Next Article in Journal
Hydrogen Atomic Positions of O–H···O Hydrogen Bonds in Solution and in the Solid State: The Synergy of Quantum Chemical Calculations with 1H-NMR Chemical Shifts and X-ray Diffraction Methods
Previous Article in Journal
Cathodic Aromatic C,C Cross-Coupling Reaction via Single Electron Transfer Pathway
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Simultaneous Determination of Three Furanocoumarins by UPLC/MS/MS: Application to Pharmacokinetic Study of Angelica dahurica Radix after Oral Administration to Normal and Experimental Colitis-Induced Rats

KM Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Korea
*
Authors to whom correspondence should be addressed.
Molecules 2017, 22(3), 416; https://doi.org/10.3390/molecules22030416
Submission received: 9 January 2017 / Revised: 21 February 2017 / Accepted: 27 February 2017 / Published: 7 March 2017
(This article belongs to the Section Natural Products Chemistry)

Abstract

:
In traditional oriental medicine, Angelica dahurica Radix (ADR) is used in the treatment of gastrointestinal, respiratory, neuromuscular, and dermal disorders. We evaluated the pharmacokinetic profiles of oxypeucedanin, imperatorin, and isoimperatorin, major active ingredients of ADR, in normal and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rats. A rapid, sensitive, and validated UPLC/MS/MS method was established for evaluating the pharmacokinetics of three furanocoumarins. After oral administration of ADR (0.5 and 1.0 g/kg), blood samples were collected periodically from the tail vein. In colitis rats, the time to reach the peak concentration (Tmax) of imperatorin and isoimperatorin was significantly delayed (p < 0.05). Lower peak plasma concentrations (Cmax) and longer mean residence times for all furanocoumarins were also observed (p < 0.05) compared with normal rats. There was no significant difference in the area under the plasma concentration–time curve or elimination half-lives. Thus, the delayed Tmax and decreased Cmax, with no influence on the elimination half-life, could be colitis-related changes in the drug-absorption phase. Therefore, the prescription and use of ADR in colitis patients should receive more attention.

1. Introduction

Angelica dahurica Radix (ADR; ‘Baek-ji’ in Korean, ‘Bai-zhi’ in Chinese) is a common ingredient in traditional formulas in Korean and Chinese pharmacopeia, such as Gumiganghwal-tang and Oyaksungi-san. In traditional oriental medicine, ADR has been used as an anti-inflammatory herbal medicine to treat respiratory diseases (e.g., common cold, nasal congestion), dermal disorders (e.g., acne, ulcer, carbuncle), pain (e.g., headache, toothache, rheumatism), and intestinal disorders (e.g., diarrhea, dysentery, chronic ulcerative colitis) [1,2]. In modern pharmacological studies, ADR has also shown anti-microbial, anti-inflammatory, anti-asthma, anti-hypertensive, and anti-cancer properties [3]. In particular, ADR improved the swelling and atrophic spots of colonic mucosal membranes in long-term clinical observations of colitis patients [2]. More than 70 coumarins have been identified from ADR [4]. Of these, furanocoumarins, including imperatorin, isoimperatorin, and oxypeucedanin, are major active constituents of ADR [5]. Similar to ADR, they have many reported biological properties. Imperatorin and isoimperatorin have anti-convulsant, vasodilatory anti-hypertension, anti-inflammatory, analgesic, anti-spasmodic, and anti-cancer activities [6,7]. Oxypeucedanin has anti-inflammatory, antibacterial, anti-fungal, and anti-cancer activities [8,9]. Therefore, the pharmacokinetics of the three furanocoumarins in ADR should be evaluated in various disease states to provide more information that could explain their efficacy and to better understand the pharmacological basis of their actions.
Several analytical methods for pharmacokinetic studies of ADR in rats have been developed. A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been reported for the determination of byakangelicin and oxypeucedanin hydrate in rat plasma [10]. A gas chromatography–mass spectrometry (GC-MS) technique was developed for the determination of oxypeucedanin and imperatorin in rat plasma [11]. Recently, two ultra-performance liquid chromatography tandem mass spectroscopy (UPLC/MS/MS) methods, with lower limits of quantification (LLOQ) of 5 ng/mL, run times exceeding 20 min, or requirements of large plasma samples, were applied to study the pharmacokinetics of coumarins derived from herbal medicine in rats [4,12]. Nevertheless, pharmacokinetic studies still require the development of more sensitive, rapid, and specific analytical methods for the simultaneous determination of the target analytes.
Therefore, this study developed and validated a sensitive UPLC/MS/MS method for the determination of oxypeucedanin, imperatorin, and isoimperatorin in rat plasma. Structural and functional alterations in the gastrointestinal tract, such as lumen pH, mobility, diarrhea, and ulceration, may affect the pharmacokinetic profiles of active constituents administered orally [13]. Therefore, we also investigated whether their pharmacokinetic profiles were altered in rats with experimentally induced colitis, because herbal medicines are most often administered orally.

