Fitoterapia 93 (2014) 88–97

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Biotransformation of isoimperatorin by rat liver microsomes and its quantification by LC–MS/MS method Tian-Li Chen a,b, You-Bo Zhang b, Wei Xu b, Ting-Guo Kang a,⁎, Xiu-Wei Yang b,⁎ a

School of Pharmaceutical Sciences, Liaoning University of Traditional Chinese Medicine, Liaoning 116600, PR China State Key Laboratory of Natural and Biomimetic Drugs (Peking University), Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, Peking University, Beijing 100191, PR China

b

a r t i c l e

i n f o

Article history: Received 19 October 2013 Accepted in revised form 18 December 2013 Available online 29 December 2013 Keywords: Isoimperatorin Metabolism Rat liver microsomes LC–QqQ–MS Qualitative analysis Quantitative analysis

a b s t r a c t The aim of the present research was to establish a comprehensive strategy to identify the metabolites of isoimperatorin after biotransformation with rat liver microsomes in vitro, and further describe metabolic kinetic characteristics of isoimperatorin and its main metabolites. Utilizing liquid chromatography with time of flight mass spectrometry (LC–TOF–MS), 18 metabolites (M 1–18) were characterized according to the typical fragment ions and literature data. Among them, M-2, 3, 5, 9, 10, and 15 were new compounds. To further verify structures of the metabolites, five main metabolites were obtained from the magnifying biotransformation incubation system, and their chemical structures were elucidated as 8-hydroxyoxypeucedanin (M-3), hydroxypeucedanin hydrate (M-4), E-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-11), Z-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-12), and oxypeucedanin (M-16) by various spectroscopy methods including IR, MS and NMR. A simple new liquid chromatography with triple quadrupole tandem mass spectrometry (LC–QqQ–MS) method was developed for the simultaneous determination of isoimperatorin and its main metabolites. The analysis was performed on a Diamonsil™ ODS C18 column with acetonitrile–water containing 0.1% formic acid as mobile phase. Total run time was 20.0 min. The results suggested that the method we exhibited was successfully applied for analysis of isoimperatorin and its metabolites. The study provides essential data for proposing metabolite pathway and further pharmacological study of isoimperatorin. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Isoimperatorin, along with its positional isomers of imperatorin, is abundant in umbelliferous vegetables, citrus fruits and several herbal medicines [1,2]. It is one of the main bioactive components of Angelica dahurica cv. Qibaizhi [3] and Angelica pubescens f. biserrata [4], as well as Glehnia littoralis Fr. Schmidt. ex Miq. [5–7], Notopterygium incisum Ting exH. T. Chang [8], and Ferulago subvelutina [9]. As an active natural furocoumarin, isoimperatorin was revealed to possess a variety of similar

⁎ Corresponding authors at: No. 38, Xueyuan Road, Haidian District, Beijing 100191, PR China. Tel.: +86 10 82805106; fax: +86 10 82802724. E-mail addresses: [email protected] (T.-L. Chen), [email protected] (T.-G. Kang), [email protected] (X.-W. Yang). 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.017

biological activity to imperatorin. Several previous reports revealed that isoimperatorin exhibited an analgesic effect through an effect on both the peripheral and central nervous systems [10], and had obviously analgesic, spasmolytic, and antitumor activities [11]; also, it could significantly repress the smooth muscle spasm of rabbit's isolate intestine caused by BaCl2 and inhibit the proliferation of tumor cells of HL-60, P388, and HELA in vitro. A bioassay-guided isolation of the root of Angelica dahurica demonstrated that isoimperatorin exhibited a significant antitumor activity, and it could inhibit the proliferation of human tumor cells as A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system), and HCT-15 (colon) in vitro [12]. Through inhibition of β-secretase, isoimperatorin was found to play an important role in treating of Alzheimer's disease [13,14]. Other

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investigations also have shown that isoimperatorin has calcium channel blocking activity, and it exhibited high inhibition of Ca2+ uptake using GH4C1 cells [15]. With the continuously increasing interest in isoimperatorin for its various activities, researchers have launched a series of experiments in vivo and in vitro to evaluate if it can be developed as a promising new drug. For pharmacokinetic and distribution study, isoimperatorin was found to have moderate elimination rate and a wide range of distribution in vivo [16,17]. Li et al. [18] researched the absorption characteristics of isoimperatorin using a model of Caco-2 cell monolayer in human intestine, and revealed that it is a well absorbed compound. In addition, a drug excretion experiment revealed that only less than 1% of the isoimperatorin as a parent drug was obtained from rat bile and urine [19], indicating that isoimperatorin tends to metabolize in vivo and was excreted mainly in the form of metabolites. Utilizing LC/TOF–MS method, Shi et al. [2] speculated in vivo metabolites in rat and in vitro microbial biotransformation products of isoimperatorin, but it was not verified by other instruments such as NMR on in vivo metabolites in rat. LC–TOF–MS is a sensitive instrument that can provide accurate mass and retention time data to identify analytes. The data of TOF–MS not only allows detecting of known compounds, but also allows searching for unknown analytes. This ability increases the confidence of metabolite identification and selectivity. It is more and more widely applied in drug metabolism research. Although the LC/MS/MS method has advantages of being simple and rapid in bioanalysis, it is incapable of distinguishing some different isomers especially in the stereoscopic chemistry. The only reliable method now for isomers is to prepare metabolite using chromatography, and identify chemical structure by spectral methods. Metabolism of a candidate drug often decides whether it is safe and effective for clinical application. Hence, candidate compounds are usually screened early of drug development. Liver microsomes are a commonly used system to measure hepatic metabolism of drugs; the system contains the main drug-metabolizing enzymes, as the cytochrome P450 (CYP450) family and flavin monooxygenase. To date, there has been no report about metabolism of isoimperatorin in vitro with liver microsomes. The aim of this study is to analyze the metabolites of isoimperatorin in vitro and further describe its metabolic kinetic characteristics. 2. Experimental 2.1. Chemicals and reagents Isoimperatorin and bergaptol were isolated and purified from the ethanol (EtOH) extract of the roots of Angelica dahurica cv. Qibaizhi [3] and Notopterygium incisum Ting ex H. T. Chang in the previous studies [8]. The chemical structures were determined by NMR and MS spectra with purity N99.8% analyzed by RP-HPLC method. Daidzein as an internal standard (IS) substance was purchased from the National Institutes for Food and Drug with purity N 99.0%. Reduced β-nicotinamide adenine dinucleotide (NADH), β-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose-6-phosphate disodium salt hydrate (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-PDH) were purchased from Sigma Chemical Co. (St. Louis,

