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J. Sep. Sci. 2014, 37, 2131–2137
Dao-quan Tang1,2 Zheng Li2 Xiang-lan Jiang1,2 Yin-jie Li2 Qian Du2 Dong-zhi Yang1,2 ∗ 1 Department
of Pharmaceutical Analysis, Xuzhou Medical College, Xuzhou, Jiangsu, China 2 Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou, Jiangsu, China Received April 1, 2014 Revised May 3, 2014 Accepted May 22, 2014
Research Article
Fingerprint analysis and multi-ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C18 column (50 × 2.1 mm id, 1.8 m) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi-ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets. Keywords: Chemical fingerprint / Quantitative analysis / Ultra high performance liquid chromatography / Xiaoyanlidan DOI 10.1002/jssc.201400362
1 Introduction Traditional Chinese medicine (TCM) played a significant role in the healthcare of Chinese people in ancient times and has become increasingly popular all over the world. Contrary to modern pharmacology that often focus on the single chemical entity aimed at a specific single target, TCM often refers to complex mixtures and one herb usually contains hundreds of chemically different components. Their curative effects are principally based on the synergic effect of their multitargeting and multi-ingredient preparations [1]. Consequently, quality control becomes a troublesome issue for crude drugs and their medical preparations. Therefore, the method that employs a few markers or pharmacologically active components to evaluate the quality and authenticity of the complex preparations is confronted with severe Correspondence: Dr. Dao-quan Tang, Department of Pharmaceutical Analysis, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China E-mail: [email protected] Fax: +86-516-83262136
Abbreviations: DAD, diode array detector; RPA, relative peak area; RRT, relative retention time; TCM, traditional Chinese medicine; XYLD, Xiaoyanlidan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
challenges [2] and better analytical strategies to assure their efficacy, safety, and consistency are in great demand. Currently, the chromatographic fingerprint technique plays an important role in the quality control of TCM, which can systematically characterize the constituents of samples and focus on the identification and assessment of the stability of the components [3]. Moreover, both the China Food and Drug Administration (CFDA) and European Medicines Agency (EMEA) have clearly denoted that appropriate fingerprint chromatograms should be applied to assess the quality consistency of botanical drug products. Although chromatographic fingerprint analysis can give an overall view of the characteristics of nearly all the components in TCM, it cannot reveal the possible content variation of each ingredient. Fortunately, quantitative analysis of active constituents as the most direct and important approach for quality control of TCM is complementary to fingerprint analysis [4–6]. Therefore, the combination of chromatographic fingerprint and quantification analysis of multi-ingredients can be used for better control of the quality of TCM products [7–9].
∗ Additional corresponding author: Dr. Dong-zhi Yang, E-mail: [email protected]
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Figure 1. Chemical structures of the seven compounds investigated.
HPLC is widely applied to analyze the chemical composition of TCM. HPLC-based quantitative methods are simple, stable, and durable [10]. The latest UHPLC technique is designed to undertake separations under the high pressure that results from a smaller particle size. This technology can take full advantages of chromatographic principles to perform separation using shorter columns or higher flow rates without loss of superior resolution and sensitivity [11]. With the strengths of highly efficient separation and shorter analysis time, UHPLC can meet the need for rapid, reproducible, and sensitive quantitative analysis of TCM [12]. Xiaoyanlidan (XYLD) tablets are a popular compound preparation of traditional Chinese herbs included in Chinese Pharmacopoeia (2010) [13], which consists of three medicinal herbs: Andrographis paniculata (sovereign drug), Linearstripe Rabdosia herb, and Picrasma quassioides, with therapeutic actions of expelling pathogenic heat, eliminating dampness, and facilitating bile excretion. This compound preparation is generally used in clinical practice for the treatment of bitter taste, hypochondriac pain, acute cholecystitis, and cholangitis caused by hepatochlic hygropyrexia [14]. Some chemicals, such as chlorogenic acid, caffeic acid, apigenin7-O-glucoside, nigakinone [15], andrographolide, neoandrographolide, and dehydroandrographolide are considered to be the main active ingredients in this formula. Chlorogenic acid, andrographolide, neoandrographolide, and dehydroandrographolide derived from Andrographis show physiological activities such as reducing fever, inflammation, ulcers, and swelling, and other protective effects [16–18]. At present, several methods have been developed for quantitative analysis of one or more chemical markers in XYLD tablets [19–21]. In the Chinese Pharmacopoeia, andrographolide and dehy-
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
droandrographolide are required to be identified using TLC and quantified by HPLC systems for the purpose of quality control. However, the methods used or reported in literature only contained one or two compounds, without consideration of other active ingredients. There are >130 pharmaceutical manufacturers that produce XYLD tablets in China. It is obvious that the current methods cannot produce a comprehensive evaluation about the quality of XYLD tablets, and there is an urgent demand to establish a more effective and reliable method for quality control. In the present study, for the first time, a combinative method was strategically established that uses UHPLC fingerprint and quantitative determination to assess the quality of XYLD tablets. Within a short analytical time, seven marker compounds including chlorogenic acid, caffeic acid, apigenin-7-O-glucoside, nigakinone, andrographolide, neoandrographolide, and dehydroandrographolide (chemical structures are shown in Fig. 1) were simultaneously separated and identified. Meanwhile, 36 common peaks were selected through fingerprint analysis. In addition, the amounts of the seven compounds in six samples from different manufacturers were compared.
