CN106841496A - A kind of composition for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation, kit and its detection method - Google Patents
A kind of composition for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation, kit and its detection method Download PDFInfo
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- CN106841496A CN106841496A CN201710004627.8A CN201710004627A CN106841496A CN 106841496 A CN106841496 A CN 106841496A CN 201710004627 A CN201710004627 A CN 201710004627A CN 106841496 A CN106841496 A CN 106841496A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a kind of composition for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation, kit and its detection method, the composition is made up of polypeptide and reference substance detection matrix, the amino acid sequence of the polypeptide is Gly Glu Pro Gly Pro Ala Gly Ser Val Gly Pro Val Gly Ala Val Gly Pro Arg, and reference substance detection matrix is fish-bone glue.Polypeptide of the invention is peculiar for dog bone, is judged whether by detecting the polypeptide containing dog derived component, and the accurate detection of dog derived component content is realized by adding reference substance to detect matrix.The 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 composition, examination for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation
Agent box and its detection method.The invention belongs to medical detection technique field.
Background technology
Dog bone is the bone of canid dog, has functions that invigorating the spleen and network, blood activating and promoting tissue regeneration, cures mainly arthralgia pain due to rheumatism, waist
Skelasthenia, numb limb, protracted dysentery, sore fistula.The preparation being made as main ingredient with dog bone has dog bone glue, dog bone glue medicinal liquor, compound dog bone
Glue, zhuanggushenjin capsule, refute bone ball etc..The discriminating of current dog bone is mainly proterties and differentiates and microscopical characters, because specificity is not strong,
Situations such as differentiating inaccurate is commonly present, cannot be differentiated for the product after being processed with dog bone, more cannot quantitative determination.In default of
Specificity discrimination method and quantitative approach, the illegal producer in part is faked or adulterated, and exorbitant profit is obtained with reduces cost.
Therefore, dog bone in glue class Chinese medicine and its compound preparation can quickly, be accurately detected in the urgent need to proposing one kind 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 it is main into
Hair is separated, it is different using the protein-contg amino acid sequence of institute, dog-bone feature peptide fragment is found out, 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.
The content 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 can quickly, 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 can quickly, precise Identification glue class Chinese medicine and its compound preparation
The method of middle dog bone derived components content.
To achieve these goals, present invention employs following technical scheme：
A kind of composition for detecting 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-Pro-Ala-Gly-
Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg (shown in SEQ ID NO.1), reference substance detects that matrix is
Fish-bone glue.
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 includes 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, plus 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, during load weighted polypeptide added into the measuring bottle, uses NH4HCO3Solution dissolves and is diluted to scale；
3) prepared by reference substance solution：With step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance it is female
Liquid is diluted, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
(2) preparation of detected sample solution
Treat that sample is placed in measuring bottle by load weighted, plus NH4HCO3Solution, ultrasound is completely dissolved testing sample, plus
NH4HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and trypsin solution, enzymolysis are added in subsequent filtrate；
(3) LC-MS instrument detection
Reference substance solution obtained in step (1), detected sample solution obtained in step (2) are taken respectively is put into LC-MS
Instrument is detected that selection m/z781.4 → 752.4,809.5,908.5 conducts detect ion pairs, wherein selection m/z781.4 →
752.4 as polypeptide quota ion pair；
(4) in sample dog bone derived components content calculating
With the content of the polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve, line are carried out for abscissa
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, is calculated as follows out dog bone in each sample
The content of derived components：
The content (%) of dog bone derived components=(weight of content of peptides/testing sample in testing sample) ÷ (5.328 ×
10-3) × 100%.
Preferably, step (1) and the hydrolysis temperature described in step (2) are 37 DEG C.
