CN106841437B - For detecting composition, kit and its detection method of donkey hide derived components content in donkey-hide gelatin - Google Patents
For detecting composition, kit and its detection method of donkey hide derived components content in donkey-hide gelatin Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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 donkey-hide gelatin.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, the amino acid sequence of polypeptide II is shown in SEQ ID NO.2, 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 donkey-hide gelatin.This method has the advantages that content detection is accurate, easy to operate, is with a wide range of applications in the donkey hide derived components context of detection of donkey-hide gelatin.
Description
Technical field
The present invention relates to the compositions and kit of donkey hide derived components content in a kind of detection donkey-hide gelatin, further include using the group
The method that object or kit carry out donkey hide derived components content detection in donkey-hide gelatin is closed, the invention belongs to medicine and food inspection technology to lead
Domain.
Background technique
Donkey-hide gelatin has thousands of years of edible history, has enriching yin of enriching blood, moisturize, stop blooding as a kind of traditional rare Chinese medicine
The effect of.Chinese Pharmacopoeia regulation, donkey-hide gelatin are that the drying skin of equid donkey Equus asinus L. or fresh hide are decocted, are concentrated
Manufactured solid gum.Pure donkey-hide gelatin must be from donkey hide very, the effect of can guaranteeing product.From raw material biological classification
It is seen on, donkey belongs to the kinds such as Perissodactyla equine donkey category, 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.Due to the herding value constantly decline of donkey, and its economic value has no raising, the quantity of donkey
Worldwide sharply decline, the supply of donkey hide is also nervous year by year, and price rises steadily.Occur many illegal points in the market
Son partially or completely replaces donkey with various other Animal Skins include horse skin, mule hide, ox-hide, pigskin etc. when producing donkey-hide gelatin
Skin is boiled, and some is even directly incorporated into industrial gelatine.These adulterants there are severe jamming market normal order, damage
Consumer's interests influence the prestige of regular product.
Donkey-hide gelatin through a long time thermophilic digestion is concentrated, and main component is very close and production technologies of different manufacturers have
Difference, 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, lack
Enough convincingnesses etc..The authenticity of hide glue more authoritative method reported at present that identifies is mainly DNA molecular identification method and inspection
Survey the LC-MS method of characteristic peptide fragment.Reported both methods can not all be compared donkey hide derived components in donkey-hide gelatin
Accurate quantitative detection, but by detection donkey-hide gelatin whether the ingredient containing donkey, and exclude other animal source components to judge
Authenticity of hide glue；But the adulterated of donkey-hide gelatin may be multifarious, it is therefore desirable to which the animal source component of exclusion can be varied, utilization
Exclusive method identifies authenticity of hide glue there are major defect, and accuracy and reliability can have a greatly reduced quality, and such case is very unfavorable
In the supervision to market donkey-hide gelatin product.Authenticity of hide glue is judged using donkey hide derived components content, can fundamentally limit donkey-hide gelatin
Imitation behavior.In reported document, donkey hide derived component is detected, substantially all without excluding horse skin derived component, though therefore
Donkey hide derived components are claimed as, are actually donkey and Ma Gongyou.
Therefore, at present there is an urgent need to propose a kind of method that can quickly, accurately detect donkey hide derived components content in donkey-hide gelatin,
And then it realizes and the true and false of donkey-hide gelatin product is identified.
Summary of the invention
It is an advantage of the invention to provide the compositions of donkey hide derived components content in quick, accurate detection donkey-hide gelatin.
It is still another object of the present invention to provide the examinations for donkey hide derived components content in quick, accurate detection donkey-hide gelatin
Agent box.
It is a further object of the invention to provide the detection sides of donkey hide derived components content in quick, accurate detection donkey-hide gelatin
Method.
To achieve the goals above, present invention employs following technical solutions:
Of the invention is a kind of for detecting the composition of donkey hide derived components content in donkey-hide gelatin, it is characterised in that: the combination
Object is made of polypeptide I, polypeptide II, polypeptide III and reference substance detection matrix, wherein the amino acid sequence of the polypeptide I is
The amino acid sequence of Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg (shown in SEQ ID NO.1), polypeptide II are His-
The amino acid sequence of Gly-His-Arg (shown in SEQ ID NO.2), polypeptide III are Gly-Val-Val-Gly-Pro-Gln-
Gly-Ala-Arg (shown in SEQ ID NO.3), it is fish glue from skin that reference substance, which detects matrix,.
Kit containing the composition is also within protection scope of the present invention.