2 Materials and methods 2.1 Chemicals and reagents Chlorogenic acid, apigenin-7-O-glucoside, andrographolide, neoandrographolide, and dehydroandrographolide were purchased from Sichuan Scvictory Biotech (Sichuan, China). Caffeic acid was obtained from Chengdu Must Biotech (Sichuan,
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J. Sep. Sci. 2014, 37, 2131–2137 Table 1. Sample information about the XYLD tablets used in the study
Sample no.
Batch no.
Manufacturer
A
120687
B C D E
111203 120602 L11K011 K1D006
F
111202
Guangdong Wannianqing Pharmaceutical Huizhou Daya Pharmaceutical Guangdong Xinfeng Pharmaceutical Guangdong Luofushan Sinopharm Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Guigang Guanfeng Pharmaceutical
China). Nigakinone was purchased from the Guangzhou University of Chinese Medicine (Guangdong, China). The purity of each standard was over 98% and suitable for HPLC determination. Six batches of commercial XYLD tablets were purchased from six Chinese manufacturers (Table 1). HPLCgrade acetonitrile was obtained from Fisher Scientific (USA). HPLC-grade formic acid was purchased from Mreda Technology (USA). Purified water was from a Milli-Q system (Millipore, Bedford, MA, USA). Other reagents were all of analytical grade. XYLD-A (Batch no. 120687) was selected to optimize chromatographic conditions and subsequent methodology.
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and acetonitrile (B) in gradient elution mode. The flow rate of the mobile phase was kept at 0.4 mL/min and the gradient of acetonitrile (B) was changed as follows: 0–5 min, 7%; 5–8 min, 7–14%; 8–16 min, 14–15%; 16–24 min, 15–18%; 24–35 min, 18–27%; 35–43 min, 27–28%; 43–47 min, 28–39%; 47– 48 min, 39–42%; 48–50 min, 42%; 50–53 min, 42–60%; 53–58 min, 60–90%; and 58–63 min, 90%. The effluents from the column were detected by the DAD where the detection wavelength was, respectively, set at 254 or 220 nm according to the absorption properties of the analyzed compounds. During fingerprint analysis, the wavelength was set at 254 nm for exhibiting the vast majority of chromatographic peaks. The column temperature was kept at 30⬚C, and the sample injection volume was 1 L.
2.3 Sample pretreatment XYLD tablets were ground into powder after their coating film was scraped off, and 1.0 g of the powder was extracted using ultrasonication with 25 mL methanol/water (70:30, v/v) for 30 min. The supernatant was filtered through a 0.22 m nylon filter membrane, and 1 L filtrate was injected into the UHPLC system.
