CN106770825A - A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method - Google Patents
A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method Download PDFInfo
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- CN106770825A CN106770825A CN201611199214.1A CN201611199214A CN106770825A CN 106770825 A CN106770825 A CN 106770825A CN 201611199214 A CN201611199214 A CN 201611199214A CN 106770825 A CN106770825 A CN 106770825A
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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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- G01N30/06—Preparation
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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 kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method, the composition is made up of polypeptide I, polypeptide II, polypeptide III and reference substance detection matrix.The amino acid sequence of the polypeptide I is that shown in SEQ ID NO.1, the amino acid sequence of polypeptide II is for shown in SEQ ID NO.2, the amino acid sequence of polypeptide III is for shown in SEQ ID NO.3, reference substance detection matrix is fish glue from skin.The detected value of polypeptide II is subtracted by the detected value of polypeptide I, and detection error is corrected using the detected value of polypeptide III, so as to realize the content detection to donkey hide derived components in animal glue.The method has the advantages that content detection is accurate, simple to operate, will be with a wide range of applications in animal glue product quality monitoring field.
Description
Technical field
The present invention relates to a kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection side
Method.The invention belongs to medical detection technique field.
Background technology
Animal glue is as raw material, by collagen contained therein is by partial hydrolysis, extraction and does with skin or bone of animal etc.
The dry protein solid content being made, there is purposes important extensively in the fields such as food, medicine and cosmetics.Common animal
Glue includes donkey-hide gelatin, oxhide gelatin, fresh Donkey-hide Glue (pig skin gelatin), sheepskin glue, horse skin glue, colla carapacis et plastri testudinis, deer horn glue etc..Wherein donkey-hide gelatin conduct
A kind of traditional rare Chinese medicine, the edible history of existing thousands of years, with enriching yin of enriching blood, moisturizes, effect of hemostasis.Pure donkey-hide gelatin
Must can ensure effect of product from donkey hide very.From raw material taxonomy, donkey belongs to Perissodactyla equine
Donkey belongs to, the kind such as including African wild donkey, Equus kiang, onager, family donkey.The family donkey that wherein China raises includes Region in Guanzhong Donkey, moral
Numerous local varieties such as state donkey, Guangling donkey, good rice donkey, Miyang donkey, Huaiyang donkey, Qingyang donkey, North China donkey, Xinjiang Donkey, southwestern donkey.By
Also nervous year by year in the supply of donkey hide, price rises steadily, and in the market occurs in that many lawless persons, when donkey-hide gelatin is produced, portion
Divide and even all replace donkey hide to be boiled including horse skin, mule hide, ox-hide, pigskin etc. with various other Animal Skins, have very
To being directly incorporated into industrial gelatine.The presence severe jamming of these adulterants market normal order, damages consumer's interests, and influence is just
Advise the prestige of product.
Animal glue is formed through the concentration of long-time thermophilic digestion, and main component is very close and production technologies of different manufacturers are deposited
It is variant, carry out the true and false to donkey-hide gelatin using methods such as infrared, near-infrared, X- diffraction, HPLC and differentiate being commonly present and judging inaccurate, scarce
Weary enough convincingnesses etc..The method for differentiating donkey hide derived components content in donkey-hide gelatin reported at present, does not all exclude horse skin substantially
Derived component, though therefore be claimed as donkey hide derived components, be actually that donkey and horse have.
Therefore, at present in the urgent need to proposing a kind of side that can quickly, accurately detect donkey hide derived components content in animal glue
Method, and then realize differentiating the true and false of the product containing donkey hide source.
The content of the invention
It is an advantage of the invention to provide one kind can quickly, donkey hide derived components content in precise Identification animal glue
Composition.
It is a further object of the invention to provide one kind can quickly, in precise Identification animal glue donkey hide derived components contain
The kit of amount.
It is still another object of the present invention to provide one kind can quickly, in precise Identification animal glue donkey hide derived components contain
The method of amount.
