WO2019034030A1 - 小儿复方鸡内金咀嚼片的鉴别和含量测定方法 - Google Patents

小儿复方鸡内金咀嚼片的鉴别和含量测定方法 Download PDF

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WO2019034030A1
WO2019034030A1 PCT/CN2018/100339 CN2018100339W WO2019034030A1 WO 2019034030 A1 WO2019034030 A1 WO 2019034030A1 CN 2018100339 W CN2018100339 W CN 2018100339W WO 2019034030 A1 WO2019034030 A1 WO 2019034030A1
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solution
methanol
chewable tablet
gold
compound chicken
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English (en)
French (fr)
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郭中华
张立壮
刘伟伟
其木格
张志平
张冰冰
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河南太龙药业股份有限公司
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Priority to US16/629,283 priority Critical patent/US11333643B2/en
Publication of WO2019034030A1 publication Critical patent/WO2019034030A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/12—Preparation by evaporation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/94—Development

Definitions

  • the invention relates to a traditional Chinese medicine quality control method, in particular to a method for identifying and contenting a chicken compound chicken chewable tablet.
  • the pediatric compound chicken inner gold chewable tablet is included in the national registration standard [YBZ03852008], and is a Chinese medicine product exclusively produced by Henan Tailong Pharmaceutical Co., Ltd.
  • the prescription Chinese herbal medicines are chicken inner gold and Liushenqu, in which the chicken inner gold is the inner wall of the dry sandbag of the animal chicken.
  • Liushenqu is a koji agent made from the co-fermentation of Spicy Scallop, Artemisia annua, Xanthium, Chixiaodou, Bitter Almond, Wheat Bran and Flour.
  • the main ingredients are rutin, artemisinin, Xanthium, ferulic acid and so on.
  • Heavy metal inspection Take about 1.0g of the test sample and inspect it according to the method of the second method of the Chinese Pharmacopoeia heavy metal inspection method. Heavy metals should not exceed 20 parts per million.
  • the object of the present invention is to provide a method for identifying and contenting a compound chicken inner chewable tablet with strong specificity, high sensitivity, good repeatability and reasonable quality control, which can comprehensively control the quality standard of the product.
  • the invention provides a method for determining and contenting a pediatric compound chicken inner chewable tablet, characterized in that the method comprises:
  • the conditions of the thin layer chromatography are as follows: a thin layer chromatography plate of silica gel G is used as a thin layer chromatography plate, and the lower layer is placed under chloroform-ethyl acetate-methanol-water (12:3:8:1) at 10 ° C or lower. Liquid is a developing agent;
  • the high performance liquid chromatography conditions are as follows: octadecylsilane bonded silica as a stationary phase, methanol-water-glacial acetic acid in a volume ratio (85:15:0.45) as mobile phase A, and acetonitrile-isopropyl
  • the alcohol-water was mobile phase B in a volume ratio (50:20:30), and the gradient elution was carried out according to the following conditions:
  • the step of identifying according to step (1) further comprises the step of using a macroporous adsorption resin column chromatography on the pediatric compound chicken inner gold chewable tablet.
  • the step of macroporous adsorption resin column chromatography comprises: mixing the pediatric compound chicken inner gold chewable tablet with a mass fraction of 75% methanol solution by weight ratio of 4:25, sonicating, and then using macroporous adsorption resin.
  • the column is preferably subjected to chromatography using an AB-8 macroporous adsorption resin column, and after elution adsorption equilibrium, the macroporous adsorption resin column is eluted with an ethanol solution having a mass fraction of 70% as an eluent, and the eluate is collected after the eluate is collected. Evaporate to dryness and add analytically pure methanol solution.
  • the step of identifying the step (1) uses a chicken-gold-methanol solution as a reference drug solution to
  • the acid-rutin-methanol solution was used as a reference solution.
  • the step of identifying by the thin layer chromatography in the step (1) comprises:
  • the step of identifying by the thin layer chromatography in the step (1) comprises:
  • the pediatric compound chicken inner chewable tablets were pulverized, mixed with a mass fraction of 75% methanol solution at a weight-to-volume ratio of 4:25, sonicated for 30 min, filtered, and the upper AB-8 macroporous adsorption resin column layer Analyze, wash with water until the washing liquid is nearly colorless, then elute with 35% ethanol and 70% ethanol in sequence, collect 70% ethanol eluent, evaporate dry, add methanol to dissolve, then get the test solution ;
  • step (2) taking chicken internal gold reference medicine, according to the method of step (1) to obtain a chicken gold-methanol reference medicine solution
  • the content determining step according to the step (2) further comprises the step of purifying the pediatric compound chicken inner gold chewable tablet by using a neutral alumina column.
  • the step of purifying the neutral alumina column comprises: mixing the pediatric compound chicken inner gold chewable tablet with the chromatographic pure methanol according to a weight ratio of 1:2.5, sonicating, and purifying through a neutral alumina column to The neutral ethanol column was eluted with an absolute ethanol solution as an eluent, and the eluent was collected and evaporated to dryness, and purified by chromatography.