2.2 Apparatus and chromatographic conditions
2.4 Preparation of standard solutions
Analyses were performed on an Agilent 1290 series UHPLC system consisting of a binary pump, an online degasser, an auto plate sampler, a column oven, and a diode array detector (DAD). All separation steps were carried out on an Agilent Eclipse plus C18 column (50 × 2.1 mm id, 1.8 m). The mobile phase was composed of 1% formic acid aqueous solution (A)
Stock solutions were prepared from the above seven standard chemicals, where appropriate amounts of the chemicals were dissolved in methanol and stored at 4⬚C before use. Working standard solutions were prepared by stepwise dilution of corresponding stock solutions with methanol/water (50:50, v/v) to the concentrations within the calibration range. The
Figure 2. Representative UHPLC–DAD chromatograms of mixed standard solutions and sample solution of XYLD tablets at 220, 254, 280, and 320 nm (3, chlorogenic acid; 4, caffeic acid; 12, apigenin-7-O-glucoside; 16, nigakinone; 17, andrographolide; 25, neoandrographolide; and 27, dehydroandrographolide).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 2. Calibration plots, LOD, and LOQ for the analyzed compounds
Compound
Linearity range (g/mL)
Calibration equation
r
LOD (g/mL)
LOQ (g/mL)
Chlorogenic acid Caffeic acid Apigenin-7-O-glucoside Nigakinone Andrographolide Neoandrographolide Dehydroandrographolide
8.00–800.00 3.19–318.95 8.88–888.00 7.80–780.00 10.30–1030.00 14.90–1490.00 19.55–1955.00
y = 4.28 x + 6.87 y = 8.90 x +15.04 y =8.96 x + 85.67 y = 20.51 x + 28.27 y = 2.70 x − 16.70 y = 3.64 x + 10.69 y = 10.28 x − 66.00
0.9992 0.9995 0.9991 0.9990 0.9995 0.9990 0.9996
0.32 0.13 0.49 0.20 0.38 0.21 0.14
0.83 0.28 1.42 0.57 1.21 0.72 0.39
Figure 3. Representative UHPLC chromatograms for fingerprint analysis of XYLD tablets.
standard solutions were filtered through 0.22 m membrane prior to analysis.
2.5 UHPLC fingerprint analysis Chromatographic fingerprint data were analyzed by the professional software Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A), which is recommended by CFDA. The correlation coefficient of the samples was calculated and similarity comparison was performed between individual chromatogram and the average chromatogram from the samples tested.
3 Results and discussion 3.1 Optimization of chromatographic conditions Several UHPLC analytical parameters including separation column, mobile phase, its elution mode and flow rate, and column temperature were optimized in order to provide suf C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ficient information about the analyzed compounds, and to achieve good resolution and reasonable analysis time. Different types of columns were tested to reach an optimum separation on the Agilent Eclipse plus C18 column. After several trials using different mobile phases including water and methanol, 0.1% formic acid water solution and methanol, 0.1% formic acid water solution and 0.1% formic acid acetonitrile solution, 0.1% formic acid water solution and acetonitrile, 0.1% acetic acid water solution and 0.1% acetic acid acetonitrile solution, 0.2% formic acid water solution and acetonitrile, and ammonium acetate water solution (pH 4.0) and acetonitrile, 0.1% formic acid water solution and acetonitrile was determined as the most appropriate eluent with a step linear gradient due to the satisfactory resolution and acceptable peak parameters it provided. The effects of temperature and flow rate were studied and 30⬚C and 0.4 mL/min were found to be optimal parameters. By reference to the absorption maxima of reference compounds in their UV spectra, different wavelengths were used for sensitive quantitative analysis of seven compounds (220 nm for neoandrographolide and 254 nm for the other six). The typical chromatograms of the compounds detected
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J. Sep. Sci. 2014, 37, 2131–2137
at 220, 254, 280, and 320 nm are shown in Fig. 2. Compared with other wavelengths, the number and area of the peaks recorded at 254 nm were larger with stable baseline. Therefore, this wavelength was adopted to obtain chromatographic fingerprint profiles.
3.2 Optimization of the extraction procedure A series of factors, including extraction method, extraction solvent, and extraction time were investigated in order to seek the most efficient extraction procedure with the highest yields of the above seven compounds. A number of extraction methods such as reflux and ultrasonic extraction were compared in this study, and it was found that ultrasonic extraction was the preferred method for its higher extraction efficiency. Solvents, including water, different concentrations (30, 50, 70, 90, and 100%) of methanol or ethanol, together with extraction duration (10, 20, 30, and 40 min) were tested. Depending on the peak areas of these seven compounds, 70% methanol (30 min) was adequate and appropriate for the extraction.
