CN106770879B - It is a kind of for detecting composition, kit and its detection method of dog bone 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 dog bone derived components content in glue class Chinese medicine and its compound preparation Download PDFInfo
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- CN106770879B CN106770879B CN201611198381.4A CN201611198381A CN106770879B CN 106770879 B CN106770879 B CN 106770879B CN 201611198381 A CN201611198381 A CN 201611198381A CN 106770879 B CN106770879 B CN 106770879B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/89—Inverse chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a kind of for detecting composition, kit and its detection method of dog bone derived components content in glue class Chinese medicine and its compound preparation, the composition is made of polypeptide and reference substance detection matrix, the amino acid sequence of the polypeptide is Gly-Glu-Pro-Gly-Val-Leu-Gly-Ala-Pro-Gly-Thr-Ala-Gly-Ala- Ser-Gly-Pro-Gly-Gly-Leu-Pro-Gly-Glu-Arg, and it is fish-bone glue that reference substance, which detects matrix,.Polypeptide of the invention is peculiar for dog bone, by detecting the polypeptide to determine whether containing dog derived component, realizes the accurate detection of dog derived component content by the way that reference substance detection matrix is added.The detection method has the advantages that content detection is accurate, swift 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 dog bone 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
Dog bone is the bone of canid dog, has effects that invigorating the spleen and network, blood activating and promoting tissue regeneration, cures mainly rheumatic arthralgia, waist
Skelasthenia, numb limb, protracted dysentery, sore fistula.There are dog bone glue, dog bone glue liquid medicine, compound dog bone by preparation made of main ingredient of dog bone
Glue, zhuanggushenjin capsule refute bone ball etc..The identification of dog bone at present is mainly character identification and microscopical characters, because specificity is not strong,
Situations such as being commonly present identification inaccuracy, for that can not be identified with the product after the processing of dog bone, it is even more impossible to quantitative detections.In default of
Specificity discrimination method and quantitative approach, the illegal producer in part carry out fraud or adulterated, obtain exorbitant profit to reduce cost.
Therefore, there is an urgent need to propose that one kind can quickly, accurately detect dog bone in glue class Chinese medicine and its compound preparation at present
The method of derived components content, and then realize the authenticity to the product of the derived components of bone containing dog.The present invention from dog bone mainly at
Hair is separated, it is different using the protein-contg amino acid sequence of institute, dog-bone feature peptide fragment is found out, is realized using the feature peptide fragment of dog bone
The detection of dog bone derived component content in glue class Chinese medicine and its compound preparation.
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 dog bone 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 dog bone 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 dog bone derived components content.
To achieve the goals above, present invention employs following technical solutions:
It is a kind of for detecting the composition of dog bone derived components content in glue class Chinese medicine and its compound preparation, the composition by
Polypeptide and reference substance detection matrix composition, the amino acid sequence of the polypeptide is Gly-Glu-Pro-Gly-Val-Leu-Gly-
Ala-Pro-Gly-Thr-Ala-Gly-Ala-Ser-Gly-Pro-Gly-Gly-Leu-Pro-Gly-Gl u-Arg(SEQ ID
Shown in NO.1), it is fish-bone glue that reference substance, which detects matrix,.
Detection kit containing the composition is also within protection scope of the present invention.
Invention further provides a kind of for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation
Method 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 surpasses
Sound is completely dissolved sample, adds NH4HCO3Solution is diluted to scale；
2) preparation of reference substance mother liquor: load weighted reference substance detection matrix is placed in measuring bottle, NH is added4HCO3Solution,
Ultrasound is completely dissolved sample, and load weighted polypeptide is added in the measuring bottle, NH is used4HCO3Solution 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
Load weighted tested animal glue is placed in measuring bottle, NH is added4HCO3Solution, ultrasound are completely dissolved animal glue, add
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymatic hydrolysis are added in subsequent filtrate；
(3) LC-MS instrument detects
Sample to be tested solution made from reference substance solution made from step (1), step (2) is taken to be put into LC-MS respectively
Instrument is detected, and is selected m/z688.1 → 997.5,926.5,839.4 as detection ion pair and is carried out MRM scanning, wherein selecting
Quota ion pair of m/z688.1 → 997.5 as polypeptide；
(4) in sample dog bone derived components content calculating
It is the drafting of abscissa progress standard curve, line using the content of the polypeptide of reference substance solution as ordinate, peak area
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, dog bone in each sample is calculated as follows out
The content of derived components:
The content (%) of dog bone derived components=(content of peptides/sample to be tested weight in sample to be tested) ÷ (7.044 ×
10-3) × 100%
Preferably, hydrolysis temperature described in step (1) and step (2) is 37 DEG C.
