mass spectrometry

Food Chemistry 141 (2013) 357–365

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Identification of in vitro and in vivo metabolites of isoimperatorin using liquid chromatography/mass spectrometry Xiaowei Shi a,1, Man Liu a,b,1, Min Zhang c, Kerong Zhang d, Songchen Liu a, Shi Qiao a, Rui Shi a, Xijuan Jiang a, Qiao Wang a,⇑ a

School of Pharmacy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, China Department of Clinical Pharmacology, Aerospace Center Hospital, Beijing 100049, China Quality Control Office, Hebei Provincial Chest Hospital, Shijiazhuang 050041, China d AB Sciex Pte. Ltd., Beijing Branch Office, Beijing 100027, China b c

a r t i c l e

i n f o

Article history: Received 19 March 2012 Received in revised form 16 November 2012 Accepted 16 February 2013 Available online 28 February 2013 Keywords: Isoimperatorin Metabolites Liquid chromatography/mass spectrometry Microbial biotransformation Coumarin

a b s t r a c t The objective of the present study was to develop a practical strategy for the identification of metabolites following the in vivo metabolism and in vitro microbial biotransformation of isoimperatorin using liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry (LC/QTRAP–MS) and liquid chromatography time of flight mass spectrometry (LC/TOF–MS). As a result, 19 metabolites were characterised in rat urine, plasma, bile and faeces after the oral administration of isoimperatorin and 13 products were identified in the sample from the transformation. Four metabolites were prepared by in vitro microbial biotransformation, and one was confirmed to be a novel compound. The side chain of isoimperatorin was found to be the primary metabolic site that underwent oxidation metabolism both in vivo and in vitro, and the metabolism of isoimperatorin in vivo and in vitro has good correlation. This is the first study of the metabolism of isoimperatorin in vivo. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Isoimperatorin is an active natural furocoumarin originating not only from lemon, lime oils and culinary herbs such as parsnip, parsley, and fennel (Marumoto & Miyazawa, 2010), but also from many traditional Chinese herbal medicines such as Angelica Dahurica and Radix Glehniae, which are also nutritional and health foods used in Chinese cooking or used as dietary supplements (Chu, Cheng, Ni, & Chen, 2009; Jiang, Zhang, Ma, Chen, & Wu, 2008; Ng, Liu, & Wang, 2004; Satoh, Narita, Endo, & Nishimura, 1996). Isoimperatorin has attracted increasing interest due to its antiinflammatory, analgesic, antispasmodic and anticancer activities (Chen, Tsai, & Wu, 1995; Moon, Jin, Son, & Chang, 2008; Moon et al., 2011; Wang, Jia, Ma, & Li, 2010). Recent pharmacological research demonstrated that isoimperatorin may have a role in Alzheimer’s disease treatment through inhibition of b-secretase (Ghosh, Hong, & Tang, 2002; John, Beck, Bienkowski, Sinha, & Heinrikson, 2003). Isoimperatorin may also enhance the inhibition of cytochrome P-448 by glutathione S-transferase, therefore, reduce ⇑ Corresponding author. Address: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, China. Tel.: +86 311 86265625; fax: +86 311 86266419. E-mail address: [email protected] (Q. Wang). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.02.068

the toxicity of the liver carcinogen aflatoxin B1 (AFB1) and incidence of liver cancer (Pokharel et al., 2006). Furthermore, isoimperatorin may inhibit the tumor necrosis factor-a (TNF-a)-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells (Moon et al., 2011). With the increasing importance of isoimperatorin in human health, it is necessary to evaluate its pharmacokinetic (PK) properties, efficacy and toxicity to establish whether it can be developed as a chemical entity, or there appears to be a risk associated with extra exposure to isoimperatorin from dietary sources. Some studies have been performed on the absorption, distribution and excretion of isoimperatorin (Feng, Ruan, & Cai, 2010; Jiang, Lv, Wang, & Yue, 2010; Li & Yang, 2011; Liu et al., 2011; Wang, Chen, & He, 2007). However, to date, no metabolic data, particularly that of isoimperatorin metabolites, are available. Previous studies investigated the absorption of isoimperatorin in a Caco-2 cell monolayer model and in rats and found that isoimperatorin was a well-absorbed compound (Li & Yang, 2011; Wang et al., 2007). Moreover, it was concluded recently in our lab that less than 1% of the isoimperatorin as a parent drug was recovered from rat urine and bile (Liu et al., 2011). All of these results indicated that isoimperatorin was probably apt to be metabolised in vivo and be excreted mainly as metabolites. Therefore, clarifying the structures of isoimperatorin metabolites is required for further understanding the efficacy and toxicity of isoimperatorin.