Further, the invention also provides it is a kind of using donkey hide source in the described compositions or agents box detection donkey-hide gelatin at
Divide the method for content, which comprises the steps of:
1) preparation of reference substance solution:
After reference substance detects matrix crushing, a certain amount of reference substance detection matrix is weighed in measuring bottle, NH is added4HCO3It is molten
Liquid, ultrasound are completely dissolved sample, obtain matrix solution, dissolve load weighted polypeptide I, polypeptide with obtained matrix solution is prepared
II and polypeptide III, shakes up, and obtains reference substance mother liquor, using the method for stepwise dilution, using matrix solution as solvent, by reference substance
Mother liquor dilutes 500-105Times, filtering with microporous membrane, in subsequent filtrate plus trypsin solution, enzymatic hydrolysis；
2) preparation of sample to be tested solution:
It takes glue sample to be measured in measuring bottle, adds NH4HCO3Solution dissolution, filtering with microporous membrane, subsequent filtrate add trypsase enzyme
Solution；
3) reference substance solution, sample to be tested solution are respectively put into LC-MS instrument, select m/z428.0 → 700.4,
556.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 carry out MRM scanning as detection ion pair,
Middle selection m/z428.0 → 700.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide I, polypeptide II and more
The quota ion pair of peptide III, the content of polypeptide I is denoted as M in donkey-hide gelatin sample to be detected1, polypeptide II content be denoted as M2, polypeptide III
Content be denoted as M3；
4) content of donkey hide derived components in donkey-hide gelatin sample is calculated as follows out:
Content (%)=(M of donkey hide derived components in sample1/853.9–M2/505.5)×840/M3× 100%.
In the present invention, it is preferred to, the NH4HCO3Solution is the NH of 1% (w/w) pH8.04HCO3Solution.
In the present invention, it is preferred to, the preparation of the reference substance solution the following steps are included:
1) preparation of matrix solution: reference substance detect matrix crush after, weigh 0.50g reference substance detection matrix in
In 250ml measuring bottle, add the NH of a small amount of 1% (w/w) pH8.04HCO3Solution, ultrasound are completely dissolved sample, with 1% (w/w)
The NH of pH8.04HCO3Solution is diluted to scale, shakes up；
2) 0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds the NH of a small amount of 1% (w/w) pH8.04HCO3It is molten
Liquid, ultrasound are completely dissolved sample, are separately added into the polypeptide III of the polypeptide II and 16.4mg of polypeptide I, 9.9mg of 16.7mg, use
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, using matrix solution as solvent, respectively by reference substance mother
Liquid dilutes 500-105Times, filtering with microporous membrane, precision measures the trypsin solution 10 μ l that 100 μ l of subsequent filtrate adds 2mg/ml, and 37
DEG C constant temperature digests 12h.
In the present invention, it is preferred to, the preparation of sample to be tested solution described in step 2) is the following steps are included: take
0.05g glue sample to be measured adds the NH of a small amount of 1% (w/w) pH8.0 in 25ml measuring bottle4HCO3Solution, ultrasound keep sample completely molten
Solution, with the NH of 1% (w/w) pH8.04HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and precision measures 100 μ of subsequent filtrate
L adds the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h.
In the present invention, it is preferred to, the liquid-phase condition that LC-MS instrument detects in step 3) are as follows: C18Reverse-phase chromatographic column, stream
Dynamic phase A is the aqueous solution containing 0.1% (v/v) formic acid, and Mobile phase B is the acetonitrile solution containing 0.1% (v/v) formic acid, flow velocity
0.3ml/min；Gradient elution: 0 → 20min, mobile phase A 100% → 25%, Mobile phase B 0% → 75%.Mass Spectrometry Conditions: electricity
Spraying positive ion mode (ESI+) carry out multiple-reaction monitoring.
Further, the invention also provides the composition and kits in detection donkey-hide gelatin donkey hide derived components
Application in content.
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 substance of content in the sterling donkey-hide gelatin of various processes, especially to remove with the closer horse of donkey affiliation,
The adulterant ingredient of mule etc.；Secondly the ingredient in donkey-hide gelatin is extremely complex, jitter when detection, therefore the substance will meet and examine
Signal response, lower noise and the interference of other substances with higher when survey, it is also contemplated that the stability of detection signal；Third
Need to set up suitable reference substance etc..In this regard, the present invention, which passes through, chooses 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, to realize to donkey in donkey-hide gelatin
The content detection of skin derived components.It is demonstrated experimentally that using method of the invention, it is possible to accurately detecting donkey hide derived components in donkey-hide gelatin
Content, and it is easy to operate, it will be with a wide range of applications in donkey-hide gelatin 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/z428.0 → 700.4, m/z253.8 →
312.2, the mass spectrogram that m/z421.0 → 684.4 is monitored；
Fig. 2 be under various sterling glue MRM scan patterns select ion pair m/z428.0 → 700.4, m/z253.8 →
312.2, the mass spectrogram that m/z421.0 → 684.4 is 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.
