- Email: [email protected]
MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract
Accepted Manuscript Title: An UPLC-MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract Author: Lan Wei Xiaobo Wang Peng Zhang Yangyang Sun Lili Jie Jingxin Zhao Shikai Dong Lixin Sun PII: DOI: Reference:
S1570-0232(15)30298-1 http://dx.doi.org/doi:10.1016/j.jchromb.2015.11.034 CHROMB 19732
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
20-6-2015 16-11-2015 19-11-2015
Please cite this article as: Lan Wei, Xiaobo Wang, Peng Zhang, Yangyang Sun, Lili Jie, Jingxin Zhao, Shikai Dong, Lixin Sun, An UPLC-MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.11.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
An UPLC-MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract Lan Weia, Xiaobo Wanga, Peng Zhangb, Yangyang Suna, Lili Jiea, Jingxin Zhaoa, Shikai Donga, Lixin Suna
a
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
b
Department of Pharmacology，Shenyang Food and Drug Inspection, Shenyang
110014, China
1
Highlights
A sensitive, selective and fast UPLC-ESI-MS/MS method has been developed for the simultaneous determination of two coumarins (umbelliferone and daphnoretin) and two flavonoids (apigenin and genkwanin) in rat plasma for the first time. The method was fully validated and successfully applied to the pharmacokinetic study of four ingredients after oral administration of Wikstroemia indica extract (WIE) to rats. The results of this study may provide some references for the apprehension of the action mechanism and safe clinical application of W. indica.
2
Abstract In this study, an ultra performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) was developed for simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in total (free and conjugated) forms in rat plasma using psoralen as internal standard. Plasma samples were protein precipitated with acetonitrile followed by liquid–liquid extracted with ethyl acetate. Four ingredients were separated on an Acquity UPLC® BEH C18 column using gradient elution with the mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile, and detected by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM). The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.99. The validated lower limit of quantification was 3 ng/mL for umbelliferone, 3 ng/mL for apigenin, 12 ng/mL for daphnoretin and 2 ng/mL for genkwanin, respectively. Intraand inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −1.1% to 15%. The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 75%. The validated method was firstly and successfully applied to investigate the pharmacokinetics of four chemical ingredients after oral administration of Wikstroemia indica extract (WIE) to rats.
Keywords: UPLC-MS/MS; Wikstroemia indica (L.) C. A. Mey.; Coumarin; Flavonoid; Pharmacokinetics
3
1. Introduction Wikstroemia indica (L.) C. A. Mey. is a member of family Thymelaeaceae and mainly distributes in middle and southeast part of China. As a traditional Chinese herb, this plant has long been employed as antipyretics, detoxicants, expectorants, vermifuges as well as aborticides in clinic practice [1]. Up to now, most studies have been focused on its pharmacological and chemical research. W. indica have antifungal, anti-inflammatory, anti-cancer, antiviral and antimalarial effects [2]. Researchers have extracted and isolated many types of constituents from W. indica, mainly including coumarins, flavonoids, lignans, anthraquinones, sterides and so on [3-12]. However, as far as we are concerned, reports regarding the pharmacokinetic study of WIE are unavailable. In recent years, researchers have discovered that the clinical effects of Traditional Chinese Medicine (TCM) were closely related to its bioactive components, so it is very essential to investigate the pharmacokinetic behavior of W. indica to illustrate the mechanism of action and supply research information for effective clinical use. Many studies have demonstrated that umbelliferone, apigenin, daphnoretin and genkwanin have a variety of biological effects and are the main bioactive ingredients of W. indica [13-17]. Several HPLC and HPLC–MS/MS methods have been reported for the determination of certain compounds mentioned above in plasma samples [15, 18-20]. Li et al. developed a HPLC method to quantify apigenin in plasma sample [15]. Lin et al. developed a HPLC method to determine daphnoretin in rat plasma [18]. Song et al. developed a HPLC–MS/MS method to determine genkwanin in rat plasma [19]. However, most of the mentioned methods employed HPLC coupled with ultraviolet detector, which suffered from a few disadvantages of long analysis time and poor specificity. Moreover, to the best of our knowledge, there is no report on the simultaneous quantification of these four compounds in biological sample. Therefore, a rapid and exclusive method is needed to detect aforesaid four components simultaneously from WIE. In the present study, a sensitive, selective and accurate ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry 4
(UPLC-ESI-MS/MS) method has been developed for the simultaneous determination of two coumarins (umbelliferone and daphnoretin) and two flavonoids (apigenin and genkwanin) in rat plasma for the first time. Their structures were showed in Figure1. The method was fully validated and successfully applied to the pharmacokinetic study after oral administration of WIE to rats. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and a meaningful basis for the clinical application of W. indica.
2. Experimental 2.1. Reagents, materials and animals W. indica was purchased from a TCM shop (Guangzhou, China) and was identified by Professor Jincai Lu (Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China). Umbelliferone and apigenin reference standards (purity ≥98%) were purchased from Phytomarker Co., Ltd (Tianjin, China) and Shilan Technology Co., Ltd (Tianjin, China), respectively. Psoralen and genkwanin reference standards (purity ≥99%) were purchased from National Institutes for Food and Drug Control. Daphnoretin reference standard was prepared in our laboratory with purity ≥98%. β-glucuronidase (type B-1, from bovine liver, containing 1,037,000 units/g) and sulfatase (type H-1, from Helix pomatia, containing 15,275 units/g) were supplied by Sigma (Company Inc, Saint Louis, USA). Acetonitrile, methanol and formic acid of HPLC-grade was obtained from Fisher Scientific (Pittsburgh, PA, USA), Shandong Yuwang Industrial Co., Ltd. (Shandong, China) and Kermel Chemical Reagent Co., Ltd (Tianjin, China), respectively. Ethyl acetate was obtained from Tianjin Huirui Chemical Technology Co., Ltd (Tianjin, China). Purified water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). All other chemicals and reagents were of analytical grade and obtained from commercial sources. Six male pathogen-free Sprague Dawley rats (SCXK 2010-0001, weighing 210–230 g) purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China) were housed in a SPF grade animal laboratory, which was kept at a 5
temperature of 22 ± 2˚C and a relative humidity of 50 ± 10%, with a natural light-dark cycle (Experimental Animal Center, Shenyang Pharmaceutical University, Shenyang, China). The animal study was carried out in accordance with the Guideline for Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution.
2.2. Instrumentations and conditions Chromatographic analysis was performed on an Acquity Ultra Performance LC system (Waters Corp., Milford, MA, USA), including a binary pump solvent manager, a column oven and an autosampler. Four analytes and IS were separated on an Acquity UPLC® BEH C18 column (2.1 × 50 mm, 1.7 μm; Waters Corp., Milford, MA, USA). The mobile phase was 0.1% formic acid aqueous solution (A) - acetonitrile (B) at the flow rate of 0.2 mL/min. The gradient program was as follows: 0-5 min, 32-60% B; 5-5.5 min, 60-32% B; 5.5-8 min, 32% B. The column and autosampler tray temperature were maintained constant at 35 °C and 4 °C, respectively. Triple-quadrupole tandem mass spectrometric detection was carried out on a Micromass Quattro Micro™ API mass spectrometric system (Waters Corp., Milford, MA, USA). The analytes and IS were all ionized by the ESI source in positive ion mode under the following conditions: capillary voltage, 3.5 kV; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow, 600 L/hr; cone gas flow, 50 L/hr. For MRM conditions the analytes and IS were optimized by infusion of reference standard, as summarized in Table 1. All data were acquired in centroid mode by Masslynx V4.1 software (Waters Corp., Milford, MA, USA).