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Mass spectrometry (MS) has proven to be a successful approach for the structural elucidation of organic species. With the continuous developments and improvements in high-performance liquid chromatography (HPLC) and MS technologies, more LC–MS methods have been applied to drug metabolite identification and structural characterization in various biological matrices (Baumann et al., 2011; Zhao et al., 2012). Generally, LC–MS using triple quadrupole or ion trap (QTRAP) mass spectrometers is able to provide molecular weight information about potential drug metabolites with additional structural information from subsequent multistage product ion scan mass spectra (Gros, Petrovic, & Barcelo, 2009). Based on our previous study (Yang et al., 2010), when information-dependent data acquisition (IDA) was performed, enhanced product ion scan (EPI) can use diversified combined scan modes such as enhanced MS (EMS)-IDA-EPI, precursor ion (PREC)-IDAEPI, multiple ion monitoring (MIM)-IDA-EPI and multiple reaction monitoring (MRM)-IDA-EPI. These modes provide information about the parent ions and fragment ions of the detected ions, and the structures of the expected and unexpected metabolites tentatively identified. However, QTRAP–MS cannot determine more accurate masses and empirical formulae of metabolites or their fragments, which is critical for confirming the structures of unknown metabolites. Time of flight (TOF) mass spectrometry is a type of high resolution MS with which the exact mass of the metabolites can be determined and a unique elemental formula based on the mass sufficiency of the constituent atoms can also be acquired. This ability increases the confidence of metabolite identification and selectivity, reduces both the matrix effects and the background interference, and also serves to differentiate the metabolites of the same nominal but differing exact masses. Therefore, a practical approach with an integrated application of low resolution LC/ QTRAP–MS and high resolution LC/TOF–MS for the identification of drug metabolites was proposed in this study. A microbial biotransformation technique using fungi is one of the most feasible alternative in vitro approaches for the in vivo drug metabolism study in mammals because some microorganisms express enzyme systems that have proved to be similar to the mammalian hepatic monooxygenases. This technique has been used as an in vitro model of drug metabolism and for the production of drug metabolites in amounts sufficient for structural confirmation (Smith & Rosazza, 1983; Sunet al., 2009; Tevell Aberg, Lofgren, Bondesson, & Hedeland, 2010). In our preliminary experiment, it was found that the transformation products of isoimperatorin with a filamentous fungus, Cunninghamella elegans, resembled the metabolites of isoimperatorin in rats. Therefore, a microbial biotransformation was performed in our study as an effective approach for studing the metabolism of isoimperatorin in rats, the approach might also be helpful in establishing metabolism of isoimperatorin in humans. Herein, we developed a protocol that combines LC/QTRAP–MS and LC/TOF–MS to identify the metabolites of isoimperatorin in vivo and in vitro. Some metabolites were first isolated and purified with microbial biotransformation, and their structures were elucidated with nuclear magnetic resonance (NMR) spectroscopy. Based on the compounds obtained, the metabolism rules (e.g., oxidation, reduction, hydrolysis, and conjugation) and the previous fragmentation rules of furocoumarin (Yang et al., 2010; Zhang et al., 2009), LC/QTRAP–MS and LC/TOF–MS methods were developed for the separation and identification of isoimperatorin and its metabolites in rats. The metabolic characteristics and profiling of isoimperatorin in rats, and in the microbial biotransformation, and the correlation of the metabolism in vivo and in vitro were demonstrated. In the present study, 19 metabolites were characterised in rats after the oral administration of isoimperatorin; 13 products were

