CN117969717A - Method for detecting total content of characteristic tripeptides in collagen peptide - Google Patents
Method for detecting total content of characteristic tripeptides in collagen peptide Download PDFInfo
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- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 claims description 6
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- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 3
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- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 2
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- UVJWCNFWEYPYSP-SNKMQCGQSA-N 2-aminoacetic acid;(2s,4r)-4-hydroxypyrrolidine-2-carboxylic acid;(2s)-pyrrolidine-2-carboxylic acid Chemical compound NCC(O)=O.OC(=O)[C@@H]1CCCN1.O[C@H]1CN[C@H](C(O)=O)C1 UVJWCNFWEYPYSP-SNKMQCGQSA-N 0.000 description 1
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- URLZCHNOLZSCCA-UHFFFAOYSA-N leu-enkephalin Chemical compound C=1C=C(O)C=CC=1CC(N)C(=O)NCC(=O)NCC(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 URLZCHNOLZSCCA-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a method for detecting and analyzing total content of characteristic tripeptides in collagen peptide, which is based on a four-level rod tandem mass spectrometry parent ion scanning mode, wherein collision energy is respectively 10-20V and 20-40V, characteristic fragment ions [ Gly-Pro-46] + (m/z 127) and [ Gly-Hyp-46] + (m/z 143), parent ion scanning ranges are 230-336 m/z and 246-352 m/z, and standard products Gly-Pro-Hyp and Gly-Hyp-Ala are used for semi-quantitative analysis of total content of the characteristic tripeptides of Gly-Pro-Yaa and Gly-Hyp-Yaa. The invention can measure the total content of characteristic tripeptide in the collagen peptide sample, compared with the content of certain or some specific characteristic peptide segments, the index can more accurately reflect the technical level of collagen peptide products obtained by various raw material sources and production processes, and the index is relatively more stable among different production batches, thereby being very suitable for being used as the technical index of the collagen peptide products.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a liquid chromatography-mass spectrometry analysis method for detecting total content of Gly-Pro-Yaa and Gly-Hyp-Yaa characteristic tripeptides in collagen peptide based on a tandem mass spectrometry parent ion scanning mode.
Background
Collagen is a connective tissue structural protein composed of Gly-X-Y repeated sequences (X and Y are respectively often proline and hydroxyproline), and is rich in livestock, poultry and fish (about 10% -25% of total protein), but is not easy to be digested and absorbed by human body after daily diet intake. The collagen can be hydrolyzed to produce bioactive peptide with joint, skin, skeleton and cardiovascular effects. The lower the molecular weight of collagen, the more easily digested and absorbed, and the bioactivity of collagen is exerted. Thus, collagen oligopeptides have become an important functional food material.
Collagen tripeptide refers to a Gly-X-Y tripeptide sequence unit in a collagen structure, japanese wine well Kang Fu in 2001 suggested the concept of a collagen tripeptide product for the first time in a research paper, and since then, collagen tripeptide (also called collagen tripeptide) appears as a collagen peptide product with high technology, high quality and high added value in the domestic and foreign large health markets. One korean scholars study showed that the exposure of collagen tripeptide (average molecular weight 246 Da) orally into blood hydroxyproline di/tripeptide was increased by a factor of ten or more compared to collagen peptide having an average molecular weight of 2000Da, and thus, the biological potency of collagen tripeptide was much higher than that of higher molecular weight collagen peptide.
The composition and content of characteristic tripeptides in collagen peptide products obtained from different animal sources and production processes vary widely, even in samples of different production batches. At present, the content of one or more characteristic peptide fragments is generally adopted as an important technical index of a collagen tripeptide product in the industry, but in the application process, the index difference among different factories, batches, raw materials and processes is found to be large, the industry consensus is difficult to obtain, and the establishment of related industry standards is not facilitated.
Aiming at the current situation of the industry, the invention develops a liquid chromatography-mass spectrometry analysis method for detecting the total content of characteristic tripeptides of two types of collagen based on a tandem mass spectrometry parent ion scanning mode, the total content of characteristic tripeptides in a collagen peptide sample can be measured, the index can more accurately reflect the technical level of collagen peptide products obtained by various raw material sources and production processes compared with the content of certain specific characteristic peptide segments, and the index is relatively more stable among different production batches, so the index is very suitable for being used as the technical index of the quality standard of the collagen tripeptide products.
