CN106153954A - A kind of method for quick of phenytoin Sodium - Google Patents
A kind of method for quick of phenytoin Sodium Download PDFInfo
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- CN106153954A CN106153954A CN201610704950.1A CN201610704950A CN106153954A CN 106153954 A CN106153954 A CN 106153954A CN 201610704950 A CN201610704950 A CN 201610704950A CN 106153954 A CN106153954 A CN 106153954A
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- 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
- G01N33/9473—Anticonvulsants, e.g. phenobarbitol, phenytoin
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Abstract
The invention discloses the method for quick of a kind of phenytoin Sodium.Phenytoin Sodium antigen and antibody prepared by the present invention have more preferable specificity.The carrier protein of phenytoin Sodium coupling is selected from hemocyanin KLH or bovine serum albumin BSA.The titer utilizing the phenytoin Sodium antibody that above-mentioned antigen goes immune mouse to obtain is higher, and sensitivity is more preferable.Utilizing the phenytoin Sodium detection card detection speed prepared fast, result is easily observed, and can realize the rapid screening to batch samples, is substantially reduced testing cost, reduces workload, has practical significance in Food and drug administration.The present invention is directed to testing sample complicated component problem, be optimized sample pretreating method, required sample size is few, it is only necessary to extracts, dilute two steps and can complete sample pre-treatments.Substantially increase detection versatility and detection efficiency.
Description
Technical field
The invention belongs to health food and medicine field of fast detection, be specifically related to the quickly side of detection of a kind of phenytoin Sodium
Method.
Background technology
Phenytoin Sodium, white powder, odorless, bitter in the mouth, molecular formula C15H11O2N2Na, is a kind of prescription for treating epilepsy
Medicine.
Epilepsy is one of modal disease of Neurology Department, a kind of chronic disease needing Long-term taking medicine to control morbidity, suffers from
Person more favors the Chinese medicine preparation of those " pure Chinese medicine " " secret prescriptions handed down in the family from generation to generation " treatment epilepsy when selecting medicine, it is believed that side effect is little, no
Method molecule make use of this psychology of patient just, illegally adds phenytoin Sodium in some Chinese medicine preparation.
In February, 2010, China epilepsy association president Li Shichuo president have submitted " about specification epilepsy diagnosis and treatment to Ministry of Public Health
The suggestion of the big problem in field three ", the most just reflect " controlling epilepsy " Chinese medicine popular in society and secretly mix Western medicine, endanger asking of patient
Topic." suggestion " provides several parts from Different hospital finding in recent years, disclose following problem:
1. the history that the existence of the phenomenon secretly mixing Western medicine in epilepsy Chinese medicine is the longest；
2. existing tens of kinds of epilepsy Chinese medicines are found to the addition of western medicine composition, are hospital preparation the most in a large number, use medicine
Monitoring (TDM) method detects epilepsy Western medicine conventional in 1-5 in 45 examples take the serum of epilepsy children of " pure Chinese medicine ", its
Middle phenytoin Sodium is at most (42 example)；
3. other doctors may repeat because not knowing the western medicine composition in Chinese medicine preparation when considering and adding with other antuepileptics to use
Medicine, is easily generated excess and poisoning.
Antiepileptic Chinese medicine adds phenytoin Sodium, and indicates the most in the description, cause patient to take medicine lack of standardization, Western medicine
Antiepileptic concentration in vivo and kind are unordered, have a strong impact on curative effect, ultimately result in epilepsy and be difficult to control to.This kind of medicine
Due to private clinic preparation, there is dosage and forbidden in thing, the uniformity is poor, easily causes somebody invalid, the situation of somebody's poisoning；
There is anaphylaxis after taking in some patient, and erythra, heating, vasculitis etc. occurs；Some patients spend a huge sum of money but due to
Underdosage and epilepsy control undesirable, some patient due to take identical antiepileptic (regular medicine) simultaneously and even
The poisoning of antiepileptic excess occurs.
