CN106770825B - It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue - Google Patents
It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue Download PDFInfo
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- CN106770825B CN106770825B CN201611199214.1A CN201611199214A CN106770825B CN 106770825 B CN106770825 B CN 106770825B CN 201611199214 A CN201611199214 A CN 201611199214A CN 106770825 B CN106770825 B CN 106770825B
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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
- G01N30/02—Column chromatography
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Abstract
The invention discloses a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue, and the composition is made of polypeptide I, polypeptide II, polypeptide III and reference substance detection matrix.The amino acid sequence of the polypeptide I is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide II is shown in SEQ ID NO.2, and the amino acid sequence of polypeptide III is shown in SEQ ID NO.3, and it is fish glue from skin that reference substance, which detects matrix,.The detected value of polypeptide II is subtracted by the detected value of polypeptide I, and corrects detection error using the detected value of polypeptide III, to realize the content detection to donkey hide derived components in animal glue.This method has the advantages that content detection is accurate, easy 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 for detecting composition, kit and its detection side of donkey hide derived components content in animal glue
Method.The invention belongs to medical detection technique fields.
Background technique
Animal glue is with the skin of animal or bone etc. for raw material, and collagen contained therein by partial hydrolysis, extraction and is done
Protein solid content made of dry, there is purposes important extensively in the fields such as food, drug 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
The effect of a kind of tradition rare Chinese medicine, has thousands of years of edible history, has enriching yin of enriching blood, moisturize, hemostasis.Pure donkey-hide gelatin
Must be from donkey hide very, the effect of can guaranteeing product.From raw material taxonomy, donkey belongs to Perissodactyla equine
The kinds such as donkey category, including African wild donkey, Equus kiang, onager, family donkey.Wherein the family donkey of China's raising 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 occurs many criminals in the market, when producing donkey-hide gelatin, portion
Divide and even all replace donkey hide to be boiled with various other Animal Skins include horse skin, mule hide, ox-hide, pigskin etc., some is very
To being directly incorporated into industrial gelatine.These adulterants there are severe jamming market normal order, damage consumer's interests, influence just
Advise the prestige of product.
Animal glue through a long time thermophilic digestion is concentrated, and main component is very close and production technologies of different manufacturers are deposited
Variant, use is infrared, the methods of near-infrared, X- diffraction, HPLC carry out true and false identification to donkey-hide gelatin and be commonly present judgement inaccuracy, scarce
Weary enough convincingnesses etc..The method of donkey hide derived components content in identification donkey-hide gelatin reported at present, substantially all without excluding horse skin
Derived component, though therefore be claimed as donkey hide derived components, be actually donkey and Ma Gongyou.
Therefore, there is an urgent need to propose a kind of side that can quickly, accurately detect donkey hide derived components content in animal glue at present
Method, and then realize and the true and false of the product in the source containing donkey hide is identified.
Summary 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 the goals above, present invention employs following technical solutions:
It is of the invention a kind of for detecting the composition of donkey hide derived components content in animal glue, 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 are 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 also provides a kind of methods for detecting donkey hide derived components content in animal glue, specifically
Include 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, adds 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, NH is used4HCO3Solution
It dissolves and is diluted to scale；
3) prepared by reference substance solution: using matrix solution made from step 1) as solvent, by the mother of reference substance made from step 2)
Liquid dilutes, filtering with microporous membrane, and trypsin solution, enzymatic hydrolysis are added in subsequent filtrate；
(2) preparation of tested animal sol solution
Load weighted tested animal glue is placed in measuring bottle, NH is added4HCO3Solution, ultrasound are completely dissolved animal glue, add
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymatic hydrolysis are added in subsequent filtrate；
(3) LC-MS instrument detects
Gelatin solution to be detected made from reference substance solution made from step (1), step (2) is respectively put into liquid matter connection
With instrument, m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,570.3 is selected to make
MRM scanning is carried out for detection ion pair, wherein m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4
Quantitative detection is carried out respectively as the quota ion pair of polypeptide I, polypeptide II and polypeptide III；
(4) in animal glue donkey hide derived components content calculating
It is the drafting of abscissa progress standard curve using the content of each polypeptide of reference substance solution as ordinate, peak area,
Thus the content of polypeptide I, polypeptide II and polypeptide III in animal glue are calculated, to calculate donkey hide derived components in animal glue
Content, the content of polypeptide I is denoted as M1 in animal glue to be detected, the content of polypeptide II is denoted as M2, the content of polypeptide III is denoted as M3,
The content of donkey hide derived components in animal glue is calculated as follows out:
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)
The 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)
The liquid-phase condition of instrument detection are as follows: C18Reverse-phase chromatographic column, mobile phase A are the aqueous solution of 0.1% (v/v) formic 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 also provides the composition or the kit in detection animal glue
Application in the content of skin source.
