CN106872614B - For detecting the composition, kit and its detection method of donkey-hide gelatin content in compound donkey-hide gelatin preparation - Google Patents
For detecting the composition, kit and its detection method of donkey-hide gelatin content in compound donkey-hide gelatin preparation Download PDFInfo
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- G—PHYSICS
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- 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 the composition, kit and its detection method of donkey-hide gelatin content in compound donkey-hide gelatin preparation.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 detection to donkey-hide gelatin content in compound donkey-hide gelatin preparation.This method has the advantages that content detection is accurate, easy to operate, will be with a wide range of applications in the quality monitoring field of donkey-hide gelatin compound preparation.
Description
Technical field
The present invention relates to the compositions and kit of donkey-hide gelatin content in a kind of detection compound donkey-hide gelatin preparation, further include using this
The method that compositions or agents box carries out donkey-hide gelatin content detection in compound donkey-hide gelatin preparation, the invention belongs to medicine and food inspection skill
Art field.
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, there is an urgent need to propose a kind of side that can quickly, accurately detect donkey-hide gelatin content in compound donkey-hide gelatin preparation at present
Method, and then realize and the true and false of compound donkey-hide gelatin preparation is identified.
Summary of the invention
It is an advantage of the invention to provide the compositions of donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation.
It is still another object of the present invention to provide for donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation
Kit.
It is a further object of the invention to provide the detections of donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation
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 gelatin content in compound donkey-hide gelatin preparation, by polypeptide I, polypeptide II, more
Peptide III and reference substance detection matrix composition, wherein the amino acid sequence of the polypeptide I is Ser-Gly-Gln-Pro-Gly-
The amino acid sequence of Thr-Val-Gly-Pro-Ala-Gly-Val-Arg (shown in SEQ ID NO.1), polypeptide II are His-Gly-
The amino acid sequence of 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,.
Detection kit containing the composition is also within protection scope of the present invention.
Further, the invention also provides a kind of using in the compositions or agents box detection compound donkey-hide gelatin preparation
The method of donkey-hide gelatin 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 compound donkey-hide gelatin preparation sample solution:
Take compound donkey-hide gelatin preparation sample to be detected in measuring bottle, weight is denoted as M1, after being dissolved in water, it is molten that trichloroacetic acid is added
Liquid, ultrasound remove supernatant, weigh after precipitating acetone washing, drying, weight is denoted as M after centrifugation2；Weigh a certain amount of precipitating
NH is added in measuring bottle in dried object4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and tryptose is added in subsequent filtrate
Enzyme solutions, enzymatic hydrolysis；
3) reference substance solution, compound donkey-hide gelatin preparation sample solution to be detected are respectively put into LC-MS instrument, select m/
Z592.2 → 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 are as detection
Ion pair carries out MRM scanning, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, the difference of m/z421.0 → 684.4
As the quota ion pair of polypeptide I, polypeptide II and polypeptide III, the content of polypeptide I is denoted as N in donkey-hide gelatin sample to be detected1, polypeptide
The content of II is denoted as N2, polypeptide III content be denoted as N3；
4) content of donkey-hide gelatin in sample is calculated as follows out:
Content (%)=(N of donkey-hide gelatin in compound donkey-hide gelatin preparation1/1182.3–N2/505.5)×840/N3×M2/M1×
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) the polypeptide III of polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg is weighed in 25ml measuring bottle, uses step
1) matrix solution prepared dissolves and 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 the compound donkey-hide gelatin preparation sample solution to be detected the following steps are included:
The compound donkey-hide gelatin preparation sample to be detected of 1~5g is weighed, weight is denoted as M1, 5ml is complemented to water, the three of 30% (w/v) are added
Monoxone 5ml, concussion mixing make final concentration of 15% (w/v), the ultrasonic 15min of trichloroacetic acid, 4 DEG C of standing 30min, centrifugation
After remove clear liquid, precipitating acetone washing, it is dry after weigh, weight is denoted as M2, the precipitating dried object of 0.05g is weighed in 25ml amount
In bottle, the NH of a small amount of 1% (w/w) pH8.0 is added4HCO3After solution dissolution, with the NH of 1% (w/w) pH8.04HCO3Solution dilution
It to scale, shakes up, 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.
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 its kits in detection compound donkey-hide gelatin preparation Ah
Application in glue content.