The detection of 1 sterling glue of embodiment
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), it is decocted obtain with donkey hide, horse skin, ox-hide, pigskin, sheepskin and fish-skin respectively.
Polypeptide I (shown in SEQ ID NO.1), polypeptide II (shown in SEQ ID NO.2), polypeptide III (SEQ ID NO.3 institute
Show), transfer to biotech firm to synthesize.
Reagent: ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2, detection method
1) preparation of matrix solution: 0.50g reference substance detection matrix is weighed in 250ml measuring bottle, adds a small amount of 1%NH4HCO3
Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor: 0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds a small amount of 1%
NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, be separately added into polypeptide I, 9.9mg of 16.7mg polypeptide II and
The polypeptide III of 16.4mg, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
3) preparation of reference substance solution: using matrix solution as solvent, reference substance mother liquor is dilute using the method for stepwise dilution
1000 times are released, filtering with microporous membrane, precision measures the 10 μ l of trypsin solution that 100 μ l of subsequent filtrate adds 2mg/ml, 37 DEG C of constant temperature
Digest 12h.
4) preparation of sterling glue sample solution to be detected: taking 0.05g sterling glue sample to be measured in 25ml measuring bottle, adds a small amount of
1%NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes
Even, filtering with microporous membrane, precision measures the 10 μ l of trypsin solution that 100 μ l of subsequent filtrate adds 2mg/ml, 37 DEG C of constant temperature enzymatic hydrolysis
12h。
5) reference substance solution, each 5 μ l of sterling glue sample solution to be detected is taken to be put into LC-MS instrument and detected respectively.Liquid
Phase condition: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are the aqueous solution of 0.1% formic acid, and Mobile phase B is
The acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution: 0 → 20min, mobile phase A 100% → 75%, flowing
Phase B 0% → 25%.Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) progress multiple-reaction monitoring, selection m/z428.0 →
700.4,556.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z428.0 → 700.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide I, more
The quota ion pair of peptide II and polypeptide III.The content of polypeptide I is denoted as M in donkey-hide gelatin sample to be detected1, polypeptide II content be denoted as
M2, polypeptide III content be denoted as M3；
6) content of donkey-hide gelatin in sample is calculated:
Fig. 1 be under reference substance solution MRM scan pattern select ion pair m/z428.0 → 700.4, m/z253.8 →
312.2, the mass spectrogram that m/z421.0 → 684.4 is monitored；Fig. 2 is selection ion pair m/ under various sterling glue MRM scan patterns
The mass spectrogram that z428.0 → 700.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are monitored.
By by the mass spectrogram peak area ratio pair of reference substance and sterling glue, calculating polypeptide I in sterling glue, polypeptide II and more
The content of peptide III, the content of polypeptide I is denoted as M in donkey-hide gelatin sample to be detected1, polypeptide II content be denoted as M2, polypeptide III content
It is denoted as M3, then, the content of donkey hide derived component in each sample is calculated as follows out:
Content (%)=(M of donkey hide derived components in sample1/853.9–M2/505.5)×840/M3× 100%.
Content (%)=(M of donkey hide derived component in donkey-hide gelatin1/853.9–M2/505.5)×840/M3× 100%=
(0.2672/853.9-0/505.5) × 840/0.2601 × 100%=101.06% ≈ 100%
Content (%)=(M of donkey hide derived component in horse skin glue1/853.9–M2/505.5)×840/M3× 100%=
(0.2608/853.9-0.1516/505.5) × 840/0.2580 × 100%=1.80% ≈ 0%
Content (%)=(M of donkey hide derived component in oxhide gelatin1/853.9–M2/505.5)×840/M3× 100%=
(0/853.9-0/505.5) × 840/0.2593 × 100%=0%
Content (%)=(M of donkey hide derived component in pig skin gelatin1/853.9–M2/505.5)×840/M3× 100%=
(0/853.9-0/505.5) × 840/0.2670 × 100%=0%
Content (%)=(M of donkey hide derived component in sheepskin glue1/853.9–M2/505.5)×840/M3× 100%=
(0/853.9-0/505.5) × 840/0.2606 × 100%=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, shows that this method can be from the angle of donkey hide derived components content
Degree accurately distinguishes donkey-hide gelatin and adulterant glue.