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Mo, USA). Sodium phenobarbital (PB) was obtained from Haidian Hospital of Beijing. LC–MS grade methanol (MeOH) and acetonitrile (MeCN) was purchased from J. T. Baker (Phillipsburg, USA). HPLC-grade formic acid was obtained from Dikma Tech. Inc. (Beijing, China). Water (H2O) was of milli-Q grade (Millipore, Billerica, MA, USA). 2.2. Instruments and chromatographic conditions Isolation of metabolites was performed on a semipreparative HPLC system. The system consisted of a LabTech P600 pump, a LC3000 UV detector, and a 7125 Rheodyne injector (Rheodyne, Cotati, CA, USA) with a loop of 5 mL. The LC Workstation is Labtech Chromsoftware (LabTech Co., Beijing, China). A preparative Phenomenex Prodigy C18 column (250 × 21.2 mm i.d., 10 μm; Phenomenex, Torrance, CA, USA) equipped with a C18 guard column (8 × 4 mm i.d., 5 μm; Dikma, China) was used for isolation and purification of the metabolites. NMR spectra were recorded on a Bruker AV 400 spectrometer with CDCl3 as solvents and TMS as internal standard. IR spectra were measured on a Thermo Nicolet Nexus 470 FT-IR spectrometer. EIMS spectra were obtained on a Finnigan TRACE 2000 GC-MS spectrometer. For qualitative analysis, Shimadzu HPLC system (consisted of a binary LC-20 AD pump, an SIL-20 AC autosampler, a CTO-20A column oven, an SPD-M20A PDA detector, and a CBM-20A system controller) coupled to an IT–TOF massspectrometer (Shimadzu, Kyoto, Japan) through an ESI interface was used for HPLC–DAD–ESI–IT–TOF–MSn analysis. All data were analyzed by Shimadzu software (Shimadzu LCMSsolution Version 3.60, Formula Predictor Version 1.2, and Accurate Mass Calculator). For mass detection, the ESI source was operated in positive ion mode and the mass spectrometer was in full scan ranges of m/z 100–800 for MS1 and m/z 50–500 for MS2 and MS3. The following parameters were set as: the heat block and curved desolvation line temperature, 200 °C; the temperature of CDL, 200 °C; the diversion ratio, 1:4; the flow of desolvation gas (N2), 1.5 L/min. For quantitative analysis, The LC–MS/MS system contained an analytical DIONEX Ultimate 3000 HPLC system (consisted of an Ultimate 3000 Pump, a DIONEX Ultimate 3000 Autosampler and a DIONEX Ultimate 3000 Compartment) and a 4000QTRAP triple quadrupole tandem mass spectrometer (Applied Biosystems/MDS Sciex, Canada) equipped with an electrospray ionization (ESI) source for the mass analysis and detection. Analyst 1.5.1 software (Applied Biosystems/MDS Sciex, Canada) was used for data collection and analysis. The chromatography conditions were the same for qualitative and quantitative analyses. The mobile phase consisted of (A) MeCN and (B) H2O containing 0.1% formic aicd (v/v) with gradient elution (0–8.0 min, 35–47%A; 8–12 min, 47–85%A; 12–20 min, 35%A). An Agilent ZORBAX SB-C18 column (150 × 4.6 mm i.d., 5 μm) was used for all separation and the flow rate was set at 1.0 mL/min. The wavelength was set at 315 nm for qualitative analysis. The inject volumes were 10 μL for qualitative analysis and 5 μL for quantitative analysis. 2.3. Experimental rat and preparation of rat liver microsomes The male Sprague–Dawley (SD) rats weighting (200 ± 10) g were obtained from the Laboratory Animal Center of the Peking

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University Health Science Center (Beijing, China). All of the experimental procedures were in compliance with the guidelines defined by the Peking University Committee on Animal Care and Use for the use of experimental animals. Liver microsomes were prepared according to the previous researches we have reported [20–22] and frozen in −80 °C before analysis. The microsomal protein concentration was determined by BCA protein quantitative method with bovine serum albumin as a standard [23]. Total CYP450 was determined using the differential spectrophotometric method as described in the previous papers [20,22]. Liver microsomes were found to contain 2.01 nmol CYPase per mg protein (20.8 mg proteins/mL).

