Abstract
An easy, quick, and sensitive approach adopting ultra-performance liquid chromatography (UPLC) equipped with diode array detector was used to analyze and systematically evaluate the quality of Pudilan tablets manufactured by 12 distinct pharmaceutical companies. In this research, 15 peaks were chosen as the common peaks to assess the similarities for different batches (S1–S43) of Pudilan tablet samples. In comparison with the control fingerprint, similarity values for 43 batches of samples exceeded 0.922. In addition, by analyzing the reference substances of epigoitrin, caffeic acid, chlorogenic acid, acetylcorynoline, baicalin and baicanshialein, the chromatogram of the 6 reference substances was established. The recoveries for the reference substances which demonstrated good regression in the linear range (r2 > 0.999) were in the range of 98.3–101.1%. The results demonstrated that the established method was highly accurate, efficient and reliable. This study provides a valid, dependable and pragmatic method to evaluate the quality of Pudilan tablet.
Introduction
Upper respiratory tract infection (URI) is a series of acute inflammatory diseases featured by mumps, lymphadenitis and tonsillitis, all of which ultimately result in the damage to lungs [1, 2]. Lymphadenitis often leads to a tender and sometimes painful swelling. Mumps primarily induces swelling of the parotid gland. Its common complications include aseptic meningitis and encephalitis, and in adults, it can cause orchitis or oophoritis [3]. Systemic complications of mumps include follicular hyperplasia and sinus hyperplasia [4]. Tonsillitis is mainly characterized by tonsillar swelling and pain with its complication including peritonsillar, parapharyngeal or retropharyngeal abscesses [5]. Pudilan preparation is composed of four traditional Chinese herbs, ie Scutellaria baicalensis Georgi, Taraxacum mongolicum Hand. – Mazz., Corydalis bungeana Turcz., Isatis indigotica Fort., and has been used to cure URI for more than 30 years since the formula was publicized by Ministry of Health of China in 1991. Clinical researches confirmed that Pudilan tablet, a typical traditional Chinese medicine (TCM), showed significant efficacy and few side effects in treating URI [6].
TCM has been used in China for thousands of years and is being recognized and accepted by more and more people in the world. Compared with western medicine that always pays more attention to a precise single target, TCM usually refers to complicated compounds and one herb often consists of more than hundreds or thousands of chemically distinct compositions. The efficacy of TCM is principally based on the synergic effect of their multi-targeting, multi-ingredient preparations [6]. Nevertheless, the difficult issue of TCM quality control is a barrier which has confined development of TCM and hampers its business recognition around the whole world. Therefore, it has great significance for TCM to establish a scientific and reasonable system for quality control. The technology of TCM chromatographic fingerprinting is playing a very important role in the quality assessment of TCM, and has proven to be scientific and comprehensive [7].
Lately, the chromatographic fingerprint technology has been introduced as a tool to assess the quality of herbal specimen or their associated products [8]. This technique has been applied by the World Health Organization (WHO), the State Food and Drug Administration of China (SFDA) as a strategic plan for the quality assessment of TCM [9, 10], and got increasing concerns because it focuses on the characterization of the total sample composition. Several ways, for instance, high-performance thin layer chromatography (HPTLC) [11–14], thin-layer chromatography (TLC) [15–17], infrared spectroscopy (IR) [18–20], mass spectrometry (MS) [21, 22], gas chromatography (GC) [23, 24], high-performance liquid chromatography (HPLC) [25–27], nuclear magnetic resonance (NMR) [28–31] and high-performance liquid chromatography (UPLC) [32–34] are utilized for fingerprinting to characterize multiple compounds in the tested herbal samples. Among the above-mentioned methods, HPLC is generally the most applied method for its high sensitivity, selectivity, and low cost [35]. As a further development of HPLC, UPLC shows better foregrounds than HPLC for its better chromatographic resolution, more sensitive performance, with considerable cost effectiveness [36].