identified in the sample from the Cunninghamella blakesleana transformation and four new transformed products of isoimperatorin were obtained. One of these products was confirmed to be novel. This is the first identification of the metabolites of isoimperatorin in vivo. The combined application of LC/QTRAP– MS and LC/TOF–MS aided by the biotransformation also offered a feasible strategy for food and drug metabolic research. 2. Experimental procedure 2.1. Materials and reagents Isoimperatorin was acquired from Tianjin Phytomarker, Ltd. (China). Psoralen, isopsoralen, imperatorin and isoimperatorin were purchased from the China Institute for the Control of Pharmaceutical and Biological Products. Xanthotoxol, xanthotoxin and bergapten were obtained from Shanghai Tauto Biotech Co., Ltd. (China). Oxypeucedanin hydrate, E-5-(4-hydroxy-3-methylbutyl2-alkenyloxy)-psoralen, Z-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen and 5-(2-hydroxy-3-methylbutyloxy)-psoralen were isolated and purified in our study. These compounds were identified by comparison of their NMR and MS data with literature data (Kuznetsova, Abyshev, Perel’son, Sheinker, & Pek, 1966; Lu, Jin, Jin, & Chen, 2007; Stavri & Gibbons, 2005). Methanol and ammonium acetate were of HPLC grade and were purchased from Fisher Scientific (USA) and Dikma Technologies Inc. (USA). Deionized water was purified using a Heal Force-PWVF Reagent Water System (Shanghai CanRex Analyses Instrument Co., Ltd., China). All other chemicals (analytical grade) were purchased from Tianjin Chemical Corporation (China). The silica gel (300– 400 mesh) for column chromatography was purchased from Qingdao Marine Chemical Corporation (China). Cunninghamella blakesleana AS 3.970, Aspergillus niger AS 3.1858, Aspergillus niger AS 3.795, Syncephalastrum racemosum AS 3.264, Rhizopus stolonofer AS 3.2050, Rhizopus arrhizus AS 3.2896, Penicillium melinii AS 3.4474, Absidia glauca AS 3.3385, Mucor subtilissimus AS 3.2454, Fusarium avenaceum AS 3.4594, Cunninghamella elegans AS 3.1207, Mucor alternate AS 3.3450, Sporotrichum sp. AS 3.2882, Beauveria bassiana AS 3.4273, Alternaria alternata AS 3.4748 were purchased from China General Microbiological Culture Collection Center (China). 2.2. Instruments and conditions The LC–MS system consisted of an Agilent 1200 HPLC system (USA) equipped with a quaternary solvent delivery system, an autosampler and a column compartment and a 3200 QTRAPTM system with an electrospray ionisation (ESI) source (AB SCIEX, CA, USA). Applied Biosystems/MDS Sciex Analyst software (versions 1.4.2) was used for the data acquisition and processing. The analytes were separated on an Agilent Zorbax Eclipse XDBC18 (150 mm  4.6 mm, 5 lm) column at a column temperature of 25 °C. The mobile phase for elution was a gradient consisting of methanol (A) and 1 mmol/L ammonium acetate (B) at a flow rate of 1 mL/min. The gradient program was as follows: 20–40% A (0– 3 min); 40–65% A (3–25 min); 65–95% A (25–30 min); 95–95% A (30–40 min). All analyses were performed in positive ion ESI with the ion spray voltage set at 5.5 kV, and the turbo spray temperature was set at 600 °C. The nebulizer gas (GS1), the heater gas (GS2) and the curtain gas were set at 4.1, 4.1 and 1.7 bar, respectively. Nitrogen was used as the nebulizer and auxiliary gas. The EPI scan rate was 4000 amu/s and the scan range was 50–800 amu. The declustering potential (DP) of EPI was set at 25 V, and the collision energy (CE) was set at 25 eV (the collision energy spread (CES) was 10 eV).

X. Shi et al. / Food Chemistry 141 (2013) 357–365

For the EMS-IDA-EPI analysis, the EMS scan ranged from m/z 100 to 800 amu with a scan rate of 4000 amu/s, and the DP and CE were set at 25 V and 10 eV, respectively. The MIM-IDA-EPI mode was performed using the MRM-EPI mode with a minimal CE in Q2, and the same ions were monitored in Q1 and Q3. The DP and CE were set at 25 V and 5 eV, respectively. The dwell time of each ion pair was 30 ms. The other parameters were identical to those used for the EMS-IDA-EPI mode. For the PREC-IDA-EPI analysis, the PREC scan was carried out with a scan range from m/z 100 to 800. Except for the characteristic ion m/z value of 203 for furanocoumarin, the precursors of the PREC scans corresponded to the specified ions monitored in the MIM-IDA-EPI mode (Yang et al., 2010). When monitoring the characteristic ion m/z value of 203, the DP and CE were set at 25 V and 25 eV and when monitoring the precursor ion, the DP and CE were set at 25 V and 5 eV. The IDA and EPI parameters were same as those used in the MIMIDA-EPI mode. A TripleTOF™ 5600 system with a DuoSpray™ source operating in the positive ESI mode was also used for the detection (AB SCIEX, CA, USA). The following parameter settings were used: ion spray voltage, 5.5 kV; ion source heater, 600 °C; curtain gas, 2.1 bar; ion source gas 1, 4.1 bar; ion source gas 2, 4.1 bar. The DP was set at 25 V. 2.3. Microbial biotransformation of isoimperatorin 15 Strains of fungi underwent a preliminary screening, and Cunninghamella blakesleana AS 3.970 was selected as the most potent strain for the biotransformation of isoimperatorin. This strain was used for the identification and preparation of isoimperatorin metabolites in vitro. 2.3.1. Preparation for sample of C. blakesleana AS 3.970 biotransformation The C. blakesleana AS 3.970 mycelia were transferred to 120 mL of liquid medium and then incubated at 25 °C with rotary shaking at 120 r/min. After the culture had incubated for 72 h, 0.2 mL of substrate (15 mg/mL, dissolved in acetone) was added. The incubation was allowed to continue for 72 h. The culture was filtered and extracted with an equal volume of ethyl acetate three times. The extract was evaporated to dryness in a rotary evaporator and dissolved in 1 mL of methanol. After the centrifugation at 12,000 rpm for 10 min, 10 lL of the supernatant was injected into the HPLC–MS system. 2.3.2. Separation, purification and characterization of biotransformation products An amount of 500 mg of isoimperatorin was used for a preparative-scale test to prepare the biotransformation products. The incubation conditions and procedures were identical to those described in Section 2.3.1 and approximately 4.3 g of ethyl acetate extract was obtained. The extract was separated by silica gel column chromatography and eluted with petroleum ether–acetone. The fractions with similar components were combined under the guidance of TLC analysis. The combined fractions were purified by preparative HPLC (Wenfen LC98-II, Beijing, China) with a SPODS column (250 mm  10 mm, 10 lm) to obtain pure compounds. The separation was performed with isocratic elution consisted of methanol–water (50:50, v/v). The flow rate was set at 17 mL/min, injection volume was 0.5 mL, the detection wavelength was set at 310 nm and the column was maintained at ambient temperature. The structures of the compounds were then identified by NOESY and 1H-1HCOSY NMR spectra recorded on a VNS600 spectrometer in trichloromethane (CDCl3). Four products, i.e., oxypeucedanin hydrate (M11), E-5-(4-hydroxy-3-methylbutyl-2alkenyloxy)-psoralen (M16), Z-5-(4-hydroxy-3-methylbutyl-2-