2. Results and Discussion

2.1. UPLC/MS/MS Analysis and Method Validation

2.1.1. Identification, Selectivity and Linearity

Under the established UPLC/MS/MS analytical conditions, there was no endogenous interference in the peak regions of oxypeucedanin (3.22 min), imperatorin (4.51 min), isoimperatorin (5.04 min), or an internal standard (IS, 3.74 min) in blank plasma with or without spiked analytes and authentic plasma samples (Figure 1). The most abundant and stable product ions of oxypeucedanin, imperatorin, isoimperatorin, and IS were m/z 203.03345 from 287.09088, 203.03336 from 271.09573, 203.03336 from 271.09573, and 163.03865 from 309.25220, respectively (Figure 2). Their calibration curves showed good linearity (r > 0.9994) and the lower limits of quantification (LLOQs) of all analytes with signal-to-noise ratios ≥20 were 1.0 ng/mL (Table 1).

2.1.2. Accuracy, Precision, Recovery, Matrix Effects and Stability

The intra- and inter-day precision and accuracy were determined in analyses of QC samples for three concentrations of the three furanocoumarins, on the same day and on three different days. The intra- and inter-day accuracy and precision of all analytes was 6.9%–6.8% and 1.3%–9.4%, respectively (Table 2).
Table 3 lists the extraction recoveries, matrix effects, and stability of the analytes from rat plasma. The recoveries of the target analytes using liquid-liquid extraction ranged from 70.3% to 97.4%. Acetonitrile, used for sample preparation, showed good extraction efficiency, and the recoveries of all analytes at various concentrations were consistent and reproducible. The matrix effects ranged from 85.2% to 100%, and there was no obvious matrix effect. The stability of the target analytes was evaluated separately during the sample storing and processing procedures. The stability ranged from 94.4% to 101.6%, indicating that all target analytes in rat plasma were stable under our experimental conditions.
In this study, we focused on developing a simple, sensitive analytical method to apply to pharmacokinetic studies of oxypeucedanin, imperatorin, and isoimperatorin. Table 4 compares the analytical characteristics of our method with reported methods [4,11,12]. In pharmacokinetic studies, the volume of plasma/blood samples collected and continuous collection in the body can influence the pharmacokinetic parameters via a decrease in blood volume with blood loss. This proposed method uses less plasma and requires shorter run times compared with the reported methods [4,11]. The sensitivities of the target analytes were improved more than four-fold compared with those of the method reported by Chen et al. [12]. In summary, we developed a more sensitive, rapid, specific UPLC/MS/MS analysis, and a simple sample preparation method, for the simultaneous determination of three furanocoumarins for pharmacokinetic studies in rats.

2.2. Application of the Analytical Method in Pharmacokinetic Study

2.2.1. Induction of Experimental Colitis

In rats, the rectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) causes acute inflammation and severe damage to the intestinal barrier within three to seven days [14]. Ethanol disrupts the mucosal barrier and TNBS haptenizes autologous colonic and microbial proteins via the host immune system [15]. The induced-colitis rats in groups 3 and 4 developed bloody stools and lost 12.5% and 13.2% of their mean body weight, respectively. Necropsy showed that TNBS caused severe ulceration at the instillation site and a significant increase in colon weight (Figure 3). These observations are consistent with those of Arab et al. [16]. Therefore, the TNBS-treated rats can be used to evaluate the effect of colitis on the pharmacokinetics of the furanocoumarins in ADR.