Accurate content detection directly is carried out to donkey hide derived components in animal glue, donkey hide derived components can be indicated by first having to find out
And the metastable substance of content in the sterling donkey-hide gelatin of various processes, it especially to remove closer with donkey affiliation
The adulterant ingredient of horse, mule etc.；Secondly the ingredient in donkey-hide gelatin is extremely complex, jitter when detection, therefore the substance will meet
Signal response with higher, lower noise and the interference of other substances when detecting, it is also contemplated that the stability of detection signal；
Third needs to set up suitable reference substance etc..
In this regard, the present invention subtracts polypeptide II's by choosing donkey hide source property characteristic polypeptide, and using the detected value of polypeptide I
Detected value, and detection error is corrected using the detected value of polypeptide III, to realize the content to donkey hide derived components in animal glue
Detection.It is demonstrated experimentally that using the content method of the invention, it is possible to accurately detect donkey hide derived components in animal glue, and operate
Simply, it will be with a wide range of applications in animal glue product quality monitoring field.
Detailed description of the invention
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to the present invention make into
The detailed description of one step, in which:
Fig. 1 be under reference substance solution MRM scan pattern select ion pair m/z393.4 → 499.3, m/z634.7 →
830.4, the mass spectrogram that m/z442.0 → 726.4 is monitored；
Fig. 2 is selection ion pair m/z393.4 → 499.3, m/ under various sterling gelatin solution MRM scan patterns
The mass spectrogram that z634.7 → 830.4, m/z442.0 → 726.4 are monitored.
Specific embodiment
Hereinafter reference will be made to the drawings, and preferred embodiment of the invention is described in detail.It is specific without indicating in preferred embodiment
The experimental method of condition, usually according to normal condition or condition proposed by manufacturer carries out.
Embodiment 1
1, 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) acquisition is decocted 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 method
(1) preparation of reference substance solution
1) preparation of matrix solution:
0.50g reference substance detection matrix is weighed in 250ml measuring bottle, adds 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 substance detection matrix is placed in 25ml measuring bottle, 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 is 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, using matrix solution made from step 1) as solvent, by reference substance made from step 2)
Mother liquor dilutes 1000 times, and filtering with microporous membrane, precision measures 100 μ l of subsequent filtrate, adds the trypsin solution 10 μ l of 2mg/ml, and 37
DEG C constant temperature digests 12h；
(2) preparation of sterling gelatin solution to be measured
It weighs sterling animal glue 0.05g to be measured to be placed in 25ml measuring bottle, adds a small amount of 1% (w/w) NH4HCO3Solution
(pH8.0), ultrasound is completely dissolved sterling animal glue, adds 1% (w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up,
Filtering with microporous membrane, precision measure 100 μ l of subsequent filtrate, add the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h；
(3) LC-MS instrument detects
5 μ l are taken to put respectively sterling gelatin solution to be measured made from reference substance solution made from step (1), step (2)
Enter the detection of LC-MS instrument, liquid-phase condition are as follows: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A 0.1%
(v/v) aqueous solution of formic acid, Mobile phase B are 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, select m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 →
726.4,570.3 MRM scanning is 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 detection 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 under reference substance solution MRM scan pattern select ion pair m/z393.4 → 499.3, m/z634.7 →
830.4, the mass spectrogram that m/z442.0 → 726.4 is monitored；Fig. 2 be selected under various sterling gelatin solution MRM scan patterns from
The mass spectrogram that son monitors m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4.
