Chemical Fingerprint and Quantitative Analysis of Cirsium setosum by LC
2009, 70, 125–131
Yanhua Lu1,&, Wei Song1, Xinhua Liang1, Dongzhi Wei1, Xiaoli Zhou2 1
2
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Box 283#, 130 Meilong Road, 200237 Shanghai, People’s Republic of China; E-Mail:
[email protected] School of Perfume and Aroma Technology, Shanghai Institute of Technology, 200235 Shanghai, People’s Republic of China
Received: 28 October 2008 / Revised: 26 March 2009 / Accepted: 27 March 2009 Online publication: 28 April 2009
Introduction
Abstract A reverse phase liquid chromatography method with diode array detection was developed to evaluate the quality of Cirsium setosum through establishing chromatographic fingerprint and simultaneous determination of six phenolic compounds, namely chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin. The chromatographic separation was performed on an Agilent SB-C18 column (250 9 4.6 mm, 5.0 lm) with a gradient elution program using a mixture of acetonitrile and 0.5% aqueous acetic acid (v/v) as mobile phase within 25 min at 326 nm wavelength. The correlation coefficients of similarity were determined from the LC fingerprints, and they shared a close similarity. The LC with electrospray ionization mass spectrometry experiment was performed to further confirm the identity of phenolic compounds. The six phenolic compounds showed good regression (R2 > 0.9995) within test ranges and the recovery of the method was in the range of 95.8–102.8%. In addition, the content of those six phenolic compounds in C. setosum growing in different locations of China was determined to establish the effectiveness of the method. The results indicated that the developed method by having a combination of chromatographic fingerprint and quantification analysis could be readily utilized as a quality control method for C. setosum and its related traditional Chinese medicinal preparations.
Keywords Column liquid chromatography Fingerprint analysis Phenolic compounds Cirsium setosum
Original DOI: 10.1365/s10337-009-1114-z 0009-5893/09/07
The aerial parts of Cirsium setosum (Willd.) MB. (field thistle; Xiaoji in Chinese) have been acknowledged by the People’s Republic of China pharmacopoeia, and have been used as an antihemorrhagic and antiphlogistic drug in traditional Chinese herbal medicines for a long time [1]. Phytochemical studies on C. setosum have revealed the presence of phenolic acids, flavonoids, triterpenes [2–5]. Pharmacological investigation demonstrated that phenolic compounds could be main bioactive constituents of C. setosum [6, 7]. These components have shown various activities, such as anti-inflammatory [8–12], antioxidant [13, 14], anti-tumor [15, 16], antibacterial [17–19] and other biological activities. Hence, the six phenolic compounds: Chlorogenic acid, caffeic acid and the flavonoids including rutin, linarin, luteolin and apigenin were selected for analyzing and evaluation of C. setosum. The development of a quality control method is an essential issue for ensuring clinical efficacy of this Chinese herbal medicine. So far, there is no specific quantitative method to control the
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C. setosum by the authors’ laboratory and their chemical structures were confirmed by UV, IR, ESI-MS, 1H NMR and 13C NMR (the dates were not mentioned). The purity of each compound was determined to be higher than 98% by normalization of the peak area detected by LC.
Apparatus and Chromatographic Conditions
Fig. 1. The chemical structures of chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin
quality of C. setosum in the Chinese Pharmacopoeia [1], and only a few chromatographic methods have been documented for the determination of one or two components present in C. setosum by LC [2, 20–22]. To our knowledge, no method is available for the co-quantification of these six phenolic components by LC. With the increasing applications of C. setosum in food and as a medicinal herb, it is essential to establish an analytical method for quality control. The strategy applied was to establishing chromatographic fingerprinting profile and simultaneous determination of six phenolic compounds including chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin for the assessment of the quality of C. setosum by LC coupled with diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS). In addition, the content of those six phenolic compounds in eleven C. setosum samples growing in different parts of China was determined to establish the effectiveness of the method.