Preferably, the liquid-phase condition that LC-MS instrument detects in step (3) are as follows: C18Reverse-phase chromatographic column, mobile phase A are
The aqueous solution of 0.1% (v/v) formic acid, Mobile phase B are the acetonitrile solution of 0.1% (v/v) formic acid, flow velocity 0.3ml/min；Gradient is washed
It is de-: 5 → 20min, mobile phase A 90% → 75%, Mobile phase B 10% → 25%, Mass Spectrometry Conditions: electron spray positive ion mode
(ESI+) carry out multiple-reaction monitoring.
Preferably, the glue class Chinese medicine be donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis, deer horn glue, donkey gelatine, bone glue, pig bone glue,
Sheep bone glue or other glue class Chinese medicines.
Further, the invention also provides the composition or the kits in detection glue class Chinese medicine and its
The application in dog bone derived components content in compound preparation.
Accurate content detection directly is carried out to dog bone derived components in glue class Chinese medicine, first have to find out can indicate dog bone source at
Point and the metastable substance of content in the glue class Chinese medicine of various processes；Secondly the ingredient of glue class Chinese medicine is extremely complex,
Jitter when detection, thus the substance to meet signal with higher response when detecting, lower noise and other
Substance interference, it is also contemplated that the stability of detection signal；Third needs to set up suitable reference substance etc..
In this regard, the present invention, which passes through, chooses dog bone source characteristic polypeptide, by detecting the polypeptide to determine whether containing dog source
Property ingredient, detect matrix by the way that reference substance is added and realize the accurate detection of dog derived component content.It is demonstrated experimentally that using this hair
Bright method can accurately detect the content of dog bone derived components in glue class Chinese medicine and its compound preparation, and easy to operate, in glue
Class tcm product quality monitoring field will be with a wide range of applications.
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 is the mass spectrogram for selecting ion pair m/z688.1 → 997.5 to monitor under reference substance solution MRM scan pattern；
Fig. 2 is the mass spectrum for selecting ion pair m/z688.1 → 997.5 to monitor under various Animal Bone sample MRM scan patterns
Figure.
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 glue class Chinese medicines, including dog bone glue, donkey gelatine, bone glue, pig bone glue, sheep bone the glue, (control of fish-bone glue
Matrix is surveyed in product examine), it is decocted obtain with dog bone, donkey bone, ox bone, pig bone, sheep bone, fish-bone respectively.
Polypeptide transfers 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
0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, and the polypeptide for weighing 40.2mg is added in the measuring bottle, with 1% (w/w) NH4HCO3Solution (pH8.0) is molten
Liquid dissolves and is diluted to scale, shakes up；
3) preparation of reference substance solution
Using the method for stepwise dilution, using matrix solution made from step 1) as solvent, by reference substance made from step 2)
Mother liquor dilutes 1000 times, and filtering with microporous membrane, precision measures 100 μ l of subsequent filtrate, adds the trypsin solution 10 μ l of 2mg/ml, and 37
DEG C constant temperature digests 12h；
(2) preparation of testing sample solution
0.05g sample to be tested is weighed in 25ml measuring bottle, a small amount of 1% (w/w) NH is added4HCO3Solution (pH8.0) dissolution
Afterwards, continue to add 1% (w/w) NH4HCO3(pH8.0) it is diluted to scale, is shaken up, filtering with microporous membrane, 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；
(3) LC-MS instrument detects
Each 5 μ l of sample to be tested solution made from reference substance solution made from step (1), step (2) is taken to be put into liquid respectively
Matter combined instrument is detected, liquid-phase condition: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are 0.1% (v/
V) aqueous solution of formic acid, Mobile phase B are the acetonitrile solution of 0.1% (v/v) formic acid, flow velocity 0.3ml/min；Gradient elution: 5 →
20min, mobile phase A 90% → 75%, Mobile phase B 10% → 25%.Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) into
Row multiple-reaction monitoring selects m/z688.1 → 997.5,926.5,839.4 as detection ion pair and carries out MRM scanning, wherein selecting
Select quota ion pair of m/z688.1 → 997.5 as polypeptide；
(4) in sample dog bone derived components content calculating
Fig. 1 is the mass spectrogram for selecting ion pair m/z688.1 → 997.5 to monitor under reference substance solution MRM scan pattern；Fig. 2
It is the mass spectrogram for selecting ion pair m/z688.1 → 997.5 to monitor under various Animal Bone sample MRM scan patterns.