In antiepileptic, the illegal phenytoin Sodium that adds seriously damages the reputation of Chinese medicine, is unfavorable for the biography of China's Culture of TCM
Holding, serious infringement epileptic's is healthy, and market in urgent need is effectively supervised, therefore, it is necessary to carry out action immediately,
Set up supervision and inspection method targetedly, provide effective instrument for strengthening the supervision of this series products, thus realize ensureing patient
Rights and interests, the development that protection Chinese medicine is in good health.
At present, the context of detection whether adding phenytoin Sodium in epilepsy Chinese patent medicine and health food mainly has following several
The method of kind:
1, thin layer chromatography
Composition to be measured in sample through methanol extraction, gained need testing solution and reference substance solution on silica gel plate through developing solvent
Launch, take out, dry, put and inspect under uviol lamp 254nm.Under the effect of developing solvent, due to different material migration velocity not
Occur with the form of speckle with, different material diverse location on chromatographic sheet, according to testing sample whether with compare
There is this experimental phenomena of speckle to judge whether contain target component in testing sample in the corresponding position of product.If test sample is molten
There is speckle in liquid thin-layer chromatogram and phenytoin Sodium reference substance relevant position, then may become containing phenytoin Sodium in prompting sample
Point.
The advantage of thin layer chromatography is the analytical tool that need not costliness.This method shortcoming is: chromatographic resolution rate is relatively low, middle one-tenth
Medicine complicated component, interference factor is many, easily produces erroneous judgement.
Development of chromatogram and to dry required time longer, is insufficient for the requirement quickly measured.Chromatographic sheet needs to dry
Dry doubling is dried to be deposited, and separating effect is relatively big by such environmental effects, is not suitable for Site Detection.Need fixing place, do not conform to
Fit and make the Site Detection that mobility is big.The professional technique of testing staff is required higher, is not suitable for basic staff execute-in-place.
2, high performance liquid chromatography
High performance liquid chromatography is conventional Modern Methods, and under identical chromatographic condition, different materials has different
Chromatographic retention.Under identical chromatographic condition, phenytoin Sodium reference substance solution is with sample extraction gained need testing solution respectively
According to chromatographic retention, sample introduction, can determine whether whether testing sample contains phenytoin Sodium composition.
The advantage of high performance liquid chromatography is that chromatographic isolation efficiency is high, highly sensitive.But currently also have the disadvantage in that instrument
Device is expensive, and time for sample pretreatment is long, chromatographic column vulnerable to pollution, and analysis cost is high；When the chromatographic peak of the composition that coexists retains
Time and phenytoin Sodium chromatographic retention close to time easily cause erroneous judgement；Instrument is high to the requirement of environment, needs fixed placement, no
It is suitable for Site Detection.
3, tablets by HPLC-MS
It is suitable for as the tablets by HPLC-MS of analyzer using high performance liquid chromatograph as separator, mass spectrograph
In complicated component, sample analysis that ambient interferences is serious, this analysis method can improve the reliability of testing result.But high-efficient liquid
Phase chromatograph-mass spectrometer coupling method is that a kind of analysis cost is higher than high performance liquid chromatography, operates more complicated analysis method.Instrument
Requirement to environment is higher, needs fixed placement, improper Site Detection.These inspections at present can only be entered in a few experiments room
OK, it is difficult to promote the use of.
In sum, in Chinese patent medicine and health food, whether add the context of detection of phenytoin Sodium at present, still lack one
Highly sensitive, simple to operate, the method that carries out rapid screening phenytoin Sodium, for pharmacy and basis medicine inspection unit, exploitation becomes
The illegal quick screening method adding phenytoin Sodium in medicine and health food, quickly analyzes sample at supervision scene, for
Chinese patent medicine and health food supervision provide evidence in time, the imitation behavior hitting lawless person and the medication peace ensureing the people
Entirely it is very important.
Summary of the invention
For above-mentioned deficiency, it is an object of the invention to provide a kind of detection easy to use, fast and convenient, highly sensitive
Adulterate in health food or medicine the quick screening method of phenytoin Sodium and detection card used thereof.
