3866 Mingqing Huang1∗ Haiyu Zhao4∗ Wei Xu1 Kedan Chu1 Zhenfeng Hong2 Jun Peng2 Lidian Chen3 1 College

of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian Province, China 2 Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian Province, China 3 College of Rehabilitation Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian Province, China 4 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

Received June 19, 2013 Revised September 26, 2013 Accepted October 8, 2013

J. Sep. Sci. 2013, 36, 3866–3873

Research Article

Rapid simultaneous determination of twelve major components in Pien Tze Huang by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry An efficient method using ultra-performance LC coupled with triple quadrupole MS was developed for the rapid determination of 12 major active components in Pien Tze Huang (PZH), a well-known traditional Chinese formula. Chromatographic separation was achieved on a Waters XBridge BEH RP18 column (50 mm × 2.1 mm id, 1.7 ␮m) with a gradient mobile phase (A: 0.1% aqueous formic acid and B: acetonitrile with 0.1% formic acid) at a flow rate of 0.8 mL/min. The chromatographic peaks of 12 components were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring was employed for the quantitative analysis. Ten batches of PZH were analyzed with a good linear regression relationship (r, 0.9987–0.9995), intraday precisions (RSD, 2.05–4.80%), interday precisions (RSD, 1.99–4.98%), repeatability (RSD, 2.21–4.20%), stability (RSD, 3.52–4.81%), and recovery (95.63–104.80%). By using this established method, the present study offered highly sensitive, specific, and speedy determination of 12 major components, which promoted the quality control investigation of PZH greatly. Keywords: Pien Tze Huang / QqQ mass spectrometry / Quantification / Traditional Chinese medicine / Ultra-performance LC DOI 10.1002/jssc.201300655



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction Recently, traditional Chinese medicines (TCM) have received great interest in the field of anticancer treatment since they have relatively few side effects compared to modern chemotherapeutics and have been used for thousands of years as important alternative remedies for a variety of diseases [1, 2]. Pien Tze Huang (PZH) is a well-known precious TCM formula, which was first prescribed by a royal physician in the Ming Dynasty (around 1555 AD). The main ingredients of PZH include moschus, calculus bovis, snake gall, and radix notoginseng. These products together confer PZH properties of heat clearing, detoxification, promotion of blood circulation, reduction of blood stasis, dissipation of hard mass, deCorrespondence: Professor Jun Peng, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, 1 Huatuo Road, Minhou Shangjie, Fuzhou, Fujian 350122, China E-mail: [email protected] Fax: +86-591-22861157

Abbreviations: MRM, multiple-reaction monitoring; TCM, traditional Chinese medicine; PZH, Pien Tze Huang; UPLC, ultraperformance LC; QqQ-MS, triple quadrupole MS  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tumescence, and analgesia [3]. Traditionally, PZH has been used to clinically treat traumatic injuries and a variety of inflammatory diseases, particularly hepatitis [3–5]. In addition, PZH has also been used in China and Southeast Asia for centuries as a folk remedy for the treatment of various cancers. Modern pharmacological studies have proposed that PZH exhibits therapeutic effects in clinical trials of tumors such as hepatocellular carcinoma and colon cancer [6, 7]. Moreover, it has been demonstrated to suppress the growth of Ehrlich– Ascites tumor, gastric carcinoma, and hepatoma in animal models [8]. Furthermore, we recently reported that PZH can inhibit colon cancer growth both in vivo and in vitro by the promotion of cancer cell apoptosis and the inhibition of cell proliferation, which is probably mediated by its inhibitory effect on activation of the STAT3 pathway [9–12]. Due to its long history and wide range of pharmacological applications, the Chinese government has now listed PZH as one of the national treasures in the catalogue of National Protected Traditional Chinese Medicines.

∗ These

authors contributed equally to this work.