3.3 Method validation of quantitative analysis A series of standard solutions comprising of seven compounds were freshly prepared to determine the linear range of quantitative analysis using the external standard method. The results of calibration were summarized in Table 2 and good correlations were found between the peak area (y) and the concentration of the tested compounds (x; r > 0.9990). LOD and LOQ, which were expressed as three- and tenfold of the ratio of the S/N, were also acquired. The LOD and LOQ values of individual compounds clearly indicated that the analytical method was acceptable with excellent sensitivity. Precision was evaluated according to the interday and intraday variability. The intraday precision was validated using low, medium, and high concentrations of mixed standard solutions under the optimum conditions three times a day. For interday precision, measurements were conducted three times a day on three consecutive days. The RSD values of the peak areas of the seven markers were found within the range of 0.1–1.6% in the intraday precision test. Similar results were obtained in interday precision assay with the range of 0.3–1.9%. The recovery was determined by accurately adding three different amounts (high, middle, and low) of the corresponding standard compounds to an XYLD tablet sample (sample A). The average recoveries were estimated according to the following formula: recovery (%) = [(amount found–original amount)/amount spiked] × 100%. The recoveries were between 95.9 and 104.3% with a RSD value of 90% of the whole area in one chromatogram. As was shown in Fig. 3, the chromatograms of XYLD tablets contained 36 distinct common peaks within 63 min. By reference to standard compounds, the seven peaks were unambiguously identified as chlorogenic acid (3), caffeic acid (44), apigenin-7-O-glucoside (1212), nigakinone (1616), andrographolide (1717), neoandrographolide (25), and dehydroandrographolide (27). www.jss-journal.com
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Table 4. Similarities of the chromatograms of six samples
Sample no.
A
B
C
D
E
F
Similarities
0.925
0.991
0.994
0.978
0.978
0.972
stability and reproducibility of the fingerprint analysis by UHPLC–DAD. The chromatographic fingerprints of XYLD tablet samples from six manufacturers were also performed. The similarity index was >0.9 (Table 4), which indicated that the XYLD tablets from different manufacturers shared a similar chromatographic pattern. However, the high RSD values of RPA indicated a wide variation in the contents of constituents, which was attributable to a number of factors, such as different origins, production process and storage conditions, and alternative environments.
3.5 Simultaneous quantification of the seven constituents in XYLD tablets The established method was applied to simultaneous determination of the seven active compounds in six commercial samples obtained from different pharmaceutical companies in China. All the contents were summarized in Table 5 and Fig. 4. The contents of andrographolide, neoandrographolide, and dehydroandrographolide derived from A. paniculata were high among the selected constituents in the six samples of XYLD tablets. There was a wide variation in the contents of the seven markers in XYLD tablet products used. The content of nigakinone derived from P. quassioides was as high as 1.95 mg/g in the products of major brands such as Wannianqing while the substance was too little to be quantified in the products of Daya, Xinfeng, and Guanfeng. Similarly, the content of apigenin-7-O-glucoside, which is one of the active ingredients of Linearstripe Rabdosia herb in the products of Wannianqing, was the highest. However, the total andrographolides including andrographolide, neoandrographolide, and dehydroandrographolide in the products of Wannianqing were the least among the six brands. So, detecting a single or only several components could not effectively control the quality of XYLD tablets. Accordingly, the combination of chromatographic fingerprint and simultaneous determination of multiple ingredients is essential.
Figure 4. The diagram of the contents of the seven targets in XYLD tablets from six companies (3, chlorogenic acid; 4, caffeic acid; 12, apigenin-7-O-glucoside; 16, nigakinone; 17, andrographolide; 25, neoandrographolide; and 27, dehydroandrographolide).
