolic routes of (-)-epicatechin in MFRHs were identified as hydroxylation, dihydroxylation and glycosylation. Conclusion: This is the first report on metabolites of (-)-epicatechin in MFRHs, and it is helpful in gaining deeper insight into the metabolism of (-)-epicatechin in vivo. The results will also provide guidance in research on the pharmacokinetics of new drugs.


INTRODUCTION
(-)-Epicatechin and its isomer (+)-catechin, which belong to the flavan-3-ol family, are ubiquitously distributed in plants [1].They have been used in many fields, such as food and medical industries due to their anti-oxidative [2], anti-microbial [3,4], and cardio-protective properties [5].Research on the metabolism of drugs is a very important step in discovery of new drugs.It is also vital for drug development, pharmacokinetics, and clinical pharmaceutics [6].However, not much is known about the metabolites of (-)-epicatechin [7].On the other hand, more than 40 metabolites of the isomer of (-)-epicatechin, (+)-catechin have been isolated and characterized through in vitro and in vivo studies [8][9][10][11].
The technique of liquid chromatography coupled with mass spectrometry (LC-MS) is a useful tool for studying the metabolites of drugs in vitro or in vivo [12].However, ultra-high performance liquid chromatography (UHPLC) can provide a higher and faster separation, and less solvent consumption than LC-MS [13], while highresolution mass spectrometry (HRMS) can provide elemental composition by accurate mass measurement.Therefore, UHPLC-HRMS has been widely used for the characterization of drug metabolites among several different LC/MS platforms [14][15].
The aim of this investigation was to isolate and characterize (-)-epicatechin metabolites from MFRHs.

EXPERIMENTAL Reagents
Authentic (-)-epicatechin standard (purity ＞98.0 %) was product of Chengdu Biopurify Phytochemicals Co, Ltd (Sichuan, China).Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Fisher, Fair Lawn, USA).Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was supplied by Zhong Sheng Rui Tai Biotech (Beijing, China), while a Milli-Q system (Millipore, MA, USA) was used for preparing distilled water.Rat liver microsomes were product of BD Biosciences (Bedford, MA, USA).Magnesium chloride and Tris-HCL buffer used in this experiment (pH 7.4) were of analytical grade.

Incubation of microsomes
Metabolic transformation was carried out in vitro in a final volume of 1 mL by incubating (-)epicatechin with RLMs in a shaking water bath at 37 o C. (-)-Epicatechin (50 µg/mL) was preincubated for 5 min in 0.1 mol/L Tris-HCl buffer (pH 7.4) containing 5mM MgCl 2 and 0.5 g/mL MFRHs.Next, NADPH (1 mM) was added to initiate the reaction.The reaction was allowed to proceed for I h, and then stopped by introduction of ice-cold acetonitrile (1 mL) to the reaction mixture.The mixture was vortexed and clarified by centrifugation at 4 °C for 10 min at 15,000 rpm.The metabolites were identified by injection of 5 μL of the supernatant into UHPLC-LTQ-Orbitrap MS.A solution prepared in a similar manner but lacking (-)-epicatechin served as blank.All analyses were done in triplicate.
Negative ion mode was used in the MS/MS operations, and mass analysis was carried out at a resolution of 30,000 in the range of 100 to 800 m/z.The capillary voltage, source voltage, capillary temperature, tube lens sheath gas flow and auxiliary gas flow rate were 35 V, 3.0 kV, 350 °C, 110 V, 30 and 10 arbitrary units, respectively.

Data processing
Data acquisition and processing were performed with Thermo X caliber 2.1 workstation (Thermo Fisher Scientific), and the results were compared with that from blank MFRH samples obtained under identical conditions.

Metabolic routes of (-)-epicatechin
In order to identify the metabolites of (-)epicatechin, the first step of this work was to study the MS 2 fragmentation pattern of (-)epicatechin.The parent ion [M-H] -was at m/z 289.0704 (-0.9 ppm，C 15 1).These product ions aided the identification of the metabolites of (-)-epicatechin.

Identified metabolites
The results from high resolution extracted ion chromatography (HREIC) are shown in Figure 2. Nine metabolites of (-)-epicatechin were tentatively characterized on the bases of high resolution mass measurements, MS spectra, and literature data in negative ion mode.The UPLC-MS data are summarized in Table 1. in their MS 2 spectra, which confirmed that they were metabolites of (-)-epicatechin.Therefore, they were tentatively characterized as hydroxylated products of (-)-epicatechin.
The compounds M1 and M2 were eluted at 6.54 and 7.38 min with the same deprotonated molecules at m/z 331.0806 (-1.9 ppm, C 17 H 15 O 7 ), which was a +36 Da (2O)-shift from that of (-)epicatechin.The MS2 spectra of M1 and M2 showed the major fragment ions at m/z 313, m/z 287, and m/z 269, due to loss of H 2 O, CO 2 , and H 2 O+ CO 2 , respectively from the parent drug which had similar fragmentation pattern with (-)epicatechin.Based on previous analyses, they were identified as dihydroxylated products of (-)epicatechin.moiety relative to the precursor ion at m/z 451 in their MS 2 spectra indicated that a glucosyl moiety was present.Thus, they were presumed to be glucosyl products of (-)-epicatechin.

DISCUSSION
The results obtained from preliminary trials with various mobile phase systems in this study showed that good chromatographic peak shapes could be achieved by using mobile system without formic acid, while much lower column pressure was afforded by the inclusion of acetonitrile in the mobile phase.Therefore, the mixture of acetonitrile and water was chosen as the mobile phase solvent system in this study.A gradient elution pattern was adopted.
(-)-Epicatechin is characterized by the presence of many hydroxyl groups on the flavanol skeleton, which make it suitable for detection by ESI in negative mode [15].To the best of our knowledge, many studies have so far focused mainly on the metabolites of (+)-catechin, while not much has been done on the elucidation of the metabolites of (-)-epicatechin.For example, 40 and 58 metabolites of (+)-catechin were found in in vitro and in vivo investigations, respectively [8][9][10][11].Among these, 7 metabolites were identified in MFRHs [16].It is worth mentioning that (-)-epicatechin has a metabolic pathway similar to that of (+)-catechin in MFRHs.Therefore, it is scientifically reasonable to assume that the in vivo metabolisms of (-)epicatechin and (+)-catechin will follow a similar route.This will be helpful in the identification of metabolic products of (-)-epicatechin in vivo.

CONCLUSION
The in vitro metabolites of (-)-epicatechin have been successfully identified based on UHPLC-LTQ-Orbitrap analysis.Based on the 9 metabolites identified and their MS data, the major metabolic pathways of (-)-epicatechin in RLMs are hydroxylation, dihydroxylation and glycosylation.This is the first report on the metabolites of (-)-epicatechin in RLMs and is considered a useful guide for understanding its metabolism in vivo.In addition, the methodology used in this study offers a new perspective for studying the metabolism and pharmacokinetics of new drugs.