The technical solution used in the present invention is:
A kind of phenytoin Sodium rapid detection card, including detection card base plate, the sample that described detection card base plate is sequentially provided with and is connected
Product pad, gold-marking binding pad, coated film and adsorptive pads, described gold-marking binding pad is provided with phenytoin Sodium antibody-colloidal gold complex
With rabbit igg-colloidal gold composite, described coated film is sequentially provided with the detection that phenytoin Sodium coupling carrier protein solution is printed
T line and the Quality Control C line printed with goat anti-rabbit igg solution.
Preferably, described gold-marking binding pad is glass fibre or polyester film, and described phenytoin Sodium antibody-colloidal gold is combined
Thing and rabbit igg-colloidal gold composite are adsorbed on glass fibre or polyester film by spraying coating process.
Preferably, described coated film is nitrocellulose filter or cellulose acetate membrane, described phenytoin Sodium coupling carrier egg
White solution and printing by spraying coating process absorption on nitrocellulose filter or cellulose acetate membrane with goat anti-rabbit igg solution.
Preferably, described sample pad is glass fibre.
Preferably, detection card base plate is PVC board.
Preferably, the consumption of described rabbit igg-colloidal gold composite is 0.3～0.5 μ g/cm2。
Preferably, the consumption of described phenytoin Sodium antibody-colloidal gold complex is 0.4-0.6 μ g/cm2。
Above-mentioned colloidal gold composite is coated with according to area, and unit is cm2。
Preferably, the consumption of described goat anti-rabbit igg is 0.03～0.05 μ g/mm.
Preferably, the consumption of described phenytoin Sodium coupling carrier albumen is: 0.05～0.1 μ g/mm.
Preferably, the carrier protein of phenytoin Sodium coupling is selected from hemocyanin KLH or bovine serum albumin BSA.
Above-mentioned antibody or antigen i.e. phenytoin Sodium coupling carrier albumen is sprayed directly on on nitrocellulose filter, comes by mm
Calculate.On above-mentioned detection card, the consumption of each component is all the optium concentration summing up out through research and development, when detecting actual sample,
If consumption too much there will be false positive, consumption is very few there will be false negative.
Preferably, phenytoin Sodium antigen and immunogen preparation method are: weigh 1 mg 5-diphenyl-phenytoin-3-
ω-positive valeric acid joins in the water of 0.6ml, and dropping pyridine few drops, to all dissolving, adds 1 mg carbodiimide, the most slowly
BSA and the KLH carbonate buffer solution of the 10mg/mL being added drop-wise to 1mL dissolves, 2 hs are stirred at room temperature, use after having reacted
The phosphate buffer of 0.01mol/L, pH7.4 is dialysed 3 days, and 8h changes a dialysis solution, prepares envelope antigen respectively and (is designated as PHT-
And immunizing antigen (being designated as PHT-KLH) BSA).
Preferably, KLH and BSA all dissolves with 0.01mol, pH9.6 carbonate buffer solution.
Preferably, the preparation method of phenytoin Sodium antibody is: with PHT-KLH immunization. Female mice, the immunity of every mice
Dosage is 120ug/0.3mL, uses quick adjuvant, dorsal sc injection, initial immunity, and immunizing antigen is complete with equal-volume Fei Shi
Adjuvant 1:1 mixes, and fully mixes, booster immunization after 3 weeks；During booster immunization, immunizing antigen and equal-volume Fei Shi Freund's incomplete adjuvant
1:1 mixes, and fully mixes, continuous immunity 2 times, every minor tick 3 weeks, and after last immunity, 10～15 days end tails are taken a blood sample also
Separating serum, use indirect elisa method to measure polyvalent antibody titer, result measures Antibody serum titer at 1:8000-1:10000
Between, collect ascites, first by ammonium sulfate method, ascites is slightly carried, then be purified with protein A-sepharose affinity chromatography.
