WO2013056572A1 - 中成药和保健食品中掺杂褪黑素的快速测定方法 - Google Patents

中成药和保健食品中掺杂褪黑素的快速测定方法 Download PDF

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WO2013056572A1
WO2013056572A1 PCT/CN2012/078069 CN2012078069W WO2013056572A1 WO 2013056572 A1 WO2013056572 A1 WO 2013056572A1 CN 2012078069 W CN2012078069 W CN 2012078069W WO 2013056572 A1 WO2013056572 A1 WO 2013056572A1
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melatonin
solution
dimethylaminocinnamaldehyde
chinese patent
added
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PCT/CN2012/078069
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English (en)
French (fr)
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王铁杰
鲁艺
肖丽和
关潇滢
韩东岐
殷果
王珏
黎雪清
闫研
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深圳市药品检验所
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Priority to US14/346,532 priority Critical patent/US9005986B2/en
Publication of WO2013056572A1 publication Critical patent/WO2013056572A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the invention relates to a method for detecting a melatonin component, in particular to a method for detecting a Chinese patent medicine and a health food, in particular for detecting whether or not melatonin is added to a sedative and hypnotic Chinese patent medicine and a health food.
  • Melatonin also known as pineal voxel, is chemically known as N-acetyl-5-methoxytryptamine, a steroid hormone secreted by the pineal gland in the human brain. Its secretion decreases as the light increases, so it is also called dark hormone. Melatonin is mainly used to regulate the body's wakefulness and sleep cycle, induce physiological sleep, and anti-aging.
  • Melatonin is an endogenous hormone. Studies have shown that the use of melatonin in large amounts may cause respiratory problems and produce a hangover effect, while melatonin may affect the action of certain sedative sleeping drugs such as clonazepam. In Europe, the cycle of melatonin, the population and dosage are strictly limited. In the United States, melatonin is marketed as a dietary supplement, and its instructions are clearly labeled and used. In China, melatonin is used as a health food. There are clear legal provisions: daily doses are l ⁇ 3mg, and other ingredients other than vitamin B6 should not be added. The precautions should indicate the driving, mechanical operation or dangerous operation. Do not use it with caution before or during operation.
  • the components to be tested in the sample are extracted by methanol, and the obtained test solution and the reference solution are spotted on a thin layer of silicone glue, developed with a developing agent, taken out, dried, and placed under ultraviolet light at 254 nm for inspection.
  • a developing agent develops different substances in the form of spots in different positions of the thin-layer chromatography plate due to the different migration speeds of different substances, and the experiment results according to whether the sample to be tested is spotted at the position corresponding to the reference substance.
  • the phenomenon is to determine whether the sample to be tested contains the target component. If the thin layer chromatogram of the test solution shows spots on the corresponding position of the melatonin reference, it may indicate that the sample may contain melatonin.
  • the advantage of thin layer chromatography is that it does not require expensive analytical instruments.
  • the disadvantages of this method are: (1) The chromatographic resolution is low, the composition of traditional Chinese medicine is complex, and there are many interference factors. It is easy to misjudge some coexisting components as melatonin. Some coexisting components have large amounts and chromatographic migration values. If the components to be tested are close, they will interfere with the detection of the target component. (2) The chromatographic development and drying time is long enough to meet the requirements of rapid measurement. (3) The TLC plate needs to be dried and stored dry. The ambient temperature and humidity have a great influence on the separation effect. (4) It is necessary to have a fixed place, and it is not suitable for on-site inspection with high liquidity. (5) The experimenter is required to have good experience.
  • High performance liquid chromatography is a commonly used modern analytical method. Different materials have different chromatographic retention times under the same chromatographic conditions. Under the same chromatographic conditions, the melatonin reference solution and the sample solution obtained from the sample extraction are separately injected, and the sample to be tested contains whether the sample to be tested contains melatonin.