To achieve these goals, present invention employs following technical scheme：
A kind of composition for detecting donkey hide derived components content in animal glue of the invention, the composition by polypeptide I,
Polypeptide II, polypeptide III and reference substance detection matrix composition, it is characterised in that：The amino acid sequence of the polypeptide I is Gly-
The amino acid sequence of Ala-Thr-Gly-Pro-Ala-Gly-Val-Arg (shown in SEQ ID NO.1), polypeptide II is Gly-Ile-
Hyp-Gly-Pro-Val-Gly-Ala-Ala-Gly-Ala-Thr-Gly-Ala-Arg (shown in SEQ ID NO.2), polypeptide III
Amino acid sequence be Gly-Val-Val-Gly-Leu-Pro-Gly-Gln-Arg (shown in SEQ ID NO.3), 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 method for detecting donkey hide derived components content in animal glue, specifically
Comprise the following steps：
(1) preparation of reference substance solution
1) preparation of matrix solution：Load weighted reference substance detection matrix is placed in measuring bottle, NH is added4HCO3Solution, surpasses
Sound is completely dissolved sample, plus NH4HCO3Solution is diluted to scale；
2) preparation of reference substance mother liquor：Load weighted reference substance detection matrix is placed in measuring bottle, NH is added4HCO3Solution,
Ultrasound is completely dissolved sample, and load weighted polypeptide I, polypeptide II and polypeptide III are placed in the measuring bottle, uses NH4HCO3Solution
Dissolve and be diluted to scale；
3) prepared by reference substance solution：With step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance it is female
Liquid is diluted, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
(2) preparation of tested animal sol solution
Load weighted tested animal glue is placed in measuring bottle, plus NH4HCO3Solution, ultrasound is completely dissolved animal glue, plus
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
(3) LC-MS instrument detection
Reference substance solution, gelatin solution to be detected obtained in step (2) obtained in step (1) are respectively put into liquid matter connection
With instrument, selection m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,570.3 are made
For detection ion pair carries out MRM scannings, wherein, m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4
Quota ion pair respectively as polypeptide I, polypeptide II and polypeptide III carries out quantitative determination；
(4) in animal glue 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,
Thus the content of polypeptide I, polypeptide II and polypeptide III in animal glue is calculated, so as to calculate donkey hide derived components in animal glue
Content, the content of polypeptide I is designated as M1, the content of polypeptide II and is designated as M2, the content of polypeptide III being designated as M3 in animal glue to be detected,
It is calculated as follows out the content of donkey hide derived components in animal glue：
The content (%) of donkey hide derived components=(M1/392.4-M2/633.7) × 880/M3 × 100%
Method for detecting donkey hide derived components content in animal glue of the invention, it is preferred that step (1) and step (2)
Described hydrolysis temperature is 37 DEG C.
Method for detecting donkey hide derived components content in animal glue of the invention, it is preferred that LC-MS in step (3)
Instrument detection liquid-phase condition be：C18Reverse-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 Spectrometry Conditions：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
Preferably, the animal glue is donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis, deer horn glue, donkey gelatine, bone glue, pig bone glue, sheep
Gelatine or other glue class Chinese medicines.
Further, the donkey the invention allows for described composition or described kit in animal glue is detected
Application in the content of skin source.
Accurate content detection directly is carried out to donkey hide derived components in animal glue, first having to find out can indicate donkey hide derived components
And in the sterling donkey-hide gelatin of various processes the metastable material of content, especially to remove nearer with donkey affiliation
The adulterant composition of horse, mule etc.；Secondly the composition in donkey-hide gelatin is extremely complex, jitter during detection, therefore the material will meet
In detection with signal response higher, relatively low noise and the interference of other materials, it is also contemplated that the stability of detection signal；
3rd needs to set up suitable reference substance etc..
In this regard, the present invention is by choosing donkey hide source property characteristic polypeptide, and detected value using polypeptide I subtracts polypeptide II's
Detected value, and detection error is corrected using the detected value of polypeptide III, so as to realize the content to donkey hide derived components in animal glue
Detection.It is demonstrated experimentally that using method of the invention, it is possible to detect the content of donkey hide derived components in animal glue, and operation exactly
Simply, will be with a wide range of applications in animal glue product quality monitoring field.
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/z393.4 → 499.3, m/z634.7 →
830.4th, the mass spectrogram of m/z442.0 → 726.4 monitoring；
Fig. 2 is selection ion pair m/z393.4 → 499.3, m/ under various sterling gelatin solution MRM scan patterns
Z634.7 → 830.4, the mass spectrogram of m/z442.0 → 726.4 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
1st, material and reagent
Material：Various sterling animal glue, including donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, fish glue from skin (control
Matrix is surveyed in product examine) 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).