Pudilan was recorded in the book Drug Standard of the Ministry of Health of China – Prepared Chinese Medicine which incorporated the preparation of Pudilan into the national medicine standard. In order to make 2,660 Pudilan tablets, 450 g root powder of S. baicalensis, 1,200 g powdered whole plant of T. mongolicum, 300 g powdered whole plant of C. bungeana and 450 g powdered root of I. indigotica are used for extraction and preparation along with designated accessories including starch and dextrin. For the final product, each tablet should contain more than 3.5 mg baicalin. To our best knowledge, there are 25 pharmaceutical companies in China that are currently producing Pudilan. Despite a number of articles describing the quality control of Pudilan preparation, such studies often focused on the concentrations of characteristic compounds in Pudilan products manufactured by individual pharmaceutical companies. Comprehensive researches on Pudilan sampled from multiple producers are needed to gain a systematic evaluation regarding its quality control. In this study, 43 batches collected from 12 pharmaceutical companies were collected and analyzed using six bioactive constituents (epigoitrin, caffeic acid, chlorogenic acid, acetylcorynoline, baicalin and baicalein) as the phytochemical markers. The results demonstrated that the UPLC-DAD approach for the fingerprint identification of Pudilan is practical, quick, and sensitive.
Experimental
Materials and reagents
Epigoitrin, caffeic acid, chlorogenic acid, acetylcorynoline, baicalin and baicalein were all purchased from Chengdu Desite Bio-Technology (Chengdu, China). The purity of these standards was above 98%, which was suitable for UPLC analysis. Pudilan samples (43 different batches) from 12 different manufacturers were listed in Table 1. Both acetonitrile and methanol were chromatographically pure (OCEANPAK, Sweden). Ultrasonic cleaner was purchased from Tianjin Aotearns Instruments Co. (Tianjin, China). Analytical grade acetic acid was purchased from Shanghai Runjie Chemical Reagent Co. (Shanghai, China).
Summary of the tested samples
| Sample no. | Manufacturer | Batch no. |
| 1 | Jiren Pharmaceutical Co., Ltd., Anhui, China | 2180712 |
| 2 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 180505 |
| 3 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 180809 |
| 4 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 181205 |
| 5 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 180506 |
| 6 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 180502 |
| 7 | Fukang Pharmaceutical Co., Ltd., Jilin, China | 180810 |
| 8 | Fangsheng Pharmaceutical Co., Ltd., Hunan, China | 190220 |
| 9 | Fangsheng Pharmaceutical Co., Ltd., Hunan, China | 190312 |
| 10 | Jianmin Pharmaceutical Co., Ltd., Hubei, China | 190519 |
| 11 | Lijun Pharmaceutical Co., Ltd., Xian, China | 180311 |
| 12 | Lijun Pharmaceutical Co., Ltd., Xian, China | 180609 |
| 13 | Lijun Pharmaceutical Co., Ltd., Xian, China | 180003 |
| 14 | Kongfu Pharmaceutical Co., Ltd., Shandong, China | 180103 |
| 15 | Kongfu Pharmaceutical Co., Ltd., Shandong, China | 180301 |
| 16 | Kongfu Pharmaceutical Co., Ltd., Shandong, China | 180304 |
| 17 | Kongfu Pharmaceutical Co., Ltd., Shandong, China | 180402 |
| 18 | Kongfu Pharmaceutical Co., Ltd., Shandong, China | 190308 |
| 19 | Longshunrong Pharmaceutical Co., Ltd., Tianjin, China | 19228 |
| 20 | Longshunrong Pharmaceutical Co., Ltd., Tianjin, China | 19222 |
| 21 | Longshunrong Pharmaceutical Co., Ltd., Tianjin, China | 19254 |
| 22 | Longfa Pharmaceutical Co., Ltd., Xianggang, China | 180415 |
| 23 | Longfa Pharmaceutical Co., Ltd., Xianggang, China | 180953 |
| 24 | Longfa Pharmaceutical Co., Ltd., Xianggang, China | 181021 |
| 25 | Longsheng Pharmaceutical Co., Ltd., Zhejiang, China | 20180210 |
| 26 | LongshengPharmaceutical Co., Ltd., Zhejiang, China | 20180509 |
| 27 | Longsheng Pharmaceutical Co., Ltd., Zhejiang, China | 20180806 |
| 28 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180401 |
| 29 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180417 |
| 30 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180419 |
| 31 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180709 |
| 32 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180827 |
| 33 | Minhai Pharmaceutical Co., Ltd., Gansu, China | 180828 |
