CN118501300A - Method for detecting total content of proline or hydroxyproline dipeptide in collagen peptide - Google Patents
Method for detecting total content of proline or hydroxyproline dipeptide in collagen peptide Download PDFInfo
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- hydroxyproline
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- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 title claims abstract description 69
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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 title claims abstract description 63
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229960002591 hydroxyproline Drugs 0.000 title claims abstract description 63
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Abstract
The invention relates to a liquid chromatography-mass spectrometry analysis method for total content of dipeptides containing proline or hydroxyproline characteristics in collagen peptide. Based on a four-level rod tandem mass spectrometry parent ion scanning mode, the method adopts collision energy of 10V-35V, characteristic fragment ions [ C 4H8N]+ (m/z 70.1) and [ C 4H8NO]+ (m/z 86.1) ] respectively, parent ion scanning ranges of 170-282 m/z and 186-298 m/z, and standard products Pro-Glu and Gly-Hyp to perform semi-quantitative analysis on total content of two types of characteristic dipeptide containing proline or hydroxyproline. The total content of proline or hydroxyproline dipeptide in the collagen peptide sample can be measured by the method, compared with the content of certain or certain specific characteristic peptide fragments, 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, so that the index is 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 detection, in particular to a liquid chromatography-mass spectrometry analysis method for detecting total content of two types of characteristic dipeptide containing proline or hydroxyproline 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.
At present, it has been ascertained that the H +/peptide cotransporter family such as PepT1 on the surface of intestinal epithelial cells can completely transport di/tripeptides, so that the di-peptides and the tripeptides in the collagen peptide can be directly absorbed by the intestinal tract, and the collagen peptide has higher biological titer. Therefore, the characteristic di/tripeptide content containing proline or hydroxyproline has become an important technical index for evaluating the quality of collagen peptide products.
The collagen peptide products obtained from different animal sources and production processes have very different compositions and contents of characteristic dipeptides containing proline or hydroxyproline, and even the compositions and contents of characteristic dipeptides in samples of different production batches are unstable. At present, the content of a certain or a plurality of characteristic peptide fragments is generally adopted as a technical index of a collagen peptide product in the industry, but in the application process, the index difference among different factories, batches, raw materials and processes is found to be very 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 dipeptide of two types of collagen containing proline or hydroxyproline based on a tandem mass spectrometry parent ion scanning mode, the total content of characteristic dipeptide in a collagen peptide sample can be measured, compared with the content of a certain or a certain specific characteristic peptide segment, 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, so the index is very suitable for being used as the technical index of the quality standard of the collagen dipeptide products.
Disclosure of Invention
The invention aims to realize the measurement of the total content of the dipeptide with the proline or the hydroxyproline characteristic in the collagen peptide, thereby providing an index which can accurately reflect the quality and the technical level of the collagen peptide product and providing technical support for the establishment of the quality standard of the collagen peptide product.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
Based on a liquid chromatography tandem mass spectrometry parent ion scanning mode, two typical peptide fragments are used as standard substances to perform semi-quantitative analysis of the dipeptide containing the two types of characteristics of proline or hydroxyproline by adopting a liquid chromatography-mass spectrometry analysis technology.
In the method, the dipeptide containing two types of proline or hydroxyproline features, wherein the proline or hydroxyproline exists in two amino acids, and the rest is any one of common amino acids.
In the above method, the liquid chromatography conditions of the liquid chromatography combined detection method are as follows:
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,5~10.0 min linearly rising from 0% b to 20%B,10.0~15.0 min from 20% b to 40%B,15.0~16.0 min from 40% b to 80% b, holding 2.0 min, and then returning to the initial mobile phase equilibrium of 4.0 min ready for the next sample injection; sample injection amount: 5.00 Mu L.