2.3. Preparation of Wikstroemia indica extract The dried W. indica were powdered to homogeneous size and sifted through a 40-mesh sieve. About 10 g of the powder was weighed accurately and reflux extracted with 200 mL of ethanol twice in a water bath, 2 h per time. The filtrate was collected and the solvent was removed using a rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, China). The contents of the four compounds in ethanol extract 6
were measured quantitatively by external standard method using the same chromatography conditions as described above.
2.4. Standard solutions, calibration curves and quality control samples The stock solutions of umbelliferone, apigenin, daphnoretin and genkwanin were prepared with proper amount of those standards dissolving into methanol, the concentrations of which were 16.1, 27.6, 56.4 and 37.6 μg/mL, respectively. The solution of IS (1.045 μg/mL) was prepared with proper amount of that standard dissolving into methanol. All stock solutions were stored in refrigerator at 4 °C. The calibration standards were prepared by the corresponding mixed standard solutions with blank rat plasma to obtain final concentrations of 3.1, 6.2, 15.5, 30.9, 61.8, 154.6, 309.1 ng/mL for umbelliferone; 2.6, 5.3, 13.2, 26.5, 53.0, 132.5, 265.0 ng/mL for apigenin; 11.7, 23.5, 58.7, 117.3, 234.6, 586.6, 1173.1 ng/mL for daphnoretin; and 2.4, 4.8, 12.0, 24.1, 48.1, 120.3, 240.6 ng/mL for genkwanin. The quality control (QC) samples were prepared separately in the same fashion (4.6, 46.4, 247.3 μg/mL for umbelliferone; 4.0, 39.7, 212.0 μg/mL for apigenin; 17.6, 176.0, 938.5 μg/mL for daphnoretin and 3.6, 36.1, 192.5 μg/mL for genkwanin).
2.5. Pretreatment of plasma sample Plasma samples (100 μL) were incubated with 200 μL of enzyme (β-glucuronidase 340 units/mL and sulfatase 340 units/mL in 0.4 mol/L acetate buffer (pH 4.6)) at 37 °C for 16 h. After termination reaction in ice bath for 5 min, the enzyme hydrolyzed samples were neutralized by 200 μL buffer solution (pH 8.98) and vortexed for 1 min, 10 μL IS and 10 μL methanol were spiked and vortexed for 1 min, the samples were spiked with 200 μL acetonitrile and vortexed for 5min to precipitate protein, and then extracted with 1 mL ethyl acetate by vortex mixing for 8 min twice. After centrifugation at 10000 rpm for 5 min, the upper layer was quantitatively transferred to another clean tube and evaporated to dryness at 37 °C under a gentle stream of nitrogen. The residue was reconstituted with 50 μL methanol, vortex-mixed for 3min, and sonicated for 1 min. After being centrifuged at 10000 rpm for 5 min, 5 7
μL of the solution was injected into the UPLC-MS/MS system for analysis.
2.6. Method validation The method was totally validated in accordance with the US FDA guidelines. Selectivity was measured by comparing the chromatograms of blank plasma from six different rats, blank plasma spiked with umbelliferone, apigenin, daphnoretin, genkwanin and IS, and plasma samples obtained after oral administration of WIE to exclude interference of endogenous substances and metabolites. Calibration curves were prepared using the standard plasma samples and constructed from peak area ratios of each analyte to IS versus plasma concentrations using a 1/x2 weighted least-squares linear regression model. The applied calibration model for all curves was y = ax + b, where y = peak area ratio of analyte to IS, x = concentration of the analyte in plasma, a = slope of the curve and b = intercept. The calibration model was accepted if the residuals were within ± 20% at the LLOQ (lower limits of quantification) and within ± 15% at all other calibration levels. The LLOQs for analytes were all found with a 10/1 signal to noise ratio and determined in six replicates with the precision less than 20% and the accuracy ranging from -20% to 20% of the spiked concentration. The LLOQ was usually defined as the lowest concentration point of the calibration curve. QC samples at low, medium and high concentrations were evaluated with six replicates at each concentration per day in a period of three days to determine intra-day, inter-day precision and accuracy, calculated with calibration curves obtained daily. The intra-day and inter-day precisions were defined as relative standard deviation (RSD, %) and the accuracy was defined as relative error (RE, %). The precision was required to be less than 15% and the accuracy ranged from −15% to 15% of the spiked concentration. The extraction recoveries of all analytes were determined by comparing the peak area of the treated plasma samples with post-extracted blank plasma sample spiked with QC sample. In addition, the extraction recoveries were evaluated at three QC levels for all analytes and at one concentration level for IS, with acceptable criteria 8
above 50%. The matrix effect was determined by comparing the peak area of the blank plasma spiked with proper amount of four analytes and IS with that of pure standard solutions containing equivalent amounts of four analytes and IS. The matrix effect values required from 85% to 115%. The stabilities of the analytes in plasma were measured in three conditions, including three freeze (−20 °C)-thaw (room temperature) cycles, 24 h storage at 4 °C and frozen (−20 °C) for 30 d at three concentrations of QC samples.
2.7. Pharmacokinetic study The developed method was used to determine umbelliferone, apigenin, daphnoretin and genkwanin in rat plasma after oral administration of WIE. The rats were fasted for 12 h with free access to water prior to oral administration of WIE with an herb dose of 8 g/kg (equivalent to umbelliferone 88 μg/kg, apigenin 40 μg/kg, daphnoretin 8600 μg/kg and genkwanin 64 μg/kg). Blood samples (approx. 0.5 mL) were collected from suborbital vein into heparinized tubes before administration and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 h after dosing, and then immediately centrifuged at 4000 rpm for 10 min. Harvested plasma samples were stored at -20 °C and analyzed within a week. The plasma concentrations of umbelliferone, apigenin, daphnoretin and genkwanin at different points were expressed as mean ± SD, which were calculated from the calibration curves obtained daily. The mean concentration-time curves were plotted and all the pharmacokinetic parameters were processed by DAS 2.1 software package (Chinese Pharmacological Society).
3. Results and discussion 3.1. Method development 3.1.1. Optimization of LC-MS/MS conditions We had researched the elution system to improve separation efficiency, and the results showed that acetonitrile-water system exhibited better resolution and peak 9
shape than methanol-water system. Because addition of small volume of formic acid into the mobile phase was helpful to enhance electrospray ionization and improve peak shape, the adding amount of formic acid (0.01%, 0.05%, 0.1% and 0.15%) was tested. Through comparing the signal intensity in four conditions, 0.1% formic acid added to water was selected. In addition, in order to shorten the running time, gradient elution programs were also investigated. The total analysis time of previous reports [18, 19, 21] was 4-10min, which only detected one of four analytes. In our study four components were determined simultaneously with the run time of 5 min (pre-equilibrium time was 3min). The optimized conditions were presented in detail in Section 2.2. During optimizing the MS parameters, it was discovered that the response observed in positive ion mode was higher than that in negative ion mode for all analytes and IS, so positive ion mode was utilized. Furthermore, precursor ions, product ions, collision energy, capillary voltage, cone voltage and other parameters were also investigated. The optimized conditions were presented in detail in Table 1 and Section 2.2.
3.1.2. Optimization of sample preparation Protein precipitation and liquid–liquid extraction were the most common methods of plasma sample preparation in previous literatures [15, 18-21]. But the extraction recoveries of PP-LLE (protein precipitation coupled with liquid–liquid extraction) were higher than aforementioned two methods for four analytes and IS in our study. Therefore, the precipitant and extractant were investigated. Among employed precipitant such as methanol and acetonitrile, extractant such as n-hexane, dichloromethane, ethyl acetate and methyl isopropyl ketone, acetonitrile and ethyl acetate were optimal, respectively. The consumption of two solvents was further tested, and the optimal conditions were presented in detail in Section 2.5.
