WO2011153861A1

WO2011153861A1 - Method for quickly detecting benzene diazepine doped in medicine and health care food

Info

Publication number: WO2011153861A1
Application number: PCT/CN2011/072085
Authority: WO
Inventors: 王铁杰; 肖丽和; 王珏; 付春旺; 钟敏; 闫研; 关潇滢
Original assignee: 深圳市药品检验所
Priority date: 2010-06-08
Filing date: 2011-03-23
Publication date: 2011-12-15
Also published as: CN101881736B; CN101881736A

Abstract

A method for quickly detecting benzene diazepine doped in a medicine and health care food consists of the following steps: (1) extracting benzene diazepine from a medicine and health care food; (2) concentrating the extracted liquid; (3) hydrolyzing the extracted benzene diazepine;(4) adding a diazotization agent; (5) neutralizing the interference matter; and (6) adding a detection agent.

Description

Rapid determination method for doping benzodiazepines in medicines and health foods This application claims to be submitted to the Chinese Patent Office on June 8, 2010, the application number is 201010195399.5, and the invention name is "dosing benzene in medicines and health foods". Priority of the Chinese Patent Application for the Rapid Determination of Diazepines, the entire contents of which are incorporated herein by reference. Technical field

The invention relates to a method for detecting benzodiazepines, in particular to detecting medicines and health foods, in particular for detecting benzodiazepines and traditional Chinese medicine preparations and nourishing heart and tranquilizing functional health foods. Method of chemical composition.

Background technique

The chemical constituents of benzodiazepines are a class of sedative hypnotics, as well as anti-anxiety, central muscle relaxation, anticonvulsant, anti-tremor and the like. The commonly used chemical components of benzodiazepines are nitrazepam, clonazepam, lorazepam, oxazepam, estazolam and chlordiazepine. Clinical pharmacology studies have shown that these drugs are significantly different from barbiturates and other sedative and hypnotic drugs in sedation and hypnosis, and that drug overdose is generally not life-threatening, so benzodiazepines have been replaced at present. Other drugs become the drug of choice for sedation, hypnosis, and anxiety.

Benzodiazepines are central nervous system inhibitors that cause inhibition of different parts of the central nervous system. The mechanism of action and its selective action on the limbic system, combined with the central benzodiazepine receptor, promotes gamma-aminobutyric acid. The release of inhibitory neurotransmitters, which leads to presynaptic and post-synaptic inhibition, has sedative, hypnotic, and anxiolytic effects. However, long-term use can produce tolerance and dependence, and common adverse reactions such as sleepiness, weakness, headache, dizziness, nausea, constipation, and even rash, liver damage, and myelosuppression.

The Chinese and Western medicine compound preparation combines the conditioning of traditional Chinese medicine with the quick-acting effect of western medicine, which is conducive to comprehensive control of the disease. The synergy between Chinese medicine and western medicine should undergo strict pharmacological and clinical verification. Due to the complex composition of traditional Chinese medicines, some of them may chemically react with certain western medicine ingredients, and the unauthorized addition of western medicine ingredients in traditional Chinese medicine may cause unpredictable adverse reactions. Benzodiazepines are prescription drugs, patients should be used under the guidance of doctors, and the State Drug Administration has strict regulations on its production, management and use. However, some lawless elements use benzodiazepines for sedation, hypnosis, and anti-focal Considering the good effect and the complex composition of Chinese patent medicines and health foods, and the small side effects, the benzodiazepine chemical composition is added to the sedative and hypnotic Chinese medicine preparations and health foods, exaggerating the publicity effect, raising the price, giving the people The physical health of the masses has caused serious harm. Especially in the vast rural and economically underdeveloped small and medium-sized cities, the lack of necessary testing equipment in these areas, coupled with the weak technical supervision ability, has given criminals more opportunities for fraud, resulting in the emergence of a large number of counterfeit drugs. This has caused confusion in the Chinese medicine preparation and health food market.

There are three main methods for testing whether chemical substances in benzodiazepines and health foods are added to benzodiazepines:

1, thin layer chromatography

The test component in the sample is extracted by decyl alcohol, and the obtained test solution and the reference solution are spotted on a silica gel GF254 thin layer plate, and dichlorosilane-anthracene-acetone-nonanol (8:8:3: 1) As a developing agent, unroll, remove, dry, and place the UV lamp at 254 nm for inspection. Under the action of the developing agent, due to the different migration speeds of different substances, different substances appear in the form of spots at different positions of the thin layer color plate, depending on whether the sample to be tested appears in the position corresponding to the reference substance. The phenomenon obtained by the experiment determines whether the sample to be tested contains the target component. If the thin-layer chromatogram of the test solution corresponds to the chemical constituents of benzodiazepines such as nitrazepam, clonazepam, lorazepam, oxazepam, estazolam and chlordiazepine Spots appearing at the location suggest that the sample may contain benzodiazepines such as nitrazepam, clonazepam, lorazepam, oxazepam, estazolam, and chlordiazepine.