It is the drafting of abscissa progress standard curve using the content of each polypeptide of reference substance solution as ordinate, peak area,
Linearly dependent coefficient R >=0.9992 of each polypeptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in animal glue,
To calculating the contents of donkey hide derived components in animal glue, the content of polypeptide I is denoted as M1 in animal glue to be detected, polypeptide II contains
Amount is denoted as M2, the content of polypeptide III is denoted as M3, and the content of donkey hide derived components in animal glue is calculated as follows out:
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 calculated result 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 this method can detect in animal glue donkey hide source at
The content divided.
Embodiment 2
1, material and reagent
Material: the donkey-hide gelatin system of different quality percentage epoxy glue sample: is separately added by the sterling glue sample in embodiment 1
At, including containing the oxhide gelatin of 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 1 method of embodiment.
Reagent: ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure).
2, detection method
(1) preparation of reference substance solution
1) preparation of matrix solution:
0.50g reference substance detection matrix is weighed in 250ml measuring bottle, adds 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 substance detection matrix is placed in 25ml measuring bottle, 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 is 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, using matrix solution made from step 1) as solvent, by reference substance made from step 2)
Mother liquor dilutes 500 times, 1000 times, 2000 times, 4000 times, 8000 times, 10000 times, filtering with microporous membrane respectively, and precision measures continuous
100 μ l of filtrate, adds the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h；
(2) preparation of tested animal sol solution
It weighs mixing animal glue 0.05g to be measured to be placed in 25ml measuring bottle, adds a small amount of 1% (w/w) NH4HCO3Solution
(pH8.0), ultrasound is completely dissolved mixing animal glue, adds 1% (w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up,
Filtering with microporous membrane, precision measure 100 μ l of subsequent filtrate, add the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h；
(3) LC-MS instrument detects
5 μ l are taken to be put into respectively gelatin solution to be detected made from reference substance solution made from step (1), step (2)
The detection of LC-MS instrument, liquid-phase condition are as follows: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are 0.1% (v/
V) aqueous solution of formic acid, Mobile phase B are 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+) into
Row multiple-reaction monitoring, select m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,
570.3 as detection ion pair progress MRM scanning, wherein m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0
→ 726.4 respectively as polypeptide I, polypeptide II and polypeptide III quota ion pair carry out quantitative detection；
(4) in animal glue donkey hide derived components content calculating
It is the drafting of abscissa progress standard curve using the content of each polypeptide of reference substance solution as ordinate, peak area,
Thus linearly dependent coefficient R >=0.9992 of each polypeptide calculates containing for polypeptide I, polypeptide II and polypeptide III in mixing animal glue
Amount, to calculate the content of donkey hide derived components in mixing animal glue, the content of polypeptide I is denoted as M1, more in animal glue to be detected
The content of peptide II is denoted as M2, the content of polypeptide III is denoted as M3, and donkey hide derived components in mixing animal glue are calculated as follows out and contain
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 calculated result 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 this method can accurately detect the content of donkey hide derived components in animal glue.
In conclusion matrix is detected using three polypeptides disclosed by the invention and reference substance, using LC-MS instrument, energy
The content of donkey hide derived components in accurate detection animal glue.
It should be noted that above embodiments are only to illustrate the technical solution of invention rather than limit, although by referring to
Invention has been described for the preferred embodiment of the present invention, but the various changes that those skilled in the art make the present invention
Or modification, without departing from spirit and scope of the invention, such equivalent forms are also fallen in the scope of the present invention.