10
3.2. Method validation 3.2.1. Selectivity, linearity and LLOQ The typical MRM chromatograms of blank rat plasma, blank rat plasma spiked with analytes and IS and the subject sample (rat plasma samples 4 h after oral administration of WIE) were presented in Fig. 2. There were no significant interferences or ion suppression observed at the retention time of umbelliferone (1.2 min), apigenin (2.1 min), daphnoretin (2.4 min), genkwanin (4.1 min) and IS (2.6 min). The calibration curves presented good linearity over the aforementioned concentration ranges. The typical calibration curves were as follows: umbelliferone, y = 5.84×10−3 x + 5.67×10−2 (r = 0.9978, n = 7); apigenin, y = 9.36×10−3 x + 3.31×10−1 (r = 0.9912, n = 7); daphnoretin, y = 9.60×10−4 x + 3.76×10−3 (r = 0.9984, n = 7); genkwanin, y = 8.73×10−3 x + 4.80×10−4 (r = 0.9983, n = 7). x referred to the concentration of the analytes in plasma (ng/mL); y referred to the peak area ratio of the analytes to IS. The LLOQs were 3.1, 2.6, 11.7 and 2.4 ng/mL for umbelliferone, apigenin, daphnoretin and genkwanin with RSD% within 20%.
3.2.2. Precision and accuracy The intra-day and inter-day precisions and accuracy of umbelliferone, apigenin, daphnoretin and genkwanin were investigated by analyzing three levels of QC samples. All the datas were shown in Table 2. All the results of the tested samples were within the acceptable criteria of ± 15%. 3.2.3. Extraction recovery and matrix effect The mean extraction recoveries of four analytes were determined by using six replicates of QC samples of each level. The results were shown in Table 2. The mean extraction recovery and matrix effect of IS were 75.01% and 108.7% at the concentration of 104.5 ng/mL. The results of the matrix effects (see Table 2）indicated that the plasma matrix peaks did not affect the quantification of the analytes in the experimental conditions.
11
3.2.4. Stability The assay had measured the stabilities of four analytes in the following conditions: three freeze (−20 °C) - thaw (room temperature) cycles, 24 h storage at 4 °C and frozen (−20 °C) for 30 d. The results were summarized in Table 3, which were well within the acceptable limit.
3.3. Pharmacokinetic study This validated UPLC-MS/MS method was successfully applied to the pharmacokinetic study after oral administration of WIE to Sprague Dawley rats. The mean plasma concentration-time profiles of umbelliferone, apigenin, daphnoretin and genkwanin in rats were illustrated by Fig.3 and the calculated pharmacokinetic parameters were listed in Table 4. Compared our findings with the previous reports [18, 19, 21], t1/2 of apigenin, daphnoretin and genkwanin in rats after administration of WIE were greater than those in a single dose of pure compounds (116.5 ± 22.59 min, 93 ± 26 min and 3.07 ± 0.90 h for apigenin, daphnoretin and genkwanin, respectively). On the one hand, other components in WIE may influence in vivo behavior of analytes. On the other hand, rats were orally administered with WIE in the form of suspension liquid, which may slow the release and absorption of drugs. So the retention time of drugs in vivo was prolonged. In addition, though the contents of daphnoretin and genkwanin in WIE were less than the dosages in literatures, AUCs were greater than pure compounds (17 ± 2 min*μg/mL and 218 ± 40 ng*h/mL for daphnoretin and genkwanin, respectively). This is probably caused by other complex ingredients in WIE, which can affect the pharmacokinetic behaviors of analytes. Moreover, the plasma concentrations of four components were detected in summation including free and metabolic forms. Overall, WIE were more efficient than pure compounds in absorption of daphnoretin and genkwanin. From the plasma concentration–time curves of four analytes, double peaks phenomenon of daphnoretin and umbelliferone were very obvious. Several reasons could explain this. Firstly, the phenomenon maybe caused with enterohepatic 12
recirculation. Moreover, daphnoretin and umbelliferone are coumarins, and there have been several reports on the enterohepatic circulation of coumarins [22]. Secondly, WIE may have reached the small intestine in two portions for the reason of gastric emptying time. Alternatively, there may have been sustained release into the blood. Thirdly, it can be inferred that herb extract could have an effect on the absorption of two compounds in rat plasma. In conclusion, the information described above might be helpful for further studies on the pharmacokinetics of W. indica, and beneficial for the application of this TCM in clinical therapy. In addition, the method developed in this study will be very practical for the analysis of large number biological samples in the future. Firstly, LLOQs of our study were low than previous reports (25.9 ng/mL, 50 ng/mL and 3.84 ng/mL for apigenin, daphnoretinfor and genkwanin, respectively). Secondly, the total analysis time decreased than previous methods expatiated in section 3.1.1. Lastly but not least, the validated method can be applied to determine the concentrations of certain ingredient or four ingredients not only in plasma, but also in urine and tissue. 4. Conclusion A selective, sensitive and fast UPLC-MS/MS method described here was developed and validated for the simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in rat plasma for the first time. The method had many advantages of simple operation, good selectivity, short analysis time and low LLOQ, making it suitable for analyzing a large number of plasma samples. The method was successfully applied to the pharmacokinetic study of four components after oral administration of WIE to Sprague Dawley rats. Through the comparision of pharmacokinetic parameters, WIE was found to be more efficient than pure compounds in the absorption of apigenin, daphnoretin and genkwanin. This study may provide some references to the apprehension of the action mechanism and safe clinical application of W. indica. Acknowledgements This work was supported by the Program for Liaoning Innovative Research Team in University (No. LT 2012018), the Project of Liaoning Distinguished Professor (2014) and the National Undergraduate Training Programs for Innovation and Entrepreneurship (No. 201410163005).
