Journal of Chromatographic Science 2015;53:736– 741 doi:10.1093/chromsci/bmu116 Advance Access publication September 11, 2014
Article
Simultaneous Determination of 10 Components in Bu-Zhong-Yi-Qi Wan by Solid Phase Extraction-High Performance Liquid Chromatography with Diode Array Detection and Evaporative Light Scattering Detection Fang Hu1, Ruijuan Zhu1, Xiaohua Liu1, Yinglai Yang1, Can Li1, Shilan Feng1* and Yingdong Li2* 1 School of Pharmacy, Lanzhou University, Gansu, 199 Donggang West Road, Lanzhou 730000, Gansu Province, China, and 2Department of Pharmacy, Gansu University of Traditional Chinese Medicine, 35 Dingxi East Road, Lanzhou 730000, Gansu Province, China
*Author to whom correspondence should be addressed. Email: [email protected]; [email protected] Received 4 August 2013; revised 20 August 2014
An effective, accurate and reliable method for the simultaneous separation and determination of 10 major components in Chinese medicine Bu-Zhong-Yi-Qi Wan (BZYQW) was developed and validated using solid phase extraction column-high performance liquid chromatography-diode array detection-evaporative light scattering detection (SPE-HPLC-DAD-ELSD). The chromatographic separation was performed on a SpursilTM C18 column (250 mm 3 4.6 mm, 5 mm) at 3088 C with an acetonitrile – water gradient as mobile phase. The DAD detection wavelength 254 nm was utilized for the quantitative analysis. The drift tube temperature and the carrier gas flow rate of the ELSD detection was set at 110.588 C and 3.1 mL/min. The total run time is 103 min, these determined components peak out within 81 min. Excellent linear behaviors over the investigated concentration ranges were observed with the values of r 2 higher than 0.9990 for all the analytes. The Linear range over hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I is 4.50 – 94.50 mg/mL, 22.75 – 364.00 mg/mL, 2.30 – 45.00 mg/mL, 11.76 – 125.14 mg/mL, 4.62–50.35 mg/mL, 1.90–28.93 mg/mL, 1.29–159.00 mg/mL, 2.90– 36.00 mg/mL, 35.40–192.40 mg/mL, 41.40–96.60 mg/mL, respectively. The method was validated by its repeatability [relative standard deviation (RSD) < 3.54%] and intra-day (RSD < 2.11%) and inter-day precision (RSD < 3.45%). The limits of detection and quantification of each component were in the ranges of 0.04 – 10.24 and 0.12 – 39.22 mg/mL, respectively. The average recovery yields of the 10 compounds ranged from 95.79 to 101.25%. The validated method was successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW from different manufacturers.
Introduction Traditional Chinese patent medicine is the essence of a valid prescription after thousands of year’s creation of medical practice by Chinese Medicine practitioners of our history. BZYQW originated from the famous Bu-Zhong-Yi-Qi Tang (BZYQT), composed of eight commonly used Chinese herbal medicines, Astragali Radix, Radix Codonopsis, Radix Glycyrrhizae, Radix Angelicae, Angelica sinensis, Rhizoma Cimicifugae, Radix Bupleuri, Pericarpium Citri Reticulatae (1). BZYQW and BZYQT are the same medicinal materials but different dosage form. Pharmacological studies and clinical practice have demonstrated that BZYQT possesses many biological functions, including
antiinflammatory effects (2) and improve immunological capacity in elderly persons (3). However, large dose, long processing time, inconvenience for carrying were the familiar disadvantages of medicinal broth in the clinical application (4). Currently, considering the convenience for storage, transport, carrying and being taken in the form of pills, BZYQW has been widely used in various departments such as medical, surgical, dermatological, gynecology, pediatrics and ENT for dozens of illnesses. BZYQW is the traditional Chinese patent medicine effective for the treatment of symptoms such as weakness caused by fatigue and weakness after illness. As many excipients joined in the process of pills, we could not use the quality control of BZYQT to evaluate BZYQW, and as there are only few reports about the quality control of BZYQW, the study of BZYQW is very necessary. HPLC is used to determine the content of astragaloside IV in BZYQW in the 2010 Chinese Pharmacopoeia. Chinese herbal medicine is a complex black box system, which contains a lot of chemical ingredients, hence it is very difficult to conduct a comprehensive quality control of traditional Chinese medicine. Pharmacy workers have been working tirelessly for the quality control of traditional Chinese medicine. Determinations of one or two components in BZYQW were largely reported: ferulic acid and isoferulic acid (5), hesperidin (6) or formononetin (7) and so on. The chemical composition becomes more complex after the combination of different traditional Chinese medicine preparations, and more interference components were produced. So the method for the determination of one or two components is not suitable for the quality control of traditional Chinese medicine. In this formula, Astragali Radix acts as the emperor herb and plays a major therapeutic role. Isoflavonoids and saponins are the main constituents of Astragali Radix, and they have pharmacological activities such as antioxidant activity (8), adjuvant with low or non-hemolytic effect (9), respectively. In order to evaluate the quality of BZYQW, DAD and ELSD were combined to simultaneously determine these components. Although Angelica sinensis plays the auxiliary role in the formula, it is rare medicinal materials that are indispensable in traditional Chinese medicine, there is a saying “nine contains of Angelica sinensis to ten prescription” in China; it is also used as a health food product for women’s care in Europe and America. Angelica sinensis has pharmacological activities such as anticoagulative (10), hematopoietic (11) and efficacy against brain tumors (12). Ligustilide and other phthalides are thought to be the biologically active components in Angelica sinensis (13 –16). So the four main components of essential oil in Angelica sinensis were
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determined. In our previous research, hesperidin was found to have greater contribution in the treatment of blood deficient and qi deficient mice, so the content of hesperidin was also determined. Therefore 10 major components: hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I in BZYQW were determined. These compounds can be divided into three categories: flavonoids, saponins, volatile oils. Although HPLC methods have been applied to determine some of the constituents in crude drugs and Chinese patented medicines (17– 24), no analytical method has been reported for simultaneous determination of these 10 major constituents in BZYQW. Hence, it is very important to establish such a method for the quality control of these major compounds, which could help to evaluate the quality of other related herbal formulas. Considering the complexity of traditional Chinese patent medicine, SPE was used to purify the sample and improve selectivity and efficiency (25). So an effective, accurate, and rapid SPEHPLC-DAD-ELSD method was developed for simultaneous quantification and identification of these 10 compounds in BZYQW.