It is the drafting of abscissa progress standard curve, line using the content of the polypeptide of reference substance solution as ordinate, peak area
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, dog bone in each sample is calculated as follows out
The content of derived components:
The content (%) of dog bone derived components=(content of peptides/sample to be tested weight in sample to be tested) ÷ (7.044 ×
10-3) × 100%.
Wherein, in each sample dog bone derived components content:
The content (%)=(0.3484 × 10 of dog bone derived components in dog bone glue-3g/0.0502g)÷(7.044×10-3)×
100%=98.53% ≈ 100%
The content (%) of dog bone derived components=(0mg/0.0506g) ÷ (7.044 × 10 in donkey gelatine-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0504g) ÷ (7.044 × 10 in bone glue-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0512g) ÷ (7.044 × 10 in pig bone glue-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0502g) ÷ (7.044 × 10 in sheep bone glue-3G) × 100%=
0%
Above-mentioned calculated result shows: the content 100% of dog bone derived components in dog bone glue, donkey gelatine, bone glue, pig bone glue,
The content of dog bone derived components is 0% in sheep bone glue.This is consistent with actual conditions, shows that this method can be detected accurately in glue class Chinese medicine
The content of dog bone derived components.
Embodiment 2
1, material and reagent
Material: various sterling glue class Chinese medicines, including dog bone glue, donkey gelatine, bone glue, pig bone glue, sheep bone glue, fish-bone glue
(reference substance detection matrix) is decocted with dog bone, donkey bone, ox bone, pig bone, sheep bone, fish-bone obtain respectively.
Polypeptide transfers to biotech firm to synthesize.
Reagent: ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure).
Glue class compound Chinese medicinal preparation: the dog bone glue that different quality percentage is added into above-mentioned sterling gelatine is made 3 again
Square glue class Chinese materia medica preparation, bone glue, the bone glue containing 30% dog bone glue, the pig containing 30% dog bone glue including containing 15% dog bone glue
Gelatine is respectively designated as preparation 1, preparation 2 and preparation 3.
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
0.05g reference substance detection matrix is weighed in 25ml measuring bottle, adds a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, weighs the polypeptide of 40.2mg in the measuring bottle, with 1% (w/w) NH4HCO3Solution (pH8.0) dissolution
And it is diluted to scale, it 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, and precision measures 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；
(2) preparation of testing sample solution
It takes 0.05g glue sample to be measured in 25ml measuring bottle, a small amount of 1% (w/w) NH is added4HCO3Solution (pH8.0), ultrasound
After being completely dissolved sample, continue to add 1% (w/w) NH4HCO3(pH8.0) it is diluted to scale, is shaken up, filtering with microporous membrane is accurate
The 10 μ l of trypsin solution that 100 μ l of subsequent filtrate adds 2mg/ml is measured, 37 DEG C of constant temperature digest 12h；
(3) LC-MS instrument detects
Each 5 μ l of sample to be tested solution made from reference substance solution made from step (1), step (2) is taken to be put into liquid respectively
Matter combined instrument is detected, liquid-phase condition: C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A are 0.1% (v/
V) aqueous solution of formic acid, Mobile phase B are the acetonitrile solution of 0.1% (v/v) formic acid, flow velocity 0.3ml/min；Gradient elution: 5 →
20min, mobile phase A 90% → 75%, Mobile phase B 10% → 25%；Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) into
Row multiple-reaction monitoring selects m/z688.1 → 997.5,926.5,839.4 as detection ion pair and carries out MRM scanning, wherein selecting
Select quota ion pair of m/z688.1 → 997.5 as polypeptide；
(4) in sample dog bone derived components content calculating
It is the drafting of abscissa progress standard curve, line using the content of the polypeptide of reference substance solution as ordinate, peak area
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, dog bone in each sample is calculated as follows out
The content of derived components:
The content (%) of dog bone derived components=(content of peptides/sample to be tested weight in sample to be tested) ÷ (7.044 ×
10-3) × 100%.