13
References [1] Y.M. Li, L. Zhu, J.G. Jiang, L. Yang, D.Y. Wang, Bioactive Components and Pharmacological Action of Wikstroemia indica (L.) C. A. Mey and Its Clinical Application, Curr. Pharm. Biotechno. 10 (2010) 743-752. [2] W.H. Huang, X.L. Zhang, Y.F. Wang, W.C. Ye, V.E.C. Ooi, H.Y. Chung, Y.L. Li, Research Antiviral biflavonoids from Radix Wikstroemiae (Liaogewanggen), Chin. Med, 5 (2010) 23-28. [3] X.L. Zhang, G.C. Wang, W.H. Huang, W.C. Ye, Y.L. Li, Biflavonoids from the roots of Wikstroemia indica, Nat. Prod. Commun. 6 (2011) 1111-1114. [4] J. Li, L.Y. Lu, L.H. Zeng, C. Zhang, J.L. Hu, X.R. Li, Sikokianin D, A New C-3/C-3"-Biflavanone from the Roots of Wikstroemia indica, Molecules. 17 (2012) 7792-7797. [5] W.H. Huang, G.X. Zhou, G.C. Wang, H.Y. Chung, W.C. Ye, Y.L. Li, A new biflavonoid with antiviral activity from the roots of Wikstroemia indica, J. Asian Nat. Prod. Res. 14 (2012) 401-406. [6] C.L. Lu, L. Zhu, J.H. Piao, J.G. Jiang, Chemical compositions extracted from Wikstroemia indica and their multiple activities, Pharm. Biol. 50 (2012) 225-231. [7] L.D. Geng, C. Zhang, Y.Q. Xiao, A new dicoumarin from stem bark of Wikstroemia indica, Chin. J. Chin. Mat. Med. 31 (2006) 43-45. [8] H.K. Yao, W.T. Zhang, Y.S. Gao, Y. Zhong, Studies on the chemical constituents of Wikstroemia indica, J. Chin. Med. Mater. 33 (2010) 1093-1095. [9] Y. Chen, W.W. Fu, L.X. Sun, Q. Wang, W. Qi, H. Yu, A new coumarin from Wikstroemia indica (L.) C. A. Mey, Chinese. Chem. Lett. 20 (2009) 592-594. [10] L.D. Geng, C. Zhang, Y.Q. Xiao, Studies on the chemical constituents in stem rind of Wikstroemia indica, Chin. J. Chin. Mat. Med. 31 (2006) 817-819. [11] L.Y. Wang, N. Unehara, S. Kitanaka, Lignans from the roots of Wikstroemia indica and their DPPH radical scavenging and nitric oxide inhibitory activities, Chem. Pharm. Bull. 53 (2005) 1348-1351. [12] L.Y. Wang, T. Unehara, S. Kitanaka, Anti-inflammatory activity of new guaiane type sesquiterpene from Wikstroemia indica, Chem. Pharm. Bull. 53 (2005) 14
137-139. [13] W.S. Ho, J.Y. Xue, S.S.M. Sun, V.E.C. Ooi, Y.L. Li, Antiviral activity of daphnoretin isolated from Wikstroemia indica, Phytother. Res. 24 (2010) 657-661. [14] C.L. Lu, Y.M. Li, G.Q. Fu, L. Yang, J.G. Jiang, L. Zhu, F.L. Lin, J. Chen, Q.S. Lin, Extraction optimisation of daphnoretin from root bark of Wikstroemia indica (L.) C.A. and its anti-tumour activity tests, Food. Chem. 124 (2011) 1500-1506. [15] L.P. Li, X.D. Wu, Z.J. Chen, S.Y. Sun, J.F. Ye, S. Zeng, H.D. Jiang, Interspecies difference of luteolin and apigenin after oral administration of Chrysanthemum morifolium extract and prediction of human pharmacokinetics, Pharmazie. 68 (2013)195-200. [16] V.P. Androutsopoulos, K. Ruparelia, R.R.J Arroo, A.M. Tsatsakis, D.A. Spandidos, CYP1-mediated antiproliferative activity of dietary flavonoids in MDA-MB-468 breast cancer cells, Toxicol. 264 (2009) 162-170. [17] A. Ishikawa, Y. Kitamura, Y. Ozeki, Y. Itoh, A. Yamada, M. Watanabe, Post-stress metabolism involves umbelliferone production in anthocyanin-producing and non-producing cells of Glehnia littoralis suspension cultures, J. Plant. Physiol. 162 (2005) 703-710. [18] L.C. Lin, K.Y. Yang, Y.F. Chen, S.C. Wang, T.H. Tsai, Measurement of daphnoretin in plasma of freely moving rat by liquid chromatography, J. Chromatogr. A. 1073 (2005) 285-289. [19] Y.Q. Song, S.X. Zhang, H. Liu, X.Q. Jin, Determination of genkwanin in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a bioavailability study, J. Pharm. Biomed. Anal. 84 (2013) 129-134. [20] R. Shi, S. Qiaoa, D.Q. Yu, X.W. Shi, M. Liua, X.J. Jiang, Q. Wang, L.T. Zhang, Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC-MS/MS and its application to pharmacokinetic study, J. Chromatogr. B. 879 (2011) 1625-1632. [21] Y. Zhang, P.L. Chang, W. Xia, K. Wu, X.J. Zhao, Study on pharmacokinetics and 15
tissue distribution of apigenin in rats by RP –HPLC, Chin. J. Dis. Control Prev. 1(2009) 79-82. [22] L. Feng, L. Wang, X.H. Jiang, Pharmacokinetics, tissue distribution and excretion of coumarin components from Psoralea corylifolia L. in rats, Arch. Pharm. Res. 33 (2010) 225-230.
16
Fig.1. Product ion scan spectra and chemical structures of umbelliferone, apigenin, daphnoretin, genkwanin and psoralen (IS).
17
Fig.2. Representative MRM chromatograms of blank rat plasma sample (A), blank rat plasma sample spiked with four analytes and IS (B), rat plasma sample obtained 4 h after oral administration of WIE (C).
18
Fig.3. Mean plasma concentration–time profiles for four analytes in rat plasma after oral administration of WIE.
19
Table 1. MRM parameters, cone voltage and collision energy for the determination of four analytes and IS. Q1 Mass
Q3 Mass
(Da)
(Da)
Cone voltage
Collision energy
(V)
(V)
Analyte Umbelliferone
163.3
107.2
38
25
Apigenin
271.5
153.1
40
35
Psoralen
187.2
131.2
32
28
Daphnoretin
353.6
179.1
50
29
Genkwanin
285.6
242.1
40
30
20
Table 2. Summary of precision, accuracy, recovery and matrix effect of four analytes in rat plasma (n = 6). Intra-da Concentratio Analytes
Recovery Inter-day
Accuracy
(RSD%)
(RE%)
y n (ng/mL)
(%,
(RSD%)
mean
SD)
Umbelliferon
Matrix effect ± (%,
mean
SD) 90.51±12.33
4.6
11.27
12.13
11.68
82.32±10.76
46.4
13.15
6.60
5.93
96.49±10.32
99.67±13.93
247.3
5.95
4.20
14.92
99.44±3.27
98.68±11.67
4.0
14.02
7.43
10.75
94.28±7.29
95.51±8.33
39.7
13.25
10.20
10.81
92.21±7.42
104.3±10.52
212.0
10.36
14.10
-1.12
101.36±6.41
110.8±8.45
17.6
9.77
13.87
10.51
75.09±10.76
109.1±9.22
176.0
11.50
13.30
-0.87
90.36±12.08
105.4±10.06
938.5
8.73
12.60
7.82
96.14±5.48
105.4±11.63
3.6
12.45
14.25
8.75
74.99±9.72
101.5±9.23
36.1
6.15
11.60
6.30
83.27±12.28
96.29±9.10
192.5
9.01
13.50
10.85
87.00±6.34
87.43±11.86
e
Apigenin
Daphnoretin
Genkwanin
21
±
Table 3. Stability of the four analytes in rat plasma at different conditions. (n = 6). 3
freeze-thaw
24 h, 4 °C
30 d, −20°C
cycles
Concentratio Analytes n (ng/mL)
RSD% RE%
RSD%
RE %
RSD RE% %
Umbellifero 4.6 ne
Apigenin
Daphnoretin
Genkwanin
2.30
3.46
6.63
6.88
-0.48
11.10
46.4
8.84
2.31
3.17
6.74
-7.92
10.71
247.3
3.84
1.68
2.57
1.23
3.22
0.86
4.0
5.97
1.10
2.07
5.00
-0.51
2.95
39.7
-11.04
1.78
8.21
3.10
-11.84
6.24
212.0
-11.20
9.69
-8.38
5.56
-5.45
4.93
17.6
-5.98
1.10
3.54
9.14
6.20
4.39
176.0
-7.80
6.18
-1.80
7.60
-12.51
1.70
938.5
-7.63
11.60
3.12
3.96
9.00
0.11
3.6
5.78
0.99
11.73
3.63
1.36
8.76
36.1
-9.83
1.15
9.06
6.39
4.14
3.58
192.5
-4.52
14.62
10.11
1.27
-2.40
0.45
22
Table 4. Noncompartmental pharmacokinetic parameters for four analytes in rat plasma after oral administration of WIE (mean ± SD, n = 6). Parameter
AUC0–t
AUC0–∞
Cmax
t1/2
Tmax
Unit
μg / L*h
μg / L*h
μg/L
h
h
878.80±62.91
1146.29±115.04
152.47±12.63
13.29±4.15
Apigenin
428.01±14.07
668.44±51.65
83.80±9.16
14.50±3.02
1.15±0.12
Daphnoretin
7151.65±646.05
9205.63±534.12
605.74±30.71
15.79±5.13
2.42±1.09
Genkwanin
776.40±80.56
1346.87±127.06
29.08±3.61
36.24±9.11
6.46±2.07
Umbellifero
0.83±0.25
ne
23
S1570-0232(15)30298-1 http://dx.doi.org/doi:10.1016/j.jchromb.2015.11.034 CHROMB 19732
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
20-6-2015 16-11-2015 19-11-2015
Please cite this article as: Lan Wei, Xiaobo Wang, Peng Zhang, Yangyang Sun, Lili Jie, Jingxin Zhao, Shikai Dong, Lixin Sun, An UPLC-MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.11.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
An UPLC-MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract Lan Weia, Xiaobo Wanga, Peng Zhangb, Yangyang Suna, Lili Jiea, Jingxin Zhaoa, Shikai Donga, Lixin Suna
a
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
b
Department of Pharmacology，Shenyang Food and Drug Inspection, Shenyang
110014, China
1
Highlights
A sensitive, selective and fast UPLC-ESI-MS/MS method has been developed for the simultaneous determination of two coumarins (umbelliferone and daphnoretin) and two flavonoids (apigenin and genkwanin) in rat plasma for the first time. The method was fully validated and successfully applied to the pharmacokinetic study of four ingredients after oral administration of Wikstroemia indica extract (WIE) to rats. The results of this study may provide some references for the apprehension of the action mechanism and safe clinical application of W. indica.