(water) with a linear gradient: 0 – 20 min (A 11%!15%), 20 – 35 min (A 15%!23%), 35 – 60 min (A 23%!37%), 60 – 64 min (A 37%!45%), 64 –80 min (A 45%!81%) with a mobile flow rate of 1 mL/min. The detection wavelength was set at 254 nm, the drift tube temperature was 110.58C and the carrier gas flow rate was 3.1 mL/min with the sample injection volume of 30 mL.
Standard solutions and calibration All standard solutions were prepared in methanol. Standards of hesperidin, senkyunolide I, senkyunolide H, ligustilide, butylene phthalide, ononin, calycosin, formononetin, astragaloside I, Astragaloside IV were accurately weighed and dissolved in volumetric flasks to obtain appropriate stock solutions, their concentration were 0.095, 0.364, 0.045, 0.159, 0.036, 0.125, 0.050, 0.029, 0.192, 0.097 mg/mL, respectively. These stock solutions were diluted with methanol to prepare a series of mixed working solutions with different concentration. All the standard solutions were stored at 48C before use; the stability was verified by re-assaying the standard solutions. Calibration curves were established based on five levels for each in duplicate.
Experimental Instrumentation and reagents The HPLC system used for the chromatographic analysis was Waters 2695 system (Waters, Milford, MA, USA), including quaternary solvent delivery pump, auto sampler manager, column compartment, photodiode array detector (Waters 2695, USA) and evaporative light scattering detector (Alltech 2000, Germany) connected to Millennium32 software. BZYQW (lot no. 83111110, 83111213, 83121124, 83121003, 83120875) were provided by Lanzhou Tai Bao Pharmaceutical Co., Ltd. BZYQW (lot no. 12J52, 12F39, 12J50, 12F40, 12J49) were from Lanzhou Foci Pharmaceutical Co., Ltd. Calycosin (111530), astragaloside IV (110781), formononetin (111703) and hesperidin (721) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Ononin, senkyunolide H, astragaloside I were obtained from Shanghai Sunzo Biology Technology Co., Ltd (Shanghai, China); Senkyunolide I was from Shanghai Yilin Biological Technology Co., Ltd (Shanghai, China); Ligustilide (12082702); Butylene phthalide (MUST-3040801). HPLC grade-acetonitrile was obtained from Merck co., Ltd (Darmstadt, Germany). Deionised water was purchased from Wahaha co., Ltd (Hangzhou, China). Analytical-grade methanol was from the northern Tianjin Tian Yi Chemical Reagent co., Ltd (Tianjin, China). Analyticalgrade ethanol was purchased from Tianjin Baishi Chemical Company co., Ltd (Tianjin, China). Waters Oasis HLB solid-phase extraction (SPE) columns (3cc, Waters, Milford, MA, USA) were used in the experiment. Lot no. 83121124 was selected as the sample for the exploration of extraction conditions and chromatographic conditions.
Methods Apparatus and chromatographic conditions The chromatographic separation was performed on a SpursilTM C18 column (250 mm 4.6 mm, 5 mm) at 308C. The mobile phase was composed of solvent A (acetonitrile) and solvent B
Preparation of sample solution BZYQW were crushed into powder, 2.5 g of the powder was accurately weighed and soaked in 60 mL methanol for 0.5 h, then extracted by ultrasonic extraction (40 KHz, 150 W) for 0.5 h, filtered and the was residue extracted with 40 mL methanol for 0.5 h, the filtrate was combined and evaporated on water bath, then dissolved with 3 mL distilled water. The dissolved sample was purified using SPE (3cc, Waters, Milford, MA, USA). The column was first conditioned with 7.5 mL methanol and then equilibrated with 7.5 mL water. After sample loading, the column was washed with 1 mL water, and then eluted with 1 mL 10% methanol, 1 mL 90% methanol and then the two parts of 10% methanol eluent and 90% methanol eluent were mixed. The mixture was evaporated under a stream of nitrogen. Then transferred to a 2-ml volumetric flask, added methanol to volume, filtered through 0.45 mm microporous membrane to yield the sample solution. Each sample in duplicate.
Method validation The validation of the method included the following parameters according to the International Conference on Harmonization (ICH) guidelines: linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery (26). The solutions of the 10 components were prepared and diluted with methanol to a suitable concentration for the construction of calibration curves. The linearity of the assay was assessed by analyzing the calibration curves using linear regression of the peak area to concentration. Their regression equations were calculated in the form of y ¼ ax þ b, for the compounds detected by DAD, y and x were peak area and compound concentration, while for ELSD detection, y and x were the logarithmic of peak area and compound concentration. The injection concentration, which could be detected at the signal-to-noise (S/N) ratio of 3, was considered to be the limit of detection (LOD). Limit of quantification (LOQ) was the Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 737
Figure 1. The HPLC-DAD chromatogram at 254 nm: blank (A), standard solution (B) and the sample solution (lot no. 83121124) (C). Peaks: 1, hesperidin; 2, senkyunolide I; 3, senkyunolide H; 4, ononin; 5, calycosin; 6, formononetin; 7, ligustilide; 8, butylene phthalide.
Figure 2. The HPLC-ELSD chromatogram: blank (D), standard solution (E) and the sample solution (lot no. 83121124) (F). Peaks: 1, hesperidin; 4, ligustilide; 6, ononin; 9, astragaloside IV; 10, astragaloside I.