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Experimental Plant Material and Chemicals The aerial parts of C. setosum were used in our experiment. Commercial herbal samples were collected from local drug stores in different provinces in China. All the voucher specimens, identified by Professor Yan-hua Lu, were deposited at the Institute of Biochemistry, East China University of Science and Technology, Shanghai, 200237, China. LC-grade methanol and acetonitrile were purchased from Caledon Laboratories, Ontario, Canada. Redistilled water was prepared in our own lab. Glacial acetic acid was of analytical grade from Shanghai Chemical Co., Shanghai, China. All the solutions were filtered through 0.45 lm membranes (Schleicher & Schuell, Dassel, Germany) and degassed by an ultrasonic bath before use. The six compounds (chlorogenic acid, caffeic acid, rutin, linarin, luteolin, apigenin; Fig. 1) were previously isolated from the aerial parts of
Quantitative analysis was performed on an Agilent 1100 liquid chromatography system, equipped with a diode array detector working in the range of 190– 400 nm, a quaternary solvent delivery system, a column temperature controller and an autosampler. The chromatographic data was recorded and processed with Agilent Chromatographic Work Station software. Analysis was carried out at 30 °C on an Agilent Eclipse SB-C18 column (250 9 4.6 mm, 5 lm), which was protected by a guard column (12.5 9 4.6 mm, 5 lm). A linear gradient elution of eluents A (0.5% (v/v) aqueous glacial acetic acid) and B (acetonitrile) was used for the separation. The elution programme was optimized and conducted as follows: a linear gradient of 13–25% B with the first 10 min, a linear gradient of 25–40% B with the range of 10–20 min and a linear gradient of 40–90% B with the range of 20–25 min. This was followed by a 10 min equilibration period prior to the injection of each sample. The peaks were recorded using UV absorbance at 326 nm. The solvent flow rate was kept at 1.0 mL min-1 and the injection volume was 10 lL.
LC–MS Analysis A Finnigan LCQ Advantage ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) was connected to the Agilent 1100 LC instrument via ESI interface for LC–MS analysis. Ultrahigh pure helium (He) was used as the collision gas and high purity nitrogen (N2) as the nebulizing gas. The optimized parameters in the positive ion
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mode were as follows: ion spray voltage, 4.5 kV; sheath gas, 50 arbitrary units; auxiliary gas, 20 arbitrary units; capillary temperature, 320 °C; capillary voltage, 18 V; tube lens offset voltage, -16 V; collision energy, ranged from 35 to 50%. For full scan MS analysis, the spectra were recorded in the range of m/z 50–800. All data were preceded by Finnigan Xcalibur TM core data system Rev. 1.2 (ThermoQuest Corporation, San Jose, CA, USA).
Sample Preparation
Fig. 2. LC chromatogram of phenolic compounds from Cirsium setosum at 326 nm wavelength. The six insets are DAD UV scan of chlorogenic acid (a), caffeic acid (b), rutin (c), linarin (d), luteolin (e) and apigenin (f) peak (190–400 nm)
All the C. setosum samples were milled into powder and oven-dried at 55 °C until constant weight was reached. Approximately 1.0 g of powder of each dried sample was extracted with 20 mL 70% methanol in an ultrasonic bath for 1 h and then cooled at room temperature. The extract was filtered through glass wool for sample clean up and diluted to 25 mL with 70% methanol. The sample solution was filtered through a 0.45 lm micropore membrane prior to LC analysis.
Standard Preparation The reference standards of the six phenolic compounds (chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin) were accurately weighed and dissolved in methanol then diluted to appropriate concentration ranges for the establishment of calibration curves. All stock and working standard solutions were stored in brown bottles at 4 °C until used for analysis.
Validation Procedure The working solutions were all prepared as described above to construct calibration curves. Each calibration curve contained six different concentrations and was performed in triplicate. An aliquot (10 lL) of each standard working solution was subjected to LC analysis. The linearity for each compound was established by plotting the peak area (y) versus concentration (x) of each analyte. Original
Fig. 3. Efficiencies of extraction for the six analytes in Cirsium setosum using different extractants and extraction methods. Values are expressed as mean ± SD (n = 3)
Stock solution containing six reference compounds was diluted to a series of appropriate concentrations with methanol and an aliquot (10 lL) of the diluted solutions was injected into LC for analysis, the limit of detection (LOD) and limit of quantification (LOQ) for each analyte was calculated with corresponding standard solution on the basis of signal-to-noise ratio (S/N) of 3 and 10, respectively. Intra-day and inter-day variations were utilized to determine the precision of the developed assay. The intra-day variation was determined by analyzing the six replicate samples (1.0 g, Zhejiang province, 070420) within 1 day and inter-day variation was determined on three consecutive days. To confirm the repeatability, five different working solutions prepared from the same sample (1.0 g, Zhejiang province, 070420) were
analyzed. Variations were expressed as relative standard deviations. The recovery test was determined by standard addition method. Six phenolic components in a mixed standard solution were spiked into the samples (0.5 g, Zhejiang province, 070420), and then extracted, processed and quantified in accordance with the established procedures, and finally the recovery rates were calculated.
