CN104090107A - Sulphonamide drug rapid screening method - Google Patents
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Abstract
The invention discloses a sulphonamide drug rapid screening method. The sulphonamide drug rapid screening method is suitable for fast detection of cream, ointment, emulsion, solutions, gel and other cosmetics, capsules, tablets, pills, powder, granules, oral liquids and other cough and asthma relieving Chinese patent medicines and health food, and greatly improves the detection generality and detection efficiency. The sulphonamide drug rapid screening method has the characteristics of simple operation, strong specificity, high sensitivity, high accuracy, suitable technology, environmental friendliness, security and the like, can be used for fast detection of illegally added chemical composition of sulphonamides in the field, and is in accordance with the relevant principles and requirements of the application. The sulphonamide drug rapid screening method can be implemented by simple training, the detection time of each sample is less than 5 minutes, and the cost of each time of detection is about 10 ~ 20 yuan. The sulphonamide drug rapid screening method detection omission rate is 0, the false detection rate is 0, the accuracy rate is 100%, and the detection limit is less than 30 mug / g.
Description
Technical field
The invention belongs to the detection technique field of sulfa drugs, particularly the general quick screening method of the illegal sulfa drugs adding of Chinese patent drug, health food and cosmetics.
Background technology
Clinical research shows, sulfa drugs has antimicrobial and anti-inflammation efficacy, in antirheumatic, anti-microbial type and relieving cough and asthma class Chinese patent drug and health food and anti-acne class (anti-acne, except mite) cosmetics, may add.China's cosmetics health specification, European Union's regulatory affairs, Pharmaceutical Affairs Law in Japan etc. have all clearly specified that sulfa drugs is banned substance in components of cosmetics.State Bureau has announced the material easily illegally adding in cosmetics in " notice of illegally adding behavior about severe strike health food cosmetics " (state's food medicine prison is checked [2011] No. 223) of issuing on 05 25th, 2011, comprising 21 kinds of sulfanilamide (SN), detecting national standard is " mensuration of 21 kinds of sulfanilamide (SN) in cosmetics " (GB/T24800.6-2009).Sulfa drugs uses commonplace in livestock and poultry cultivation anti-infective therapy process, illegal situation of adding few in health-oriented products, and domestic and foreign literature is seldom reported.Because sulfa drugs action time and the metabolism time is in vivo longer, long-term use may cause accumulating in human body, and then function of human body is produced to harm, and causes pathogen to develop immunity to drugs, and is necessary to set up the quick screening method of illegal interpolation.
The residual component aspect that mainly concentrates at present the samples such as food, biology, environment about the research report of sulfa drugs fast detecting, comprises thin-layered chromatography, spectroscopic methodology, immunoassay etc.The method for quick that the Ministry of Agriculture of China is given for sulfa drug residue in monitoring pork is euzymelinked immunosorbent assay (ELISA), and the method influence factor is more, easily occurs false positive results.Aspect illegal interpolation, Shantou City has developed in institute for drug control the physics and chemistry quick screening method of sulfamido composition in cosmetics.Physics and chemistry quick screening method be utilize chemical composition to be measured the physics such as solubleness, functional diagram or and chemical property and matrix components different, get rid of matrix interference by Sample Pretreatment Technique, then carry out qualitative analysis according to the physicochemical property of chemical composition to be measured.Because physics and chemistry quick screening method great majority utilize the functional group of composition to be measured that certain class special chemical occurs to react to reach the object of discriminating, affected by matrix, pre-treating method is had relatively high expectations, be applicable to macro-analysis, generally can not the too low composition to be measured of detectable concentration.
Different from physics and chemistry quick screening method, colloid gold immune technology has high specificity and high sensitivity, is applicable to trace and trace analysis.Because colloid gold immune analytical technology is simple and quick, result is accurate, specificity is strong, Environmental Safety, be applicable to execute-in-place, be one of main development direction of quick screening method.Sulfamido colloid gold immune analytical approach is widely used at food residue thing detection field, have not been reported but illegally add detection field at Chinese patent drug, health food and cosmetics.Up to the present, the illegal quick screening method adding is all according to researching and developing after Function Classification, a kind of quick screening method of a class chemical composition of class health-oriented products.Develop the residue detection colloid gold test paper of a large amount of single sulfanilamide (SN) compositions both at home and abroad, as Sulfamethoxazole, sulphadiazine, 5-methoxysulfadiazine, fanasil, sulfaquinoxaline etc., the specificity of single sulfanilamide (SN) component glue gold test paper is stronger, and range of application is wideless.