In the method, the mass spectrum conditions of the liquid chromatography-mass spectrometry detection method are as follows: ion source: electrospray ion source (positive ion scan); spray voltage: 4.0 A kV; drying gas temperature: 300 ℃; spray pressure: 40 psi, dry gas flow rate: 10 L/min; scanning mode: parent ion scanning (Precursor Scan); collision energy: the dipeptide containing two types of characteristics of proline or hydroxyproline is selected from 10V-35V; characteristic fragment ions: the dipeptide containing the proline type characteristic is selected from [ C 4H8N]+ (m/z 70.1) ] and the dipeptide containing the hydroxyproline type characteristic is selected from [ C 4H8NO]+ (m/z 86.1); parent ion scan range: the proline type characteristic dipeptide is 170-282 m/z, and the hydroxyproline type characteristic dipeptide is 186-298 m/z.
In the method, the typical peptide standard product adopts Pro-Glu as a standard product, and the hydroxyproline-containing characteristic dipeptide adopts Gly-Hyp as a standard product.
Compared with the prior art, the invention has the beneficial effects that:
1) Can realize the determination of the total content of characteristic dipeptide in the collagen peptide sample. 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 [ C 4H8N]+ (m/z 70.1) and the [ C 4H8NO]+ (m/z 86.1) are respectively selected as characteristic fragment ions, and typical peptide segments Pro-Glu and Gly-Hyp are used as standard substances, so that semi-quantitative analysis of total quantity of two types of characteristic dipeptide containing proline or hydroxyproline is realized.
2) At present, the industry generally adopts the content of one or more characteristic peptide fragments as an important technical index of a collagen peptide 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 dipeptide with two types of characteristics of proline or hydroxyproline is used as the technical index of the collagen peptide product, the quality and the technical level of the product obtained by various raw material sources and production processes can be reflected more accurately, and the index is relatively more stable among different production batches, so that the total content is very suitable for being used as the technical index of the quality standard of the collagen peptide product.
Drawings
FIG. 1 fragmentation path in mass spectrum of proline-containing characteristic dipeptide; a is a Pro-X characteristic dipeptide fragmentation pathway; b is the X-Pro characteristic dipeptide fragmentation pathway.
FIG. 2 contains the fragmentation path in a hydroxyproline-characterized dipeptide mass spectrum; a is the Hyp-X characteristic dipeptide fragmentation pathway; b is the X-Hyp characteristic dipeptide fragmentation pathway.
FIG. 3 proline containing characteristic dipeptide collision energy optimisation; a is Gly-Pro; b is Pro-Ala; c is Pro-Hyp; d is Pro-Glu.
FIG. 4 hydroxyproline-containing characteristic dipeptide collision energy optimization; a is Gly-Hyp; b is Hyp-Ala; c is Hyp-Ser; d is Pro-Hyp; e is Hyp-Hyp.
FIG. 5 is a total ion flow diagram of a typical collagen peptide sample in parent ion scan mode; a is a total ion flow diagram in parent ion scan mode with [ C 4H8N]+ (m/z 70.1) as a characteristic fragment ion; b is the total ion flow diagram in parent ion scan mode with [ C 4H8NO]+ (m/z 86.1) as the characteristic fragment ion.
FIG. 6 is a 8-feature dipeptide formula; wherein a is Gly-Hyp; b is Hyp-Ser; c is Hyp-Ala; d is Hyp-Hyp; e is Pro-Hyp; f is Pro-Ala; g is Gly-Pro; h is Pro-Glu.
Figure 7 is a dipeptide analysis of the type of proline and hydroxyproline contained in a typical commercial collagen peptide product.
Fig. 8 shows the content of characteristic dipeptides with proline type in the commercial products of 25 collagen peptides in parent ion scan mode.
Fig. 9 shows the characteristic dipeptides content of hydroxyproline-containing type in 25 commercial collagen peptides in parent ion scan mode.
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, thermo Scientific, usa); 8 characteristic dipeptide standard substances (the purity is equal to or more than 95 percent, shanghai Chu peptide biotechnology Co., ltd.); 25 commercial collagen peptide products (each of the major vendors).
