CN103630622A - Detection substance for tortoise-shell glue and products thereof and LC-MS (Liquid Chromatography-Mass Spectrometry) detection method thereof - Google Patents
Detection substance for tortoise-shell glue and products thereof and LC-MS (Liquid Chromatography-Mass Spectrometry) detection method thereof Download PDFInfo
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Abstract
The invention provides a method for detecting tortoise-shell glue and products thereof. The method provided by the invention comprises the step of detecting by using the difference between the specific nucleotide sequence or amino acid sequence of genomes of the tortoise-shell glue products and the specific nucleotide sequence or amino acid sequence of genomes of other imitations, wherein the specific amino acid sequence is shown in SEQ ID No. 1. According to the method provided by the invention, fake ingredients, such as donkeys, horses, cattle, pigs, sheep and deer, in the tortoise-shell glue and products thereof can be detected rapidly, so as to distinguish the authenticity. The invention further relates a polypeptide and a composition formed by the polypeptide and a corresponding carrier. Furthermore, the invention relates to application of the polypeptide and composition thereof in detection of donkey, horse, cattle, pig, sheep or deer-derived ingredients in the tortoise-shell glue and products thereof.
Description
Technical field
The present invention relates to detection thing and the detection method thereof of a breeding ass, horse, ox, pig, sheep or deer derived component, specifically can be applicable to the authenticity of colla carapacis et plastri testudinis and goods thereof, belong to Chinese medicine detection field.
Background technology
Tortoise plastron, carapace and the plastron of Testudinidae animal tortoise (Chinemys reevesii), belong to traditional rare Chinese medicine.Chinese Pharmacopoeia according to latest edition is recorded, and taking tortoise plastron can be nourishing and suppressing Yang, the strong bone of kidney-nourishing, the bushing of nourishing blood.In the last few years, along with the raising of biology level, the correlative study of tortoise plastron also deepened continuously.There are some researches show, tortoise plastron powder and extract thereof not only can strengthen liver function, release the pressure, and also have effect anticancer and that regulate Immunoresistance.The two series products Guiling Jellies that the tortoise plastron of take is made as raw material and colla carapacis et plastri testudinis on whole Market of Chinese Materia Medica in occupation of important position.
According to version Chinese Pharmacopoeia in 2010, record, colla carapacis et plastri testudinis is to take tortoise plastron as raw material, through decocting, boils, concentrates the solid gum of making, and property is salty, sweet, cool, returns liver, kidney, the heart channel of Hang-Shaoyin.Can enriching yin, nourish blood, stop blooding, be mainly used in deficiency of Yin hectic fever, hectic fever due to yin night sweat, soreness and weakness of waist and knees, the deficiency of blood is sallow, uterine bleeding band is inferior.Long-term taking colla carapacis et plastri testudinis also helps and strengthens body self-regulation, can promote longevity, and is also beneficial to hyperactivity of yang due to yin deficiency patient's physical rehabilitation.Along with improving constantly of living standard, similar with donkey-hide gelatin, colla carapacis et plastri testudinis also becomes one of first-selection that people nourish.
Because tortoise plastron self is famousr and precious, medical value is remarkable, and tortoise meat is also usually edible for people for cook various kinds tonic, in the last few years to various Chelonians to catch and kill phenomenon very serious, some Chelonian kind is endangered even.Therefore, the supply of tortoise plastron raw material is very limited, and price is high, has also directly had influence on the production of colla carapacis et plastri testudinis.On colla carapacis et plastri testudinis market, have some counterfeit and shoddy goods, it makes raw material is not tortoise plastron, but is replaced by the assorted skin of other animals, broken bone, comprises pigskin, ox-hide, the skin of animals died of illness even, dirty skin, rotten skin etc.Once such counterfeit and shoddy goods are shaped, outward appearance, color and luster, smell all to certified products colla carapacis et plastri testudinis and similar, true and false difficulty is distinguished.Take these adulterants not only without any curative effect, also probably harmful to health, consequence is very serious.