The present invention develops the general collaurum quick screening method of efficient sulfa drugs, and a kind of quick screening method detects the same class chemical composition of adding in dissimilar health-oriented products simultaneously.General quick screening method is matrix difference and target component concentration difference for detection of the subject matter that in dissimilar health-oriented products, certain class chemical composition is faced.For sulfamido chemical composition quick screening method, just there is obviously difference in the matrix of Chinese patent drug and health food and the matrix of cosmetics, and also difference is larger for sulfanilamide (SN) concentration therein.The main matrix of cosmetics is grease, surfactant etc., and the matrix of Chinese patent drug and health food is compared much complicated.As oral formulations, taking Sulfamethoxazole sheet and compound sulfamethoxazole and trimethoprim tablets as example, be 500mg/ time and 400mg/ time for the clinical RD of antibacterial functions, the concentration in capsule and tablet generally exceedes 25%, generally exceedes 1% in the concentration of pill.As external preparation, taking silver sulfadiazine ointment as example, be 1% for the clinical recommendation specification of antibacterial functions.Both sulfanilamide (SN) concentration differs more than 25 times.Therefore, Chinese patent drug, health food need to be taked respectively different pre-treating methods from cosmetics.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the general quick screening method of the illegal sulfa drugs adding of a kind of Chinese patent drug, health food and cosmetics is provided.
The technical solution used in the present invention is:
The quick screening method of sulfa drugs, comprises the steps:
1) sampling;
2) sample to be checked is added in extraction agent, jolting mixes, and leaves standstill to upper solution and clarifies;
3) draw upper solution, drop in PBS solution, jolting mixes, and leaves standstill to upper solution and clarifies;
4) draw the upper solution that step 3) obtains, drip in the well of sulfa drugs colloidal-gold detecting-card;
5) application of sample is after 3～5 minutes, according to the C line of test card and T line colour developing situation sentence read result.
Preferably, sampling method is as follows:
1. cosmetics: solid, semisolid dosage form, get 0.1～0.5g; Aqua, gets 0.1～0.5mL;
2. Chinese patent drug or health food: capsule, get 1/4～1 intragranular tolerant; Tablet, is crushed to powder, gets 1/4～1 amount; Pill, granule, powder, get each serving 1/8 of consumption; Oral liquid, gets 0.1～0.5mL.
Preferably, the hydrochloric acid that described extraction agent is 0.05～0.2mol/L or 1%～5% sodium hydroxide solution.
Preferably, the hydrochloric acid that described extraction agent is 0.05～0.2mol/L.
Preferably, step 2), sample to be checked is added in 1.0～2.0mL extraction agent.
Preferably, step 3), drips 2～3 and drops in 5～10mL PBS solution.
Preferably, step 4), drips 2～3 in well.
Preferably, step 5), testing result interpretation method is as follows:
1. negative: T line and C line all develop the color;
2. positive: T line is without colour developing, the colour developing of C line;
3. invalid: C line does not develop the color, will after Sample Dilution to be checked, again detect.
The preparation method of sulfa drugs colloidal-gold detecting-card, comprises the following steps:
1) respectively sulfamido antigen, sheep anti-mouse igg are sprayed on nitrocellulose filter, as T line and C line, dry, obtain coated film;
2) prepare the sulfanilamide (SN) monoclonal anti nanocrystal composition of colloid gold label, be coated on polyester film or glass fibre, obtain gold-marking binding pad;
3) prepare sample pad;
4) assembling of test card: sulfa drugs colloidal-gold detecting-card is made up of PVC template and PVC backboard is stained with in order sample pad, gold-marking binding pad, coated film and adsorptive pads on PVC backboard.
The invention has the beneficial effects as follows:
1) sulfa drugs quick screening method of the present invention is applicable to the fast detecting of the relieving cough and asthma class Chinese patent drugs such as the cosmetics such as creme, paste, emulsion, solution, gel and capsule, tablet, pill, powder, granule, oral liquid and health food, greatly improve detection versatility and detection efficiency, also provide exemplary investigation for being based upon the illegal general quick screening method that adds the chemical compositions such as microbiotic, steroids in multiclass health-oriented products simultaneously.