  • the content determination step described in the step (2) uses an artemisinin-p-hydroxybenzaldehyde-Xan Erting-methanol solution as a reference solution.
  • the step of determining the content by the high performance liquid chromatography in the step (2) comprises:
  • the step of determining the content by the high performance liquid chromatography in the step (2) comprises:
  • the pediatric compound chicken inner chewable tablets were pulverized, mixed with the chromatographic pure methanol solution at a weight-to-volume ratio of 1:2.5, sonicated for 30 minutes, allowed to cool, filtered, and passed through a neutral alumina column (100 ⁇ ). Purification by 120 mesh), elution with absolute ethanol, collecting the eluent, concentrating under reduced pressure, dissolving in chromatographically pure methanol, and filtering to obtain a test solution;
  • the high performance liquid chromatography conditions are: using octadecylsilane bonded silica as a stationary phase, the column temperature of the column is 38 ° C, with methanol - Water-glacial acetic acid was mobile phase A in a volume ratio (85:15:0.45), and mobile phase B in a volume ratio (50:20:30) in acetonitrile-isopropanol-water. Gradient elution was carried out according to the following conditions. The flow rate of the mobile phase is 1 ml / min;
  • the specific thin layer chromatography conditions of the present invention are used for identification, and specific high performance liquid chromatography conditions are used for content determination, and the specificity and accuracy are strong, which can fully reflect the chemical composition of the product, high sensitivity, good reproducibility, and operation. Simple, more objective, comprehensive and sensitive reflects the changes in product quality, thus overall control of drug quality, to achieve comprehensive monitoring of the quality of traditional Chinese medicine.
  • Figure 1 is a linear view of the artemisinin reference substance
  • Figure 2 is a linear view of the p-hydroxybenzaldehyde reference product
  • Figure 3 is a linear view of the Xan Erting reference.
  • Reagents methanol, ethanol, chloroform, ethyl acetate, sulfuric acid are analytically pure, AB-8 macroporous adsorption resin column, silica gel G thin layer plate.
  • Sample to be tested Pediatric compound chicken inner chewing tablets (batch number: 1610222; 1.2g / piece): Henan Tailong Pharmaceutical Co., Ltd. provided.
  • Negative sample 1 (excluding chicken gold): provided by Henan Tailong Pharmaceutical Co., Ltd.
  • Negative sample 2 (excluding Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.
  • Blank sample 3 (excluding chicken inner gold, Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.
  • Reference substance chicken internal gold reference medicine (batch number: 121153-201002), ferulic acid reference substance (batch number: 110773-201614), rutin reference substance (batch number: 100080-201610), all provided by China Food and Drug Administration Institute .
  • test solution Take 10 pieces of puer compound chicken inner chewable tablets, grind finely, weigh about 4g, add 75% methanol 25ml, ultrasonic for 30min, filter, upper AB-8 macroporous adsorption resin column (The column is 1.8cm, the column height is 18cm, and the column is packed by wet method), washed with water until the water washing liquid is nearly colorless, and then eluted with 60ml of 705% ethanol and 70% ethanol in sequence, and 70% ethanol eluent is collected. , evaporated to dryness, and dissolved in 5 mL of methanol to prepare a test solution.
  • the test solution has corresponding spots in the spot of the chicken gold reference drug solution and the reference solution.
  • the negative sample solution 1 had no corresponding spots at the spot of the chicken-gold reference drug solution.
  • the negative sample solution 2 had no corresponding spots at the spots of the ferulic acid and rutin reference solution.
  • the blank sample solution 3 had no corresponding spots in the spot of the chicken internal gold reference drug solution, ferulic acid and rutin reference solution.
  • the specific thin-layer chromatographic identification conditions of the present invention were used to identify the chicken chewable tablets in the chicken compound, and the identification method had no blank interference, and the components did not interfere with each other, and the specificity was strong and the operability was strong.
  • Reagents methanol, acetonitrile, isopropanol for chromatographic purity, ethanol, glacial acetic acid for analytical purity. Neutral alumina, chromatography column.
  • Sample to be tested Pediatric compound chicken inner chewing tablets (batch number: 1610222, 1.2g / piece): provided by Henan Tailong Pharmaceutical Co., Ltd.
  • Negative sample (excluding Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.
  • Control substance artemisinin reference substance (batch number: 100202-201606), p-hydroxybenzaldehyde reference substance (batch number: 201613), Xan Erting reference substance (batch number: 201609).
  • test solution Take the chicken chewable tablets in the compound chicken, grind and smash, accurately weigh 10g, place it in a conical flask, add 25ml of methanol precisely, close the plug, weigh the weight, and sonicize (power 250W, frequency 40kHz) 30 minutes, take out, let cool, then weigh the weight, make up the lost weight with methanol, shake, filter, and accurately measure 10ml of the filtrate, add to the neutral alumina column (100 ⁇ 120 mesh, weight 10 g, inner diameter 1 cm), eluted with 50 ml of absolute ethanol, the eluate was collected, concentrated to dryness under reduced pressure at 50 ° C, dissolved in a suitable amount of purified methanol, transferred to a 2 ml volumetric flask, and added. Chromatographically pure methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.