Glycine-hydroxyproline with molecular formula and molecular weight of C 7H12N2O4/188.0797 and structural formula a shown in figure 6;
hydroxyproline-serine has a molecular formula and a molecular weight of C 8H14N2O5/218.0903, and a structural formula of b in figure 6;
hydroxyproline-alanine has molecular formula and molecular weight of C 8H14N2O4/202.0954, and structural formula of C in figure 6;
Hydroxyproline-hydroxyproline with molecular formula and molecular weight of C 10H16N2O5/244.1059 and structural formula d shown in figure 6;
Proline-hydroxyproline with molecular formula and molecular weight of C 10H16N2O4/228.111 and structural formula of e shown in figure 6;
proline-alanine with molecular formula and molecular weight of C 8H14N2O3/186.1004 and structural formula f shown in figure 6; glycine-proline with molecular formula and molecular weight of C 7H12N2O3/172.0848 and structural formula g shown in figure 6;
Proline-glutamic acid with molecular formula and molecular weight of C 10H16N2O5/244.1059 and structural formula of h shown in figure 6;
3. preparing a standard working solution:
Preparing a stock solution: accurately weighing the characteristic dipeptide standard substances 20mg respectively, placing the characteristic dipeptide standard substances in 20mL volumetric flasks respectively, dissolving the characteristic dipeptide standard substances with ultrapure water, fixing the volume to the scale, shaking the solution uniformly to prepare 1.0 mg/mL standard stock solution, keeping the stock solution in a sealed state in a dark place below-18 ℃ environment, and keeping the stock solution for 3 months.
Preparing a mixed working solution: 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 8 series of mixed standard solutions with the concentration of 1.0 ng/mL, 5.0 ng/mL, 10.0 ng/mL, 20.0 ng/mL, 50.0 ng/mL, 100.0 ng/mL, 200 ng/mL and 500.0 ng/mL of standard substances, wherein the solutions need to be prepared in situ.
Example 1
The embodiment provides a method for detecting the content of characteristic dipeptide containing proline or hydroxyproline 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 ×2.1 mm, 5 μm) or other chromatographic columns 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: 0.35 mL/min;
Mobile phase gradient elution procedure: 0~5 min 0%B,5~10.0 min linearly rises from 0% b to 20%B,10.0~15.0 min from 20% b to 40%B,15.0~16.0 min from 40% b to 80% b, holds 2.0 min, and then returns to the initial mobile phase equilibrium of 4.0 min ready 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: 4.0 A kV;
Desolventizing gas temperature: 325 deg.c;
drying gas flow rate: 10 L/min nitrogen;
Taper hole voltage: 100V;
Atomization pressure: 40 Nitrogen at psig;
collision energy: the dipeptides containing the characteristics of proline or hydroxyproline are 10V-35V;
Parent ion scan (Precursor Scan) mode is adopted, parent ion scan range: the dipeptide containing the proline type characteristic is selected from 170-282 m/z, and the dipeptide containing the hydroxyproline type characteristic is selected from 186-298 m/z. To ensure Mass accuracy, leucine enkephalin (Mass concentration 50 ng/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 dipeptide containing the proline type characteristic is selected from [ C 4H8N]+ (m/z 70.1) ] and the dipeptide containing the hydroxyproline type characteristic is selected from [ C 4H8NO]+ (m/z 86.1); wherein, regarding the selection of characteristic fragment ions: in order to determine characteristic sub-ions, a secondary mass spectrum scanning mode is adopted to determine the characteristic sub-ions, and 70.1 fragment ion peaks appear in the proline-containing type characteristic dipeptide standard substance, and the ion intensity is the strongest; the standard dipeptide with hydroxyproline type characteristic shows a fragment ion peak of 86.1, and the ionic strength is the strongest.
2. Selection of characteristic ion fragments in characteristic dipeptidomimetic ion mode containing proline and hydroxyproline types
Accurately preparing eight standard solutions (Gly-Pro, pro-Ala, pro-Hyp, pro-Glu, gly-Hyp, hyp-Ala, hyp-Ser, pro-Hyp and Hyp-Hyp) containing proline and hydroxyproline characteristic dipeptide with the concentration of 100ng/mL respectively, filtering with a 0.22 μm nylon filter membrane, transferring the filtrate into a sample injection vial, and performing mass spectrometry in a full scanning mode and a sub-ion scanning mode;
3. optimizing collision energy of dipeptides with proline and hydroxyproline type characteristics
Eight standard solutions (Gly-Pro, pro-Ala, pro-Hyp, pro-Glu, gly-Hyp, hyp-Ala, hyp-Ser, pro-Hyp and Hyp-Hyp) containing proline and hydroxyproline characteristic dipeptide with the concentration of 100 ng/mL are accurately prepared respectively, and under the conditions that the fragmentation voltage is 100V and the sample injection amount is consistent, characteristic ion fragments are arranged in a parent ion scanning mode, and the abundance of the characteristic ion fragments when the collision energy is 10V, 15V, 20V, 25V, 30V and 35V is respectively examined.
