Patent ID: 12196762

DETAILED DESCRIPTION

The present invention will be further described in detail hereinafter with reference to the specific embodiments, but the present invention is not limited to these embodiments.

The trypsin used in the following embodiments was purchased from Promega Company.

Embodiment 1

A deer-derived specific peptide had two specific peptide sequences, as shown in sequence table 1:peptide 1:Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Hyp-Gly-Ala-Val-Gly-Ala-Lys; andpeptide 2:Gly-Asn-Asp-Gly-Ala-Thr-Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Hyp-Gly-Phe-Pro-Gly-Ala-Val-Gly-Ala-Lys.

The polypeptides above were prepared by Nanjing GenScript Biotech Corporation using a solid phase synthesis method.

Embodiment 2 Proportional relationship between peptide 1 and peptide 2 of deer hide gelatin

batches of commercially available deer hide gelatin samples were taken, with each batch of about 10 mg, added with 5 ml of phosphate buffer solution (pH=7.8), the samples were completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 μl of supernatant were placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly, and enzymolyzed at a constant temperature of 37° C. for 12 hours. After enzymolysis, 60 μl of 10% v/v TFA solution was added to stop the reaction, and then centrifuged at 12,000 rpm for 20 minutes to obtain the enzymatic hydrolysate of the deer hide gelatin medical material, which was stored at −20° C. for later use.

The enzymatic hydrolysates of each batch of deer hide gelatin were injected into the liquid chromatograph/mass spectrometer for detection, wherein a sample size was 1 μg, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer were as follows: a chromatographic column was a 1.7 μm C18 reversed phase column (2.1 μm×100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasts for 3.5 minutes to 4 minutes, and 10% A elution lasts for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500° C.; an ionization voltage of 5,500 V; a desolvent temperature of 500° C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown inFIG.2andFIG.3. The ion pair conditions corresponding to the specific peptides were set as follows:peptide 1: M/z 850.4 (triple charge)→515.4, DP=162.49, CE=32.87; andpeptide 2: m/z 845.0 (triple charge)→507.3, DP=162.52, CE=31.51.

The values of Apeptide 1/Apeptide 2in the 10 batches of deer hide gelatin were shown in Table 1. The average value of Apeptide 1/Apeptide 2was 0.0.612±0.282.

TABLE 1Results of Apeptide 1/Apeptide 2in deer hide gelatinAverage valueApeptide 1/of Apeptide 1/BatchApeptide 1Apeptide 2Apeptide 2Apeptide 2Deer hide1179722850710.4140.612 ± 0.282gelatin-1Deer hide945341970730.480gelatin-2Deer hide1470073358230.438gelatin-3Deer hide93660889061.053gelatin-4Deer hide1248931465080.852gelatin-5Deer hide1034892829730.366gelatin-6Deer hide1286781157501.112gelatin-7Deer hide734541819280.404gelatin-8Deer hide778331556400.500gelatin-9Deer hide994191973610.504gelatin-10

Embodiment 3 Proportional relationship between peptide 1 and peptide 2 of deer antler gelatin

batches of deer antler samples were taken and prepared into deer antler gelatin samples according to the method of preparing deer antler gelatin in Chinese Pharmacopoeia 2020, with each batch of 10 mg, added with 5 ml of phosphate buffer solution (pH=7.8), the samples were completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 μl of supernatant were placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly, and enzymolyzed by ultrasound for 10 minutes. After enzymolysis, 60 μl of 10% v/v TFA solution was added to stop the reaction, and then centrifuged at 12,000 rpm for 20 minutes to obtain the enzymatic hydrolysate of the deer antler gelatin, which was stored at −20° C. for later use.

The enzymatic hydrolysates of each batch of deer antler gelatin were injected into the liquid chromatograph/mass spectrometer for detection, wherein a sample size was 1 μg, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer were as follows: a chromatographic column was a 1.7 μm C18 reversed phase column (2.1 μm×100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasted for 3.5 minutes to 4 minutes, and 10% A elution lasted for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500° C.; an ionization voltage of 5,500 V; a desolvent temperature of 500° C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown inFIG.2andFIG.3. The ion pair conditions corresponding to the specific peptides were set as follows:peptide 1: M/z 850.4 (triple charge)→515.4, DP=162.49, CE=32.87; andpeptide 2: m/z 845.0 (triple charge)→507.3, DP=162.52, CE=31.51.

The values of Apeptide 1/Apeptide 2in the 10 batches of deer antler gelatin were shown in Table 2. The average value of Apeptide 1/Apeptide 2was 7.428±1.617.