738 Hu et al.
Table I Calibration Curves, LOD, LOQ of the 10 Analytes in BZYQW
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Regression equation y ¼ ax þ b
r2
Liner range (mg/mL)
LOD (mg/mL)
LOQ (mg/mL)
y ¼ 1.40 107x21.59 104 y ¼ 3.80 107x22.90 105 y ¼ 4.40 107x26.12 104 y ¼ 1.10 108x23.28 104 y ¼ 9.40 107x24.72 103 y ¼ 1.20 108x21.17 104 y ¼ 1.50 108x29.52 104 y ¼ 1.40 108x21.00 105 y ¼ 1.74x þ 7.09 y ¼ 1.51x þ 6.61 y ¼ 1.67x þ 7.54 y ¼ 1.61x þ 7.23
0.9990 0.9992 0.9990 1.0000 1.0000 0.9998 0.9993 0.9992 0.9991 0.9991 0.9995 0.9993
4.50 –94.50 22.75 –364.00 3.70 –45.00 1.29 –159.00 2.90 –36.00 19.76 –125.14 4.62 –50.35 1.90 –28.93 41.40 –96.60 40.20 –185.00 19.50 –94.50 15.76 –125.14
0.263 0.329 0.648 0.131 0.204 0.0418 0.0731 0.320 10.2 11.8 5.77 4.59
0.864 1.10 1.92 0.427 0.672 0.116 0.244 1.08 31.0 39.2 19.2 15.3
E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
Table III Recovery Test of the Developed Method (n ¼ 5)
Table II Precision and Repeatability of the 10 Analytes Repeatability (n ¼ 6)
Precision
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Intra-assay (n ¼ 6)
Inter-assay (n ¼ 3)
Mean (mg/g)
RSD (%)
Mean (mg/g)
RSD (%)
32.60 65.20 4.10 10.65 5.22
1.46 1.08 2.11 1.36 1.82
30.80 61.22 3.98 10.95 4.99
3.10 2.98 3.12 3.45 2.21
10.93 73.35 1.42 20.69 3.97
1.52 3.11 3.42 3.28 3.39
21.89 9.85 3.46 45.02 54.89 30.88 25.09
1.65 1.21 1.22 1.02 1.34 1.32 1.06
20.79 9.56 3.37 45.58 53.90 31.02 24.87
2.03 2.01 2.28 1.87 2.35 3.45 2.87
24.41 19.95 6.23 69.62 48.31 31.95 38.44
2.07 1.84 1.66 2.58 3.28 3.54 3.06
Mean (mg/g)
RSD (%)
E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
injection concentration corresponding to the peak heights with S/N ¼ 10. The precision was examined by performing the intra-day and inter-day assays by six replicate injections of the mixture standard solutions. Intra-day variations at three times within one day and inter-day variations for three consecutive days were chosen to determine the precision of the developed method. Six same samples of BZYQW were analyzed to confirm the repeatability of the method, and the RSD was taken as a measure of reproducibility. The same sample solution was stored at 48C and analyzed every 6 h over 2 days to investigate the stability of the solution. Its accuracy was also evaluated by a recovery test. The recovery of the 10 components were determined at middle levels with five replicates by comparing the peak areas from extracted samples with those in post-extracted samples spiked with the analytes at the same concentration. The SPE extraction recovery was calculated by comparing the peak area of the analyte spiked to solvent followed by sample extraction to that of the analyte spiked to the already extracted solvent. Results The DAD and ELSD chromatographic profiles of the blank, standard solution and the sample solution are showed in Figure 1 and Figure 2.
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I
Original concentration (mg/mL)
Quantity added (mg/mL)
Quantity found (mg/mL)
Recovery (%)
R.S.D. (%)
30.60 64.20 3.98 10.35 5.08
25.52 57.87 4.38 10.14 4.89
56.70 119.51 8.25 19.94 9.55
101.03 97.90 98.68 97.32 95.79
1.63 2.86 2.68 2.98 2.19
20.89 9.55 3.06 43.02 51.89
20.54 9.03 2.99 42.08 52.07
41.94 18.55 5.96 83.33 105.26
101.23 99.84 98.51 97.92 101.25
1.77 1.65 2.38 2.10 2.01
Linearity and limit of detection Standard stock solutions of the 10 components were prepared and diluted to appropriate concentrations for plotting the calibration curves. All calibration curves showed good linearity (r 2 . 0.9990) between the peak area and concentration within test ranges; the LODs (S/N ¼ 3) and the LOQs (S/N ¼ 10) for the eight components detected in DAD are ,0.65 and 1.92 mg/mL at 254 nm and ,11.76 and 39.22 mg/mL, respectively, for the four saponins in ELSD. All analytes were established to be stable. The results of regression analysis for each of the 10 compounds, together with the LOQ and LOD values, are shown in Table I. Precision and accuracy Table II shows the results of the precision tests of the 10 analytes and the repeatability of the method; it was shown that the RSD was lower than 3.45% both for the intra-assay and inter-assay precision. Besides, validation studies of this method proved that this assay has good repeatability with RSD in the range of 1.52– 3.54%. Table III shows that the developed analytical method has good accuracy with the overall recovery from 95.79 to 101.25% for the analytes concerned, and the RSD values were ,3.00%. Extraction efficiency The extraction efficiency determined for 10 analytes are shown in Table IV, which were all 90.99%, with relative standard deviation values 2.89%. Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 739
Table IV Determination of the Active Components in BZYQW by the Developed HPLC Method Content (mg/g)
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Batch 6
Batch 7
Batch 8
Batch 9
Batch 10
34.26 74.71 Nd 9.30 5.05 12.40 27.83 7.88 68.61 70.24 46.28 15.91
78.80 133.15 2.06 1.89 0.74 16.80 6.05 1.36 33.52 44.80 79.83 20.33
31.87 64.35 2.18 10.42 5.02 23.29 9.72 3.53 44.98 55.84 32.18 24.64
36.71 73.58 4.16 26.76 8.53 30.92 16.63 7.92 75.81 59.04 37.34 31.92
40.10 74.77 2.30 2.93 3.98 16.43 9.23 4.26 71.78 59.84 41.23 17.38
42.94 94.25 3.14 34.58 5.98 30.54 7.95 5.20 47.13 166.82 44.78 32.06
60.35 120.88 2.32 16.45 4.23 24.78 6.34 2.42 52.80 35.12 61.35 23.07
65.82 132.80 2.94 27.19 4.17 37.18 9.55 4.58 57.42 137.76 66.47 38.69
50.04 115.95 3.00 11.57 2.68 21.64 5.24 1.72 30.98 47.60 55.98 25.92
54.38 136.28 1.89 37.98 8.04 26.44 13.21 3.80 148.64 88.24 68.14 27.93
nd: not detected; E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
Table V Extract Recovery of 10 Constituents in BZYQW by SPE (n ¼ 5) Spiked (mg/mL)
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
32.60 65.20 4.10 10.65 5.22 21.89 9.85 3.46 45.02 54.89 30.88 25.09
Extract recovery Relative recovery (%)
RSD (%)
95.96 94.01 93.28 90.99 94.03 96.04 96.87 97.32 96.10 95.88 95.08 95.98
2.13 2.57 2.90 2.89 2.78 2.09 1.98 1.95 1.78 2.08 2.19 2.23
Sample determination The target compounds were identified by comparison of LC retention time and UV absorption with the reference compounds. The proposed HPLC method was applied to analyze the 10 compounds in 10 sample batches of BZYQW; each sample was analyzed two times. Table V shows the results of determination of the 10 compounds. Among the 10 compounds, senkyunolide I, astragaloside I, astragaloside IV and hesperidin are the most abundant, and the concentration of compound senkyunolide I is the highest in all samples except the sample 4, 6, 8, 10. In sample 4 and 10, the concentration of astragaloside IV is up to 75.81 and 148.64 mg/g, respectively. In sample 6 and 8, the concentration of astragaloside I is up to 166.82 and 137.76 mg/g, respectively.