4. Pretreatment of commercial collagen peptide samples
Respectively accurately weighing 25 collagen peptide products on the market 10.00 mg, accurately adding 10 mL ultrapure water for constant volume, vortex oscillating, fully dissolving a sample solution prepared into 1 mg/mL, diluting the sample concentration to 100 ug/mL, filtering by using a 0.22 mu m nylon filter membrane, and transferring the filtrate into a sample injection vial for ultra-high performance liquid-tandem mass spectrometry detection;
5. Experimental method
The standard solution of characteristic dipeptide with different concentrations is used as a recovery indicator, and the low, medium and high levels of the characteristic dipeptide with proline and hydroxyproline types are respectively 10 ng/mL, 20 ng/mL and 40 ng/mL, and 5 parallel samples are prepared for each addition concentration.
6. Calculation of relative response factor of two characteristic dipeptide 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.
（1）
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.
7. 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) Parent ion scan mode feature fragment ion selection
The experiment adopts ESI ion source, the peak time and the accurate mass number of the parent ion of various standard substances are determined through mass spectrum full scanning mode, then the fragment sub-ions of various standard substances are determined through sub-ion scanning mode analysis, and the fragment ion with highest sensitivity and stable response of each substance is selected as the characteristic ion fragment in the parent ion scanning mode. By analyzing ion fragments generated by various standard substances, due to different front and rear positions of proline, determining that characteristic dipeptide containing proline is cracked according to a path shown as a in fig. 1 and b in fig. 1 in a mass spectrum, wherein the first mode of proline is subjected to the cracking of hydroxyl groups and carbon monoxide groups in sequence when the proline is in the front, and finally characteristic fragments [ C 4H8N]+ (m/z 70.1) are formed, and the second mode of proline is subjected to the direct cracking through peptide bonds when the proline is in the rear, and then decarboxylation is performed, and finally characteristic fragments [ C 4H8N]+ (m/z 70.1) are formed, so that the two modes are different in process, but the formed characteristic fragment ions are the same; a in fig. 2 and b in fig. 2 show the hydroxyproline-containing characteristic dipeptide fragmentation pathway, which proceeds similarly to the proline-containing characteristic dipeptide fragmentation pathway, resulting in the formation of stable [ C 4H8NO]+ (m/z 86.1) fragment ions.
(2) Selection of the optimal collision energy for dipeptides featuring proline and hydroxyproline types
The collision energy is an important factor influencing the measurement result, and too low energy can lead to insufficient obvious parent ion fragmentation, so that the generated characteristic fragments are too few and low in abundance, and the quantitative measurement of the target substance is not facilitated; if the energy is too high, fragments which are easy to generate are too much and too small, and the quantitative and qualitative effects are not facilitated. Therefore, the proper collision energy is selected to generate the characteristic ion fragment signal with higher intensity, and the interference of other fragment ions is avoided, so that the accuracy of quantitative analysis is improved. The result is shown in fig. 3, and as can be seen from fig. 3, the response abundance of the parent ion shows a trend of increasing and then decreasing under the collision energy of 10V-35V of the 4 characteristic dipeptide standard solutions containing proline, and when the collision energy is 30V, the response abundance is highest, the interference of other fragment ions is avoided, the chromatographic peak is sharp, the separation effect is good, and the method can be applied to the subsequent quantitative analysis and measurement. The collision energy optimization results of the 5 characteristic dipeptide standards containing hydroxyproline are shown in fig. 4, in the collision range, the response abundance of parent ions shows a trend of rising first and then decreasing, and the response abundance is highest when the collision energy is 25V, so that the optimal collision energy of the characteristic dipeptide containing proline is 30V, and the optimal collision energy of the characteristic dipeptide containing hydroxyproline is 25V.