Preferably, the liquid-phase condition of LC-MS instrument detection is in step (3)：C18Reverse-phase chromatographic column, mobile phase A is
0.1% (v/v) first aqueous acid, Mobile phase B is 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 allows for described composition or described kit 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 into
Divide and the metastable material of content in the glue class Chinese medicine of various processes；Secondly the composition of glue class Chinese medicine is extremely complex,
Jitter during detection, thus the material to meet in detection with signal response higher, relatively low noise and other
Material is disturbed, it is also contemplated that the stability of detection signal；3rd needs to set up suitable reference substance etc..
In this regard, the present invention judges whether to contain dog source by choosing dog bone source characteristic polypeptide by detecting the polypeptide
Property composition, realize the accurate detection of dog derived component content by adding reference substance to detect matrix.It is demonstrated experimentally that using this hair
Bright method, can exactly detect the content of dog bone derived components in glue class Chinese medicine and its compound preparation, and simple to operate, in glue
Class tcm product quality monitoring field will be with a wide range of applications.
Brief description of the drawings
In order that the object, technical solutions and advantages of the present invention are clearer, below in conjunction with accompanying drawing the present invention is made into
The detailed description of one step, wherein：
Fig. 1 is the mass spectrogram of selection ion pair m/z781.4 → 752.4 monitoring under reference substance solution MRM scan patterns；
Fig. 2 is the mass spectrum of selection ion pair m/z781.4 → 752.4 monitoring under various sterling animal gelatine MRM scan patterns
Figure.
Specific embodiment
Hereinafter with reference to accompanying drawing, preferred embodiment of the invention is described in detail.It is specific without indicating in preferred embodiment
The experimental technique of condition, is generally carried out according to the condition proposed by normal condition or manufacturer.
Embodiment 1
1st, 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), 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).
2nd, detection method
(1) preparation of reference substance solution
1) preparation of matrix solution
Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus 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
Weigh 0.05g reference substances and detect matrix in 25ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, and the polypeptide for weighing 30.4mg adds 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, with step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance
Mother liquor dilutes 1000 times, and filtering with microporous membrane, precision measures the μ l of subsequent filtrate 100, plus 2mg/ml trypsin solution 10 μ l, 37
DEG C constant temperature enzymolysis 12h；
(2) preparation of testing sample solution
Weigh 0.05g various glue class samples to be measured to be placed in 25ml measuring bottles, add a small amount of 1% (w/w) NH4HCO3(pH8.0)
After solution dissolving, continuation (w/w) NH of Jia 1%4HCO3(pH8.0) scale is diluted to, is shaken up, filtering with microporous membrane, precision measures continuous
The μ l of filtrate 100 add the μ l of trypsin solution 10 of 2mg/ml, 37 DEG C of constant temperature to digest 12h；
(3) LC-MS instrument detection
Reference substance solution obtained in step (1), each 5 μ l of detected sample solution obtained in step (2) are taken respectively is put into liquid
Matter combined instrument detected, liquid-phase condition：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% (v/
V) first aqueous acid, Mobile 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+) enter
Row multiple-reaction monitoring, selection m/z781.4 → 752.4,809.5,908.5 carry out MRM scannings as detection ion pair, wherein selecting
Select quota ion pair of m/z781.4 → 752.4 as polypeptide；
(4) in sample dog bone derived components content calculating
Fig. 1 is the mass spectrogram of selection ion pair m/z781.4 → 752.4 monitoring under reference substance solution MRM scan patterns；Fig. 2
It is the mass spectrogram that the monitoring of ion pair m/z781.4 → 752.4 is selected under various sterling animal gelatine MRM scan patterns.
With the content of the polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve, line are carried out for abscissa
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, is calculated as follows out dog bone in each sample
The content of derived components：
The content (%) of dog bone derived components=(weight of content of peptides/testing sample in testing sample) ÷ (5.328 ×
10-3) × 100%.