Disclosure of Invention
The invention aims to realize the measurement of the total content of characteristic tripeptides in collagen peptide, thereby providing an index which can accurately reflect the quality and technical level of collagen tripeptide products and providing technical support for the establishment of the quality standards of the collagen tripeptide products.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
and (3) performing semi-quantitative analysis on the tripeptides with the two types of characteristics of Gly-Pro-Yaa and Gly-Hyp-Yaa by adopting a liquid chromatography-mass spectrometry analysis technology and based on a tandem mass spectrometry parent ion scanning mode and taking 2 typical peptide fragments as standard substances.
In the above method, the tripeptides characterized by Gly-Pro-Yaa and Gly-Hyp-Yaa are characterized in that Gly represents glycine, pro represents proline, hyp represents hydroxyproline and Yaa represents any one of amino acids.
In the above method, the liquid chromatography-mass spectrometry detection method is characterized in that the liquid chromatography conditions are as follows: chromatographic column: ATLANTIS T3C 18 column (15 cm. Times.2.1 mm,5 μm) or other column of comparable performance; column temperature: 40 ℃; mobile phase: phase A is 0.1% formic acid aqueous solution (V/V), phase B is 95% acetonitrile aqueous solution containing 0.1% formic acid; flow rate: 0.35mL/min; elution procedure: 0-5 min 0% B, 5-10.0 min from 0% B to 20% B, 10.0-15.0 min from 20% B to 40% B, 15.0-17.0 min from 40% B to 80% B, holding for 2.0min, and returning to the initial mobile phase balance for 4.0min to prepare for the next sample injection; sample injection amount: 5.00. Mu.L.
In the above method, the liquid chromatography-mass spectrometry detection method is characterized in that mass spectrometry conditions are as follows: ion source: electrospray ion source (positive ion scan); spray voltage: 4.0kV; drying gas temperature: 300 ℃; spray pressure: 35psi; drying gas flow rate: 8L/min; scanning mode: parent ion scanning (Precursor Scan); collision energy: the Gly-Pro-Yaa type is 10-20V, and the Gly-Hyp-Yaa type is 20-40V; characteristic fragment ions: the Gly-Pro-Yaa type is [ Gly-Pro-46] + (m/z 127), and the Gly-Hyp-Yaa type is [ Gly-Hyp-46] + (m/z 143); parent ion scan range: the Gly-Pro-Yaa type is selected from 230 to 336m/z, and the Gly-Hyp-Yaa type is selected from 246 to 352m/z.
In the above method, the typical peptide standard is characterized in that Gly-Pro-Yaa type is selected from Gly-Pro-Hyp as standard, and Gly-Hyp-Yaa type is selected from Gly-Hyp-Ala as standard.
Compared with the prior art, the invention has the technical advantages that:
1) Can realize the measurement of the total content of characteristic tripeptides in collagen peptide samples. At present, a tandem mass spectrum multi-reaction monitoring (MRM) mode is commonly adopted in the industry to quantitatively determine one or more characteristic peptide fragments, and the determination of the total characteristic peptide is difficult to achieve. According to the invention, through a parent ion scanning mode for the first time, the [ Gly-Pro-46] + and the [ Gly-Hyp-46] + are respectively selected as characteristic fragment ions, and typical peptide segments Gly-Pro-Hyp and Gly-Hyp-Ala are used as standard substances, so that semi-quantitative analysis of total quantity of the two types of characteristic tripeptides Gly-Pro-Yaa and Gly-Hyp-Yaa is realized.
2) At present, the industry generally adopts the content of one or more characteristic peptide fragments as an important technical index of the collagen tripeptide product, but in the application process, the index difference among different factories, batches, raw materials and processes is found to be very large, so that the industry consensus is difficult to obtain, and the establishment of related industry standards is not facilitated. The total content of the two types of characteristic tripeptides Gly-Pro-Yaa and Gly-Hyp-Yaa is used as the technical index of the collagen tripeptide product, can more accurately reflect the quality and technical level of the product obtained by various raw material sources and production processes, and is relatively more stable among different production batches, so the index is very suitable for being used as the technical index of the quality standard of the collagen tripeptide product.
Drawings
FIG. 1 is a total ion flow diagram of a typical collagen peptide sample with [ Gly-Pro-46] + as a characteristic fragment ion in parent ion scan mode.