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 misidentify some coexisting components as benzodiazepines. Some coexisting components are large and chromatographic. If the migration value is close to the component to be tested, it will interfere with the detection of the target component. (2) The long time for color language to unfold and dry is insufficient to meet the requirements of rapid measurement. (3) The thin layer color plate needs to be dried and stored dry, and 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.

2, high performance liquid chromatography

High performance liquid chromatography is a commonly used modern analytical method. Under the same chromatographic conditions, different substances have different chromaticity retention times. Under the following conditions: Color column: Agilent Zorbax SB-C 18 (4.6 mmx250 mm, 5μιη); Mobile phase: sterol-acetonitrile-volume fraction 0.5% citric acid ρΗ (2.8 ± 0.1) (30: 15: 55); Detection wavelength: 254nm; Column temperature: 35 °C; Flow rate: 1.3ml'min; Injection volume: 20μ1, The solution of nitrazepam, clonazepam, lorazepam, oxazepam, estazolam and chlordiazepoxide and the sample solution obtained by sample extraction are separately injected, and can be judged according to the retention time of the chromatographic peak. Test samples for the components of nitrazepam, clonazepam, lorazepam, oxazepam, estazolam and chlordiazep.

The advantages of high performance liquid chromatography are high chromatographic separation efficiency and high sensitivity. The 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) when the coexisting peak retention time is related to nitrazepam, clonazepam, lorazepam It is easy to make a wrong judgment when the chromatographic peak retention time of oxazepam, estazolam and chlordiazepoxide is close; (3) The instrument has high requirements on the use environment and needs to be fixedly placed, which is not suitable for a site with high fluidity. Detection.

3, high performance liquid chromatography - mass spectrometry

High performance liquid chromatography-mass spectrometry using a high performance liquid chromatograph as a separator and a mass spectrometer as an analyzer is suitable for sample analysis with complex composition and severe background interference, which improves the reliability of the test results. Gao Qing et al. established a high performance liquid chromatography-mass spectrometry method for the examination of illegal addition of sedative and hypnotic drugs in traditional Chinese medicine preparations and health foods: Waters Sunfire C18 column, gradient elution, mobile phase A was 20 mmol/L ammonium acetate solution The mobile phase B is sterol, the detection wavelength is 230 nm, and the flow rate is 1 ml/min (split ratio 4:1).

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.

Before performing high performance liquid chromatography-mass spectrometry analysis, high performance liquid chromatography is generally used as a screening method to reduce the cost of analysis. These tests can only be carried out in a small number of laboratories and are not readily available for use.

At present, there is no release of a rapid test method for benzodiazepines in the pharmaceutical and health food industries. For pharmacies and primary drug testing units, rapid screening methods for the unauthorized addition of chemical ingredients in proprietary Chinese medicines and health foods are developed. Samples are taken at the regulatory site. Rapid analysis, it is necessary to provide evidence for drug supervision in a timely manner, to crack down on the fraud of criminals and to ensure the safety of the people. Summary of the invention

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 pharmaceuticals and health foods whether or not benzodiazepine is added. A quick test method for substances. Another object of the present invention is to provide an application of the above method for preparing a rapid test kit for benzodiazepines.

Another object of the present invention is to provide an application of the above method for screening test for the presence or absence of the addition of benzodiazepines in pharmaceuticals and health foods.

The rapid determination method for doping benzodiazepines in the medicines and health foods of the invention comprises the following steps:

(1) extracting benzodiazepines from medicines and health foods;

(2) concentrated extract;

(3) hydrolyzing the extracted benzodiazepines;

(4) adding a diazotization reagent;

(5) neutralizing interfering substances;

(6) Add a detector.

The clinically common chemical components of benzodiazepines are nitrazepam, clonazepam, lorazepam, oxazepam, estazolam and chlordiazepine.

The purpose of adding ethyl acetate to pharmaceuticals and health foods is to extract the benzodiazepines from the matrix and to reduce the interference of the traditional Chinese pigments themselves with the color reaction. The extraction process can be performed by shaking, sonicating or vortexing, filtering, and collecting the filtrate.

