CN106770766B - It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in glue class Chinese medicine and its compound preparation - Google Patents
It is a kind of for detecting composition, kit and its detection method of donkey hide derived components content in glue class Chinese medicine and its compound preparation Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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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 glue class Chinese medicine and its compound preparation, 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 glue class Chinese medicine and its compound preparation.This method has the advantages that content detection is accurate, easy to operate, will be with a wide range of applications in glue class Chinese medicine and its compound preparation product quality monitoring field.
Description
Technical field
The present invention relates to a kind of for detecting the composition of donkey hide derived components content in glue class Chinese medicine and its compound preparation, examination
Agent box and its detection method.The invention belongs to medical detection technique fields.
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.Pure donkey-hide gelatin must be from donkey hide very, the effect of can guaranteeing product.From raw material taxonomy
It sees, donkey belongs to the kinds such as Perissodactyla equine donkey category, including African wild donkey, Equus kiang, onager, family donkey.Wherein China's raising
Family donkey include Region in Guanzhong Donkey, Dezhou donkey, Guangling donkey, good rice donkey, Miyang donkey, Huaiyang donkey, Qingyang donkey, North China donkey, Xinjiang Donkey, southwest
Numerous local varieties such as donkey.Since the supply of donkey hide is also nervous year by year, price rises steadily, and occurs 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, there is an urgent need to propose that one kind can quickly, accurately detect donkey hide in glue class Chinese medicine and its compound preparation at present
The method of derived components content, and then realize and the true and false of the product of the derived components containing donkey hide is identified.
Summary of the invention
It is an advantage of the invention to provide one kind can quickly, in precise Identification glue class Chinese medicine and its compound preparation
The composition of donkey hide derived components content.
It is a further object of the invention to provide one kind being capable of quick, precise Identification glue class Chinese medicine and its compound preparation
The kit of middle donkey hide derived components content.
It is still another object of the present invention to provide one kind being capable of quick, precise Identification glue class Chinese medicine and its compound preparation
The method of middle donkey hide derived components content.
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 and its compound preparation, described group
It closes object to be made of polypeptide I, polypeptide II, polypeptide III and reference substance detection matrix, it is characterised in that: the amino acid of the polypeptide I
Sequence is Gly-Ala-Thr-Gly-Pro-Ala-Gly-Val-Arg (shown in SEQ ID NO.1), the amino acid sequence of polypeptide II
It is classified as Gly-Glu-Pro-Gly-Pro-Ala-Gly-Leu-Pro-Gly-Pro-Hyp-Gly-Glu- Arg (SEQ ID NO.2
It is shown), the amino acid sequence of polypeptide III is Gly-Val-Val-Gly-Pro-Gln-Gly-Ala-Arg (SEQ ID NO.3 institute
Show), it is fish glue from skin that reference substance, which detects matrix,.
Detection kit containing the composition is also within the scope of the present invention.
Further, the invention also provides one kind contains for detecting donkey hide derived components in glue class Chinese medicine and its compound preparation
The method of amount, specifically comprises 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 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 added4HCO3It is molten
Liquid, ultrasound are completely dissolved sample, and load weighted polypeptide I, polypeptide II and polypeptide III are added in the measuring bottle, NH is used4HCO3
Solution 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 sample to be tested solution
Sample to be tested is taken, weight is denoted as N1, solution of trichloroacetic acid is added, ultrasound stands, removes supernatant after centrifugation, sinks
It weighs after shallow lake acetone washing, drying, weight is denoted as N2, weigh and be deposited in measuring bottle, NH is added4HCO3Solution is dissolved and is diluted
It to scale, shakes up, filtering with microporous membrane, trypsin solution is added in subsequent filtrate, digest；
(3) LC-MS instrument detects
Reference substance solution, sample to be tested solution is taken to be put into LC-MS instrument and detected respectively, selection m/z393.4 →
499.3,643.3, m/z702.8 → 725.4,838.4, m/z421.0 → 684.4,528.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z393.4 → 499.3, m/z702.8/725.4, m/z421.0 → 684.4 are respectively as polypeptide I, more
The quota ion pair of peptide II and polypeptide III carry out quantitative detection；
(4) in sample 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,
Linearly dependent coefficient R >=0.9992 of each polypeptide, thus calculates the content of polypeptide I, polypeptide II and polypeptide III in sample, to
The content of polypeptide I is denoted as M in test sample1, polypeptide II content be denoted as M2, polypeptide III content be denoted as M3, then, press
Formula calculates the content of donkey hide derived components in each sample:
The content (%) of donkey hide derived components=(M1/392.4-M2/1403.5) × 525/M3 × N2/N1× 100%.