Colour Online: See the article online to view Fig. 3 in colour. www.jss-journal.com

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Although the global demand of TCM has been increasing year by year, the problem of quality control remains one of the major obstacles for its internationalization. TCM formulas are a complex combination of many natural products, each of which contains numerous chemical compounds. TCM formulas therefore are considered to be multicomponent agents exerting their therapeutic function in a more holistic way. The efficacy of PZH should be associated with the synergistic or interactive work of numerous chemicals, including bile acids from calculus bovis, saponins from panax notoginseng, muscone from moschus and conjugated bile acids from snake gall [13–19]. In other words, there is no single active constituent that is responsible for the overall function of this formula. Three saponin components, notoginsenoside R1, ginsenoside Rb1, and ginsenoside Rg1 in PZH were analyzed by HPLC with a long analysis time (100 min), and their LOQs were in the range of 0.3464–0.4224 ␮g/mL. Only ginsenoside Rg1 in PZH was analyzed by TLC after complicated and time-consuming alumina column purification, and its LOQ was 0.5 ␮g/mL [20, 21]. However, for PZH and other TCM formulas, HPLC with UV detection cannot provide sufficient structural information of the analytes, and the TLC method has even more shortcomings such as poor repeatability and low sensitivity [22]. Actually, the present quality control severely restricts the clinical application and in-depth study of PZH. Therefore, it is of great significance to develop a more sensitive and efficient analytical method for the determination of the active components in PZH for its quality assurance. The major compounds in the aforementioned four ingredients of PZH should be selected for the quality control analysis. Along with the development of analytical technology, ultra-performance LC (UPLC) coupled with triple quadrupole MS (QqQ-MS) can effectively avoid false-positive results with high sensitivity and provide a reliable quantification of different compounds [23–25]. Obviously, UPLC–QqQ-MS is suitable for the determination of active constituents in PZH samples. In present study, we report here for the first time a UPLC–QqQ-MS method for the rapid simultaneous determination of 12 major active components in PZH, including notoginsenoside R1, ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rg3, cholic acid, deoxycholic acid, hyodeoxycholic acid, ursodesoxycholic acid, chenodeoxycholic acid, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, and muscone. Ten batches of PZH were collected for the analysis. The general content ranges of these active components are given in this study, which benefits the quality control and clinical usage of PZH.

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Province, China) and stored at 4⬚C until analysis. Voucher specimens were deposited in the College of Pharmacy, Fujian University of Traditional Chinese Medicine.

2.2 Reagents and standards Acetonitrile, methanol, and formic acid (HPLC grade) for UPLC analysis were bought from Merck (Darmstadt, Germany). Deionized water was prepared using a Millipore MilliQ purification system (Millipore, Bedford, MA, USA). Standards of notoginsenoside R1 (1), ginsenoside Rb1 (2), ginsenoside Rg1 (3), ginsenoside Rg3 (4), cholic acid (5), deoxycholic acid (6), hyodeoxycholic acid (7), ursodesoxycholic acid (8), chenodeoxycholic acid (9), sodium taurochenodeoxycholate (10), sodium tauroursodeoxycholate (11), muscone (12), astragaloside IV (internal standard 1, IS-1), and cinobufagin (internal standard 2, IS-2) were bought from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The chemical structures of these standards are shown in Fig. 1. The purity of all compounds was more than 98% (determined by HPLC or GC).

2.3 Preparation of standard solutions and samples Twelve standards were dissolved in methanol (approx. 1 mg/mL) individually. The stock solution of each analyte was further diluted with methanol to achieve a series of standard working solutions that were used to establish the calibration curves. All solutions were stored at 4⬚C in the dark and brought to room temperature, then filtered through a 0.22 ␮m micropore membrane before use. The powder of each PZH sample was precisely weighed (0.050 g), transferred into dark brown calibrated flasks, and then extracted with 50 mL of methanol in an ultrasonic bath for 30 min. Additional methanol was added to make up the lost weight. The extracted solution was centrifuged at 15 000 rpm for 10 min, and the supernatant was filtered through a 0.22 ␮m micropore membrane for analysis. Internal standards of astragaloside IV and cinobufagin were dissolved together in methanol to the final concentration of about 0.5 mg/mL. Ten microliters of the IS working solution and 440 ␮L of methanol were added to 450 ␮L of the mixed standards solution or sample solution, then mixed and filtered through a 0.22 ␮m micropore membrane prior to injection.