| 34 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 180124 |
| 35 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 180728 |
| 36 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 190211 |
| 37 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 190321 |
| 38 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 180522 |
| 39 | Shuanglong Pharmaceutical Co., Ltd., Tangshan, China | 181002 |
| 40 | Yunnan Baiyao Co., Ltd., Yunnan, China | zbb1913 |
| 41 | Yunnan Baiyao Co., Ltd., Yunnan, China | zbb1908 |
| 42 | Yunnan Baiyao Co., Ltd., Yunnan, China | zcb1801 |
| 43 | Yunnan Baiyao Co., Ltd., Yunnan, China | zhb1809 |
Chromatographic conditions for Pudilan analysis
The instrument was an Agilent 1290 (Agilent Instruments, USA) ultra-high performance liquid chromatograph. Separation of chemicals was performed on an Agilent TC-C18 (4.6 mm × 100 mm, 2.7 μm) column. The mobile phase was 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution. The mobile phase flow rate was maintained at 0.5 mL min−1 and the following gradient of acetonitrile (B) was applied: 0–4 min, 4%; 4–8 min, 4–13%; 8–25 min, 13–22%; 25–35 min, 22–25%; 35–45 min, 25–41%; 45–50 min, 41–65%; 50–65 min, 65–100%; 65–66 min, 100–5%; and 66–70 min, 5%. In the fingerprinting process, the wavelength was adjusted to 289 nm. The column temperature was set to 30 °C and the sample injection volume was 3 μL.
Preparation of the standard and sample solutions
The six reference compounds were accurately weighed and dissolved in absolute methanol (95%, v/v) at the following concentrations epigoitrin 1.103 mg mL−1, chlorogenic acid 1.191 mg mL−1, caffeic acid 1.072 mg mL−1, baicalin 1.016 mg mL−1, baicalein 1.042 mg mL−1, and acetylcorynoline 1.098 mg mL−1 to acquire stock standard solutions. Store the solutions at 4 °C and keep it in darkness.
After sugarcoat of Pudilan tablets from different batches was scraped off, samples were then ground into powder. The powders were measured and then put in conical flasks with caps (50 mL) containing 35 mL methanol with the total weight recorded as M1. The extraction was performed in an ultrasonic water bath for an hour at normal atmospheric temperature. After sonication, the weight was measured again (M2) and the missing weight was supplemented with methanol. After filtration through a 0.22 μm membrane, all the sample solutions (Nos. from 1 to 43) were stored at 4 °C in a dark environment before use. Prior to UPLC analysis, the solutions were passed through a 0.22 μm organic filter membrane.
Data analysis
The chromatographic fingerprint data of the samples were evaluated by the specialized program Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A), which was recommended by China Food and Drug Administration (CFDA). Correlation coefficients were calculated for the samples and similarities between individual chromatograms and common chromatograms of the tested samples were compared.
Results and discussion
Optimization of chromatographic conditions
The mobile phase of different compositions (methanol–ultrapure water, acetonitrile–ultrapure water, methanol–0.1% phosphoric acid, methanol–0.2% phosphoric acid, acetonitrile–0.1% phosphoric acid, acetonitrile–0.2% phosphoric acid) were tested for an optimal condition. The experimental result indicated that acetonitrile–0.2% phosphoric acid was suitable due to the steadier baseline, better separation and the more characteristic peaks in the chromatograms compared with other conditions. Pudilan tablet contains various constituents, for example, chlorogenic acid as well as caffeic acid. Addition of suitable amount of certain acid into the mobile phase would adjust its pH value for the purpose of achieving better peak shape.
Moreover, three column temperatures (30, 35, and 40 °C) were tested too. Based on the data acquired, the temperature was set at 30 °C. Concerning detection wavelength, absorption profiles at 245, 254, 280, 289, 310, and 330 nm were evaluated due to the multiple phytochemical markers used. At 289 nm, the six phytochemical makers exhibited better and more chromatographic peaks were detected compared with that of other wavelengths. For the purpose of reflecting both the chemical properties of Pudilan tablet and its chemical properties, 289 nm was chosen as the best wavelength.