  • neutral alumina column 100 ⁇ 120 mesh, weight 10 g, inner diameter 1 cm
  • Assay method accurately draw 20 ⁇ l of each of the reference solution and the test solution, and inject into a liquid chromatograph, and measure, that is, obtain.
  • Chromatographic conditions octadecylsilane bonded silica as the stationary phase, methanol-water-glacial acetic acid (85:15:0.45) as mobile phase A, acetonitrile-isopropanol-water (50:20:30)
  • mobile phase B gradient elution was carried out according to the following table. The column temperature was 38 ° C, the mobile phase flow rate was 1 ml per minute; the evaporative light scattering detector was used to detect; the theoretical plate number should be no less than 6000 in terms of artemisinin.
  • Each tablet contains 0.016 mg of artemisinin, 0.104 mg of p-hydroxybenzaldehyde, and 0.050 mg of Xanthium.
  • Preparation of negative sample solution Take a negative sample (excluding Liushen), and prepare a negative sample solution lacking Liushen according to the preparation method of 2.2. Step (1).
  • test solution had the corresponding peak at the same time as the reference solution retention time; the negative test solution lacking Liushen had no corresponding peak at the same retention time as the reference solution. It indicates that the detected components are more prominent, that is, from the six divine songs, the method is specific.
  • step 2.2 Prepare the test solution according to the preparation method of step 2.2 (1), and place the test solution for 0, 2, 4, 6, 8, 12, 24 h at room temperature, respectively, at the above time point, according to step 2.2 ( 3)
  • the chromatographic conditions determine the content of artemisinin, p-hydroxybenzaldehyde, and chlorhexidine in the sample, and calculate the RSD value. The results are shown in Table 4.
  • test solution prepared by the method of the present invention has good stability within 24 hours at room temperature.
  • the method for determining the content of the compound chicken inner chewable tablet of the invention is strong in specificity, high in precision and good in reproducibility, and can effectively control product quality.