  • the advantages of high performance liquid chromatography are high chromatographic separation efficiency and high sensitivity. Disadvantages are: (1) the instrument is expensive, the sample preparation time is long, the column is susceptible to contamination, and the analysis cost is high; (2) it is easy to make mistakes when the coexisting peak retention time is close to the melatonin peak retention time. (3) The instrument has high requirements on the use environment and needs to be fixedly placed, which is not suitable for on-site inspection with high fluidity.
  • High performance liquid chromatography-mass spectrometry with high performance liquid chromatography as a separator and mass spectrometer as an analyzer is suitable for sample analysis with complex composition and severe background interference. This analysis method can improve the detection junction. The reliability of the fruit.
  • High performance liquid chromatography-mass spectrometry is an analytical method that is more expensive to analyze than HPLC and more complex to operate.
  • the instrument has higher requirements on the use environment and needs to be fixedly placed, which is not suitable for on-site inspection with high fluidity. These tests can only be carried out in a small number of laboratories and are not easy to promote.
  • the object of the present invention is to provide a defect that overcomes the existing detection technology, has low analysis cost, does not require the use of valuable analytical instruments, has strong specificity, high sensitivity, and is suitable for on-site inspection of whether Chinese medicine and health foods are added with melatonin. Quick test method.
  • Another object of the present invention is to provide an application of the above method for preparing a melatonin rapid test kit.
  • a further object of the present invention is to provide an application of the above method for screening or screening for the addition of melatonin in Chinese medicines and health foods.
  • the rapid determination method for doping melatonin in the medicine and health food of the invention comprises the following steps:
  • the melatonin chemical name is N-acetyl-5-methoxytryptamine, which belongs to the guanamine hormone. It has been found through experiments that after adding a solution of p-dimethylaminocinnamaldehyde hydrochloride to the melatonin solution, the solution becomes In blue and green.
  • p-dimethylaminocinnamaldehyde hydrochloride ethanol solution Take 0.01-0.1 g of p-dimethylaminocinnamaldehyde and add 10 ml of a 40% ⁇ 70% hydrochloric acid ethanol solution to dissolve it.
  • the concentration of the hydrochloric acid ethanol solution is preferably 50% to 60%; the amount of the dimethylaminocinnamaldehyde added and the addition volume of the hydrochloric acid ethanol solution can be selected and adjusted according to the actual situation, so that the finally obtained p-dimethylaminocinnamaldehyde
  • the concentration of the hydrochloric acid ethanol solution is 1 to 10 mg/ml.
  • the hydrochloric acid-ethanol ratio is preferably in the range of 1.5:1 to 1:1, and preferably the hydrochloric acid-ethanol ratio is 1:1 to prepare a color developing agent.
  • the color development effect of the concentration of p-dimethylaminocinnamaldehyde is 4 to 6 mg/ml, and the concentration of the aqueous solution of dimethylaminocinnamaldehyde in ethanol is preferably 4 mg/ml.
  • a melatonin rapid test kit can be prepared.
  • the present invention can be applied to a screening test in which a proprietary Chinese medicine and a health food are incorporated into melatonin. Compared with the prior art, the present invention has the following beneficial effects:
  • Sensitive reaction The minimum detection limit of the substance to be tested in this experiment reaches 2% of the clinical minimum effective dose (the average sample detection limit is 0.025 mg/ml), so this method is used to check Chinese patent medicines and health foods. Whether or not the melatonin component is added has sufficient sensitivity.
  • This method can be applied to liquid samples and solid samples. This method can be used for both traditional Chinese medicine preparations and health food products.