FIG. 2 is a total ion flow diagram of a typical collagen peptide sample with [ Gly-Hyp-46] + as a characteristic fragment ion in parent ion scan mode.
FIG. 3 is a structural formula of 6 characteristic tripeptide standards.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples relate to test analysis equipment, reagents and conditions as follows:
1. Instrument and apparatus: agilent 1290 Infinity II ultra-high performance liquid chromatograph (Agilent company, usa), agilent 6410 triple quadrupole mass spectrometer (Agilent company, usa), milli-Q ultra-pure water machine (Millipore company, usa), vortex-Genie 2 Vortex oscillator (SCIENTIFIC INDUSTRIES company, usa); electronic balance (Sartorius company, germany), sonicator (EMERSON company, usa), 0.22 μm microfiltration membrane (nylon 6, navigator).
2. Reagent: acetonitrile (LC-MS grade, merck limited, germany); water (LC-MS grade, merck libo limited); formic acid (99% purity, acros, usa); 6 characteristic tripeptide standard (purity is equal to or higher than 95 percent, shanghai Chu peptide biotechnology Co., ltd.); 25 commercial collagen peptide products (each of the major vendors).
Glycine-proline-hydroxyproline with molecular formula and molecular weight of C 12H19N3O5/285.13 and structural formula shown in figure 3 (1);
glycine-proline-arginine with molecular formula and molecular weight of C 13H24N6O4/328.19 and structural formula shown in figure 3 (2);
Glycine-proline-alanine with molecular formula and molecular weight of C 10H17N3O4/243.12, and structural formula shown in figure 3 (3);
Glycine-hydroxyproline-lysine with molecular formula and molecular weight of C 13H24N4O5/316.17, and structural formula shown in figure 3 (4);
glycine-hydroxyproline-arginine with molecular formula and molecular weight of C 13H24N6O5/344.18, and structural formula shown in figure 3 (5);
Glycine-hydroxyproline-alanine with molecular formula and molecular weight of C 10H17N3O5/259.12, and structural formula shown in figure 3 (6);
3. Preparing a standard working solution:
Preparing a stock solution: respectively accurately weighing 10mg of each characteristic tripeptide standard, respectively placing in 10mL volumetric flasks, dissolving with ultrapure water, fixing the volume to scale, shaking uniformly to prepare 1.0mg/mL standard stock solution, keeping in a sealed state at-18 ℃ in a dark place, and keeping for 3 months.
Preparing a mixed working solution: and accurately transferring a proper amount of standard stock solution, preparing each standard stock solution into mixed working solution with the concentration of 10 mug/mL by using ultrapure water, adding water for dilution, and preparing a series of mixed standard solutions with the concentration of Gly-Pro-Hyp and Gly-Hyp-Ala of 10.0ng/mL, 15.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL, 150ng/mL and 200.0ng/mL, wherein the solutions need to be prepared in an on-demand mode.
Example 1
The embodiment provides a method for detecting the content of characteristic tripeptides in collagen peptide.
1. Instrument working conditions:
chromatographic conditions of ultra-high performance liquid chromatograph:
Sample injection volume: 5.00. Mu.L;
The chromatographic column is ATLANTIS T C18 chromatographic column (15 cm×2.1mm,5 μm) or other chromatographic column with equivalent performance.
Column temperature: 40 ℃;
mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution, and phase B is 95% acetonitrile aqueous solution containing 0.1% formic acid;
flow rate: 0.35mL/min;
Mobile phase gradient elution procedure: 0-5 min 0% B, 5-10.0 min from 0% B to 20% B, 10.0-15.0 min from 20% B to 40% B, 15.0-17.0 min from 40% B to 80% B, holding for 2.0min, and returning to the initial mobile phase balance for 4.0min to prepare for the next sample injection.
Mass spectrometry conditions for triple quadrupole mass spectrometer:
Ion source: an electrospray ion source;
Positive ion scan mode (ESI +);
Drying gas temperature: 300 ℃;
Spray voltage: 4.0kV;
Desolventizing gas temperature: 325 deg.c;
Drying gas flow rate: 8L/min nitrogen;
Taper hole voltage: 65V;
Atomization pressure: 35psig nitrogen;
collision energy: the Gly-Pro-Yaa type is 10-20V, and the Gly-Hyp-Yaa type is 20-40V;
Parent ion scan (Precursor Scan) mode is adopted, parent ion scan range: the Gly-Pro-Yaa type is selected from 230 to 336m/z, and the Gly-Hyp-Yaa type is selected from 246 to 352m/z. In order to ensure the accuracy of Mass numbers, leucine enkephalin (Mass concentration 50ng/mL, m/z:556.2771 in positive ion mode) was used for real-time calibration, and data acquisition processing was performed by Mass Hunter software developed by Agilent corporation.