Sequence table
<110>Donga donkey-hide gelatin limited liability company
<120>a kind of for detecting composition, kit and its detection method of donkey hide derived components content in animal glue
<130> KLPI161254
<160> 3
<170>PatentIn 3.5
<210> 1
<211> 9
<212> PRT
<213>polypeptide I
<400> 1
GlyAlaThrGlyProAlaGlyValArg 9
<210> 2
<211> 15
<212> PRT
<213>polypeptide II
<400> 2
GlyIleHypGlyProValGlyAlaAlaGlyAlaThrGlyAlaArg 15
<210> 3
<211> 9
<212> PRT
<213>polypeptide III
<400> 3
GlyValValGlyLeuProGlyGlnArg 9
Claims (3)
1. a kind of method for detecting donkey hide derived components content in animal glue, which comprises the steps of:
(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 make
Sample is completely dissolved, and adds 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
It is completely dissolved sample, load weighted polypeptide I, polypeptide II and polypeptide III are put into the measuring bottle, NH is used4HCO3Solution dissolution
And it is diluted to scale；
The amino acid sequence of the polypeptide I is Gly-Ala-Thr-Gly-Pro-Ala-Gly-Val-Arg, the amino acid of polypeptide II
Sequence is
Gly-Ile-Hyp-Gly-Pro-Val-Gly-Ala-Ala-Gly-Ala-Thr-Gly-Ala- Arg, the amino of polypeptide III
Acid sequence is Gly-Val-Val-Gly-Leu-Pro-Gly-Gln-Arg, and it is fish glue from skin that reference substance, which detects matrix,；
3) prepared by reference substance solution: using matrix solution made from step 1) as solvent, reference substance mother liquor made from step 2) is dilute
It releases, filtering with microporous membrane, trypsin solution, enzymatic hydrolysis is added in subsequent filtrate；
(2) preparation of tested animal sol solution
Load weighted tested animal glue is placed in measuring bottle, NH is added4HCO3Solution, ultrasound are completely dissolved animal glue, add NH4HCO3
Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymatic hydrolysis are added in subsequent filtrate；
(3) LC-MS instrument detects
Gelatin solution to be detected made from reference substance solution made from step (1), step (2) is respectively put into LC-MS
Instrument selects m/z393.4 → 499.3,643.3, m/z634.7 → 830.4,927.5, m/z442.0 → 726.4,570.3 conducts
It detects ion pair and carries out MRM scanning, wherein m/z393.4 → 499.3, m/z634.7 → 830.4, m/z442.0 → 726.4 point
Quota ion pair not as polypeptide I, polypeptide II and polypeptide III carries out quantitative detection；
(4) in animal glue donkey hide derived components content calculating
It is the drafting of abscissa progress standard curve using the content of each polypeptide of reference substance solution as ordinate, peak area, thus
The content of polypeptide I, polypeptide II and polypeptide III in animal glue are calculated, so that the content of donkey hide derived components in animal glue is calculated,
The content of polypeptide I is denoted as M1 in animal glue to be detected, the content of polypeptide II is denoted as M2, the content of polypeptide III is denoted as M3, 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%.
2. the method as described in claim 1, which is characterized in that hydrolysis temperature described in step (1) and step (2) is 37 DEG C.
3. the method as described in claim 1, which is characterized in that the liquid-phase condition that LC-MS instrument detects in step (3) are as follows: C18
Reverse-phase chromatographic column, mobile phase A are the aqueous solution of 0.1%v/v formic 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: electricity
Spraying positive ion mode (ESI+) carry out multiple-reaction monitoring.
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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 |
| CN110824083B (en) * | 2019-11-06 | 2022-04-12 | 东阿阿胶股份有限公司 | Application of donkey-bone glue characteristic polypeptide in detection of donkey-bone glue components in animal skin glue and products thereof |
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