359

alkenyloxy)-psoralen (M17) and 5-(2-hydroxy-3-methylbutyloxy)-psoralen (M19, Pranferol), were prepared and purified, and M16 was identified as a novel compound. The detailed structural elucidation procedure for these compounds is described in the Supplementary data section. 2.4. Preparation of standard solutions The appropriate amount of each standard (xanthotoxol, psoralen, xanthotoxin, isopsoralen, bergapten, imperatorin and isoimperatorin) was weighed and dissolved in methanol to make seven individual stock solutions. Then, each stock solution was mixed with methanol to prepare the final mixed standard solution 1. The appropriate amount of the prepared products of the C. blakesleana AS 3.970 biotransformation was weighed and dissolved in methanol. Then, each stock solution was diluted and mixed with methanol to prepare the final mixed standard solution 2. All solutions were stored at 4 °C. 2.5. Animal, biological sample collection and preparation Male Sprague–Dawley rats (250 g body weight) were provided by the Lab Animal Center of Hebei Medical University. The protocols for the animal experiments were approved by the Animal Center of Hebei Medical University. The animals were maintained at ambient temperature (22–24 °C) and 60% relative humidity with a 12 h light/dark cycle. The animals were kept in an environmentally controlled breeding room for at least one week before starting the experiments. The animals were provided with food and water ad libitum and fasted overnight before biological sample collections, but they still had access to deionized water. Groups of rats received a single oral administration of isoimperatorin in 0.5% carboxymethylcellulose sodium (CMC-Na) at a concentration of 80 mg/kg. Urine and faeces were collected into containers at 72 h after dosing. For bile sampling, rats were anaesthetised with urethane (1.0 g/kg, i.p.), and the abdominal incision was made, and the common bile duct was cannulated with PE-10 tubing for the collection of the bile samples 0–36 h after dosing. The blood samples were collected in heparinized tubes 30, 45, 60, 90, 120, 150, 180 and 210 min after the oral administration of isoimperatorin. The rats immediately were euthanized by overdose of urethane (4.0 g/kg, i.p.) at the end of collection of the bile or blood samples. The blood was centrifuged at 4000 rpm for 10 min to separate the red blood cells and plasma and then combined to obtain the mixture plasma. All samples were stored at 20 °C until further extraction and analysis. A liquid–liquid extraction was used for pre-treating all of the biological samples. The urine, bile and plasma samples were independently extracted three times with an equal volume of ethyl acetate. The extract was evaporated to dryness in a rotary evaporator and dissolved in 1 mL of methanol. The faeces sample was ground and extracted three times with ethyl acetate in an ultrasonic bath for 20 min and then filtered. The filtrate was evaporated to dryness, and the residue was dissolved in 1 mL of methanol. 3. Results and discussion 3.1. HPLC-MS analysis strategy Based on the fragmentation rules of furocoumarins from our previous study (Yang et al., 2010) and the literature (Zhang et al., 2009) and using six known furocoumarins and four products prepared by microbial transformation as references, we first analysed the metabolites of isoimperatorin (M0) in vivo and in vitro using LC/QTRAP–MS, and then we further verified the structures of the

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X. Shi et al. / Food Chemistry 141 (2013) 357–365

Fig. 1. Workflow for the identification of the metabolites of isoimperatorin.