TABLE 2Results of Apeptide 1/Apeptide 2in deer antler gelatinAverage valueApeptide 1/of Apeptide 1/BatchApeptide 1Apeptide 2Apeptide 2Apeptide 2Deer antler17333472384177.2707.428 ± 1.617gelatin-1Deer antler18740772050649.139gelatin-2Deer antler17365682899735.989gelatin-3Deer antler12174321829216.656gelatin-4Deer antler17390912894096.009gelatin-5Deer antler18590762462967.548gelatin-6Deer antler18021541869939.638gelatin-7Deer antler19451713118106.238gelatin-8Deer antler175812417520710.035gelatin-9Deer antler14189182463545.760gelatin-10

Embodiment 4 Proportional relationships between peptide 1 and peptide 2 in mixed samples of deer hide gelatin and deer antler gelatin with different proportions

The deer antler gelatin and the deer hide gelatin were mixed according to proportions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, respectively added with 5 ml of phosphate buffer solution (pH-7.8) into about 10 mg of mixed gelatin sample in each batch, the sample was completely dissolved by ultrasound, centrifuged at 12,000 rpm for 20 minutes, 150 μl of supernatant was placed into a 2 ml centrifuge tube, diluted with 1 ml of 50 mM PBS, added with 1 wt % trypsin, shaken evenly for microwave enzymolysis for 30 minutes, added with 60 μl of 10% v/v TFA solution to stop the reaction after enzymolysis, centrifuged at 12,000 rpm for 20 minutes to obtain enzymolysis solutions of mixed gelatin samples with different proportions, which were stored at −20° C. for later use.

The enzymatic hydrolysates of the mixed gelatin samples with different proportions were injected into the liquid chromatograph/mass spectrometer, wherein a sample size was 1 μg, and liquid phase conditions for detection by the liquid chromatograph/mass spectrometer are as follows: a chromatographic column was a 1.7 μm C18 reversed phase column (2.1 μm×100 mm) with a flow rate of 0.3 ml/min, a mobile phase A was acetonitrile, a mobile phase B was 0.1% formic acid, 10% to 30% A linear gradient elution lasted for 0 to 3.5 minutes, 30% to 10% A linear gradient elution lasted for 3.5 minutes to 4 minutes, and 10% A elution lasted for 4 minutes to 6 minutes. A mass spectrometry condition for detection by the liquid chromatograph/mass spectrometer was: an electrospray positive ion mode ESI+, and mass spectrometry parameters comprised: an ion source temperature of 500° C.; an ionization voltage of 5,500 V; a desolvent temperature of 500° C.; an ion source gas 1 of 60 psi; and an ion source gas 2 of 60 psi. The mass spectra were shown inFIG.2andFIG.3. The ion pair conditions corresponding to the specific peptides were set as follows:peptide 1: M/z 850.4 (triple charge)→515.4, DP=162.49, CE=32.87; andpeptide 2: m/z 845.0 (triple charge)→507.3, DP=162.52, CE=31.51.

Values of Apeptide 1/Apeptide 2of mixed gelatin samples with different proportions were shown in Table 3, and a numerical relationship between the proportion of the deer antler gelatin in the mixed gelatin and Apeptide 1/Apeptide 2was shown inFIG.1. A graph was drawn by using the proportion of the deer antler gelatin in the mixed gelatin as an abscissa and using the value of Apeptide 1/Apeptide 2as an ordinate, and the proportion of the deer antler gelatin had a linear relationship to Apeptide 1/Apeptide 2, y=4.7903x+0.4106, and R2=0.9669. It was indicated that the proportion of the deer antler gelatin adulterated was related to the peak areas of the peptide 1 and the peptide 2. Therefore, the mixing proportion of the deer antler gelatin and the deer hide gelatin could be determined according to the ratio of Apeptide 1/Apeptide 2.

TABLE 3Relationship between the mixing proportion of the deer antlergelatin/deer hide gelatin mixed sample and the peak areaMixed gelatin proportionProportionProportionPeak areaof deerof deerratioantlerhidePeak areaApeptide 1/gelatin %gelatin %Peptide 1Peptide 2Apeptide 201003410044150230.82210903290573706890.88820805134343873551.32530707557374194401.80240607709953855692.000505010766574437202.426604011400663616423.152703014492463969463.651802017555113595934.882901017102173603634.746100021795604216025.170

Embodiment 5

Mixed control of the peptide 1 and the peptide 2 with a certain concentration were taken, and fed for six times continuously under the above-mentioned chromatography-mass spectrometry conditions, to determine the peak areas of the control of the peptide 1 and the peptide 2, and calculate the RSD of the peak areas of the control. The results were shown in Table 4, and the RSD of the peptide 1 and the peptide 2 were respectively 1.10% and 1.67%, indicating that the method had excellent precision.

A corresponding concentration when a signal-to-noise ratio (S/N) of the peptide 1 and the peptide 2 was about 3, was taken as a limit of detection (LOD), and a corresponding concentration when the signal-to-noise ratio (S/N) of the peptide 1 and the peptide 2 was about 10, was taken as a limit of quantitation (LOQ). The results were shown in Table 4, and the LOQ and the LOD of the peptide 1 were 0.72 ng/ml and 0.24 ng/ml respectively. The LOQ and the LOD of the peptide 2 were 2.40 ng/ml and 0.80 ng/ml respectively.

TABLE 4Precision, limit of detection and limit of quantitation ofpeptide 1 and peptide 2PrecisionLOQ(RSD, %)(ng/ml)LOD (ng/ml)Peptide 11.100.720.24Peptide 21.672.400.80