Preferably, the preparation method of coated film is: with pH7.4,0.01M PBS, 10%-20% sucrose by phenytoin
Sodium antigen PHT-BSA is diluted to 0.5～1.0mg/ml, is sprayed onto on nitrocellulose membrane, and the discharge rate of every 300mm is 30ul, for T line；
Goat-anti rabbit is diluted to 0.3mg/ml～0.5mg/ml, and the discharge rate of every 300mm is 30ul, and running speed is 80 mm/second, pump pressure
100 pump pressures, movable length 290cm is C line, and the spacing of C, T line is 4.5mm, puts 45 DEG C of oven overnight 12～18 hours.
Preferably, the preparation method of gold-marking binding pad is:
Take the colloid gold particle that radius is 15nm～40nm, adjust pH value to 7.0-7.4, under agitation press 10 μ g antigens/ml colloid
Gold adds phenytoin Sodium antibody, and the BSA that last every 1ml adds 100ul 10% stabilizes it, and is centrifuged 30min at 12000rpm/s,
Abandon supernatant, with the Tris-HCl buffer of the 0.01M pH8.5 of 100ul, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰-
0.5‰NaN3Resuspended, obtain phenytoin Sodium antibody-colloidal gold complex；
Phenytoin Sodium antibody-colloidal gold complex and rabbit igg-colloidal gold composite re-suspension liquid Tris-of 0.01M pH8.0
HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TWEEN-20,0.1 ‰-0.5 ‰ NaN3It is diluted to 8%～12%,
Take the glass fibre that area is 25 × 30cm, every coating 20ml～30ml, put baking oven, place 18～24 hours at 37 DEG C；Or
Person phenytoin Sodium antibody-colloidal gold complex and the PBS of rabbit igg-colloidal gold composite every 100ul 0.01M pH8.0,5%
BSA, 1 ‰-0.5 ‰ NaN3It is diluted to 150～300ul, then adds 20% sucrose, dissolve mixing, be sprayed onto with metal-spraying equipment processed
On the polyester film crossed or glass fibre；Equipment pressure 0.015～0.02MPa used, metal spraying parameter is 0.05～0.2ul/mm,
Running speed is 80 mm/second, is sprayed onto on processed polyester film or glass fibre, polyester film or glass fibre processing method
For: it is 3 ‰～5 ‰ Tris, 5%BSA, 0.1 ‰～0.5 ‰ NaN that polyester film or glass fibre are placed on treatment fluid3Middle immersion 2
Hour, liquid should not have polyester film or glass fibre completely, puts 37 DEG C of baking ovens 12～18 hours.
Preferably, the preparation method of sample pad is: taking 25 × 30cm glass fibre, every coating 20ml～30ml coating is molten
Liquid is PBS, 1%BSA, 1%TritonX-100,0.1 ‰-0.5 ‰ NaN of 0.01M pH7.43, put baking oven 37 degree 18～24 little
Time.
The method for quick of a kind of phenytoin Sodium, comprises the steps:
1) detected sample is carried out dissolution process；
2) phenytoin Sodium rapid detection card described above is utilized to detect.
Preferably, in step 1), when detected sample is solid, takes the amount of 0.5-2 grain or sheet or ball or capsule, add alcohols
Organic solvent 1-3mL dissolves, then adds slow releasing agent and be diluted to after cumulative volume 10ml filter, and shaking, no less than 30 seconds, collects filtrate；Treat
When detection sample is liquid, it is not necessary to pre-treatment, directly detect.
Preferably, when step 1) detected sample is solid, as follows according to different dosage form sample dissolving method: hard capsule:
Outward winding capsule shells, take about 1 intragranular tolerant；Soft capsule: cut off capsule shells with shears, extrudes 1 intragranular tolerant；Tablet: be crushed to powder
End, takes 1 amount；Pill: take 1/2 each serving consumption, crushes or shreds.
Preferably, the organic solvent in step 1) is ethanol.
Preferably, in step 1), concentration of alcohol is 50%v/v, and addition is that 2mL dissolves detected sample.
Preferably, the slow releasing agent in step 1) is the PBS of 0.01M, pH7.4.