  • Example 1 Commercial health food A (capsule, labeled as 1 to 2 capsules per time, high performance liquid chromatography detection results, this product contains melatonin lmg) Take one of the contents of this product, placed 2 ml of ethyl acetate was added to the sample tube, shaken for 1 min, filtered, and the filtrate was used as a test solution; 0.04 g of p-dimethylaminocinnamaldehyde was added, and 10 ml of a 50% hydrochloric acid ethanol solution was added to dissolve it, as a color developing reagent; To the test solution, 1 ml of p-dimethylaminocinnamaldehyde hydrochloride solution was added, and the solution turned blue-green within 30 s, thereby judging that the sample contained illegally added melat
  • Example 2 Commercially available health food B (capsule, labeled as 1 ⁇ 2 capsules per time, high performance liquid chromatography detection results, this product contains melatonin 0.9mg) Take one of the contents of this product, placed in the sample tube 3 ml of ethyl acetate was added, shaken for 1 min, filtered, and the filtrate was used as a test solution; 0.08 g of p-dimethylaminocinnamaldehyde was added, and 10 ml of a 60% hydrochloric acid ethanol solution was added to dissolve it, as a color developing reagent; 0.5 ml of p-dimethylaminocinnamaldehyde hydrochloride solution was added to the test solution, and the solution turned blue-green within 30 s, thereby judging that the sample contained illegally added melatonin.
  • Example 3 Commercial health food C (capsule, labeled as 1 to 6 capsules per time, high performance liquid chromatography detection results, this product contains melatonin lmg) Take one of the contents of this product, placed In the sample tube, 3 ml of ethyl acetate was added, shaken for 1 min, filtered, and the filtrate was used as a test solution; 0.1 g of p-dimethylaminocinnamaldehyde was added, and 10 ml of a 40% hydrochloric acid ethanol solution was added to dissolve it, as a color developing reagent; 0.5 ml of p-dimethylaminocinnamaldehyde hydrochloride solution was added to the test solution, and the solution turned blue-green within 30 s, thereby judging that the sample contained illegally added melatonin.
  • Example 4 Commercial health food D (capsule, labeled as 2 capsules per time, high performance liquid chromatography detection results, this product contains melatonin 1.8mg per capsule) Take one of the contents of this product, placed in the sample Add 3 ml of ethyl acetate to the tube, shake for 1 min, filter, and use the filtrate as the test solution; take p-dimethylaminocinnamaldehyde O.Olg, add 10 ml of 40% hydrochloric acid ethanol solution, dissolve it, as a color reagent Add 0.5 ml of p-dimethylaminocinnamaldehyde hydrochloride solution to the test solution, and the solution turns blue-green within 30 s, thereby judging that the sample contains illegally added melatonin.
  • Example 5 Commercial health food E (capsule, labeled as 2 capsules per time, high performance liquid chromatography detection results containing 0.9mg of melatonin per capsule) Take one part of the product, place it in the sample tube, add 1 ml of ethyl acetate, shake for 1 min, filter, and use the filtrate as the test solution; take 0.05 g of p-dimethylaminocinnamaldehyde and add 70% hydrochloric acid in ethanol solution.
  • Example 6 Commercial health food F (tablet, labeled as 2 tablets each time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain melatonin component, as a negative control test) Take this product, Grinding, placed in a sample tube, adding 2 ml of ethyl acetate, shaking for 1 min, filtering, and using the filtrate as a test solution; taking 0.04 g of p-dimethylaminocinnamaldehyde, adding 10 ml of a 50% hydrochloric acid solution to dissolve it.
  • Example 7 Commercially available proprietary Chinese medicine G (capsule, labeled as 4 capsules per time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain melatonin, used as a negative control test) Take this product one The contents were placed in a sample tube, 3 ml of ethyl acetate was added, shaken for 1 min, filtered, and the filtrate was used as a test solution; 0.04 g of p-dimethylaminocinnamaldehyde was added, and 10 ml of a 50% hydrochloric acid ethanol solution was added to dissolve it.