(3) Linear range, limit of detection and limit of quantification of analytical methods
In order to avoid the influence of matrix effect, the method adopts matrix matching standard solution to establish a standard curve so as to eliminate or weaken the matrix effect. The mixed standard working solution prepared above is measured according to the conditions of the embodiment, the concentration is measured from low to high, an external standard method is adopted to prepare a matrix matching calibration curve, the concentration (ng/mL) of a standard substance is taken as an abscissa, the chromatographic peak area of the standard substance is taken as an ordinate, a linear regression equation is constructed, and a standard correlation curve is drawn. The content of each type of characteristic dipeptide in a commercially available sample was calculated from the calibration curve. Typically, a concentration corresponding to a signal-to-noise ratio of 3 times is used as the detection limit, and a concentration corresponding to a signal-to-noise ratio of 10 times is used as the quantification limit. The linear equation, correlation coefficient, linear range and detection limit of the dipeptides with proline and hydroxyproline type characteristics in this example are shown in table 1:
TABLE 1 Linear equation, correlation coefficient, linear range and detection limit for dipeptides characterized by proline and hydroxyproline types
The result shows that the mass concentration of the two types of characteristic dipeptide is in a linear range of 0.01-200 mug/mL, a good linear relation is shown, the correlation coefficient of the analytes is more than 0.990, and the characteristic dipeptide containing proline and hydroxyproline in a commercial sample can be rapidly, conveniently, stably and accurately quantitatively measured. The detection limit of the dipeptide containing the proline type characteristic is 3 mug/kg, and the quantitative limit is 10 mug/mL; the detection limit of the dipeptide containing hydroxyproline type characteristics is 1 mug/kg, and the quantitative limit is 5 mug/kg, which all meet the quantitative requirement.
(4) Analytical methods in-day precision and daytime precision and stability
In this example, the precision and stability of two types of characteristic dipeptides were also examined: precision is the proximity between a series of measurements obtained from multiple samples of the same sample, stability 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 solution containing proline and hydroxyproline type characteristic dipeptide is prepared into low concentration and high concentration for daily precision experiments, the same sample is measured three times in one day to obtain daily precision, the same sample is measured 1 time in 1, 3 and 5 days to obtain daily precision, the measurement is carried out according to the method conditions of the embodiment, and the precision of each added level is shown in table 2 for 5 parallel samples.
TABLE 2 within-day precision and daytime precision and accuracy of dipeptides characterized by the proline and hydroxyproline types
The peak areas of the low-concentration and high-concentration quality control samples are analyzed, the quality control samples are quantitatively combined with an external standard method matrix matching standard curve, and the precision and accuracy results of each quality control sample are obtained through calculation, wherein the accuracy between the average value and the theoretical value of the acceptance standard is 85.0% -115.0%, and the relative standard deviation is less than or equal to 15%. The results are shown in Table 2, and the results show that the daily precision of the dipeptide containing the proline type characteristic is between 96.86% and 102.02%, the daily precision is between 96.39% and 98.99%, and the Relative Standard Deviation (RSD) is less than 10%. The daily precision of the characteristic dipeptide containing the hydroxyproline type is 93.60-101.92%, the daily precision is 92.30-96.87%, and the Relative Standard Deviation (RSD) is less than 10%, so that the characteristic dipeptide meets the standard requirements. The experimental result shows that the detection method has high precision, strong stability and reliable result, and is suitable for the detection of two types of characteristic dipeptide.
(5) Method recovery rate results analysis
Collagen peptide samples with characteristic dipeptide content lower than detection limit are taken as blank matrixes, a proper amount of standard working solution containing proline and hydroxyproline type characteristic dipeptide is taken, 3 concentrations of low concentration, medium concentration and high concentration are prepared by using blank matrix sample solutions, sample adding recovery experiments are carried out, measurement is carried out according to the method of the embodiment, 5 parallel samples are measured at each adding level, and sample adding recovery (recovery% = (S Actual measurement -S Original sample ）/S Scalar addition is 100%) and relative standard deviation (RSD%) are calculated, wherein the recovery is shown in table 3.