The detection of 2 epoxy glue sample of embodiment
1, material and reagent
Material: epoxy glue sample is made of the donkey-hide gelatin that the sterling glue sample in embodiment 1 is separately added into different quality, including
Oxhide gelatin containing 15% (w/w) donkey-hide gelatin, the oxhide gelatin containing 60% (w/w) donkey-hide gelatin, contains the oxhide gelatin containing 30% (w/w) donkey-hide gelatin
The horse skin glue of 30% (w/w) donkey-hide gelatin, the pig skin gelatin for containing 30% (w/w) donkey-hide gelatin, respectively as sample 1-5.
Polypeptide I (shown in SEQ ID NO.1, purity 99.2%), polypeptide II (shown in SEQ ID NO.2, purity 99.5%),
Polypeptide III (shown in SEQ ID NO.3, purity 99.2%), transfers to biotech firm to synthesize.
Reagent: ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure).
2, detection method
1) preparation of matrix solution: 0.50g reference substance detection matrix is weighed in 250ml measuring bottle, adds a small amount of 1%NH4HCO3
Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor: 0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds a small amount of 1%
NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, be separately added into polypeptide I, 9.9mg of 16.7mg polypeptide II and
The polypeptide III of 16.4mg, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
3) preparation of reference substance solution: using the method for stepwise dilution, using matrix solution as solvent, respectively by reference substance mother
Liquid dilution 103Again, 104Again, 2 × 104Again, 4 × 104Again, 6 × 104Again, filtering with microporous membrane, precision measure 100 μ l of subsequent filtrate and add
The 10 μ l of trypsin solution of 2mg/ml, 37 DEG C of constant temperature digest 12h.
4) preparation of epoxy glue sample solution to be detected: taking 0.05g epoxy glue sample to be measured in 25ml measuring bottle, adds a small amount of
1%NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes
Even, filtering with microporous membrane, precision measures the 10 μ l of trypsin solution that 100 μ l of subsequent filtrate adds 2mg/ml, 37 DEG C of constant temperature enzymatic hydrolysis
12h。
5) reference substance solution, each 5 μ l of epoxy glue sample solution to be detected is taken to be put into LC-MS instrument and detected respectively.Liquid
Phase condition: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are the aqueous solution of 0.1% formic acid, and Mobile phase B is
The acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution: 0 → 20min, mobile phase A 100% → 75%, flowing
Phase B 0% → 25%.Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) progress multiple-reaction monitoring, selection m/z428.0 →
700.4,556.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z428.0 → 700.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide I, more
The quota ion pair of peptide II and polypeptide III.
6) content of donkey hide derived component in sample is calculated:
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 epoxy glue,
The content of polypeptide I is denoted as M in epoxy glue sample to be detected1, polypeptide II content be denoted as M2, polypeptide III content be denoted as M3, so
Afterwards, the content of donkey hide derived component in each sample is calculated as follows out:
Content (%)=(M of donkey hide derived component in sample1/853.9–M2/505.5)×840/M3× 100%
Content (%)=(M of donkey hide derived component in sample 11/853.9–M2/505.5)×840/M3× 100%=
(0.0406/853.9-0/505.5) × 840/0.2598 × 100%=15.37%
Content (%)=(M of donkey hide derived component in sample 21/853.9–M2/505.5)×840/M3× 100%=
(0.0812/853.9-0/505.5) × 840/0.2612 × 100%=30.58%
Content (%)=(M of donkey hide derived component in sample 31/853.9–M2/505.5)×840/M3× 100%=
(0.1616/853.9-0/505.5) × 840/0.2608 × 100%=60.95%
Content (%)=(M of donkey hide derived component in sample 41/853.9–M2/505.5)×840/M3× 100%=
(0.2632/853.9-0.1071/505.5) × 840/0.2585 × 100%=31.31%
Content (%)=(M of donkey hide derived component in sample 51/853.9–M2/505.5)×840/M3× 100%=
(0.0811/853.9-0/505.5) × 840/0.2652 × 100%=30.08%
Above-mentioned calculated result shows: donkey hide derived components content 15.37% in the oxhide gelatin sample containing 15% donkey-hide gelatin contains 30%
Donkey hide derived components content 30.58%% in the oxhide gelatin sample of donkey-hide gelatin, donkey hide derived components in the oxhide gelatin sample containing 60% donkey-hide gelatin
Donkey hide derived components content 31.31%, the pigskin containing 30% donkey-hide gelatin in content 60.95%%, the horse skin glue sample containing 30% donkey-hide gelatin
Donkey hide derived components content 30.08% in glue sample.This is consistent with actual conditions, shows that this method can accurately detect donkey hide in donkey-hide gelatin
The content of derived components.
