Journal of Chromatography B, 947–948 (2014) 57–61
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of xanthatin by ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry: Application to pharmacokinetic study of xanthatin in rat plasma Cuiping Yan a,b,1 , Huan Li a,b,1 , Yu Wu d , Donghao Xie c , Zebin Weng a,b , Baochang Cai a,b , Xiao Liu a,b , Weidong Li a,b,∗ , Zhipeng Chen a,b,∗∗ a Nanjing University of Chinese Medicine, Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing 210023, China b Pharmacy College of Nanjing University of TCM, Nanjing 210023, China c Shanghai Dahua Hospital of Shanghai Xuhui District, Shanghai 200031, China d Nantong Hospital of Traditional Chinese Medicine, Nantong 226001, China
a r t i c l e
i n f o
Article history: Received 28 May 2013 Accepted 5 December 2013 Available online 18 December 2013 Keywords: Xanthatin Pharmacokinetics Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–ESI-MS/MS)
a b s t r a c t A sensitive, specific and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been established to study pharmacokinetic properties of xanthatin. Xanthatin, a compound which belongs to sesquiterpene lactone group, was determined in rat plasma with psoralen as internal standard. Chromatographic separation was performed on an Agilent Zorbax Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 m) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase was composed of methanol and 0.1% formic acid water solution. Analysis was performed under a triple-quadruple tandem mass-spectrometer with an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM) mode to determine xanthatin at [M+H]+ m/z 247.3 → m/z 205.2 and that of IS at [M+H]+ m/z 187.1 → m/z 143.0 within 5 min. The assay method exhibited good separation of xanthatin from the interference of endogenous substances. The lower limit of quantification (LLOQ) was 1 ng/mL, with a good linearity within the concentration range of 1–5000 ng/mL (r = 0.9990). Intra-day and inter-day precision RSD was less than 9.27%; intra-day and inter-day accuracy was 88.48% and 102.25% respectively. The extraction recoveries of xanthatin range from 82.12% to 89.55%, and the extraction RSD was less than 9.01%. The established LC–ESI-MS/MS method is rapid and sensitive, which has been successfully applied to quantify xanthatin in rat plasma for the first time. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Xanthium strumarium L. (Asteraceae), a traditional medical plant in Asia and Africa, was widely used for the treatment of rhinitis, rheumatism, eczema, cancer, ulcer and malaria [1–3]. Xanthatin, with an ␣-methylene-␥-butrolactone structure, was one of the major active components isolated from the leaves of X. strumarium L. [4]. In recent years, various biological activities of this compound
∗ Corresponding author at: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing 210023, China. Tel.: +86 86798281. ∗∗ Corresponding author. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Chen). 1 Both these authors contributed equally to the project and are considered co-first authors.
have been reported, such as anti-inflammation [5], antileishmanial, antifungal [6], antioxidant [7], trypanocidal and cytotoxic properties [8]. Particularly, xanthatin can significantly inhibit the growth of various animal and human tumors [9–11]. The molecular mechanisms of its anticancer properties are attracting more and more attention. Previous studies have shown that xanthatin exhibited anti-proliferative effects by inducing the expression of the GADD45␥ gene in MDA-MB-231 cells [12], thus induce cell cycle arrest and apoptosis via disrupting NF-B pathway in A549 Cells [13]. Xanthatin also induces human gastric carcinoma MKN-45 cells apoptosis by activating p53 pathway [14]. In addition, our previous study demonstrated that xanthatin could significantly induce apoptosis of murine melanoma B16-F10 by activating Wnt/-catenin signaling in vitro. It is obviously that xanthatin could inhibit B16-F10 tumor growth in the murine melanoma model by intraperitoneal injection at designated concentration of 4.8 mg/200 g [15]. On the other hand, scientists are focusing on
1570-0232/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.006
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developing an efficient method to synthesize xanthatin and investigating its structure-activity relationship [16]. All of these results suggest that xanthatin maybe a promising drug entity, which can be developed for treating human cancers. In the process of developing a new drug, relative pharmacokinetic parameters are necessary to be explored. To the best of the authors’ knowledge, the pharmacokinetic characters of xanthatin have not been reported yet. Therefore, it is necessary to develop a method to determine the concentration of xanthin in plasma and investigate its pharmacokinetic properties. The results will contribute to the following pharmacological research of xanthatin. The purpose of this paper was to establish a sensitive, rapid and specific UHPLC–MS/MS method for the determination of xanthatin in rat plasma. 2. Materials and methods 2.1. Reagents and materials Methanol (Calepure, Canada, HPLC grade). The water used in this research was purified with an EPED water purification system from Nanjing EPED system (Nanjing, China), and the other reagents were HPLC grade. The air-dried aerial parts of X. strumarium L. were collected from the suburbs of Xuzhou, Jiangsu province of China. The herb was authenticated by Professor Jianwei Chen of Nanjing University of Chinese Medicine. Xanthatin (Fig. 1a, purity ≥98%) was isolated from the air-dried aerial parts of X. strumarium L. in the authors’ laboratory. Its chemical structure was confirmed by using 1 H NMR, DEPT, 1 H, 1 H-COSY, NOESY, HSQC, and HMBC. Psoralen (Fig. 1b, purity ≥99%) was obtained from Shanghai U-sea Biotech Co. Ltd. (Shanghai, China). Other reagents and solvents were of the highest quality available. 2.2. Equipments All separations were performed on a DGU-20A 5R series UHPLC system equipped with a LC-30AD binary pump (Shimadzu Corporation UFLC XR, Kyoto, Japan). Mass spectrometry was conducted by using a 5500 triple quad tandem mass spectrometer equipped with electrospray ionization (ESI) source (AB SCIEX, Foster City, CA, USA).