Data Analysis Similarity analysis was performed by professional the software Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A), which was recommended by SFDA (State Food and Drug Administration of China). The
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Fig. 4. The chromatographic fingerprints of eleven C. setosum samples. The reference fingerprint was developed with the mean of fusion vectors of all samples. The denotations from 1 to 14 are the corresponding chemicals as listed. Peak numbers: 4 = chlorogenic, 5 = caffeic acid, 6 = rutin, 11 = linarin, 12 = luteolin, 13 = apigenin. Sample numbers: S1 Zhejiang province (061207); S2 Shanghai (061223); S3 Shanghai (061115); S4 Anhui province (061210); S5 Jiangsu province (060802); S6 Shandong province (061205); S7 Shanxi province (070312); S8 Zhejiang province (070420); S9 Jiangsu province (070320); S10 Hebei province (070413); S11 Anhui province (070216); R Simulative mean chromatogram
software was to employ the correlative coefficient in evaluating the similarities of different chromatograms.
Results and Discussion Optimization of Chromatographic Conditions Several mobile phases, including methanol–water and acetonitrile–water in combination with glacial acetic acid, were tested. It was found that an acetonitrile–water system containing 0.5% glacial acetic acid (v/v) gave the best separation of the six phenolic compounds. Figure 2 demonstrates the separation obtained for a typical sample of
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the six phenolic compounds and the six insets are DAD UV scan of chlorogenic acid (a), caffeic acid (b), rutin (c), linarin (d), luteolin (e) and apigenin (f) peak (190–400 nm). The retention time of chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin was 5.61, 7.91, 10.41, 17.57, 19.28 and 22.51 min, respectively. It can be seen that good separation was achieved within 25 min using the conditions described.
Optimization of Extraction Procedure In order to obtain quantitative extraction, the variables involved in the procedure such as the extraction solvent, extraction
method and extraction time were optimized. The C. setosum samples (1.0 g, Zhejiang province, 070420) were extracted by ultrasonication for 1 h with 25 mL water, 30% methanol, 50% methanol, 70% methanol and 100% methanol to analyze the effects of the solvent on extraction efficiency. Pure water could not extract the flavonoids efficiently and methanol was unfavorable for the extraction of chlorogenic acid and caffeic acid. Comparing the extraction efficiency of the different solvents, it was found that 70% methanol was the most suitable extraction solvent. After the solvent was ascertained, the extraction methods such as refluxing for 3 h, ultrasonication for 1 h and soxhlet extraction for 24 h at room temperature were
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investigated. Ultrasonication was found to be the most suitable extraction method for extracting the phenolic components completely (Fig. 3). Finally, the effect of extraction time on the content of each analyte was investigated using 70% aqueous methanol as the solvent for 30, 60, 90 and 120 min, respectively. It was found that 60 min of sonication was sufficient to extract the analytes.
LC–UV Fingerprint Analysis To perform fingerprinting analysis, the chromatograms of different samples have to be standardized. The process of standardization included the selection of ‘‘common peaks’’ in chromatograms and the normalization of retention times of all the common peaks. Here, the extracts of eleven samples of C. setosum collected from different parts of China served as the sample set. The LC chromatographic fingerprints were shown in Fig. 4. The peak of linarin (11) at retention time 17.57 min indicated the highest content in all the eleven peaks. Therefore, it was selected as a reference peak. Among all the peaks observed, 14 of them (denoted from 1 to 14) were defined as common peaks because they showed up in all samples (Fig. 4). The relative retention time (RRT) and relative retention area (RPA) of these 14 peaks with respect to peak 11 in 11 samples were shown in Table 1. Based on the comparisons with standard compounds, six peaks were unambiguously identified chlorogenic acid (4), caffeic acid (5), rutin (6), linarin (11), luteolin (12) and apigenin (13). In addition, similarity analysis was performed for the eleven chosen C. setosum. The similarity indexes were calculated by mean fusion vector method. As listed in Table 2, the similarity index of ten samples was higher than 0.963, which suggested that the samples from different habitats shared the similar chromatographic patterns. Detailed analysis of the LC profile of each sample indicated that the common peak abundance varies from each other (data not shown), which could be caused by the difference of plant origin, the effect of environment, season of collection, drying process and storage conditions, etc. Original
Table 1. The relative retention time (RRT) and relative retention area (RPA) of these 14 peaks with respect to peak 11 in eleven C. setosum samples Peak no.