metabolites using LC/TOF–MS. The workflow for the metabolite identification is presented in Fig. 1. When analysing with QTRAP, three scan modes including EMSIDA-EPI, PREC-IDA-EPI and MIM-IDA-EPI were used. First, because of the similarity amongst the metabolites, and the prototype and the fragmentation rules of isoimperatorin (Fig. 2 Supplementary data) and other furanocoumarins, the characteristic diagnostic fragment ions (e.g. ion with an m/z value of 203) was used in the PREC-IDA-EPI analysis to identify potential metabolites. Meanwhile, EMS-IDA-EPI mode was also performed. By comparing the fragment ions and the retention times of the unknown metabolites in the PREC-IDA-EPI and EMS-IDA-EPI modes, the molecular weight and fragment ions of the expected metabolites were initially acquired. Then, the PREC-IDA-EPI analysis using each parent ion as a scan ion was performed to further confirm the molecular weight of the expected metabolites. The fragment ions and the retention times of the specified ions were also provided. The MIM-IDA-EPI analysis of different parent ions further supplied information about the fragment ions. The structures of the metabolites were tentatively elucidated according to the molecular weight and the information about the fragment ions produced by the three scan modes. As a result, a total of 21 metabolites were accurately identified. The retention times, molecular weights, fragment ions and parent ions of the 21 metabolites are presented in Table 1. The chromatograms of the 21 metabolites in the urine, faeces, bile, plasma and biotransformation samples are shown in Fig. 2. 3.2. Identification of the metabolites of isoimperatorin in rat urine sample by QTRAP system A total of 18 metabolites were characterised in the urine samples from rats after the oral administration of isoimperatorin.

Using a combination of the EMS-IDA-EPI mode and the PRECIDA-EPI mode, the molecular weights of the unknown metabolites were identified, and the [M+H]+, [M+Na]+, [M+NH4]+, [2M+H]+, [2M+Na]+ and [2M+NH4]+ ions were highly relevant. We use M2 to describe the detailed analysis procedure. In the first PREC-IDAEPI mode, the characteristic diagnostic ion with an m/z value of 203 was first used as the monitored ion. The molecular weight of M2 was identified with the simultaneous appearance of [M+H]+ with m/z = 301, [M+NH4]+ with m/z = 318, [2M+H]+ with m/ z = 601 and [2M+NH4]+ with m/z = 618, which indicated that M2 had a molecular weight of 300 Da. Then, the PREC-IDA-EPI of the parent ion of M2 (m/z = 300) was applied to further confirm the molecular weight of M2. In the EMS-IDA-EPI mode, the molecular weight of M2 was identified based on the significance of [M+H]+ with m/z = 301, [M+Na]+ with m/z = 323, [2M+H]+ with m/z = 601 and [2M+Na]+ with m/z = 623, which further suggested that M2 had a molecular weight of 300 Da. Thus, the molecular weight of M2 was unambiguously identified. The PREC spectrum and the EMS spectrum of M2 are presented in Fig. 3. Similarly, the molecular weights of the other metabolites were also obtained, and the molecular weights are summarized in Table 1. The MIM-IDA-EPI mode was then used for each expected metabolite. By comparing the retention times obtained from the MIM-IDAEPI chromatograms of the urine sample, the molecular weights and fragment ions with standards and the peaks of M1, M7, M9, M10 and M21 were unambiguously identified as xanthotoxol, psoralen, xanthotoxin, isopsoralen and imperatorin. Meanwhile, the peak with retention time of approximately 30.8 min was confirmed as the prototype isoimperatorin. Additionally, the peaks of M11, M16, M17 and M19 were identified as oxypeucedanin hydrate (product 1), E-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (product 2), Z-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (product 3) and 5-(2-hydroxy-3-methylbutyloxy)-psoralen

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X. Shi et al. / Food Chemistry 141 (2013) 357–365 Table 1 Isoimperatorin metabolites detected and structurally characterised on a LC/QTRAP–MS instrument. No.a

RTb

Compound name

MWb

MFb

MS1

MS2

M0

30.79

Isoimperatorin

270

C16H14O4

9.81

Xanthotoxol

202

C11H6O4

271, 288, 293, 541, 558 203, 225

M2

11.30

300

C16H12O6

M3

12.0

300

C16H12O6

M4

11.67

E-5-(2-Methyl-2-butylalkenyl acid-4-oxy)psoralen Z-5-(2-Methyl-2-butylalkenyl acid-4-oxy)psoralen 5-Hydroxypsoralen

202

C11H6O4

301, 618, 301, 618, 203,

271, 203, 175, 159, 147, 131, 119, 103, 91, 69 203, 175, 159, 147, 131, 103, 91, 77 301, 202, 174, 146, 118, 90

M5

12.44

5-Methoxy-8-hydroxypsoralen

232

C12H8O5

233, 250

M6

13.66

5-(2-Methylbutyl acid-4-oxy)-psoralen

302

C16H14O6

M7⁄ M8 M9⁄ M10⁄ M11⁄ M12

14.08 14.19 14.57 15.32 15.55 15.66

186 232 216 186 304 302

C11H6O3 C12H8O5 C12H8O4 C11H6O3 C16H16O6 C17H18O5

M13

16.31

302

C17H18O5

303, 320, 325

M14⁄ M15 M16⁄

18.61 19.64 20.52

216 316 286

C12H8O4 C17H16O6 C16H14O5

M17⁄

22.03

286

C16H14O5

217, 317, 287, 590, 287,

M18

23.42

Psoralen 8-Methoxy-5-hydroxypsoralen Xanthotoxin Isopsoralen Oxypeucedanin hydrate 5-(4-Hydroxy-3-hydroxy methylbutyl-2alkenyloxy)–psoralen 5-(4-Hydroxy-3-methyl-2-oxobutyloxy)– psoralen Bergapten Apaensin E-5-(4-Hydroxy-3-methylbutyl-2alkenyloxy)-psoralen Z-5-(4-Hydroxy-3-methylbutyl-2alkenyloxy)-psoralen Pabulenol