Glue class Chinese medicine forms gelatin substance by skin, bone, first-class the boiling of animal, and comprises donkey-hide gelatin, colla carapacis et plastri testudinis, deer horn glue etc., has the traditional Chinese medicine of typical national characters.Glue class Chinese medicine is general to be concentrated and forms through long-time thermophilic digestion, principal ingredient production technology very close and different manufacturers there are differences, and adopts the methods such as infrared, near infrared, X-diffraction to carry out the true and false to glue class Chinese medicine sterling and differentiates the normal situations such as judgement is inaccurate that exist.The discriminating of glue class Chinese medicament turtle-derived component, has scholar and differentiates from DNA angle, but just qualitative, has false negative and false positive phenomenon, and operation is comparatively complicated.Have and report and utilize Mass Spectrometer Method glue class Chinese medicament turtle-derived component from the angle of polypeptide, result accurately and reliably.But also there is no at present the angle from collagen polypeptide, the detection method of adulterant composition in disposable discriminating colla carapacis et plastri testudinis and goods thereof.
In glue class Chinese medicine, principal ingredient is collagen polypeptide, the collagen, amino acid sequence of different animals there are differences, in different plant species except tortoise, the same polypeptide of the stable existence containing can be used as the characteristic polypeptide of adulterant composition in colla carapacis et plastri testudinis, by digestion with restriction enzyme, utilize the mass spectrum completely can be to the colla carapacis et plastri testudinis evaluation of tracing to the source.
Summary of the invention
The present invention is directed to the problems referred to above, a kind of detection thing and the authenticity method thereof of colla carapacis et plastri testudinis and goods thereof is provided, the method is simple to operate, and characteristic is strong, highly sensitive, can be used for the evaluation of tracing to the source of adulterant composition in colla carapacis et plastri testudinis and goods thereof.
Technical scheme of the present invention is as follows:
First, the invention provides a kind of method that detects colla carapacis et plastri testudinis and goods thereof, it utilizes colla carapacis et plastri testudinis goods and in other Counterfeit Item genomes, has the different of specificity nucleotide sequence or amino acid sequence and detect, and described specific amino acid is as shown in SEQ.ID No1.
Preferably, described colla carapacis et plastri testudinis goods are selected from a kind of in food, health products or the medicine that colla carapacis et plastri testudinis makes, and do not contain donkey, horse, ox, pig, sheep, deer derived component in formula.
Preferably, described other Counterfeit Items refer to that one or more in donkey, horse, ox, pig, sheep or deer boil the gelatin substance forming.
More specifically, under, comprise the steps:
(1) choose specific amino acid or amino acid sequence in colla carapacis et plastri testudinis goods and other Counterfeit Item genomes, described specific amino acid is as shown in SEQ.ID No1;
(2) colla carapacis et plastri testudinis or its goods are dissolved, or extract the polypeptide constituents of described colla carapacis et plastri testudinis or its goods and dissolve, add subsequently restriction enzyme to carry out enzyme and cut;
(3) solution after enzyme step (2) being obtained is cut is put into LC-MS instrument, take colla carapacis et plastri testudinis sterling as matrix, adds the described amino acid sequence of step (1) in contrast, and selects parent ion m/z553.3 and the daughter ion thereof of this amino acid sequence to monitor.
If it is consistent with reference substance to detect the retention time of this ion, and its daughter ion is consistent with the daughter ion of reference substance, in described colla carapacis et plastri testudinis or its goods, contains this adulterant composition; If this ion consistent with reference substance retention time, does not contain this adulterant composition.
Preferably, described in step (2), dissolving reagent used is that mass percent is 1%, the NH of pH value 8.0
4hCO
3solution; Described restriction enzyme is trypsase.
Further, the invention provides a kind of molecular marked compound that detects colla carapacis et plastri testudinis and goods thereof, the amino acid sequence of this molecular marked compound is as shown in SEQ.ID No1.
Further, the invention provides the gene of the described albumen of coding.
Further, the invention provides described molecular marked compound and whether contain the application in donkey, horse, ox, pig, sheep or deer derived component in detecting colla carapacis et plastri testudinis.
Further, the invention provides a kind of pharmaceutical composition, its described albumen that comprises pharmacy effective dose and one or more pharmaceutically acceptable carriers.
Further, the invention provides described pharmaceutical composition and whether contain the application in donkey, horse, ox, pig, sheep or deer derived component in detecting colla carapacis et plastri testudinis.