2
Abstract In this study, an ultra performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) was developed for simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in total (free and conjugated) forms in rat plasma using psoralen as internal standard. Plasma samples were protein precipitated with acetonitrile followed by liquid–liquid extracted with ethyl acetate. Four ingredients were separated on an Acquity UPLC® BEH C18 column using gradient elution with the mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile, and detected by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM). The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.99. The validated lower limit of quantification was 3 ng/mL for umbelliferone, 3 ng/mL for apigenin, 12 ng/mL for daphnoretin and 2 ng/mL for genkwanin, respectively. Intraand inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −1.1% to 15%. The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 75%. The validated method was firstly and successfully applied to investigate the pharmacokinetics of four chemical ingredients after oral administration of Wikstroemia indica extract (WIE) to rats.
Keywords: UPLC-MS/MS; Wikstroemia indica (L.) C. A. Mey.; Coumarin; Flavonoid; Pharmacokinetics
3
1. Introduction Wikstroemia indica (L.) C. A. Mey. is a member of family Thymelaeaceae and mainly distributes in middle and southeast part of China. As a traditional Chinese herb, this plant has long been employed as antipyretics, detoxicants, expectorants, vermifuges as well as aborticides in clinic practice [1]. Up to now, most studies have been focused on its pharmacological and chemical research. W. indica have antifungal, anti-inflammatory, anti-cancer, antiviral and antimalarial effects [2]. Researchers have extracted and isolated many types of constituents from W. indica, mainly including coumarins, flavonoids, lignans, anthraquinones, sterides and so on [3-12]. However, as far as we are concerned, reports regarding the pharmacokinetic study of WIE are unavailable. In recent years, researchers have discovered that the clinical effects of Traditional Chinese Medicine (TCM) were closely related to its bioactive components, so it is very essential to investigate the pharmacokinetic behavior of W. indica to illustrate the mechanism of action and supply research information for effective clinical use. Many studies have demonstrated that umbelliferone, apigenin, daphnoretin and genkwanin have a variety of biological effects and are the main bioactive ingredients of W. indica [13-17]. Several HPLC and HPLC–MS/MS methods have been reported for the determination of certain compounds mentioned above in plasma samples [15, 18-20]. Li et al. developed a HPLC method to quantify apigenin in plasma sample [15]. Lin et al. developed a HPLC method to determine daphnoretin in rat plasma [18]. Song et al. developed a HPLC–MS/MS method to determine genkwanin in rat plasma [19]. However, most of the mentioned methods employed HPLC coupled with ultraviolet detector, which suffered from a few disadvantages of long analysis time and poor specificity. Moreover, to the best of our knowledge, there is no report on the simultaneous quantification of these four compounds in biological sample. Therefore, a rapid and exclusive method is needed to detect aforesaid four components simultaneously from WIE. In the present study, a sensitive, selective and accurate ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry 4
(UPLC-ESI-MS/MS) method has been developed for the simultaneous determination of two coumarins (umbelliferone and daphnoretin) and two flavonoids (apigenin and genkwanin) in rat plasma for the first time. Their structures were showed in Figure1. The method was fully validated and successfully applied to the pharmacokinetic study after oral administration of WIE to rats. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and a meaningful basis for the clinical application of W. indica.
2. Experimental 2.1. Reagents, materials and animals W. indica was purchased from a TCM shop (Guangzhou, China) and was identified by Professor Jincai Lu (Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China). Umbelliferone and apigenin reference standards (purity ≥98%) were purchased from Phytomarker Co., Ltd (Tianjin, China) and Shilan Technology Co., Ltd (Tianjin, China), respectively. Psoralen and genkwanin reference standards (purity ≥99%) were purchased from National Institutes for Food and Drug Control. Daphnoretin reference standard was prepared in our laboratory with purity ≥98%. β-glucuronidase (type B-1, from bovine liver, containing 1,037,000 units/g) and sulfatase (type H-1, from Helix pomatia, containing 15,275 units/g) were supplied by Sigma (Company Inc, Saint Louis, USA). Acetonitrile, methanol and formic acid of HPLC-grade was obtained from Fisher Scientific (Pittsburgh, PA, USA), Shandong Yuwang Industrial Co., Ltd. (Shandong, China) and Kermel Chemical Reagent Co., Ltd (Tianjin, China), respectively. Ethyl acetate was obtained from Tianjin Huirui Chemical Technology Co., Ltd (Tianjin, China). Purified water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). All other chemicals and reagents were of analytical grade and obtained from commercial sources. Six male pathogen-free Sprague Dawley rats (SCXK 2010-0001, weighing 210–230 g) purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China) were housed in a SPF grade animal laboratory, which was kept at a 5
temperature of 22 ± 2˚C and a relative humidity of 50 ± 10%, with a natural light-dark cycle (Experimental Animal Center, Shenyang Pharmaceutical University, Shenyang, China). The animal study was carried out in accordance with the Guideline for Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution.
2.2. Instrumentations and conditions Chromatographic analysis was performed on an Acquity Ultra Performance LC system (Waters Corp., Milford, MA, USA), including a binary pump solvent manager, a column oven and an autosampler. Four analytes and IS were separated on an Acquity UPLC® BEH C18 column (2.1 × 50 mm, 1.7 μm; Waters Corp., Milford, MA, USA). The mobile phase was 0.1% formic acid aqueous solution (A) - acetonitrile (B) at the flow rate of 0.2 mL/min. The gradient program was as follows: 0-5 min, 32-60% B; 5-5.5 min, 60-32% B; 5.5-8 min, 32% B. The column and autosampler tray temperature were maintained constant at 35 °C and 4 °C, respectively. Triple-quadrupole tandem mass spectrometric detection was carried out on a Micromass Quattro Micro™ API mass spectrometric system (Waters Corp., Milford, MA, USA). The analytes and IS were all ionized by the ESI source in positive ion mode under the following conditions: capillary voltage, 3.5 kV; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow, 600 L/hr; cone gas flow, 50 L/hr. For MRM conditions the analytes and IS were optimized by infusion of reference standard, as summarized in Table 1. All data were acquired in centroid mode by Masslynx V4.1 software (Waters Corp., Milford, MA, USA).
2.3. Preparation of Wikstroemia indica extract The dried W. indica were powdered to homogeneous size and sifted through a 40-mesh sieve. About 10 g of the powder was weighed accurately and reflux extracted with 200 mL of ethanol twice in a water bath, 2 h per time. The filtrate was collected and the solvent was removed using a rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, China). The contents of the four compounds in ethanol extract 6
were measured quantitatively by external standard method using the same chromatography conditions as described above.