2nd, detection method
(1) preparation of reference substance solution
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：
Load weighted 0.05g reference substances detection matrix is placed in 25ml measuring bottles, a small amount of 1% (w/w) NH is added4HCO3It is molten
Liquid (pH8.0), ultrasound is completely dissolved sample, weighs the polypeptide of the polypeptide II and 17.2mg of polypeptide I, 24.7mg of 15.3mg
III in the measuring bottle, with 1% (w/w) NH4HCO3Solution (pH8.0) dissolves and is diluted to scale, shakes up；
3) preparation of reference substance solution：
Using the method for stepwise dilution, with step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance
Mother liquor dilutes 1000 times, and filtering with microporous membrane, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml trypsin solution 10 μ l, 37
DEG C constant temperature enzymolysis 12h；
(2) preparation of sterling gelatin solution to be measured
Sterling animal glue 0.05g to be measured is weighed to be placed in 25ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution
(pH8.0), ultrasound is completely dissolved sterling animal glue, (w/w) NH of plus 1%4HCO3Solution (pH8.0) is diluted to scale, shakes up,
Filtering with microporous membrane, 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；
(3) LC-MS instrument detection
Sterling gelatin solution to be measured obtained in reference substance solution, step (2) obtained in step (1) is taken into 5 μ l respectively to put
Enter the detection of LC-MS instrument, liquid-phase condition is：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1%
(v/v) first aqueous acid, 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+)
Carry out multiple-reaction monitoring, selection m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 →
726.4th, 570.3 MRM scannings are carried out as detection ion pair, wherein, m/z393.4 → 499.3, m/z634.7 → 830.4, m/
Z442.0 → 726.4 carry out quantitative determination respectively as the quota ion pair of polypeptide I, polypeptide II and polypeptide III；
(4) in animal glue donkey hide derived components content calculating
Fig. 1 be select under reference substance solution MRM scan patterns ion pair m/z393.4 → 499.3, m/z634.7 →
830.4th, the mass spectrogram of m/z442.0 → 726.4 monitoring；Fig. 2 be select under various sterling gelatin solution MRM scan patterns from
The mass spectrogram that son is monitored to m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4.
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 >=0.9992 of each polypeptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in animal glue,
So as to calculating the content of donkey hide derived components in animal glue, the content of polypeptide I is designated as M1, polypeptide II and contains in animal glue to be detected
Amount is designated as M2, the content of polypeptide III and is designated as M3, is calculated as follows out the content of donkey hide derived components in animal glue：
The content (%) of donkey hide derived components=(M1/392.4-M2/633.7) × 880/M3 × 100%
Wherein, in each sample donkey hide derived components content：
Content (%)=(0.2411/392.4-0/633.7) × 880/0.5418 × 100% of donkey hide derived components in donkey-hide gelatin
=99.79% ≈ 100%
The content (%) of donkey hide derived components=(0/392.4-0/633.7) × 880/0.5367 × 100%=in horse skin glue
0%
The content (%) of donkey hide derived components=(0.2420/392.4-0.3896/633.7) × 880/0.5422 in oxhide gelatin
× 100%=0.31% ≈ 0%
The content (%) of donkey hide derived components=(0/392.4-0/633.7) × 880/0.5486 × 100%=in pig skin gelatin
0%
The content (%) of donkey hide derived components=(0.2697/392.4-0.4333/633.7) × 880/0.5592 in sheepskin glue
× 100%=0.56% ≈ 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, show the method can detect in animal glue donkey hide source into
The content divided.
Embodiment 2
1st, material and reagent
Material：Epoxy glue sample：The donkey-hide gelatin system of different quality percentage is separately added into by the sterling glue sample in embodiment 1
Into, including the oxhide gelatin containing 15% donkey-hide gelatin, the oxhide gelatin containing 30% donkey-hide gelatin, the oxhide gelatin containing 60% donkey-hide gelatin, containing 30% donkey-hide gelatin
Horse skin glue, the pig skin gelatin containing 30% donkey-hide gelatin, are respectively designated as sample 1, sample 2, sample 3, sample 4 and sample 5.
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).