  • Example 8 Commercially available proprietary Chinese medicine H (pill, labeled as about 28 pills each time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain melatonin, used as a negative control test) Take a pill of this product, Grinding, placed in a sample tube, add 1 ml of ethyl acetate, shake for 1 min, filter, the filtrate is used as a test solution; take 0.04 g of p-dimethylaminocinnamaldehyde, add 10 ml of 50% hydrochloric acid ethanol solution, Dissolve it as a coloring reagent; add 1 ml of p-dimethylaminocinnamaldehyde hydrochloride solution to the test solution, and the solution does not change color within 30 s, thereby judging that the sample does not contain illegally added melatonin.
  • Example 9 Commercially available proprietary Chinese medicine 1 (oral liquid, the dosage is 10 ⁇ 20ml each time, the result of high performance liquid chromatography-mass spectrometry detection does not contain melatonin component, as a negative control test) Take this product 2ml , placed in a sample tube, add 2ml of ethyl acetate, shake for 1min, after standing, take the ethyl acetate layer, filter, the filtrate as the test solution; take p-dimethylaminocinnamaldehyde 0.04g, add 50% hydrochloric acid 10ml of solution, dissolved, as a coloring reagent; add 1ml of p-dimethylaminocinnamaldehyde hydrochloride solution to the test solution, the solution does not change color within 30s, it can be judged that the sample does not contain illegal addition of melatonin ingredient.
  • Example 10 Commercially available proprietary Chinese medicine J (granules, labeled as 5 g each time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain melatonin, used as a negative control test) Take 0.5g of this product, The mixture was placed in a sample tube, added with 3 ml of ethyl acetate, shaken for 1 min, filtered, and the filtrate was used as a test solution; 0.04 g of p-dimethylaminocinnamaldehyde was added, and 10 ml of a 50% hydrochloric acid ethanol solution was added to dissolve it.