2.3. Chromatographic and mass-spectroscopic conditions The analytical column was Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 m). The mobile phase was composed of methanol (B) and 0.1% formic acid water solution (A) with gradient elution system (0–1.5 min, 5–50% B; 1.5–3.5 min, 50–70% B; 3.5–4 min, 70–85% B; 4–4.5 min, 85–50% B; 4.5–5 min, 50–5% B) at a flow rate of 0.3 mL/min. Injection volume was 5 L and the column temperature was 40 ◦ C. Mass analysis was performed in the positive ion electrospray ionization mode with an electrospray ionization (ESI) source. The ion spray voltage was set at 5.5 kV. The optimized parameters were recorded as following data: Ion source temperature, 600 ◦ C; curtain gas (CUR), 241.33 kPa; ion source gas 1 (GAS1), 344.75 kPa; ion source gas 2 (GAS2), 413.70 kPa; ion spray voltage (IS), 5500 V; declustering potential (DP), 69 V; collision energy (CE), 14 V; entrance potential (EP), 10 V and collision exit potential (CXP), 14 V. Multiple reaction monitoring (MRM) mode was applied for the quantitation at [M+H]+ m/z 247.3 → m/z 205.2 for xanthatin and at [M+H]+ m/z 187.1 → m/z 143.0 for psoralen (IS). The chemical structure and the mass spectrum of xanthatin and psoralen were shown in Fig. 1.
2.4. Preparation of stock solutions and quality control samples Stock solution was prepared by dissolving xanthatin and psoralen in methanol at concentration of 200 g/mL each. The internal standard (IS) working solution (10 ng/mL) was obtained by dilute the stock solution with methanol. Calibration samples were prepared by spiking 90 L blank plasma with 10 L xanthatin working solutions to produce final concentrations of 1, 10, 50, 100, 500, 1000 and 5000 ng/mL. The quality control (QC) samples were prepared at concentrations of 2, 50, 500 and 3000 ng/mL respectively. All solutions were kept in 4 ◦ C refrigerator and brought to room temperature before use. 2.5. Sample preparation To each sample, 10 L of IS solution (10 ng/mL) was added into 100 L of rat plasma. The mixture was extracted with 1 mL of ethyl acetate by vortexing for 3 min. The aqueous and organic layer was separated by centrifugation at 4000 r/min for 5 min and the organic layer was transferred to another centrifuge tube and evaporated to dry under a gentle stream of nitrogen gas at 40 ◦ C. The residue was reconstituted with 100 L of mobile phase (0.1% formic acidmethanol at ratio of 5: 95), then centrifuged at 4000 r/min for 5 min. For analyzing, 5 L of the supernatant was injected into the LC–MS/MS system. 2.6. Method validation 2.6.1. Specificity Specificity of the method was evaluated by comparing chromatograms of blank plasma samples, samples spiked with IS plus xanthatin and treated plasma samples. 2.6.2. Linearity and quantification Various concentrations of xanthatin calibration standard solution (1, 10, 50, 100, 500, 1000, 5000 ng/mL) with IS (10 ng/mL) were prepared as plasma sample preparation method and assayed by UHPLC–MS/MS system. Peak area ratios of xanthatin/IS plotted against the corresponding concentrations were calculated to construct calibration curve. The calibration curve was established via 1/x2 weighted linear least-squares regression model. The lower limit of quantification (LLOQ) was determined as the lowest concentration on the QC samples. 2.6.3. Precision and accuracy Accuracy and precision of the method were determined by analyzing six replicates of the QC and LLOQ samples. The intra-day precision and accuracy of the method were assessed by determining the QC samples six times within a single day, while the inter-day precision and accuracy were estimated by determining the QC samples over three consecutive days. 2.6.4. Recovery and matrix effects The extraction recovery was determined by comparing the concentration of extracted QC samples with the same concentration of xanthatin reference standard solutions. Matrix effects of the method were calculated by comparing the peak areas of reference standard in extracts of blank plasma with that of neat standard solution. Recovery and matrix effects experiments at four QC concentrations for plasma were examined (n = 5). 2.6.5. Stability The stability of xanthatin in plasma was investigated by analyzing of four QC samples (n = 5). The short-term stability was investigated by analyzing plasma samples kept at room temperature for 4 h. Freezing-thawing stability was determined after
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Fig. 1. Chemical structures and MS/MS spectra of xanthatin (a) and psoralen (b).