2) the inventive method has simple to operate, the feature such as specificity is strong, highly sensitive, accuracy is high, technology is applicable, Environmental Safety, can carry out fast detecting to the sulfamido chemical composition of illegal interpolation at the scene, meet correlation principle and the requirement applied.
3) the invention belongs to portable field detection technique, can turn to fast screening reagent kit by product.Can implement the method through simple training, be less than 5 minutes the detection time of each sample, and each testing cost is about 10～20 yuan.
4) the method handling safety, without carcinogenic, hypertoxic, high poison, inflammable and explosive reagent, not with an organic solvent.
5) the method loss is 0, and false drop rate is 0, and accuracy is 100%.
6) quick screening method detection limit of the present invention be less than 30 μ g/g(be less than clinical RD 1/300).
Brief description of the drawings
Fig. 1 is the operation chart of sulfa drugs colloidal-gold detecting-card;
Fig. 2 is that sulfa drugs colloidal-gold detecting-card screening results is judged schematic diagram.
Embodiment
Below in conjunction with specific embodiment, further set forth content of the present invention.
Sulfamido detects the preparation of khaki associated materials:
Sulfamido haptens preparation method is:
Take N-acetylsulfanilyl chloride 8.5g and Methyl anthranilate 3.5g in the conical flask of 50mL, add pyridine 28mL, stir it is fully dissolved, reflux is installed in fuming cupboard, and temperature reaches 100 while spending, backflow 1h, after room temperature is cooling, ice bath continues to stir 1h, has yellow mercury oxide to produce.Collection reactant liquor is centrifugal, will precipitate with after 20mL 2mol/L NaOH dissolving, boils pyridine, after continuation backflow 1h room temperature is cooling, adjusts pH to 4 with 4mol/l hydrochloric acid, has gray precipitate to produce.Regather reactant liquor centrifugal, centrifugal 15 minutes of 10000r/s, resuspended with the distilled water of 40mL, ethyl acetate extraction 3 times, collects ethyl acetate layer, and decompression is dry.
Synthesizing of sulfanilamide (SN) parent nucleus holoantigen:
Get sulfamido haptens 4mg and be dissolved in 0.4mL dimethyl formamide, then add the EDC of 20mg, stirring and dissolving, is divided into two parts of equal-volumes; BSA and OVA respectively take 10mg, dissolve respectively, then add respectively above in two parts of solution with the PBS of the 0.01mol/L pH7.4 of 1.5mL, and with the PBS dialysis of 0.01mol/L PH7.4 3 days, 8h changed dislysate one time, makes envelope antigen and immunizing antigen respectively.
The preparation of sulfamido antibody:
By the immunizing antigen immunity female BALB/c mouse in 6-8 age in week of sulfamido carrier protein combination, immunizing dose is 200ug/ time, adopts quick adjuvant, back hypodermic injection.First immunisation, uses sulfamido carrier protein immunizing antigen to mix with equal-volume Freund's adjuvant complete 1:1, fully mixes booster immunization after 3 weeks; Booster immunization, mix with equal-volume Fei Shi Freund's incomplete adjuvant 1:1 with sulfamido carrier protein immunizing antigen, fully mix, continuous immunity 2 times, every minor tick 3 weeks, the blood sampling of 10～15 days end tails separation of serum after last immunity, adopt indirect elisa method to measure polyvalent antibody and tire, and result is measured antibody serum and tired between 1:8000～1:10000.
Get immune mouse spleen cell and myeloma cell and merge, filter out the positive cell strain of stably excreting sulfamido and expand and cultivate, be injected into induction ascites in Mice Body; After extracting ascites, adopt sad ammonium sulfate method purifying ,-20 degree are preserved.
The preparation of sheep anti-mouse igg:
Using goat as immune animal, the goat using mouse source antibody as immunogen immune health, obtains sheep anti-mouse igg.
Sulfa drugs colloidal-gold detecting-card is characterised in that: on PVC plate, be connected successively sample pad, gold-marking binding pad, coated film and adsorptive pads.