2.4. Standard solutions, calibration curves and quality control samples The stock solutions of umbelliferone, apigenin, daphnoretin and genkwanin were prepared with proper amount of those standards dissolving into methanol, the concentrations of which were 16.1, 27.6, 56.4 and 37.6 μg/mL, respectively. The solution of IS (1.045 μg/mL) was prepared with proper amount of that standard dissolving into methanol. All stock solutions were stored in refrigerator at 4 °C. The calibration standards were prepared by the corresponding mixed standard solutions with blank rat plasma to obtain final concentrations of 3.1, 6.2, 15.5, 30.9, 61.8, 154.6, 309.1 ng/mL for umbelliferone; 2.6, 5.3, 13.2, 26.5, 53.0, 132.5, 265.0 ng/mL for apigenin; 11.7, 23.5, 58.7, 117.3, 234.6, 586.6, 1173.1 ng/mL for daphnoretin; and 2.4, 4.8, 12.0, 24.1, 48.1, 120.3, 240.6 ng/mL for genkwanin. The quality control (QC) samples were prepared separately in the same fashion (4.6, 46.4, 247.3 μg/mL for umbelliferone; 4.0, 39.7, 212.0 μg/mL for apigenin; 17.6, 176.0, 938.5 μg/mL for daphnoretin and 3.6, 36.1, 192.5 μg/mL for genkwanin).
2.5. Pretreatment of plasma sample Plasma samples (100 μL) were incubated with 200 μL of enzyme (β-glucuronidase 340 units/mL and sulfatase 340 units/mL in 0.4 mol/L acetate buffer (pH 4.6)) at 37 °C for 16 h. After termination reaction in ice bath for 5 min, the enzyme hydrolyzed samples were neutralized by 200 μL buffer solution (pH 8.98) and vortexed for 1 min, 10 μL IS and 10 μL methanol were spiked and vortexed for 1 min, the samples were spiked with 200 μL acetonitrile and vortexed for 5min to precipitate protein, and then extracted with 1 mL ethyl acetate by vortex mixing for 8 min twice. After centrifugation at 10000 rpm for 5 min, the upper layer was quantitatively transferred to another clean tube and evaporated to dryness at 37 °C under a gentle stream of nitrogen. The residue was reconstituted with 50 μL methanol, vortex-mixed for 3min, and sonicated for 1 min. After being centrifuged at 10000 rpm for 5 min, 5 7
μL of the solution was injected into the UPLC-MS/MS system for analysis.
2.6. Method validation The method was totally validated in accordance with the US FDA guidelines. Selectivity was measured by comparing the chromatograms of blank plasma from six different rats, blank plasma spiked with umbelliferone, apigenin, daphnoretin, genkwanin and IS, and plasma samples obtained after oral administration of WIE to exclude interference of endogenous substances and metabolites. Calibration curves were prepared using the standard plasma samples and constructed from peak area ratios of each analyte to IS versus plasma concentrations using a 1/x2 weighted least-squares linear regression model. The applied calibration model for all curves was y = ax + b, where y = peak area ratio of analyte to IS, x = concentration of the analyte in plasma, a = slope of the curve and b = intercept. The calibration model was accepted if the residuals were within ± 20% at the LLOQ (lower limits of quantification) and within ± 15% at all other calibration levels. The LLOQs for analytes were all found with a 10/1 signal to noise ratio and determined in six replicates with the precision less than 20% and the accuracy ranging from -20% to 20% of the spiked concentration. The LLOQ was usually defined as the lowest concentration point of the calibration curve. QC samples at low, medium and high concentrations were evaluated with six replicates at each concentration per day in a period of three days to determine intra-day, inter-day precision and accuracy, calculated with calibration curves obtained daily. The intra-day and inter-day precisions were defined as relative standard deviation (RSD, %) and the accuracy was defined as relative error (RE, %). The precision was required to be less than 15% and the accuracy ranged from −15% to 15% of the spiked concentration. The extraction recoveries of all analytes were determined by comparing the peak area of the treated plasma samples with post-extracted blank plasma sample spiked with QC sample. In addition, the extraction recoveries were evaluated at three QC levels for all analytes and at one concentration level for IS, with acceptable criteria 8
above 50%. The matrix effect was determined by comparing the peak area of the blank plasma spiked with proper amount of four analytes and IS with that of pure standard solutions containing equivalent amounts of four analytes and IS. The matrix effect values required from 85% to 115%. The stabilities of the analytes in plasma were measured in three conditions, including three freeze (−20 °C)-thaw (room temperature) cycles, 24 h storage at 4 °C and frozen (−20 °C) for 30 d at three concentrations of QC samples.
2.7. Pharmacokinetic study The developed method was used to determine umbelliferone, apigenin, daphnoretin and genkwanin in rat plasma after oral administration of WIE. The rats were fasted for 12 h with free access to water prior to oral administration of WIE with an herb dose of 8 g/kg (equivalent to umbelliferone 88 μg/kg, apigenin 40 μg/kg, daphnoretin 8600 μg/kg and genkwanin 64 μg/kg). Blood samples (approx. 0.5 mL) were collected from suborbital vein into heparinized tubes before administration and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 h after dosing, and then immediately centrifuged at 4000 rpm for 10 min. Harvested plasma samples were stored at -20 °C and analyzed within a week. The plasma concentrations of umbelliferone, apigenin, daphnoretin and genkwanin at different points were expressed as mean ± SD, which were calculated from the calibration curves obtained daily. The mean concentration-time curves were plotted and all the pharmacokinetic parameters were processed by DAS 2.1 software package (Chinese Pharmacological Society).
3. Results and discussion 3.1. Method development 3.1.1. Optimization of LC-MS/MS conditions We had researched the elution system to improve separation efficiency, and the results showed that acetonitrile-water system exhibited better resolution and peak 9
shape than methanol-water system. Because addition of small volume of formic acid into the mobile phase was helpful to enhance electrospray ionization and improve peak shape, the adding amount of formic acid (0.01%, 0.05%, 0.1% and 0.15%) was tested. Through comparing the signal intensity in four conditions, 0.1% formic acid added to water was selected. In addition, in order to shorten the running time, gradient elution programs were also investigated. The total analysis time of previous reports [18, 19, 21] was 4-10min, which only detected one of four analytes. In our study four components were determined simultaneously with the run time of 5 min (pre-equilibrium time was 3min). The optimized conditions were presented in detail in Section 2.2. During optimizing the MS parameters, it was discovered that the response observed in positive ion mode was higher than that in negative ion mode for all analytes and IS, so positive ion mode was utilized. Furthermore, precursor ions, product ions, collision energy, capillary voltage, cone voltage and other parameters were also investigated. The optimized conditions were presented in detail in Table 1 and Section 2.2.
3.1.2. Optimization of sample preparation Protein precipitation and liquid–liquid extraction were the most common methods of plasma sample preparation in previous literatures [15, 18-21]. But the extraction recoveries of PP-LLE (protein precipitation coupled with liquid–liquid extraction) were higher than aforementioned two methods for four analytes and IS in our study. Therefore, the precipitant and extractant were investigated. Among employed precipitant such as methanol and acetonitrile, extractant such as n-hexane, dichloromethane, ethyl acetate and methyl isopropyl ketone, acetonitrile and ethyl acetate were optimal, respectively. The consumption of two solvents was further tested, and the optimal conditions were presented in detail in Section 2.5.