QC samples through three freezing-thawing cycles by freezing at −20 ◦ C and thawing at 37 ◦ C water bath. The long-term stability was assessed after the QC samples were stored at −20 ◦ C for 14 days. 2.7. Pharmacokinetic study Eighteen male Sprague-Dawley rats, weighing 220–250 g, were obtained from the Experimental Animal Center (Nanjing University of Chinese Medicine, China). The rats were housed in a room with controlled temperature and humidity, and allowed free
access to food and water. They were fasted overnight before the experiment. Rats were divided into three groups administered xanthatin via intravenous at provided concentrations (2.4 mg/200 g, 4.8 mg/200 g and 9.6 mg/200 g), respectively. Xanthatin was completely dissolved in the mixture solution, which composed of 85% physiological saline, 10% Pluronic F-68 and 5% DMSO, facilitated by ultrasonication. Blood samples (approximately 0.15 mL) were collected from the oculi chorioideae vein at each time point (0, 2, 5, 15, 30, 60, 120, 240, 360 min) after administration. The blood samples were centrifuged at 12,000 rev/min for 10 min at 4 ◦ C and
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3.2.2. Linearity and sensitivity The calibration curve of xanthatin showed a good linearity in the range of 1–5000 ng/mL. The regression equation of xanthatin in plasma was y = 0.01013x + 0.10364 (r = 0.9990). The LLOD of xanthatin in rat plasma was 1 ng/mL. 3.2.3. Precision and accuracy The QC and LLOQ samples were analyzed for accuracy and precision. The RSD of inter-day precision of QC samples were 5.67%, 7.24%, 9.18% and 7.34%, and the LLOQ sample was 8.24%. The interday precision RSD of QC samples were 9.03%, 9.27%, 9.13% and 8.78%, and the LLOQ sample was 9.35% respectively. The accuracy for xanthatin was 88.48–102.25%. The accuracy and precision of the method are in accordance with the requirements of the determination of biological samples.
Fig. 2. SIM chromatograms of blank plasma (a); blank plasma spiked with xanthatin (5 ng/mL) and IS (b); treated plasma (c). Xanthatin: Rt = 3.47 min; IS: Rt = 3.37 min.
the supernatants were stored at −20 ◦ C freezer. After analyzing, the obtained data was processed by using Win Nonlin53 software (Pharsight Corporation, Mountain View, USA). 3. Results 3.1. Method optimization In this study, a new UHPLC–MS/MS method was developed to quantify xanthatin in rat plasma. A gradient elution system was applied in order to get rid of interference of endogenous compounds. The concentration of formic acid in mobile phase (0.1%) was optimized for the best signal response of the analytes. The mass parameter of DP, CE, EP, and CXP was optimized to enhance the ionization efficiency of xanthatin. Table 1 3.2. Method validation 3.2.1. Assay specificity The typical chromatograms of the blank plasma samples, blank plasma samples added with xanthatin or IS and treated plasma samples were shown in Fig. 2. The retention time of xanthatin and IS was 3.47 and 3.37 min, respectively. The results indicated that the endogenous ingredients did not interfere with the analyzing of xanthatin or the IS in the plasma samples.