The preparation of sulfa drugs colloidal-gold detecting-card:
Preparation method is as follows:
1) preparation of coated film
Use 0.01M Tris-HCl(pH8.0) sulfamido envelope antigen is diluted to 0.5～1.0mg/mL by damping fluid, is sprayed onto on nitrocellulose membrane, and the discharge rate of every 300mm is 30 μ L, is detection line (T line); Sheep anti-mouse igg is diluted to 0.3mg/mL～0.5mg/mL, is sprayed onto on nitrocellulose membrane, and the discharge rate of every 300mm is 30 μ L, is control line (C line), and the spacing of C, T line is 4.5mm; In 45 DEG C, baking oven, dry, obtain coated film;
2) preparation of gold-marking binding pad
Gold chloride is diluted to concentration 0.01%(volume ratio with high purity water), magnetic agitation heating is boiled, in every 100mL gold chloride, add rapidly the trisodium citrate of 0.7mL, continue to boil, no longer change and stop heating to hue preserving redness, be cooled to the high purity water that supplements loss after room temperature, obtain colloidal gold solution;
Get the colloidal gold solution that particle radius is 20nm～40nm, adjust pH to 7.5～8.0, under agitation add sulfamido monoclonal antibody by 10 μ g antibody/mL colloidal gold solutions, last every 1mL adds 100 μ L 10%BSA to make it stable, the centrifugal 30min of 12000rpm, abandon supernatant, will precipitate solution (containing 0.01M Tris(pH8.5), 2%～5% sucrose, 2%BSA, 0.1 ‰～0.5 ‰ NaN with 1/10th times of initial collaurum volumes
3) resuspended, obtain sulfanilamide (SN) monoclonal antibody-colloidal gold composite;
By sulfanilamide (SN) monoclonal antibody-colloidal gold composite 0.01M Tris-HCl(pH8.0), 2%～5% sucrose, 5% BSA, 1 ‰～5 ‰ TritonX-100,0.5%～1% cholic acid, 0.1 ‰～0.5 ‰ NaN
3solution dilution to 5%～10%, gets glass fibre, and every coating 30mL, dries, and obtains gold-marking binding pad; Or every sulfanilamide (SN) monoclonal antibody-colloidal gold composite 100 μ L 0.01M Tris-Hcl(pH8.0) damping fluid, 5%BSA, 0.1 ‰～0.5 ‰ NaN
3be diluted to 150～300 μ L, then add 20% sucrose, dissolving mixes, and is sprayed onto on the polyester film or glass fibre of processed mistake with metal-spraying equipment, dries, and obtains gold-marking binding pad;
3) preparation of sample pad
Get glass fibre, coating 20～30mL containing 0.01M Tris-HCl(PH8.0), 1% BSA, 1% TWEEN-20,0.1 ‰～0.5 ‰ NaN
3solution, 37 DEG C of oven dry.
4) assembling of test card
The assembling of test card: sulfa drugs colloidal-gold detecting-card is made up of PVC template and PVC backboard is stained with in order sample pad, gold-marking binding pad, coated film and adsorptive pads on PVC backboard; Sample pad is pressed gold-marking binding pad 2-4cm, gold-marking binding pad press mold 1-2cm, thieving paper press mold 1-2cm.On coated film, be sprayed with successively detection line, control line from sample pad to adsorptive pads direction.With being connected adhesive tape, sample pad, gold-marking binding pad and NC film are linked up to adhesive tape press mold 2-3cm.The plate posting is cut into the wide bar of 3mm with cutting machine.PVC template comprises well (S), detection zone, control zone, and it is placed on PVC backboard, and well (S) is corresponding to sample pad, and detection zone can show detection line (T), and control zone can show control line (C).The test card of making is put into the aluminium foil bag sealed storage with drying agent.
The quick screening method (referring to Fig. 1) of sulfa drugs, comprises the steps:
1) sampling;
2) sample to be checked is added in extraction agent, jolting mixes, and leaves standstill to upper solution and clarifies;
3) draw upper solution, drop in PBS solution, jolting mixes, and leaves standstill to upper solution and clarifies;
4) draw the upper solution that step 3) obtains, drip in the well of sulfa drugs colloidal-gold detecting-card;
5) application of sample is after 3～5 minutes, according to the C line of test card and T line colour developing situation sentence read result.
Sampling method is as follows:
1. cosmetics: solid, semisolid dosage form, get 0.1～0.5g; Aqua, gets 0.1～0.5mL;
2. Chinese patent drug or health food: capsule, get 1/4～1 intragranular tolerant; Tablet, is crushed to powder, gets 1/4～1 amount; Pill, granule, powder, get each serving 1/8 of consumption; Oral liquid, gets 0.1～0.5mL.