Peak 27 (dehydroandrographolide) indicated the highest content all the peaks. Therefore, it was selected as a reference peak to calculate the relative retention time (RRT) and relative peak area (RPA) of all common peaks according to the following formulas: RRT = RTpeak /RTpeak27 and RPA = PApeak /PApeak27 . The RRT and RPA of the common peaks in the eleven samples were shown in Table 3. The RSD values of the RRT were
J. Sep. Sci. 2014, 37, 2131–2137
Dao-quan Tang1,2 Zheng Li2 Xiang-lan Jiang1,2 Yin-jie Li2 Qian Du2 Dong-zhi Yang1,2 ∗ 1 Department
of Pharmaceutical Analysis, Xuzhou Medical College, Xuzhou, Jiangsu, China 2 Key Laboratory of New Drug and Clinical Application, Xuzhou Medical College, Xuzhou, Jiangsu, China Received April 1, 2014 Revised May 3, 2014 Accepted May 22, 2014
Research Article
Fingerprint analysis and multi-ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C18 column (50 × 2.1 mm id, 1.8 m) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi-ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets. Keywords: Chemical fingerprint / Quantitative analysis / Ultra high performance liquid chromatography / Xiaoyanlidan DOI 10.1002/jssc.201400362
1 Introduction Traditional Chinese medicine (TCM) played a significant role in the healthcare of Chinese people in ancient times and has become increasingly popular all over the world. Contrary to modern pharmacology that often focus on the single chemical entity aimed at a specific single target, TCM often refers to complex mixtures and one herb usually contains hundreds of chemically different components. Their curative effects are principally based on the synergic effect of their multitargeting and multi-ingredient preparations [1]. Consequently, quality control becomes a troublesome issue for crude drugs and their medical preparations. Therefore, the method that employs a few markers or pharmacologically active components to evaluate the quality and authenticity of the complex preparations is confronted with severe Correspondence: Dr. Dao-quan Tang, Department of Pharmaceutical Analysis, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China E-mail: [email protected] Fax: +86-516-83262136
Abbreviations: DAD, diode array detector; RPA, relative peak area; RRT, relative retention time; TCM, traditional Chinese medicine; XYLD, Xiaoyanlidan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
challenges [2] and better analytical strategies to assure their efficacy, safety, and consistency are in great demand. Currently, the chromatographic fingerprint technique plays an important role in the quality control of TCM, which can systematically characterize the constituents of samples and focus on the identification and assessment of the stability of the components [3]. Moreover, both the China Food and Drug Administration (CFDA) and European Medicines Agency (EMEA) have clearly denoted that appropriate fingerprint chromatograms should be applied to assess the quality consistency of botanical drug products. Although chromatographic fingerprint analysis can give an overall view of the characteristics of nearly all the components in TCM, it cannot reveal the possible content variation of each ingredient. Fortunately, quantitative analysis of active constituents as the most direct and important approach for quality control of TCM is complementary to fingerprint analysis [4–6]. Therefore, the combination of chromatographic fingerprint and quantification analysis of multi-ingredients can be used for better control of the quality of TCM products [7–9].
∗ Additional corresponding author: Dr. Dong-zhi Yang, E-mail: [email protected]
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Figure 1. Chemical structures of the seven compounds investigated.
HPLC is widely applied to analyze the chemical composition of TCM. HPLC-based quantitative methods are simple, stable, and durable [10]. The latest UHPLC technique is designed to undertake separations under the high pressure that results from a smaller particle size. This technology can take full advantages of chromatographic principles to perform separation using shorter columns or higher flow rates without loss of superior resolution and sensitivity [11]. With the strengths of highly efficient separation and shorter analysis time, UHPLC can meet the need for rapid, reproducible, and sensitive quantitative analysis of TCM [12]. Xiaoyanlidan (XYLD) tablets are a popular compound preparation of traditional Chinese herbs included in Chinese Pharmacopoeia (2010) [13], which consists of three medicinal herbs: Andrographis paniculata (sovereign drug), Linearstripe Rabdosia herb, and Picrasma quassioides, with therapeutic actions of expelling pathogenic heat, eliminating dampness, and facilitating bile excretion. This compound preparation is generally used in clinical practice for the treatment of bitter taste, hypochondriac pain, acute cholecystitis, and cholangitis caused by hepatochlic hygropyrexia [14]. Some chemicals, such as chlorogenic acid, caffeic acid, apigenin7-O-glucoside, nigakinone [15], andrographolide, neoandrographolide, and dehydroandrographolide are considered to be the main active ingredients in this formula. Chlorogenic acid, andrographolide, neoandrographolide, and dehydroandrographolide derived from Andrographis show physiological activities such as reducing fever, inflammation, ulcers, and swelling, and other protective effects [16–18]. At present, several methods have been developed for quantitative analysis of one or more chemical markers in XYLD tablets [19–21]. In the Chinese Pharmacopoeia, andrographolide and dehy-
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
droandrographolide are required to be identified using TLC and quantified by HPLC systems for the purpose of quality control. However, the methods used or reported in literature only contained one or two compounds, without consideration of other active ingredients. There are >130 pharmaceutical manufacturers that produce XYLD tablets in China. It is obvious that the current methods cannot produce a comprehensive evaluation about the quality of XYLD tablets, and there is an urgent demand to establish a more effective and reliable method for quality control. In the present study, for the first time, a combinative method was strategically established that uses UHPLC fingerprint and quantitative determination to assess the quality of XYLD tablets. Within a short analytical time, seven marker compounds including chlorogenic acid, caffeic acid, apigenin-7-O-glucoside, nigakinone, andrographolide, neoandrographolide, and dehydroandrographolide (chemical structures are shown in Fig. 1) were simultaneously separated and identified. Meanwhile, 36 common peaks were selected through fingerprint analysis. In addition, the amounts of the seven compounds in six samples from different manufacturers were compared.