3.2.4. Recovery and matrix effects The extraction recoveries of xanthatin at concentrations of 2, 50, 500 and 3000 ng/mL from rat plasma were 87.10%, 84.23%, 82.12% and 89.55% respectively. The RSD of extraction recoveries were 7.73%, 9.01%, 8.10% and 8.27% correspondently. In addition, the recoveries of the IS from the plasma were 81.18% and the RSD was 8.20%. Under the UHPLC–MS/MS conditions, the matrix effects of four QC samples were 91.89%, 92.23%, 94.76% and 97.18% respectively. The RSD of the matrix effects for the QC samples were 5.13%, 7.29%, 7.65% and 8.92%. In addition, the matrix effects of the IS was 103.61% and the RSD was 6.89%. 3.2.5. Stability The QC samples were used for stability experiments. The shortterm temperature stability was 6.25%, 7.28%, 6.90% and 5.16% for all four QC levels respectively, with the highest RSD not more than 7.18%. The freezing-thawing stability was 7.14%, 7.38%, 8.15%, and 6.88% for correspondent concentration, with the highest RSD not more than 8.96%. The long-term stability was 8.47%, 6.25%, 6.82% and 9.45% at each concentration with the highest RSD not more than 8.74%. Results manifested that xanthatin was not easily degradable under experimental conditions. 3.3. Pharmacokinetics study Various doses of xanthatin (2.4 mg/200 g, 4.8 mg/200 g and 9.6 mg/200 g), based on previous study, were intravenously injected into rats. The plasma concentration-time profiles of xanthatin were shown in Fig. 3. The main pharmacokinetic parameters were 14,340.20 (±7122.41), 32,149.52 (±11,259.44), 49,524.28 (±28,520.88). The plasma concentrations of xanthatin were rapidly dropped within 30 min after intravenous administration. The t1/2 of three concentrations was found to be 108.58 (±32.82), 123.50 (±66.69), 181.71 (±148.26) min, respectively. As the dose increased, the AUC0–∞ and AUC0–t were gradually enlarged. The total body CL were 0.13 (±0.14), 0.17 (±0.11), 0.22 (±0.13) and
Table 1 Pharmacokinetic data of xanthatin in rats (n = 6). Parameter
Dose (mg/200 g) 2.4 (iv)
Cmax (ng/mL) AUC0–t (ng h/mL) AUC0–∞ (ng h/mL) T1/2 (min) CL (mL/min) MRT (min) Vd (mL)
418.72 14,340.20 15,538.97 108.58 0.13 225.10 46.85
4.8 (iv) ± ± ± ± ± ± ±
137.51 7122.41 7733.12 32.82 0.14 187.70 20.19
904.89 32,149.52 36,431.22 123.50 5 0.17 159.99 25.13
9.6 (iv) ± ± ± ± ± ± ±
193.53 11,259.44 14,498.16 66.69 0.11 30.49 7.41
1773.46 49,524.28 61,885.45 181.71 0.22 208.22 48.35
± ± ± ± ± ± ±
1733.10 28,520.88 30,704.80 148.26 0.13 85.97 11.03
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Fig. 3. Mean plasma concentration–time curves for xanthatin in rat plasma (n = 6).
Vd were 46.85 (±20.19), 159.99 (±30.49), 208.22 (±85.97) of three concentrations. 4. Discussion This research was concentrated in the investigation of pharmacokinetic properties of xanthatin. A sensitive and specific UHPLC–MS/MS method was established for the determination of low-level concentrations of xanthatin in plasma samples for the first time. Xanthatin, a high lipophilic compound, exhibited promising drug like characters. However, its hydrophobic property will cause poor absorption when oral administrated. Thus, intravenous administration at low, medium and high doses of xanthatin was applied to increase the bioavailability. The t1/2 obtained at low, medium and high doses had no significant difference after Student T-test was used for comparison, which indicated it is safe when applied to clinical therapies. Cmax and AUC0–t were exhibited dose-dependent at three concentrations. The pharmacokinetic properties of xanthatin exhibited linear trend. All of the obtained data indicated that xanthatin could be metabolized and rapidly eliminated from the body of rat after administration. The method that developed in this research proved reliable for pharmacokinetic studies of xanthatin. It can be served as a useful method for further exploration of the pharmacological activity and clinical potential of xanthatin.