Beneficial effect of the present invention is as follows:
Adopt method of the present invention, can fast detecting colla carapacis et plastri testudinis and goods in the adulterant composition such as donkey, horse, ox, pig, sheep, deer, thereby minute evident, although collagen polypeptide has certain hydrolysis and destruction in glue class Chinese medicine, but the present invention is based on the otherness polypeptide in its principal ingredient collagen, its content is high, and the degree that is damaged is very little, can identify whether containing the adulterant compositions such as donkey, horse, ox, pig, sheep or deer in colla carapacis et plastri testudinis and goods thereof.
The invention still further relates to polypeptide, its amino acid sequence is Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg, and the composition being comprised of described polypeptide and respective carrier.In addition, also comprise the application in donkey, horse, ox, pig, sheep or deer derived component in detecting colla carapacis et plastri testudinis and goods thereof of described polypeptide and composition thereof.
Accompanying drawing explanation
Fig. 1 selects the mass spectrogram of ion pair m/z553.3 → 667.1,764.4,821.3 monitorings under synthetic polypeptide and sterling glue multiple-reaction monitoring scan pattern;
Fig. 2 selects the mass spectrogram of ion pair m/z553.3 → 667.1,764.4,821.3 monitorings under synthetic polypeptide and the epoxy glue multiple-reaction monitoring scan pattern that adds 5% adulterant glue;
Fig. 3 is the mass spectrogram that synthetic polypeptide and colla carapacis et plastri testudinis goods (colla carapacis et plastri testudinis particle) are selected ion pair m/z553.3 → 667.1,764.4,821.3 monitorings under multiple-reaction monitoring scan pattern.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, scope of the present invention is not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The acquisition of specificity peptide chain in the present invention:
1 material and reagent
Material: sterling glue comprises donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, colla carapacis et plastri testudinis, deer horn glue, sheepskin glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn, sheepskin respectively.
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg.
The detection pre-treatment of 2 samples
(1) preparation of glue sample enzymolysis solution
Get 1.0g glue sample to be measured in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of synthetic polypeptide control sample enzymolysis solution
Take a certain amount of synthetic polypeptide, add 1%NH
4hCO
3solution (pH8.0), shakes up, and be mixed with concentration and be about 0.3 μ g/mL, filtering with microporous membrane, standby.
The discovery of 3 polypeptide
(1) discovery of ion
Get 5 μ L enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution [volume fraction], Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0～40min, 2%～50% Mobile phase B (the percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) carry out one-level and entirely sweep, sweep limit m/z400-m/z1000.
From the mass spectrum of glue sample separately, entirely sweep and figure, extract ion m/z553.3, by contrast, find in 7.65min left and right only has colla carapacis et plastri testudinis not this quasi-molecular ions, and all contain in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue and deer horn glue.Detection and checking by multiple batches of sample show that above-mentioned conclusion is correct.Illustrate: can be by detecting the ion m/z553.3 in colla carapacis et plastri testudinis, the adulterant compositions such as the donkey that determines whether to adulterate, horse, ox, pig, sheep or deer source property.
(2) amino acid sequence of polypeptide is tentatively inferred
Utilize triple quadrupole bar mass spectrum, the above-mentioned ion of finding out is carried out to daughter ion scanning, confirm this ion band double charge, by sequence assembly, infer the partial sequence that polypeptide.Partial sequence by inference, at NCBI(http: //www.ncbi.nlm.nih.gov/) database carries out sequence retrieval, feature in conjunction with it (does not have in tortoise, and all have in donkey, horse, ox, pig, sheep or deer, and be about 1105.6 by molecular weight after tryptic digestion), derive the possible sequence Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg of this polypeptide.According to the CID fracture theory of peptide chain, calculate the possible daughter ion of this polypeptide as follows:
| a | b | c" | Res: | x | y" | z |
| 30.0 | 58.0 | 77.1 | Gly | - | - | - |
| 129.1 | 157.1 | 176.1 | Val | 1072.5 | 1048.6 | 1029.5 |
| 257.2 | 285.2 | 304.2 | Gln | 973.5 | 949.5 | 930.4 |
| 314.2 | 342.2 | 361.2 | Gly | 845.4 | 821.4 | 802.4 |
| 411.2 | 439.2 | 458.3 | Pro | 788.4 | 764.4 | 745.4 |
| 524.3 | 552.3 | 571.3 | Hyp | 691.3 | 667.4 | 648.3 |
| 581.3 | 609.3 | 628.3 | Gly | 578.3 | 554.3 | 535.3 |
| 678.4 | 706.4 | 725.4 | Pro | 521.2 | 497.3 | 478.2 |
| 749.4 | 777.4 | 796.4 | Ala | 424.2 | 400.2 | 381.2 |
| 806.4 | 834.4 | 853.5 | Gly | 353.2 | 329.2 | 310.2 |
| 903.5 | 931.5 | 950.5 | Pro | 296.1 | 272.2 | 253.1 |
| - | - | - | Arg | 199.1 | 175.1 | 156.1 |
Can entirely sweep coincideing in figure with the daughter ion of peculiar ion m/z553.3 in donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue and deer horn glue.