10
3.2. Method validation 3.2.1. Selectivity, linearity and LLOQ The typical MRM chromatograms of blank rat plasma, blank rat plasma spiked with analytes and IS and the subject sample (rat plasma samples 4 h after oral administration of WIE) were presented in Fig. 2. There were no significant interferences or ion suppression observed at the retention time of umbelliferone (1.2 min), apigenin (2.1 min), daphnoretin (2.4 min), genkwanin (4.1 min) and IS (2.6 min). The calibration curves presented good linearity over the aforementioned concentration ranges. The typical calibration curves were as follows: umbelliferone, y = 5.84×10−3 x + 5.67×10−2 (r = 0.9978, n = 7); apigenin, y = 9.36×10−3 x + 3.31×10−1 (r = 0.9912, n = 7); daphnoretin, y = 9.60×10−4 x + 3.76×10−3 (r = 0.9984, n = 7); genkwanin, y = 8.73×10−3 x + 4.80×10−4 (r = 0.9983, n = 7). x referred to the concentration of the analytes in plasma (ng/mL); y referred to the peak area ratio of the analytes to IS. The LLOQs were 3.1, 2.6, 11.7 and 2.4 ng/mL for umbelliferone, apigenin, daphnoretin and genkwanin with RSD% within 20%.
3.2.2. Precision and accuracy The intra-day and inter-day precisions and accuracy of umbelliferone, apigenin, daphnoretin and genkwanin were investigated by analyzing three levels of QC samples. All the datas were shown in Table 2. All the results of the tested samples were within the acceptable criteria of ± 15%. 3.2.3. Extraction recovery and matrix effect The mean extraction recoveries of four analytes were determined by using six replicates of QC samples of each level. The results were shown in Table 2. The mean extraction recovery and matrix effect of IS were 75.01% and 108.7% at the concentration of 104.5 ng/mL. The results of the matrix effects (see Table 2）indicated that the plasma matrix peaks did not affect the quantification of the analytes in the experimental conditions.
11
3.2.4. Stability The assay had measured the stabilities of four analytes in the following conditions: three freeze (−20 °C) - thaw (room temperature) cycles, 24 h storage at 4 °C and frozen (−20 °C) for 30 d. The results were summarized in Table 3, which were well within the acceptable limit.
3.3. Pharmacokinetic study This validated UPLC-MS/MS method was successfully applied to the pharmacokinetic study after oral administration of WIE to Sprague Dawley rats. The mean plasma concentration-time profiles of umbelliferone, apigenin, daphnoretin and genkwanin in rats were illustrated by Fig.3 and the calculated pharmacokinetic parameters were listed in Table 4. Compared our findings with the previous reports [18, 19, 21], t1/2 of apigenin, daphnoretin and genkwanin in rats after administration of WIE were greater than those in a single dose of pure compounds (116.5 ± 22.59 min, 93 ± 26 min and 3.07 ± 0.90 h for apigenin, daphnoretin and genkwanin, respectively). On the one hand, other components in WIE may influence in vivo behavior of analytes. On the other hand, rats were orally administered with WIE in the form of suspension liquid, which may slow the release and absorption of drugs. So the retention time of drugs in vivo was prolonged. In addition, though the contents of daphnoretin and genkwanin in WIE were less than the dosages in literatures, AUCs were greater than pure compounds (17 ± 2 min*μg/mL and 218 ± 40 ng*h/mL for daphnoretin and genkwanin, respectively). This is probably caused by other complex ingredients in WIE, which can affect the pharmacokinetic behaviors of analytes. Moreover, the plasma concentrations of four components were detected in summation including free and metabolic forms. Overall, WIE were more efficient than pure compounds in absorption of daphnoretin and genkwanin. From the plasma concentration–time curves of four analytes, double peaks phenomenon of daphnoretin and umbelliferone were very obvious. Several reasons could explain this. Firstly, the phenomenon maybe caused with enterohepatic 12
recirculation. Moreover, daphnoretin and umbelliferone are coumarins, and there have been several reports on the enterohepatic circulation of coumarins [22]. Secondly, WIE may have reached the small intestine in two portions for the reason of gastric emptying time. Alternatively, there may have been sustained release into the blood. Thirdly, it can be inferred that herb extract could have an effect on the absorption of two compounds in rat plasma. In conclusion, the information described above might be helpful for further studies on the pharmacokinetics of W. indica, and beneficial for the application of this TCM in clinical therapy. In addition, the method developed in this study will be very practical for the analysis of large number biological samples in the future. Firstly, LLOQs of our study were low than previous reports (25.9 ng/mL, 50 ng/mL and 3.84 ng/mL for apigenin, daphnoretinfor and genkwanin, respectively). Secondly, the total analysis time decreased than previous methods expatiated in section 3.1.1. Lastly but not least, the validated method can be applied to determine the concentrations of certain ingredient or four ingredients not only in plasma, but also in urine and tissue. 4. Conclusion A selective, sensitive and fast UPLC-MS/MS method described here was developed and validated for the simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in rat plasma for the first time. The method had many advantages of simple operation, good selectivity, short analysis time and low LLOQ, making it suitable for analyzing a large number of plasma samples. The method was successfully applied to the pharmacokinetic study of four components after oral administration of WIE to Sprague Dawley rats. Through the comparision of pharmacokinetic parameters, WIE was found to be more efficient than pure compounds in the absorption of apigenin, daphnoretin and genkwanin. This study may provide some references to the apprehension of the action mechanism and safe clinical application of W. indica. Acknowledgements This work was supported by the Program for Liaoning Innovative Research Team in University (No. LT 2012018), the Project of Liaoning Distinguished Professor (2014) and the National Undergraduate Training Programs for Innovation and Entrepreneurship (No. 201410163005).
