[Abstract] Objective To establish a high performance liquid chromatography (HPLC) method for determining the content of components in Shuanghuanglian injection. Methods Methanol-water (10% phosphoric acid solution adjusted to pH 2.7) was used as the mobile phase, the flow rate was 1.0ml / min, the detection wavelength was 326nm, and it was washed on the Diamonsil (diamond) C18 (5μm, 4.6mm × 150mm) chromatographic column It can be quantified by external standard method and the content of baicalin can be determined. Results Baicalin showed a good linear relationship in the range of 5.1 ～ 81.6μg / ml, r = 0.9998, the average recovery rate was 100.34%, RSD = 0.82% (n = 5). Conclusion The method is simple, sensitive, reproducible, accurate, high-precision, good linear relationship, and high recovery. The results show that baicalin is stable within 12 hours, which is suitable for the quality control of Shuanghuanglian injection.
ã€Key words】 Shuanghuanglian injection Baicalin high performance liquid chromatography
Shuanghuanglian injection is a traditional Chinese medicine preparation developed in recent years. It is mainly used clinically for acute upper respiratory tract infections, acute bronchitis, acute tonsillitis, pneumonia, and acute urinary tract infections. Its efficacy is accurate, safe, and widely used. Shuanghuanglian injection is made from extracts of three traditional Chinese medicines: honeysuckle, baicalin, and forsythia. The main components are baicalin, chlorogenic acid, and forsythin [1]. Pharmacological studies have confirmed that baicalin has bacteriostatic, heat-clearing, antihypertensive, sedative, diuretic, choleretic, anti-inflammatory, anti-allergic, detoxification and other effects; chlorogenic acid has antibacterial and anti-inflammatory, clearing heat and detoxification effects; and forsythin It has strong antibacterial and antiviral activity.
The methods for determining the content of baicalin in Shuanghuanglian injection mainly include high performance liquid chromatography, thin layer scanning, ultraviolet spectrophotometry, etc. High performance liquid chromatography has the characteristics of high separation efficiency, fast analysis speed and precision, which is suitable for the composition of traditional Chinese medicine Diversity and complexity are also the most widely used in the quality control of Shuanghuanglian preparations. In this paper, the content of baicalin was determined by optimizing the chromatographic conditions, in order to lay a foundation for further study of chlorogenic acid and other components in Shuanghuanglian injection, and provide a theoretical basis for the technological research and quality control of Shuanghuanglian injection.
1 Instruments and reagents
1.1 Instrument Agilent1100 high-performance liquid chromatograph (Agilent, USA); TU-1800 UV-Vis spectrophotometer (Beijing General Analysis General Instrument Co., Ltd.); KQ-100 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Electronics Balance (Shanghai Jingke Balance); Yuhua SHZ-D (Ⅲ) circulating water vacuum pump (Gongyi Yingyu Yuhua Instrument Factory, Henan Province); Diamonsil (diamond) C18 column (5μm, 4.6mm × 150mmCat.no: 9990 Ser. No: 8011571 Dikma Technologies).
1.2 Reagent Shuanghuanglian Injection (Lot No. Z23021166 Heilongjiang Wusulijiang Jiada Pharmaceutical Co., Ltd.); Baicalin Chemical Standards (Lot No. 110715-200514, China National Institute for Pharmaceutical and Biological Products); Chromatography Methanol (Tianjin Damao Chemical Reagent Factory) Analytical pure methanol (Tianjin North Tianyi Chemical Reagent Factory); phosphoric acid (Hebei Baoding Chemical Reagent Factory).
2 Methods and results
2.1 Chromatographic conditions Diamonsil (diamond) C18 column (5μm, 4.6mm × 150mm), mobile phase is A-methanol (chromatographically pure): B-distilled water (50:50), flow rate is 1.0ml / min, detection wavelength is 326nm , Injection volume 20μl. The separation chromatogram is shown in Figures 1-2.
2.2 Preparation of the solution
2.2.1 Preparation of standard solution Precisely weigh 0.005100g of baicalin standard into a 50ml volumetric flask, dilute to the mark with methanol (analytical purity), and sonicate until it is completely dissolved. Prepare a stock solution with a concentration of 0.102mg / ml and put it in the refrigerator for low temperature storage.
2.2.2 Preparation of test solution Use a 0.1ml pipette to accurately draw 0.05ml to 10ml volumetric flask from Shuanghuanglian injection, dilute to the mark with methanol (analytical purity), and dissolve by sonication for 10min. Then filter with a 0.45μm filter membrane to a 10ml colorimetric tube soaked with chromic acid washing solution for 3h and dried, and tighten the stopper.