  • the above-mentioned rapid screening method is used for detecting illegally added melatonin components in health products, and has the characteristics of simple operation, strong specificity, high sensitivity, high accuracy, and the like, and is suitable for inspectors to operate on site, Meet the needs of supervision and inspection at the grassroots level.

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Abstract

一种中成药和保健食品中掺杂褪黑素的快速测定方法，包括（1）用乙酸乙酯提取中成药或保健食品中的褪黑素；（2）向提取液中加入对二甲基氨基肉桂酸，观察颜色。该方法快速、简便，专属性强、准确率高，反应灵敏，适用范围广，适用于现场检验中成药和保健食品中是否掺入了褪黑素。

Description

中成药和保健食品中掺杂褪黑素的快速测定方法 本申请要求于 2011 年 10 月 18 日提交中国专利局、 申请号为 201110316788.3、发明名称为"中成药和保健食品中掺杂褪黑素的快速测定方法" 的中国专利申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明涉及褪黑素成分的检测方法， 具体是检测中成药和保健食品中， 尤 其是用于检测镇静安眠类中成药和保健食品中是否添加褪黑素的方法。 背景技术
目前市场上存在许多种解决失眠问题的中成药和保健食品， 宣传其成分主 要为纯植物或者纯中药， 不含有助眠功效的西药成分或者激素类成分等。
褪黑素又称松果体素， 化学名称为 N-乙酰基 -5-甲氧基色胺， 是人体大脑内 松果体分泌的一种吲哚胺类激素。 其分泌随着光照的增加而减少， 故又被称为 黑暗荷尔蒙。 褪黑素主要用于调节人体的清醒和睡眠周期， 诱导生理睡眠， 以 及抗衰老等。
褪黑素是一种内源性激素， 有研究表明， 大量使用褪黑素可能会引发呼吸 问题， 产生宿醉效应， 同时褪黑素会影响某些镇静安眠药物如氯硝西泮的作用。 在欧洲， 褪黑素的服用周期、 适用人群以及剂量均有严格限制。 在美国， 褪黑 素以膳食补充剂的形式销售， 其说明书上需明确标示含量及使用剂量。 在我国， 褪黑素作为保健食品使用， 有明确的法律规定： 日服用剂量为 l〜3mg， 不得添 加除维生素 B6以外的其他成分， 注意事项中应注明从事驾驶、机械作业或危险 操作者， 不要在操作前或操作中食用和自身免疫症 （类风湿等） 及甲亢患者慎 用。 故服用褪黑素最好遵循医嘱， 明确使用剂量， 儿童使用更需要谨慎。 但是， 一些不法分子利用褪黑素的镇静、 催眠的良好作用和中成药和保健食品成分复 杂的特点， 在镇静催眠类中成药和保健食品中擅自添加褪黑素， 夸大宣传功效， 抬高价格； 而使得食用者无法明确意识到自己的服用剂量， 从而引发不良后果， 给人民群众的身体健康造成了严重危害。 特别在广大的农村和经济欠发达的中 小城市， 由于这些地区缺乏必要的检验仪器， 加上技术监管能力薄弱， 给了不 法分子更多的造假和添加之机， 导致大量假药劣药出现， 造成了中药制剂和保 健食品市场的混乱。
检验镇静安神类中成药和保健食品中是否添加褪黑素的现有方法主要有以 下四种：
1、 薄层色谱法
样品中的待测成分经过甲醇提取， 所得的供试品溶液与对照品溶液点在硅 胶薄层板上， 用展开剂展开， 取出， 晾干， 置紫外灯 254 nm下检视。 在展开剂 的作用下， 由于不同物质的迁移速度不同， 不同物质在薄层色谱板的不同位置 以斑点的形式出现， 根据待测样品是否在与对照品相应的位置上出现斑点这一 实验所得的现象来判断待测样品是否含有目标成分。 如果供试品溶液薄层色谱 图与褪黑素对照品相应的位置上出现斑点， 则提示样品中可能含有褪黑素成分。
薄层色谱法的优点是不需要昂贵的分析仪器。 该法缺点是： （1 ) 色谱分辨 率较低， 中药制剂成分复杂， 干扰因素多， 易将某些共存成分误判为褪黑素成 分， 某些共存成分如果量较大且色谱迁移值与待测成分接近， 则会干扰目标成 分的检出。 （2 ) 色谱展开和晾干的时间长， 不足以满足快速测定的要求。 （3 ) 薄层色谱板需要烘干并干燥存放， 环境温度和湿度对分离效果的影响较大。 （4 ) 需要有固定的场所， 不适合作流动性大的现场检测。 （5 ) 要求实验人员有较好 的经验。