  • the pediatric compound chicken inner chewable tablets were identified according to the method described in 1.2 of Example 1, except that the developing agent used in Example 1 was replaced with the following developing agent:
  • the content of pediatric compound chicken inner chewable tablets was determined according to the method described in 2.2 of Example 1, except that the mobile phase and elution conditions used in Example 1 were replaced by the following mobile phases and elution conditions:

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Abstract

一种中成药质量控制方法，具体涉及小儿复方鸡内金咀嚼片鉴别和含量测定方法。采用特定的薄层色谱鉴别条件进行鉴别，以及特别的高效液相色谱条件进行含量测定，专属性和准确性强，能够全面反映产品的化学成分，灵敏度高，重现性好，操作简单，更加客观、全面及灵敏地反映产品质量变化情况，从而对产品质量进行整体控制，实现中药质量的全面监测。

Description

小儿复方鸡内金咀嚼片的鉴别和含量测定方法 技术领域
本发明涉及一种中药质量控制方法，具体涉及小儿复方鸡内金咀嚼片的鉴别和含量测定方法。
背景技术
在现有技术中，小儿复方鸡内金咀嚼片收入于国家注册标准[YBZ03852008]，为河南太龙药业股份有限公司独家生产的中药产品。处方：鸡内金85g，六神曲165g。制法：以上二味，粉碎成细粉，过筛，加入辅料，混匀，制成颗粒，干燥，压制成1000片，即得。性状：本品为着色异型咀嚼片；气香，味甜。处方中药材分别为鸡内金和六神曲，其中鸡内金为动物家鸡的干燥沙囊内壁。六神曲为辣蓼、青蒿、苍耳草、赤小豆、苦杏仁、麦麸、面粉共同发酵制成的曲剂，主要成分有芦丁、青蒿素、苍耳亭、阿魏酸等。
目前，现有的对小儿复方鸡内金咀嚼片进行质量检测，主要按照以下标准：
(1)鉴别：取小儿复方鸡内金咀嚼片含量测定项下细粉，置显微镜下观察：石细胞橙黄色，贝壳形，壁较厚，较宽一边纹孔明显。
(2)含量测定：取小儿复方鸡内金咀嚼片20片，精密称定，研细，取粉末约2g，精密称定，照中国药典氮测定法第一法进行消化，照中国药典氮测定法第二法进行测定。每片含氮不得低于12.0mg。
(3)重金属检査：取供试品约1.0g，照中国药典重金属检査法第二法项下方法进行检査。含重金属不得过百万分之二十。
(4)其他应符合中国药典片剂项下有关的各项规定。
上述质量标准存在缺点是：鉴别过于简单，显微鉴别仅为六神曲的质量控制指标，没有对鸡内金进行控制；含量测定氮元素为控制大类成分，专属性、重复性差，不能定量全面的检测其有效成份。
目前，关于两味药材的国家现行标准中均未涉及薄层鉴别和成分定量检验的项目。
发明内容
本发明的目的是针对上述不足，提供一种专属性强，灵敏度高，重复性好，质量控 制合理的小儿复方鸡内金咀嚼片鉴别和含量测定方法，该方法能够全面控制产品的质量标准。
本发明提供一种小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，所述方法包括：
(一)采用薄层色谱法进行鉴别的步骤，其中，
所述薄层色谱的条件为：以硅胶G薄层板为检测用薄层色谱板，以三氯甲烷-乙酸乙酯-甲醇-水(12:3:8:1)10℃以下放置的下层液为展开剂；
(二)采用高效液相色谱法进行含量测定的步骤，其中，
所述高效液相色谱的条件为：以十八烷基硅烷键合硅胶为固定相，以甲醇-水-冰醋酸按体积比(85:15:0.45)为流动相A，以乙腈-异丙醇-水按体积比(50:20:30)为流动相B，按照下述条件进行梯度洗脱：
时间(分钟) 流动相A(％) 流动相B(％)
0 0 100
12 0 100
20 30 70
32 85 15
34 15 85
46 15 85
优选地，步骤(一)所述的鉴别步骤还包括将小儿复方鸡内金咀嚼片采用大孔吸附树脂柱层析的步骤。
优选地，所述大孔吸附树脂柱层析的步骤包括：将小儿复方鸡内金咀嚼片与质量分数为75％甲醇溶液按照重量体积比4:25混合，超声处理，然后采用大孔吸附树脂柱，优选采用AB-8大孔吸附树脂柱进行层析，洗脱吸附平衡后，以质量分数为70％的乙醇溶液为洗脱剂对大孔吸附树脂柱进行洗脱，收集洗脱液后蒸干，加分析纯的甲醇溶液。
优选地，步骤(一)所述的鉴别步骤以鸡内金-甲醇溶液为对照药材溶液，以阿魏
酸-芦丁-甲醇溶液为对照品溶液。
优选地，步骤(一)所述薄层色谱法进行鉴别的步骤包括：
(1)将小儿复方鸡内金咀嚼片按所述大孔吸附树脂柱层析的步骤制得供试品溶液；
(2)将鸡内金对照药材按所述大孔吸附树脂柱层析的步骤制得鸡内金-甲醇对照药材溶液；
(3)取阿魏酸对照品和芦丁对照品，加甲醇制得阿魏酸-芦丁-甲醇对照品溶液；
(4)吸取上述三种溶液，按照所述薄层色谱条件进行鉴别。