impurities became more when extracted with methanol, so SPE was used to purify the sample, which gave rise to optimum extraction of all the 10 components with broad range of polarity in high yield. In order to investigate extraction time, powdered BZYQW were extracted with methanol for 0.5, 1, 1.5 h, respectively. The results suggested that all the 10 components were almost completely extracted by extracting with methanol two times for 0.5 h each. Among the detection of the compounds, flavonoids have response both in the DAD and in the ELSD. But for the low content of calycosin, within this range does not linearly standard of calibration curves in ELSD. Formononetin only has response in DAD for the low content. The concentration of hesperidin and ononin were both detected by DAD and ELSD: the results in Table V indicate that the concentrations are significantly different. The concentration of each compound obtained from ELSD is higher than that from DAD. Although the concentrations of hesperidin and ononin obtained from ELSD are higher, the peak areas of ELSD detection are lower than that of DAD detection, which indicates the poor sensitivity, and also because of the high detection limit of ELSD, DAD is commonly used than ELSD (27). It is obvious that DAD detection at 254 nm showed a very good sensitivity with significantly lower LODs and wider calibration ranges than that achieved by ELSD for the detection of hesperidin and ononin (marked E hesperidin and E ononin in ELSD). These data indicate that the developed method was precise, accurate and sensitive enough for the quantitation of the major constituents in BZYQW.
Conclusion Discussion In order to optimize the extraction conditions for achievement of quantitative extraction, variables involved in the procedure such as extraction method, solvent and extraction time were optimized. Compared with refluxing extraction and soxhlet extraction, the ultrasonic treatment procedure was found to be the better extraction method for all the 10 components. Pure and aqueous methanol or ethanol solutions were tried as the extraction solvent, the best solvent was found to be methanol. But 740 Hu et al.
In the present study, an accurate and reliable analytical method for the simultaneous determination of 10 major components in Chinese medicine BZYQW was developed. The method was developed using SPE-HPLC-DAD-ELSD, which makes it possible to comprehensively evaluate the quality of BZYQW. Good linearity, repeatability, intra-day and inter-day precision, accuracy and reliability were presented in the method validation procedure. The developed method has been successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online. Acknowledgments The study was financially supported by National Science and Technology Supporting Program for the 12th Five-Year Plan (NO.2011BAI05B02). References 1. The State Commission of Chinese Pharmacopoeia; Pharmacopoeia of People’s Republic of China, Chinese Medical Science and Technology Press, (2010); Part I: 780– 781. 2. Yang, S.H., Yu, C.L.; Antiinflammatory effects of Bu-zhong-yi-qi-tang in patients with perennial allergic rhinitis; Journal of Ethnopharmacology, (2008); 115: 104–109. 3. Kuroiwa, A., Liou, S., Yan, H., Eshita, A., Naitoh, S., Nagayama, A.; Effect of a traditional Japanese herbal medicine, Hochu-ekki-to (Bu-Zhong-Yi-Qi Tang), on immunity in elderly persons; International Immunopharmacology, (2004); 4: 317–324. 4. Xue, R., Cao, Y.F., Han, N., Lin, X.W., Liu, Z.H., Yin, J.; Activity of DBXX granules on anti-gastric ulcer and decreasing the side effect of chemotherapy in S180 tumor-bearing mice; Journal of Ethnopharmacology, (2011); 137: 1156– 1160. 5. Ke, X.H., Chen, W., Chen, J.F., Hua, R.F., Yang, X.C.; Determination of ferulic acid and isoferulic acid in Buzhongyiqi pill by HPLC; Anhui Medical and Pharmaceutical Journal, (2010); 14: 768–769. 6. Xu, D.Q.; Determination of hesperidin in Buzhongyiqi Pills by HPLC; Strait Pharmaceutical Journal, (2007); 19: 48 –50. 7. Dong, X.L., Li, H.J.; Content determination of formononetin in Buzhong Yiqi Pills by HPLC; China Pharmacy, (2012); 23: 3440–3441. 8. Zhao, B.S., Fu, Y.J., Wang, W., Zu, Y.G., Gu, C.B., Luo, M., et al.; Enhanced extraction of isoflavonoids from Radix Astragali by incubation; Innovative Food Science and Emerging Technologies, (2011); 12: 577–585. 9. Yang, Z.G., Sun, H.X., Fang, W.H.; Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune responses to ovalbumin in mice; Vaccine, (2005); 23: 5196– 5203. 10. Zhou, G.S., Yang, N.Y., Tang, Y.P., Duan, J.A., Jiang, S., Yan, H., et al.; Chemical constituents from the aerial parts of Angelica sinensis and their bioactivities; Chinese Journal of Natural Medicines, (2012); 10: 295– 298. 11. Lee, J.G., Hsieh, W.T., Chen, S.U., Chiang, B.H.; Hematopoietic and myeloprotective activities of an acidic Angelica sinensis polysaccharide on human CD34þ stem cells; Journal of Ethnopharmacology, (2012); 139: 739– 745. 12. Lin, P.C., Liu, P.Y., Lin, S.Z., Harn, H.J.; Angelica sinensis: a Chinese herb for brain cancer therapy; BioMedicine, (2012); 2: 30 –35. 13. Yang, J.Y., Chen, H.H., Wu, J., Gong, S.X., Chen, C.Q., Zhang, T.J., et al.; Advances in studies on pharmacological functions of ligustilide and their mechanisms; Chinese Herbal Medicines, (2012); 4: 26– 32. 14. Chen, L.L., Qi, J., Chang, Y.X., Zhu, D.N., Yu, B.Y.; Identification and determination of the major constituents in traditional Chinese medicinal formula Danggui-Shaoyao-San by HPLC-DAD-ESI-MS/MS; Journal of Pharmaceutical and Biomedical Analysis, (2009); 50: 127– 137.