Method for detecting donkey hide derived components content in glue class Chinese medicine and its compound preparation of the invention, it is preferred that step
Suddenly hydrolysis temperature described in (1) and step (2) is 37 DEG C.
Method for detecting donkey hide derived components content in glue class Chinese medicine and its compound preparation of the invention, it is preferred that step
Suddenly the liquid-phase condition that LC-MS instrument detects in (3) are as follows: C18Reverse-phase chromatographic column, mobile phase A are the water-soluble of 0.1% (v/v) formic acid
Liquid, 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.
Preferably, the glue class Chinese medicine is donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis, deer horn glue or other glue class Chinese medicines.
Further, the invention also provides the composition or the kits in detection glue class Chinese medicine and its
The application in donkey hide derived components content in compound preparation.
Accurate content detection directly is carried out to donkey hide derived components in glue class Chinese medicine, first have to find out can indicate donkey hide source at
Point and the metastable substance of content in the sterling donkey-hide gelatin of various processes, especially to remove closer with donkey affiliation
The adulterant ingredient of horse, mule etc.；Secondly the ingredient of donkey-hide gelatin is extremely complex, jitter when detection, therefore the substance will meet
Signal response, lower noise and the interference of other substances with higher when detection, it is also contemplated that the stability of detection signal；The
Three need 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 to donkey in glue class Chinese medicine and its compound preparation
The content detection of skin derived components.It is demonstrated experimentally that using method of the invention, it is possible to accurately detecting glue class Chinese medicine and its compound system
The content of donkey hide derived components in agent, and it is easy to operate, before glue class tcm product quality monitoring field will have a wide range of applications
Scape.
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/z702.8 →
725.4, the mass spectrogram that m/z421.0 → 684.4 is monitored；
Fig. 2 is selection ion pair m/z393.4 → 499.3, m/z702.8 under various sterling glue class Chinese medicine MRM scan patterns
→ 725.4, 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.
Embodiment 1
1, material and reagent
Material: various sterling glue class Chinese medicines, including donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, colla carapacis et plastri testudinis, deer
Angle glue, fish glue from skin (reference substance detection matrix) are decocted with donkey hide, horse skin, ox-hide, pigskin, sheepskin, tortoise plastron, deer horn, fish-skin respectively
It obtains.
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 17.3mg of polypeptide I, 27.4mg 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 10 μ l of trypsin solution of 2mg/ml,
37 DEG C of constant temperature digest 12h；
(2) preparation of testing sample solution
Sterling glue class traditional Chinese medicine sample 0.5g (being denoted as N1) to be measured is weighed, the solution of trichloroacetic acid 5ml of 30% (w/w) is added,
Ultrasonic 15min, 4 DEG C of standing 30min remove supernatant, weigh (being denoted as N2), claim after precipitating acetone washing, drying after centrifugation
The NH of a small amount of 1% (w/w) is added in 25ml measuring bottle in sediment after taking 0.05g dry4HCO3After solution (pH8.0) dissolution,
With the NH of 1% (w/w)4HCO3Solution (pH8.0) is diluted to scale, shakes up, filtering with microporous membrane, and precision measures 100 μ l of subsequent filtrate
Add the 10 μ l of trypsin solution of 2mg/ml, 37 DEG C of constant temperature digest 12h.
(3) LC-MS instrument detects
5 μ l are taken to be put into liquid matter respectively testing sample solution made from reference substance solution made from step (1), step (2)
Combined instrument detection, liquid-phase condition are as follows: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are 0.1% (v/v) first
The aqueous solution of 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 it is how anti-
It should monitor, select m/z393.4 → 499.3,643.3, m/z702.8 → 725.4,838.4, m/z421.0 → 684.4,528.3
As detection ion pair carry out MRM scanning, wherein selection m/z393.4 → 499.3, m/z702.8 → 725.4, m/z421.0 →
684.4 respectively as polypeptide I, polypeptide II and polypeptide III quota ion pair, carry out quantitative detection；
(4) in sterling glue class Chinese medicine 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/z702.8 →
725.4, the mass spectrogram that m/z421.0 → 684.4 is monitored；Fig. 2 is to select ion under various sterling glue class Chinese medicine MRM scan patterns
The mass spectrogram that m/z393.4 → 499.3, m/z702.8 → 725.4, m/z421.0 → 684.4 are monitored.