The filtrate is evaporated or dried, and the residue is the test product. Evaporation or drying of the ethyl acetate is carried out by concentrating the sample and dispersing the benzodiazepines from the ethyl acetate.

The hydrochloric acid solution is added to the residue, vortexed, ultrasonicated or shaken to hydrolyze the benzodiazepine in a hydrochloric acid solution to a compound having an aromatic primary amine structure (illustrated by clonazepam as an example):

The diazotization reagent is sodium nitrite, and a sodium nitrite solution is added dropwise to the product obtained by hydrolysis after adding a hydrochloric acid solution to react a compound having an aromatic primary amine structure with a sub-reaction to form a diazotized product.

+ H ₂ 0 + NaCl

Add urea to neutralize excess nitrous acid, otherwise the excess nitrous acid will nitrate the developer basic β-naphthol to form 1-nitro-2-naphthol, which will turn the solution into scarlet and interfere with the coupling. Observation of the color reaction.

2 NaNO, + 2HC1 > 2N ₂ t + C0 ₂ t +3H ₂ 0 + 2NaCl

The alkaline β-naphthol test solution is added dropwise, and the diazotized product undergoes a coupling color reaction to form a pink to orange red

The specific technical solutions are as follows:

1. Take solid medicine or health food, grind the sample or cut it, add 3~5ml of ethyl acetate, vortex it by vortexing or vortex for 3~5 minutes, filter it with filter paper moistened with ethyl acetate in advance. The filtrate is collected, and the filtrate is evaporated to dryness with a water bath of 70 to 80 ° C or blown with a hair dryer. The residue is the test sample; or the appropriate amount of liquid medicine or health food is placed, placed in a separatory funnel, and ethyl acetate is added. ~5ml, shake and extract for 1~3 minutes, let stand for layering, separate the ethyl acetate layer, filter through filter paper, and drain the filtrate with 70~80 °C water bath or blow dry with hair dryer, the residue is for test Product.

2. Add 2~4ml of hydrochloric acid solution with a mass fraction of 15%~25% to the residue, vortex for 3~5min, and disperse it as the test solution. The vortex operation can be performed in a vortex, and the vortex operation can be replaced by ultrasonic or shaking.

For most pharmaceutical or health food products currently on the market, the recommended sampling quantities are as follows.

3. Adding a sodium nitrite solution having a molar concentration of 0.05 to 0.2 mol/L to the solution after adding hydrochloric acid 0.1~0.3 ml (2~6 drops), vortex, sonicate or shake for l~3min.

4. Add a molar concentration of 0.1~0.3mol/L urea 0.5~1.5ml to the test solution, vortex, sonicate or shake for 2~4min.

5. Add 0.25~0.5ml (5~10 drops) of alkaline β-naphthol test solution to the filtrate. If the solution appears turbid from pink to orange red (or insoluble matter with pink to orange red), then It can be judged that the sample contains benzodiazepines; if pink or orange-red turbidity or insoluble matter is not observed after standing, it can be judged that the sample does not contain benzodiazepines.

Preparation of alkaline β-naphthol test solution: Take 0.25 g of β-naphthol, add 10 ml of NaOH solution (1→10) to dissolve it. Should be kept away from light.

According to the present invention, a rapid test kit for benzodiazepines can be prepared.

According to the present invention, it can be applied to a screening test for whether a pharmaceutical or a health food is incorporated into a benzodiazepine substance.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Fast and simple: The whole operation process includes ethyl acetate leaching for 3 minutes, filtration for 1 minute, evaporation of the filtrate or drying for 2 minutes, hydrolysis of hydrochloric acid for 3 minutes, diazotization for 1 minute, removal of nitrous acid for 2 minutes, The developer is then added and the entire process is completed in 12 minutes. Compared to liquid chromatography and LC/MS (at least 3 hours), it is much simpler and faster, and no reference is needed.

(2) Strong specificity and high accuracy: According to the method verification results, the common excipients of tablets, capsules, pills, granules, oral agents and other dosage forms have no interference on the detection of benzodiazepines.

(3) Sensitive reaction: The minimum detection limit of the substance to be tested in this experiment reaches one tenth of the clinical minimum effective dose. Therefore, it is necessary to check whether or not the benzodiazepine component is added to the drug or health food by using this method. .

(4) Wide range of applications: 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. detailed description

The following examples are intended to aid those of ordinary skill in the art to further understand the invention, but are not intended to limit the invention in any way.

