CN106706783A - Composition for identifying authenticity of donkey-hide gelatin and compound preparation thereof, kit and detection method thereof - Google Patents
Composition for identifying authenticity of donkey-hide gelatin and compound preparation thereof, kit and detection method thereof Download PDFInfo
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- CN106706783A CN106706783A CN201611197888.8A CN201611197888A CN106706783A CN 106706783 A CN106706783 A CN 106706783A CN 201611197888 A CN201611197888 A CN 201611197888A CN 106706783 A CN106706783 A CN 106706783A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a composition for identifying the authenticity of donkey-hide gelatin and a compound preparation thereof, a kit and a detection method thereof. The composition is prepared from a polypeptide I, a polypeptide II, a polypeptide III and a reference substance detection matrix, and accordingly the authenticity of the donkey-hide gelatin is judged by utilizing donkey-hide source ingredient contents in the donkey-hide gelatin and the compound preparation thereof. An amino acid sequence of the polypeptide I is shown as SEQIDNO.1, an amino acid sequence of the polypeptide II is shown as SEQIDNO.2, an amino acid sequence of the polypeptide III is shown as SEQIDNO.3, and the reference substance detection matrix is fish skin glue. The detection of the donkey-hide source ingredient contents in the donkey-hide gelatin and the compound preparation thereof is achieved by subtracting a polypeptide II detection value from a polypeptide I detection value and utilizing a polypeptide III detection value to correct detection deviations. The method has the advantages of being accurate in content detection and simple in operation and has a wide application prospect in the field of donkey-hide gelatin product quality monitoring.
Description
Technical field
The present invention relates to a kind of composition, kit and its detection method for identifying donkey-hide gelatin and its compound preparation true and false.This
Invention belongs to medical detection technique field.
Background technology
Donkey-hide gelatin with enriching yin of enriching blood, is moisturized, is stopped blooding as a kind of traditional rare Chinese medicine, the edible history of existing thousands of years
Effect.Chinese Pharmacopoeia specifies that donkey-hide gelatin dries skin or fresh hide through decocting, concentrating for equine species donkey Equus asinus L.'s
The solid gum being made.Pure donkey-hide gelatin must can ensure effect of product from donkey hide very.From raw material biological classification
Seen on, donkey belongs to Perissodactyla equine donkey category, the kind such as including African wild donkey, Equus kiang, onager, family donkey.It is wherein Chinese
The family donkey of raising include Region in Guanzhong Donkey, Dezhou donkey, Guangling donkey, good rice donkey, Miyang donkey, Huaiyang donkey, Qingyang donkey, North China donkey, Xinjiang Donkey,
Numerous local varieties such as southwestern donkey.Because the herding value of donkey constantly declines, and its economic worth has no raising, the quantity of donkey
Worldwide drastically decline, the supply of donkey hide is also nervous year by year, and price rises steadily.In the market occurs in that many illegal point
Son, when donkey-hide gelatin is produced, partially or completely replaces donkey with various other Animal Skins including horse skin, mule hide, ox-hide, pigskin etc.
Skin is boiled, and what is had is even directly incorporated into industrial gelatine.Often with donkey-hide gelatin as main composition in the compound preparation of donkey-hide gelatin,
If what is added in the compound preparation of production donkey-hide gelatin is cheap adulterant donkey-hide gelatin, its drug effect is well imagined.In a word, these adulterants
Presence not only compromise consumer's interests, and severe jamming market normal order have impact on the prestige of regular product.
Donkey-hide gelatin is formed through the concentration of long-time thermophilic digestion, and main component is very close and production technologies of different manufacturers have
Difference, using the methods such as infrared, near-infrared, X- diffraction, HPLC donkey-hide gelatin is carried out the true and false differentiate be commonly present judge it is inaccurate, shortage
Enough convincingnesses etc..The method mainly DNA molecular authentication method and the inspection that differentiate that authenticity of hide glue is more authoritative reported at present
Survey the LC-MS method of characteristic peptide fragment.The both approaches reported cannot all be compared to donkey hide derived components in donkey-hide gelatin
Accurate quantitative determination, but by detect in donkey-hide gelatin whether the composition containing donkey, and exclude other animal derived components and judge
Authenticity of hide glue；But the adulterated of donkey-hide gelatin may be multifarious, it is therefore desirable to which the animal derived components of exclusion can be varied, utilize
There is major defect differentiating authenticity of hide glue in exclusive method, its accuracy and reliability can have a greatly reduced quality, and such case is very unfavorable
In the supervision to market donkey-hide gelatin product.Judge authenticity of hide glue using donkey hide derived components content, can fundamentally limit donkey-hide gelatin
Imitation behavior.In the document reported, donkey hide derived component is detected, horse skin derived component is not all excluded substantially, though therefore
Donkey hide derived components are claimed as, are actually that donkey and horse have.