303, 622, 187, 233, 217, 187, 305, 303,

286

C16H14O5

287, 304, 309, 573

M19⁄ M20 M21⁄

23.50 25.60 28.20

Pranferol 5-(3-Hydroxy-3-methylbutyloxy)-psoralen Imperatorin

288 288 270

C16H16O5 C16H16O5 C16H14O4

289, 306 289, 306 271, 288, 293, 541, 558

M1

a b c

⁄

318, 323, 601, 623 318, 323, 601, 623 225

320, 325,605, 627 204 250 334 204 322, 609, 626 320, 325

334 334, 633, 650 304, 309, 573, 595 304,309, 573

301, 202, 174, 146, 118, 90 203, 175, 159, 147, 131, 119, 103, 91, 77 233, 218, 215, 190, 162, 134, 106, 78 303, 203, 175, 159, 147, 131, 101, 91 187, 159, 143, 131, 115, 103, 77 233, 218, 190, 162, 134, 106 217, 202, 185, 174, 161, 118, 90 187, 159, 143, 131, 115, 103, 77 305, 203, 159, 147, 131, 103 303, 203, 175, 159, 147, 131, 101, 91 303, 203, 175, 159, 147, 131, 101, 91 217, 202, 174, 161, 146, 131,118 317, 233, 218, 190, 173, 162, 145 287, 203, 175, 159, 147, 131, 103, 91, 77 287, 203, 175, 159, 147, 131, 103, 91, 77 287, 269, 203, 175, 159, 147, 131, 103, 91, 77 289, 203, 175, 159, 147, 131, 91 289, 203, 175, 159, 147, 131, 91 271, 203, 185, 175, 159, 147, 131, 103, 69

Uc p

Fc p

Bc p

Pc p

Mc p

p

p

p

p

p

p

p

p

p

p

p

p p p

p

p p p p p p

p p p p p

p

p

p

p

p

p

p

p p

p p p

p

p

p

p p

p

p

p

p p p

p

p

p

p

p

p

p

p

p p p

p p p

p p

p

p

The standards have been marked by ⁄. RT: relation time; MW: molecular weight; MF: molecular formula. p U: Urine; F: Faeces; B: Bile; P: Plasma; M: Microbial transformation. The compounds that existed in the sample have been marked by .

(product 4) by comparing the chromatograms of these species with those of the products identified from the C. blakesleana AS 3.970 transformation. The chromatograms of the mixed standard solutions are shown in Fig. 2. The process used for the structural characterization of the metabolites for which we could not acquire standards was as follows. For the MIM-IDA-EPI analysis of the m/z = 301 ion, the structures of M2 and M3 were detected. The two metabolites have the same fragment ions, and the fragmentation characteristics were consistent with the known reference standard bergapten. The typical fragment ions were [M+H–C5H9O]+ with m/z = 202, [M+H– C5H9O–CO]+ with m/z = 174, [M+H–C5H9O–2CO]+ with m/z = 146, [M+H–C5H9O–3CO]+ with m/z = 118 and [M + H–C5H9O–4CO]+ with m/z = 90 (Yang et al., 2010; Zhang et al., 2009). Imperatorin (the location isomer of isoimperatorin, with an isopentenyl moiety in the C-8 position) can generate oxidative metabolites and then further generate the carboxylic metabolites of imperatorin (Teng, Huang, Huang, Chung, & Chen, 2004). Similarly, M16 and M17, the oxidative metabolites of isoimperatorin, further generated the carboxylic metabolites of isoimperatorin. Based on the elution order of the isomers M16 and M17, M2 and M3 were identified as E-5-(2-methyl-2-butylalkenyl acid-4-oxy)-psoralen and Z-5-(2methyl-2-butylalkenyl acid-4-oxy)-psoralen. For the MIM-IDA-EPI analysis of the ion with m/z = 233, the structural characterization of M5 and M8 was completed. M5 and M8 had the same molecular weight of 232 Da, and the characteristic fragment ions had m/z values of 233, 218, 215, 190, 162, 134, 106 and 78. Upon comparing the EPI spectra of M5 and M8 with that of cnidilin (a furocoumarin), it was found that the EPI