RRT
RSD (%)
RPA
RSD (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0.15 0.19 0.32 0.45 0.59 0.64 0.69 0.73 0.78 0.82 1.00 1.10 1.28 1.37
0.10 0.17 0.27 0.17 0.19 0.17 0.16 0.15 0.17 0.09 0.05 0.11 0.15 0.07
0.07 0.02 0.34 0.05 0.02 0.02 0.03 0.02 0.18 0.06 1.00 0.02 0.15 0.04
0.28 0.07 2.93 0.40 0.11 0.14 0.18 0.11 1.07 0.51 0.00 0.05 0.76 0.15
Identification of Six Phenolic Components from C. setosum The compounds in samples were confirmed by overlapping their spectra with those of the standards at the same retention time. Spiking sample with reference compounds performed a further confirmation assay. LC–ESI-MS experiments were performed to further confirm the identity of compounds. For ESI-MS analysis, positive ionization mode was used. Positive MS spectra were dominated by the [M + H]+ ion for six phenolic compounds (Table 3). The retention times and mass spectra of products exactly matched with the corresponding standard compounds, which are shown in Table 3.
Validation of the Method Results from the calibration curve, limits of detection and quantification for the six phenolic compounds are summarized in Table 4. All calibration curves showed good linear regression (R2 > 0.9995) within test ranges. The LODs (S/N = 3) and the LOQs (S/N = 10) for the analytes were less than 15 and 45 ng mL-1. Table 5 shows the results of the tests of precision and repeatability. It indicates that the RSD values of the overall intra-day and inter-day variations were less than 3.2% for the analytes. Besides, The RSD of repeatability test was not over 2.0% for all analytes interested. The results of recovery test are summarized in
Table 6. The average recoveries of the six phenolic compounds were 95.8– 102.8% and their RSD values were less than 3.2%. Therefore, the LC-DAD method is precise, accurate and sensitive enough for simultaneously quantitative evaluation of six active phenolic compounds in C. setosum.
Quantitative Determination of C. setosum The developed analytical method was successfully applied to the simultaneous determination of chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin in eleven samples of C. setosum, which were obtained from various provinces and cities in China (Fig. 4). Each sample was determined in triplicate. Peaks in the chromatograms were identified by comparing the retention times and on-line UV spectra with those of the standards. The LC-DAD profiles are illustrated in Fig. 4. Table 7 shows the content of the six phenolic compounds in eleven samples of C. setosum. It was found that the content of each analyte varied greatly among the different samples. In the majority of cases linarin was the main component, whose content varied from 1.92 to 4.32 mg g-1. Similar variation could also be found for the other phenolic components. The variation in content of constituents could certainly lead to the variation of therapeutic effects. Hence, each procedure involved should be standardized.
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Table 2. The similarities of eleven C. setosum samples (Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine, version 2004A) No.