Therefore, donkey-hide gelatin content in donkey-hide gelatin and its compound preparation can quickly, be accurately detected in the urgent need to proposing one kind at present
Method, and then realize differentiating the true and false of donkey-hide gelatin product.
The content of the invention
It is an advantage of the invention to provide it is a kind of can quickly, precise Identification donkey-hide gelatin and its compound preparation true and false
Composition.
It is a further object of the invention to provide one kind can quickly, precise Identification donkey-hide gelatin and its compound preparation true and false
Kit.
It is still another object of the present invention to provide one kind can quickly, precise Identification donkey-hide gelatin and its compound preparation true and false
Method.
To achieve these goals, present invention employs following technical scheme：
Of the invention a kind of for donkey-hide gelatin and its composition of compound preparation authenticity, the composition is by polypeptide I, many
Peptide II, polypeptide III and reference substance detection matrix composition, wherein：The amino acid sequence of the polypeptide I is Ala-Gly-Glu-
Thr-Gly-Ala-Ser-Gly-Pro-Hyp-Gly-Phe-Ala-Gly-Glu-Lys (SEQ ID NO.1), the amino of polypeptide II
Acid sequence is Gly-Ala-Ser-Gly-Pro-Ala-Gly-Val-Arg (SEQ ID NO.2), the amino acid sequence of polypeptide III
It is Gly-Ser-Glu-Gly-Pro-Gln-Gly-Val-Arg (SEQ ID NO.3) that reference substance detection matrix is fish glue from skin.
Detection kit containing the composition is also within protection scope of the present invention.
Further, the invention allows for a kind of donkey-hide gelatin and its method for compound preparation authenticity, including following step
Suddenly：
1) preparation of reference substance solution：
Weigh reference substance and detect that matrix in measuring bottle, adds NH4HCO3Solution, ultrasound is completely dissolved sample, adds and weighs
Good polypeptide I, polypeptide II and polypeptide III, is completely dissolved, and shakes up, filtering with microporous membrane, and trypsin solution is added in subsequent filtrate,
Enzymolysis；
2) preparation of detected sample solution：
Testing sample is taken, weight is designated as N1, after being dissolved in water, solution of trichloroacetic acid being added, ultrasound removes supernatant after centrifugation
Liquid, precipitation washed with acetone, dry after weigh, weight is designated as N2；A certain amount of precipitation dried object is weighed in measuring bottle, is added
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution is added in subsequent filtrate, enzymolysis；
3) LC-MS instrument detection
Reference substance solution, detected sample solution is taken respectively to be put into LC-MS instrument and detected；Selection m/z725.3 →
962.5th, 818.4, m/z386.4 → 499.3,643.3, m/z444.0 → 331.2,613.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z725.3 → 962.5, m/z386.4 → 499.3, m/z444.0 → 331.2 are respectively as polypeptide I, many
The quota ion pair of peptide II and polypeptide III carries out quantitative determination；
4) in sample donkey hide derived components content calculating
With the content of each polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve is carried out for abscissa,
Linearly dependent coefficient R >=0.998 of each polypeptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in epoxy glue, treats
The content of polypeptide I is designated as M in detection donkey-hide gelatin sample1, polypeptide II content be designated as M2, polypeptide III content be designated as M3, then, press
Following formula calculates the content of donkey hide derived components in each sample：
Content (%)=(M of donkey hide derived components in donkey-hide gelatin1/1448.5–M2/770.8)×1772/M3×N2/N1。
In authenticity identification method of the present invention, it is preferred that step 1) described in the preparation of reference substance solution include
Following steps：
1) preparation of matrix solution：
Weigh 0.50g reference substances detect matrix in 250ml measuring bottles, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution,
Ultrasound is completely dissolved sample, with the NH of 1% (w/w) pH8.04HCO3Solution is diluted to scale, shakes up；
2) preparation of reference substance mother liquor：
Weigh 0.05g reference substances detect matrix in 25ml measuring bottles, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution,
Ultrasound is completely dissolved sample, is separately added into the polypeptide III of the polypeptide II and 17.3mg of polypeptide I, 15.1mg of 28.3mg, uses
The NH of 1% (w/w) pH8.04HCO3Solution is diluted to scale, shakes up；
3) preparation of reference substance solution：
Using the method for stepwise dilution, with matrix solution as solvent, reference substance mother liquor is diluted into 500~10000 times, micropore
Membrane filtration, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h.