15. Wang, Z.J., Wo, S.K., Wang, L., Lau, C.B.S., Lee, V.H.L., Chow, M.S.S., et al.; Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography-mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis, (2009); 50: 232– 244. 16. Yeh, J.C., Garrard, I.J., Chantal Cho, C.W., Annie Bligh, S.W., Lu, G.H., Fan, T.P., et al.; Bioactivity-guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles; Journal of Chromatography A, (2012); 1236: 132–138. 17. Feng, C., Cai, Y.L., Ruan, J.L.; Simultaneous determination of 10 active components in traditional Chinese medicine “YIGONG” capsule by RP-HPLC-DAD; Journal of Pharmaceutical and Biomedical Analysis, (2008); 47: 442–447. 18. Yang, D.Z., An, Y.Q., Jiang, X.L., Tang, D.Q., Gao, Y.Y., Zhao, H.T., et al.; Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional Chinese medicine preparation; Talanta, (2011); 85: 885– 890. 19. Ouyang, L.Q., Wu, H.L., Nie, J.F., Zhang, Y., Zou, H.Y., Fu, H.Y., et al.; Simultaneous determination of psoralen and isopsoralen in plasma and Chinese medicine Xian Ling Gu Bao capsule by using HPLC-DAD coupled with alternating trilinear decomposition algorithm; Analytica Chimica Acta, (2009); 650: 160–166. 20. Liu, H., Su, J., Liang, X., Zhang, X., He, Y.J., Huang, H.Q., et al.; Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-DAD-ESI-MS; Journal of Pharmaceutical Analysis, (2011); 1: 1–7. 21. Li, H., Wang, S.W., Zhang, B.L., Xie, Y.H., Yang, Q., Cao, W., et al.; Simultaneous quantitative determination of 9 active components in traditional Chinese medicinal preparation ShuangDan oral liquid by RP-HPLC coupled with photodiode array detection; Journal of Pharmaceutical and Biomedical Analysis, (2011); 56: 820–824. 22. Yin, L.H., Lu, B.N., Qi, Y., Xu, L.N., Han, X., Xu, Y.W., et al.; Simultaneous determination of 11 active components in two wellknown traditional Chinese medicines by HPLC coupled with diode array detection for quality control; Journal of Pharmaceutical and Biomedical Analysis, (2009); 49: 1101–1108. 23. Yang, X.X., Zhang, X.X., Chang, R.M., Wang, Y.W., Li, X.N.; Simultaneous quantification of five major active components in capsules of the traditional Chinese medicine “Shu-Jin-Zhi-Tong” by high performance liquid chromatography; Journal of Pharmaceutical Analysis, (2011); 1: 284– 290. 24. Chen, Y.Y., Guo, J., Fan, H., Huang, L.H.; Simultaneous determination of seven alkaloids in Fufang Zhenzhu Tiaozhi capsule by HPLC coupled with DAD; Chinese Herbal Medicines, (2012); 4: 230–236. 25. Zhang, P.X., Jin, Y.R., Chen, J.F., Yao, H., Zhang, H.Q., Yu, A., et al.; Ultrasonic-assisted nebulization extraction coupled with SPE and HPLC for the determination of triterpenoids in root of Euphorbia pekinensis Rupr.; Chromatographia, (2013); 76: 967–974. 26. Wei, H., Sun, L., Tai, Z., Gao, S., Xu, W., Chen, W.; A simple and sensitive HPLC method for the simultaneous determination of eight bioactive components and fingerprint analysis of Schisandra sphenanthera; Analytica Chimica Acta, (2010); 662: 97– 104. 27. Qi, L.W., Yu, Q.T., Li, P., Li, S.L., Wang, Y.X., Sheng, L.H., et al.; Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by highperformance liquid chromatography coupled with diode array and evaporative light scattering detectors; Journal of Chromatography A, (2006); 1134: 162– 169.
Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 741
Article
Simultaneous Determination of 10 Components in Bu-Zhong-Yi-Qi Wan by Solid Phase Extraction-High Performance Liquid Chromatography with Diode Array Detection and Evaporative Light Scattering Detection Fang Hu1, Ruijuan Zhu1, Xiaohua Liu1, Yinglai Yang1, Can Li1, Shilan Feng1* and Yingdong Li2* 1 School of Pharmacy, Lanzhou University, Gansu, 199 Donggang West Road, Lanzhou 730000, Gansu Province, China, and 2Department of Pharmacy, Gansu University of Traditional Chinese Medicine, 35 Dingxi East Road, Lanzhou 730000, Gansu Province, China
*Author to whom correspondence should be addressed. Email: [email protected]; [email protected] Received 4 August 2013; revised 20 August 2014
An effective, accurate and reliable method for the simultaneous separation and determination of 10 major components in Chinese medicine Bu-Zhong-Yi-Qi Wan (BZYQW) was developed and validated using solid phase extraction column-high performance liquid chromatography-diode array detection-evaporative light scattering detection (SPE-HPLC-DAD-ELSD). The chromatographic separation was performed on a SpursilTM C18 column (250 mm 3 4.6 mm, 5 mm) at 3088 C with an acetonitrile – water gradient as mobile phase. The DAD detection wavelength 254 nm was utilized for the quantitative analysis. The drift tube temperature and the carrier gas flow rate of the ELSD detection was set at 110.588 C and 3.1 mL/min. The total run time is 103 min, these determined components peak out within 81 min. Excellent linear behaviors over the investigated concentration ranges were observed with the values of r 2 higher than 0.9990 for all the analytes. The Linear range over hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I is 4.50 – 94.50 mg/mL, 22.75 – 364.00 mg/mL, 2.30 – 45.00 mg/mL, 11.76 – 125.14 mg/mL, 4.62–50.35 mg/mL, 1.90–28.93 mg/mL, 1.29–159.00 mg/mL, 2.90– 36.00 mg/mL, 35.40–192.40 mg/mL, 41.40–96.60 mg/mL, respectively. The method was validated by its repeatability [relative standard deviation (RSD) < 3.54%] and intra-day (RSD < 2.11%) and inter-day precision (RSD < 3.45%). The limits of detection and quantification of each component were in the ranges of 0.04 – 10.24 and 0.12 – 39.22 mg/mL, respectively. The average recovery yields of the 10 compounds ranged from 95.79 to 101.25%. The validated method was successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW from different manufacturers.