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 donkey-hide gelatin.
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>for detecting composition, kit and its detection method of donkey hide derived components content in donkey-hide gelatin
<130> KLPI161260
<160> 3
<170> PatentIn 3.5
<210> 1
<211> 9
<212> PRT
<213>polypeptide I
<400> 1
Gly Pro Ser Gly Pro Gln Gly Val Arg
<210> 2
<211> 4
<212> PRT
<213>polypeptide II
<400> 2
His Gly His Arg
<210> 3
<211> 9
<212> PRT
<213>polypeptide III
<400> 3
Gly Val Val Gly Pro Gln Gly Ala Arg
Claims (5)
1. a kind of method of donkey hide derived components content in detection donkey-hide gelatin, which comprises the steps of:
1) preparation of reference substance solution:
After reference substance detects matrix crushing, a certain amount of reference substance detection matrix is weighed in measuring bottle, NH is added4HCO3Solution surpasses
Sound is completely dissolved sample, obtains matrix solution, with prepare obtained matrix solution dissolve load weighted polypeptide I, polypeptide II and
Polypeptide III, shakes up, and obtains reference substance mother liquor, using the method for stepwise dilution, using matrix solution as solvent, by reference substance mother liquor
Dilute 500-105Times, filtering with microporous membrane, in subsequent filtrate plus trypsin solution, enzymatic hydrolysis；
Wherein, the amino acid sequence of the polypeptide I is Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg, polypeptide II's
Amino acid sequence is His-Gly-His-Arg, and the amino acid sequence of polypeptide III is Gly-Val-Val-Gly-Pro-Gln-Gly-
Ala-Arg, it is fish glue from skin that reference substance, which detects matrix,；
2) preparation of sample to be tested solution:
It takes glue sample to be measured in measuring bottle, adds NH4HCO3Solution dissolution, filtering with microporous membrane, subsequent filtrate add trypsin digestion；
3) reference substance solution, sample to be tested solution are respectively put into LC-MS instrument, select m/z428.0 → 700.4,
556.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 carry out MRM scanning as detection ion pair,
Middle selection m/z428.0 → 700.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide I, polypeptide II and more
The quota ion pair of peptide III, the content of polypeptide I is denoted as M in donkey-hide gelatin sample to be detected1, polypeptide II content be denoted as M2, polypeptide III
Content be denoted as M3；
4) content of donkey hide derived components in sample is calculated as follows out:
Content %=(the M of donkey hide derived components in sample1/853.9–M2/505.5)×840/M3× 100%.
2. the method as described in claim 1, which is characterized in that the NH4HCO3Solution is the NH of 1%w/w pH8.04HCO3
Solution.
3. the method as described in claim 1, which is characterized in that the preparation of the reference substance solution the following steps are included:
1) preparation of matrix solution: after reference substance detects matrix crushing, the reference substance detection matrix of 0.50g is weighed in 250ml amount
In bottle, add the NH of a small amount of 1%w/w pH8.04HCO3Solution, ultrasound are completely dissolved sample, with 1%w/w pH8.0's
NH4HCO3Solution is diluted to scale, shakes up；
2) 0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds the NH of a small amount of 1%w/w pH8.04HCO3Solution, ultrasound
It is completely dissolved sample, the polypeptide III of the polypeptide II and 16.4mg of polypeptide I, 9.9mg of 16.7mg is separately added into, uses 1%w/w
The NH of pH8.04HCO3Solution is diluted to scale, shakes up；
3) preparation of reference substance solution: respectively that reference substance mother liquor is dilute using matrix solution as solvent using the method for stepwise dilution
Release 500-105Times, filtering with microporous membrane, precision measures the 10 μ l of trypsin solution that 100 μ l of subsequent filtrate adds 2mg/ml, 37 DEG C of perseverances
Temperature enzymatic hydrolysis 12h.
4. the method as described in claim 1, which is characterized in that the preparation of sample to be tested solution described in step 2) includes
Following steps: it takes 0.05g glue sample to be measured in 25ml measuring bottle, adds the NH of a small amount of 1%w/w pH8.04HCO3Solution, ultrasound make
Sample is completely dissolved, with the NH of 1%w/w pH8.04HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and precision measures
100 μ l of subsequent filtrate adds the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h.
5. 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 containing 0.1%v/v formic acid, and Mobile phase B is the acetonitrile containing 0.1%v/v formic acid
Solution, flow velocity 0.3ml/min；Gradient elution: 0 → 20min, mobile phase A 100% → 25%, Mobile phase B 0% → 75%；Matter
Spectral condition: electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
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