2、 高效液相色谱法
高效液相色谱法是常用的现代分析方法， 在相同的色谱条件下， 不同的物 质有不同的色谱保留时间。 在相同的色谱条件下褪黑素对照品溶液与样品提取 所得供试品溶液分别进样， 根据色谱峰保留时间可判断待测样品是否含有褪黑 素成分。
高效液相色谱法的优点是色谱分离效率高、 灵敏度高。 缺点是： （1 ) 仪器 价格昂贵， 样品前处理时间长， 色谱柱易受污染， 分析成本高； （2 ) 当共存的 色谱峰保留时间与褪黑素色谱峰保留时间接近时容易做出错误的判断； （3 ) 仪 器对使用环境的要求高， 需要固定放置， 不适合流动性大的现场检测。
3、 高效液相色谱 -质谱联用法
以高效液相色谱仪作为分离器、 质谱仪作为分析器的高效液相色谱-质谱联 用法适合于成分复杂、 背景干扰严重的样品分析， 这种分析方法可提高检测结 果的可靠性。
高效液相色谱-质谱联用法是一种分析成本比高效液相色谱更高、 操作更加 复杂的分析方法。 仪器对使用环境的要求更高， 需要固定放置， 不适合做流动 性大的现场检测。 这些检验只能在少数实验室中进行， 不易推广使用。
目前中成药和保健食品行业尚无利用理化反应快速筛查褪黑素的方法发 布， 对于药店和基层药检单位， 开发中成药和保健食品中擅自添加褪黑素的快 速筛查方法， 在监管现场对样品进行快速分析， 为中成药和保健食品监管及时 提供证据， 打击不法分子的造假行为和保障人民群众的用药安全是非常必要的。 发明内容
本发明的目的在于提供一种克服现有检测技术的缺陷， 分析成本低、 不需 要使用贵重分析仪器、 专属性强、 灵敏度高、 适用于现场检验中成药和保健食 品中是否添加褪黑素的快速检验方法。
本发明的另一目的在于提供上述方法在制备褪黑素快速检验试剂盒的应 用。
本发明的再一目的在于提供上述方法在检验中成药和保健食品中是否添加 褪黑素的筛查检验中的应用。
本发明中成药和保健食品中掺杂褪黑素的快速测定方法， 依次由以下歩骤 组成：
( 1 ) 用乙酸乙酯提取中成药和保健食品中的褪黑素；
(2 ) 加入显色剂对二甲基氨基肉桂醛。
褪黑素化学名称为 N-乙酰基 -5-甲氧基色胺， 属于吲哚胺类激素， 经过试验 发现， 向褪黑素溶液中加入对二甲基氨基肉桂醛盐酸乙醇溶液后， 溶液变成蓝 绿色。
Figure imgf000004_0001
具体的技术方案如下：
1、 取固态的中成药或保健食品， 研细， 加入 l〜3ml乙酸乙酯， 振摇 lmin, 过滤， 作为供试液； 或量取液态的中成药或保健食品， 加入 l〜3ml 乙酸乙酯， 振摇 lmin, 静置后取乙酸乙酯层， 过滤， 作为供试液。
针对目前市场上的大多数中成药和保健食品， 推荐取样量如下：
Figure imgf000005_0001
2、 对二甲基氨基肉桂醛盐酸乙醇溶液的配制： 取对二甲基氨基肉桂醛 0.01〜0.1g， 加入质量浓度为 40%〜70%的盐酸乙醇溶液 10ml， 使之溶解， 即得。 其中， 盐酸乙醇溶液浓度优选 50%〜60%； 对二甲基氨基肉桂醛的加入量和盐酸 乙醇溶液的加入体积可以根据实际情况进行选择和调整， 使最终得到的对二甲 基氨基肉桂醛盐酸乙醇溶液的浓度为 l〜10mg/ml。
其中，盐酸 -乙醇比例以 1.5: 1至 1： 1的范围为佳，优选盐酸 -乙醇比例为 1： 1配制显色剂。 以对二甲基氨基肉桂醛浓度为 4〜6mg/ml的显色效果为佳， 优选 二甲基氨基肉桂醛的盐酸乙醇溶液浓度为 4mg/ml。
3、 向供试液中加入 0.5〜lml的对二甲基氨基肉桂醛盐酸乙醇溶液， 如果溶 液迅速变为蓝绿色（出现明显蓝色至绿色），则可判断样品含有褪黑素；如果 30s 后仍未见到明显蓝绿色（未出现明显蓝色至绿色），则可判断样品不含有褪黑素。
根据本发明， 可以制成褪黑素快速检验试剂盒。
根据本发明， 可以应用于中成药和保健食品是否掺入褪黑素的筛查检验中。 同现有技术相比， 本发明具有如下有益效果：
( 1 ) 快速、 简便： 提取过程 1分钟、 显色反应 30秒， 整个操作过程在 2 分钟内完成，相对于液相色谱、液质联用方法（至少 2-3个小时）简便快捷得多， 并且无需对照品。 (2 ) 专属性强、 准确率高： 根据方法验证结果， 片剂、 胶囊剂、 丸剂、 颗 粒剂、 口服剂等剂型的常用辅料对褪黑素物质的检出均无干扰。
(3 ) 反应灵敏： 本实验待检物质的最低检出限达到临床最低有效剂量的二 百分之一 （平均样品检出限为 0.025mg/ml) ， 因此用该方法检查中成药和保健 食品中是否添加褪黑素成分具有足够的灵敏度。