Introduction Traditional Chinese patent medicine is the essence of a valid prescription after thousands of year’s creation of medical practice by Chinese Medicine practitioners of our history. BZYQW originated from the famous Bu-Zhong-Yi-Qi Tang (BZYQT), composed of eight commonly used Chinese herbal medicines, Astragali Radix, Radix Codonopsis, Radix Glycyrrhizae, Radix Angelicae, Angelica sinensis, Rhizoma Cimicifugae, Radix Bupleuri, Pericarpium Citri Reticulatae (1). BZYQW and BZYQT are the same medicinal materials but different dosage form. Pharmacological studies and clinical practice have demonstrated that BZYQT possesses many biological functions, including
antiinflammatory effects (2) and improve immunological capacity in elderly persons (3). However, large dose, long processing time, inconvenience for carrying were the familiar disadvantages of medicinal broth in the clinical application (4). Currently, considering the convenience for storage, transport, carrying and being taken in the form of pills, BZYQW has been widely used in various departments such as medical, surgical, dermatological, gynecology, pediatrics and ENT for dozens of illnesses. BZYQW is the traditional Chinese patent medicine effective for the treatment of symptoms such as weakness caused by fatigue and weakness after illness. As many excipients joined in the process of pills, we could not use the quality control of BZYQT to evaluate BZYQW, and as there are only few reports about the quality control of BZYQW, the study of BZYQW is very necessary. HPLC is used to determine the content of astragaloside IV in BZYQW in the 2010 Chinese Pharmacopoeia. Chinese herbal medicine is a complex black box system, which contains a lot of chemical ingredients, hence it is very difficult to conduct a comprehensive quality control of traditional Chinese medicine. Pharmacy workers have been working tirelessly for the quality control of traditional Chinese medicine. Determinations of one or two components in BZYQW were largely reported: ferulic acid and isoferulic acid (5), hesperidin (6) or formononetin (7) and so on. The chemical composition becomes more complex after the combination of different traditional Chinese medicine preparations, and more interference components were produced. So the method for the determination of one or two components is not suitable for the quality control of traditional Chinese medicine. In this formula, Astragali Radix acts as the emperor herb and plays a major therapeutic role. Isoflavonoids and saponins are the main constituents of Astragali Radix, and they have pharmacological activities such as antioxidant activity (8), adjuvant with low or non-hemolytic effect (9), respectively. In order to evaluate the quality of BZYQW, DAD and ELSD were combined to simultaneously determine these components. Although Angelica sinensis plays the auxiliary role in the formula, it is rare medicinal materials that are indispensable in traditional Chinese medicine, there is a saying “nine contains of Angelica sinensis to ten prescription” in China; it is also used as a health food product for women’s care in Europe and America. Angelica sinensis has pharmacological activities such as anticoagulative (10), hematopoietic (11) and efficacy against brain tumors (12). Ligustilide and other phthalides are thought to be the biologically active components in Angelica sinensis (13 –16). So the four main components of essential oil in Angelica sinensis were
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determined. In our previous research, hesperidin was found to have greater contribution in the treatment of blood deficient and qi deficient mice, so the content of hesperidin was also determined. Therefore 10 major components: hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I in BZYQW were determined. These compounds can be divided into three categories: flavonoids, saponins, volatile oils. Although HPLC methods have been applied to determine some of the constituents in crude drugs and Chinese patented medicines (17– 24), no analytical method has been reported for simultaneous determination of these 10 major constituents in BZYQW. Hence, it is very important to establish such a method for the quality control of these major compounds, which could help to evaluate the quality of other related herbal formulas. Considering the complexity of traditional Chinese patent medicine, SPE was used to purify the sample and improve selectivity and efficiency (25). So an effective, accurate, and rapid SPEHPLC-DAD-ELSD method was developed for simultaneous quantification and identification of these 10 compounds in BZYQW.
(water) with a linear gradient: 0 – 20 min (A 11%!15%), 20 – 35 min (A 15%!23%), 35 – 60 min (A 23%!37%), 60 – 64 min (A 37%!45%), 64 –80 min (A 45%!81%) with a mobile flow rate of 1 mL/min. The detection wavelength was set at 254 nm, the drift tube temperature was 110.58C and the carrier gas flow rate was 3.1 mL/min with the sample injection volume of 30 mL.
Standard solutions and calibration All standard solutions were prepared in methanol. Standards of hesperidin, senkyunolide I, senkyunolide H, ligustilide, butylene phthalide, ononin, calycosin, formononetin, astragaloside I, Astragaloside IV were accurately weighed and dissolved in volumetric flasks to obtain appropriate stock solutions, their concentration were 0.095, 0.364, 0.045, 0.159, 0.036, 0.125, 0.050, 0.029, 0.192, 0.097 mg/mL, respectively. These stock solutions were diluted with methanol to prepare a series of mixed working solutions with different concentration. All the standard solutions were stored at 48C before use; the stability was verified by re-assaying the standard solutions. Calibration curves were established based on five levels for each in duplicate.
Experimental Instrumentation and reagents The HPLC system used for the chromatographic analysis was Waters 2695 system (Waters, Milford, MA, USA), including quaternary solvent delivery pump, auto sampler manager, column compartment, photodiode array detector (Waters 2695, USA) and evaporative light scattering detector (Alltech 2000, Germany) connected to Millennium32 software. BZYQW (lot no. 83111110, 83111213, 83121124, 83121003, 83120875) were provided by Lanzhou Tai Bao Pharmaceutical Co., Ltd. BZYQW (lot no. 12J52, 12F39, 12J50, 12F40, 12J49) were from Lanzhou Foci Pharmaceutical Co., Ltd. Calycosin (111530), astragaloside IV (110781), formononetin (111703) and hesperidin (721) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Ononin, senkyunolide H, astragaloside I were obtained from Shanghai Sunzo Biology Technology Co., Ltd (Shanghai, China); Senkyunolide I was from Shanghai Yilin Biological Technology Co., Ltd (Shanghai, China); Ligustilide (12082702); Butylene phthalide (MUST-3040801). HPLC grade-acetonitrile was obtained from Merck co., Ltd (Darmstadt, Germany). Deionised water was purchased from Wahaha co., Ltd (Hangzhou, China). Analytical-grade methanol was from the northern Tianjin Tian Yi Chemical Reagent co., Ltd (Tianjin, China). Analyticalgrade ethanol was purchased from Tianjin Baishi Chemical Company co., Ltd (Tianjin, China). Waters Oasis HLB solid-phase extraction (SPE) columns (3cc, Waters, Milford, MA, USA) were used in the experiment. Lot no. 83121124 was selected as the sample for the exploration of extraction conditions and chromatographic conditions.