Optimization of sample extraction
The extraction solutions, extraction means and extraction time were studied. Both methanol and ethanol are common for the extraction of polar and certain non-polar metabolites. In this study, methanol solvent was observed to yield more peaks and better peak pattern than ethanol. Subsequently, different solvent percentages of methanol (60, 70, 80 and 100%) were assessed and 70% methanol was selected as it showed highest extraction rate. At last, ultrasonic extraction methods (40, 60, and 80 min) were investigated. The results indicated that the 60 min sonication was most efficient based on the peak areas of tested samples in UPLC chromatograms. Therefore, 70% methanol ultrasonic extraction for 60 min was affirmed as the most suitable extraction method.
Method validation of quantitative analysis
For the four ingredients used to produce Pudilan tablets, epigoitrin is the characteristic compound for Isatis indigotica, and acetylcorynoline for Corydalis bungeana. Baicalin and baicalein are related to the quality of Scutellaria baicalensis, and chlorogenic acid as well as caffeic acid to Taraxacum mongolicum. Therefore, we chose to adopt these compounds for quantitative analysis of Pudilan products. Methanol stock solutions for the six phytochemical markers were prepared, and their chemical structures were demonstrated in Fig. 1. After determination of the six makers with different concentrations, respective standard curves were created according to peak area (y axis) versus the concentrations of each marker (x axis). The standard curves as well as ranges of the six markers were shown in Table 2 and Fig. 2. Good linearity (r2 > 0.999) was achieved for all six markers within tested ranges. The results regarding instrumental precision of the six markers and their relative standard deviation (RSD) values were all less than 2.75% (n = 6). In addition, the repeatability evaluation of this approach demonstrated that the detection has good reproducibility with RSD less than 2.86% (n = 6) for the six markers. Concerning stability, sample solutions stored at 25 °C were analyzed at 0, 3, 6, 12, 24, 36, and 48 h. It was confirmed that Pudilan sample solutions were stable for at least 48 h (RSD < 2.90%).
The chemical structures of the phytochemical markers
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01084
Data for linear regression assessment (n = 6)
| Chemicals | Regression equation | Linear range (μg) | Correlation coefficient (r2) |
| epigoitrin | y = 9.4251x + 2.4693 | 0.001–0.03 | 0.9996 |
| chlorogenic acid | y = 2.9868x + 1.3551 | 3–16 | 0.9999 |
| caffeic acid | y = 9.0782x + 0.5217 | 1–6 | 0.9998 |
| baicalin | y = 2812.9x + 262.99 | 1–12 | 0.9998 |
| acetylcorynoline | y = 67.572x − 2.4617 | 2–12 | 1 |
| baicalein | y = 273.03x + 281.18 | 1–16 | 0.9995 |
Calibration curves of phytochemical markers of epigoitrin, caffeic acid, chlorogenic acid, acetylcorynoline, baicalin and baicalein
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01084
Evaluation on accuracy of the current method was carried out by recovery test. Six markers at different levels (high, medium and low) were added to 2.0 g Pudilan powder (Jiren 2180712). Subsequently, the samples were processed and analyzed by the suggested method. The experiments were conducted in triplicate at each level. The formula for estimating average recovery rate was as follows: [(amount determined − original amount)/amount added] × 100%. As a result, the average recovery rates for the six markers ranged from 98.3 to 101.1%, with RSD < 2.63%. Thus, the above data indicated that the method was reliable for the simultaneous quantification of the six phytochemical markers.
Quantification of chemicals in Pudilan samples
In order to simultaneously determine the contents of six compounds in distinct batches of Pudilan tablets from different manufacturers, retention times and the online UV spectra of the established UPLC method were compared for determination of the target markers whose UV absorption profiles were also taken into consideration (Fig. 3). The concentrations of the 6 markers in the Pudilan samples were then calculated. As seen in Table 3, the results showed that the variability of their contents was high. The concentration of epigoitrin ranged from 0.0001 to 0.0024 μg μL−1, chlorogenic acid 0.0010–0.015 μg μL−1, caffeic acid 0.0004–0.0640 μg μL−1, baicalin 0.2699–3.3092 μg μL−1, acetylcorynoline 0.0011–0.0319 μg μL−1 and baicalein 0.6080–8.9294 μg μL−1. The results demonstrated that quality of Pudilan tablets from different manufacturers was highly instable. The final product is determined by both active pharmaceutical ingredients as well as productive process. To achieve better Pudilan products, standardization of Isatis indigotica, Corydalis bungeana, Scutellaria baicalensis and Taraxacum mongolicum that are used as raw materials must be realized.