Colloid gold immune reaction needed is carried out in aqueous solution, therefore needs from sample to be checked, to extract chemical composition to be measured by pre-treating method, then in suitable solution medium, reacts.Inventor's discovery, due to illegal sulfamido chemical composition of adding in sample to be checked, often concentration is higher, therefore only need to select suitable solvent that constant in sample to micro-sulfanilamide (SN) material is extracted and be diluted to the micro-concentration to trace.The present invention preferably the hydrochloric acid of 0.05～0.2mol/L or 1%～5% sodium hydroxide solution as extraction agent.Preferred, using 0.05～0.2mol/L dilute hydrochloric acid solution as extraction agent.
Preferably, step 2), sample to be checked is added in 1.0～2.0mL extraction agent.
Preferably, step 3), drips 2～3 and drops in 5～10mL PBS solution.
Preferably, step 4), drips 2～3 in well.
sulfa drugs colloidal-gold detecting-card screening results interpretation method following (referring to Fig. 2):
1. negative (–): T line and C line all develop the color, represents in sample not containing sulfamido chemical composition (being that sulfonamides substrate concentration is lower than detection limit).
2. positive (+): T line, without colour developing, the colour developing of C line, represents in sample containing sulfamido chemical composition (being that sulfonamides substrate concentration is higher than detection limit).
3. invalid: not occur C line, show that test card lost efficacy or sample substrate overload, change test card after sample solution can being diluted to 10 times again to measure.
Points for attention:
(1) storage of colloidal-gold detecting-card and service condition are 4～30 DEG C.
(2), when result judges, if drip sample solution rear C line and T line of occurring in the well of test card, get final product judged result negative at once; If only there is C line, to wait 3 minutes after reading result again.
(3) sensitivity of collaurum quick screening method is very high, in operating process, will avoid sample cross pollution, and the aid such as test card and suction pipe relating to is disposable use.
embodiment 1 extracts the selection of solvent
Sulfamido chemical composition is generally soluble in watery hydrochloric acid or sodium hydroxide solution, is insoluble in water.The detectability of sulfamido colloid gold immune analytical approach is lower than 0.1 μ g/mL(100ng/L), therefore select respectively water, dilute hydrochloric acid solution, diluted sodium hydroxide solution as extraction agent, the PB(phosphate buffer of different pH values), PBS(phosphate buffered saline) and PBST(phosphate buffered saline in add tween) damping fluid tests.Draw and adopt 0.05～0.2 mol/L dilute hydrochloric acid solution as extraction agent through test, the PBS of 0.01mol/L, pH7.2 is as buffer solution medium, and the colour developing result of colloid gold immune reaction is better compared with other medium reappearances, and stable, the results are shown in Table 1.
The comparing result table of the selection of table 1 extraction agent to the T line colour developing degree of depth
Remarks: "-" represents negative findings; "+" represents positive findings, and " ± " represents weak positive findings; " L0～L10 " represents colour developing concentration situation.Owing to adopting A competitive inhibition method, when developing the color simultaneously, T line and C line be with negative result, positive result while only having C line colour developing band.In the time that extract is different with dilution, the colour developing of T line is deep mixed, and upper table " L0～L10 " represents T line colour developing degree of depth situation, and the larger expression colour developing of numeral concentration is darker, and " L0 " represents not aobvious band.
embodiment 2
Sample to be checked: commercially available being denoted as " certain angel's acne eliminating cream ".
1) get sample 0.1g to be checked, add in No. 1 extraction tube that contains 2 mL 0.1mol/L dilute hydrochloric acid solutions, tighten bottle cap, jolting approximately 30 seconds energetically, leaves standstill to upper solution and clarifies;
2) draw upper solution, drip 3 and drop in No. 2 extraction tubes that contain 5 mL 0.01mol/L, pH7.2 PBS solution, jolting approximately 30 seconds, mixes it, leaves standstill to be measured;
3) draw No. 2 extraction tubes solution at the middle and upper levels, vertically drip 3 solution in the well of above-mentioned sulfa drugs colloidal-gold detecting-card;
4) after application of sample, start timing, after 3～5 minutes, colour developing result is positive.
Find that by quick screening method of the present invention it has illegally added sulfamido chemical composition, added Sulfamethoxazole by Liquid Chromatography/Mass Spectrometry and liquid phase chromatography confirmation, content is 0.6%.
embodiment 3
Sample to be checked: commercially available being denoted as " certain brand breath is breathed heavily peaceful ball ".