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 polypeptide I, polypeptide II and polypeptide III in sterling glue class Chinese medicine
Content, so that the content of donkey hide derived components in sterling glue class Chinese medicine is calculated, the content of polypeptide I in sterling glue class Chinese medicine to be detected
Be denoted as M1, the content of polypeptide II is denoted as M2, the content of polypeptide III is denoted as M3, donkey hide in sterling glue class Chinese medicine is calculated as follows out
The content of derived components:
The content (%) of donkey hide derived components=(M1/392.4-M2/1403.5) × 525/M3 × N2/N1× 100%
Wherein, in each sample donkey hide derived components content:
The content (%)=525/ (0.2411/392.4-0/1403.5) × 525/0.2601 of donkey hide derived components in donkey-hide gelatin
× 0.4035/0.5012 × 100%=99.84%
The content (%) of donkey hide derived components=(0/392.4-0/1403.5) × 525/0.2580 in horse skin glue ×
0.4012/0.5008 × 100%=0%
The content (%) of donkey hide derived components=(0.2506/392.4-0.8833/1403.5) × 525/ in oxhide gelatin
0.2593 × 0.4028/0.5021 × 100%=1.51% ≈ 0%
The content (%) of donkey hide derived components=(0/392.4-0/1403.5) × 525/0.2670 in pig skin gelatin ×
0.4068/0.5022 × 100%=0%
The content (%) of donkey hide derived components=(0.2442/392.4-0.8712/1403.5) × 525/ in sheepskin glue
0.2606 × 0.4038/0.5012 × 100%=0.26% ≈ 0%
The content (%) of donkey hide derived components=(0/392.4-0/1403.5) × 525/0.2488 in colla carapacis et plastri testudinis ×
0.4056/0.5008 × 100%=0%
The content (%) of donkey hide derived components=(0.2432/392.4-0.8706/1403.5) × 525/ in deer horn glue
0.2567 × 0.4066/0.5022 × 100%=-0.09% ≈ 0%
Above-mentioned calculated result shows: the content 100% of donkey hide derived components, horse skin glue, oxhide gelatin, pig skin gelatin, sheep in donkey-hide gelatin
Hide glue, colla carapacis et plastri testudinis, the content of donkey hide derived components is 0% in deer horn glue.This is consistent with actual conditions, shows that this method can detect glue
The content of donkey hide derived components in class Chinese medicine.
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 colla carapacis et plastri testudinis, the colla carapacis et plastri testudinis containing 30% donkey-hide gelatin, the deer horn glue containing 30% donkey-hide gelatin including containing 15% donkey-hide gelatin.
Compound glue class Chinese materia medica preparation: in complex prescription glue mucilage production process vegetable drug extract concentrate solution, respectively plus
Enter above-mentioned epoxy glue sample, extracts concentrate solution according to every 9g vegetable drug and 1.0g epoxy glue sample is added, 3 compound glue are made
Class Chinese materia medica preparation, is respectively designated as preparation 1, preparation 2 and preparation 3, donkey hide derived components content is respectively 1.5%, 3.0%,
3.0%.
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 17.3mg of polypeptide I, 27.4mg 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 testing sample solution
Glue class traditional Chinese medicine sample 5.0g (being denoted as N1) to be measured is weighed, the solution of trichloroacetic acid 5ml of 30% (w/w), ultrasound is added
15min, 4 DEG C of standing 30min remove supernatant after centrifugation, weighing is denoted as N2 after precipitating acetone washing, drying, weighs 0.05g
The NH of a small amount of 1% (w/w) is added in 25ml measuring bottle in sediment after drying4HCO3After solution (pH8.0) dissolution, with 1%
(w/w) NH4HCO3Solution (pH8.0) is diluted to scale, shakes up, filtering with microporous membrane, and precision measures 100 μ l of subsequent filtrate and adds
The 10 μ l of trypsin solution of 2mg/ml, 37 DEG C of constant temperature digest 12h.