2.3 Investigation of linear relationship Precisely measure the concentration of 0.102mg / ml baicalin standard stock solutions 0.5, 1, 2, 4, 6, 8ml to 10ml volumetric flasks respectively, dilute to the mark with methanol (analytical purity), and prepare After a series of standard solutions with concentrations of 5.1, 10.2, 20.4, 40.8, 61.2, and 81.6 μg / ml, filter them with a 0.45 μm filter membrane to 6 10 ml colorimetric tubes that have been dried after being soaked with chromic acid for 3 hours. , Tighten the stopper. According to the above chromatographic conditions, the concentration of baicalin standard solution was measured 3 times. Based on the average of the three peak areas, the standard curve of baicalin was obtained. The results are shown in Table 1. Baicalin showed a good linear relationship in the concentration range of 5.1-81.6 μg / ml. The regression equation is Y = 41.409X-132.35, r = 0.9998.
2.4 Determination of recovery rate of sample addition A 1.0ml pipette was used to accurately pipette 1ml to 100ml volumetric flask from Shuanghuanglian injection, make up to volume with methanol (analytical purity), and ultrasonically dissolve it for 10min. Draw 5ml to 10ml volumetric flasks from it, a total of 5 portions, each precision added baicalin standard stock solutions 0.051, 0.102, 0.204, 0.306, 0.357, 0.510mg, dilute to the mark with methanol (analytical purity), and sonicate for 10min It was completely dissolved, measured according to the above chromatographic conditions, calculated the content of baicalin based on the peak area, and calculated the average sample recovery rate of baicalin. The results are shown in Table 2. Table 1 Examination data of linear relationship of baicalin Table 2 Experimental results of baicalin recovery
2.5 Precision experiment Take 1 Shuanghuanglian injection, prepare 1 sample solution according to the preparation method of the test sample solution, measure according to the above chromatographic conditions, and inject 5 times in total. The results are shown in Table 3. Table 3 Precision experimental results of baicalin
2.6 Reproducibility experiment Take 5 Shuanghuanglian injections of the same batch number, prepare 5 sample solutions according to the preparation method of the test solution, and measure according to the above chromatographic conditions. The results are shown in Table 4. Table 4 Reproducibility experimental results of baicalin
2.7 Stability experiment A sample solution is prepared according to the preparation method of the test solution, and placed at room temperature, and the peak area is measured at 0, 3, 6, 9, 12 h according to the above chromatographic conditions. The experimental result RSD = 1.22% , Indicating that baicalin is stable within 12h, the results are shown in Table 5. Table 5 Stability test results of baicalin
2.8 Determination of sample content Prepare the sample solution according to the preparation method of the test solution, and measure according to the above chromatographic conditions. The content of baicalin in Shuanghuanglian injection was 7.1mg / ml. The results are shown in Figures 1-2.
3 Discussion
The adverse reactions of Shuanghuanglian injection originate from the complex components of Chinese patent medicines, such as chlorogenic acid and isochlorogenic acid contained in honeysuckle, which not only have antibacterial and antiviral effects, but also have allergenic effects, which can cause allergic reactions and cause One of the main causes of allergic reactions. Tong Lu [2] reported that the allergic reaction caused by Shuanghuanglian injection is related to baicalin, and the toxic reaction is related to another component in Scutellaria baicalensis.
The polarities of baicalin and chlorogenic acid are very different. In the quality standard, only the content of the two is determined separately, and the content of the two is measured by HPLC method. The gradient elution method is used to gradually reduce the polarity of the mobile phase. The absorption wavelength is generally selected at 326nm to improve the sensitivity of chlorogenic acid. The HPLC determination of baicalin showed that the mobile phase was methanol: water: glacial acetic acid (5: 50: 1); methanol: water: phosphoric acid (50: 50: 0.2); isopropanol: methanol: 3% acetic acid (5: 30:65); methanol: 0.5% triethylamine (75:25) system. The chromatographic conditions and system suitability test of baicalin in the 2005 edition of "Chinese Pharmacopoeia" are: using octadecylsilane bonded silica gel as a filler; using methanol: water: glacial acetic acid (50: 50: 1) as the flow Phase, the detection wavelength is 274nm, the number of theoretical plates should be no less than 1500 calculated by the baicalin peak. However, in the process of exploring the experimental conditions, we found that the mobile phase uses methanol: water (adjust the pH to 2.7 with 10% phosphoric acid), the ratio is set to 50:50, baicalin is well separated from other components in the injection, and can reduce phosphoric acid Salt erosion to the pump, etc.
There are many reports on the analysis methods of the main components in Shuanghuanglian injection [3 ～ 5], but there are rare reports on the analysis methods of other components in the preparation. This article aims to find out the analysis and separation methods of various components through the analysis and determination of baicalin Causes of adverse reactions in Shuanghuanglian injection.
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