Characteristic fragment ions: the Gly-Pro-Yaa type is [ Gly-Pro-46] + (m/z 127), and the Gly-Hyp-Yaa type is [ Gly-Hyp-46] + (m/z 143); wherein, regarding the selection of characteristic fragment ions: in order to determine characteristic ion, a secondary mass spectrum scanning mode is adopted to determine the characteristic ion, fragment ion peaks of 127 appear in Gly-Pro-Yaa type characteristic tripeptides, and the ion intensity is the strongest; the Gly-Hyp-Yaa type characteristic tripeptide has 143 fragment ion peaks and the ion intensity is strongest.
2. Pretreatment of commercially available collagen peptide products
Respectively accurately weighing 10mg of 25 collagen peptide products sold in the market, accurately adding 10mL of ultrapure water for constant volume, vortex shaking, fully dissolving the sample solution prepared into 1mg/mL, diluting the concentration of the sample to 100ng/mL, filtering by using a nylon filter membrane with the thickness of 0.22 mu m, and transferring the filtrate into a sample injection vial for ultra-high performance liquid-tandem mass spectrometry detection;
3. Experimental method
The standard solution of characteristic tripeptides with different concentrations is used as a recovery indicator, the addition levels of Gly-Pro-Yaa type characteristic tripeptides are respectively 30ng/mL, 60ng/mL and 120ng/mL, and the addition levels of Gly-Hyp-Yaa type characteristic tripeptides are respectively 50ng/mL, 100ng/mL and 200ng/mL, and 5 parallel samples are prepared for each addition concentration.
4. Calculation of relative response factor of tripeptide of two characteristics in parent ion scanning mode
Five mixed standard solutions of different concentration gradients were prepared and analyzed by triple quadrupole mass spectrometer parent ion scan mode. The relative response factor for each analyte is then calculated by the following equation.
Wherein P s and P x represent chromatographic peak areas of the reference and analyte, respectively; c s and C x represent the concentrations of the reference and analyte, respectively; i represents the analyte concentration gradient level.
5. Analysis of experimental results
The detection results of this embodiment are further analyzed to verify the feasibility of the method provided by the present invention;
(1) Linear range, limit of detection and limit of quantification of analytical methods
The above prepared mixed standard working solution was measured according to the conditions of this example, the concentration was measured from low to high, an external standard method was used to prepare a calibration curve, the standard concentration (ng/mL) was used as the abscissa, and the chromatographic peak area of the standard was used as the ordinate, a linear regression equation was constructed, and a standard correlation curve was drawn. The content of each type of characteristic tripeptide in the commercial sample was calculated from the calibration curve. Typically, a 3-fold signal-to-noise ratio is used as the detection limit and a 10-fold signal-to-noise ratio is used as the quantification limit. In this example, the linear equation, correlation coefficient, linear range and detection limit of the Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic tripeptides are shown in Table 1:
TABLE (1) Linear equation, correlation coefficient, linear Range and detection Limit of Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic tripeptides
The characteristic tripeptides of the two types show good linear relation in the linear range, the correlation coefficient of the analytes is larger than 0.990, the quantitative requirement is met, and the characteristic tripeptides in the commercial samples can be rapidly, conveniently, stably and accurately quantitatively measured. Gly-Pro-Yaa type characteristic tripeptide detection limit is 5 mug/kg, and quantitative limit is 10 mug/mL; gly-Hyp-Yaa type characteristic tripeptide detection limit is 15 mug/kg and quantification limit is 50 mug/mL.
(2) Analytical methods in-day precision and daytime precision and accuracy
In this example, the precision and accuracy of two types of characteristic tripeptides were also examined: precision is the proximity between a series of measurements obtained from multiple samples of the same sample, and accuracy is the mean value of the measurements (n=5) calculated for each level repetition, the precision of which is expressed in RSD. The standard working solutions of Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic tripeptide were prepared to have high, medium and low concentrations for daily daytime precision experiments, the same sample was measured three times a day for daily precision, the same sample was measured 1 time a day for 1,3 and 5 days respectively, and 5 parallel samples were measured for each addition level according to the method conditions of the present example, and the precision is shown in tables 2 and 3.