(3) LC-MS instrument detects
5 μ l are taken to be put into liquid matter respectively testing sample solution made from reference substance solution made from step (1), step (2)
Combined instrument detection, liquid-phase condition are as follows: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are 0.1% (v/v) first
The aqueous solution of 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 it is how anti-
It should monitor, select m/z393.4 → 499.3,643.3, m/z702.8 → 725.4,838.4, m/z421.0 → 684.4,528.3
As detection ion pair carry out MRM scanning, wherein selection m/z393.4 → 499.3, m/z702.8 → 725.4, m/z421.0 →
684.4 respectively as polypeptide I, polypeptide II and polypeptide III quota ion pair, carry out quantitative detection；
(4) in glue class Chinese medicine 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 plastic emitting class Chinese medicine
Amount, to calculate the content of donkey hide derived components in plastic emitting class Chinese medicine, the content of polypeptide I is denoted as M1, more in glue class Chinese medicine to be detected
The content of peptide II is denoted as M2, the content of polypeptide III is denoted as M3, and the content of donkey hide derived components in plastic emitting class Chinese medicine is calculated as follows:
Wherein, in each sample donkey hide derived components content:
Content (%)=(0.0322/392.4-0/1403.5) × 525/ 0.2296 of donkey hide derived components in 1 sample of preparation
× 0.4016/5.00 × 100%=1.51%
Content (%)=(0.0728/392.4-0/1403.5) × 525/0.2436 of donkey hide derived components in 2 sample of preparation
× 0.4041/5.00 × 100%=3.23%
The content (%) of donkey hide derived components=(0.2512/392.4-0.6326/1403.5) × 525/ in 3 sample of preparation
0.2460 × 0.4051/5.00 × 100%=3.28%
Above-mentioned calculated result shows: the donkey hide source detection level of the preparation 1-3 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 glue class Chinese medicine and its compound preparation.
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 glue class Chinese medicine and its compound preparation.
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, the kit of donkey hide derived components content in glue class Chinese medicine and its compound preparation
And its detection method
<130> KLPI161258
<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
GlyGluProGlyProAlaGlyLeuProGlyProHypGlyGluArg 15
<210> 3
<211> 9
<212> PRT
<213>polypeptide III
<400> 3
GlyValValGlyProGlnGlyAlaArg 9
Claims (3)
1. a kind of method for detecting donkey hide derived components content in glue class Chinese medicine and its compound preparation, which is characterized in that including
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 make
Sample is completely dissolved, and adds NH4HCO3Solution is diluted to scale；The reference substance detection matrix is fish glue from skin；
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 added in 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-Glu-Pro-Gly-Pro-Ala-Gly-Leu-Pro-Gly-Pro-Hyp-Gly-Glu- Arg, the amino of polypeptide III
Acid sequence is Gly-Val-Val-Gly-Pro-Gln-Gly-Ala-Arg；
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 sample to be tested solution
Sample to be tested is taken, weight is denoted as N1, solution of trichloroacetic acid is added, ultrasound stands, removes supernatant after centrifugation, precipitates with third
It weighs after ketone washing, drying, weight is denoted as N2, weigh and be deposited in measuring bottle, NH is added4HCO3Solution dissolves and is diluted to scale,
It shakes up, filtering with microporous membrane, trypsin solution is added in subsequent filtrate, digest；
(3) LC-MS instrument detects
Reference substance solution, sample to be tested solution is taken to be put into LC-MS instrument and detected respectively, selection m/z393.4 →
499.3,643.3, m/z702.8 → 725.4,838.4, m/z421.0 → 684.4,528.3 carry out MRM as detection ion pair
Scanning, wherein selection m/z393.4 → 499.3, m/z702.8 → 725.4, m/z421.0 → 684.4 are respectively as polypeptide I, more
The quota ion pair of peptide II and polypeptide III carry out quantitative detection；
(4) in sample 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 sample are calculated, the content of polypeptide I is denoted as M in sample to be tested1, polypeptide II
Content be denoted as M2, polypeptide III content be denoted as M3, then, the content of donkey hide derived components in each sample is calculated as follows out:
The content (%) of donkey hide derived components=(M1/392.4-M2/1403.5) × 525/M3 × N2/N1× 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 0.1%, the aqueous solution of v/v formic acid, Mobile phase B 0.1%, the acetonitrile solution of v/v formic acid, stream
Fast 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.
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