13
References [1] Y.M. Li, L. Zhu, J.G. Jiang, L. Yang, D.Y. Wang, Bioactive Components and Pharmacological Action of Wikstroemia indica (L.) C. A. Mey and Its Clinical Application, Curr. Pharm. Biotechno. 10 (2010) 743-752. [2] W.H. Huang, X.L. Zhang, Y.F. Wang, W.C. Ye, V.E.C. Ooi, H.Y. Chung, Y.L. Li, Research Antiviral biflavonoids from Radix Wikstroemiae (Liaogewanggen), Chin. Med, 5 (2010) 23-28. [3] X.L. Zhang, G.C. Wang, W.H. Huang, W.C. Ye, Y.L. Li, Biflavonoids from the roots of Wikstroemia indica, Nat. Prod. Commun. 6 (2011) 1111-1114. [4] J. Li, L.Y. Lu, L.H. Zeng, C. Zhang, J.L. Hu, X.R. Li, Sikokianin D, A New C-3/C-3"-Biflavanone from the Roots of Wikstroemia indica, Molecules. 17 (2012) 7792-7797. [5] W.H. Huang, G.X. Zhou, G.C. Wang, H.Y. Chung, W.C. Ye, Y.L. Li, A new biflavonoid with antiviral activity from the roots of Wikstroemia indica, J. Asian Nat. Prod. Res. 14 (2012) 401-406. [6] C.L. Lu, L. Zhu, J.H. Piao, J.G. Jiang, Chemical compositions extracted from Wikstroemia indica and their multiple activities, Pharm. Biol. 50 (2012) 225-231. [7] L.D. Geng, C. Zhang, Y.Q. Xiao, A new dicoumarin from stem bark of Wikstroemia indica, Chin. J. Chin. Mat. Med. 31 (2006) 43-45. [8] H.K. Yao, W.T. Zhang, Y.S. Gao, Y. Zhong, Studies on the chemical constituents of Wikstroemia indica, J. Chin. Med. Mater. 33 (2010) 1093-1095. [9] Y. Chen, W.W. Fu, L.X. Sun, Q. Wang, W. Qi, H. Yu, A new coumarin from Wikstroemia indica (L.) C. A. Mey, Chinese. Chem. Lett. 20 (2009) 592-594. [10] L.D. Geng, C. Zhang, Y.Q. Xiao, Studies on the chemical constituents in stem rind of Wikstroemia indica, Chin. J. Chin. Mat. Med. 31 (2006) 817-819. [11] L.Y. Wang, N. Unehara, S. Kitanaka, Lignans from the roots of Wikstroemia indica and their DPPH radical scavenging and nitric oxide inhibitory activities, Chem. Pharm. Bull. 53 (2005) 1348-1351. [12] L.Y. Wang, T. Unehara, S. Kitanaka, Anti-inflammatory activity of new guaiane type sesquiterpene from Wikstroemia indica, Chem. Pharm. Bull. 53 (2005) 14
137-139. [13] W.S. Ho, J.Y. Xue, S.S.M. Sun, V.E.C. Ooi, Y.L. Li, Antiviral activity of daphnoretin isolated from Wikstroemia indica, Phytother. Res. 24 (2010) 657-661. [14] C.L. Lu, Y.M. Li, G.Q. Fu, L. Yang, J.G. Jiang, L. Zhu, F.L. Lin, J. Chen, Q.S. Lin, Extraction optimisation of daphnoretin from root bark of Wikstroemia indica (L.) C.A. and its anti-tumour activity tests, Food. Chem. 124 (2011) 1500-1506. [15] L.P. Li, X.D. Wu, Z.J. Chen, S.Y. Sun, J.F. Ye, S. Zeng, H.D. Jiang, Interspecies difference of luteolin and apigenin after oral administration of Chrysanthemum morifolium extract and prediction of human pharmacokinetics, Pharmazie. 68 (2013)195-200. [16] V.P. Androutsopoulos, K. Ruparelia, R.R.J Arroo, A.M. Tsatsakis, D.A. Spandidos, CYP1-mediated antiproliferative activity of dietary flavonoids in MDA-MB-468 breast cancer cells, Toxicol. 264 (2009) 162-170. [17] A. Ishikawa, Y. Kitamura, Y. Ozeki, Y. Itoh, A. Yamada, M. Watanabe, Post-stress metabolism involves umbelliferone production in anthocyanin-producing and non-producing cells of Glehnia littoralis suspension cultures, J. Plant. Physiol. 162 (2005) 703-710. [18] L.C. Lin, K.Y. Yang, Y.F. Chen, S.C. Wang, T.H. Tsai, Measurement of daphnoretin in plasma of freely moving rat by liquid chromatography, J. Chromatogr. A. 1073 (2005) 285-289. [19] Y.Q. Song, S.X. Zhang, H. Liu, X.Q. Jin, Determination of genkwanin in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a bioavailability study, J. Pharm. Biomed. Anal. 84 (2013) 129-134. [20] R. Shi, S. Qiaoa, D.Q. Yu, X.W. Shi, M. Liua, X.J. Jiang, Q. Wang, L.T. Zhang, Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC-MS/MS and its application to pharmacokinetic study, J. Chromatogr. B. 879 (2011) 1625-1632. [21] Y. Zhang, P.L. Chang, W. Xia, K. Wu, X.J. Zhao, Study on pharmacokinetics and 15
tissue distribution of apigenin in rats by RP –HPLC, Chin. J. Dis. Control Prev. 1(2009) 79-82. [22] L. Feng, L. Wang, X.H. Jiang, Pharmacokinetics, tissue distribution and excretion of coumarin components from Psoralea corylifolia L. in rats, Arch. Pharm. Res. 33 (2010) 225-230.
16
Fig.1. Product ion scan spectra and chemical structures of umbelliferone, apigenin, daphnoretin, genkwanin and psoralen (IS).
17
Fig.2. Representative MRM chromatograms of blank rat plasma sample (A), blank rat plasma sample spiked with four analytes and IS (B), rat plasma sample obtained 4 h after oral administration of WIE (C).
18
Fig.3. Mean plasma concentration–time profiles for four analytes in rat plasma after oral administration of WIE.
19
Table 1. MRM parameters, cone voltage and collision energy for the determination of four analytes and IS. Q1 Mass
Q3 Mass
(Da)
(Da)
Cone voltage
Collision energy
(V)
(V)
Analyte Umbelliferone
163.3
107.2
38
25
Apigenin
271.5
153.1
40
35
Psoralen
187.2
131.2
32
28
Daphnoretin
353.6
179.1
50
29
Genkwanin
285.6
242.1
40
30
20
Table 2. Summary of precision, accuracy, recovery and matrix effect of four analytes in rat plasma (n = 6). Intra-da Concentratio Analytes
Recovery Inter-day
Accuracy
(RSD%)
(RE%)
y n (ng/mL)
(%,
(RSD%)
mean
SD)
Umbelliferon
Matrix effect ± (%,
mean
SD) 90.51±12.33
4.6
11.27
12.13
11.68
82.32±10.76
46.4
13.15
6.60
5.93
96.49±10.32
99.67±13.93
247.3
5.95
4.20
14.92
99.44±3.27
98.68±11.67
4.0
14.02
7.43
10.75
94.28±7.29
95.51±8.33
39.7
13.25
10.20
10.81
92.21±7.42
104.3±10.52
212.0
10.36
14.10
-1.12
101.36±6.41
110.8±8.45
17.6
9.77
13.87
10.51
75.09±10.76
109.1±9.22
176.0
11.50
13.30
-0.87
90.36±12.08
105.4±10.06
938.5
8.73
12.60
7.82
96.14±5.48
105.4±11.63
3.6
12.45
14.25
8.75
74.99±9.72
101.5±9.23
36.1
6.15
11.60
6.30
83.27±12.28
96.29±9.10
192.5
9.01
13.50
10.85
87.00±6.34
87.43±11.86
e
Apigenin
Daphnoretin
Genkwanin
21
±
Table 3. Stability of the four analytes in rat plasma at different conditions. (n = 6). 3
freeze-thaw
24 h, 4 °C
30 d, −20°C
cycles
Concentratio Analytes n (ng/mL)
RSD% RE%
RSD%
RE %
RSD RE% %
Umbellifero 4.6 ne
Apigenin
Daphnoretin
Genkwanin
2.30
3.46
6.63
6.88
-0.48
11.10
46.4
8.84
2.31
3.17
6.74
-7.92
10.71
247.3
3.84
1.68
2.57
1.23
3.22
0.86
4.0
5.97
1.10
2.07
5.00
-0.51
2.95
39.7
-11.04
1.78
8.21
3.10
-11.84
6.24
212.0
-11.20
9.69
-8.38
5.56
-5.45
4.93
17.6
-5.98
1.10
3.54
9.14
6.20
4.39
176.0
-7.80
6.18
-1.80
7.60
-12.51
1.70
938.5
-7.63
11.60
3.12
3.96
9.00
0.11
3.6
5.78
0.99
11.73
3.63
1.36
8.76
36.1
-9.83
1.15
9.06
6.39
4.14
3.58
192.5
-4.52
14.62
10.11
1.27
-2.40
0.45
22
Table 4. Noncompartmental pharmacokinetic parameters for four analytes in rat plasma after oral administration of WIE (mean ± SD, n = 6). Parameter
AUC0–t
AUC0–∞
Cmax
t1/2
Tmax
Unit
μg / L*h
μg / L*h
μg/L
h
h
878.80±62.91
1146.29±115.04
152.47±12.63
13.29±4.15
Apigenin
428.01±14.07
668.44±51.65
83.80±9.16
14.50±3.02
1.15±0.12
Daphnoretin
7151.65±646.05
9205.63±534.12
605.74±30.71
15.79±5.13
2.42±1.09
Genkwanin
776.40±80.56
1346.87±127.06
29.08±3.61
36.24±9.11
6.46±2.07
Umbellifero
0.83±0.25
ne
23