The ingredient of donkey-hide gelatin compound preparation is increasingly complex compared with donkey-hide gelatin, therefore carries out content inspection to donkey-hide gelatin in donkey-hide gelatin compound preparation
It surveys, difficulty is bigger than detecting in simple donkey-hide gelatin.This not only to find out can indicate donkey hide derived components and different process Ah
The metastable substance of content in glue, the adulterant ingredient of removal and the closer horse of donkey affiliation, mule etc.；It is also contemplated that detection letter
Number stability, meet signal with higher response when detecting, lower noise and the interference of other substances etc..These are all needed
Want innovative work.In this regard, the present invention is subtracted by choosing donkey hide source property characteristic polypeptide, and using the detected value of polypeptide I
The detected value of polypeptide II, and detection error is corrected using the detected value of polypeptide III, to realize to donkey in donkey-hide gelatin 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 donkey hide in donkey-hide gelatin compound preparation
The content of derived components, and it is easy to operate, it will be with a wide range of applications in donkey-hide gelatin compound preparation 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/z592.2 → 756.4, m/z253.8 →
312.2, the mass spectrogram that m/z421.0 → 684.4 is monitored.
Fig. 2 be compound donkey-hide gelatin preparation sample and its adulterant selected under MRM scan pattern ion pair m/z592.2 →
756.4, the mass spectrogram that m/z253.8 → 312.2, m/z421.0 → 684.4 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
1) material
Various sterling glue, including donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, (reference substance detects base to fish glue from skin
Matter), it decocted with donkey hide, horse skin, ox-hide, pigskin, sheepskin and fish-skin, be concentrated, obtained after drying 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.
Compound donkey-hide gelatin preparation sample: in complex prescription glue mucilage production process vegetable drug extract concentrate solution, respectively plus
Enter above-mentioned sterling glue, extracts concentrate solution addition 1.0g sterling glue sample according to every 9g vegetable drug and the compound containing donkey-hide gelatin is made
Corii asini pulp (preparation 1), the complex prescription glue mucilage adulterant (preparation 2) containing horse skin the glue, (system of the complex prescription glue mucilage adulterant containing oxhide gelatin
Agent 3), the complex prescription glue mucilage adulterant (preparation 4) containing pig skin gelatin, the complex prescription glue mucilage adulterant (preparation 5) containing sheepskin glue.
2) reagent
Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2 detection methods
1) preparation of matrix solution: 0.50g reference substance detection matrix is weighed in 250ml 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 up.
2) preparation of reference substance mother liquor: weigh the polypeptide III of the polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg in
In 25ml measuring bottle, scale is dissolved and be diluted to the matrix solution of above-mentioned preparation, is shaken 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 compound donkey-hide gelatin preparation sample solution: weighing 5g, (weight is denoted as M1) compound donkey-hide gelatin preparation sample to be detected
Product complement to 5ml with water, and the trichloroacetic acid 5ml of 30% (w/v) is added, and concussion mixing makes final concentration of the 15% of trichloroacetic acid
(w/v), ultrasonic 15min, 4 DEG C of standing 30min remove clear liquid after centrifugation, weighing (weight note after precipitating acetone washing, drying
For M2), the precipitating dried object of 0.05g is weighed in 25ml measuring bottle, and a small amount of 1% NH is added4HCO3After solution (pH8.0) dissolution,
With 1% 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；
5) it detects: reference substance solution, each 5 μ l of compound donkey-hide gelatin preparation sample solution to be detected being taken to be put into LC-MS instrument respectively
It is detected.Liquid-phase condition: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are the water-soluble of 0.1% formic acid
Liquid, Mobile phase B are the acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution: 0 → 20min, mobile phase A 100%
→ 25%, Mobile phase B 0% → 75%.Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) multiple-reaction monitoring is carried out, select m/
Z592.2 → 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 are as detection
Ion pair carries out MRM scanning, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, the difference of m/z421.0 → 684.4
Quota ion pair as polypeptide I, polypeptide II and polypeptide III.
6) content of donkey-hide gelatin in sample is calculated:
Fig. 1 be under reference substance solution MRM scan pattern select ion pair m/z592.2 → 756.4, m/z253.8 →
312.2, the mass spectrogram that m/z421.0 → 684.4 is monitored.Fig. 2 is that compound donkey-hide gelatin preparation and its adulterant are selected under MRM scan pattern
Select ion pair m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 monitor mass spectrogram.
