Abstract: Honeysuckle FlosLonicerae, also known as Honeysuckle and Shuanghua, is a commonly used Chinese medicine in clinical practice. It has the effect of clearing away heat and detoxifying, cooling wind and heat. Wait. In Chinese patent medicines, honeysuckle is used either directly as fine powder or in the form of extracts, but most of the preparations are used in the latter form.
Objective To establish a HPLC method for the determination of chlorogenic acid in honeysuckle extract. Methods The domestic C18 column (4.6mm × 250mm, 7μm); the mobile phase was acetonitrile: 0.4% phosphoric acid (12:88); the detection wavelength was 327nm; the flow rate was 1ml · min-1; the injection volume was 10μl; the column temperature was 25 ℃. Results Chlorogenic acid had a good linear relationship in the range of 0.0716 ～ 0.6444μg, r = 0.9996; average recovery rate was 99.99%, RSD = 1.61% (n = 6). Conclusion The method is simple, rapid and accurate, and can be used for the content determination and quality control of honeysuckle extract.
There are two forms of honeysuckle medicine, one is direct feeding of whole grass, and the medicine is decoction, that is, the decoction (ie, the extract of the medicinal material) is used as the semi-finished product (intermediate); the other is to extract the effective parts first, and then extract The material is fed, that is, the extract is used as an intermediate (raw material) (Liu Yan'e "Master Thesis of the Second Military Medical University"). In order to effectively control the intrinsic quality of honeysuckle extract (intermediate), this experiment refers to the relevant literature [2, 3] and the 2005 edition of the Chinese Pharmacopoeia [4] method, using high-performance liquid chromatography to determine the chlorogen in honeysuckle extract Acid content.
1 Instruments and reagents
Agilent1100series high performance liquid chromatograph (G1322A exhaust valve, G1311A pump, G1316A column thermostat, G1314A UV detector), chemistation chromatography workstation, KromasilC18 chromatography column, ultrasonic extractor (KQ3200DE CNC ultrasonic instrument Kunshan Ultrasonic Instrument Co., Ltd.) ; Chlorogenic acid reference substance (Chinese Institute of Pharmaceutical and Biological Products, batch number 110753-200212), honeysuckle extract (made in this laboratory), acetonitrile, phosphoric acid are analytically pure; water is double distilled water.
2 Methods and results [5]
2.1 Establishment of analytical methods-selection of separation and detection conditions
2.1.1 Chromatographic conditions The chromatographic column is a domestic Kromasil C18 column (250mm × 4.6mm, 7μm); the column temperature is 25 ℃; the mobile phase is acetonitrile-0.4% phosphoric acid (12:88); the flow rate is 1ml · min-1; the detection wavelength is 327nm .
2.1.2 The effect of pH on the peak shape Chlorogenic acid is an organic acid. It is easy to cause frontier or tailing peaks in HPLC measurement. Adding acid in the mobile phase can improve this phenomenon. This experiment compares the effect of water, acetic acid, and phosphoric acid on the peak shape. When 0.4% phosphoric acid is used, a better peak shape can be obtained.
2.1.3 Chromatography system adaptability experiment Under selected conditions, chlorogenic acid and other components in the sample can be separated by baseline, the resolution of chlorogenic acid and its adjacent chromatographic peak is greater than 1.5, and the number of theoretical plates (N) is 5000 the above.
a-Control product b-Test product
2.2 Preparation of reference substance solution Precisely weigh the appropriate amount of chlorogenic acid reference substance, put it in a brown measuring bottle, add 50% methanol to make a solution containing 35.8 μg per ml, that is, you get it. 2.3 Preparation of test solution Weigh precisely 10mg of this extract, put it in a 100ml volumetric flask, dissolve it in double-distilled water, after sonication for 2min, dilute to the mark with double-distilled water, shake well, and pass 0.45μg microporous membrane , Obtained after taking the filtrate.
2.4 Examination of the linear range and the drawing of the standard curve Precisely draw 2, 4, 6, 10, 14, 18 μl of the reference solution and inject them into the liquid chromatograph. The peak area was determined according to the above chromatographic conditions. The peak area integral value (A) was used to regress the injection volume, and the regression curve equation was obtained: Y = 2468.4X-16.205, r = 0.9996. The results show that chlorogenic acid has a good linear relationship with the peak area in the range of 0.0716 ～ 0.6444μg.
2.5 Precision experiment Pipette 10 μl of the reference solution accurately, repeat the injection 6 times according to the above chromatographic conditions, and record the peak area value of chlorogenic acid in each group. The average peak area was 840.5, and the RSD was 0.69% (n = 6).
2.6 Stability experiment Take the same test solution and inject 10μl at 0, 2, 4, 6, and 8h respectively. The average peak area value of chlorogenic acid was 680.24, RSD: 0.38% (n = 5) , Indicating that the test solution is stable within 8h.
2.7 Repeatability experiment: Take 6 samples of the same batch number (070204) and determine according to the content determination method. As a result, the average content of chlorogenic acid was 28.01%, and the RSD was 1.07% (n = 6).
2.8 Sample recovery rate experiment Weigh 6 parts of honeysuckle extract with known content, add the appropriate amount of chlorogenic acid reference substance accurately, prepare and measure according to the method of test solution, and calculate the recovery rate.
2.9 Sample determination Take 3 batches of samples, prepare the test solution according to the above method, inject 10μl, determine according to the determined chromatographic conditions, and calculate the content of chlorogenic acid in the sample by external standard method according to the peak area value.
3 Discussion
The honeysuckle extract uses organic acids as the active ingredient. In this experiment, the chlorogenic acid content was used as the quality control index of the honeysuckle extract. At the same time, the content of chlorogenic acid in the three batches of honeysuckle extract was determined. Through calculation, the content was 20% the above. The method established in this paper is simple, fast, and has good repeatability and stability. It can be used for the quality control of the extract in actual production. The work done in this article provides a quality monitoring method for the development of honeysuckle related products.
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