TABLE 3 recovery and relative standard deviation of dipeptides characterized by the proline and hydroxyproline types
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). The specific results are shown in table 3, and the results show that the recovery rate of the dipeptide containing the proline type characteristic under the conditions of low, medium and high standard addition concentrations ranges from 91.44% to 101.99%, and the relative standard deviation RSD (n=5) ranges from 0.61% to 2.98%. The recovery rate of the dipeptides with hydroxyproline type characteristics ranges from 88.49% to 100.87%, the Relative Standard Deviation (RSD) ranges from 4.00% to 6.23%, and the detection method meets the standard requirements.
(6) Relative response factor calculation
A large amount of characteristic dipeptides of the proline and hydroxyproline type cannot be obtained as a standard, and therefore, the present invention quantifies the characteristic dipeptide content of this type by using one standard. The use of ƒ values generated from chromatographic peak areas of standard and analyte improves 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 dipeptides were calculated according to equation (1). The results of the specific calculation of the relative response factor are shown in tables 4 and 5.
TABLE 4 relative response factor of dipeptides with proline type characteristics
Table 5 relative response factor of dipeptides with hydroxyproline type characteristics
The results are shown in tables 4 and 5, and show that Pro-Glu and Gly-Hyp are the first choice of two types of characteristic dipeptide, the relative response factors of the two standards are in the middle position in 8 standards, meanwhile, the change corresponding to each analyte is smaller, and the combination of the analytes is closest to 1, so that the quantitative error of the rest analytes can be greatly reduced.
Example 2
The overall performance of the 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 commercial samples from commercial products for characteristic dipeptide analysis, and the semi-quantitative analysis of the total content of the characteristic dipeptide containing proline and hydroxyproline is realized by calculating by using the matrix matching calibration curve of two types of single standard substances in the method.
(1) Qualitative analysis of characteristic dipeptides in commercially available collagen peptide products
The commercial 25 collagen peptide products are used as test objects, and through a parent ion scanning mode, [ C 4H8N]+ (m/z 70.1) and [ C 4H8NO]+ (m/z 86.1) are respectively selected as characteristic fragment ions, and the parent ion scanning ranges are respectively 170-282 m/z and 186-298 m/z, so that qualitative analysis of collagen characteristic dipeptide is carried out. As shown in fig. 5, which is a total ion flow diagram of a typical collagen peptide sample in a parent ion scanning mode, respectively selecting [ C 4H8N]+ and [ C 4H8NO]+ ] as characteristic fragment ions, and analyzing a in fig. 5 and b in fig. 5, and finding that 17 dipeptides containing proline exist in a parent ion scanning product by using 70.1 as characteristic ion fragments; b 17 hydroxyproline-containing dipeptides were present in the parent ion scan product with 86.1 as the characteristic ion fragment. The results are shown in fig. 7, and the results show that the characteristic dipeptide in 25 commercial collagen peptide products is basically identical with other amino acids except for proline or hydroxyproline, and the qualitative analysis of the method is accurate, so that the subsequent quantification of the two types of characteristic dipeptide is facilitated.
(2) Calculation of total content of dipeptides characterized by proline and hydroxyproline types in commercial collagen peptide
In this example, according to the detection method provided in example 1, 25 commercial collagen peptides are respectively prepared accurately to have a concentration of 100 ug/mL, and are analyzed by a liquid chromatography mass spectrometry system according to the step method provided in example 1, and the total content of the characteristic dipeptide containing proline and hydroxyproline is calculated by using [ C 4H8N]+ and [ C 4H8NO]+ ] as characteristic fragment ions, and using the parent ion scanning ranges of 170-282 m/z and 186-298 m/z, respectively, and using typical peptide segments Pro-Glu and Gly-Hyp as standard substances. Specific results of qualitative and quantitative analysis of dipeptides characterized by the presence of proline and hydroxyproline in 25 commercial collagen peptide products are shown in fig. 8 and 9; the results show that the content of the characteristic dipeptide containing proline type in the 25 collagen peptide products on the market is in the range of 0.05 mg/g-70.77 mg/g, the Pro-Lys is far higher than that of other 16 dipeptides, about 70.77 mg/g and Pro-Arg times, the highest content is 16.83 mg/g, and the Pro-His content is the lowest content, about 0.45 mg/g. The commercial 25 collagen peptide products contain hydroxyproline type characteristic dipeptides in the content range of 0.007 mg/g-11.18 mg/g, the Hyp-Lys content is far higher than other 16 dipeptides, the highest content is 11.18 mg/g, the highest content is 7.6 mg/g after Hyp-Ala, and the Hyp-His content is overall lower, about 0.17 mg/g.