2nd, detection method
(1) preparation of reference substance solution
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：
Load weighted 0.05g reference substances detection matrix is placed in 25ml measuring bottles, a small amount of 1% (w/w) NH is added4HCO3It is molten
Liquid (pH8.0), ultrasound is completely dissolved sample, weighs the polypeptide of the polypeptide II and 17.2mg of polypeptide I, 24.7mg of 15.3mg
III in the measuring bottle, with 1% (w/w) NH4HCO3Solution (pH8.0) dissolves and is diluted to scale, shakes up；
3) preparation of reference substance solution：
Using the method for stepwise dilution, with step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance
Mother liquor dilutes 500 times, 1000 times, 2000 times, 4000 times, 8000 times, 10000 times respectively, and filtering with microporous membrane, precision measures continuous
The μ l of filtrate 100, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h；
(2) preparation of tested animal sol solution
Mixing animal glue 0.05g to be measured is weighed to be placed in 25ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution
(pH8.0), ultrasound is completely dissolved mixing animal glue, (w/w) NH of plus 1%4HCO3Solution (pH8.0) is diluted to scale, shakes up,
Filtering with microporous membrane, 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；
(3) LC-MS instrument detection
Reference substance solution, gelatin solution to be detected obtained in step (2) obtained in step (1) are taken 5 μ l and be put into respectively
LC-MS instrument detects that liquid-phase condition is：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% (v/
V) first aqueous acid, 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+) enter
Row multiple-reaction monitoring, selection m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,
570.3 carry out MRM scannings as detection ion pair, wherein, m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0
→ 726.4 carry out quantitative determination respectively as the quota ion pair of polypeptide I, polypeptide II and polypeptide III；
(4) in animal glue 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 >=0.9992 of each polypeptide, polypeptide I, polypeptide II and polypeptide III contain in thus calculating mixing animal glue
Amount, so as to calculate the content of donkey hide derived components in mixing animal glue, the content of polypeptide I is designated as M1, many in animal glue to be detected
The content of peptide II is designated as M2, the content of polypeptide III and is designated as M3, is calculated as follows out containing for donkey hide derived components in mixing animal glue
Amount：
The content (%) of donkey hide derived components=(M1/392.4-M2/633.7) × 880/M3 × 100%
The content (%) of donkey hide derived components=(0.2493/392.4-0.3425/633.7) in oxhide gelatin containing 15% donkey-hide gelatin
× 880/0.5369 × 100%=15.55%
The content (%) of donkey hide derived components=(0.2467/392.4-0.2732/633.7) in oxhide gelatin containing 30% donkey-hide gelatin
× 880/0.5473 × 100%=31.77%
The content (%) of donkey hide derived components=(0.2430/392.4-0.1564/633.7) in oxhide gelatin containing 60% donkey-hide gelatin
× 880/0.5416 × 100%=60.52%
Content (%)=(0.0749/392.4-0/633.7) of donkey hide derived components in horse skin glue containing 30% donkey-hide gelatin ×
880/0.5587 × 100%=30.06%
Content (%)=(0.0719/392.4-0/633.7) of donkey hide derived components in pig skin gelatin containing 30% donkey-hide gelatin ×
880/0.5391 × 100%=29.91%
Above-mentioned result of calculation shows：The donkey hide source detection level of the sample 1-5 obtained using detection method of the invention with
Actual conditions are consistent, and show that the method can accurately detect the content of donkey hide derived components in animal glue.
In sum, using three polypeptides disclosed by the invention and reference substance detection matrix, using LC-MS instrument, energy
The content of donkey hide derived components in accurate detection animal glue.
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 detecting donkey hide derived components content in animal glue, kit and its detection method
<130> KLPI161254
<160> 3
<170>PatentIn 3.5
<210> 1
<211> 9
<212> PRT
<213>Polypeptide I
<400> 1
Gly Ala Thr Gly Pro Ala Gly Val Arg 9
<210> 2
<211> 15
<212> PRT
<213>Polypeptide II
<400> 2
Gly Ile Hyp Gly Pro Val Gly Ala Ala Gly Ala Thr Gly Ala Arg 15
<210> 3
<211> 9
<212> PRT
<213>Polypeptide III
<400> 3
Gly Val Val Gly Leu Pro Gly Gln Arg 9
Claims (7)
1. a kind of composition for detecting donkey hide derived components content in animal glue, the composition is by polypeptide I, polypeptide II, many
Peptide III and reference substance detection matrix composition, it is characterised in that：The amino acid sequence of the polypeptide I is Gly-Ala-Thr-
The amino acid sequence of Gly-Pro-Ala-Gly-Val-Arg, polypeptide II is Gly-Ile-Hyp-Gly-Pro-Val-Gly-Ala-
The amino acid sequence of Ala-Gly-Ala-Thr-Gly-Ala-Arg, polypeptide III is Gly-Val-Val-Gly-Leu-Pro-Gly-
Gln-Arg, reference substance detection matrix is fish glue from skin.