Methods Apparatus and chromatographic conditions The chromatographic separation was performed on a SpursilTM C18 column (250 mm 4.6 mm, 5 mm) at 308C. The mobile phase was composed of solvent A (acetonitrile) and solvent B
Preparation of sample solution BZYQW were crushed into powder, 2.5 g of the powder was accurately weighed and soaked in 60 mL methanol for 0.5 h, then extracted by ultrasonic extraction (40 KHz, 150 W) for 0.5 h, filtered and the was residue extracted with 40 mL methanol for 0.5 h, the filtrate was combined and evaporated on water bath, then dissolved with 3 mL distilled water. The dissolved sample was purified using SPE (3cc, Waters, Milford, MA, USA). The column was first conditioned with 7.5 mL methanol and then equilibrated with 7.5 mL water. After sample loading, the column was washed with 1 mL water, and then eluted with 1 mL 10% methanol, 1 mL 90% methanol and then the two parts of 10% methanol eluent and 90% methanol eluent were mixed. The mixture was evaporated under a stream of nitrogen. Then transferred to a 2-ml volumetric flask, added methanol to volume, filtered through 0.45 mm microporous membrane to yield the sample solution. Each sample in duplicate.
Method validation The validation of the method included the following parameters according to the International Conference on Harmonization (ICH) guidelines: linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery (26). The solutions of the 10 components were prepared and diluted with methanol to a suitable concentration for the construction of calibration curves. The linearity of the assay was assessed by analyzing the calibration curves using linear regression of the peak area to concentration. Their regression equations were calculated in the form of y ¼ ax þ b, for the compounds detected by DAD, y and x were peak area and compound concentration, while for ELSD detection, y and x were the logarithmic of peak area and compound concentration. The injection concentration, which could be detected at the signal-to-noise (S/N) ratio of 3, was considered to be the limit of detection (LOD). Limit of quantification (LOQ) was the Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 737
Figure 1. The HPLC-DAD chromatogram at 254 nm: blank (A), standard solution (B) and the sample solution (lot no. 83121124) (C). Peaks: 1, hesperidin; 2, senkyunolide I; 3, senkyunolide H; 4, ononin; 5, calycosin; 6, formononetin; 7, ligustilide; 8, butylene phthalide.
Figure 2. The HPLC-ELSD chromatogram: blank (D), standard solution (E) and the sample solution (lot no. 83121124) (F). Peaks: 1, hesperidin; 4, ligustilide; 6, ononin; 9, astragaloside IV; 10, astragaloside I.
738 Hu et al.
Table I Calibration Curves, LOD, LOQ of the 10 Analytes in BZYQW
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Regression equation y ¼ ax þ b
r2
Liner range (mg/mL)
LOD (mg/mL)
LOQ (mg/mL)
y ¼ 1.40 107x21.59 104 y ¼ 3.80 107x22.90 105 y ¼ 4.40 107x26.12 104 y ¼ 1.10 108x23.28 104 y ¼ 9.40 107x24.72 103 y ¼ 1.20 108x21.17 104 y ¼ 1.50 108x29.52 104 y ¼ 1.40 108x21.00 105 y ¼ 1.74x þ 7.09 y ¼ 1.51x þ 6.61 y ¼ 1.67x þ 7.54 y ¼ 1.61x þ 7.23
0.9990 0.9992 0.9990 1.0000 1.0000 0.9998 0.9993 0.9992 0.9991 0.9991 0.9995 0.9993
4.50 –94.50 22.75 –364.00 3.70 –45.00 1.29 –159.00 2.90 –36.00 19.76 –125.14 4.62 –50.35 1.90 –28.93 41.40 –96.60 40.20 –185.00 19.50 –94.50 15.76 –125.14
0.263 0.329 0.648 0.131 0.204 0.0418 0.0731 0.320 10.2 11.8 5.77 4.59
0.864 1.10 1.92 0.427 0.672 0.116 0.244 1.08 31.0 39.2 19.2 15.3
E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
Table III Recovery Test of the Developed Method (n ¼ 5)
Table II Precision and Repeatability of the 10 Analytes Repeatability (n ¼ 6)
Precision
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Intra-assay (n ¼ 6)
Inter-assay (n ¼ 3)
Mean (mg/g)
RSD (%)
Mean (mg/g)
RSD (%)
32.60 65.20 4.10 10.65 5.22
1.46 1.08 2.11 1.36 1.82
30.80 61.22 3.98 10.95 4.99
3.10 2.98 3.12 3.45 2.21
10.93 73.35 1.42 20.69 3.97
1.52 3.11 3.42 3.28 3.39
21.89 9.85 3.46 45.02 54.89 30.88 25.09
1.65 1.21 1.22 1.02 1.34 1.32 1.06
20.79 9.56 3.37 45.58 53.90 31.02 24.87
2.03 2.01 2.28 1.87 2.35 3.45 2.87
24.41 19.95 6.23 69.62 48.31 31.95 38.44
2.07 1.84 1.66 2.58 3.28 3.54 3.06
Mean (mg/g)
RSD (%)
E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
injection concentration corresponding to the peak heights with S/N ¼ 10. The precision was examined by performing the intra-day and inter-day assays by six replicate injections of the mixture standard solutions. Intra-day variations at three times within one day and inter-day variations for three consecutive days were chosen to determine the precision of the developed method. Six same samples of BZYQW were analyzed to confirm the repeatability of the method, and the RSD was taken as a measure of reproducibility. The same sample solution was stored at 48C and analyzed every 6 h over 2 days to investigate the stability of the solution. Its accuracy was also evaluated by a recovery test. The recovery of the 10 components were determined at middle levels with five replicates by comparing the peak areas from extracted samples with those in post-extracted samples spiked with the analytes at the same concentration. The SPE extraction recovery was calculated by comparing the peak area of the analyte spiked to solvent followed by sample extraction to that of the analyte spiked to the already extracted solvent. Results The DAD and ELSD chromatographic profiles of the blank, standard solution and the sample solution are showed in Figure 1 and Figure 2.