2.4 LC

2 Materials and methods 2.1 Samples Ten batches of PZH samples were collected from Zhangzhou PienTzeHuang Pharmaceutical (Zhangzhou City, Fujian  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Chromatographic analysis was performed on a Waters UPLC system (Waters, USA) equipped with an online vacuum degasser, a binary pump, an autosampler, and a thermostatted column compartment. Chromatographic separation was carried out at 25⬚C on an Waters XBridge BEH RP18 column (2.1× 50 mm, 1.7 ␮m). The mobile phases consisted of 0.1% www.jss-journal.com

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Figure 1. Chemical structures of the 14 investigated analytes.

formic acid in water (A) and acetonitrile with 0.1% formic acid (B). The gradient elution program was as follows: 10–30% B at 0–3 min, 30–35% B at 3–8.5 min, 35–40% B at 8.5–8.6 min, 40% B at 8.6–9.8 min, 40–60% B at 9.8–10.6 min, 60–100% B at 10.6–11.5 min, 100% B at 11.5–13 min, 100–10% B at 13–13.1 min, and 10% B at 13.1–14 min. The flow rate was kept at 0.8 mL/min, and the sample volume injected was 3 ␮L.

mized to achieve maximum sensitivity. Nitrogen was used as curtain gas (CUR), nebulizer gas (GS1), heater gas (GS2), and collision gas. The optimized MS conditions were fixed as follows: capillary voltage 5500 V, source temperature 500⬚C, and dwell time 50 ms. The most proper collision energy and declustering potential were selected according to each analyte (Table 1).

2.6 Method validation 2.5 MS MS/MS was performed on an AB Sciex API 4000 triple quadrupole mass spectrometer with an ESI source (Applied Biosystems, Toronto, Canada). The MS spectra were acquired in the positive ion multiple-reaction monitoring (MRM) mode, which was carried out by optimization of the product ion obtained from the fragment of the isolated precursor ion for each analyte. Once the product ions were chosen, the MRM conditions for each standard were further opti C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The quantification method was validated for linearity, LOQs and LODs, precision, repeatability, stability, and accuracy. At least six concentrations of calibration standard solution were made and analyzed in triplicate, and then the calibration curves were constructed by plotting the ratios of the peak areas of each standard to IS versus the concentration of each analyte. Linear regression analysis was used to calculate the slope, intercept, and the correlation coefficient of each calibration line. Typically, LOD and LOQ are three times www.jss-journal.com

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Table 1. Retention time, related MS data of 14 investigated analytes detected on the LC-MS

Compounds

RT (min)

MW

Quantification transition (m/z)

DP (V)

CE (V)

CXP (V)

Notoginsenoside R1 Ginsenoside Rb1 Ginsenoside Rg1 Ginsenoside Rg3 Cholic acid Deoxycholic acid Hyodeoxycholic acid Ursodesoxycholic acid Chenodeoxycholic acid Sodium taurochenodeoxycholate Sodium tauroursodeoxycholate Muscone Astragaloside IV Cinobufagin

2.24 3.67 2.40 9.11 5.27 9.33 6.98 6.61 9.21 6.66 4.49 11.56 3.82 6.09

933 1108 800 784 408 392 392 392 392 521 521 238 784 442

956 → 956 1131 → 789 823 → 643 807 → 807 391 → 355 357 → 161 357 → 161 357 → 161 357 → 161 522 → 486 522 → 486 239 → 95 807 → 807 443 → 365