优选地，步骤(一)所述薄层色谱法进行鉴别的步骤包括：
(1)将小儿复方鸡内金咀嚼片粉碎，将其与质量分数为75％甲醇溶液按照重量体积比为4:25混合，超声处理30min，滤过，上AB-8大孔吸附树脂柱层析，以水洗至水洗液近无色，然后依次采用质量分数为35％乙醇、70％乙醇洗脱，收集70％乙醇洗脱液，蒸干，加甲醇使之溶解，即得供试品溶液；
(2)取鸡内金对照药材，按步骤(1)的方法制得鸡内金-甲醇对照药材溶液；
(3)取阿魏酸对照品和芦丁对照品，加甲醇制成每1ml含阿魏酸0.5mg，芦丁0.5mg的混合溶液作为对照品溶液；
(4)吸取上述三种溶液，分别点于高效硅胶G薄层板上，以三氯甲烷-乙酸乙酯-甲醇-水按体积比12:3:8:1为展开剂展开，取出晾干，喷以质量分数为10％硫酸乙醇溶液，加热105℃至斑点显色清晰，分别在日光和紫外光(365nm)下检视。
优选地，步骤(二)所述的含量测定步骤还包括将小儿复方鸡内金咀嚼片采用中性氧化铝柱进行纯化的步骤。
优选地，所述中性氧化铝柱进行纯化的步骤包括：将小儿复方鸡内金咀嚼片与色谱纯甲醇按照重量体积比为1:2.5混合，超声处理，通过中性氧化铝柱纯化，以无水乙醇溶液为洗脱剂对中性氧化铝柱进行洗脱，收集洗脱液蒸干，加色谱纯甲醇。
优选地，步骤(二)所述的含量测定步骤以青蒿素-对羟基苯甲醛-苍耳亭-甲醇溶液为对照品溶液。
优选地，步骤(二)所述高效液相色谱法进行含量测定的步骤包括：
(1)将小儿复方鸡内金咀嚼片按所述中性氧化铝柱进行纯化的步骤制得供试品溶液；
(2)取青蒿素、对羟基苯甲醛和苍耳亭对照品，加甲醇制成青蒿素-对羟基苯甲醛-苍耳亭-甲醇的混合溶液为对照品溶液；
(3)吸取上述两种溶液，注入高效液相色谱仪，按照所述高效液相色谱条件进行含量测定。
优选地，步骤(二)所述高效液相色谱法进行含量测定的步骤包括：
(1)将小儿复方鸡内金咀嚼片粉碎，将其与色谱纯甲醇溶液按照重量体积比为1:2.5混合，超声处理30分钟，放冷，滤过，通过中性氧化铝柱(100～120目)纯化， 用无水乙醇洗脱，收集洗脱液，减压浓缩，用色谱纯甲醇溶解，滤过，即得供试品溶液；
(2)取青蒿素、对羟基苯甲醛和苍耳亭对照品，加甲醇制成每1ml含青蒿素0.3mg、对羟基苯甲醛0.2mg、苍耳亭0.1mg的混合溶液为对照品溶液；
(3)吸取上述两种溶液，注入高效液相色谱仪测定，所述高效液相色谱条件为：以十八烷基硅烷键合硅胶为固定相，色谱柱柱温为38℃，以甲醇-水-冰醋酸按体积比(85:15:0.45)为流动相A，以乙腈-异丙醇-水按体积比(50:20:30)为流动相B，按照下述条件进行梯度洗脱；所述流动相的流速为1ml/min；
时间(分钟) 流动相A(％) 流动相B(％)
0 0 100
12 0 100
20 30 70
32 85 15
34 15 85
46 15 85
本发明的效果：
采用本发明特定的薄层色谱鉴别条件进行鉴别，以及特定的高效液相色谱条件进行含量测定，专属性和准确性强的，能够全面反映产品的化学成份，灵敏度高，重现性好，操作简单，更加客观、全面及灵敏地反映出产品质量变化情况，从而对药品质量进行整体控制，实现中药质量的全面监测。
附图说明
图1是青蒿素对照品线性考察图；
图2是对羟基苯甲醛对照品线性考察图；
图3是苍耳亭对照品线性考察图。
具体实施方式
以下通过实施例对本发明进行详细说明。应当理解的是，此处实施例仅用于示例性对本发明进行说明，并不用于对本发明进行限制。
实施例1
1.小儿复方鸡内金咀嚼片薄层鉴别方法
1.1仪器与试药
试剂：甲醇、乙醇、三氯甲烷、乙酸乙酯、硫酸均为分析纯，AB-8大孔吸附树脂柱，硅胶G薄层板。
待测样品-小儿复方鸡内金咀嚼片(批号：161022；1.2g/片)：河南太龙药业股份有限公司提供。
阴性样品1(不含鸡内金)：河南太龙药业股份有限公司提供。
阴性样品2(不含六神曲)：河南太龙药业股份有限公司提供。
空白样品3(不含鸡内金、六神曲)：河南太龙药业股份有限公司提供。
对照品：鸡内金对照药材(批号：121153-201002)，阿魏酸对照品(批号：110773-201614)，芦丁对照品(批号：100080-201610)，均由中国食品药品检定研究院提供。
仪器：ZF7C三用紫外分析仪，AL-204型电子分析天平，HQ-500DE型超声波清洗器；真空抽滤装置、(1cm×30cm)层析柱、薄层显色喷雾装置。
1.2方法
(1)供试品溶液的制备：取小儿复方鸡内金咀嚼片10片，研细，称取约4g，加入75％甲醇25ml，超声30min，滤过，上AB-8大孔吸附树脂柱(柱内经1.8cm，柱高18cm，湿法装柱)，以水洗至水洗液近无色，然后依次采用质量分数为35％乙醇、70％乙醇各60ml洗脱，收集70％乙醇洗脱液，蒸干，加5mL甲醇使之溶解，作为供试品溶液。
(2)阴性样品溶液的制备：分别取阴性样品1、阴性样品2、空白样品3各10片，按照供试品溶液制备方法制成阴性样品溶液1、阴性样品溶液2和空白样品溶液3。
(3)对照品溶液制备：取鸡内金对照药材1g，同上述供试品溶液制备方法制成对照药材溶液。
(4)取阿魏酸对照品和芦丁对照品，加甲醇制成每l ml含阿魏酸0.5mg，芦丁0.5mg的混合溶液作为对照品溶液。