The invention has the beneficial effects as follows:
Phenytoin Sodium antigen and antibody prepared by the present invention have more preferable specificity.The carrier protein choosing of phenytoin Sodium coupling
From hemocyanin KLH or bovine serum albumin BSA.Utilize the titer of the phenytoin Sodium antibody that above-mentioned antigen goes immune mouse to obtain
Higher, sensitivity is more preferable.
Operating procedure of the present invention is simple, and detection speed is fast, and result is easily observed, and can realize the quick sieve to batch samples
Look into, be substantially reduced testing cost, reduce workload, Food and drug administration has practical significance.
The present invention is directed to testing sample complicated component problem, be optimized sample pretreating method, required sample size is few,
Only need to extract, dilute two steps and can complete sample pre-treatments.Substantially increase detection versatility and detection efficiency.There is operation letter
The features such as single, specificity is strong, highly sensitive, accuracy is high, technology is applicable, Environmental Safety, can be at the scene to the illegal benzene added
Appropriate English Sodium chemistry composition is used for quickly detecting, and meets correlation principle and the requirement of popularization and application.Can implement through simple training
The method, the detection time of each sample, each testing cost was about 15～20 yuan less than 15 minutes.The method loss is 0,
False drop rate is 0, and accuracy is 100%, and detection limit is less than 10 μ g/g.Be applicable to the Chinese patent medicines such as capsule, tablet, pill, liquid agent and
The quick detection of health food.
Accompanying drawing explanation
Fig. 1 is the detection examination card structure schematic diagram that the present invention provides；
Fig. 2 be the detection card result of determination that provides of the present invention be schematic diagram time negative；
Fig. 3 be the detection card result of determination that provides of the present invention be schematic diagram time positive；
Fig. 4 is the detection card result of determination that the present invention provides schematic diagram when being invalid.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the claim of invention is described in further detail, but does not constitute this
Any restriction of invention, the amendment of anyone limited number of time made within the scope of the invention as claimed, still in the power of the present invention
In the range of profit is claimed.
Embodiment 1 phenytoin Sodium antigen, immunogen and the preparation of antibody
1) phenytoin Sodium antigen and immunogen preparation method are:
Weighing in the water that 1 mg 5-diphenyl-phenytoin-3-ω-positive valeric acid joins 0.6ml, dropping pyridine few drops is to entirely
Portion dissolves, and adds 1 mg carbodiimide, and in BSA and KLH of the 10mg/mL being slowly dropped to 1mL respectively, (BSA and KLH all uses
0.01mol, pH9.6 carbonate buffer solution dissolves), 2 hs are stirred at room temperature, with the phosphoric acid of 0.01mol/L, pH7.4 after having reacted
Salt buffer is dialysed 3 days, and 8h changes a dialysis solution, prepares envelope antigen and immunizing antigen respectively.
2) phenytoin Sodium preparation method for antibody is:
With phenytoin Sodium immunizing antigen immunization. Female mice, the immunizing dose of every mice is 120ug/0.3mL, uses quickly assistant
Agent, dorsal sc injection, initial immunity, immunizing antigen mixes with equal-volume Freund's adjuvantcomplete 1:1, fully mixes, after 3 weeks
Booster immunization；During booster immunization, immunizing antigen mixes with equal-volume Fei Shi Freund's incomplete adjuvant 1:1, fully mixes, continuous immunity 2
Secondary, every minor tick 3 weeks, after last immunity, 10～15 days end tails are taken a blood sample and separate serum, use indirect elisa method to survey
Determining polyvalent antibody titer, result mensuration Antibody serum titer, between 1:8000-1:10000, is collected ascites, is first used ammonium sulfate method
Ascites is slightly carried, then is purified with protein A-sepharose affinity chromatography.