The practical applicability of the liquid chromatography-mass spectrometry analysis method for detecting the content of the dipeptide containing the proline and the hydroxyproline type characteristics based on the tandem mass spectrum parent ion scanning mode is further verified through the detection of the actual collagen peptide sample. 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 dipeptides containing proline and hydroxyproline type characteristics in collagen peptide 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 10V-35V, characteristic fragment ions [ C 4H8N]+ (m/z 70.1) and [ C 4H8NO]+ (m/z 86.1) ] are adopted, parent ion scanning ranges are 170-282 m/z and 186-298 m/z, typical peptide segments Pro-Glu and Gly-Hyp are used as standard substances, and semi-quantitative analysis of total content of characteristic dipeptide containing proline and hydroxyproline 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 the characteristic dipeptide, greatly improves the detection flux and reduces the detection cost. Through methodological verification, the method has better precision and recovery rate, the total content of the characteristic dipeptide in the collagen peptide sample can be measured in actual sample measurement, 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 or some specific characteristic peptide segments, and the index is relatively more stable in 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 (1)
1. The detection method is characterized by detecting the total content of two types of characteristic dipeptides containing proline or hydroxyproline in the collagen peptide based on a tandem mass spectrometry parent ion scanning mode, and specifically comprises a liquid chromatography tandem mass spectrometry analysis method; the liquid chromatography conditions were:
Chromatographic column: ATLANTIS T3C 18 chromatography column;
Column temperature: 40 ℃;
mobile phase: the phase A is formic acid aqueous solution with the volume ratio of 0.1 percent, and the phase B is acetonitrile aqueous solution with the volume ratio of 95 percent and containing 0.1 percent of 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 15.0-16.0 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;
The mass spectrum conditions are as follows:
ion source: an electrospray ion source;
Spray voltage: 4.0 A kV;
drying gas temperature: 300 ℃;
Spray pressure: 40 A psi;
Drying gas flow rate: 10 L/min;
Scanning mode: scanning parent ions;
collision energy: the dipeptides containing proline or hydroxyproline type characteristics are 10V-35V;
Characteristic fragment ions: the dipeptide with proline type characteristics is C 4H8N]+ with m/z of 70.1, and the dipeptide with hydroxyproline type characteristics is C 4H8NO]+ with m/z of 86.1;
parent ion scan range: the dipeptide containing the proline type characteristic is selected from 170-282 m/z, and the dipeptide containing the hydroxyproline type characteristic is selected from 186-298 m/z;
Pro-Glu is selected as a standard substance, and Gly-Hyp is selected as a standard substance.
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| US20110166365A1 (en) * | 2008-09-30 | 2011-07-07 | Nitta Gelatin Inc. | Collagen peptide, dipeptide and malady inhibitor |
| CN115804456A (en) * | 2021-09-13 | 2023-03-17 | 株式会社爱茉莉太平洋 | Use of gluconolactone and liquid composition containing collagen peptide |
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| US20110166365A1 (en) * | 2008-09-30 | 2011-07-07 | Nitta Gelatin Inc. | Collagen peptide, dipeptide and malady inhibitor |
| CN102177173A (en) * | 2008-09-30 | 2011-09-07 | 新田明胶株式会社 | Collagen peptide, dipeptide, and agent for prevention of disease |
| CN115804456A (en) * | 2021-09-13 | 2023-03-17 | 株式会社爱茉莉太平洋 | Use of gluconolactone and liquid composition containing collagen peptide |
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