2.2.2. Pharmacokinetics of ADR in Normal and Colitis-Induced Rats

Figure 4 plots the mean plasma concentration–time curves of oxypeucedanin, imperatorin, and isoimperatorin after oral administration of ADR, and Table 5 summarizes their main pharmacokinetic parameters. The plasma concentrations of the three furanocoumarins in both the normal and TNBS-treated rats varied with the dose of ADR administered (0.5 or 1.0 g/kg). Our UPLC-MS/MS method was applied successfully to pharmacokinetic studies of oral oxypeucedanin, imperatorin, and isoimperatorin in normal and induced-colitis rats (n = 6), and the three furanocoumarins were confirmed to be bioavailable active components of ADR.
All three furanocoumarins reached peak plasma levels at 40–75 min in the normal rats, indicating rapid oral absorption. However, the times to reach the peak concentration (Tmax) of the three furanocoumarins were faster than in previous reports [11,12]. Zhao et al. [11] suggested that the pharmacokinetic profile of a single compound and the ADR extract after oral administration could differ as a result of the dosage form and composition. The doses of ADR used in this study were 4.5 and 9.0 times lower than those used by Chen et al. [12]. Excessive doses in pharmacokinetic studies could influence the pharmacokinetic parameters in the absorption phases [17]. In this regard, the difference in Tmax might be due to differences in the amounts of furanocoumarin and non-furanocoumarin components as a result of the ADR preparation method and the dose of the test substance. In the colitis rats, the Tmax of imperatorin and isoimperatorin was markedly delayed at 113–144 min (p < 0.05). After ADR administration, the peak plasma concentrations (Cmax) of oxypeucedanin, imperatorin, and isoimperatorin decreased significantly to approximately 50% (p < 0.05). Conversely, the mean residence times (MRT) of oxypeucedanin, imperatorin, and isoimperatorin were prolonged by 40%~65% (p < 0.05). The area under the plasma concentration–time curve (AUC0→∞) and the elimination half-lives (t1/2) of the three furanocoumarins were not significantly different between normal and colitis-induced rats. In colitis-induced rats, the delayed Tmax and decreased Cmax could be influenced in the drug absorption phase.
The changes in the physiological environment with gastrointestinal tract disorders, including colitis, can affect intestinal absorption with the oral administration of therapeutic agents, and can lead to therapeutic failure or adverse effects [18]. In colitis, functional disorders such as dyspepsia, intestinal hypomotility, and delayed gastric emptying are accompanied by structural changes resulting from inflammatory infiltrates, tissue swelling, and ulceration [19]. In particular, dyspeptic symptoms and gastroparesis, leading to impaired and delayed gastric emptying, occur frequently in colitis patients [20], and delayed gastric emptying can influence the pharmacokinetic parameters of oral drugs, such as Tmax and Cmax [21]. De Schepper et al. [22] reported that gastric emptying was delayed without a change in the upper intestinal transit via a neuronal pathway involving pelvic afferent nerve hyperactivity in TNBS-induced colitis rats. In addition, changes in CYP expression and the altered metabolic activity due to colitis could alter the plasma concentrations of drugs metabolized by CYP. Masubuchi et al. [23] found that the contents and activities of hepatic microsomes, such as CYP1A2, CYP2E1, CYP2C11, and CYP3A2, were decreased in TNBS-treated rats. Imperatorin and isoimperatorin are metabolized to xanthotoxol and heraclenin, respectively, via demethylation, hydrolysis, methylation, demethylation, and oxidation by liver microsomes, and these metabolites were identified in rat plasma, bile, and urine [24,25,26]. In the present study, TNBS-induced colitis resulted in a decrease in Cmax and a delay in Tmax without AUC0→∞, indicating impaired oral absorption of the three furanocoumarins. In this regard, further mechanistic studies of factors influencing their oral absorption, including under the above-mentioned conditions, are required in pathophysiological states.

3. Materials and Methods

3.1. Materials

Imperatorin, isoimperatorin, and oxypeucedanin were purchased from ChemFaces (Wuhan, China; Figure 5). Warfarin as an IS, formic acid, ethanol, and TNBS were obtained from Sigma-Aldrich (St. Louis, MO, USA). “MS-grade” water and acetonitrile (Fischer Scientific, Loughborough, UK) were used for the MS analysis and plasma preparation. Raw ADR material was purchased from a local herb market (Yeongcheon, Korea) and stored in the herbarium (registration number #58) of the KM Application Center at the Korea Institute of Oriental Medicine. An ethanolic extract of ADR, a lyophilized brownish power, was prepared according to Jeong et al. [27]. The oxypeucedanin, imperatorin, and isoimperatorin contents in ADR were 6.67, 2.34, and 0.99 mg/g extract, respectively.

3.2. UPLC-MS/MS Analysis and Method Validation

Plasma concentrations of the three furanocoumarins were determined using an UltiMate 3000 (Dionex, Sunnyvale, CA, USA)—equipped Thermo Q-Exactive (Thermo Fisher Scientific, Bremen, Germany). A Hypersil GOLD column (50 mm × 2.1 mm, 1.9 μm, San Jose, CA, USA) and gradient method were used for chromatographic separation. A gradient elution (A, 0.1% formic acid in water; B, acetonitrile) with a 0.3 mL/min flow rate was programmed as follows: 35% B, 0–1 min, 35%–60% B, 1–3.5 min, and 60% B, 3.5–6 min. In the MS/MS analysis, positive ion mode and a parallel reaction monitoring (PRM) method were applied. The spray voltage (3.0 kV), capillary temperature (320 °C), sheath gas pressure (40 arbitrary units), auxiliary gas pressure (five arbitrary units), and resolution (17,500) were set to achieve optimal sensitivity and selectivity with the PRM method. Normalized collision energies for each analyte are shown in Table 6. To clean the plasma, each plasma sample (100 μL) was deproteinized with acetonitrile (200 μL) with IS (20.0 ng/mL), vortexed vigorously (5 min), and centrifuged (12,000 rpm, 10 min). Then, an aliquot of the filtered supernatant (0.22 μm, 3 μL) was injected into the UPLC/MS/MS system. Method validation was performed for linearity, selectivity, accuracy, precision, recovery, matrix effects, and stability, according to Zhou et al. [28]. Quality control (QC) samples (n = 6) at three concentration (2, 50, and 150 ng/mL) were used for method validation.