Example 1 Tianxingjian Good Night Gelatin (gelatin, labeled as 2 capsules per time, high performance liquid chromatography-mass spectrometry detection results of this product containing chlordiazepam 0.353mg)

Take two contents, place in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 3 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; 2.5ml of hydrochloric acid solution, vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to hydrochloric acid solution, vortex for 1 minute; add 0.2 mol/L urea 1 ml to vortex for 2 minutes; 5 drops of the β-naphthol test solution, and a red precipitate gradually appeared in the solution, thereby judging that the sample contained illegally added benzodiazepines.

Example 2

Tianxingjian Good Night Gelatin (gelatin, labeled as 2 capsules each time, high performance liquid chromatography-mass spectrometry detection results) This product contains nitricepsin 0.557mg

Take two contents, place in a 10 ml sample tube, add 3 ml of ethyl acetate, vortex for 5 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; 2 ml of hydrochloric acid solution, vortex for 5 minutes; 6 drops of 0.05 mol/L sodium nitrite solution was added dropwise to the hydrochloric acid solution, vortexed for 3 minutes; 0.1 mol/L urea 1.5 ml was added and vortexed for 4 minutes; 5 drops of β-naphthol test solution, the solution gradually appeared red precipitate, which can be judged that the sample contains illegally added benzodiazepines.

Example 3

Baizi Jingxin capsule (gelatin, labeled as 2 capsules per time, high performance liquid chromatography-mass spectrometry detection results, this product contains eszolate 0.572mg per capsule)

Take two contents of the sample, place in a 10 ml sample tube, add 4 ml of ethyl acetate, vortex for 4 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; 4ml of hydrochloric acid solution, vortex for 3 minutes; 2 drops of 0.2 mol/L nitrous sodium S sodium solution was added dropwise to the hydrochloric acid solution, vortex for 2 minutes; 0.3 mol/L urea 0.5 ml vortex was added dropwise for 2 minutes; Five drops of the alkaline β-naphthol test solution, and a red precipitate gradually appeared in the solution, thereby judging that the sample contained illegally added benzodiazepines.

Example 4

Sleeping tablets (tablets, labeled as 2 tablets each time, high performance liquid chromatography-mass spectrometry results in the product containing chlordiazepine 0.870mg per capsule)

Take two samples, crush them, place them in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 3 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; Add 3 ml of hydrochloric acid solution and vortex for 4 minutes; add 4 drops of 0.15 mol/L sodium nitrite S solution to the hydrochloric acid solution, vortex 1 minute; add 0.2 mol/L urea 1 ml vortex for 2 minutes; add 5 drops of alkaline β-naphthol test solution, and the solution gradually appears red precipitate, which can be judged that the sample contains illegal addition of benzodiazepine Class of substances.

Example 5

Wild wine flower dream Corning capsule (gelatin, labeled as 2 capsules each time, high performance liquid chromatography-mass spectrometry detection results of this product containing lorazepam 0.093mg)

Take 1 volume of the contents, place in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 4 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; 2.5ml of hydrochloric acid solution, vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to hydrochloric acid solution, vortex for 1 minute; add 0.2 mol/L urea 1 ml to vortex for 2 minutes; 5 drops of the β-naphthol test solution, and a red precipitate gradually appeared in the solution, thereby judging that the sample contained illegally added benzodiazepines.

Example 6

Anshen Bunao Liquid (oral solution, labeled as 10ml each time, high performance liquid chromatography-mass spectrometry detection results) This product does not contain benzodiazepines, simulated addition of oxazepam reference substance 0.30mg)

Take 5 ml of Anshen Bunao Liquid, place it in a separatory funnel, add 0.30 mg of the nitrazepam reference substance, add 5 ml of ethyl acetate, shake and extract for 1 minute, let stand for stratification, and separate the ethyl acetate layer. Evaporate dry with a water bath at 80 ° C or blow dry with a hair dryer. The residue is the test sample. Add 2.5 ml of hydrochloric acid solution to the residue and vortex for 3 minutes. Add 0.1 mol/L sodium nitrite solution to the hydrochloric acid solution. Drop, vortex for 1 minute; add 0.2 mol/L urea 1 ml vortex for 2 minutes; add 5 drops of alkaline β-naphthol test solution, and the solution gradually appears red precipitate, which can be judged that the sample contains illegally added benzene. Diazepines.