TABLE 2 Gly-Pro-Yaa type characteristic tripeptides in-day precision and daytime precision and accuracy
TABLE 3 Gly-Hyp-Yaa type characteristic tripeptides in-day precision and daytime precision and accuracy
The peak areas of high, medium and low concentration quality control samples are used, quantification is carried out through an external standard method standard curve, and the precision and accuracy result of each quality control sample is obtained through calculation, wherein the acceptance standard is that the accuracy between the measured value average value and the theoretical value is 85.0% -115.0%, and the relative standard deviation is less than or equal to 15%. The results are shown in tables 2 and 3, and the results show that the Gly-Pro-Yaa type characteristic tripeptide has daily precision of 94.78-112.49%, daily precision of 96.15-107.92% and Relative Standard Deviation (RSD) of less than 15%. The Gly-Hyp-Yaa type characteristic tripeptide has the daily precision of 97.08-110.57 percent, the daily precision of 100.05-114.36 percent and the Relative Standard Deviation (RSD) of less than 15 percent, and meets the standard requirements. The experimental result shows that the detection method has high precision and reliable result, and is suitable for detecting the two types of characteristic tripeptides.
(3) Analytical method recovery results
The sample recovery rate experiments were performed by taking Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic tripeptide standard working solutions to prepare high, medium and low 3 concentrations, and the samples were measured according to the method of the present example with the commercial collagen peptide substrate as the test object, and 5 parallel samples were measured at each addition level, and the recovery rates are shown in tables 4 and 5.
TABLE 4 Gly-Pro-Yaa type characteristic tripeptide recovery and relative standard deviation
TABLE 5 Gly-Hyp-Yaa type characteristic tripeptide recovery and relative standard deviation
The recovery results should be accurate at the LOQ level of 70% -120% (20% relative standard deviation) and at higher measured concentrations of 80% -115% (15% relative standard deviation). Specific results are shown in tables 4 and 5, and the results show that the recovery rate of Gly-Pro-Yaa type characteristic tripeptides under the conditions of high, medium and low standard addition concentration ranges from 89.08% to 118.70%, and the relative standard deviation RSD (n=5) ranges from 1.07% to 6.76%. The recovery rate of Gly-Hyp-Yaa type characteristic tripeptide ranges from 101.30% to 112.86%, the relative standard deviation RSD (n=5) ranges from 2.23% to 7.31%, and the standard requirements are met. The above shows that the detection method has good recovery rate.
(5) Relative response factor calculation
A large number of Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic tripeptides cannot be obtained as a standard, and therefore, the invention quantifies the characteristic tripeptide content of the type by using a standard substance. The f-number generated by the chromatographic peak areas of the standard and analyte is used to improve the accuracy of the method. Each analyte was selected in turn as a reference and the relative response factors for the two types of characteristic tripeptides were calculated according to equation (1). The results of the specific calculation of the relative response factor are shown in tables 6 and 7.
TABLE 6 Gly-Pro-Yaa type characteristic tripeptide relative response factors
TABLE 7 Gly-Hyp-Yaa type characteristic tripeptide relative response factor
The results are shown in tables 6 and 7, and show that Gly-Pro-Hyp and Gly-Hyp-Ala are the first choice of the two types of characteristic tripeptides, and their relative response factors are located at the middle positions in the two types of polypeptides, so that the quantitative errors of the residual analytes can be greatly reduced.
Example 2
The utility of this method in the detection of actual samples was further evaluated by detecting actual samples of commercially available collagen peptides. The method is applied to 25 product samples from commercial products for characteristic tripeptide analysis, and the calculation is carried out by using a single standard substance matrix matching calibration curve in the method, so that the semi-quantitative analysis of the total content of the two types of characteristic tripeptides of Gly-Pro-Yaa and Gly-Hyp-Yaa is realized.