The rapid detection card of embodiment 2 phenytoin Sodium
The rapid detection card of phenytoin Sodium, including the sample pad 1 being connected successively in PVC board and PVC board 5, gold-marking binding pad 2, bag
Tunicle 3 and adsorptive pads 4 form, and see that Fig. 1, sample pad 1 are pressed in the 1/2～about 1/3 of gold-marking binding pad 2, and gold-marking binding pad 2 is pressed
At the 0.1～0.2cm of coated film 3, T linear distance coated film 3 lower end 7mm～7.5mm, C linear distance coated film 3 upper end 7mm～
7.3mm, C, T spacing are 4.5mm, and adsorptive pads is pressed at coated film 1～2mm.
Wherein: gold-marking binding pad is absorption phenytoin Sodium antibody-colloidal gold complex and the glass of rabbit-colloidal gold composite
Cellucotton, the orthoscopic recessiveness detection line T line printed with the solution of phenytoin Sodium coupling carrier albumen successively on coated film, use
The orthoscopic recessiveness nature controlling line C line that goat anti-rabbit igg solution is printed, two lines is arranged in parallel.
Described coated film is nitrocellulose filter or cellulose acetate membrane, the use of described rabbit igg-colloidal gold composite
Amount is 0.3～0.5 μ g/cm2, preferably 0.25～0.45 μ g/cm2, the consumption of described phenytoin Sodium antibody-colloidal gold complex
It is 0.4～0.6 μ g/ cm2, preferably 0.4 μ g/mm, the concentration of described goat anti-rabbit igg is 4mg/ml, described goat anti-rabbit igg
Consumption is 0.03～0.05 μ g/mm.
The preparation method of the rapid detection card of embodiment 3 phenytoin Sodium
The preparation method of this detection card is sample pad, gold-marking binding pad, coated film and the adsorptive pads being connected successively in PVC board.
Comprise the following steps:
Wherein:
1) preparation method of coated film is:
With pH7.4,0.01M PBS, 10%-20% sucrose by phenytoin Sodium antigen diluent to 0.5～1.0mg/ml, it is sprayed onto
On nitrocellulose membrane, the discharge rate of every 300mm is 30ul, for T line；Goat-anti rabbit is diluted to 0.3mg/ml～0.5mg/ml, every 300mm
Discharge rate be 30ul, running speed is 80 mm/second, pump pressure 100 pump pressure, and movable length 290cm is C line, between C, T line
Away from for 4.5mm, put 45 DEG C of oven overnight 12～18 hours.
2) preparation method of gold-marking binding pad is:
With high purity water, the gold chloride of 1% is diluted to 0.01%, puts into magnetic stir bar, be placed in temperature constant magnetic stirring heating mantle and boil
Boiling, acutely after boiling, every 100ml is disposably rapidly added the trisodium citrate of the 0.1mol/L of 0.7ml, continues to boil, to color
Keeping redness no longer to change and stop heating, supply the water of loss after being cooled to room temperature, qualified gold colloidal should be bright, nothing
Impurity and floating thing, can preserve one month and not change.
Take the colloid gold particle that radius is 15nm～40nm, adjust pH value to 7.0-7.4, under agitation press 10 μ g antigens/ml
Gold colloidal adds phenytoin Sodium antibody, and the BSA that last every 1ml adds 100ul 10% stabilizes it, and is centrifuged at 12000rpm/s
30min, abandons supernatant, with 1/10th of 100ul(initial colloid gold volume) 0.01M pH8.5 Tris-HCl buffering
Liquid, 1 ‰～5 ‰ PVP-40,5%BSA, 0.1 ‰-0.5 ‰ NaN3Resuspended, obtain phenytoin Sodium antibody-colloidal gold complex,
Put 4 DEG C can put about 2 months.