(5)照薄层色谱法试验，吸取上述六种溶液各5μl，分别点于同一高效硅胶G薄层板上，以三氯甲烷-乙酸乙酯-甲醇-水按体积比(12:3:8:1)，10℃以下放置的下层溶液为展开剂，展开，取出晾干，喷以10％硫酸乙醇溶液，在105℃加热至斑点显色清晰，分别在日光和紫外光(365nm)下检视。
1.3结果及结论
供试品溶液在鸡内金对照药材溶液和对照品溶液的斑点处均有对应斑点。阴性样品溶液1在鸡内金对照药材溶液斑点处无对应斑点。阴性样品溶液2在阿魏酸和芦丁对照品溶液斑点处无对应斑点。空白样品溶液3在鸡内金对照药材溶液、阿魏酸和芦丁对照品溶液斑点处均无对应斑点。
采用本发明特定的薄层色谱鉴别条件对小儿复方鸡内金咀嚼片进行鉴别，鉴别方法无空白干扰，成分之间无相互干扰，专属性强，可操作性强。
2.小儿复方鸡内金咀嚼片的含量测定方法
2.1仪器与试药
试剂：甲醇、乙腈、异丙醇为色谱纯，乙醇、冰醋酸为分析纯。中性氧化铝，层析柱。
仪器：Agilent1260高效液相色谱仪；Agilent 1290Infinity ELSD；AL-204型电子分析天平；HQ-500DE型超声波清洗器；DK-96-Ⅱ型恒温水浴锅；真空浓缩装置，(1cm×30cm)层析柱等。
待测样品-小儿复方鸡内金咀嚼片(批号：161022，1.2g/片)：河南太龙药业股份有限公司提供。
阴性样品(不含六神曲)：河南太龙药业股份有限公司提供。
对照品：青蒿素对照品(批号：100202-201606)，对羟基苯甲醛对照品(批号：201613)，苍耳亭对照品(批号：201609)。
2.2方法与结果
(1)供试品溶液的制备：取小儿复方鸡内金咀嚼片，研细粉碎，精密称定10g，置具塞锥形瓶中，精密加入甲醇25ml，密塞，称定重量，超声处理(功率250W，频率40kHz)30分钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，精密量取续滤液10ml，加在中性氧化铝柱(100～120目，重量为10g，内径为1cm)上，用无水乙醇50ml洗脱，收集洗脱液，50℃减压浓缩至干，用色谱纯甲醇适量溶解，转移至2ml容量瓶中，加色谱纯甲醇至刻度，摇匀，滤过，取续滤液，即得供试品溶液。
(2)对照品溶液的制备：取青蒿素、对羟基苯甲醛和苍耳亭对照品适量，精密称定，加色谱纯甲醇制成每1ml含青蒿素0.1mg、对羟基苯甲醛0.2mg、苍耳亭0.1mg的混合对照品溶液。
(3)测定法：分别精密吸取对照品溶液与供试品溶液各20μl，注入液相色谱仪，测定，即得。色谱条件：以十八烷基硅烷键合硅胶为固定相，以甲醇-水-冰醋酸(85∶15∶0.45)为流动相A，以乙腈-异丙醇-水(50:20:30)为流动相B，按下表中的规定进行梯度洗脱，柱温38℃，流动相流速为每分钟1ml；蒸发光散射检测器检测；理论板数以青蒿素计算应不低于6000。
表1
时间(分钟) 流动相A(％) 流动相B(％)
0 0 100
12 0 100
20 30 70
32 85 15
34 15 85
46 15 85
(4)结果本品每片含青蒿素0.016mg，对羟基苯甲醛0.104mg，苍耳亭0.050mg。
2.3含量测定方法学考察
2.3.1线性关系试验
取青蒿素、对羟基苯甲醛和苍耳亭对照品适量，精密称定，加甲醇分别制备混合对照品溶液1-5，每个对照品溶液中青蒿素、对羟基苯甲醛和苍耳亭各自的浓度见表1，分别注入液相色谱仪，按照2.2步骤(3)所述色谱条件进行测定。
以对照品浓度为横坐标(X)，以峰面积为纵坐标(Y)进行线性回归，分别得青蒿素、对羟基苯甲醛、苍耳亭线性回归方程。
结果青蒿素在1.5ug～24ug范围内呈良好的线性关系，线性回归方程为Y＝5004X-39396；对羟基苯甲醛在1ug～16ug范围内呈良好的线性关系，线性回归方程为Y＝9426.5x+33214；苍耳亭在0.5ug～8ug范围内呈良好的线性关系，线性回归方程方程为Y＝3118.9x-2778.1。结果分别见表2和图1、图2、图3。
表2
Figure PCTCN2018100339-appb-000001
Figure PCTCN2018100339-appb-000002
2.3.2专属性
阴性样品溶液的制备：取阴性样品(不含六神曲)，按照2.2.步骤(1)供试品制备方法制成缺少六神曲的阴性样品溶液。
分别精密吸取2.2步骤(1)所得供试品溶液、步骤(2)所得对照品溶液、及缺少六神曲的阴性供试品溶液各20ul，注入液相色谱仪，按照2.2步骤(3)所述色谱条件进行测定。
结果：供试品溶液在与对照品溶液保留时间相同处有相应的色谱峰；缺少六神曲的阴性供试品溶液在与对照品溶液保留时间相同处没有相应的色谱峰。表明所检测的成分较突出的即来源于六神曲，该方法专属性强。
2.3.3精密度
按照2.2步骤(1)所述方法制备小儿复方鸡内金咀嚼片供试品溶液，重复进样6次注入液相色谱仪，按照2.2步骤(3)所述色谱条件分别进行测定，结果见表3。
表3精密度结果
Figure PCTCN2018100339-appb-000003
由以上结果可知，本发明的小儿复方鸡内金咀嚼片的含量测试方法具有良好的精密度。
2.3.4待测样品溶液稳定性
按照2.2步骤(1)制备方法制备供试品溶液，在室温条件下，将供试品溶液放置0、2、4、6、8、12、24h时间，分别于上述时间点，按照2.2步骤(3)所述色谱条件测定样品中青蒿素、对羟基苯甲醛、苍耳亭含量，并计算RSD值。结果见表4。