3.3. Animal Experiments

The animal study was approved by the Institutional Animal Care and Use Committee of Korea Institute of Oriental Medicine (Daejeon, Korea). Male Sprague-Dawley rats (245–260 g) were obtained from Samtako (Osan, Korea), housed under standard laboratory conditions with free access to food and water, and acclimated for seven days. Experimental colitis was induced using TNBS under isoflurane anesthesia, according to Arab et al. [16]. Before inducing colitis, the rats were fasted for 24 h. Under isoflurane anesthesia, the rats were given a single rectal dose of TNBS (50 mg/kg in 50% ethanol) through a medical grade polyurethane catheter (2 mm, external diameter) in the descending colon 8 cm from the anus sphincter. After slowly injecting the TNBS over 1 min, the rats were kept in a supine Trendelenburg position for 3 min to avoid intracolonic TNBS leakage. Normal rats received only 50% ethanol instead of TNBS, and the animals were used for the pharmacokinetic study on day 5 of the TNBS or 50% ethanol treatment. Before the pharmacokinetic study, no medication for pain control was administered, to avoid interrupting the colitis induction. Normal and induced-colitis rats were divided randomly into four groups (n = 6 each) (group 1, ADR 0.5 g/kg in normal rats; group 2, ADR 1.0 g/kg in normal rats; group 3, ADR 0.5 g/kg in induced-colitis rats; and group 4, ADR 1.0 g/kg in induced-colitis rats). ADR was prepared in distilled water, and administered orally by single gavage (5 mL/kg of dosing volume). Blood samples were collected periodically from the tail vein over 480 min without anesthesia. To obtain plasma, blood samples were centrifuged (12,000 rpm, 10 min), with the plasma stored at −80 °C before use.

3.4. Data Analysis

All values are presented as means ± standard deviation. A non-compartmental pharmacokinetic analysis for the time-concentration data of the three furanocoumarins was performed using the program PKSolver [29]. Comparisons of pharmacokinetic parameters between normal and TNBS-treated rats were performed using analysis of variance (ANOVA) or Student’s t-test with Prism 5.0. p-Values < 0.05 were considered to indicate statistical significance.

4. Conclusions

A rapid, sensitive UPLC/MS/MS method for the determination of three furanocoumarins was developed and used to evaluate the effects of colitis in a pharmacokinetic rat study. We first demonstrated that the colitis altered the rate and extent of oral absorption of major bioactive constituents after ADR administration, although the mechanisms are not clear. Thus, the prescription and use of ADR in colitis patients should receive more attention, and further studies are needed to clarify whether the altered pharmacokinetic profile is clinically relevant for therapeutic dosing.

Acknowledgments

This work was supported by the grant (No. K16281) from the Korea Institute of Oriental Medicine funded by the Ministry of Education, Science and Technology (MEST), the Republic of Korea.

Author Contributions

Participated in the research design: Y.H.H., H.J.Y. and J.Y.M.; Conducted experiments: Y.H.H. and H.J.Y.; Performed data analyses: Y.H.H. and H.J.Y.; Wrote the manuscript: Y.H.H.

Conflicts of Interest

The authors declare no conflicts of interests.