2 Materials and methods 2.1 Chemicals and reagents Chlorogenic acid, apigenin-7-O-glucoside, andrographolide, neoandrographolide, and dehydroandrographolide were purchased from Sichuan Scvictory Biotech (Sichuan, China). Caffeic acid was obtained from Chengdu Must Biotech (Sichuan,
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J. Sep. Sci. 2014, 37, 2131–2137 Table 1. Sample information about the XYLD tablets used in the study
Sample no.
Batch no.
Manufacturer
A
120687
B C D E
111203 120602 L11K011 K1D006
F
111202
Guangdong Wannianqing Pharmaceutical Huizhou Daya Pharmaceutical Guangdong Xinfeng Pharmaceutical Guangdong Luofushan Sinopharm Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Guigang Guanfeng Pharmaceutical
China). Nigakinone was purchased from the Guangzhou University of Chinese Medicine (Guangdong, China). The purity of each standard was over 98% and suitable for HPLC determination. Six batches of commercial XYLD tablets were purchased from six Chinese manufacturers (Table 1). HPLCgrade acetonitrile was obtained from Fisher Scientific (USA). HPLC-grade formic acid was purchased from Mreda Technology (USA). Purified water was from a Milli-Q system (Millipore, Bedford, MA, USA). Other reagents were all of analytical grade. XYLD-A (Batch no. 120687) was selected to optimize chromatographic conditions and subsequent methodology.
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and acetonitrile (B) in gradient elution mode. The flow rate of the mobile phase was kept at 0.4 mL/min and the gradient of acetonitrile (B) was changed as follows: 0–5 min, 7%; 5–8 min, 7–14%; 8–16 min, 14–15%; 16–24 min, 15–18%; 24–35 min, 18–27%; 35–43 min, 27–28%; 43–47 min, 28–39%; 47– 48 min, 39–42%; 48–50 min, 42%; 50–53 min, 42–60%; 53–58 min, 60–90%; and 58–63 min, 90%. The effluents from the column were detected by the DAD where the detection wavelength was, respectively, set at 254 or 220 nm according to the absorption properties of the analyzed compounds. During fingerprint analysis, the wavelength was set at 254 nm for exhibiting the vast majority of chromatographic peaks. The column temperature was kept at 30⬚C, and the sample injection volume was 1 L.
2.3 Sample pretreatment XYLD tablets were ground into powder after their coating film was scraped off, and 1.0 g of the powder was extracted using ultrasonication with 25 mL methanol/water (70:30, v/v) for 30 min. The supernatant was filtered through a 0.22 m nylon filter membrane, and 1 L filtrate was injected into the UHPLC system.