The UV spectrum of six markers in Pudilan tablet
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01084
Quantification of chemicals in Pudilan tablets from different manufacturers
| No of batches | epigoitrin (μg μL−1) | chlorogenic acid (μg μL−1) | caffeic acid (μg μL−1) | baicalin (μg μL−1) | acetylcorynoline (μg μL−1) | baicalein (μg μL−1) |
| Fukang180502 | 0.0005 | 0.0074 | 0.0027 | 2.0504 | 0.0140 | 2.9569 |
| Fukang180505 | 0.0005 | 0.0120 | 0.0023 | 2.9034 | 0.0140 | 3.5112 |
| Fukang180809 | 0.0004 | 0.0062 | 0.0011 | 1.7421 | 0.0215 | 3.6228 |
| Fukang181205 | 0.0012 | 0.0088 | 0.0027 | 1.9058 | 0.0192 | 3.9154 |
| Fukang180506 | 0.0005 | 0.0107 | 0.0021 | 1.4644 | 0.0118 | 2.9729 |
| Fukang180810 | 0.0011 | 0.0096 | 0.0020 | 1.7837 | 0.0200 | 3.8635 |
| Jiren2180712 | 0.0014 | 0.0052 | 0.0034 | 2.1734 | 0.0011 | 2.5337 |
| Lijun180311 | 0.0002 | 0.0025 | 0.0009 | 1.9318 | 0.0232 | 1.1082 |
| Lijun180609 | 0.0024 | 0.0028 | 0.0021 | 1.8278 | 0.0186 | 0.8010 |
| Lijun180003 | 0.0001 | 0.0036 | 0.0004 | 2.2525 | 0.0134 | 2.7422 |
| Jianmin190519 | 0.0009 | 0.0049 | 0.0050 | 2.2843 | 0.0241 | 1.4291 |
| Fangsheng190312 | 0.0002 | 0.0052 | 0.0030 | 2.2985 | 0.0268 | 1.8177 |
| Fangsheng190220 | 0.0007 | 0.0101 | 0.0082 | 2.3401 | 0.0238 | 2.4481 |
| Kongfu180103 | 0.0007 | 0.0043 | 0.0034 | 2.5138 | 0.0151 | 2.9451 |
| Kongfu180301 | 0.0008 | 0.0108 | 0.0090 | 2.3950 | 0.0233 | 2.0644 |
| Kongfu180304 | 0.0009 | 0.0093 | 0.0085 | 2.0384 | 0.0188 | 4.2773 |
| Kongfu180402 | 0.0006 | 0.0094 | 0.0640 | 2.6588 | 0.0186 | 1.2366 |
| Kongfu190308 | 0.0005 | 0.0114 | 0.0105 | 2.9318 | 0.0202 | 1.1689 |
| Longshunrong19228 | 0.0009 | 0.0056 | 0.0063 | 0.7915 | 0.0076 | 6.5571 |
| Longshunrong19222 | 0.0009 | 0.0108 | 0.0151 | 0.7922 | 0.0077 | 8.7705 |
| Longshunrong19254 | 0.0011 | 0.0126 | 0.0169 | 3.3092 | 0.0016 | 8.9294 |
| Longfa181021 | 0.0006 | 0.0019 | 0.0011 | 1.1968 | 0.0101 | 3.3145 |
| Longfa180415 | 0.0006 | 0.0035 | 0.0035 | 1.5106 | 0.0104 | 3.7422 |
| Longfa180953 | 0.0013 | 0.0029 | 0.0018 | 0.8306 | 0.0068 | 3.5602 |
| Longsheng20180509 | 0.0004 | 0.0017 | 0.0031 | 2.7483 | 0.0269 | 1.6741 |
| Longsheng20180210 | 0.0001 | 0.0034 | 0.0042 | 2.5486 | 0.0081 | 0.6080 |
| Longsheng20180806 | 0.0013 | 0.0022 | 0.0017 | 2.9033 | 0.0281 | 2.0499 |
| Minhai180417 | 0.0003 | 0.0016 | 0.0004 | 0.7470 | 0.0044 | 3.2754 |
| Minhai180401 | 0.0004 | 0.0024 | 0.0009 | 0.7845 | 0.0057 | 2.7984 |
| Minhai180419 | 0.0005 | 0.0024 | 0.0007 | 0.6910 | 0.0042 | 3.9524 |
| Minhai180709 | 0.0005 | 0.0010 | 0.0008 | 0.6558 | 0.0043 | 4.0328 |
| Minhai180827 | 0.0003 | 0.0016 | 0.0021 | 0.5962 | 0.0035 | 4.0041 |
| Minhai180828 | 0.0004 | 0.0015 | 0.0015 | 0.5086 | 0.0027 | 4.7735 |
| Shuanglong190321 | 0.0003 | 0.0026 | 0.0015 | 1.8492 | 0.0162 | 1.0860 |
| Shuanglong180124 | 0.0004 | 0.0033 | 0.0023 | 1.9205 | 0.0202 | 0.9407 |