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I
Original concentration (mg/mL)
Quantity added (mg/mL)
Quantity found (mg/mL)
Recovery (%)
R.S.D. (%)
30.60 64.20 3.98 10.35 5.08
25.52 57.87 4.38 10.14 4.89
56.70 119.51 8.25 19.94 9.55
101.03 97.90 98.68 97.32 95.79
1.63 2.86 2.68 2.98 2.19
20.89 9.55 3.06 43.02 51.89
20.54 9.03 2.99 42.08 52.07
41.94 18.55 5.96 83.33 105.26
101.23 99.84 98.51 97.92 101.25
1.77 1.65 2.38 2.10 2.01
Linearity and limit of detection Standard stock solutions of the 10 components were prepared and diluted to appropriate concentrations for plotting the calibration curves. All calibration curves showed good linearity (r 2 . 0.9990) between the peak area and concentration within test ranges; the LODs (S/N ¼ 3) and the LOQs (S/N ¼ 10) for the eight components detected in DAD are ,0.65 and 1.92 mg/mL at 254 nm and ,11.76 and 39.22 mg/mL, respectively, for the four saponins in ELSD. All analytes were established to be stable. The results of regression analysis for each of the 10 compounds, together with the LOQ and LOD values, are shown in Table I. Precision and accuracy Table II shows the results of the precision tests of the 10 analytes and the repeatability of the method; it was shown that the RSD was lower than 3.45% both for the intra-assay and inter-assay precision. Besides, validation studies of this method proved that this assay has good repeatability with RSD in the range of 1.52– 3.54%. Table III shows that the developed analytical method has good accuracy with the overall recovery from 95.79 to 101.25% for the analytes concerned, and the RSD values were ,3.00%. Extraction efficiency The extraction efficiency determined for 10 analytes are shown in Table IV, which were all 90.99%, with relative standard deviation values 2.89%. Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 739
Table IV Determination of the Active Components in BZYQW by the Developed HPLC Method Content (mg/g)
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Batch 6
Batch 7
Batch 8
Batch 9
Batch 10
34.26 74.71 Nd 9.30 5.05 12.40 27.83 7.88 68.61 70.24 46.28 15.91
78.80 133.15 2.06 1.89 0.74 16.80 6.05 1.36 33.52 44.80 79.83 20.33
31.87 64.35 2.18 10.42 5.02 23.29 9.72 3.53 44.98 55.84 32.18 24.64
36.71 73.58 4.16 26.76 8.53 30.92 16.63 7.92 75.81 59.04 37.34 31.92
40.10 74.77 2.30 2.93 3.98 16.43 9.23 4.26 71.78 59.84 41.23 17.38
42.94 94.25 3.14 34.58 5.98 30.54 7.95 5.20 47.13 166.82 44.78 32.06
60.35 120.88 2.32 16.45 4.23 24.78 6.34 2.42 52.80 35.12 61.35 23.07
65.82 132.80 2.94 27.19 4.17 37.18 9.55 4.58 57.42 137.76 66.47 38.69
50.04 115.95 3.00 11.57 2.68 21.64 5.24 1.72 30.98 47.60 55.98 25.92
54.38 136.28 1.89 37.98 8.04 26.44 13.21 3.80 148.64 88.24 68.14 27.93
nd: not detected; E hesperidin: hesperidin detected in ELSD; E ononin: ononin detected in ELSD.
Table V Extract Recovery of 10 Constituents in BZYQW by SPE (n ¼ 5) Spiked (mg/mL)
Hesperidin Senkyunolide I Senkyunolide H Ligustilide Butylene phthalide Ononin Calycosin Formononetin Astragaloside IV Astragaloside I E hesperidin E ononin
32.60 65.20 4.10 10.65 5.22 21.89 9.85 3.46 45.02 54.89 30.88 25.09
Extract recovery Relative recovery (%)
RSD (%)
95.96 94.01 93.28 90.99 94.03 96.04 96.87 97.32 96.10 95.88 95.08 95.98
2.13 2.57 2.90 2.89 2.78 2.09 1.98 1.95 1.78 2.08 2.19 2.23
Sample determination The target compounds were identified by comparison of LC retention time and UV absorption with the reference compounds. The proposed HPLC method was applied to analyze the 10 compounds in 10 sample batches of BZYQW; each sample was analyzed two times. Table V shows the results of determination of the 10 compounds. Among the 10 compounds, senkyunolide I, astragaloside I, astragaloside IV and hesperidin are the most abundant, and the concentration of compound senkyunolide I is the highest in all samples except the sample 4, 6, 8, 10. In sample 4 and 10, the concentration of astragaloside IV is up to 75.81 and 148.64 mg/g, respectively. In sample 6 and 8, the concentration of astragaloside I is up to 166.82 and 137.76 mg/g, respectively.
impurities became more when extracted with methanol, so SPE was used to purify the sample, which gave rise to optimum extraction of all the 10 components with broad range of polarity in high yield. In order to investigate extraction time, powdered BZYQW were extracted with methanol for 0.5, 1, 1.5 h, respectively. The results suggested that all the 10 components were almost completely extracted by extracting with methanol two times for 0.5 h each. Among the detection of the compounds, flavonoids have response both in the DAD and in the ELSD. But for the low content of calycosin, within this range does not linearly standard of calibration curves in ELSD. Formononetin only has response in DAD for the low content. The concentration of hesperidin and ononin were both detected by DAD and ELSD: the results in Table V indicate that the concentrations are significantly different. The concentration of each compound obtained from ELSD is higher than that from DAD. Although the concentrations of hesperidin and ononin obtained from ELSD are higher, the peak areas of ELSD detection are lower than that of DAD detection, which indicates the poor sensitivity, and also because of the high detection limit of ELSD, DAD is commonly used than ELSD (27). It is obvious that DAD detection at 254 nm showed a very good sensitivity with significantly lower LODs and wider calibration ranges than that achieved by ELSD for the detection of hesperidin and ononin (marked E hesperidin and E ononin in ELSD). These data indicate that the developed method was precise, accurate and sensitive enough for the quantitation of the major constituents in BZYQW.
Conclusion Discussion In order to optimize the extraction conditions for achievement of quantitative extraction, variables involved in the procedure such as extraction method, solvent and extraction time were optimized. Compared with refluxing extraction and soxhlet extraction, the ultrasonic treatment procedure was found to be the better extraction method for all the 10 components. Pure and aqueous methanol or ethanol solutions were tried as the extraction solvent, the best solvent was found to be methanol. But 740 Hu et al.
In the present study, an accurate and reliable analytical method for the simultaneous determination of 10 major components in Chinese medicine BZYQW was developed. The method was developed using SPE-HPLC-DAD-ELSD, which makes it possible to comprehensively evaluate the quality of BZYQW. Good linearity, repeatability, intra-day and inter-day precision, accuracy and reliability were presented in the method validation procedure. The developed method has been successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW.
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Simultaneous Determination of 10 Components in BZYQW by SPE-HPLC-DAD-ELSD 741