2.2 Apparatus and chromatographic conditions
2.4 Preparation of standard solutions
Analyses were performed on an Agilent 1290 series UHPLC system consisting of a binary pump, an online degasser, an auto plate sampler, a column oven, and a diode array detector (DAD). All separation steps were carried out on an Agilent Eclipse plus C18 column (50 × 2.1 mm id, 1.8 m). The mobile phase was composed of 1% formic acid aqueous solution (A)
Stock solutions were prepared from the above seven standard chemicals, where appropriate amounts of the chemicals were dissolved in methanol and stored at 4⬚C before use. Working standard solutions were prepared by stepwise dilution of corresponding stock solutions with methanol/water (50:50, v/v) to the concentrations within the calibration range. The
Figure 2. Representative UHPLC–DAD chromatograms of mixed standard solutions and sample solution of XYLD tablets at 220, 254, 280, and 320 nm (3, chlorogenic acid; 4, caffeic acid; 12, apigenin-7-O-glucoside; 16, nigakinone; 17, andrographolide; 25, neoandrographolide; and 27, dehydroandrographolide).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 2. Calibration plots, LOD, and LOQ for the analyzed compounds
Compound
Linearity range (g/mL)
Calibration equation
r
LOD (g/mL)
LOQ (g/mL)
Chlorogenic acid Caffeic acid Apigenin-7-O-glucoside Nigakinone Andrographolide Neoandrographolide Dehydroandrographolide
8.00–800.00 3.19–318.95 8.88–888.00 7.80–780.00 10.30–1030.00 14.90–1490.00 19.55–1955.00
y = 4.28 x + 6.87 y = 8.90 x +15.04 y =8.96 x + 85.67 y = 20.51 x + 28.27 y = 2.70 x − 16.70 y = 3.64 x + 10.69 y = 10.28 x − 66.00
0.9992 0.9995 0.9991 0.9990 0.9995 0.9990 0.9996
0.32 0.13 0.49 0.20 0.38 0.21 0.14
0.83 0.28 1.42 0.57 1.21 0.72 0.39
Figure 3. Representative UHPLC chromatograms for fingerprint analysis of XYLD tablets.
standard solutions were filtered through 0.22 m membrane prior to analysis.
2.5 UHPLC fingerprint analysis Chromatographic fingerprint data were analyzed by the professional software Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A), which is recommended by CFDA. The correlation coefficient of the samples was calculated and similarity comparison was performed between individual chromatogram and the average chromatogram from the samples tested.
3 Results and discussion 3.1 Optimization of chromatographic conditions Several UHPLC analytical parameters including separation column, mobile phase, its elution mode and flow rate, and column temperature were optimized in order to provide suf C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ficient information about the analyzed compounds, and to achieve good resolution and reasonable analysis time. Different types of columns were tested to reach an optimum separation on the Agilent Eclipse plus C18 column. After several trials using different mobile phases including water and methanol, 0.1% formic acid water solution and methanol, 0.1% formic acid water solution and 0.1% formic acid acetonitrile solution, 0.1% formic acid water solution and acetonitrile, 0.1% acetic acid water solution and 0.1% acetic acid acetonitrile solution, 0.2% formic acid water solution and acetonitrile, and ammonium acetate water solution (pH 4.0) and acetonitrile, 0.1% formic acid water solution and acetonitrile was determined as the most appropriate eluent with a step linear gradient due to the satisfactory resolution and acceptable peak parameters it provided. The effects of temperature and flow rate were studied and 30⬚C and 0.4 mL/min were found to be optimal parameters. By reference to the absorption maxima of reference compounds in their UV spectra, different wavelengths were used for sensitive quantitative analysis of seven compounds (220 nm for neoandrographolide and 254 nm for the other six). The typical chromatograms of the compounds detected
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J. Sep. Sci. 2014, 37, 2131–2137
at 220, 254, 280, and 320 nm are shown in Fig. 2. Compared with other wavelengths, the number and area of the peaks recorded at 254 nm were larger with stable baseline. Therefore, this wavelength was adopted to obtain chromatographic fingerprint profiles.
3.2 Optimization of the extraction procedure A series of factors, including extraction method, extraction solvent, and extraction time were investigated in order to seek the most efficient extraction procedure with the highest yields of the above seven compounds. A number of extraction methods such as reflux and ultrasonic extraction were compared in this study, and it was found that ultrasonic extraction was the preferred method for its higher extraction efficiency. Solvents, including water, different concentrations (30, 50, 70, 90, and 100%) of methanol or ethanol, together with extraction duration (10, 20, 30, and 40 min) were tested. Depending on the peak areas of these seven compounds, 70% methanol (30 min) was adequate and appropriate for the extraction.