References [1] Y.T. Ma, M.C. Huang, F.L. Hsu, H.F. Chang, Phytochemistry 48 (1998) 1083–1085. [2] M.H. Yin, D.G. Kang, D.H. Choi, T.O. Kwon, H.S. Lee, J. Ethnopharmacol. 99 (2005) 113–117. [3] R. Gautam, A. Saklani, S.M. Jachak, J. Ethnopharmacol. 110 (2007) 200–234. [4] T.A. Geissman, P. Deuel, E.K. Bonde, F.A. Addicott, J. Am. Chem. Soc. 76 (1954) 685–687. [5] I.T. Kim, Y.M. Park, J.H. Won, H.J. Jung, H.J. Park, J.W. Choi, K.T. Lee, Biol. Pharm. Bull. 28 (2005) 94–100. [6] M. Lavault, A. Landreaua, G. Larcher, J.P. Bouchara, F. Pagniez, P.L. Pape, P. Richomme, Fitoterapia 76 (2005) 363–366. [7] J.H. Yoon, H.J. Lim, H.J. Lee, H.D. Kim, R. Jeon, J.H. Ryu, Bioorg. Med. Chem. Lett. 18 (2008) 2179–2182. [8] E. Nibret, M. Youns, R.L. Krauth-Siegel, M. Wink, Phytother. Res. 25 (2011) 1883–1890. [9] A.L. Demain, P. Vaishnav, Microb. Biotechnol. 4 (2011) 687–699. [10] I. Ramirez-Erosa, Y. Huang, R.A. Hickie, R.G. Sutherland, B. Barl, Can. J. Physiol. Pharmacol. 85 (2007) 1160–1172. [11] A. Kovacs, A. Vasas, P. Forgo, B. Rethy, I. Zupko, J. Z. Naturforsch. C 64 (2009) 343–349. [12] S. Takeda, K. Matsuo, K. Yaji, S. Okajima-Miyazaki, M. Harada, H. Miyoshi, Y. Okamoto, T. Amamoto, M. Shindo, C.J. Omiecinski, H. Aramaki, Toxicology 24 (2011) 855–865. [13] L. Zhang, J.S. Ruan, L.G. Yan, W.D. Li, Y. Wu, L. Tao, F. Zhang, S.Z. Zheng, A.Y. Wang, Y. Lu, Molecules 17 (2012) 3736–3750. [14] L. Zhang, L. Tao, J.S. Ruan, W.D. Li, Y. Wu, L.G. Yan, F. Zhang, F.T. Fan, S.Z. Zheng, A.Y. Wang, Y. Lu, Planta Med. 78 (2012) 890–895. [15] W.D. Li, Y. Wu, L. Zhang, L.G. Yan, F.Z. Yin, J.S. Ruan, Z.P. Chen, G.M. Yang, C.P. Yan, D. Zhao, Y. Lu, B.C. Cai, Phytomedicine 20 (2013) 865–873. [16] K. Matsumoto, K. Koyachi, M. Shindo, Tetrahedron 69 (2013) 1043–1049.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of xanthatin by ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry: Application to pharmacokinetic study of xanthatin in rat plasma Cuiping Yan a,b,1 , Huan Li a,b,1 , Yu Wu d , Donghao Xie c , Zebin Weng a,b , Baochang Cai a,b , Xiao Liu a,b , Weidong Li a,b,∗ , Zhipeng Chen a,b,∗∗ a Nanjing University of Chinese Medicine, Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing 210023, China b Pharmacy College of Nanjing University of TCM, Nanjing 210023, China c Shanghai Dahua Hospital of Shanghai Xuhui District, Shanghai 200031, China d Nantong Hospital of Traditional Chinese Medicine, Nantong 226001, China
a r t i c l e
i n f o
Article history: Received 28 May 2013 Accepted 5 December 2013 Available online 18 December 2013 Keywords: Xanthatin Pharmacokinetics Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–ESI-MS/MS)
a b s t r a c t A sensitive, specific and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been established to study pharmacokinetic properties of xanthatin. Xanthatin, a compound which belongs to sesquiterpene lactone group, was determined in rat plasma with psoralen as internal standard. Chromatographic separation was performed on an Agilent Zorbax Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 m) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase was composed of methanol and 0.1% formic acid water solution. Analysis was performed under a triple-quadruple tandem mass-spectrometer with an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM) mode to determine xanthatin at [M+H]+ m/z 247.3 → m/z 205.2 and that of IS at [M+H]+ m/z 187.1 → m/z 143.0 within 5 min. The assay method exhibited good separation of xanthatin from the interference of endogenous substances. The lower limit of quantification (LLOQ) was 1 ng/mL, with a good linearity within the concentration range of 1–5000 ng/mL (r = 0.9990). Intra-day and inter-day precision RSD was less than 9.27%; intra-day and inter-day accuracy was 88.48% and 102.25% respectively. The extraction recoveries of xanthatin range from 82.12% to 89.55%, and the extraction RSD was less than 9.01%. The established LC–ESI-MS/MS method is rapid and sensitive, which has been successfully applied to quantify xanthatin in rat plasma for the first time. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Xanthium strumarium L. (Asteraceae), a traditional medical plant in Asia and Africa, was widely used for the treatment of rhinitis, rheumatism, eczema, cancer, ulcer and malaria [1–3]. Xanthatin, with an ␣-methylene-␥-butrolactone structure, was one of the major active components isolated from the leaves of X. strumarium L. [4]. In recent years, various biological activities of this compound
∗ Corresponding author at: Nanjing University of Chinese Medicine, Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing 210023, China. Tel.: +86 86798281. ∗∗ Corresponding author. E-mail addresses: [email protected] (W. Li), [email protected] (Z. Chen). 1 Both these authors contributed equally to the project and are considered co-first authors.