(4 )适用范围广： 该方法可以用于液体样品和固体样品， 无论是中药制剂、 保健食品均可使用本方法。
( 5 ) 经济实用： 本方法样品平均成本较低， 适宜于市场的普及与大规模生 产。 本发明检测褪黑素的方法， 也适用于其它具有治疗失眠、 辅助睡眠功效的 食品中掺入褪黑素的快速检验。
具体实施方式 以下实施例将有助于本领域的普通技术人员进一歩理解本发明， 但不以任 何形式限制本发明。 实施例 1 市售保健食品 A (胶囊剂， 标示服用量为每次 1〜2粒， 高效液相色谱法检 测结果本品每粒含褪黑素 lmg) 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 2ml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐酸乙醇溶液 10ml, 使之溶解，作为显色试剂；向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 lml， 溶液在 30s内变为蓝绿色， 由此可判断样品中含有非法添加褪黑素成分。 实施例 2 市售保健食品 B (胶囊剂， 标示服用量为每次 1〜2粒， 高效液相色谱法检 测结果本品每粒含褪黑素 0.9mg) 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 3ml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.08g， 加入 60%盐酸乙醇溶液 10ml, 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 0.5ml, 溶液在 30s内变为蓝绿色， 由此可判断样品中含有非法添加褪黑素成分。 实施例 3 市售保健食品 C (胶囊剂， 标示服用量为每次 1〜6粒， 高效液相色谱法检 测结果本品每粒含褪黑素 lmg) 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 3ml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.1g， 加入 40%盐酸乙醇溶液 10ml， 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 0.5ml, 溶液在 30s内变为蓝绿色， 由此可判断样品中含有非法添加褪黑素成分。 实施例 4 市售保健食品 D (胶囊剂， 标示服用量为每次 2粒， 高效液相色谱法检测 结果本品每粒含褪黑素 1.8mg) 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 3 ml, 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 O.Olg, 加入 40%盐酸乙醇溶液 10ml, 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 0.5ml, 溶液在 30s内变为蓝绿色， 由此可判断样品中含有非法添加褪黑素成分。 实施例 5 市售保健食品 E (胶囊剂， 标示服用量为每次 2粒， 高效液相色谱法检测结 果本品每粒含褪黑素 0.9mg) 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 1 ml, 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.05g， 加入 70%盐酸乙醇溶液 10ml, 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 0.6ml, 溶液在 30s内变为蓝绿色， 由此可判断样品中含有非法添加褪黑素成分。 实施例 6 市售保健食品 F (片剂， 标示服用量为每次 2片， 高效液相色谱-质谱法检 测结果本品不含褪黑素成分， 作为阴性对照试验用） 取本品一片， 研碎， 置于样品管中， 加入乙酸乙酯 2ml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐酸乙醇溶液 10ml, 使之溶解，作为显色试剂；向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 lml， 溶液在 30s内没有变色， 由此可判断样品中不含有非法添加褪黑素成分。 