Phenytoin Sodium antibody-colloidal gold complex and rabbit igg-colloidal gold composite re-suspension liquid with 0.01M pH8.0's
Tris-HCl buffer, 1 ‰～5 ‰ PVP-40,5%BSA, 1 ‰～5 ‰ TWEEN-20,0.1 ‰-0.5 ‰ NaN3It is diluted to 8%
～12%, take the glass fibre that area is 25 × 30cm, every coating 20ml～30ml, put baking oven, place 18～24 at 37 DEG C little
Time；Or phenytoin Sodium antibody-colloidal gold complex and rabbit igg-colloidal gold composite every 100ul 0.01M pH8.0
PBS, 5%BSA, 1 ‰-0.5 ‰ NaN3It is diluted to 150～300ul, then adds 20% sucrose, dissolve mixing, be sprayed onto with metal-spraying equipment
On processed polyester film or glass fibre；Equipment pressure 0.015～0.02MPa used, metal spraying parameter be 0.05～
0.2ul/mm, running speed is 80 mm/second, is sprayed onto on processed polyester film or glass fibre, polyester film or glass fibers
Dimension processing method is: it is 3 ‰～5 ‰ Tris, 5%BSA, 0.1 ‰～0.5 ‰ that polyester film or glass fibre are placed on treatment fluid
NaN3Middle immersion 2 hours, liquid should not have polyester film or glass fibre completely, puts 37 DEG C of baking ovens 12～18 hours.
3) sample pad is prepared
Taking 25 × 30cm glass fibre, every coating 20ml～30ml coating solution is the PBS, 1%BSA, 1% of 0.01M pH7.4
TritonX-100,0.1 ‰-0.5 ‰ NaN3, put 37 degree of baking oven 18～24 hours.
The method for quick of embodiment 4 phenytoin Sodium
1) method for quick of this phenytoin Sodium, comprises the steps: successively
The product that need to detect adds alcohol organic solvent dissolve, then add filtration after the dilution of PBS slow releasing agent, collect filtrate.
Wherein: different dosage form sample dissolving method is as follows:
Hard capsule: capsule shells of outwarding winding, takes about 1 intragranular tolerant；
Soft capsule: cutting off capsule shells with shears, extrusion about 1 intragranular is tolerant；
Tablet: be crushed to powder, takes about 1 amount；
Pill: take about 1/2 each serving consumption, crushes or shreds.
Adding organic solvent in any one of above-mentioned sample, the usage amount of organic solvent with submergence testing sample is
Preferably, the present invention selects the usage amount of 2mL, substantially can cover the needs of different dosage form sample.Preferably alcohol organic solvent is
Ethanol, concentration is at about 50%v/v.The dissolving of product to be detected can be promoted, do not disturb subsequent experimental simultaneously.
For ensureing to extract completely, the shaking time need to be no less than 30 seconds, to adapt to the needs of various dosage form.
It is found through experiments, drops to the filtrate on phenytoin Sodium colloidal gold strip, if the final concentration of organic solvent is big
In 50%, then phenytoin Sodium colloidal gold strip can not normally use, thus the PBS that need to add more than 1mL carries out dilute
Release.Simultaneously, it is contemplated that extracting solution needs to be lost certain solution in filter process, therefore the present invention adds PBS and carries out dilute
Releasing to cumulative volume is 10mL, has both guaranteed the sensitivity of reagent paper, ensures again have enough extracting solution to be loaded.
Liquid dosage form: without pre-treatment, directly detects.
2) filtrate added drop-wise step 1) collected is on phenytoin Sodium colloidal gold chromatographic detection card, beginning timing, and 3～5min
After, according to detection T line and the colour developing of control C line of colloidal gold strip, it is judged that testing sample is positive or negative.
3) step 2) described in colloidal gold test on control C line and detection T line all develop the color, then be judged as feminine gender
(see figure 2), i.e. testing sample are not added with phenytoin Sodium chemical composition；Control line C line colour developing on colloidal gold test, inspection
Survey line T does not develops the color, then be judged as in positive (see figure 3), i.e. testing sample containing phenytoin Sodium chemical composition；Gold colloidal detection examination
Control line C on paper and detection line T does not develops the color, or detection line T colour developing only occurs, then invalid (see figure 4), application are tested in explanation
New colloidal gold test detects again.
The inventive method have simple to operate, specificity is strong, highly sensitive, accuracy is high, technology is applicable, Environmental Safety etc.