(1) Qualitative analysis of characteristic oligopeptides in commercially available collagen peptide products
The commercial 25 collagen peptide products are used as test objects, and [ Gly-Pro-46] + and [ Gly-Hyp-46] + are respectively selected as characteristic fragment ions through a parent ion scanning mode, and the parent ion scanning range is set to be 100-700 m/z, so that qualitative analysis of collagen characteristic oligopeptides is carried out. FIG. 1 is a total ion flow diagram of a typical collagen peptide sample in a parent ion scanning mode, wherein [ Gly-Pro-46] + is selected as a characteristic fragment ion, and analysis on FIG. 1 shows that the product contains characteristic oligopeptides such as tripeptides, tetrapeptides, pentapeptides and the like, and the main characteristic oligopeptides are tripeptides; as shown in FIG. 2, which is a total ion flow diagram of a typical collagen peptide sample in a parent ion scanning mode, and [ Gly-Hyp-46] + is selected as a characteristic fragment ion, analysis on FIG. 2 shows that tripeptides, tetrapeptides, pentapeptides and other short peptides exist in the product, and the tripeptides are mainly used. The qualitative analysis results of the characteristic oligopeptides in the 25 commercial collagen peptide products are shown in table 8, and the results show that 18 characteristic oligopeptides coexist in the 25 commercial collagen peptide products; gly-Hyp-Yaa-Xaa of the tetrapeptides, which is predominantly Gly-Hyp-Ser-Lys and Gly-Hyp-Asn-Pro, was present in 21 commercial collagen peptide samples; gly-Pro-Yaa-Xaa this type is prevalent as Gly-Pro-Hyp-Arg, and is present in 20 commercially available collagen peptide samples; gly-Pro-Yaa-Xaa-Haa, which is a type of pentapeptide, was found in 20 commercial collagen peptide samples, with Gly-Pro-Lys-Leu-Pro predominating.
TABLE 8 short peptide analysis of 25 commercially available collagen peptide products
(2) Calculation of total tripeptide content of commercial collagen peptide Gly-Pro-Yaa type and Gly-Hyp-Yaa type characteristic
In this example, according to the detection method provided in example 1, 25 commercial collagen peptides were prepared accurately to have a concentration of 100ng/mL, and analyzed by a liquid chromatography mass spectrometry system according to the procedure provided in example 1, and the total content of tripeptides of two types, gly-Pro-Yaa and Gly-Hyp-Yaa, was calculated by using the parent ion scanning mode, respectively, using [ Gly-Pro-46] + and [ Gly-Hyp-46] +, as characteristic fragment ions, the parent ion scanning ranges of 230-336 m/z and 246-352 m/z, and the typical peptide fragments Gly-Pro-Hyp and Gly-Hyp-Ala, as standard substances. Specific results of qualitative and quantitative analysis of the two types of characteristic tripeptides Gly-Pro-Yaa and Gly-Hyp-Yaa in the 25 commercial collagen peptide products are shown in tables 9-1 and 9-2; the result shows that the content of Gly-Pro-Yaa type characteristic tripeptides in 25 commercial collagen peptide products is in the range of 6.60mg/g-256.30mg/g, and the two characteristic tripeptides Gly-Pro-Hyp and Gly-Pro-Arg are the most, wherein the Gly-Pro-Hyp ratio in 25 commercial products can reach 7.69%, namely 76.94mg/g; the Gly-Pro-Arg ratio can reach 4.05% at maximum, namely 40.49mg/g. The content of Gly-Hyp-Yaa type characteristic tripeptides in 25 commercial collagen peptide products is in the range of 30.21mg/g-228.10mg/g, and three characteristic tripeptides Gly-Hyp-Ala, gly-Hyp-Arg and Gly-Hyp-Lys are the most, wherein the Gly-Hyp-Ala ratio in 25 commercial products can reach 2.42% at most, namely 24.22mg/g; the Gly-Hyp-Arg ratio can reach 7.52% at the highest, namely 75.22mg/g; the Gly-Hyp-Lys ratio can reach 3.26% at the highest, namely 32.63mg/g.
Table 9-1 qualitative and quantitative analysis of two types of characteristic tripeptides Gly-Pro-Yaa and Gly-Hyp-Yaa in 25 commercially available collagen peptide products
Table 9-2 qualitative and quantitative analysis of two types of characteristic tripeptides Gly-Pro-Yaa and Gly-Hyp-Yaa in 25 commercially available collagen peptide products
Through the detection of an actual collagen peptide sample, the practicability of the liquid chromatography-mass spectrometry analysis method for detecting the content of the tripeptide of the two types of collagen characteristics of Gly-Pro-Yaa and Gly-Hyp-Yaa based on a tandem mass spectrometry parent ion scanning mode is further verified. Therefore, in the collagen peptide production process, the method can more accurately reflect the technical level of collagen peptide products obtained by various raw material sources and production processes, and the index is relatively more stable among different production batches, thereby being beneficial to strengthening the control of the product quality by the supervision department.