In authenticity identification method of the present invention, it is preferred that step 2) described in detected sample solution preparation
Comprise the following steps：0.5-5g compound donkey-hide gelatin preparation samples to be measured are taken, weight is designated as N1, it is 4-5ml to add water to cumulative volume, is mixed
After conjunction, solution of trichloroacetic acid 5ml, the ultrasonic 15min of 30% (w/w) are added, 4 DEG C of standing 30min remove clear liquid after centrifugation, heavy
Shallow lake washed with acetone, dry after weigh, weight is designated as N2；The precipitation dried object of 0.05g is weighed in 25ml measuring bottles, is added a small amount of
The NH of 1% (w/w)4HCO3After solution (pH8.0) dissolving, with the NH of 1% (w/w)4HCO3Solution (pH8.0) is diluted to scale, shakes
Even, filtering with microporous membrane, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, 37 DEG C of constant temperature enzymolysis
12h。
In authenticity identification method of the present invention, it is preferred that step 5) in LC-MS instrument detection liquid-phase condition
For：C18Reverse-phase chromatographic column, mobile phase A is 0.1% (v/v) first aqueous acid, and Mobile phase B is the acetonitrile of 0.1% (v/v) formic acid
Solution, flow velocity 0.3ml/min；Gradient elution：0 → 40min, mobile phase A 100% → 50%, Mobile phase B 0% → 50%, matter
Spectral condition：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
Further, the invention allows for described composition or described kit in detection donkey-hide gelatin and its compound
The application in donkey-hide gelatin content in preparation.
Accurate content detection directly is carried out to donkey hide derived components in donkey-hide gelatin, first have to find out can indicate donkey hide derived components and
The metastable material of content in the sterling donkey-hide gelatin of various processes, especially to remove the horse nearer with donkey affiliation,
The adulterant composition of mule etc.；Secondly the composition in donkey-hide gelatin is extremely complex, jitter during detection, therefore the material will be met in inspection
Responded with signal higher during survey, relatively low noise and other materials are disturbed, it is also contemplated that the stability of detection signal；3rd
Needs set up suitable reference substance etc..In this regard, the present invention is by choosing donkey hide source property characteristic polypeptide, and using the inspection of polypeptide I
Measured value subtracts the detected value of polypeptide II, and corrects detection error using the detected value of polypeptide III, thus realize to donkey-hide gelatin and its
The content detection of donkey hide derived components in compound preparation.It is demonstrated experimentally that using method of the invention, it is possible to detect exactly donkey-hide gelatin and
The content of donkey hide derived components in its compound preparation, and it is simple to operate, will be answered with extensive in donkey-hide gelatin product quality monitoring field
Use prospect.
Brief description of the drawings
In order that the object, technical solutions and advantages of the present invention are clearer, below in conjunction with accompanying drawing the present invention is made into
The detailed description of one step, wherein：
Fig. 1 be select under reference substance solution MRM scan patterns ion pair m/z725.3 → 962.5, m/z386.4 →
499.3rd, the mass spectrogram of m/z444.0 → 331.2 monitoring.
Fig. 2 be select under various sterling glue MRM scan patterns ion pair m/z725.3 → 962.5, m/z386.4 →
499.3rd, the mass spectrogram of m/z444.0 → 331.2 monitoring.
Specific embodiment
Hereinafter with reference to accompanying drawing, preferred embodiment of the invention is described in detail.It is specific without indicating in preferred embodiment
The experimental technique of condition, is generally carried out according to the condition proposed by normal condition or manufacturer.