表4小儿复方鸡内金咀嚼片含量测定样品溶液稳定性试验
Figure PCTCN2018100339-appb-000004
结果表明，本发明方法制备得到的供试品溶液在室温下，24小时内稳定性良好。
2.3.5重现性试验
取小儿复方鸡内金咀嚼片161022批六份，按照2.2步骤(1)的方法制备供试品溶液，按照2.2步骤(3)所述色谱条件，分别测定含量。结果见表5。
表5小儿复方鸡内金咀嚼片含量测定重现性试验
Figure PCTCN2018100339-appb-000005
Figure PCTCN2018100339-appb-000006
结果表明，本发明的含量测定方法重现性良好。
2.3.6回收率试验
分别精密称取已知含量(青蒿素0.151mg/片，对羟基苯甲醛0.093mg/片，苍耳亭0.0487mg/片)的小儿复方鸡内金咀嚼片样品六份，均按照2.2步骤(1)的方法制成六份供试品溶液。精密称取青蒿素对照品、对羟基苯甲醛对照品、苍耳亭对照品适量，制成每1ml含青蒿素0.2521mg，对羟基苯甲醛0.1559mg，苍耳亭0.0813mg的对照品混合溶液100ml，分别向六份样品中各加入对照品2ml。按照2.2步骤(3)所述色谱条件进行含量测定，并按照《中国药典》2015年版四部9101药品质量标准分析方法验证指导原则进行回收率计算。结果见表6、7、8。
计算公式：
Figure PCTCN2018100339-appb-000007
表6青蒿素加样回收率结果
Figure PCTCN2018100339-appb-000008
Figure PCTCN2018100339-appb-000009
表7对羟基苯甲醛加样回收率结果
Figure PCTCN2018100339-appb-000010
表8苍耳亭加样回收率结果
Figure PCTCN2018100339-appb-000011
综上所述，本发明小儿复方鸡内金咀嚼片的含量测定方法专属性强，精密度高，重现性好，能够有效控制产品质量。
对比例1-6
按照实施例1中1.2所述方法对小儿复方鸡内金咀嚼片进行鉴别，所不同的是，将实施例1所用展开剂分别采用以下展开剂进行替换：
a.乙酸乙酯-甲酸-水(8:1：1)
b.三氯甲烷-丙酮(95:5)
c.正丁醇-冰醋酸-水(7:1:1)
d.正丁醇-冰醋酸-水(8:2:1)
e.正丁醇-乙醇-冰醋酸-水(4:1:1:2)
f.三氯甲烷-乙酸乙酯-甲醇-甲酸(40:5:10:0.2)
结果在b展开剂的条件下有阴性干扰，在a、c、d、e、f展开剂条件下斑点无法彻底有效分离。
对比例7-10
按照实施例1中2.2所述方法对小儿复方鸡内金咀嚼片进行含量测定，所不同的是，将实施例1所用流动相以及洗脱条件分别采用以下流动相以及洗脱条件进行替换：
a.以十八烷基硅烷键合硅胶为填充剂，以甲醇-水-冰醋酸(85∶15∶0.45)为流动相A，以正己烷-异丙醇-水(20:50:30)为流动相B，按下表中的规定进行梯度洗脱。
时间(分钟) 流动相A(％) 流动相B(％)
0 0 100
10 0 100
60 100 0
b.以十八烷基硅烷键合硅胶为填充剂，甲醇为流动相A，以0.4％磷酸为流动相B，按下表中的规定进行梯度洗脱。
时间(分钟) 流动相A(％) 流动相B(％)
0 100 0
10 80 20
60 55 45
c.以十八烷基硅烷键合硅胶为填充剂，乙腈：0.2％乙酸(60:40)为流动相。
d.以十八烷基硅烷键合硅胶为填充剂，以甲醇-水-冰醋酸(85∶15∶0.45)为流动相A，以乙腈-异丙醇-水(50:20:30)为流动相B，按下表中的规定进行梯度洗脱。
时间(分钟) 流动相A(％) 流动相B(％)
0 0 100
10 0 100
60 100 0
在以上a-c流动相情况下，分别进行对照品溶液和供试品溶液的检测，结果方法b和c均有未显现出的主峰，方法a的保留时间比较晚，流动相跨度梯度大，且峰形不佳。在方法d情况下，各成分主峰虽明显，但峰形欠佳。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。

Claims (11)

  1. 一种小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，所述方法包括：
    (一)采用薄层色谱法进行鉴别的步骤，其中，
    所述薄层色谱的条件为：以硅胶G薄层板为检测用薄层色谱板，以三氯甲烷-乙酸乙酯-甲醇-水(12:3:8:1)10℃以下放置的下层液为展开剂；
    (二)采用高效液相色谱法进行含量测定的步骤，其中，
    所述高效液相色谱的条件为：以十八烷基硅烷键合硅胶为固定相，以甲醇-水-冰醋酸按体积比(85:15:0.45)为流动相A，以乙腈-异丙醇-水按体积比(50:20:30)为流动相B，按照下述条件进行梯度洗脱：
    时间(分钟) 流动相A(％) 流动相B(％) 0 0 100 12 0 100 20 30 70 32 85 15 34 15 85 46 15 85