Wherein, in each sample dog bone derived components content:
The content (%)=(0.3562 × 10 of dog bone derived components in dog bone glue-3g/0.0506g)÷(7.044×10-3)×
100%=99.94% ≈ 100%
The content (%)=(0.0540 × 10 of dog bone derived components in bone glue containing 15% dog bone glue-3g/0.0502g)÷
(7.044×10-3) × 100%=15.27%
The content (%)=(0.1104 × 10 of dog bone derived components in bone glue containing 30% dog bone glue-3g/0.0508g)÷
(7.044×10-3) × 100%=30.85%
The content (%)=(0.1082 × 10 of dog bone derived components in pig bone glue containing 30% dog bone glue-3g/0.0506g)÷
(7.044×10-3) × 100%=30.36%
Above-mentioned calculated result shows: the dog bone source detection level in the preparation 1-3 obtained using detection method of the invention
It is consistent with actual conditions, shows that this method can accurately detect the content of dog bone derived components in glue class Chinese medicine and its compound preparation.
In conclusion matrix is detected using polypeptide disclosed by the invention and reference substance, it, can be accurate using LC-MS instrument
Detect the content of dog bone derived components in 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 dog bone derived components content in glue class Chinese medicine and its compound preparation
And its detection method
<130> KLPI161250
<160> 1
<170>PatentIn 3.5
<210> 1
<211> 24
<212> PRT
<213>polypeptide
<400> 1
GlyGluProGlyValLeuGlyAlaProGlyThrAlaGlyAlaSerGlyProGlyGlyLeuProGlyGluArg 24
Claims (6)
1. a kind of for detecting the composition of dog bone derived components content in glue class Chinese medicine and its compound preparation, the composition is by more
Peptide and reference substance detection matrix composition, it is characterised in that: the amino acid sequence of the polypeptide is Gly-Glu-Pro-Gly-Val-
Leu-Gly-Ala-Pro-Gly-Thr-Ala-Gly-Ala-Ser-Gly-Pro-Gly- Gly-Leu-Pro-Gly-Glu-Arg,
It is fish-bone glue that reference substance, which detects matrix,.
2. it is as described in claim 1 for detecting the composition of dog bone derived components content in glue class Chinese medicine and its compound preparation,
It is characterized in that, the glue class Chinese medicine is donkey-hide gelatin, oxhide gelatin, colla carapacis et plastri testudinis, deer horn glue, donkey gelatine, bone glue, pig bone glue or sheep bone
Glue.
3. a kind of detection kit containing composition described in claim 1.
4. a kind of method for detecting dog bone 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；
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 is added in the measuring bottle, NH is used4HCO3Solution dissolves and is diluted to scale；It is described more
Peptide is polypeptide described in claim 1；
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
Load weighted sample to be tested glue is placed in measuring bottle, NH is added4HCO3Solution, ultrasound are completely dissolved sample glue, add NH4HCO3
Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymatic hydrolysis are added in subsequent filtrate；
(3) LC-MS instrument detects
Take respectively sample to be tested solution made from reference substance solution made from step (1), step (2) be put into LC-MS instrument into
Row detection selects m/z688.1 → 997.5,926.5,839.4 as detection ion pair and carries out MRM scanning, wherein selecting m/
Quota ion pair of z688.1 → 997.5 as polypeptide；
The liquid-phase condition of LC-MS instrument detection are as follows: C18Reverse-phase chromatographic column, mobile phase A are the aqueous solution of 0.1%v/v formic acid, flowing
Phase B is the acetonitrile solution of 0.1%v/v formic acid, flow velocity 0.3ml/min；Gradient elution: 5 → 20min, mobile phase A 90% → 75%,
Mobile phase B 10% → 25%, Mass Spectrometry Conditions: electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring；
(4) in sample dog bone derived components content calculating
It is the drafting of abscissa progress standard curve, linear phase using the content of the polypeptide of reference substance solution as ordinate, peak area
Close coefficients R >=0.9990, thus calculate the content of polypeptide in sample, then, be calculated as follows out in each sample dog bone source at
The content divided:
The content (%) of dog bone derived components=(content of peptides/sample to be tested weight in sample to be tested) ÷ (7.044 × 10-3) × 100%.
5. method as claimed in claim 4, which is characterized in that hydrolysis temperature described in step (1) and step (2) is 37 DEG C.
6. composition described in claim 1 or kit as claimed in claim 3 are in detection glue class Chinese medicine and its compound preparation
In dog bone derived components content in application.
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