2.4. Isoimperatorin biotransformation by rat liver microsomes The experimental procedure was conducted with the previous method we have reported [20,22]. For qualitative analysis, a typical biotransformation incubation mixture consisted of 100 mmol/L potassium phosphate buffer (pH 7.4), 2 mg/mL protein (rat liver microsomes), 1 mmol/L isoimperatorin, and 1 mmol/L NADPH. The final volume was 1.0 mL. The reaction was initiated by adding the NADPH generating system. After incubation at 37 °C for 30 min, the reaction was terminated by adding 2.0 mL of MeOH. Then, the mixture was centrifugated at 16,000 g for 15 min. The upper layer was transferred to a glass pipes and dried under a gental N2 gas at 37 °C. The residue was redissolved with 200 μL MeOH and submitted to LC–IT–TOF–MSn system for analysis. Eighteen metabolites (named M1–18) were detected and identified. To gain metabolites of isoimperatorin, a total of 1 L biotransformation incubation system consisted of 2 g protein (rat liver microsomes), 100 mmol/L potassium phosphate buffer (pH 7.4) and 300 mg of isoimperatorin as substrate was prepared. The reaction was initiated by NADPH-generating system. After incubation at 37 °C for 2 h, the reaction was terminated and extracted by adding 1 L of ice-cold cyclohexane. Then the system was extracted with 2 L ethyl acetate (EtOAc) for five times. The supernatant was combined and evaporated to dryness by rotary evaporator. The residue (3.4 g) was applied to a silica gel column chromatography and eluted with cyclohexane–EtOAc and EtOAc–MeOH. The eluant was collected and divided to five fractions. Then, the fractions were submitted to semi-preparative HPLC for separation and purification of the metabolites. The monitor wavelength was set at 325 nm. Fractions 1 and 2 were eluted with MeCN–H2O (30: 70, v/v) to yield metabolites 3 (M3, yield 13.33 mg) and 4 (M4, 1.95 mg). Fractions 3 and 4 were eluted with MeCN–H2O (35: 65, v/v) to yield metabolite 11 (M11, 5.33 mg). Fractions 5 was eluted with MeCN–H2O (40: 60, v/v) to gain metabolites 12 (M12, 4.23 mg) and 16 (M16, 30.03 mg). For quantitative analysis, the biotransformation incubation system consisted of 100 mmol/L potassium phosphate buffer (pH 7.4), 2 mg/mL protein (rat liver microsomes), 20 μmol/L isoimperatorin, and 1 mmol/L NADPH. The final volume of biotransformation incubation mixture was 200 μL and the reaction was carried out in 96-well plates. The reaction was initiated by adding the NADPH generating system after preincubation for 5 min and terminated at 0, 30, 60, 90, and 120 min by imbibing 150 μL reaction liquid to 200 μL ice-cold MeOH which contained 20 μL daidzein (IS)

solution. After centrifugation at 16,000 g for 15 min, the supernate was submitted to LC–MS system for analysis. 2.5. Spectroscopic data of main metabolites 2.5.1. 8-Hydroxyoxypeucedanin (M3) Pale yellow crystallization (EtOAc); IRνKBr maxcm−1: 3434, 2924, 2853, 1738, 1720, 1692, 1589, 1433, 1381, 1352, 1054; EI–MS m/z 302 [M]+, 260, 231 (100), 217, 203, 175, 160, 149, 123, 95, 85, 69; HR–ESI–MS m/z 303.0894 [M + H]+ (calcd. for C16H15O6, 303.0869); 1H NMR (CDCl3, 400 MHz) δ: 8.19 (1H, d, J = 8.6 Hz, H-4), 7.83 (1H, d, J =1.8 Hz, H-2′), 6.93 (1H, d, J = 1.8 Hz, H-3′), 6.32 (1H, d, J = 9.6 Hz, H-3), 4.60 (1H, dd, J = 10.8, 4.0 Hz, Ha-1″), 4.25 (1H, dd, J = 10.8, 6.5 Hz, Hb-1″), 3.20(1H, dd, J = 4.0, 6.4 Hz, H-2″), 1.41 (3H, s, 3″– CH3), 1.38 (3H, s, 3″–CH3). The 1H NMR spectrum of M3 was similar with M16, but the signal of H-8 could not be observed. The molecular weight of M3 was 302 Da according to the EI–MS spectrum, which is larger 16 Da than that of M16. The data indicated that there was one hydroxyl group was at C-8 position of M3. Thus, M3 was named 8-hydroxyoxypeucedanin, it is a new compound. 2.5.2. Oxypeucedanin hydrate (M4) Pale yellow crystallization (EtOAc); IRνKBr maxcm−1: 3400, 2969, 2917, 2849, 1720, 1622, 1578, 1355, 1133; EI–MS m/z 304 [M]+, 289, 271, 244, 229, 215, 203, 202 (100), 174, 157, 145, 89, 59; HR–ESI–MS m/z 305.1016 [M + H]+ (calcd. for C16H17O6, 305.1025); 1H NMR (CDCl3, 400 MHz) δ: 8.13 (1H, d, J = 9.6 Hz, H-4), 7.57 (1H, d, J = 2.5 Hz, H-2′), 7.12 (1H, s, H-8), 6.98 (1H, d, J = 2.5 Hz, H-3′),6.25 (1H, d, J = 9.6 Hz, H-3), 4.51 (1H, dd, J = 9.6, 2.5 Hz, Ha-1″), 4.41 (1H, dd, J = 9.6 Hz, 7.5 Hz, Hb-1″), 3.88 (1H, dd, J = 7.5, 2.6 Hz, H-2″), 1.29 (3H, s, 3″–CH3), 1.33 (3H, s, 3″–CH3); 13C NMR (CDCl3, 100 MHz) δ: 25.1 (3″–CH3), 26.6 (3″–CH3), 71.6 (C-1″), 74.3 (C-2″), 76.4 (C-3″), 94.6 (C-8), 104.5 (C-3′), 107.1 (C-10), 113.3 (C-3), 114.1 (C-6), 139.2 (C-4), 145.1 (C-2′), 148.4 (C-5), 152.4 (C-9), 158.0 (C-7), 161.2 (C-2). The above data were in agreement with those in literature [3]. 2.5.3. E-5-(4-Hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (M11) Pale yellow crystallization (EtOAc); IRνKBr maxcm−1: 3438, 2954, 2916, 2848, 1719, 1622, 1579 1359, 1071; EI–MS m/z 286 [M]+, 270, 268, 203, 202 (100), 174, 157, 149, 145, 118, 89; HR–ESI–MS m/z 287.0919 [M + H]+ (calcd. for C16H15O5, 287.0919), 309.0739 [M + Na]+ 309.0739 (calcd. for C16H14NaO5, 309.0739), 325.0476 (calcd. for C16H14KO5, 325.0478); 1H NMR (CDCl3, 400 MHz) δ: 8.16 (1H, d, J = 9.8 Hz, H-4), 7.60(1H, d, J = 2.4 Hz, H-2′), 7.17 (1H, s, H-8), 6.96 (1H, d, J = 1.5 Hz, H-3′),6.28 (1H, d, J = 9.8 Hz, H-3), 5.85(1H, t, J = 6.7Hz, H-2″), 5.01 (2H, d, = 6.5Hz, H-1″), 4.10 (2H, s, 4″–CH2OH), 1.73 (3H, s, 5″–CH3); 13C NMR (CDCl3, 100 MHz) δ: 13.7 (5″–CH3), 67.4 (4″–CH2), 69.2 (C-1″),94.4 (C-8), 104.9 (C-3′), 107.4 (C-10), 112.8 (C-3), 114.1 (C-6), 120.9 (C-2″), 139.4(C-4), 141.4(C-3″), 145.0 (C-2′), 148.7(C-5), 152.7 (C-9), 158.2 (C-7), 161.2 (C-2). The 1H and 13C NMR spectra of M11 and M12 were similar to each other. However, some small differences in chemical shifts were observed. In the 1 H NMR spectrum, the signals at δH 4.20 (2H, s, 4″–CH2OH) and 1.89 (3H, s, 5″–CH3) in M12 showed an up-field shift to δH 4.10