(3) sequence is determined
Amino acid sequence by inference, entrusts polypeptide Synesis Company (the biochemical company limited of gill) to carry out the synthetic of this polypeptide.Then the daughter ion that utilizes LC-MS instrument to synthesize polypeptide is swept entirely, and in its testing conditions and glue sample, the daughter ion of corresponding polypeptide is swept term harmonization entirely.By contrasting ion appearance time, the daughter ion of synthetic polypeptide and donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue or deer horn glue, entirely sweep figure, find that both fit like a glove, thereby determine that this ion m/z553.3 is polypeptide Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg.
Embodiment 1
1 material and reagent
Material: sterling glue comprises donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, colla carapacis et plastri testudinis, deer horn glue, sheepskin glue, decocts and obtains with donkey hide, horse skin, ox-hide, pigskin, tortoise plastron, deer horn, sheepskin respectively.
Epoxy glue sample (containing the colla carapacis et plastri testudinis of 5% oxhide gelatin, the colla carapacis et plastri testudinis of the colla carapacis et plastri testudinis of 10% oxhide gelatin, 20% oxhide gelatin, the colla carapacis et plastri testudinis of 40% oxhide gelatin) by above-mentioned sterling glue, be accurately mixed and made into (percentage is massfraction).
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling colla carapacis et plastri testudinis sample in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1mL and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ L of 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of glue sample enzymolysis solution
Get 1.0g glue sample to be measured in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ L enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution [volume fraction], Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0～40min, 2%～50% Mobile phase B (the percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) select to carry out many reaction detection, select m/z553.3 → 667.1,764.4,821.3 as detecting ion pair.
The results are shown in Figure 1,7.65min place and only have in colla carapacis et plastri testudinis sterling and do not detect corresponding quasi-molecular ions, other all detect.The detection of epoxy glue sample, the percentage composition of oxhide gelatin of take is horizontal ordinate, take m/z553.3 → 821.3, to extract chromatographic peak area in ion flow graph be ordinate, drawing standard curve, R
2be more than 0.999, linear good.The adulterant composition of visible this method in can specific detection colla carapacis et plastri testudinis, thus colla carapacis et plastri testudinis is carried out to authenticity.
1 material and reagent
Material: the colla carapacis et plastri testudinis sterling of epoxy glue sample in embodiment 1 adds respectively 5% adulterant glue to make, comprise containing the colla carapacis et plastri testudinis of 5% deer horn glue, the colla carapacis et plastri testudinis of the colla carapacis et plastri testudinis of 5% donkey-hide gelatin, 5% horse skin glue, containing the colla carapacis et plastri testudinis of 5% oxhide gelatin, the colla carapacis et plastri testudinis of the colla carapacis et plastri testudinis of 5% pig skin gelatin, 5% sheepskin glue (percentage is massfraction).
Reagent: ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g sterling colla carapacis et plastri testudinis to be measured sample in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1mL and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ L of 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of glue sample enzymolysis solution
Get 1.0g testing sample in 500mL measuring bottle, add a small amount of 1%NH
4hCO
3solution, the ultrasonic sample that makes dissolves completely, uses 1%NH
4hCO
3solution (pH8.0) is diluted to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ L enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution [volume fraction], Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0～40min, 2%～50%B(percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) select to carry out many reaction detection, select m/z553.3 → 667.1,764.4,821.3 as detecting ion pair.
The results are shown in Figure the synthetic polypeptide control sample in 2,7.65min place, add in the epoxy glue sample of 5% adulterant glue and all detect corresponding quasi-molecular ions, and do not detect corresponding quasi-molecular ions in sterling colla carapacis et plastri testudinis in case study on implementation 1.The adulterant composition of visible this method in can specific detection colla carapacis et plastri testudinis.