References

  1. Lee, K.; Shin, M.S.; Ham, I.; Choi, H.Y. Investigation of the mechanisms of Angelica dahurica root extract-induced vasorelaxation in isolated rat aortic rings. BMC Complement. Altern. Med. 2015, 15, 395. [Google Scholar] [CrossRef] [PubMed]
  2. Liu, C.; Tseng, A.; Yang, S. Chinese Herbal Medicine: Modern Applications of Traditional Formulas; CRC Press (Taylor & Francis Group): Boca Raton, FL, USA, 2004; p. 446. [Google Scholar]
  3. Wei, W.; Wu, X.W.; Deng, G.G.; Yang, X.W. Anti-inflammatory coumarins with short- and long-chain hydrophobic groups from roots of Angelica dahurica cv. Hangbaizhi. Phytochemistry 2016, 123, 58–68. [Google Scholar] [CrossRef] [PubMed]
  4. Zhao, A.H.; Zhang, Y.B.; Yang, X.W. Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats. Phytomedicine 2016, 23, 1029–1036. [Google Scholar] [CrossRef] [PubMed]
  5. Kang, S.W.; Kim, C.Y.; Song, D.G.; Pan, C.H.; Cha, K.H.; Lee, D.U.; Um, B.H. Rapid identification of furanocoumarins in Angelica dahurica using the online LC-MMR-MS and their nitric oxide inhibitory activity in RAW 264.7 cells. Phytochem. Anal. 2010, 21, 322–327. [Google Scholar] [CrossRef] [PubMed]
  6. Koziol, E.; Skalicka-Wozniak, K. Imperatorin-pharmacological meaning and analytical clues: Profound investigation. Phytochem. Rev. 2016, 15, 627–649. [Google Scholar] [CrossRef] [PubMed]
  7. Ranjbar, S.; Shokoohinia, Y.; Ghobadi, S.; Bijari, N.; Gholamzadeh, S.; Moradi, N.; Ashrafi-Kooshk, M.R.; Aghaei, A.; Khodarahmi, R. Studies of the Interaction between Isoimperatorin and Human Serum Albumin by Multispectroscopic Method: Identification of Possible Binding Site of the Compound Using Esterase Activity of the Protein. Sci. World J. 2013, 2013, 305081. [Google Scholar] [CrossRef] [PubMed]
  8. Fujioka, T.; Furumi, K.; Fujii, H.; Okabe, H.; Mihashi, K.; Nakano, Y.; Matsunaga, H.; Katano, M.; Mori, M. Antiproliferative constituents from umbelliferae plants. V. A new furanocoumarin and falcarindiol furanocoumarin ethers from the root of Angelica japonica. Chem. Pharm. Bull. (Tokyo) 1999, 47, 96–100. [Google Scholar] [CrossRef] [PubMed]
  9. Choi, J.S.; Shin, H.Y.; Kwon, K.S.; Shin, S.; Choung, S.Y.; Kwon, Y.S.; Lee, J.W.; Choi, B.H.; Lee, C.K. Effects of oxypeucedanin on global gene expression and MAPK signaling pathway in mouse neuroblastoma Neuro-2A cells. Planta Med. 2011, 77, 1512–1518. [Google Scholar] [CrossRef] [PubMed]
  10. Ishihara, K.; Kushida, H.; Yuzurihara, M.; Wakui, Y.; Yanagisawa, T.; Kamei, H.; Ohmori, S.; Kitada, M. Interaction of drugs and Chinese herbs: Pharmacokinetic changes of tolbutamide and diazepam caused by extract of Angelica dahurica. J. Pharm. Pharmacol. 2000, 52, 1023–1029. [Google Scholar] [CrossRef] [PubMed]
  11. Zhao, G.; Peng, C.; Du, W.; Wang, S. Pharmacokinetic study of eight coumarins of Radix Angelicae Dahuricae in rats by gas chromatography-mass spectrometry. Fitoterapia 2013, 89, 250–256. [Google Scholar] [CrossRef] [PubMed]
  12. Chen, L.; Jian, Y.; Wei, N.; Yuan, M.; Zhuang, X.; Li, H. Separation and simultaneous quantification of nine furanocoumarins from Radix Angelicae dahuricae using liquid chromatography with tandem mass spectrometry for bioavailability determination in rats. J. Sep. Sci. 2015, 38, 4216–4224. [Google Scholar] [CrossRef] [PubMed]
  13. Bassotti, G.; Antonelli, E.; Villanacci, V.; Salemme, M.; Coppola, M.; Annese, V. Gastrointestinal motility disorders in inflammatory bowel diseases. World J. Gastroenterol. 2014, 20, 37–44. [Google Scholar] [CrossRef] [PubMed]
  14. Qin, H.Y.; Xiao, H.T.; Wu, J.C.; Berman, B.M.; Sung, J.J.; Bian, Z.X. Key factors in developing the trinitrobenzene sulfonic acid-induced post-inflammatory irritable bowel syndrome model in rats. World J. Gastroenterol. 2012, 18, 2481–2492. [Google Scholar] [CrossRef] [PubMed]
  15. Randhawa, P.K.; Singh, K.; Singh, N.; Jaggi, A.S. A review on chemical-induced inflammatory bowel disease models in rodents. Korean J. Physiol. Pharmacol. 2014, 18, 279–288. [Google Scholar] [CrossRef] [PubMed]