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(2H, s, 4″–CH2OH) and 1.73 (3H, s, 5″–CH3) in M11, In the 13C NMR spectrum, δC 21.4 (5″–CH3) and 61.9 (4″–CH2OH) showed an up-field shift to δC 13.7 (5″–CH3) and a down-field shift to δC 67.4 (4″–CH2). The molecular formula of M11 was C16H15O5 according to the HR–ESI–MS and EI–MS spectra. The above data demonstrated that M11 and M12 were the cis-trans-isomers. 2.5.4. Z-5-(4-Hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (M12) Pale yellow crystallization (EtOAc); IRνKBr maxcm−1: 3428, 2923, 2852, 1731, 1624, 1579, 1455, 1367, 1351, 1131; EI–MS m/z 286 [M]+, 268, 203, 202 (100), 201, 175, 174, 157, 146, 145, 118, 89; HR–ESI–MS m/z 287.0914 [M + H]+ (calcd. for C16H15O5, 287.0920); 1H NMR (CDCl3, 400 MHz) δ: 8.16 (1H, d, J = 9.7 Hz, H-4), 7.60(1H, d, J = 2.4 Hz, H-2′), 7.17 (1H, s, H-8), 6.96 (1H, d, J = 1.5 Hz, H-3′),6.29 (1H, d, J = 9.8 Hz, H-3), 5.70(1H, t, J = 6.8Hz, H-2″), 5.03 (2H, d, = 6.9Hz, H-1″), 4.20 (2H, s, 4″–CH2OH), 1.89 (3H, s, 5″–CH3); 13 C NMR (CDCl3, 100 MHz) δ: 21.4 (5″–CH3), 61.9 (4″– CH2OH), 68.9 (C-1″),94.5 (C-8), 104.9 (C-3′), 107.5 (C-10), 112.8 (C-3), 114.1 (C-6), 122.1 (C-2″), 139.4(C-4), 141.4(C-3″), 145.1 (C-2′), 148.6 (C-5), 152.6 (C-9), 158.1 (C-7), 161.2 (C-2). The above data were in agreement with the literature data [24]. 2.5.5. Oxypeucedanin (M16) Pale yellow crystallization (EtOAc); 3435, 2963, 2923, 2858, 1727, 1625, 1579, 1385, 1345, 1129; EI–MS m/s 286 [M]+, 215, 202, 201, 187, 173, 157, 145, 129, 89, 85, 59 (100), 57; HR–ESI–MS m/z 287.0919 [M + H]+ (calcd. for C16H15O5, 287.0920); 1H NMR (CDCl3, 400 MHz) δ: 8.20 (1H, d, J = 9.8 Hz, H-4), 7.61 (1H, d, J = 2.4 Hz, H-2′), 7.19 (1H, s, H-8), 6.95 (1H, d, J = 2.2 Hz, H-3′), 6.31 (1H, d, J = 9.8 Hz, H-3), 4.59 (1H, dd, J = 10.8, 4.3 Hz, Ha-1″), 4.43 (1H, dd, J = 10.8, 6.5 Hz, Hb-1″), 3.23 (1H, dd, J = 4.4, 6.4 Hz, H-2″), 1.41 (3H, s, 3″–CH3), 1.33 (3H, s, 3″–CH3); 13C NMR (CDCl3, 100 MHz) δ: 19.0 (3″–CH3), 24.6 (3″–CH3), 58.3 (C-3″), 61.1 (C-2″), 72.3 (C-1″), 94.8 (C-8), 104.7 (C-3′), 107.4 (C-10), 113.2 (C-3), 113.5 (C-6), 138.9 (C-4), 145.3 (C-2′), 148.3 (C-5), 152.5 (C-9), 158.0 (C-7), 161.9 (C-2). The above data were in agreement with those in literature [25].