have been reported, such as anti-inflammation [5], antileishmanial, antifungal [6], antioxidant [7], trypanocidal and cytotoxic properties [8]. Particularly, xanthatin can significantly inhibit the growth of various animal and human tumors [9–11]. The molecular mechanisms of its anticancer properties are attracting more and more attention. Previous studies have shown that xanthatin exhibited anti-proliferative effects by inducing the expression of the GADD45␥ gene in MDA-MB-231 cells [12], thus induce cell cycle arrest and apoptosis via disrupting NF-B pathway in A549 Cells [13]. Xanthatin also induces human gastric carcinoma MKN-45 cells apoptosis by activating p53 pathway [14]. In addition, our previous study demonstrated that xanthatin could significantly induce apoptosis of murine melanoma B16-F10 by activating Wnt/-catenin signaling in vitro. It is obviously that xanthatin could inhibit B16-F10 tumor growth in the murine melanoma model by intraperitoneal injection at designated concentration of 4.8 mg/200 g [15]. On the other hand, scientists are focusing on
1570-0232/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.12.006
58
C. Yan et al. / J. Chromatogr. B 947–948 (2014) 57–61
developing an efficient method to synthesize xanthatin and investigating its structure-activity relationship [16]. All of these results suggest that xanthatin maybe a promising drug entity, which can be developed for treating human cancers. In the process of developing a new drug, relative pharmacokinetic parameters are necessary to be explored. To the best of the authors’ knowledge, the pharmacokinetic characters of xanthatin have not been reported yet. Therefore, it is necessary to develop a method to determine the concentration of xanthin in plasma and investigate its pharmacokinetic properties. The results will contribute to the following pharmacological research of xanthatin. The purpose of this paper was to establish a sensitive, rapid and specific UHPLC–MS/MS method for the determination of xanthatin in rat plasma. 2. Materials and methods 2.1. Reagents and materials Methanol (Calepure, Canada, HPLC grade). The water used in this research was purified with an EPED water purification system from Nanjing EPED system (Nanjing, China), and the other reagents were HPLC grade. The air-dried aerial parts of X. strumarium L. were collected from the suburbs of Xuzhou, Jiangsu province of China. The herb was authenticated by Professor Jianwei Chen of Nanjing University of Chinese Medicine. Xanthatin (Fig. 1a, purity ≥98%) was isolated from the air-dried aerial parts of X. strumarium L. in the authors’ laboratory. Its chemical structure was confirmed by using 1 H NMR, DEPT, 1 H, 1 H-COSY, NOESY, HSQC, and HMBC. Psoralen (Fig. 1b, purity ≥99%) was obtained from Shanghai U-sea Biotech Co. Ltd. (Shanghai, China). Other reagents and solvents were of the highest quality available. 2.2. Equipments All separations were performed on a DGU-20A 5R series UHPLC system equipped with a LC-30AD binary pump (Shimadzu Corporation UFLC XR, Kyoto, Japan). Mass spectrometry was conducted by using a 5500 triple quad tandem mass spectrometer equipped with electrospray ionization (ESI) source (AB SCIEX, Foster City, CA, USA).
2.3. Chromatographic and mass-spectroscopic conditions The analytical column was Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 3.5 m). The mobile phase was composed of methanol (B) and 0.1% formic acid water solution (A) with gradient elution system (0–1.5 min, 5–50% B; 1.5–3.5 min, 50–70% B; 3.5–4 min, 70–85% B; 4–4.5 min, 85–50% B; 4.5–5 min, 50–5% B) at a flow rate of 0.3 mL/min. Injection volume was 5 L and the column temperature was 40 ◦ C. Mass analysis was performed in the positive ion electrospray ionization mode with an electrospray ionization (ESI) source. The ion spray voltage was set at 5.5 kV. The optimized parameters were recorded as following data: Ion source temperature, 600 ◦ C; curtain gas (CUR), 241.33 kPa; ion source gas 1 (GAS1), 344.75 kPa; ion source gas 2 (GAS2), 413.70 kPa; ion spray voltage (IS), 5500 V; declustering potential (DP), 69 V; collision energy (CE), 14 V; entrance potential (EP), 10 V and collision exit potential (CXP), 14 V. Multiple reaction monitoring (MRM) mode was applied for the quantitation at [M+H]+ m/z 247.3 → m/z 205.2 for xanthatin and at [M+H]+ m/z 187.1 → m/z 143.0 for psoralen (IS). The chemical structure and the mass spectrum of xanthatin and psoralen were shown in Fig. 1.