Wherein, in each sample dog bone derived components content：
The content (%)=(0.2682 × 10 of dog bone derived components in dog bone glue-3g/0.0502g)÷(5.328×10-3)×
100%=100.27% ≈ 100%
The content (%) of dog bone derived components=(0mg/0.0506g) ÷ (5.328 × 10 in donkey gelatine-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0504g) ÷ (5.328 × 10 in bone glue-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0512g) ÷ (5.328 × 10 in pig bone glue-3) × 100%=0%
The content (%) of dog bone derived components=(0mg/0.0502g) ÷ (5.328 × 10 in sheep bone glue-3G) × 100%=
0%
Above-mentioned result of calculation 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, in showing that the method can accurately detect glue class Chinese medicine
The content of dog bone derived components.
Embodiment 2
1st, 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：3 are made to the dog bone glue of different quality percentage is added in above-mentioned sterling glue class Chinese medicine
Individual compound glue class Chinese medicine preparation, including the bone glue containing 15% dog bone glue, the bone glue containing 30% dog bone glue, containing 30% dog bone glue
Pig bone glue, be respectively designated as preparation 1, preparation 2 and preparation 3.
2nd, detection method
(1) preparation of reference substance solution
1) preparation of matrix solution
Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus 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
Weigh 0.05g reference substances and detect matrix in 25ml measuring bottles, plus a small amount of 1% (w/w) NH4HCO3Solution (pH8.0),
Ultrasound is completely dissolved sample, weighs the polypeptide of 30.4mg in the measuring bottle, with 1% (w/w) NH4HCO3Solution (pH8.0) dissolves
And scale is diluted to, shake up；
3) preparation of reference substance solution
Using the method for stepwise dilution, with step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance
Mother liquor dilutes 500 times, 1000 times, 2000 times, 4000 times, 8000 times, 10000 times, and filtering with microporous membrane, precision measures subsequent filtrate
100 μ l, plus 2mg/ml the μ l of trypsin solution 10,37 DEG C of constant temperature digest 12h；
(2) preparation of testing sample solution
0.05g glue samples to be measured are taken in 25ml measuring bottles, a small amount of 1% (w/w) NH is added4HCO3(pH8.0) solution, ultrasound
After being completely dissolved sample, continuation (w/w) NH of Jia 1%4HCO3(pH8.0) scale is diluted to, is shaken up, filtering with microporous membrane is accurate
The μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml are measured, 37 DEG C of constant temperature digest 12h；
(3) LC-MS instrument detection
Reference substance solution obtained in step (1), each 5 μ l of detected sample solution obtained in step (2) are taken respectively is put into liquid
Matter combined instrument detected, liquid-phase condition：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% (v/
V) first aqueous acid, Mobile 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+) enter
Row multiple-reaction monitoring, selection m/z781.4 → 752.4,809.5,908.5 carry out MRM scannings as detection ion pair, wherein selecting
Select quota ion pair of m/z781.4 → 752.4 as polypeptide；
(4) in sample dog bone derived components content calculating
With the content of the polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve, line are carried out for abscissa
Property coefficient R >=0.9990, thus calculates the content of polypeptide in sample, then, is calculated as follows out dog bone in each sample
The content of derived components：
The content (%) of dog bone derived components=(weight of content of peptides/testing sample in testing sample) ÷ (5.328 ×
10-3) × 100%.
Wherein, in each sample dog bone derived components content：
The content (%)=(0.2688 × 10 of dog bone derived components in dog bone glue-3g/0.0506g)÷(5.328×10-3)×
100%=99.70% ≈ 100%
The content (%)=(0.0411 × 10 of dog bone derived components in bone glue containing 15% dog bone glue-3g/0.0502g)÷
(5.328×10-3) × 100%=15.36%
The content (%)=(0.0816 × 10 of dog bone derived components in bone glue containing 30% dog bone glue-3g/0.0508g)÷
(5.328×10-3) × 100%=30.15%
The content (%)=(0.0830 × 10 of dog bone derived components in pig bone glue containing 30% dog bone glue-3g/0.0506g)÷
(5.328×10-3) × 100%=30.79%
Above-mentioned result of calculation shows：Using the dog bone source detection level in the preparation 1-3 that detection method of the invention is obtained
It is consistent with actual conditions, shows that the method can accurately detect the content of dog bone derived components in glue class Chinese medicine and its compound preparation.