80 80 100 80 60 80 80 80 80 80 80 60 80 80

25 75 50 25 20 33 33 33 33 32 32 24 25 22

5 5 5 5 5 5 5 5 5 5 5 5 5 5

CE, collision energy; CXP, collision cell exit potential; DP, declustering potential.

and ten times the noise level, respectively. For each target constituent, the LODs and LOQs were determined by serial dilution of standard solution under the described UPLC–QqQ-MS conditions. Intra- and interday variations were chosen to determine the precision of the developed method. For intraday precision test, the standards solutions were analyzed for six replicates within one day, while for interday precision test, the solutions were examined in duplicate on three consecutive days. Both assays were determined by performing four different concentration levels of the standards. Variations were expressed as RSD. To confirm the repeatability, six samples of PZH (S3004) were extracted and analyzed on three separate days. The RSD value was calculated as a measurement of method repeatability. In order to investigate the stability of the samples, each sample solution was analyzed every 4 h within 12 h and stored at 25⬚C. The recovery was used to evaluate the accuracy of the method and determine by adding the mixed standard solutions with three different concentration levels (120, 100, and 80%) to the known amounts of PZH sample. The concentrations for each standard at 100% level were 41.67, 41.67, 41.67, 0.21, 41.67, 1.25, 2.08, 2.08, 20.83, 0.83, 2.08, and 4.17 ␮g/mL, respectively. The mixture was extracted and analyzed. Three replicates were performed at each level. The percentage recoveries were calculated according to the following equation: (detected amount–original amount) × 100% / added amount.

3 Results and discussion 3.1 Optimization of UPLC–MS/MS conditions In order to obtain optimal chromatograms with good peak shapes and high sensitivity, different mobile phases includ C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ing water/methanol and water/acetonitrile were examined. As a result, the good separation for the analytes was achieved by using water/acetonitrile. In addition, it was found that formic acid was not only a benefit for the improvement of chromatographic separation, but also for enhancing the abundance of [M + H]+ in the positive model. Besides, because of the similar structures, retention time and ionization response in ESI-MS, astragaloside IV, and cinobufagin were chosen as internal standards of notoginseng saponins, bile acids and muscone, respectively. To develop a sensitive and accurate quantitative method, the MS/MS fragmentation for each analyte was investigated by direct infusion of the single standard solution into the mass spectrometer, and their product ion scan mass spectra were recorded (Fig. 2). All the analytes were characterized according to their mass spectra to ascertain their precursor ions and to select proper product ions for the MRM analysis. According to the relevant legislation of the EU Commission, at least two product ions should be selected to confirm the precursor ion of investigated analyte in the MS/MS analysis [26]. As can be seen, all notoginseng saponins revealed precursor ion [M+Na]+ in the MS spectrum, and product ions of [M–Glc+Na]+ and [M–Glc–Glc+H2 O+Na]+ were also observed. The precursor ion [M+H]+ was observed in these bile acids, as well as [M–H2 O+H]+ , [M–2H2 O+H]+ , and [M–3H2 O+H]+ . In addition, muscone generated a precursor ion [M+H]+ at m/z 239, and the diagnostic ions of [M– CO+H]+ were detected. Meanwhile, the continuous lost of CH2 groups generated the fragments at m/z 151, 137, 123, 109, 95, and 81 in the MS/MS fragmentation. Different parameters including declustering potential, collision energy, and collision cell exit potential were studied with the purpose of achieving the richest relative abundance of precursor ions and product ions, and at last the most sensitive transition in MRM was selected. The optimum results are shown in Table 1 and MRM chromatogram of 14 markers are shown in Fig. 3. www.jss-journal.com

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Figure 2. Chemical characterization of the 14 investigated analytes by product ion scan in MS/MS.

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Figure 2. Continued.

3.2 Method validation

quantitative determination of major components in PZH samples (Supporting Information Table S1).