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
Similarities
0.991
0.993
0.994
0.985
0.791
0.963
0.979
0.995
0.987
0.978
0.991
Calculated by the mean of fusion vectors of all samples as the reference fingerprint Table 3. Identity confirmation of the six phenolic components in C. setosum Retention time (min)
Identification
UV kmax (nm)
MS (m/z)
5.61 7.91 10.41 17.57 19.28 22.51
Chlorogenic acid Caffeic acid Rutin Linarin Luteolin Apigenin
222, 222, 258, 269, 252, 268,
355.2 181.0 611.2 593.3 287.4 271.4
306, 324 306, 324 361 326 344 338
([M ([M ([M ([M ([M ([M
+ + + + + +
H]+) H]+) H]+) H]+) H]+) H]+)
Table 4. Calibration curves, detection limits and quantification limits of the analytes (n = 5) Analyte
Calibration curve
Chlorogenic acid Caffeic acid Rutin Linarin Luteolin Apigenin
y y y y y y
= = = = = =
10.269x 24.658x 4.2626x 8.1807x 12.665x 22.231x
+ 4.3868 + 22.479 + 0.0033 + 8.3579 - 0.1987 + 12.647
Linear range (lg mL-1)
R2
LOD (ng mL-1)
LOQ (ng mL-1)
1.20–120 0.40–40 0.55–55 2.9–290 1.1–110 1.4–140
0.9997 0.9995 1.0000 0.9998 0.9999 0.9999
10 5 10 15 15 10
30 15 30 45 45 30
y peak area, x the concentration of each reference compound (lg mL-1), R2 correlation coefficient of regression equations, LOD limit of detection (S/N = 3), LOQ limit of quantification (S/N = 10)
Table 5. Analytical results of intra- and inter-day variability for the analytes in C. setosum Analyte
Chlorogenic acid Caffeic acid Rutin Linarin Luteolin Apigenin
Intra-day (n = 6)
Inter-day (n = 6)
Repeatability (n = 6)
Content (mg g-1)
RSD (%)
Content (mg g-1)
RSD (%)
Content (mg g-1)
RSD (%)
0.806 0.022 0.138 3.770 0.046 0.275
1.7 1.5 1.4 1.1 1.6 1.8
0.798 0.022 0.141 3.767 0.047 0.268
3.2 2.2 2.1 1.7 2.5 2.0
0.801 0.022 0.145 3.760 0.045 0.269
1.6 2.0 1.3 1.4 1.5 1.8
Table 6. Recovery of each analyte determined by standard addition method (n = 3) Analytes
Original (mg)
Spiked (mg)
Found (mg)
Recovery (%)
RSD (%)
Chlorogenic acid Caffeic acid Rutin Linarin Luteolin Apigenin
0.403
0.400
0.788
96.3
2.6
0.012 0.071 1.887 0.023 0.145
0.012 0.070 1.890 0.025 0.140
0.024 0.143 3.729 0.047 0.288
101.0 102.8 97.5 95.8 102.1
2.5 2.7 3.2 2.4 2.9
The data was present as average of three determinations Recovery (%) = 100 9 (amount found - original amount)/amount spiked
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Table 7. Content of each analyte in different C. setosum samples (n = 3) Samples
Chlorogenic acid (mg g-1)
Caffeic acid (mg g-1)
Rutin (mg g-1)
Linarin (mg g-1)
Luteolin (mg g-1)
Apigenin (mg g-1)
Zhejiang province (061207) Shanghai (061223) Shanghai (061115) Anhui province (061210) Jiangsu province (060802) Shandong province (061205) Shanxi province (070312) Zhejiang province (070420) Jiangsu province (070320) Hebei province (070413) Anhui province (070216)
0.510 0.702 0.924 0.701 1.977 0.700 0.946 0.803 0.422 0.271 0.490
0.028 0.016 0.020 0.014 0.090 0.017 0.027 0.022 0.013 0.011 0.070
0.100 0.119 0.135 0.107 0.207 0.126 0.099 0.140 0.091 0.058 0.131
3.584 4.315 3.216 3.340 1.920 3.135 3.338 3.771 3.291 2.724 2.891
0.038 0.043 0.039 0.037 0.023 0.056 0.021 0.046 0.036 0.029 0.050
0.208 0.240 0.221 0.132 0.050 0.335 0.062 0.277 0.117 0.062 0.223
Conclusions Combination of fingerprint with quantitative analysis of several marker compounds for quality control of traditional Chinese herbal medicines is definitely an improvement over the old methodology. The chromatographic fingerprint has predominance in showing the authenticity of this herb, while the quantification of several marker compounds can better reflect the quality of TCM. In the present study, a simple, accurate and reliable LC method was developed to evaluate the quality of C. setosum through establishing chromatographic fingerprint and simultaneous determination of six phenolic compounds, namely chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin. The LC–ESI-MS experiment was performed to further confirm the identity of peaks of samples. The results demonstrate that the developed method is accurate and reproducible and could be readily utilized as a suitable quality control method for the quantification of the aerial parts of C. setosum, derived extracts and phytomedicines. The results of the analysis on the eleven C. setosum samples suggested that the content of the six phenolic compounds varied significantly in the aerial parts of C. setosum from different locations of
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China. Therefore, the evaluation of data might be useful in quality assurance as well as for determination of adulteration of the crude drug.
Acknowledgment This work is supported by Shanghai Leading Academic Discipline Project (B505).
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