  16. Arab, H.H.; Al-Shorbagy, M.Y.; Abdallah, D.M.; Nassar, N.N. Telmisartan attenuates colon inflammation, oxidative perturbations and apoptosis in a rat model of experimental inflammatory bowel disease. PLoS ONE 2014, 9, e97193. [Google Scholar]
  17. Ploemen, J.P.H.T.M.; Kramer, H.; Krajnc, E.I.; Martin, I. The use of toxicokinetic data in preclinical safety assessment: A toxicologic pathologist perspective. Toxicol. Pathol. 2007, 35, 834–837. [Google Scholar] [CrossRef] [PubMed]
  18. Cai, H.D.; Su, S.L.; Li, Y.; Zhu, Z.; Guo, J.; Zhu, Y.; Guo, S.; Qian, D.; Duan, J. Simultaneous Determination of Four Tanshinones by UPLC-TQ/MS and Their Pharmacokinetic Application after Administration of Single Ethanol Extract of Danshen Combined with Water Extract in Normal and Adenine-Induced Chronic Renal Failure Rats. Molecules 2016, 21, 1630. [Google Scholar] [CrossRef] [PubMed]
  19. Quigley, E.M. Review article: Gastric emptying in functional gastrointestinal disorders. Aliment. Pharmacol. Ther. 2004, 20, 56–60. [Google Scholar] [CrossRef] [PubMed]
  20. Pallotta, N.; Pezzotti, P.; Calabrese, E.; Baccini, F.; Corazziari, E. Relationship between gastrointestinal and extra-gastrointestinal symptoms and delayed gastric emptying in functional dyspeptic patients. World J. Gastroenterol. 2005, 11, 4375–4381. [Google Scholar] [CrossRef] [PubMed]
  21. Yanez, J.A.; Remsberg, C.M.; Sayre, C.L.; Forrest, M.L.; Davies, N.M. Flip-flop pharmacokinetics—Delivering a reversal of disposition: Challenges and opportunities during drug development. Ther. Deliv. 2011, 2, 643–672. [Google Scholar] [CrossRef] [PubMed]
  22. De Schepper, H.U.; de Man, J.G.; van Nassauw, L.; Timmermans, J.P.; Herman, A.G.; Pelckmans, P.A.; de Winter, B.Y. Acute distal colitis impairs gastric emptying in rats via an extrinsic neuronal reflex pathway involving the pelvic nerve. Gut 2007, 56, 195–202. [Google Scholar] [CrossRef] [PubMed]
  23. Masubuchi, Y.; Enoki, K.; Horie, T. Down-regulation of hepatic cytochrome P450 enzymes in rats with trinitrobenzene sulfonic acid-induced colitis. Drug Metab. Dispos. 2008, 36, 597–603. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, J.; Zhang, M.; Fu, S.; Li, T.; Wang, S.; Zhao, M.M.; Ding, W.J.; Wang, C.Y.; Wang, Q. Simultaneous determination of imperatorin and its metabolite xanthotoxol in rat plasma by using HPLC-ESI-MS coupled with hollow fiber liquid phase microextraction. J. Chromatogr. B 2014, 945, 185–192. [Google Scholar] [CrossRef] [PubMed]
  25. Song, G.; Jin, M.; Du, Y.; Cao, L.; Xu, H. UPLC-QTOF-MS/MS based screening and identification of the metabolites in rat bile after oral administration of imperatorin. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2016, 1022, 21–29. [Google Scholar] [CrossRef] [PubMed]
  26. Chen, T.L.; Zhang, Y.B.; Xu, W.; Kang, T.G.; Yang, X.W. Biotransformation of isoimperatorin by rat liver microsomes and its quantification by LC-MS/MS method. Fitoterapia 2014, 93, 88–97. [Google Scholar] [CrossRef] [PubMed]
  27. Jeong, Y.H.; Oh, Y.C.; Cho, W.K.; Lee, B.; Ma, J.Y. Anti-Inflammatory Effects of Melandrii Herba Ethanol Extract via Inhibition of NF-kappaB and MAPK Signaling Pathways and Induction of HO-1 in RAW 264.7 Cells and Mouse Primary Macrophages. Molecules 2016, 21, 818. [Google Scholar] [CrossRef] [PubMed]
  28. Zhou, W.; Tam, K.Y.; Meng, M.; Shan, J.; Wang, S.; Ju, W.; Cai, B.; Di, L. Pharmacokinetics screening for multi-components absorbed in the rat plasma after oral administration of traditional Chinese medicine Flos Lonicerae Japonicae-Fructus Forsythiae herb couple by sequential negative and positive ionization ultra-high-performance liquid chromatography/tandem triple quadrupole mass spectrometric detection. J. Chromatogr. A 2015, 1376, 84–97. [Google Scholar] [PubMed]
  29. Zhang, Y.; Huo, M.R.; Zhou, J.P.; Xie, S.F. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 2010, 99, 306–314. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the compounds are not available from the authors.