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linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), accuracy and precision, extraction recovery, and matrix effects. Specificity was determined from the lack of interfering peaks of endogenous substances at the retention times of isoimperatorin and the main metabolites. Linearity was detected at six level of concentrations that cover the ranges of the analytes with r ≥ 0.99. The LLOD and LLOQ were set at the lowest concentration giving a signal–noise (S/N) ratio of 3:1 and 10:1, respectively. The intra- and inter-day precisions were presented as the relative standard deviations (RSDs) and accuracy was the percentage of calculated concentration and added concentration of the analytes at low, medium and high concentrations. The extraction recoveries of the analytes were measured by comparing peak areas extracted from HILM with those of the same amounts in MeOH at three concentrations. Stability test of the analytes in HILM was examined with QC samples stored at room temperature (22 °C) for 48 h. All of the samples were assayed at low, medium and high concentrations in triplicate. Matrix effect (ME) was assessed by comparing corresponding peak areas of isoimperatorin and the metabolites in pre-extracted HILM to those of the analytes directly dissolved in the mobile phase. 3. Results and discussion 3.1. The strategy for chemical structure identification of the metabolites

2.6. Method validation for quantification

The products of isoimperatorin biotransformation with rat liver microsomes in vitro were first analyzed using comprehensive spectroscopy technology as LC–IT–TOF–MSn, NMR, IR, EI–MS and UV, etc. Based on the fragment rules of furocoumarins and the literature reports [2,27], we speculated structures of the metabolites by LC–TOF–MSn in positive-ion mode utilizing Shimadzu LC–MS solution software. Then, the five main metabolites were prepared and isolated by amplification of the biotransformation incubation system, and their structures were verified by above-mentioned spectroscopies. In turn, the structures of other metabolites were further determined by analysis fragmentation rules of the main metabolites.

The stock solutions of isoimperatorin (0.7 mg/mL), M4 (0.5 mg/mL), M7 (0.5 mg/mL), M11 (0.5 mg/mL) M12 (0.5 mg/mL), and M16 (0.5 mg/mL) were all prepared in MeOH and kept at 4 °C. In order to construct calibration curves, a series of standard solutions of the six compounds above was prepared by appropriately diluting the stock solutions. The stock solution of IS was prepared in MeOH and diluted to a final concentration of 50 ng/mL. Six calibration curves were constructed using peak area ratios of the analytes and IS vs the concentrations of the analytes. Quality control (QC) samples were prepared at low, medium and high concentrations by adding the six analytes to drug-free heat-inactivated liver microsomes (HILM) and dealing with the mixture as the analytical samples, respectively. The method was validated according to the industrial guidelines of the US Food and Drug Administration for bioanalytical method validation [26]. It includes specificity,

3.1.1. Identification of the metabolites of isoimperatorin by LC–IT–TOF–MSn A total of 18 metabolites were identified based on the retention times, molecular weights, parent ions and fragment ions which are presented in Table 1. The fragment rules of the main metabolites were shown in Fig. 1. The fragment rules of the parent compound (isoimperatorin) were first analyzed. In the full-scan mass spectrum, isoimperatorin (M0) yielded [M + H]+ ion at m/z 271.0969 with 13.92 min of retention time (Rt). The typical fragment ions were at m/z 203, 175, 159, 147, 131, 119 and 91, and the relative abundance of m/z 159 and 131 was higher than that of m/z 175 and 147, which was identical with the literature reports [2,27]. In the full-scan mass spectrum, M1 yielded [M + H]+ ion at m/z 303.0872 with Rt = 3.27 min. Its molecular formula was determined to be C16H14O6 (as shown in Table 1)

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according to the HR–MS data, which indicated that M1 was an oxidative metabolite of isoimperatorin. The daughter ion with m/z 203.0372 of M1 was the typical characteristic fragment ion of the mono-oxysubstituted furocoumarins [27], suggesting that the metabolic reaction was occurred only on the isopentenoxyl group of isoimperatorin. By comparing the Rt and daughter ions of M1 with literature [2], the structure of M1 was identified as 5-(2-methylbutyl acid-4-oxy)-psoralen. The molecular formulae of M2 and M3 were C16H16O7 and C16H14O6, respectively, according to the HR–MS data. In MS2 spectra, the two metabolites have the same fragment ion at m/z 219 which was formed by the neutral loss of 102 Da and 84 Da (C5H10O2 and C5H8O) from M2 and M3. Contrasted with the neutral loss (68 Da of C5H8) of isoimperatorin, it could be deduced that the isopentenoxyl group of isoimperatorin was metabolized to 4-hydroxy-3-hydroxymethylbutyl group to form M2 and metabolized to epoxyisopentenyl group to form M3. The daughter ions of M3 with m/z 287 further confirmed our speculation. The typical fragment ion at m/z 219 of M2 and M3 suggested that the oxidation also occurred in the linear type furocoumarin nucleus. According to the reports [24,28], the hydroxylation was probably at C-8 position. So the chemical structures of M2 and M3 were identified as 5-(4-hydroxy-3-hydroxymethylbutyloxy)-8hydroxy-psoralen and 8-hydroxyoxypeucedanin. M3 was obtained by magnifying liver microsomal biotransformation in the next experiment. Its chemical structure was further determined by NMR, UV and IR spectrum. M2 and M3 were new compounds. The molecular formulae of M4, M5 and M10 were determined as C16H16O6 according to the HR–MS spectra. They have the same quasi-molecular ion [M + H]+ of m/z 305 on MS1 spectra. M4 and M5 have the typical fragment ion at m/z 203 which was followed by loss of CO or CO2 and the successive loss of CO, yielding the typical product ions at m/z