  2. 根据权利要求1所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(一)所述的鉴别步骤还包括将小儿复方鸡内金咀嚼片采用大孔吸附树脂柱层析的步骤。
  3. 根据权利要求2所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，所述大孔吸附树脂柱层析的步骤包括：将小儿复方鸡内金咀嚼片与质量分数为75％甲醇溶液按照重量体积比4:25混合，超声处理，然后采用大孔吸附树脂柱，优选采用AB-8大孔吸附树脂柱进行层析，洗脱吸附平衡后，以质量分数为70％的乙醇溶液为洗脱剂对大孔吸附树脂柱进行洗脱，收集洗脱液后蒸干，加分析纯的甲醇溶液。
  4. 根据权利要求1-3任一项所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(一)所述的鉴别步骤以鸡内金-甲醇溶液为对照药材溶液，以阿魏酸-芦丁-甲醇溶液为对照品溶液。
  5. 根据权利要求1-4中任一项所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(一)所述薄层色谱法进行鉴别的步骤包括：
    (1)将小儿复方鸡内金咀嚼片按所述大孔吸附树脂柱层析的步骤制得供试品溶液；
    (2)将鸡内金对照药材按所述大孔吸附树脂柱层析的步骤制得鸡内金-甲醇对照药材溶液；
    (3)取阿魏酸对照品和芦丁对照品，加甲醇制得阿魏酸-芦丁-甲醇对照品溶液；
    (4)吸取上述三种溶液，按照所述薄层色谱条件进行鉴别。
  6. 根据权利要求5所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(一)所述薄层色谱法进行鉴别的步骤包括：
    (1)将小儿复方鸡内金咀嚼片粉碎，将其与质量分数为75％甲醇溶液按照重量体积比为4:25混合，超声处理30min，滤过，上AB-8大孔吸附树脂柱层析，以水洗至水洗液近无色，然后依次采用质量分数为35％乙醇、70％乙醇洗脱，收集70％乙醇洗脱液，蒸干，加甲醇使之溶解，即得供试品溶液；
    (2)取鸡内金对照药材，按步骤(1)的方法制得鸡内金-甲醇对照药材溶液；
    (3)取阿魏酸对照品和芦丁对照品，加甲醇制成每1ml含阿魏酸0.5mg，芦丁0.5mg的混合溶液作为对照品溶液；
    (4)吸取上述三种溶液，分别点于高效硅胶G薄层板上，以三氯甲烷-乙酸乙酯-甲醇-水按体积比12:3:8:1为展开剂展开，取出晾干，喷以质量分数为10％的硫酸乙醇溶液，加热105℃至斑点显色清晰，分别在日光和紫外光(365nm)下检视。
  7. 根据权利要求1-6任一项所述的小儿复方鸡内金咀嚼片的鉴别和测定方法，其特征在于，步骤(二)所述的含量测定步骤还包括将小儿复方鸡内金咀嚼片采用中性氧化铝柱进行纯化的步骤。
  8. 根据权利要求7所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，所述中性氧化铝柱进行纯化的步骤包括：将小儿复方鸡内金咀嚼片与色谱纯甲醇按照重量体积比为1:2.5混合，超声处理，通过中性氧化铝柱纯化，以无水乙醇溶液为洗脱剂对中性氧化铝柱进行洗脱，收集洗脱液蒸干，加色谱纯甲醇。
  9. 根据权利要求1-8任一项所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(二)所述的含量测定步骤以青蒿素-对羟基苯甲醛-苍耳亭-甲醇 溶液为对照品溶液。
  10. 根据权利要求1-9任一项所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(二)所述高效液相色谱法进行含量测定的步骤包括：
    (1)将小儿复方鸡内金咀嚼片按所述中性氧化铝柱进行纯化的步骤制得供试品溶液；
    (2)取青蒿素、对羟基苯甲醛和苍耳亭对照品，加甲醇制成青蒿素-对羟基苯甲醛-苍耳亭-甲醇的混合溶液为对照品溶液；
    (3)吸取上述两种溶液，注入高效液相色谱仪，按照所述高效液相色谱条件进行含量测定。
  11. 根据权利要求1-10任一项所述的小儿复方鸡内金咀嚼片的鉴别和含量测定方法，其特征在于，步骤(二)所述高效液相色谱法进行含量测定的步骤包括：
    (1)将小儿复方鸡内金咀嚼片粉碎，将其与色谱纯甲醇溶液按照重量体积比为1:2.5混合，超声处理30分钟，放冷，滤过，通过中性氧化铝柱(100～120目)纯化，用无水乙醇洗脱，收集洗脱液，减压浓缩，用色谱纯甲醇溶解，滤过，即得供试品溶液；
    (2)取青蒿素、对羟基苯甲醛和苍耳亭对照品，加甲醇制成每1ml含青蒿素0.3mg、对羟基苯甲醛0.2mg、苍耳亭0.1mg的混合溶液为对照品溶液；
    (3)吸取上述两种溶液，注入高效液相色谱仪测定，所述高效液相色谱条件为：以十八烷基硅烷键合硅胶为固定相，色谱柱柱温为38℃，以甲醇-水-冰醋酸按体积比(85:15:0.45)为流动相A，以乙腈-异丙醇-水按体积比(50:20:30)为流动相B，按照下述条件进行梯度洗脱；所述流动相的流速为1ml/min；
    Figure PCTCN2018100339-appb-100001
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