Embodiment 3
1 material and reagent
Material: colla carapacis et plastri testudinis, colla carapacis et plastri testudinis particle (Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.), add oxhide gelatin colla carapacis et plastri testudinis particle (self-control), add the colla carapacis et plastri testudinis particle (self-control) of donkey-hide gelatin
Reagent: trichloroacetic acid (analyzing pure), ammonium bicarbonate (analyzing pure), trypsase (sequence is pure, purchased from National Institute for Food and Drugs Control), synthetic polypeptide Gly-Val-Gln-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Pro-Arg.
2 detection methods
(1) preparation of synthetic polypeptide control sample enzymolysis solution
Get 1.0g colla carapacis et plastri testudinis particle and add 1%NH
4hCO
3solution 24mL dissolves, and adds 4g trichloroacetic acid, places 10min for 4 ℃, and the centrifugal 5min of 10000rpm, abandons supernatant, and sediment is proceeded in 100mL measuring bottle, uses 1%NH
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, and filtering with microporous membrane, precision measures subsequent filtrate 1mL and adds synthetic polypeptide 0.3 μ g, then adds the trypsin solution 100 μ L of 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(2) preparation of testing sample enzymolysis solution
Get 1.0g testing sample and add 1%NH
4hCO
3solution 25mL dissolves, and adds 4g trichloroacetic acid, places 10min for 4 ℃, and the centrifugal 5min of 10000rpm, abandons supernatant, and sediment is proceeded in 100mL measuring bottle, uses 1%NH
4hCO
3solution (pH8.0) dissolves and is settled to scale, shakes up, and filtering with microporous membrane, precision measures the trypsin solution 100 μ L that subsequent filtrate 1mL adds 2mg/mL, 37 ℃ of constant temperature enzymolysis 6h.
(3) detect
Get 5 μ L enzymolysis solution and put into the detection of LC-MS instrument.Liquid-phase condition: C
18reverse-phase chromatographic column (2.1mm * 100mm, 1.8 μ m), mobile phase A is 0.1% formic acid solution [volume fraction], Mobile phase B is acetonitrile, flow velocity 0.3mL/min; Gradient elution: 0～40min, 2%～50%B(percentage here represents from 0 minute to the 40th minute, flow B by 2% linear gradient be 50%, mobile phase A fades to 50% by 98%).Mass spectrum condition: electron spray positive ion mode (ESI
+) carry out many reaction detection, select m/z553.3 → 667.1,764.4,821.3 as detecting ion pair.
The results are shown in Figure the synthetic polypeptide control sample in 3,7.65min place, add oxhide gelatin colla carapacis et plastri testudinis particle, add the colla carapacis et plastri testudinis particle of donkey-hide gelatin all to detect corresponding quasi-molecular ions, other do not detect, as seen the adulterant composition of this method in can specific detection colla carapacis et plastri testudinis goods.
Sequence table
<110> Dong-E donkey-hide Gelatin Co., Ltd., Shandong Prov.
Detection thing and the LC-MS detection method thereof of <120> colla carapacis et plastri testudinis and goods thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> artificial sequence
<400> 1
Gly Val Gln Gly Pro Hyp Gly Pro Ala Gly Pro Arg
1 5
Claims (10)
1. detect a method for colla carapacis et plastri testudinis and goods thereof, it utilizes colla carapacis et plastri testudinis goods and in other Counterfeit Item genomes, has the different of specificity nucleotide sequence or amino acid sequence and detect, and described specific amino acid is as shown in SEQ.ID No1.
2. method according to claim 1, is characterized in that, described colla carapacis et plastri testudinis goods are selected from a kind of in food, health products or the medicine that colla carapacis et plastri testudinis makes, and do not contain donkey, horse, ox, pig, sheep, deer derived component in formula.
3. method according to claim 1, is characterized in that, described other Counterfeit Items refer to that one or more in donkey, horse, ox, pig, sheep or deer boil the gelatin substance forming.
4. according to the method described in any one in claim 1～3, it specifically comprises the steps:
(1) choose specific amino acid or amino acid sequence in colla carapacis et plastri testudinis goods and other Counterfeit Item genomes, described specific amino acid is as shown in SEQ.ID No1;
(2) colla carapacis et plastri testudinis or its goods are dissolved, or extract the polypeptide constituents of described colla carapacis et plastri testudinis or its goods and dissolve, add subsequently restriction enzyme to carry out enzyme and cut;
(3) solution after enzyme step (2) being obtained is cut is put into LC-MS instrument, take colla carapacis et plastri testudinis sterling as matrix, adds the described amino acid sequence of step (1) in contrast, and selects parent ion m/z553.3 and the daughter ion thereof of this amino acid sequence to monitor.