203, 175, 159, 147, 131 and 119. The mass data suggested that M4 and M5 were oxidized only on the side-chain of isoimperatorin with the isopentenoxyl group substituted by two oxygen atoms and two hydrogen atoms. M4 was obtained by magnifying liver microsomal biotransformation, and its chemical structure was determined as oxypeucedanin hydrate by NMR, UV and IR spectroscopy. As for the chemical structure of M5, two hydroxyl groups were probably linked at the two methyl groups of the isopentenoxyl group and the olefinic linkage was substituted by two hydrogen atoms. Thus, the chemical structure of M5 was deduced as 5-(4-hydroxy-3-hydroxymethylbutyloxy)-psoralen. The typical fragment ion of M10 was m/z 219 in MS2 spectrum, suggesting that one hydroxyl group was linked on the linear type furocoumarin nucleus. Compared with the chemical structure of M5, one hydroxyl group linked at the methyl group of the isopentenoxyl group probably moved to C-8 position. The chemical structure of M10 was identified as 5-(4-hydroxy-3-methylbutyl-oxy)-8-hydroxy-psoralen. M5 and M10 were new compounds. In MS1 spectra, M6, M8 and M9 displayed the same quasi-molecular ion [M + H]+ with m/z 303, their molecular formulae were determined as C16H14O6 by HR–MS spectra. As shown in Table 1, the three metabolites have identical molecular weight of 302 Da and bigger 32 Da than that of isoimperatorin. The typical fragment ion of M6 and M8 was m/z 203 as well as a series of characteristic fragment ions of m/z 175, 159, 147 and 131 Da. The fragment ions indicated that the oxidation occurred only on the side chain with two oxygen atoms added. The retention times of M6 and M8 were 4.96 and 5.22 min, their chemical structures were identified as 5-(4-hydroxy-3-hydroxymethylbutyl-2-alkenyloxy)-psoralen and 5-(4-hydroxy-3-methyl-2-oxobutyloxy)-psoralen according to the previous research [2]. The typical fragment ion of M9 was m/z 219 in MS/MS spectrum, indicating that one hydroxyl group was linked at the furocoumarin nucleus and the epoxidation

Table 1 LC–ESI–IT–TOF–MS data obtained in positive ion detection mode for identification of 18 metabolites of isoimperatorin. No.

tR

Formula

M0 13.92 C16H14O4 M1 3.27 C16H14O6 M2 3.47 C16H16O7 M3 3.52 C16H14O6 M4 3.85 C16H16O6 M5 4.09 C16H16O6 M6 4.96 C16H14O6 M7 5.06 C11H6O4 M8 5.22 C16H14O6 M9 6.09 C16H14O6 M10 6.25 C16H16O6 M11 7.25 C16H14O5 M12 7.74 C16H14O5 M13 8.19 C16H16O5 M14 9.73 C16H16O5 M15 10.92 C16H14O5 M16 11.18 C16H14O5 M17 11.34 C16H14O5 M18 13.87 C16H14O4

Major fragments ions in positive mode 271.0969, 303.0872, 321.0978, 303.0894, 305.1016, 305.1033, 303.0881, 203.0329, 303.0854, 303.0859, 305.1014, 287.0920, 287.0914, 289.1057, 289.1088, 287.0926, 287.0919, 77.0353 287.0921, 91.0513 271.0971,

203.0392, 203.0372, 219.0281, 287.0686, 203.0349, 203.0334, 203.0334, 175.0312, 203.0345, 287.0971, 237.0413, 269.0769, 203.0354, 203.0341, 221.0420, 219.0248, 271.0961,

175.0536, 159.0374, 159.0380, 147.0406, 191.0338, 190.0317, 219.0275, 173.0423, 175.0981, 159.0340, 191.0381, 175,1350, 201.0145, 175.0410, 159.0394, 147.0436, 175.0410, 147.0449, 237.0385, 219.0345, 219.0294, 191.0344, 203.0361, 175.0419, 175.0467, 159.0619, 175.0661, 159.0479, 203.0349, 159.0435, 203.1472, 199.1705, 203.0339, 175.0410,

Calculated Measured Diff (m/z) (m/z) (ppm) 147.0403, 131.0513 175.0410, 130.0837, 147.0460, 159.0479, 159.0389, 131.0402, 159.0413, 191.0369, 174.0402, 159.0449, 147.0354, 147.0449, 147.0497, 173.0305, 159.0420,

131.0518, 119.0410, 91.0604

270.0892 302.0790 320.0896 302.0790 304.0947 304.0947 302.0790 202.0266 302.0790 302.0790 304.0947 286.0841 286.0841 288.0998 288.0998 286.0841 286.0841

270.0896 302.0799 320.0905 302.0804 304.0943 304.0960 302.0808 202.0256 302.0781 302.0790 304.0941 286.0847 286.0841 288.0983 288.1014 286.0848 286.0846

1.48 2.97 2.80 4.62 −1.31 4.26 5.94 −4.93 −2.97 0.00 −1.97 2.09 0.00 −5.19 5.53 2.44 1.74

271.0872, 203.0349, 201.0204, 174.0402, 159.0481, 147.0465, 131.0502, 103.0610,

286.0841

286.0848

2.44

203.0333, 175.0460, 159.0467, 147.0450, 131.0321, 119.0465, 91.0560

270.0892

270.0898

2.21

163.0394, 123.0555 131.0392, 147.0449, 131.0467 119.0459, 131.0508 147.0679 173.0423, 147.0434, 131.0514, 131.0555, 131.0338 162.1218 147.0444,