Feature, can be used for quickly detecting the illegal phenytoin Sodium chemical composition added at the scene, meet the correlation principle of popularization and application
With require.Can implement the method through simple training, the detection time of each sample is less than 15 minutes, and each testing cost is about
It it is 15～20 yuan.The method loss is 0, and false drop rate is 0, and accuracy is 100%, and detection limit is less than 10 μ g/g.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. a phenytoin Sodium rapid detection card, it is characterised in that: include detecting card base plate, described detection card base plate sets successively
Sample pad, gold-marking binding pad, coated film and the adsorptive pads having and be connected, described gold-marking binding pad be provided with phenytoin Sodium antibody-
Colloidal gold composite and rabbit igg-colloidal gold composite, be sequentially provided with phenytoin Sodium coupling carrier albumen molten on described coated film
Liquid print detection T line and with goat anti-rabbit igg solution printing Quality Control C line.
Detection card the most according to claim 1, it is characterised in that: the consumption of described rabbit igg-colloidal gold composite is
0.3～0.5 μ g/cm2。
Detection card the most according to claim 1, it is characterised in that: described phenytoin Sodium antibody-colloidal gold complex
Consumption is 0.4-0.6 μ g/cm2。
Detection card the most according to claim 1, it is characterised in that: the consumption of described goat anti-rabbit igg is 0.03～0.05 μ g/
mm。
Detection card the most according to claim 1, it is characterised in that: the consumption of described phenytoin Sodium coupling carrier albumen is
0.05～0.1 μ g/mm.
6. the method for quick of a phenytoin Sodium, it is characterised in that comprise the steps:
(1) detected sample is carried out dissolution process；
(2) the phenytoin Sodium rapid detection card described in any one of claim 1-5 is utilized to detect.
Method for quick the most according to claim 6, it is characterised in that: in step 1), when detected sample is solid,
Take the amount of 0.5-2 grain or sheet or ball or capsule, add alcohol organic solvent 1-3mL and dissolve, then add slow releasing agent and be diluted to cumulative volume
Filtering after 10ml, shaking, no less than 30 seconds, collects filtrate；When detected sample is liquid, it is not necessary to pre-treatment, directly detection is i.e.
Can.
Method for quick the most according to claim 6, it is characterised in that: when step 1) detected sample is solid, root
As follows according to different dosage form sample dissolving method: hard capsule: capsule shells of outwarding winding, take about 1 intragranular tolerant；Soft capsule: cut off with shears
Capsule shells, extrudes 1 intragranular tolerant；Tablet: be crushed to powder, takes 1 amount；Pill: take 1/2 each serving consumption, crushes or cuts
Broken.
Method for quick the most according to claim 6, it is characterised in that: the organic solvent in step 1) is ethanol.
Method for quick the most according to claim 6, it is characterised in that: the slow releasing agent in step 1) be 0.01M,
The PBS of pH7.4.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610704950.1A CN106153954A (en) | 2016-08-22 | 2016-08-22 | A kind of method for quick of phenytoin Sodium |
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|---|---|---|---|
| CN201610704950.1A CN106153954A (en) | 2016-08-22 | 2016-08-22 | A kind of method for quick of phenytoin Sodium |
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| CN106153954A true CN106153954A (en) | 2016-11-23 |
Family
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| CN201610704950.1A Pending CN106153954A (en) | 2016-08-22 | 2016-08-22 | A kind of method for quick of phenytoin Sodium |
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| CN107389563A (en) * | 2017-07-25 | 2017-11-24 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A kind of quality determining method of slow controlled release pharmaceutic adjuvant sustained release performance |
| CN108918851A (en) * | 2018-07-16 | 2018-11-30 | 夏泉 | A kind of preparation method of Lamotrigine colloidal gold strip |
| CN109490537A (en) * | 2018-12-14 | 2019-03-19 | 武汉上成生物科技有限公司 | A kind of clenbuterol hydrochloride test strips and preparation method thereof |
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| CN109490537A (en) * | 2018-12-14 | 2019-03-19 | 武汉上成生物科技有限公司 | A kind of clenbuterol hydrochloride test strips and preparation method thereof |
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