By by the mass spectrogram peak area ratio pair of reference substance and compound donkey-hide gelatin preparation sample and its adulterant, calculate compound Ah
The content of polypeptide I, polypeptide II and polypeptide III in glue formulation samples and its adulterant, the content note of polypeptide I in donkey-hide gelatin sample to be detected
For N1, polypeptide II content be denoted as N2, polypeptide III content be denoted as N3；Then, containing for donkey-hide gelatin in each sample is calculated as follows out
Amount:
Content (%)=(N of donkey-hide gelatin in sample1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
Content (%)=(N of donkey-hide gelatin in preparation 11/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.3245/1182.3-0/505.5) × 840/0.2319 × 0.5209/5.0810 × 100%=10.19%
Content (%)=(N of donkey-hide gelatin in preparation 21/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.3212/1182.3-0.1388/505.5) × 840/0.2331 × 0.5493/5.0415 × 100%=-0.11% ≈ 0%
Content (%)=(N of donkey-hide gelatin in preparation 31/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2322 × 0.5581/5.0370 × 100%=0%
Content (%)=(N of donkey-hide gelatin in preparation 41/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2306 × 0.5260/5.0561 × 100%=0%
Content (%)=(N of donkey-hide gelatin in preparation 51/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2352 × 0.0.5144/5.0505 × 100%=0%
Above-mentioned calculated result shows: the donkey-hide gelatin content detected in the compound ass-hide gelatin slurry samples containing donkey-hide gelatin and additional amount phase
Symbol, and the donkey-hide gelatin content detected in the complex prescription glue mucilage adulterant without donkey-hide gelatin is 0%, this is consistent with actual conditions, shows the party
Method can detect the donkey-hide gelatin content in donkey-hide gelatin compound preparation.
Embodiment 2
1 material and reagent
1) material
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.
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 contains
The oxhide gelatin of 15% donkey-hide gelatin, the oxhide gelatin containing 30% donkey-hide gelatin, the oxhide gelatin containing 60% donkey-hide gelatin.
Compound donkey-hide gelatin preparation sample: in complex prescription glue mucilage production process vegetable drug extract concentrate solution, respectively plus
Enter above-mentioned epoxy glue sample, according to every 9g vegetable drug extract concentrate solution be added 1.0g epoxy glue sample be made 3 compound Ah
Rubber cement adulterant, is respectively designated as preparation 1, preparation 2, preparation 3, and donkey-hide gelatin content is respectively 1.5%, 3.0%, 6.0%.
2) reagent
Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2 detection methods
1) preparation of matrix solution: 0.50g reference substance detection matrix is weighed in 250ml 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 up.
2) preparation of reference substance mother liquor: weigh the polypeptide III of the polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg in
In 25ml measuring bottle, scale is dissolved and be diluted to the matrix solution of above-mentioned preparation, is shaken up.
3) preparation of reference substance solution: using the method for stepwise dilution, using matrix solution as solvent, by reference substance mother liquor point
Not Xi Shi 500 times, 1000 times, 2000 times, 4000 times, 8000 times, 10000 times, filtering with microporous membrane, precision measure subsequent filtrate
100 μ l add the 10 μ l of trypsin solution of 2mg/ml, and 37 DEG C of constant temperature digest 12h.
4) preparation of compound donkey-hide gelatin preparation sample solution: weighing 5g, (weight is denoted as M1) compound donkey-hide gelatin preparation sample to be detected
Product complement to 5ml with water, and the trichloroacetic acid 5ml of 30% (w/v) is added, and concussion mixing makes final concentration of the 15% of trichloroacetic acid
(w/v), ultrasonic 15min, 4 DEG C of standing 30min remove clear liquid after centrifugation, weighing (weight note after precipitating acetone washing, drying
For M2), the precipitating dried object of 0.05g is weighed in 25ml measuring bottle, and a small amount of 1% NH is added4HCO3After solution (pH8.0) dissolution,
With 1% 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；
5) it detects: taking each 5 μ l of reference substance solution, sample to be tested solution to be put into LC-MS instrument respectively and detected.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% → 25%, flowing
Phase B 0% → 75%.Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) progress multiple-reaction monitoring, selection m/z592.2 →
756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 as detection ion pairs into
Row MRM scanning, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide
I, the quota ion pair of polypeptide II and polypeptide III.