The invention develops a liquid chromatography-mass spectrometry analysis method for detecting total content of tripeptides characteristic of two types of collagen proteins, namely Gly-Pro-Yaa and Gly-Hyp-Yaa based on a tandem mass spectrometry parent ion scanning mode. The method disclosed by the invention is based on a four-level rod tandem mass spectrometry parent ion scanning mode, collision energy is respectively 10-20V and 20-40V, characteristic fragment ions [ Gly-Pro-46] + (m/z 127) and [ Gly-Hyp-46] + (m/z 143) are adopted, parent ion scanning ranges are 230-336 m/z and 246-352 m/z, and typical peptide segments Gly-Pro-Hyp and Gly-Hyp-Ala are used as standard substances, so that semi-quantitative analysis of total content of Gly-Pro-Yaa and Gly-Hyp-Yaa characteristic tripeptides in a commercial collagen peptide product is realized.
The method breaks through the mode that the traditional method only aims at target objects one by one to detect respectively, realizes multiple cluster analysis detection of characteristic tripeptides, greatly improves detection flux and reduces detection cost. Through methodological verification, the method has better precision and recovery rate, the total content of the characteristic tripeptide in the collagen peptide sample can be measured in actual sample measurement, compared with the content of a certain or some specific characteristic peptide segments, the index can more accurately reflect the technical level of collagen peptide products obtained by various raw material sources and production processes, and the index is relatively more stable among different production batches, thereby providing technical support for ensuring the quality of the collagen peptide.
While the foregoing description of the embodiments of the present application has been presented with reference to the drawings, it is not intended to limit the scope of the application, and on the basis of the technical solutions of the present application, a person skilled in the art may make various modifications or variations without any inventive effort. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (6)
1. The application of the total content of the tripeptides with two types of characteristics, namely Gly-Pro-Yaa and Gly-Hyp-Yaa, in the technical index of the reaction collagen peptide product.
2. The use according to claim 1, wherein in the tripeptides of both Gly-Pro-Yaa and Gly-Hyp-Yaa, gly stands for glycine, pro stands for proline, hyp stands for hydroxyproline and Yaa stands for any amino acid.
3. The detection method is characterized by comprising a liquid chromatography-tandem mass spectrometry parent ion scanning mode-based detection method for the total content of characteristic tripeptides in the collagen peptide.
4. The method according to claim 3, wherein the liquid chromatography conditions are:
Chromatographic column: ATLANTIS T3C 18 column (15 cm X2.1 mm, 5 μm) or other column of comparable performance;
Column temperature: 40 ℃;
Mobile phase: phase A is 0.1% formic acid aqueous solution (V/V), phase B is 95% acetonitrile aqueous solution containing 0.1% formic acid;
Flow rate: 0.35 mL/min;
elution procedure: 0-5 min 0% B; linearly rising from 0% B to 20% B for 5-10.0 min; linearly rising from 20% B to 40% B within 10.0-15.0 min; linearly increasing from 40% B to 80% B for 15.0-17.0 min, and keeping 2.0 min; then returning to the initial mobile phase balance of 4.0 min, and preparing for the next sample injection;
Sample injection amount: 5.00 Mu L.
5. The method of claim 3, wherein the mass spectrometry conditions are:
ion source: electrospray ion source (positive ion scan);
spray voltage: 4.0 A kV;
Drying gas temperature: 300 ℃;
Spray pressure: 35A psi;
Drying gas flow rate: 8L/min;
scanning mode: parent ion scanning (Precursor Scan);
Collision energy: the Gly-Pro-Yaa type is 10-20V, and the Gly-Hyp-Yaa type is 20-40V;
Characteristic fragment ions: the Gly-Pro-Yaa type is [ Gly-Pro-46] + (m/z 127), and the Gly-Hyp-Yaa type is [ Gly-Hyp-46] + (m/z 143);
Parent ion scan range: the Gly-Pro-Yaa type is selected from 230 to 336 m/z, and the Gly-Hyp-Yaa type is selected from 246 to 352 m/z.
6. The method according to claim 3, wherein Gly-Pro-Hyp is used as a standard, and Gly-Hyp-Yaa is used as a standard.
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