3.2.1 Linearity and detection limit 3.2.2 Precision, repeatability, and stability The calibration curves, plotted with a series of concentrations of standard solutions, were constructed from the peak areas ratios of each standard to IS versus concentrations of each analyte. Acceptable linear correlation at these conditions was confirmed by correlation coefficients (r, 0.9987–0.9995). The LODs (S/N = 3) and LOQs (S/N = 10) for all standard analytes were in the range of 0.001–0.02 and 0.001–0.02 ␮g/mL, respectively, indicating that this method is sensitive for the  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The intra- and interday precisions were investigated to evaluate the precision of this method by performing four different concentration levels of the standards under the optimized conditions. The RSDs of intraday precision were in the range of 2.77–4.9, 3.24–4.78, 1.51–4.45, and 1.33–4.13%, respectively. The RSDs of interday precision were in the range of 3.28–4.97, 3.32–4.97, 3.85–4.99, and 3.35–4.96%, respectively. www.jss-journal.com

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Table 2. Recoveries of 12 analytes with n = 3

Compounds

Figure 3. LC–MS/MS MRM chromatogram of the 14 target standards: (1) notoginsenoside R1; (2) ginsenoside Rb1; (3) ginsenoside Rg1; (4) ginsenoside Rg3; (5) cholic acid; (6) deoxycholic acid; (7) hyodeoxycholic acid; (8) ursodesoxycholic acid; (9) chenodeoxycholic acid; (10) sodium taurochenodeoxycholate; (11) sodium tauroursodeoxycholate; (12) muscone; (IS-1) astragaloside IV; (IS-2) cinobufagin.

Original (␮g)

Notoginsenoside R1

385.14

Ginsenoside Rb1

743.91

Ginsenoside Rg1

1376.37

Ginsenoside Rg3

1.76

Cholic acid

823.20

Deoxycholic acid

307.41

Added (␮g)

Detected (␮g)

Recovery (%)

RSD (%)

80 100 120 80 100 120 80 100 120 0.4 0.5 0.6 80 100 120 2.4 3.0 3.6 4 5 6 4 5 6 40 50 60 1.6 2.0 2.4 4 5 6 8 10 12

467.23 481.91 507.93 821.63 846.26 860.93 1460.21 1474.19 1498.64 2.17 2.28 2.38 906.69 925.38 944.95 309.84 310.38 310.96 13.61 14.57 15.56 9.90 10.78 11.65 151.26 159.99 169.25 5.17 5.57 5.92 14.93 16.07 16.96 42.13 43.75 46.16

102.62 96.77 102.33 97.16 102.35 97.52 104.80 97.81 101.89 103.49 103.05 103.05 104.36 102.18 101.45 101.16 98.98 98.49 99.12 98.51 98.69 100.43 97.81 96.07 100.00 97.46 96.65 98.91 99.39 97.38 95.63 99.21 97.52 103.27 98.86 104.51

4.70 3.84 4.70 4.21 3.11 4.10 3.61 3.09 4.29 2.19 2.12 2.24 4.75 2.35 4.99 4.32 2.70 3.72 4.25 3.19 3.54 3.45 4.46 4.30 2.73 4.35 4.78 4.64 4.03 3.88 3.62 4.40 4.42 3.36 3.71 3.38

Hyodeoxycholic acid

9.64

Ursodesoxycholic acid

5.89

Chenodeoxycholic acid

111.26

Sodium taurochenodeoxycholate

3.58

Sodium tauroursodeoxycholate

11.11

The accuracy of the method was determined by a recovery test. Mixed standard solutions were prepared at three different concentration levels (80, 100, and 120% of the known amounts of PZH samples (S3018)), and then added to the sample for analysis. Triplicate experiments were conducted at each level. As shown in Table 2, the recovery rate of 12 standards varied from 95.63–104.80% (RSD≤4.78%), revealing the acceptable recovery and accuracy of this method.