实施例 7 市售中成药 G (胶囊剂， 标示服用量为每次 4粒， 高效液相色谱-质谱法检 测结果本品不含褪黑素成分， 作为阴性对照试验用） 取本品一粒内容物， 置于样品管中， 加入乙酸乙酯 3ml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐酸乙醇溶液 10ml, 使之溶解，作为显色试剂；向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 lml， 溶液在 30s内没有变色， 由此可判断样品中不含有非法添加褪黑素成分。 实施例 8 市售中成药 H (丸剂， 标示服用量为每次约 28丸， 高效液相色谱-质谱法检 测结果本品不含褪黑素成分， 作为阴性对照试验用） 取本品一丸， 研碎， 置于样品管中， 加入乙酸乙酯 lml， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐酸乙醇溶液 10ml, 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 1 ml, 溶液在 30s内没有变色， 由此可判断样品中不含有非法添加褪黑素成分。 实施例 9 市售中成药 1 (口服液， 标示服用量为每次 10〜20ml， 高效液相色谱-质谱法 检测结果本品不含褪黑素成分， 作为阴性对照试验用） 取本品 2ml， 置于样品管中， 加入 2ml乙酸乙酯， 振摇 lmin, 静置后取乙 酸乙酯层， 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐 酸乙醇溶液 10ml， 使之溶解， 作为显色试剂； 向供试液中加对二甲基氨基肉桂 醛盐酸乙醇溶液 lml， 溶液在 30s内没有变色， 由此可判断样品中不含有非法添 加褪黑素成分。 实施例 10 市售中成药 J (颗粒剂， 标示服用量为每次 5g， 高效液相色谱 -质谱法检测 结果本品不含褪黑素成分， 作为阴性对照试验用） 取本品 0.5g， 研碎， 置于样品管中， 加入 3ml乙酸乙酯， 振摇 lmin, 过滤， 滤液作为供试液； 取对二甲基氨基肉桂醛 0.04g， 加入 50%盐酸乙醇溶液 10ml, 使之溶解，作为显色试剂；向供试液中加对二甲基氨基肉桂醛盐酸乙醇溶液 lml， 溶液在 30s内没有变色， 由此可判断样品中不含有非法添加褪黑素成分。 快速筛查方法评价
收集具有镇静安神功效的中成药、 改善睡眠功能的保健食品以及标示具有 上述作用的健康产品共 80批样品供实验用， 检测结果以相关验证方法进行了验 证， 结果见表 1 :
表 1 验证结果表
快速筛查结果 最终确证结果 合计
添加 未添加
阳性 真阳性 A (25 ) 假阳性 B ( 1 ) 26 阴性 假阴性 C (0) 真阴性 D ( 54 ) 54 合计 25 55 80
根据广东省药品检验所制定的 《中成药和保健食品中擅自添加化学成分的 快速筛查方法指导原则和技术要求 （试行）》 的要求， 对快速筛查方法总结， 见 表 2:
表 2 快速筛查方法总结表
Figure imgf000010_0001
可以看出， 采用上述快速筛查方法对健康产品中非法添加的褪黑素成分进 行检测， 具有操作简单、 专属性强、 灵敏度高、 准确度高等特点， 适用于检验 人员在现场进行操作， 能够满足基层的监督检验需要。

Claims

权 利 要 求
1、 一种中成药和保健食品中掺杂褪黑素的快速测定方法， 依次由以下歩骤 组成：
( 1 ) 用乙酸乙酯提取中成药或保健食品中的褪黑素；
(2 ) 向提取液中加入对二甲基氨基肉桂醛， 观察颜色。
2、 如权利要求 1所述的测定方法， 其特征在于， 所述对二甲基氨基肉桂醛 用盐酸乙醇溶液溶解。
3、 如权利要求 2所述的测定方法， 其特征在于， 所述盐酸乙醇溶液的浓度 为 40%〜70%。
4、 如权利要求 2或 3所述的测定方法， 其特征在于， 所述对二甲基氨基肉 桂醛盐酸乙醇溶液的浓度为 l〜10mg/ml。
5、 如权利要求 1所述的测定方法， 依次由以下歩骤组成：
( 1 ) 取中成药或保健食品， 研细， 加入乙酸乙酯， 振荡， 过滤， 滤液作为 供试液；
(2 )取对二甲基氨基肉桂醛 0.01〜0.1g，加入 40%〜70%盐酸乙醇溶液 10ml, 使之溶解， 作为显色试剂；
(3 ) 向所述供试液中加入对二甲基氨基肉桂醛盐酸乙醇溶液显色试剂， 观 察颜色。
6、 权利要求 1所述的测定方法在制备褪黑素快速检验试剂盒中的应用。
7、 权利要求 1所述的测定方法在中成药和保健食品是否掺入褪黑素的筛查 检验中的应用。
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