135.0373 119.0569, 103.0417 131.0400 103.0509

163.0273, 147.0449, 135.0428 131.0523, 119.0569 103.0610 103.0610

131.0517, 103.0518, 91.0549,

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Fig. 1. MS1 and MS/MS spectra as well as the typical fragmentation pathway of the major metabolites (A-M11, B-M16) of isoimperatorin.

reaction was occurred on the isopentenoxyl group. M9 was a positional isomer of M3 and fragment ions with m/z 173 and 145 indicated that the hydroxyl group linked at the furocoumarin nucleus was probably on C-3 position according to the rules of the metabolic rule of coumarin [22,28]. The chemical structure of M9 was identified as 3-hydroxy-oxypeucedanin and it was a new compound. The quasi-molecular ion [M + H]+ of M7 was m/z 203.0329 and its molecular formula was determined as C11H6O4 by HR–MS. In MS/MS spectrum, it showed a series of characteristic fragment ions at m/z 175, 159, 147, 131, 119 and 103, suggesting that M7 was obtained by oxidative cleavage of the isopentenoxyl group from the structure of isoimperatorin to form mono-hydroxysubstituted furocoumarins. Further, the relative abundance of m/z 159 was larger than that of m/z 175, which indicated that the hydroxyl group was at C-5 position

[27]. Thus, the chemical structure of M7 was determined as bergaptol. Its fragmentation rule was identical with the reference bergaptol we isolated at the previous research [8]. M11, M12 and M16 have the same quasi-molecular ion [M + H]+ of m/z 287, their molecular formulae were determined as C16H14O5 as shown in Table 1. The three metabolites exhibited the typical fragment ions at m/z 203, 189, 175, 159, 147, 131 and 103. In MS/MS spectra, the relative abundance of m/z 159 was higher than that of m/z 175, which indicated that the side-chain of the metabolites was at C-5 position. M11 and M12 have the identical fragment ions of [M + H-H2O]+ with m/z = 269, suggesting that one hydroxyl group was linked at the side-chain. Considering the retention times and literature data [2], M11 and M12 were cis-trans-isomers and were identified as 5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen. M11

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and M12 were then obtained by magnifying liver microsomal biotransformation in the next experiment. Their chemical structures were further determined by NMR, UV and IR spectra. M11 and M12 were identified as E-5-(4hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen and Z-5-(4hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen, respectively. Fragment ion [M + H-16]+ with m/z = 271 of M16 in MS/MS spectrum indicated that an epoxidation reaction may occurred at the side-chain. Thus, the chemical structure of M16 was identified as oxypeucedanin [24]. Then, we prepared M16 in magnifying liver microsomal biotransformation experiment. Its structure was further confirmed by NMR, EI–MS and IR spectra. M13 and M14 have the same quasi-molecular ion [M + H]+ of m/z 289, their molecular formulae were determined as C16H16O5 as shown in Table 1. The two metabolites exhibited the typical fragment ions at m/z 203, 175, 159, 147, and 131 in MS/MS spectra. The relative abundance of m/z 159 was higher than that of m/z 175, which indicated that the side chain of the metabolites was at C-5 position. In the previous research, Shi, et al. [2] have prepared and identified two metabolites which have the same molecular weights with M13 and M14. According to the retention times and the MS/MS data, we identified the

two metabolites as 5-(2-hydroxy-3-methylbutyloxy)-psoralen and 5-(3-hydroxy-3-methylbutyloxy)-psoralen. M15 and M17 have the same quasi-molecular ion [M + H]+ of m/z 287. Their molecular formulae were determined as C16H14O5 according to the HR–MS spectra. Their molecular weights were bigger than that of isoimperatorin for 16 Da. M15 exhibited similar fragment ions with M9 at m/z 219, 203, 173, 162 and 113 in MS/MS spectrum, indicating that the oxygen atom was probably linked at C-3 position. M15 was identified as 3-hydroxyisoimperatorin and it was a new compound. The typical fragment ions of M17 at m/z 203 and 271 suggested that the oxygen atom was linked in the side-chain and M17 was an epoxide of isoimperatorin. M17 has similar fragment ions with M16 at m/z 271, 203, 175, 159, 147, 131, 103 and 91, but the relative abundances of m/z 175 and 147 were higher than that of m/z 159 and 131, which indicated that the side-chain of the metabolites was at C-8 position. M17 was a position isomer of M16 and was identified as heraclenin [29]. The molecular formula of M18 was the same as isoimperatorin determined by the HR–MS data as shown in Table 1. The typical fragment ions of M18 were at m/z 203, 175, 159,147, 131, 119, 91, and 90, which was almost the same as isoimperatorin. The only obvious difference of the

Fig. 2. The proposed metabolic pathways of isoimperatorin.

T.-L. Chen et al. / Fitoterapia 93 (2014) 88–97

two spectra was that the relative abundances of m/z 175 and 147 were higher than that of m/z 159 and 131. According to the previous report [2,27], M18 was identified as imperatorin [3]. 3.1.2. Metabolic pathway of isoimperatorin The present research suggested that there were various metabolic sites of isoimperatorin in the isopentenoxyl group and the furocoumarin nucleus. As shown in Fig. 2, the proposed metabolic pathway suggested that main metabolic site was on the isopentenoxyl group of isoimperatorin to form hydroxylation or epoxidation of metabolites. In addition, hydroxylation reaction was also occurred on the furocoumarin nucleus and this type of metabolites can easily be identified on the base of distinctive ion at m/z 219 in MS/MS spectrum. Fission of the isopentenoxyl group from the furocoumarin nucleus was also observed, but it is not the main types of biotransformation pathways.