2.4. Preparation of stock solutions and quality control samples Stock solution was prepared by dissolving xanthatin and psoralen in methanol at concentration of 200 g/mL each. The internal standard (IS) working solution (10 ng/mL) was obtained by dilute the stock solution with methanol. Calibration samples were prepared by spiking 90 L blank plasma with 10 L xanthatin working solutions to produce final concentrations of 1, 10, 50, 100, 500, 1000 and 5000 ng/mL. The quality control (QC) samples were prepared at concentrations of 2, 50, 500 and 3000 ng/mL respectively. All solutions were kept in 4 ◦ C refrigerator and brought to room temperature before use. 2.5. Sample preparation To each sample, 10 L of IS solution (10 ng/mL) was added into 100 L of rat plasma. The mixture was extracted with 1 mL of ethyl acetate by vortexing for 3 min. The aqueous and organic layer was separated by centrifugation at 4000 r/min for 5 min and the organic layer was transferred to another centrifuge tube and evaporated to dry under a gentle stream of nitrogen gas at 40 ◦ C. The residue was reconstituted with 100 L of mobile phase (0.1% formic acidmethanol at ratio of 5: 95), then centrifuged at 4000 r/min for 5 min. For analyzing, 5 L of the supernatant was injected into the LC–MS/MS system. 2.6. Method validation 2.6.1. Specificity Specificity of the method was evaluated by comparing chromatograms of blank plasma samples, samples spiked with IS plus xanthatin and treated plasma samples. 2.6.2. Linearity and quantification Various concentrations of xanthatin calibration standard solution (1, 10, 50, 100, 500, 1000, 5000 ng/mL) with IS (10 ng/mL) were prepared as plasma sample preparation method and assayed by UHPLC–MS/MS system. Peak area ratios of xanthatin/IS plotted against the corresponding concentrations were calculated to construct calibration curve. The calibration curve was established via 1/x2 weighted linear least-squares regression model. The lower limit of quantification (LLOQ) was determined as the lowest concentration on the QC samples. 2.6.3. Precision and accuracy Accuracy and precision of the method were determined by analyzing six replicates of the QC and LLOQ samples. The intra-day precision and accuracy of the method were assessed by determining the QC samples six times within a single day, while the inter-day precision and accuracy were estimated by determining the QC samples over three consecutive days. 2.6.4. Recovery and matrix effects The extraction recovery was determined by comparing the concentration of extracted QC samples with the same concentration of xanthatin reference standard solutions. Matrix effects of the method were calculated by comparing the peak areas of reference standard in extracts of blank plasma with that of neat standard solution. Recovery and matrix effects experiments at four QC concentrations for plasma were examined (n = 5). 2.6.5. Stability The stability of xanthatin in plasma was investigated by analyzing of four QC samples (n = 5). The short-term stability was investigated by analyzing plasma samples kept at room temperature for 4 h. Freezing-thawing stability was determined after
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Fig. 1. Chemical structures and MS/MS spectra of xanthatin (a) and psoralen (b).
QC samples through three freezing-thawing cycles by freezing at −20 ◦ C and thawing at 37 ◦ C water bath. The long-term stability was assessed after the QC samples were stored at −20 ◦ C for 14 days. 2.7. Pharmacokinetic study Eighteen male Sprague-Dawley rats, weighing 220–250 g, were obtained from the Experimental Animal Center (Nanjing University of Chinese Medicine, China). The rats were housed in a room with controlled temperature and humidity, and allowed free
access to food and water. They were fasted overnight before the experiment. Rats were divided into three groups administered xanthatin via intravenous at provided concentrations (2.4 mg/200 g, 4.8 mg/200 g and 9.6 mg/200 g), respectively. Xanthatin was completely dissolved in the mixture solution, which composed of 85% physiological saline, 10% Pluronic F-68 and 5% DMSO, facilitated by ultrasonication. Blood samples (approximately 0.15 mL) were collected from the oculi chorioideae vein at each time point (0, 2, 5, 15, 30, 60, 120, 240, 360 min) after administration. The blood samples were centrifuged at 12,000 rev/min for 10 min at 4 ◦ C and
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3.2.2. Linearity and sensitivity The calibration curve of xanthatin showed a good linearity in the range of 1–5000 ng/mL. The regression equation of xanthatin in plasma was y = 0.01013x + 0.10364 (r = 0.9990). The LLOD of xanthatin in rat plasma was 1 ng/mL. 3.2.3. Precision and accuracy The QC and LLOQ samples were analyzed for accuracy and precision. The RSD of inter-day precision of QC samples were 5.67%, 7.24%, 9.18% and 7.34%, and the LLOQ sample was 8.24%. The interday precision RSD of QC samples were 9.03%, 9.27%, 9.13% and 8.78%, and the LLOQ sample was 9.35% respectively. The accuracy for xanthatin was 88.48–102.25%. The accuracy and precision of the method are in accordance with the requirements of the determination of biological samples.
Fig. 2. SIM chromatograms of blank plasma (a); blank plasma spiked with xanthatin (5 ng/mL) and IS (b); treated plasma (c). Xanthatin: Rt = 3.47 min; IS: Rt = 3.37 min.
the supernatants were stored at −20 ◦ C freezer. After analyzing, the obtained data was processed by using Win Nonlin53 software (Pharsight Corporation, Mountain View, USA). 3. Results 3.1. Method optimization In this study, a new UHPLC–MS/MS method was developed to quantify xanthatin in rat plasma. A gradient elution system was applied in order to get rid of interference of endogenous compounds. The concentration of formic acid in mobile phase (0.1%) was optimized for the best signal response of the analytes. The mass parameter of DP, CE, EP, and CXP was optimized to enhance the ionization efficiency of xanthatin. Table 1 3.2. Method validation 3.2.1. Assay specificity The typical chromatograms of the blank plasma samples, blank plasma samples added with xanthatin or IS and treated plasma samples were shown in Fig. 2. The retention time of xanthatin and IS was 3.47 and 3.37 min, respectively. The results indicated that the endogenous ingredients did not interfere with the analyzing of xanthatin or the IS in the plasma samples.