1) get sample to be checked, get each serving approximately 1/8 of consumption, crush, add in No. 1 extraction tube that contains 2 mL 0.1 mol/L dilute hydrochloric acid solutions, tighten bottle cap, jolting approximately 30 seconds energetically, leaves standstill to upper solution and clarifies;
2) draw upper solution, drip 3 and drop in No. 2 extraction tubes that contain 10 mL 0.01mol/L, pH7.2 PBS solution, jolting approximately 30 seconds, mixes it, leaves standstill to be measured;
3) draw No. 2 extraction tubes solution at the middle and upper levels, vertically drip 3 solution in the well of above-mentioned sulfa drugs colloidal-gold detecting-card;
4) after application of sample, start timing, after 3～5 minutes, colour developing result is positive.
Find that by quick screening method of the present invention it has illegally added sulfamido chemical composition, added Sulfamethoxazole by Liquid Chromatography/Mass Spectrometry and liquid phase chromatography confirmation, content is 1.1%, takes in 26.4mg at every turn, takes in 52.8mg every day.Inquiry State Bureau database, finds that this product is the counterfeit drug of usurping Heilungkiang Kang Yuan pharmaceutcal corporation, Ltd authentication code.
embodiment 4 sensitivity tests
1, the detection sensitivity of sample: get the about 15mg of Sulfamethoxazole reference substance, put in 50mL measuring bottle, be settled to scale with 0.1mol/L dissolve with hydrochloric acid solution dilution.Precision measures above-mentioned solution 0.1mL, gets respectively a kind of negative Chinese patent drug and cosmetics as the about 0.9g of blank matrix, mixes, and makes sample 1 and sample 2.
1) the 1(Chinese patent drug of materialsing) 0.4 g, sample 2(cosmetics) 0.2 g;
2) sample 1, sample 2 are added respectively in No. 1 extraction tube that contains 2 mL 0.1 mol/L dilute hydrochloric acid solutions, tighten bottle cap, energetically jolting approximately 30 seconds, leave standstill to upper solution and clarify, tighten bottle cap, jolting approximately 30 seconds energetically, leaves standstill to upper solution and clarifies;
3) draw upper solution, sample 1～3 drops in No. 2 extraction tubes that contain 10 mL 0.01mol/L, pH7.2 PBS solution, and jolting approximately 30 seconds, mixes it, leaves standstill to be measured; Sample 2～3 drops in No. 2 extraction tubes that contain 5 mL 0.01mol/L, pH7.2 PBS solution, and jolting approximately 30 seconds, mixes it, leaves standstill to be measured;
4) draw No. 2 extraction tubes solution at the middle and upper levels, vertically drip 3 solution in the well of above-mentioned sulfa drugs colloidal-gold detecting-card;
5) after application of sample, start timing, after 3 minutes, colour developing result is positive.
Detection limit is less than 30 μ g/g, lower than 1/300 of the minimum clinical RD (1%, i.e. 10000 μ g/g) of sulfa drugs.
2, the detection sensitivity of reference substance:
The detection that records sulfamido chemical composition reference substance solution is limited to 10～80 μ g/L, and wherein the detection of Sulfamethoxazole is limited to 80 μ g/L.
embodiment 5 specificity tests
With the method for embodiment 4 steps 1, in the time there is the chemical compositions such as metacortandracin, theophylline, aminophylline, chlorphenamine, metronidazole, chloromycetin, clindamycin class, quinolones in sample, testing result is negative, shows that this colloid gold test paper is to said medicine composition no cross reaction.
embodiment 6 serviceability tests
With the method for embodiment 4 steps 1, investigated respectively temperature (5 DEG C, 10 DEG C, 20 DEG C and 30 DEG C), extracted solvent pH(pH7.1,7.2 and 7.3), the factor such as colloidal-gold detecting-card (two lot numbers), different sampling amount (0.05g, 0.1g and 0.2g) and different operating personnel, measurement result is unaffected.
embodiment 7 accuracy testings
Buy, supervise the modes such as sampling by market and collect altogether 110 batch samples (the 76 batches of anti-acne class cosmetics and the 34 batches of relieving cough and asthma class Chinese patent drugs and health food.Wherein, comprise relieving cough and asthma class Chinese patent drug and the health foods such as capsule, tablet, pill, powder, granule, oral liquid, the cosmetics such as creme, paste, emulsion, solution, gel.) checking the inventive method accuracy.In relieving cough and asthma class Chinese patent drug and health food, the confirmation of Sulfamethoxazole and referee method are the supplementary method of inspection 2009031 of State Food and Drug Administration's approval.In cosmetics, the confirmation of sulfanilamide (SN) and referee method are " mensuration of 21 kinds of sulfanilamide (SN) in GB-T 24800.6-2009 cosmetics ".