2. a kind of detection kit containing composition described in claim 1.
3. a kind of method for detecting donkey hide derived components content in animal glue, it is characterised in that comprise the following steps：
(1) preparation of reference substance solution
1) preparation of matrix solution：Load weighted reference substance detection matrix is placed in measuring bottle, NH is added4HCO3Solution, ultrasound makes
Sample is completely dissolved, plus NH4HCO3Solution is diluted to scale；
2) preparation of reference substance mother liquor：Load weighted reference substance detection matrix is placed in measuring bottle, NH is added4HCO3Solution, ultrasound
Sample is completely dissolved, load weighted polypeptide I, polypeptide II and polypeptide III are put into the measuring bottle, use NH4HCO3Solution dissolves
And it is diluted to scale；
3) prepared by reference substance solution：With step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance mother liquor it is dilute
Release, filtering with microporous membrane, trypsin solution, enzymolysis are added in subsequent filtrate；
(2) preparation of tested animal sol solution
Load weighted tested animal glue is placed in measuring bottle, plus NH4HCO3Solution, ultrasound is completely dissolved animal glue, plus NH4HCO3
Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
(3) LC-MS instrument detection
Reference substance solution, gelatin solution to be detected obtained in step (2) obtained in step (1) are respectively put into LC-MS
Instrument, selection m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,570.3 conducts
Detection ion pair carries out MRM scannings, wherein, m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4 point
Quantitative determination is not carried out as the quota ion pair of polypeptide I, polypeptide II and polypeptide III；
(4) in animal glue 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, thus
The content of polypeptide I, polypeptide II and polypeptide III in animal glue is calculated, so as to calculate the content of donkey hide derived components in animal glue,
The content of polypeptide I is designated as M1, the content of polypeptide II and is designated as M2, the content of polypeptide III being designated as M3 in animal glue to be detected, as the following formula
Calculate the content of donkey hide derived components in animal glue：
The content (%) of donkey hide derived components=(M1/392.4-M2/633.7) × 880/M3 × 100%.
4. method as claimed in claim 3, it is characterised in that the hydrolysis temperature described in step (1) and step (2) is 37 DEG C.
5. method as claimed in claim 3, it is characterised in that the liquid-phase condition of LC-MS instrument detection is in step (3)：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.
6. it is used to detect the composition of donkey hide derived components content in animal glue as claimed in claim 1, it is characterised in that described
Animal glue is donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis, deer horn glue, donkey gelatine, bone glue, pig bone glue, sheep bone glue or other glue class Chinese medicines.
7. the kit described in the composition or claim 2 described in claim 1 detect animal glue in donkey hide source content
In application.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611199214.1A CN106770825B (en) | 2016-12-22 | 2016-12-22 | It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue |
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|---|---|---|---|
| CN201611199214.1A CN106770825B (en) | 2016-12-22 | 2016-12-22 | It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue |
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| Publication Number | Publication Date |
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| CN106770825A true CN106770825A (en) | 2017-05-31 |
| CN106770825B CN106770825B (en) | 2019-12-03 |
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| CN109521104A (en) * | 2018-08-03 | 2019-03-26 | 西北工业大学 | It is a kind of for an identification method for emerald green historical relic adhesive |
| CN110824083A (en) * | 2019-11-06 | 2020-02-21 | 东阿阿胶股份有限公司 | Donkey-bone glue characteristic polypeptide and application thereof in detection of donkey-bone glue components in animal skin glue and products thereof |
| CN113880916A (en) * | 2021-08-19 | 2022-01-04 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109521104A (en) * | 2018-08-03 | 2019-03-26 | 西北工业大学 | It is a kind of for an identification method for emerald green historical relic adhesive |
| CN110824083A (en) * | 2019-11-06 | 2020-02-21 | 东阿阿胶股份有限公司 | Donkey-bone glue characteristic polypeptide and application thereof in detection of donkey-bone glue components in animal skin glue and products thereof |
| CN113880916A (en) * | 2021-08-19 | 2022-01-04 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
| CN113880916B (en) * | 2021-08-19 | 2023-06-30 | 青海瑞肽生物科技有限公司 | Yak skin antioxidant polypeptide and preparation method and application thereof |
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