5. method according to claim 4, is characterized in that, dissolves reagent used and be 1%, the NH of pH value 8.0 described in step (2)
4hCO
3solution; Described restriction enzyme is trypsase.
6. detect a molecular marked compound for colla carapacis et plastri testudinis and goods thereof, the amino acid sequence of this molecular marked compound is as shown in SEQ.ID No1.
Coding albumen claimed in claim 6 gene.
8. whether molecular marked compound claimed in claim 6 contains the application in donkey, horse, ox, pig, sheep or deer derived component in detecting colla carapacis et plastri testudinis.
9. a pharmaceutical composition, is characterized in that, the albumen claimed in claim 6 that comprises pharmacy effective dose and one or more pharmaceutically acceptable carriers.
10. whether pharmaceutical composition claimed in claim 9 contains the application in donkey, horse, ox, pig, sheep or deer derived component in detecting colla carapacis et plastri testudinis.
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|---|---|---|---|
| CN201310518572.4A CN103630622A (en) | 2013-10-28 | 2013-10-28 | Detection substance for tortoise-shell glue and products thereof and LC-MS (Liquid Chromatography-Mass Spectrometry) detection method thereof |
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|---|---|---|---|
| CN201310518572.4A CN103630622A (en) | 2013-10-28 | 2013-10-28 | Detection substance for tortoise-shell glue and products thereof and LC-MS (Liquid Chromatography-Mass Spectrometry) detection method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN103630622A true CN103630622A (en) | 2014-03-12 |
Family
ID=50211893
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| CN113063952A (en) * | 2015-03-30 | 2021-07-02 | 国立大学法人神户大学 | Method for identifying animal species |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1987401A (en) * | 2006-12-15 | 2007-06-27 | 中国科学院植物研究所 | Method for protein gel internal enzymolysis |
| CN103383383A (en) * | 2013-07-12 | 2013-11-06 | 山东东阿阿胶股份有限公司 | Method for detection of swine-derived component in glue traditional Chinese medicine and its products |
-
2013
- 2013-10-28 CN CN201310518572.4A patent/CN103630622A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1987401A (en) * | 2006-12-15 | 2007-06-27 | 中国科学院植物研究所 | Method for protein gel internal enzymolysis |
| CN103383383A (en) * | 2013-07-12 | 2013-11-06 | 山东东阿阿胶股份有限公司 | Method for detection of swine-derived component in glue traditional Chinese medicine and its products |
Non-Patent Citations (5)
| Title |
|---|
| VAN DER WERF, INEZ D.; CALVANO, COSIMA D.; LAVIANO, ROCCO: "Multi-technique chemical characterisation of a 12-13th-century painted Crucifix", 《MICROCHEMICAL JOURNAL》, vol. 106, 26 May 2012 (2012-05-26), pages 87 - 94, XP028956751, DOI: 10.1016/j.microc.2012.05.011 * |
| 刘涛, 张贵锋, 孟庆强, 马光辉, 苏志国: "阿胶中特异性多肤的分离与液质联用分析", 《质谱学报》, vol. 27, 31 July 2006 (2006-07-31), pages 181 - 182 * |
| 王前: "明胶制备工艺及溯源方法研究", 《中国优秀硕士论文全文数据库》, no. 7, 15 July 2010 (2010-07-15), pages 47 - 65 * |
| 程显隆 等: "UPLC － QTOF －MS 结合主成分分析法用于龟甲胶、鹿角胶中添加牛皮源成分的检测研究", 《药物分析杂志》, vol. 32, no. 6, 30 June 2012 (2012-06-30), pages 931 - 935 * |
| 魏锋，程显隆，石岩，肖新月，马双成，林瑞超: "UPLC-QTOF-MS 技术用于胶类药材的专属性检测方法研究及应用", 《2011年中国药学大会暨第11届中国药师周论文集》, 4 November 2011 (2011-11-04), pages 1 - 8 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113063952A (en) * | 2015-03-30 | 2021-07-02 | 国立大学法人神户大学 | Method for identifying animal species |
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