Embodiment 1
1 material and reagent
Material：Various sterling glue, including donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, fish glue from skin (control product examine
Survey matrix) decoct acquisition with donkey hide, horse skin, ox-hide, pigskin, sheepskin, fish-skin respectively.
Polypeptide I, polypeptide II, polypeptide III transfer to biotech firm to synthesize.
Reagent：Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2 detection methods
1) preparation of matrix solution：
Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, with 1% (w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor：
Weigh 0.05g reference substances and detect matrix in 25ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, is separately added into the polypeptide III of the polypeptide II and 17.3mg of polypeptide I, 15.1mg of 28.3mg, uses
1% (w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
3) preparation of reference substance solution：
Using the method for stepwise dilution, with matrix solution as solvent, reference substance mother liquor is diluted into 1000 times, miillpore filter mistake
Filter, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h.
4) preparation of detected sample solution：
(weight is designated as N to take 0.5g sterling glue samples to be measured1), it is 5ml to add water to cumulative volume, after dissolving, adds 30%
(w/w) solution of trichloroacetic acid 5ml, ultrasonic 15min, 4 DEG C stand 30min, and supernatant is removed after centrifugation, and precipitation is washed with acetone
Wash, dry after weigh that (weight is designated as N2), the precipitation dried object of 0.05g is weighed in 25ml measuring bottles, add a small amount of 1%
NH4HCO3After solution (pH8.0) dissolving, with the NH of 1% (w/w)4HCO3Solution (pH8.0) is diluted to scale, shakes up, micropore filter
Membrane filtration, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h.
5) LC-MS instrument detection
Each 5 μ l of reference substance solution, detected sample solution are taken respectively be put into LC-MS instrument detected.Liquid-phase condition：
C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% (v/v) first aqueous acid, and Mobile phase B is
The acetonitrile solution of 0.1% (v/v) formic acid, flow velocity 0.3ml/min；Gradient elution：0 → 40min, mobile phase A 100% → 50%,
Mobile phase B 0% → 50%.Mass Spectrometry Conditions：Electron spray positive ion mode (ESI+) multiple-reaction monitoring is carried out, select m/z725.3
→ 962.5,818.4, m/z386.4 → 499.3,643.3, m/z444.0 → 331.2,613.3 are carried out as detection ion pair
MRM scan, wherein selection m/z725.3 → 962.5, m/z386.4 → 499.3, m/z444.0 → 331.2 respectively as polypeptide I,
The quota ion pair of polypeptide II and polypeptide III carries out quantitative determination.
6) in sample donkey hide derived components content calculating
Fig. 1 be select under reference substance solution MRM scan patterns ion pair m/z725.3 → 962.5, m/z386.4 →
499.3rd, the mass spectrogram of m/z444.0 → 331.2 monitoring.Fig. 2 is selection ion pair m/ under various sterling glue MRM scan patterns
Z725.3 → 962.5, m/z386.4 → 499.3, the mass spectrogram of m/z444.0 → 331.2 monitoring.
With the content of the polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve is carried out for abscissa, it is many
Linearly dependent coefficient R >=0.998 of peptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in epoxy glue, to be detected
The content of polypeptide I is designated as M in donkey-hide gelatin sample1, polypeptide II content be designated as M2, polypeptide III content be designated as M3, then, as the following formula
Calculate the content of donkey hide derived components in each sample：
Content (%)=(M of donkey hide derived components in donkey-hide gelatin1/1448.5–M2/770.8)×1772/M3×N2/N1=
(0.4633%/1448.5-0%/770.8) × 1772/0.4392% × 0.4035/0.5012 × 100%=103.9% ≈
100%
Content (%)=(M of donkey hide derived components in horse skin glue1/1448.5–M2/770.8)×1772/M3×N2/N1=
(0.4712%/1448.5-0.2508%/770.8) × 1772/0.4408% × 0.4012/0.5008 × 100% ≈ 0%
Content (%)=(M of donkey hide derived components in oxhide gelatin1/1448.5–M2/770.8)×1772/M3×N2/N1=
(0%/1448.5-0%/770.8) × 1772/0.4233% × 0.4028/0.5021 × 100%=0%
Content (%)=(M of donkey hide derived components in pig skin gelatin1/1448.5–M2/770.8)×1772/M3×N2/N1=
(0%/1448.5-0%/770.8) × 1772/0.4567% × 0.4068/0.5022 × 100%=0%
Content (%)=(M of donkey hide derived components in sheepskin glue1/1448.5–M2/770.8)×1772/M3×N2/N1=
(0%/1448.5-0%/770.8) × 1772/0.4322% × 0.4038/0.5012 × 100%=0%
Above-mentioned result of calculation shows：The content 100% of donkey hide derived components, horse skin glue, oxhide gelatin, pig skin gelatin and sheep in donkey-hide gelatin
The content of donkey hide derived components is 0% in hide glue.This is consistent with actual conditions, shows that the method can be from the angle of donkey hide derived components content
Degree accurately distinguishes donkey-hide gelatin and adulterant glue.