spectrum of cnidilin contained all of the characteristic ions of M5 and M8. Therefore, we deduced that the structures of M5 and M8 might be 5-methoxy-8-hydroxypsoralen or 8-methoxy-5-hydroxypsoralen, the same structure as the fragment ions of cnidilin after losing an isopentenyl moiety with an intermediate ion having an eight-membered ring. According to the observed retention times of 12.44 and 14.19 min, a methyl as a hydrophobic group should be at the C-8 position for M8. Therefore, M5 might be 5-methoxy-8-hydroxypsoralen and M8 might be 8-methoxy-5hydroxypsoralen. For the MIM-IDA-EPI analysis of the ion with m/z = 303, the structures of M6, M12 and M13 were identified. These three metabolites contained the typical fragment ions at m/z = 203, 175, 159, 147, 131, 101 and 91. Moreover, the relative abundance of the m/z = 159 ion was higher than that of the m/z = 175 ion, which suggested that the isopentenoxy group of the three metabolites was at the C-5 position. The molecular weight of M6 was 2 Da greater than those of M2 and M3, indicating that M6 was the hydrogenated product of M2 and M3. Thus, M6 was tentatively identified as 5-(2-methylbutyl acid-4-oxy)-psoralen. The fragment ions of M12 and M13 were identical, and, according to the molecular weight and elution order of M12 and M13, M12 and M13 were tentatively identified as 5-(4-hydroxy-3-hydroxymethylbutyl-2alkenyloxy)-psoralen and 5-(4-hydroxy-3-methyl-2-oxobutyloxy)-psoralen. For the MIM-IDA-EPI analysis of the m/z = 287 ion, the characterization of M16, M17 and M18 was completed while monitoring the ion with m/z = 287. The molecular weights of the three metabolites were all 286 Da. The three metabolites contained the typical

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Fig. 2. The chromatograms of the 7 standards (A1), the 4 products from the microbial transformation (A2) and the metabolites in the urine (B1, B2, B3), faeces (C1, C2), bile (D), plasma (E1, E2) and biotransformation (F1, F2) samples. A1, A2: The total ion chromatograms (TIC) of the MIM spectra; B1, C1, D1, E1, F1: The TIC of the PREC spectra; B2: The extract ion chromatogram (XIC) of the MIM chromatogram of 233.1/233.1; B3, C2, E2, F2: The XIC of the MIM chromatogram of 187.1/187.1.

fragment ions at m/z = 203, 175, 159, 147, 131, 103 and 91. Moreover, the relative abundance of the m/z = 159 ion was higher than that of m/z = 175 ion, which suggested that the isopentenoxy group of the three metabolites was at the C-5 position. M16 and M17 had the same characteristic fragmentation pathway, which had been identified as E-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psora-

len and Z-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen. The fragment ion of M18, including [M+H–H2O]+ with m/z = 269, suggested that the side chain might contain one hydroxyl group. Moreover, considering the molecular weight of 286 Da, the retention time and literature data, M18 was tentatively identified as pabulenol (Kang, Zhou, Sun, Han, & Guo, 2008).

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molecular weight of the metabolite was 202 Da, and the fragment ions of the metabolite were at m/z = 203, 175, 159, 147, 131, 119, 103, 91 and 77. Moreover, the relative abundance of m/z = 159 ion was higher than that of m/z = 175 ion, which suggested that the substituent group of the metabolite was at the C-5 position. M4 and M1 had the same fragment ions, but the retention time of M4 was less than that of M1. According to the elution rule of isomers, the retention times of linear-type furocoumarins substituted with a substituent group at C-8 were less than those of species with substitution at C-5. Thus, M4 was identified as 5-hydroxypsoralen (bergaptol). For the MIM-IDA-EPI analysis of the m/z = 317 ion, the structural characterization of M15 was completed. The characteristic fragment ions of M15 had m/z values of 233, 218, 190, 173, 162 and 145. Upon comparing the EPI spectra of M15 with that of cnidilin (a furocoumarin), it was found that the EPI spectrum of cnidilin contained all of the characteristic ions of M5 and M8. Based on the literature (Kang et al., 2008), we deduced that M15 was apaensin. As a result, a total of 21 metabolites were accurately identified. The retention times, molecular weights, fragment ions and parent ions of the 21 metabolites are presented in Table 1. 3.5. Identification of the metabolites by the TOF system

Fig. 3. Typical PREC spectrum (A) and EMS spectrum (B) of M2.