Muscone

33.87

3.2.4 Sample determination

4 Concluding remarks

This developed analytical method was successfully applied for the identification and quantification of 12 target compounds in ten batches of PZH. The contents of the investigated 12 compounds, based on their respective calibration curves, are summarized in Table 3. There were great variations among the contents of some analytes in different batches of PZH. For instance, sample S2026 had the lowest contents of notoginsenoside R1, ginsenoside Rb1, and ginsenoside Rg1, as well as sample S3004 in the contents of deoxycholic acid, hyodeoxycholic acid, ursodesoxycholic acid, and chenodeoxycholic acid. Real sample data demonstrate that UPLC–QqQ-

In this study, a UPLC–QqQ–MS method for the simultaneous determination of three types of constituents in PZH has been developed and validated for the first time, which enabled the quality control of PZH. The quantitative ranges of these components in this method are 0.04–64, 0.02–64, 0.002–64, 0.02–1.28, 0.02–64, 0.04–64, 0.02–1.28, 0.004–1.28, 0.02–64, 0.02–1.28, 0.004–1.28, and 0.02–6.4 mg/g. Compared with the current published HPLC and TLC methods [20, 21], the MRM mode of QqQ-MS of the developed method enabled identification of target compounds with high selectivity even at low concentration by comparison with standards, and rapid

Six independent samples of PZH (S3004) were analyzed in parallel by the above-established method for the evaluation of method repeatability. The RSD of 12 standards were within the range of 2.21–4.20%. The storage stability of the measurements for sample solution was 3.52–4.81% within 12 h at room temperature. In conclusion, the developed method had good precision, repeatability, and stability (Supporting Information Table S2). 3.2.3 Recovery

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MS is suitable for the analysis of these active components in PZH samples.

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Table 3. Contents of 12 analytes in different PZH samples

Samples

Content of each compound in ten batches of samples (mg/g)

No.

1

2

3

4

5

6

7

8

9

10

11

12

S2017 S2026 S3004 S3010 S3018 S3027 S4002 S4011 S4018 S4023 Aver.

13.61 9.02 14.08 10.82 16.27 11.48 12.10 10.56 13.72 13.39 12.51

11.90 8.07 12.39 9.58 14.29 10.08 10.60 9.34 12.02 11.79 11.01

36.71 24.04 30.46 29.45 33.53 28.55 31.31 29.48 34.36 32.87 31.08

0.024 0.035 0.019 0.029 0.035 0.027 0.031 0.032 0.033 0.038 0.030

15.01 18.06 15.68 19.93 17.46 17.67 19.71 21.41 21.04 20.28 18.63

1.57 1.91 1.22 1.47 2.03 1.51 1.59 1.50 1.59 1.90 1.63

0.312 0.263 0.262 0.313 0.327 0.264 0.287 0.269 0.310 0.331 0.294

0.210 0.181 0.130 0.177 0.162 0.154 0.119 0.137 0.189 0.130 0.159

3.82 3.19 2.71 3.99 3.54 3.03 3.27 2.97 3.42 3.30 3.32

0.156 0.177 0.228 0.184 0.217 0.209 0.191 0.207 0.229 0.222 0.202

0.077 0.066 0.077 0.072 0.093 0.077 0.089 0.100 0.108 0.092 0.085

0.493 0.517 0.704 0.627 0.697 0.555 0.612 0.667 0.574 0.717 0.616

analysis performed within 14 min facilitated the efficient quantification of the target compounds in PZH. Also, it was particularly suitable for the simultaneous quantitative analysis of different compounds. This method will be applied for further pharmacological, pharmacokinetic, and clinical studies.

[11] Zhuang, Q., Hong, F., Shen, A., Zheng, L., Zeng, J., Lin, W., Chen, Y., Sferra, T. J., Hong, Z., Peng, J., Int. J. Oncol. 2012, 40, 1569–1574.

The work was supported by the National Natural Science Foundation of China (81073097, 81202904 and 81373940) and the Provincial Natural Science Foundation of Fujian (2012J05152 and 2012Y4005).

[14] Ng, T. B., J. Pharm. Pharmacol. 2006, 58, 1007–1019.

The authors have declared no conflict of interest.

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