Embodiment 2
1 material and reagent
Material：
Epoxy glue sample is made up of the donkey-hide gelatin that the sterling glue sample in embodiment 1 is separately added into different quality, including contains
The oxhide gelatin of 15% donkey-hide gelatin, the oxhide gelatin containing 30% donkey-hide gelatin, the oxhide gelatin containing 60% donkey-hide gelatin.
Donkey-hide gelatin compound preparation sample：Concentrate solution is extracted with the vegetable drug in complex prescription glue mucilage production process, is added respectively
Enter above-mentioned epoxy glue, extracting concentrate solution according to every 9g vegetable drugs adds 1.0g epoxy glue samples to be made in 3 compound glue classes
Medicine preparation, is respectively designated as preparation 1, preparation 2, preparation 3, and donkey hide derived components content is respectively 1.5%, 3.0%, 6.0%.
Polypeptide I, polypeptide II, polypeptide III, fish glue from skin (reference substance detection matrix) are prepared according to the method for embodiment 1.
Reagent：Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure).
2 detection methods
1) preparation of matrix solution：
Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, with 1% (w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor：
The polypeptide III of polypeptide II and 17.3mg of polypeptide I, 15.1mg of 28.3mg is weighed in 25ml measuring bottles, with above-mentioned
The matrix solution of preparation dissolves and is diluted to scale, shakes up.
3) preparation of reference substance solution：
Using the method for stepwise dilution, with matrix solution as solvent, respectively by reference substance mother liquor dilute 500 times, 1000 times,
2000 times, 4000 times, 8000 times, 10000 times, filtering with microporous membrane, precision measures the tryptose of subsequent filtrate 100 μ l plus 2mg/ml
The μ l of enzyme solutions 10,37 DEG C of constant temperature digest 12h.
4) preparation of detected sample solution：
(weight is designated as N to take 5.0g1) donkey-hide gelatin compound preparation sample to be measured, it is 5ml to add water to cumulative volume, after mixing, plus
Enter 30% solution of trichloroacetic acid 5ml, ultrasonic 15min, 4 DEG C of standing 30min, remove clear liquid after centrifugation, precipitation washed with acetone,
Being weighed after drying, (weight is designated as N2), the precipitation dried object of 0.05g is weighed in 25ml measuring bottles, add a small amount of 1% (w/w's)
NH4HCO3After solution (pH8.0) dissolving, with the NH of 1% (w/w)4HCO3Solution (pH8.0) is diluted to scale, shakes up, micropore filter
Membrane filtration, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, and 37 DEG C of constant temperature digest 12h.
5) LC-MS instrument detection
Each 5 μ l of reference substance solution, donkey-hide gelatin compound preparation sample solution are taken respectively be put into LC-MS instrument detected.Liquid phase
Condition：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% first aqueous acid, and Mobile phase B is
The acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution：0 → 40min, mobile phase A 100% → 50%, flowing
Phase B 0% → 50%.Mass Spectrometry Conditions：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring, selection m/z725.3 →
962.5th, 818.4, m/z386.4 → 499.3,643.3, m/z444.0 → 331.2,613.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z725.3 → 962.5, m/z386.4 → 499.3, m/z444.0 → 331.2 are respectively as polypeptide I, many
The quota ion pair of peptide II and polypeptide III carries out quantitative determination.