For the MIM-IDA-EPI analysis of the m/z = 289, the structures of M19 and M20 were identified. The two metabolites had identical fragment ions, and both of them were characteristic of 5-hydroxy-psoralen. M19 has been identified as 5-(2-hydroxy-3-methylbutyloxy)-psoralen which was prepared from C. blakesleana AS 3.970 transformation, and M20 was tentatively assigned as 5-(3hydroxy-3-methylbutyloxy)-psoralen, which was the position isomer of M19. 3.3. Identification of metabolites in the rat faeces, bile and plasma samples using the QTRAP system After completion of the analysis of the metabolites in the rat urine sample following the oral administration of isoimperatorin, the faeces, bile and plasma samples were analysed using the same approach. Most of the metabolites that were found in the rat faeces, bile and plasma samples were also in the urine sample except bergapten (M14), which was only detected in the faecal samples (according to a comparison of the retention time, molecular weight and fragment ions with those of the standard). The other metabolites were identified based on the analysis of urine sample. A total of 19 metabolites were characterised and their detailed results are summarized in Table 1. 3.4. Identification of the microbial transformation products of isoimperatorin in vitro 13 Products (Table 1) were detected by analysing the sample that was prepared from the C. blakesleana AS 3.970 transformation of isoimperatorin, and 11 products also existed in the in vivo biological samples, which indicated that there is a good correlation between the isoimperatorin metabolites from in vivo samples and those from the microbial transformation. The other two microbial transformation products, M4 and M15, were detected with the MIM-IDA-EPI scan mode. The detailed analysis is described below. For the MIM-IDA-EPI analysis of the m/z = 203 ion in addition to for the known metabolites, the structure of M4 was identified. The

In the TOF/MS spectra of the metabolites, the elementary composition and exact molecular weights of the ions can be automatically calculated, and the molecular formulas of the compounds can be inferred. After analysing the data of the metabolites obtained by the Triple TOF system, the protonated molecular weights of all the target compounds were calculated within an error of 3 ppm. The mass determination accuracy results of the metabolites obtained by the Triple TOF system are shown in Table 2. These results indicated that there were adequate data to accurately identify the structure of the metabolites. 3.6. Metabolic pathway The proposed metabolic pathways of isoimperatorin are shown in Fig. 4. These pathways indicated that the side chain of isoimperatorin was the primary metabolic site. Additionally, isoimperatorin mainly underwent oxidation metabolism, i.e., hydroxylation on the side-chain, both in vivo and in vitro. After hydroxylation, the

Table 2 LC/TOF–MS data of the metabolites of isoimperatorin. No.

MF

Calculated (m/z)

Experimental (m/z)

ppm

M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21

C16H14O4 C11H6O4 C16H12O6 C16H12O6 C11H6O4 C12H8O5 C16H14O6 C11H6O3 C12H8O5 C12H8O4 C11H6O3 C16H16O6 C16H14O6 C16H14O6 C12H8O4 C17H16O6 C16H14O5 C16H14O5 C16H14O5 C16H16O5 C16H16O5 C16H14O4

271.0965 203.0339 301.0707 301.0707 203.0339 233.0445 303.0863 187.0390 233.0445 217.0495 187.0390 305.1020 303.0863 303.0863 217.0495 317.1020 287.0914 287.0914 287.0914 289.1071 289.1071 271.0965

271.0963 203.0339 301.0708 301.0708 203.0339 233.0440 303.0864 187.0385 233.0439 217.0492 187.0385 305.1017 303.0865 303.0863 217.0492 317.1025 287.0912 287.0912 287.0912 289.1069 289.1068 271.0962

0.70 0.05 0.47 0.47 0.05 1.93 0.30 2.51 2.36 1.57 2.51 0.85 0.63 0.03 1.57 1.58 0.70 0.70 0.70 0.52 0.86 1.07

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Fig. 4. The proposed metabolic pathways of isoimperatorin.

polarity of the isoimperatorin metabolites became stronger than that of the parent drug, and the increase in polarity allowed the metabolites to be more easily excreted from the body. This conclusion was further supported by the relatively short retention time of the metabolites in the chromatograms. In addition, a preliminary conclusion could be drawn by analysing the height and area of the peaks in the chromatograms. It appeared that E-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (M16) and Z-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen (M17) were the dominant species both in vivo and in vitro.

4. Conclusions In this study, highly sensitive and effective LC/QTRAP–MS and LC/TOF–MS methods for the in vivo identification of the metabolites of isoimperatorin in conjunction with an in vitro microbial biotransformation have been developed. This is the first report of the structural identification of the metabolites of isoimperatorin. It is also the first study of the co-application of in vivo and in vitro metabolism of isoimperatorin. The present study demonstrated that the metabolites of isoimperatorin in vivo and in the microbial transformation had good correlation. There is no doubt that the association of various LC–MS and microbial transformation techniques offered more valuable information about the chemical structures. The LC/QTRAP–MS methods provided abundant information about the parent ions, fragment ions and retention times of the monitored ions, and the LC/TOF–MS method provided the exact molecular weights and molecular formulas of the metabolites. All of these data demonstrated that this integrated method was an easy and accurate approach for the characterization of metabolites. This approach may play an important role not only in the process of drug research and development, but also in the study of the metabolism of bioactive constituents of foods.

Acknowledgements We are grateful for financial support from the National Natural Science Foundation of China (81102412), the Ministry of Education Key Project of Science and Technology Foundation of China (211021) and the Natural Science Foundation of Hebei Province of China (C2011206158, 08B031).

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