3.3 Method validation of quantitative analysis A series of standard solutions comprising of seven compounds were freshly prepared to determine the linear range of quantitative analysis using the external standard method. The results of calibration were summarized in Table 2 and good correlations were found between the peak area (y) and the concentration of the tested compounds (x; r > 0.9990). LOD and LOQ, which were expressed as three- and tenfold of the ratio of the S/N, were also acquired. The LOD and LOQ values of individual compounds clearly indicated that the analytical method was acceptable with excellent sensitivity. Precision was evaluated according to the interday and intraday variability. The intraday precision was validated using low, medium, and high concentrations of mixed standard solutions under the optimum conditions three times a day. For interday precision, measurements were conducted three times a day on three consecutive days. The RSD values of the peak areas of the seven markers were found within the range of 0.1–1.6% in the intraday precision test. Similar results were obtained in interday precision assay with the range of 0.3–1.9%. The recovery was determined by accurately adding three different amounts (high, middle, and low) of the corresponding standard compounds to an XYLD tablet sample (sample A). The average recoveries were estimated according to the following formula: recovery (%) = [(amount found–original amount)/amount spiked] × 100%. The recoveries were between 95.9 and 104.3% with a RSD value of 90% of the whole area in one chromatogram. As was shown in Fig. 3, the chromatograms of XYLD tablets contained 36 distinct common peaks within 63 min. By reference to standard compounds, the seven peaks were unambiguously identified as chlorogenic acid (3), caffeic acid (44), apigenin-7-O-glucoside (1212), nigakinone (1616), andrographolide (1717), neoandrographolide (25), and dehydroandrographolide (27). www.jss-journal.com
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Table 4. Similarities of the chromatograms of six samples
Sample no.
A
B
C
D
E
F
Similarities
0.925
0.991
0.994
0.978
0.978
0.972
stability and reproducibility of the fingerprint analysis by UHPLC–DAD. The chromatographic fingerprints of XYLD tablet samples from six manufacturers were also performed. The similarity index was >0.9 (Table 4), which indicated that the XYLD tablets from different manufacturers shared a similar chromatographic pattern. However, the high RSD values of RPA indicated a wide variation in the contents of constituents, which was attributable to a number of factors, such as different origins, production process and storage conditions, and alternative environments.
3.5 Simultaneous quantification of the seven constituents in XYLD tablets The established method was applied to simultaneous determination of the seven active compounds in six commercial samples obtained from different pharmaceutical companies in China. All the contents were summarized in Table 5 and Fig. 4. The contents of andrographolide, neoandrographolide, and dehydroandrographolide derived from A. paniculata were high among the selected constituents in the six samples of XYLD tablets. There was a wide variation in the contents of the seven markers in XYLD tablet products used. The content of nigakinone derived from P. quassioides was as high as 1.95 mg/g in the products of major brands such as Wannianqing while the substance was too little to be quantified in the products of Daya, Xinfeng, and Guanfeng. Similarly, the content of apigenin-7-O-glucoside, which is one of the active ingredients of Linearstripe Rabdosia herb in the products of Wannianqing, was the highest. However, the total andrographolides including andrographolide, neoandrographolide, and dehydroandrographolide in the products of Wannianqing were the least among the six brands. So, detecting a single or only several components could not effectively control the quality of XYLD tablets. Accordingly, the combination of chromatographic fingerprint and simultaneous determination of multiple ingredients is essential.
Figure 4. The diagram of the contents of the seven targets in XYLD tablets from six companies (3, chlorogenic acid; 4, caffeic acid; 12, apigenin-7-O-glucoside; 16, nigakinone; 17, andrographolide; 25, neoandrographolide; and 27, dehydroandrographolide).
Peak 27 (dehydroandrographolide) indicated the highest content all the peaks. Therefore, it was selected as a reference peak to calculate the relative retention time (RRT) and relative peak area (RPA) of all common peaks according to the following formulas: RRT = RTpeak /RTpeak27 and RPA = PApeak /PApeak27 . The RRT and RPA of the common peaks in the eleven samples were shown in Table 3. The RSD values of the RRT were