Figure 1. PRM chromatogram of the analytes and internal standard (IS) in plasma: (A) blank plasma; (B) spiked blank plasma in LLOQ; (C) plasma sample after oral administration of Angelica dahurica Radix.
Figure 1. PRM chromatogram of the analytes and internal standard (IS) in plasma: (A) blank plasma; (B) spiked blank plasma in LLOQ; (C) plasma sample after oral administration of Angelica dahurica Radix.
Molecules 22 00416 g001
Figure 2. Mass spectrum of the analytes and internal standard (IS). Production m/z transitions for oxypeucedanin (A); imperatorin (B); isoimperatorin (C); and warfarin (IS, D) were selected 287.09088 → 203.03345, 271.09573 → 203.03336, and 309.25220 → 163.03865, respectively.
Figure 2. Mass spectrum of the analytes and internal standard (IS). Production m/z transitions for oxypeucedanin (A); imperatorin (B); isoimperatorin (C); and warfarin (IS, D) were selected 287.09088 → 203.03345, 271.09573 → 203.03336, and 309.25220 → 163.03865, respectively.
Molecules 22 00416 g002
Figure 3. TNBS-induced experimental colitis in rats. (A) Macroscopic appearance of normal and damaged colon; (B) Weight of explanted colon.
Figure 3. TNBS-induced experimental colitis in rats. (A) Macroscopic appearance of normal and damaged colon; (B) Weight of explanted colon.
Molecules 22 00416 g003
Figure 4. Plasma concentration–time curves of oxypeucedanin (A); imperatorin (B); and isoimperatorin (C) after oral administration of Angelica dahurica Radix (ADR, 0.5 and 1.0 g/kg, open and closed symbols, respectively) to normal (circle) and experimental colitis (square)-induced rats.
Figure 4. Plasma concentration–time curves of oxypeucedanin (A); imperatorin (B); and isoimperatorin (C) after oral administration of Angelica dahurica Radix (ADR, 0.5 and 1.0 g/kg, open and closed symbols, respectively) to normal (circle) and experimental colitis (square)-induced rats.
Molecules 22 00416 g004
Figure 5. Chemical structures of oxypeucedanin (A); imperatorin (B); isoimperatorin (C); and warfarin (internal standard, D).
Figure 5. Chemical structures of oxypeucedanin (A); imperatorin (B); isoimperatorin (C); and warfarin (internal standard, D).
Molecules 22 00416 g005
Table 1. Linearity and calibration curves of oxypeucedanin, imperatorin, and isoimperatorin.
Table 1. Linearity and calibration curves of oxypeucedanin, imperatorin, and isoimperatorin.
ComponentRange (ng/mL)Linear Regression EquationCorrelation CoefficientLLOQ (ng/mL)
Oxypeucedanin1.0–200y = 0.0444x − 0.03810.99961.0
Imperatorin1.0–200y = 0.0857x + 0.01360.99941.0
Isoimperatorin1.0–200y = 0.0431x − 0.02950.99951.0
Table 2. Accuracy and precision of oxypeucedanin, imperatorin, and isoimperatorin in rat plasma.
Table 2. Accuracy and precision of oxypeucedanin, imperatorin, and isoimperatorin in rat plasma.
ComponentsNominal Concentration (ng/mL)Intra-Day (n = 6)Inter-Day (n = 6)
Measured Concentration (ng/mL)RE (%)RSD (%)Measured Concentration(ng/mL)RE (%)RSD (%)
Oxypeucedanin22.1 ± 0.26.89.42.1 ± 0.26.88.1
5049.8 ± 1.7−0.73.449.1 ± 2.6−3.75.3
150152.4 ± 6.51.64.3149.1 ± 1.9−0.21.3
Imperatorin21.9 ± 0.2−6.98.31.9 ± 0.2−4.98.7
5049.4 ± 1.9−2.23.949.9 ± 2.3−0.54.5
150149.1 ± 5.7−0.63.8151.7 ± 4.71.23.1
Isoimperatorin21.9 ± 0.1−5.17.02.0 ± 0.1−4.14.4
5050.3 ± 1.81.03.648.6 ± 1.3−5.62.8
150150.2 ± 3.00.11.9151.4 ± 2.70.91.8
Table 3. Extract recovery, matrix effects, and stability of oxypeucedanin, imperatorin, and isoimperatorin in rat plasma.
Table 3. Extract recovery, matrix effects, and stability of oxypeucedanin, imperatorin, and isoimperatorin in rat plasma.
Components/Nominal Concentration (ng/mL)Recovery (%)Matrix (%)Stability (%)
Free-Thaw CyclesAt −70 °C for 30 DaysAt Room Temperature for 24 h
Oxypeucedanin
297.4 ± 7.9100.2 ± 6.794.4 ± 6.497.7 ± 6.394.7 ± 7.6
5084.6 ± 7.699.9 ± 2.299.9 ± 4.398.0 ± 0.897.8 ± 3.6
15077.6 ± 7.4100.8 ± 2.195.0 ± 8.899.6 ± 1.497.8 ± 6.6