6) 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 linearly dependent coefficient R >=0.998 of each polypeptide calculates polypeptide I in compound donkey-hide gelatin preparation sample, polypeptide II and polypeptide
The content of III, the content of polypeptide I is denoted as N in compound donkey-hide gelatin preparation sample to be detected1, polypeptide II content be denoted as N2, polypeptide III
Content be denoted as N3, then, the content of donkey-hide gelatin in each sample is calculated as follows out:
Content (%)=(N of donkey-hide gelatin in sample1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
Content (%)=(N of donkey-hide gelatin in preparation 11/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.0496/1182.3-0/505.5) × 840/0.2341 × 0.5065/5.0521 × 100%=1.51%
Content (%)=(N of donkey-hide gelatin in preparation 21/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.0992/1182.3-0/505.5) × 840/0.2413 × 0.5256/5.0535 × 100%=3.04%
Content (%)=(N of donkey-hide gelatin in preparation 31/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.1968/1182.3-0/505.5) × 840/0.2398 × 0.5116/5.0445 × 100%=5.91%
Above-mentioned calculated result shows: matrix is detected using three polypeptides disclosed by the invention and reference substance, using liquid matter
Combined instrument can accurately detect the content of donkey-hide gelatin in donkey-hide gelatin 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>for detecting the composition, kit and its detection method of donkey-hide gelatin content in compound donkey-hide gelatin preparation
<130> KLPI161261
<160> 3
<170> PatentIn 3.5
<210> 1
<211> 13
<212> PRT
<213>polypeptide I
<400> 1
Ser Gly Gln Pro Gly Thr Val Gly Pro Ala 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 gelatin content in detection compound donkey-hide gelatin preparation, 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 Ser-Gly-Gln-Pro-Gly-Thr-Val-Gly-Pro-Ala-Gly-
The amino acid sequence of Val-Arg, polypeptide II are 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 compound donkey-hide gelatin preparation sample solution:
Take compound donkey-hide gelatin preparation sample to be detected in measuring bottle, weight is denoted as M1, after being dissolved in water, solution of trichloroacetic acid is added, surpasses
Sound removes supernatant, weighs after precipitating acetone washing, drying, weight is denoted as M after centrifugation2；Weigh a certain amount of precipitating dried object
In measuring bottle, NH is added4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution is added in subsequent filtrate,
Enzymatic hydrolysis；
3) reference substance solution, compound donkey-hide gelatin preparation sample solution to be detected are respectively put into LC-MS instrument, select m/z592.2
→ 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 conduct detection ion pairs
MRM scanning is carried out, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as more
The quota ion pair of peptide I, polypeptide II and polypeptide III, the content of polypeptide I is denoted as N in donkey-hide gelatin sample to be detected1, polypeptide II contains
Amount is denoted as N2, polypeptide III content be denoted as N3；
4) content of donkey-hide gelatin in sample is calculated as follows out:
Content %=(the N of donkey-hide gelatin in compound donkey-hide gelatin preparation1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
2. the method as described in claim 1, which is characterized in that the NH4HCO3Solution is the NH of 1%w/wpH8.04HCO3
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) the polypeptide III of polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg is weighed in 25ml measuring bottle, is matched with step 1)
The matrix solution of system dissolves and 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 compound donkey-hide gelatin preparation sample to be detected described in step 2) is molten
The following steps are included: weighing 1~5g compound donkey-hide gelatin preparation sample to be detected, weight is denoted as M for the preparation of liquid1, complemented to water
5ml, is added the trichloroacetic acid 5ml of 30%w/v, and concussion mixing makes the final concentration of 15%w/v of trichloroacetic acid, ultrasonic 15min, 4
DEG C stand 30min, remove supernatant after centrifugation, precipitating acetone washing, it is dry after weigh, weight is denoted as M2, weigh 0.05g's
Dried object is precipitated in 25ml measuring bottle, the NH of a small amount of 1%w/w pH8.0 is added4HCO3After solution dissolution, with 1%w/w pH8.0
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and precision measures the tryptose that 100 μ l of subsequent filtrate adds 2mg/ml
10 μ l of enzyme solutions, 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%；
Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
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| CN104280468A (en) * | 2013-07-12 | 2015-01-14 | 山东东阿阿胶股份有限公司 | Method for detecting horse-breed and mule-breed derived ingredients in gelatin-type traditional Chinese medicine and products |
| CN105842375A (en) * | 2016-03-29 | 2016-08-10 | 山东出入境检验检疫局检验检疫技术中心 | Polypeptide group for identification of donkey components in gelatin and products thereof, |
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| CN105842375A (en) * | 2016-03-29 | 2016-08-10 | 山东出入境检验检疫局检验检疫技术中心 | Polypeptide group for identification of donkey components in gelatin and products thereof, |
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| "NCBI： XP_001492989, Collagen alpha-2(I) chain [Equus caballus]";Uniprot;《Uniprot》;20151120;第973-976位 * |
| "UniProtKB：C01A2-ORYAF，Collagen alpha-2(I) chain of Orycteropus after（Aardvark）";Uniprot;《Uniprot》;20150722;第36-44位 * |
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