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the peak areas obtained from mobile phase and post-extraction blank sample spiked with the analytes at the same concentrations. As shown in Table S2, the ratios of the analytes ranged from 91.32 to 101.3%, suggesting that there were no matrix effects for each analytes. The data summarized in Table S2 indicated that the method established in this assay conformed to the guidelines of the US Food and Drug Administration for bioanalytical method validation.

3.1.3. Preparation and identification of the main metabolites Following magnifying incubation with rat liver microsomes in vitro, isoimperatorin was metabolized and five main metabolites were isolated. The structures of these main products were determined as M3, M4, M11, M12 and M16 by various spectroscopic methods. Their spectral data were as follows. 3.2. Time course of biotransformation and quantification of the main metabolites A separate study for quantitative analysis of isoimperatorin and its main metabolites in different reaction time was carried out. In this paper, an agilent ZORBAX SB-C18 column (250 × 4.6 mm i.d., 5 μm) was used as the stationary phase. MeCN and H2O containing 0.1% formic aicd were used as the mobile phase with gradient elution in 20 min (0–8.0 min, 35–47%A; 8–12 min, 47–85%A; 12–20 min, 35%A). As shown in Fig. 3, The LC–MS/MS chromatograms of the samples showed no interfering peaks from endogenous substances. The retention times for M0, M4, M7, M11, M12, M16 and IS were 13.82, 3.05, 3.15, 4.90, 6.85, 7.25, and 10.88 min, respectively. 3.2.1. Linearity, LLOD and LLOQ Calibration curves for each analytes were constructed by peak area ratios of the analytes and IS vs the concentrations of the analytes. As shown in Table S1, all of the calibration curves showed good linearity with correlation coefficients (r) ≥ 0.9967. The LLODs and LLOQs for analytes were summarized in Table S1, the data suggested that the obtained results are sufficient to support the study. 3.2.2. Precision, accuracy, extraction recovery, analyte stability and matrix effects QC samples at low, medium and high concentrations of each analytes were analyzed for method validation. As shown in Table S2, the intra- and inter-day accuracies ranged from 86.04% to 114.4% with intra- and inter-day precisions (RSDs) less than 14.15%. The extraction recoveries of the analytes ranged from 75.45 to 90.70%. The analytes were found to be stable at room temperature for 48 h with no significant degradation observed, the accuracies were in the range of 87.64% and 111.0%. Matrix effects were evaluated by analyzing

Fig. 3. Representative MRM chromatograms of the analytes and daidzein (IS) in rat liver microsomes: (A) blank sample; (B) blank HILM spiked with the analytes and daidzein (M0 287.5 ng/mL, M4 3.75 ng/mL, M7 46.88 ng/mL, M11 22.5 ng/mL, M12 3.75 ng/mL, M16 75 ng/mL); the retention times were 3.05 min, 3.15 min, 4.90 min, 6.85 min, 7.25 min, 10.88 min and 13.82 min for IS, M4, M7, M11, M12, M16, and M0, respectively. (C) Liver microsomal supernatant obtained after incubation of M0 with rat liver microsomes for 30 min.

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Concentration (ng/ug protein)

2000

Acknowledgment

1800 M0

1600

This research was partly supported by the National Key Technology R & D Program of China (2011BAI07B08).

M4

1400

M11

1200

M12

1000

Appendix A. Supplementary data

M16

800

M7

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.12.017.

600 400 200

References

0 0

20

40

60

80

100

120

140

Time (min) Fig. 4. Metabolite formation from isoimperatorin incubated with liver microsomes from PB-pretreated rats.

3.2.3. Time course study of isoimperatorin and its main metabolites The metabolic profile of isoimperatorin in PB-treated male SD rat liver microsomes on a 2 h of incubation period was demonstrated in Fig. 4. The metabolic formation of the five polar metabolites showed a time-dependent increase in 2 h of incubation period and is followed by the significant decrease of isoimperatorin. 4. Conclusions This is the first report for biotransformation of isoimperatorin with rat liver microsomes in vitro although biotransformation of imperatorin by fungi Cunninghamella blakesleana [2] and Aspergillus flavus [30] had been reported. In this study, a highly sensitive LC–IT–TOF–MS method was developed and 18 metabolites were detected according to the exact molecular weights and the characteristic fragment ions provided by TOF– MS data. As shown in Fig. 2, the metabolic sites of isoimperatorin were mainly on the isopentenoxyl group to form hydroxylation or epoxidation of metabolites homologously with imperatorin [28,30,31]. Furthermore, isoimperatorin could be modified to imperatorin by liver drug enzyme in the presence of NADPH, which was accordance with the previous report [2]. The results demonstrated that the LC–TOF–MS method was an easy and accurate approach for the characterization of metabolites. To verify structures of the metabolites, five main metabolites were further prepared by magnifying liver microsomal incubation system and their chemical structures were elucidated by various spectroscopy methods in this study. For metabolic kinetic characteristics study, a simple new liquid chromatography with LC–QqQ–MS method was developed for the simultaneous determination of isoimperatorin and its main metabolites. The results suggested that the method can satisfy the requirement of qualitative analysis for metabolism study of isoimperatorin and its main metabolites in rat liver microsomes. The results provided essential data for further pharmacological and clinical studies of isoimperatorin. Conflict of interest statement The authors declare that there is no conflict of interest.

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MS method.

The aim of the present research was to establish a comprehensive strategy to identify the metabolites of isoimperatorin after biotransformation with r...
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