Imperatorin
284.9 ± 9.891.8 ± 7.697.4 ± 7.199.0 ± 6.497.6 ± 7.8
5083.4 ± 3.792.3 ± 2.0100.2 ± 3.3101.0 ± 3.7100.9 ± 5.0
15079.8 ± 4.285.2 ± 3.1101.1 ± 3.894.9 ± 9.7101.6 ± 1.6
Isoimperatorin
285.3 ± 12.294.9 ± 12.9101.2 ± 4.698.3 ± 6.595.9 ± 7.4
5075.6 ± 4.796.2 ± 6.998.8 ± 3.398.6 ± 3.6100.3 ± 5.2
15070.3 ± 7.694.6 ± 7.795.6 ± 9.297.1 ± 6.397.5 ± 7.2
Table 4. Comparison of the developed method and the previous method for determination of three furanocoumarins.
Table 4. Comparison of the developed method and the previous method for determination of three furanocoumarins.
MethodsLC/MS/MS
[4]
LC/MS/MS
[12]
GC-MS
[11]
UPLC-MS/MS
(This Work)
LLOQ (ng/mL)
Oxypeucedanin1.165.007.001.00
Imperatorin0.145.00 1.00
Isoimperatorin0.144.0010.001.00
Plasma sample amount (μL)3005020050
Run time (min)27.05.550.06.0
Flow rate (mL/min)1.00.31.20.3
Table 5. Pharmacokinetic parameters of oxypeucedanin, imperatorin, and isoimperatorin after oral administration of Angelica dahurica Radix to normal and experimental TNBS-treated rats.
Table 5. Pharmacokinetic parameters of oxypeucedanin, imperatorin, and isoimperatorin after oral administration of Angelica dahurica Radix to normal and experimental TNBS-treated rats.
ParametersNormalTNBS-Treated
0.5 g/kg1.0 g/kg0.5 g/kg1.0 g/kg
Oxypeucedanin
t1/2 (min)78.1 ± 7.573.5 ± 7.891.9 ± 44.872.9 ± 7.7
Tmax (min)43.2 ± 11.249.1 ± 11.054.0 ± 22.842.4 ± 14.3
Cmax (ng/mL)38.5 ± 1.6101.2 ± 21.229.0 ± 4.0 *61.2 ± 11.9 *
AUC0→∞ (min·ng/mL)5753.4 ± 649.810,866.7 ± 1720.67745.0 ± 1576.013,126.2 ± 2382.3
MRT (min)125.9 ± 13.6118.9 ± 11.0203.7 ± 54.2 *175.7 ± 7.8 *
Imperatorin
t1/2 (min)59.5 ± 7.261.5 ± 6.865.2 ± 11.556.1 ± 13.9
Tmax (min)54.0 ± 7.772.0 ± 6.7113.7 ± 13.4 *127.1 ± 18.4 *
Cmax (ng/mL)94.5 ± 19.7201.4 ± 18.827.7 ± 6.3 *118.5 ± 32.5 *
AUC0→∞ (min·ng/mL)11,860.3 ± 2832.523,889.0 ± 4003.15196.0 ± 1349.724,845.0 ± 6485.8
MRT (min)121.1 ± 10.4127.6 ± 11.5172.6 ± 12.8 *187.6 ± 9.5 *
Isoimperatorin
t1/2 (min)63.8 ± 21.978.1 ± 2.488.0 ± 12.968.6 ± 24.2
Tmax (min)72.0 ± 6.767.5 ± 6.3120.0 ± 16.7 *144.0 ± 16.3 *
Cmax (ng/mL)72.1 ± 12.4128.8 ± 8.721.1 ± 4.9 *71.3 ± 16.2 *
AUC0→∞ (min·ng/mL)9445.4 ± 2157.117,778.9 ± 2519.34197.9 ± 919.717,881.7 ± 4111.3
MRT (min)143.2 ± 21.5145.2 ± 11.6204.3 ± 15.2 *225.3 ± 33.5 *
t1/2, elimination half-life; Tmax, time to reach peak concentration; Cmax, peak plasma concentration; AUC0→∞, area under the plasma concentration–time curve from zero to infinity time; MRT, mean residence time. * p < 0.05, compared to normal rats treated with same dose.
Table 6. The information of PRM parameters.
Table 6. The information of PRM parameters.
ComponentsChemical FormulaPrecursor ion [M + H]+ProductionNormalize Collision Energy
CalculatedMeasured
OxypeucedaninC16H14O5287.09140287.09088203.0334535
ImperatorinC16H14O4271.09649271.09573203.0333620
IsoimperatorinC16H14O4271.09649271.09619203.0333620
Wafarin (IS)C19H16O4309.11214309.25220251.0699250

Share and Cite

MDPI and ACS Style

Hwang, Y.-H.; Yang, H.J.; Ma, J.Y. Simultaneous Determination of Three Furanocoumarins by UPLC/MS/MS: Application to Pharmacokinetic Study of Angelica dahurica Radix after Oral Administration to Normal and Experimental Colitis-Induced Rats. Molecules 2017, 22, 416. https://doi.org/10.3390/molecules22030416

AMA Style

Hwang Y-H, Yang HJ, Ma JY. Simultaneous Determination of Three Furanocoumarins by UPLC/MS/MS: Application to Pharmacokinetic Study of Angelica dahurica Radix after Oral Administration to Normal and Experimental Colitis-Induced Rats. Molecules. 2017; 22(3):416. https://doi.org/10.3390/molecules22030416

Chicago/Turabian Style

Hwang, Youn-Hwan, Hye Jin Yang, and Jin Yeul Ma. 2017. "Simultaneous Determination of Three Furanocoumarins by UPLC/MS/MS: Application to Pharmacokinetic Study of Angelica dahurica Radix after Oral Administration to Normal and Experimental Colitis-Induced Rats" Molecules 22, no. 3: 416. https://doi.org/10.3390/molecules22030416

Article Metrics

Back to TopTop