6) in sample donkey hide derived components content calculating
With the content of each polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve is carried out for abscissa,
Linearly dependent coefficient R >=0.998 of each polypeptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in epoxy glue, treats
The content of polypeptide I is designated as M in detection donkey-hide gelatin sample1, polypeptide II content be designated as M2, polypeptide III content be designated as M3, then, press
Following formula calculates the content of donkey hide derived components in each sample：
Content (%)=(M of donkey hide derived components in preparation 11/1448.5–M2/770.8)×1772/M3×N2/N1
(0.0670%/1448.5-0%/770.8) × 1772/0.4392% × 0.4102/5.02 × 100%=1.52%
Content (%)=(M of donkey hide derived components in preparation 21/1448.5–M2/770.8)×1772/M3×N2/N1
(0.1327/1448.5-0%/770.8) × 1772/0.4412% × 0.4166/5.05 × 100%=3.04%
Content (%)=(M of donkey hide derived components in preparation 31/1448.5–M2/770.8)×1772/M3×N2/N1
(0.2601/1448.5-0%/770.8) × 1772/0.4322 × 0.4187/5.05 × 100%=6.10%
Above-mentioned result of calculation shows：Detection level is consistent with actual conditions, shows that the method can accurately detect compound preparation
The content of middle donkey-hide gelatin composition, so as to realize differentiating the purpose of donkey-hide gelatin and its compound preparation true and false.
In sum, using three polypeptides disclosed by the invention and reference substance detection matrix, using LC-MS instrument, energy
The content of donkey-hide gelatin in accurate detection donkey-hide gelatin and its compound preparation.
It should be noted that above example is only used to illustrate the technical scheme of invention and unrestricted, although by referring to
Invention has been described for the preferred embodiments of the present invention, but the various changes that those skilled in the art make to the present invention
Or modification, without departing from spirit and scope of the invention, these equivalent form of values are also fallen within the scope of the present invention.
Sequence table
<110>Donga donkey-hide gelatin limited company
<120>A kind of composition for donkey-hide gelatin and its compound preparation authenticity, kit and its detection method
<130> KLPI161263
<160> 3
<170>PatentIn 3.5
<210> 1
<211> 16
<212> PRT
<213>Polypeptide I
<400> 1
Ala Gly Glu Thr Gly Ala Ser Gly Pro Hyp Gly Phe Ala Gly Glu Lys
<210> 2
<211> 9
<212> PRT
<213>Polypeptide II
<400> 2
Gly Ala Ser Gly Pro Ala Gly Val Arg
<210> 3
<211> 9
<212> PRT
<213>Polypeptide III
<400> 3
Gly Ser Glu Gly Pro Gln Gly Val Arg
Claims (8)
1. be used for the composition of donkey-hide gelatin and its compound preparation authenticity, the composition by polypeptide I, polypeptide II, polypeptide III with
And reference substance detection matrix composition, it is characterised in that：The amino acid sequence of the polypeptide I is Ala-Gly-Glu-Thr-Gly-
The amino acid sequence of Ala-Ser-Gly-Pro-Hyp-Gly-Phe-Ala-Gly-Glu-Lys, polypeptide II is Gly-Ala-Ser-
The amino acid sequence of Gly-Pro-Ala-Gly-Val-Arg, polypeptide III is Gly-Ser-Glu-Gly-Pro-Gln-Gly-Val-
Arg, reference substance detection matrix is fish glue from skin.
2. the detection kit of composition described in claim 1 is contained.