3.2.4. Recovery and matrix effects The extraction recoveries of xanthatin at concentrations of 2, 50, 500 and 3000 ng/mL from rat plasma were 87.10%, 84.23%, 82.12% and 89.55% respectively. The RSD of extraction recoveries were 7.73%, 9.01%, 8.10% and 8.27% correspondently. In addition, the recoveries of the IS from the plasma were 81.18% and the RSD was 8.20%. Under the UHPLC–MS/MS conditions, the matrix effects of four QC samples were 91.89%, 92.23%, 94.76% and 97.18% respectively. The RSD of the matrix effects for the QC samples were 5.13%, 7.29%, 7.65% and 8.92%. In addition, the matrix effects of the IS was 103.61% and the RSD was 6.89%. 3.2.5. Stability The QC samples were used for stability experiments. The shortterm temperature stability was 6.25%, 7.28%, 6.90% and 5.16% for all four QC levels respectively, with the highest RSD not more than 7.18%. The freezing-thawing stability was 7.14%, 7.38%, 8.15%, and 6.88% for correspondent concentration, with the highest RSD not more than 8.96%. The long-term stability was 8.47%, 6.25%, 6.82% and 9.45% at each concentration with the highest RSD not more than 8.74%. Results manifested that xanthatin was not easily degradable under experimental conditions. 3.3. Pharmacokinetics study Various doses of xanthatin (2.4 mg/200 g, 4.8 mg/200 g and 9.6 mg/200 g), based on previous study, were intravenously injected into rats. The plasma concentration-time profiles of xanthatin were shown in Fig. 3. The main pharmacokinetic parameters were 14,340.20 (±7122.41), 32,149.52 (±11,259.44), 49,524.28 (±28,520.88). The plasma concentrations of xanthatin were rapidly dropped within 30 min after intravenous administration. The t1/2 of three concentrations was found to be 108.58 (±32.82), 123.50 (±66.69), 181.71 (±148.26) min, respectively. As the dose increased, the AUC0–∞ and AUC0–t were gradually enlarged. The total body CL were 0.13 (±0.14), 0.17 (±0.11), 0.22 (±0.13) and
Table 1 Pharmacokinetic data of xanthatin in rats (n = 6). Parameter
Dose (mg/200 g) 2.4 (iv)
Cmax (ng/mL) AUC0–t (ng h/mL) AUC0–∞ (ng h/mL) T1/2 (min) CL (mL/min) MRT (min) Vd (mL)
418.72 14,340.20 15,538.97 108.58 0.13 225.10 46.85
4.8 (iv) ± ± ± ± ± ± ±
137.51 7122.41 7733.12 32.82 0.14 187.70 20.19
904.89 32,149.52 36,431.22 123.50 5 0.17 159.99 25.13
9.6 (iv) ± ± ± ± ± ± ±
193.53 11,259.44 14,498.16 66.69 0.11 30.49 7.41
1773.46 49,524.28 61,885.45 181.71 0.22 208.22 48.35
± ± ± ± ± ± ±
1733.10 28,520.88 30,704.80 148.26 0.13 85.97 11.03
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Fig. 3. Mean plasma concentration–time curves for xanthatin in rat plasma (n = 6).
Vd were 46.85 (±20.19), 159.99 (±30.49), 208.22 (±85.97) of three concentrations. 4. Discussion This research was concentrated in the investigation of pharmacokinetic properties of xanthatin. A sensitive and specific UHPLC–MS/MS method was established for the determination of low-level concentrations of xanthatin in plasma samples for the first time. Xanthatin, a high lipophilic compound, exhibited promising drug like characters. However, its hydrophobic property will cause poor absorption when oral administrated. Thus, intravenous administration at low, medium and high doses of xanthatin was applied to increase the bioavailability. The t1/2 obtained at low, medium and high doses had no significant difference after Student T-test was used for comparison, which indicated it is safe when applied to clinical therapies. Cmax and AUC0–t were exhibited dose-dependent at three concentrations. The pharmacokinetic properties of xanthatin exhibited linear trend. All of the obtained data indicated that xanthatin could be metabolized and rapidly eliminated from the body of rat after administration. The method that developed in this research proved reliable for pharmacokinetic studies of xanthatin. It can be served as a useful method for further exploration of the pharmacological activity and clinical potential of xanthatin.
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