110 batch samples (1～57
#, 92～110
#for cosmetics, 58～91
#for Chinese patent drug and health food) fast sieve result and confirmed results in table 2, have 17 batch samples positive, detect composition be Sulfamethoxazole (wherein 10 batches, cosmetics, content is 0.1%～0.6%; 7 batches of Chinese patent drug and health foods, content is 1.1%～25.3%).
Watch 2 samples sieve result and confirmed results soon
With the result comparison of confirmation method, the accuracy rate of quick screening method of the present invention is 100%.
Having confirmed the sulfamido chemical composition that the inventive method can detect comprises: 14 kinds of Sulfamethoxazole, NU-445, sulphadiazine, sulfamethyldiazine, sulfamethazine, sulfaquinoxaline, arnosulfan yl pyrimidines, sulfametoxydiazine, daimeton, fanasil, cistosulfa, sulfamethoxypyridazine, sulfabenzamide, sulfadoxines (sulfamethoxine) etc.
Claims (9)
1. the quick screening method of sulfa drugs, comprises the steps:
1) sampling;
2) sample to be checked is added in extraction agent, jolting mixes, and leaves standstill to upper solution and clarifies;
3) draw upper solution, drop in PBS solution, jolting mixes, and leaves standstill to upper solution and clarifies;
4) draw the upper solution that step 3) obtains, drip in the well of sulfa drugs colloidal-gold detecting-card;
5) application of sample is after 3～5 minutes, according to the C line of test card and T line colour developing situation sentence read result.
2. quick screening method according to claim 1, is characterized in that: sampling method is as follows:
1. cosmetics: solid, semisolid dosage form, get 0.1～0.5g; Aqua, gets 0.1～0.5mL;
2. Chinese patent drug or health food: capsule, get 1/4～1 intragranular tolerant; Tablet, is crushed to powder, gets 1/4～1 amount; Pill, granule, powder, get each serving 1/8 of consumption; Oral liquid, gets 0.1～0.5mL.
3. quick screening method according to claim 1, is characterized in that: the hydrochloric acid that described extraction agent is 0.05～0.2mol/L or 1%～5% sodium hydroxide solution.
4. quick screening method according to claim 3, is characterized in that: the hydrochloric acid that described extraction agent is 0.05～0.2mol/L.
5. according to the quick screening method described in claim 1～4 any one, it is characterized in that: step 2), sample to be checked is added in 1.0～2.0mL extraction agent.
6. quick screening method according to claim 1, is characterized in that: step 3), drips 2～3 and drop in 5～10mL PBS solution.
7. quick screening method according to claim 1, is characterized in that: step 4), drips 2～3 in well.
8. quick screening method according to claim 1, is characterized in that: step 5), and testing result interpretation method is as follows:
1. negative: T line and C line all develop the color;
2. positive: T line is without colour developing, the colour developing of C line;
3. invalid: C line does not develop the color, will after Sample Dilution to be checked, again detect.
9. according to the quick screening method described in claim 1～8 any one, it is characterized in that: the preparation method of sulfa drugs colloidal-gold detecting-card, comprises the following steps:
1) respectively sulfamido antigen, sheep anti-mouse igg are sprayed on nitrocellulose filter, as T line and C line, dry, obtain coated film;
2) prepare the sulfanilamide (SN) monoclonal anti nanocrystal composition of colloid gold label, be coated on polyester film or glass fibre, obtain gold-marking binding pad;
3) prepare sample pad;
4) assembling of test card: sulfa drugs colloidal-gold detecting-card is made up of PVC template and PVC backboard is stained with in order sample pad, gold-marking binding pad, coated film and adsorptive pads on PVC backboard.
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| CN108761093A (en) * | 2018-07-19 | 2018-11-06 | 河南百奥生物工程有限公司 | A kind of antibodies against foot-and-mouth disease virus evaluation test strips |
| CN108931644A (en) * | 2018-07-19 | 2018-12-04 | 河南省农业科学院 | A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection |
| CN108761093B (en) * | 2018-07-19 | 2020-12-25 | 河南百奥生物工程有限公司 | Test strip for evaluating foot-and-mouth disease virus antibody |
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