| Shuanglong180728 | 0.0003 | 0.0025 | 0.0014 | 1.9098 | 0.0188 | 0.7680 |
| Shuanglong190211 | 0.0004 | 0.0046 | 0.0039 | 1.9409 | 0.0207 | 1.1272 |
| Shuanglong180522 | 0.0003 | 0.0030 | 0.0017 | 1.8174 | 0.0183 | 0.8768 |
| Shuanglong181002 | 0.0004 | 0.0035 | 0.0017 | 1.9506 | 0.0204 | 0.8926 |
| Yunnanzbb1908 | 0.0002 | 0.0086 | 0.0040 | 0.2699 | 0.0319 | 1.2826 |
| Yunnanzbb1913 | 0.0007 | 0.0144 | 0.0085 | 2.8683 | 0.0280 | 1.5996 |
| Yunnanzbb1801 | 0.0006 | 0.0106 | 0.0052 | 2.5362 | 0.0266 | 1.3033 |
| Yunnanzbb1809 | 0.0006 | 0.0150 | 0.0110 | 2.6322 | 0.0293 | 1.2815 |
Method validation of fingerprint
Evaluation of the apparatus precision was performed by testing the mixed solution containing 6 markers 6 times in a row in a day. The experimental results were termed as RSD and the RSD values for relative retention time (RRT) as well as relative peak area (RPA) were less than 0.43% and 2.18%, respectively.
To assess the reproducibility of the method, six independently prepared solutions from the same batch (Jiren 2180712) were tested. It was found that the RSD values were less than 0.69 and 5.16% for RRT and RPA, separately.
In order to test the stability, sample solutions stored at 25 °C were tested at 0, 3, 6, 12, 24, 36, and 48 h. The RSD values of RRT and RPA did not exceed 0.85% and 2.46%, respectively, indicating that the sample solutions were stable within 48 h under room temperature.
UPLC-DAD fingerprint analysis
For fingerprinting purposes, chromatograms of various batches of samples must be standardized. The standardization process involved selection of “shared peaks” within the resulted chromatograms as well as normalization of retention times for all shared peaks. For standardizing the fingerprint, 43 samples collected from different manufacturers were tested using the developed approach. By designating the peaks shown in each batch of samples as “shared peaks” for Pudilan tablets, the fingerprints of these specimens were generated and displayed in Fig. 4A. The reference chromatogram of Pudilan tablets was demonstrated in Fig. 4B. More than 30 peaks were presented in the fingerprint of Pudilan tablets and we selected 15 of them as shared peaks due to their universal presence in all samples and relatively strong chromatographic signals. The peaks 1, 2, 3, 6, 9 and 15 were identified as the respective phytochemical markers by comparing to the standard solutions (Fig. 4B). Correlation coefficient was applied as an indicator for similarity analysis. In this study, the chromatograms of each sample were compared with other samples and the reference chromatograms using Chromatographic Similarity Evaluation software. As shown in Table 4, the similarity ranged from 0.922 to 0.999, which indicated similar chemical profiles in different batches.