3. the method for donkey-hide gelatin and its compound preparation authenticity, it is characterised in that comprise the following steps：
1) preparation of reference substance solution：
Weigh reference substance and detect that matrix in measuring bottle, adds NH4HCO3Solution, ultrasound is completely dissolved sample, adds load weighted
Polypeptide I, polypeptide II and polypeptide III, are completely dissolved, and shake up, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
2) preparation of detected sample solution：
Testing sample is taken, weight is designated as N1, after being dissolved in water, solution of trichloroacetic acid being added, ultrasound removes supernatant, sinks after centrifugation
Shallow lake washed with acetone, dry after weigh, weight is designated as N2；A certain amount of precipitation dried object is weighed in measuring bottle, NH is added4HCO3It is molten
Liquid is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution is added in subsequent filtrate, enzymolysis；
3) LC-MS instrument detection
Reference substance solution, detected sample solution is taken respectively to be put into LC-MS instrument and detected；Selection m/z725.3 →
962.5th, 818.4, m/z386.4 → 499.3,643.3, m/z444.0 → 331.2,613.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z725.3 → 962.5, m/z386.4 → 499.3, m/z444.0 → 331.2 are respectively as polypeptide I, many
The quota ion pair of peptide II and polypeptide III carries out quantitative determination；
4) in sample donkey hide derived components content calculating
With the content of each polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve is carried out for abscissa, it is each many
Linearly dependent coefficient R >=0.998 of peptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in epoxy glue, to be detected
The content of polypeptide I is designated as M in donkey-hide gelatin sample1, polypeptide II content be designated as M2, polypeptide III content be designated as M3, then, as the following formula
Calculate the content of donkey hide derived components in each sample：
Content (%)=(M of donkey hide derived components in donkey-hide gelatin1/1448.5–M2/770.8)×1772/M3×N2/N1。
4. method as claimed in claim 3, it is characterised in that step 1) described in the preparation of reference substance solution include following step
Suddenly：
1) preparation of matrix solution：
Weigh 0.50g reference substances detect matrix in 250ml measuring bottles, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution, ultrasound
It is completely dissolved sample, with the NH of 1% (w/w) pH8.04HCO3Solution is diluted to scale, shakes up；
2) preparation of reference substance mother liquor：
Weigh 0.05g reference substances detect matrix in 25ml measuring bottles, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution, ultrasound
Sample is completely dissolved, the polypeptide III of the polypeptide II and 17.3mg of polypeptide I, 15.1mg of 28.3mg is separately added into, with 1% (w/
W) NH of pH8.04HCO3Solution is diluted to scale, shakes up；
3) preparation of reference substance solution：
Using the method for stepwise dilution, with matrix solution as solvent, reference substance mother liquor is diluted into 500~10000 times, miillpore filter
Filtering, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h.
5. method as claimed in claim 3, it is characterised in that step 2) described in detected sample solution preparation include with
Lower step：0.5-5g compound donkey-hide gelatin preparation samples to be measured are taken, weight is designated as N1, it is 4-5ml to add water to cumulative volume, after mixing,
Solution of trichloroacetic acid 5ml, the ultrasonic 15min of 30% (w/w) are added, 4 DEG C stand 30min, and clear liquid is removed after centrifugation, and precipitation uses third
Weighed after ketone washing, drying, weight is designated as N2, the precipitation dried object of 0.05g is weighed in 25ml measuring bottles, add a small amount of 1% (w/
W) NH of pH8.04HCO3After solution dissolving, with the NH of 1% (w/w) pH8.04HCO3Solution is diluted to scale, shakes up, micropore filter
Membrane filtration, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, and 37 DEG C of constant temperature digest 12h.
6. method as claimed in claim 3, it is characterised in that step 5) in the liquid-phase condition of LC-MS instrument detection be：C18
Reverse-phase chromatographic column, mobile phase A is 0.1% (v/v) first aqueous acid, and Mobile phase B is the acetonitrile solution of 0.1% (v/v) formic acid,
Flow velocity 0.3ml/min；Gradient elution：0 → 40min, mobile phase A 100% → 50%, Mobile phase B 0% → 50%, mass spectrum bar
Part：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
7. the application in donkey-hide gelatin content of the composition described in claim 1 in detection donkey-hide gelatin and its compound preparation.
8. the application in donkey-hide gelatin content of the kit described in claim 2 in detection donkey-hide gelatin and its compound preparation.
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| CN103630623A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Substance and method for detecting tortoise-shell glue and products thereof |
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| CN104280468A (en) * | 2013-07-12 | 2015-01-14 | 山东东阿阿胶股份有限公司 | Method for detecting horse-breed and mule-breed derived ingredients in gelatin-type traditional Chinese medicine and products |
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| CN104280468A (en) * | 2013-07-12 | 2015-01-14 | 山东东阿阿胶股份有限公司 | Method for detecting horse-breed and mule-breed derived ingredients in gelatin-type traditional Chinese medicine and products |
| CN103630623A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Substance and method for detecting tortoise-shell glue and products thereof |
| CN103630644A (en) * | 2013-10-31 | 2014-03-12 | 山东东阿阿胶股份有限公司 | LC-MS (Liquid Chromatography-Mass Spectrometry) detection method for tortoise-derived ingredients in glue type traditional Chinese medicines and products thereof |
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