In sum, using polypeptide disclosed by the invention and reference substance detection matrix, using LC-MS instrument, can be accurate
The content of dog bone derived components in detection glue class Chinese medicine and its compound preparation.
It should be noted that above example is only used to illustrate the technical scheme of invention and unrestricted, although by referring to
Invention has been described for the preferred embodiments of the present invention, but the various changes that those skilled in the art make to the present invention
Or modification, without departing from spirit and scope of the invention, these equivalent form of values are also fallen within the scope of the present invention.
Sequence table
<110>Donga donkey-hide gelatin limited company
<120>A kind of composition for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation, kit and its
Detection method
<130> KLPI161252
<160> 1
<170>PatentIn 3.5
<210> 1
<211> 18
<212> PRT
<213>Polypeptide
<400> 1
Gly Glu Pro Gly Pro Ala Gly Ser Val Gly Pro Val Gly Ala Val Gly Pro Arg 18
Claims (7)
1. a kind of composition for detecting 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-Pro-
Ala-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg, reference substance detection matrix is fish-bone glue.
2. a kind of detection kit containing composition described in claim 1.
3. a kind of method for detecting dog bone derived components content in glue class Chinese medicine and its compound preparation, it is characterised 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 makes
Sample is completely dissolved, plus 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
Sample is completely dissolved, during load weighted polypeptide added into the measuring bottle, NH is used4HCO3Solution dissolves and is diluted to scale；
3) prepared by reference substance solution：With step 1) obtained in matrix solution be solvent, by step 2) obtained in reference substance mother liquor it is dilute
Release, filtering with microporous membrane, trypsin solution, enzymolysis are added in subsequent filtrate；
(2) preparation of detected sample solution
Load weighted testing sample is placed in measuring bottle, plus NH4HCO3Solution, ultrasound is completely dissolved sample, plus NH4HCO3Solution
Scale is diluted to, is shaken up, filtering with microporous membrane adds trypsin solution, enzymolysis in subsequent filtrate；
(3) LC-MS instrument detection
Take reference substance solution obtained in step (1), detected sample solution obtained in step (2) respectively and be put into LC-MS instrument
Row detection, selection m/z781.4 → 752.4,809.5,908.5 conduct detection ion pairs, wherein selection m/z781.4 → 752.4
As the quota ion pair of polypeptide；
(4) in sample dog bone derived components content calculating
With the content of the polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve, linear phase are carried out for abscissa
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 into
The content divided：
The content (%) of dog bone derived components=(weight of content of peptides/testing sample in testing sample) ÷ (5.328 × 10-3)×
100%.
4. method as claimed in claim 3, it is characterised in that the hydrolysis temperature described in step (1) and step (2) is 37 DEG C.
5. method as claimed in claim 3, it is characterised in that the liquid-phase condition of LC-MS instrument detection is in step (3)：C18
Reverse-phase chromatographic column, mobile phase A is 0.1% (v/v) first aqueous acid, and Mobile 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 spectrum bar
Part：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
6. it is used to detect the composition of dog bone derived components content in glue class Chinese medicine and its compound preparation as claimed in claim 1,
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, sheep bone
Glue or other glue class Chinese medicines.
7. the kit described in the composition or claim 2 described in claim 1 is in detection glue class Chinese medicine and its compound preparation
In dog bone derived components content in application.
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| CN201710004627.8A CN106841496B (en) | 2017-01-04 | 2017-01-04 | 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 |
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| CN201710004627.8A CN106841496B (en) | 2017-01-04 | 2017-01-04 | 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 |
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| CN103630621A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Method for detecting sheep-derived ingredients in glue type traditional Chinese medicines and products thereof |
| CN103630620A (en) * | 2013-10-28 | 2014-03-12 | 山东东阿阿胶股份有限公司 | Method for detecting deer-derived ingredients in glue type traditional Chinese medicines and products thereof |
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