UPLC fingerprint of 43 Pudilan batches A), and the reference chromatogram B) obtained by using the fingerprint similarity evaluation system of traditional Chinese medicine. All chromatograms were obtained at 289 nm. The peaks labeled 1–15 in the chromatogram represented the 15 common peaks. The peaks marked with 1, 2, 3, 6, 9 and 15 were epigoitrin, chlorogenic acid, caffeic acid, baicalin, acetylcorynoline and baicalein, respectively
Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01084
The similarities of Pudilan tablets from different manufacturers
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | Reference | |
| 1 | 1.000 | 0.996 | 0.997 | 0.999 | 0.977 | 0.998 | 0.998 | 0.978 | 0.986 | 0.963 | 0.992 | 0.992 | 0.998 |
| 2 | 0.996 | 1.000 | 0.990 | 0.997 | 0.982 | 0.990 | 0.989 | 0.988 | 0.970 | 0.979 | 0.981 | 0.982 | 0.996 |
| 3 | 0.997 | 0.990 | 1.000 | 0.995 | 0.966 | 0.997 | 0.997 | 0.966 | 0.982 | 0.950 | 0.992 | 0.993 | 0.993 |
| 4 | 0.999 | 0.997 | 0.995 | 1.000 | 0.981 | 0.996 | 0.995 | 0.982 | 0.983 | 0.968 | 0.989 | 0.990 | 0.998 |
| 5 | 0.977 | 0.982 | 0.966 | 0.981 | 1.000 | 0.968 | 0.965 | 0.992 | 0.954 | 0.985 | 0.972 | 0.972 | 0.988 |
| 6 | 0.998 | 0.990 | 0.997 | 0.996 | 0.968 | 1.000 | 1.000 | 0.966 | 0.991 | 0.950 | 0.993 | 0.993 | 0.994 |
| 7 | 0.998 | 0.989 | 0.997 | 0.995 | 0.965 | 1.000 | 1.000 | 0.963 | 0.991 | 0.946 | 0.993 | 0.993 | 0.993 |
| 8 | 0.978 | 0.988 | 0.966 | 0.982 | 0.992 | 0.966 | 0.963 | 1.000 | 0.943 | 0.995 | 0.961 | 0.963 | 0.987 |
| 9 | 0.986 | 0.970 | 0.982 | 0.983 | 0.954 | 0.991 | 0.991 | 0.943 | 1.000 | 0.922 | 0.985 | 0.982 | 0.981 |
| 10 | 0.963 | 0.979 | 0.950 | 0.968 | 0.985 | 0.950 | 0.946 | 0.995 | 0.922 | 1.000 | 0.944 | 0.946 | 0.974 |
| 11 | 0.992 | 0.981 | 0.992 | 0.989 | 0.972 | 0.993 | 0.993 | 0.961 | 0.985 | 0.944 | 1.000 | 0.999 | 0.991 |
| 12 | 0.992 | 0.982 | 0.993 | 0.990 | 0.972 | 0.993 | 0.993 | 0.963 | 0.982 | 0.946 | 0.999 | 1.000 | 0.992 |
| Reference | 0.998 | 0.996 | 0.993 | 0.998 | 0.988 | 0.994 | 0.993 | 0.987 | 0.981 | 0.974 | 0.991 | 0.992 | 1.000 |
Conclusions
The technique has been successfully used to determine contents of six phytochemical markers in a total of 43 samples collected from 12 different manufacturers. Similar chemical profiles were confirmed for all the tested samples with dramatically varying concentrations of the six phytochemical markers. This indicated that the Chinese government should strengthen the requirements in order to achieve high quality of Pudilan products nationwide. The validation results showed that the established method had satisfactory reproducibility and sensitivity to be used for quality evaluation of Pudilan tablets. The 43 batches of Pudilan products were evaluated by a combination of fingerprinting and similarity analysis. The results indicated that the UPLC method used in this study is applicable for quality control of Pudilan tablets.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (82204710), Anhui Natural Science Foundation (2208085QH271), Education Department of Anhui Province (KJ2020A0385) and Anhui University of Chinese Medicine (2021qnyc06, 2021LCTH04).
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