Example 7

Sleeping Sleeping Gelatin (gelatin, labeled as 2 capsules per time, high performance liquid chromatography-mass spectrometry detection results. This product does not contain benzodiazepines, as a negative control test)

Take 1 volume of the contents, place in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 3 minutes, filter, and collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is the test sample; 2.5ml of hydrochloric acid solution, vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to hydrochloric acid solution, vortex for 1 minute; add 0.2 mol/L urea 1 ml to vortex for 2 minutes; 5 drops of β-naphthol test solution, no red precipitate appeared after the solution, and it can be judged that the sample does not contain benzodiazepines.

Example 8

Sleeping melatonin tablets (tablets, labeled as 2 tablets each time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain benzodiazepines, as a negative control test) Take a piece of melatonin tablets, crush, place in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 3 minutes, filter, collect the filtrate; the filtrate is blown dry with a hair dryer, and the residue is tested. Add 2.5 ml of hydrochloric acid solution to the residue and vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to the hydrochloric acid solution, vortex for 1 minute; add 0.2 mol/L urea 1 ml vortex 2 minutes; 5 drops of alkaline β-naphthol test solution were added dropwise, and no red precipitate appeared in the solution after standing, thereby judging that the sample contained no benzodiazepines.

Example 9

Naoxinshu Oral Liquid (oral solution, labeled as 10ml each time, high performance liquid chromatography-mass spectrometry detection results, this product does not contain benzodiazepines, as a negative control test)

Take 5 ml of Naoxinshu oral solution, place in a separatory funnel, add 5 ml of ethyl acetate, shake for 1 minute, let stand for stratification, separate the ethyl acetate layer, and drain the filtrate with a water bath at 80 ° C or Dry the hair dryer, the residue is the test sample; add 2.5ml hydrochloric acid solution to the residue, vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to the hydrochloric acid solution, vortex for 1 minute; Add 0.2 mol/L urea 1 ml to vortex for 2 minutes; add 5 drops of alkaline β-naphthol test solution, and the solution does not show red precipitate after the solution, so that the sample can be judged to be free of benzodiazepines.

Example 10

Zishen Ningshen Pills (Pills, labeled as 10g each time, high performance liquid chromatography-mass spectrometry detection results) This product does not contain benzodiazepines, as a negative control test)

Take Wuji Ningshen Pills 5.0g, crush, place in a 10 ml sample tube, add 5 ml of ethyl acetate, vortex for 3 minutes, filter, collect the filtrate; the filtrate is blown dry with a hair dryer, the residue is the test sample Add 2.5ml of hydrochloric acid solution to the residue and vortex for 3 minutes; add 5 drops of 0.1 mol/L sodium nitrite solution to the hydrochloric acid solution, vortex for 1 minute; add 0.2 mol/L urea 1 ml vortex 2 Minutes; 5 drops of alkaline β-naphthol test solution were added dropwise, and no red precipitate appeared in the solution, so that no benzodiazepines were added to the sample.

Claims

Rights request

1. A rapid determination method for doping benzodiazepines in medicines and health foods, which in turn consists of the following steps:

(1) extracting benzodiazepines from medicines and health foods;

(2) concentrated extract;

(3) hydrolyzing the extracted benzodiazepines;

(4) adding a diazotization reagent;

(5) neutralizing interfering substances;

(6) Add a detector.

The assay method according to claim 1, wherein the benzodiazepine substance is extracted from a medicine and a health food with ethyl acetate.

The method according to claim 1, wherein the benzodiazepine substance is hydrolyzed with a hydrochloric acid solution.

The measuring method according to claim 1, wherein the diazotizing reagent is a nitrous acid solution.

5. The assay method according to claim 1, characterized in that the interference substance is neutralized with urea.

The measuring method according to claim 5, wherein the urea molar concentration is 0.1 to 0.3 mol/L.

The measuring method according to claim 1, wherein the detecting agent is an alkaline β-naphthol test solution.

8. The method according to claim 1, comprising the following steps in sequence:

(1) Take medicine or health food, add ethyl acetate, ultrasonically, vortex or shake, filter, collect Filtrate

(2) The filtrate is evaporated to dryness to obtain a test sample;

(3) adding a hydrochloric acid solution, vortexing, and hydrolyzing the extracted benzodiazepines;

(4) dropping sodium nitrite solution into the test solution and vortexing;

(5) adding urea, vortexing, neutralizing interfering substances;

(6) Drip the alkaline β-naphthol test solution.

9. The use of the assay of claim 1 in the preparation of a rapid test kit for benzodiazepines.

10. The method according to claim 1, wherein the application of the drug or the health food to the screening test of the benzodiazepine substance is carried out.