Quantitative detection method of porcine I-type collagen

Abstract

The invention relates to the technical field of analysis and detection, in particular to a quantitative detection method of porcine I-type collagen, which comprises the following steps: S1, determining the macromolecular purity of porcine I-type collagen by volume exclusion chromatography; s2, measuring the contents of moisture, ash, anions and volatile organic components in the pig type I collagen; s3, according to the data obtained in S1 and S2, obtaining the content of the pig type I collagen by adopting a mass balance method; and S4, correcting the pig type I collagen content measured by the mass balance method by adopting an isotope dilution mass spectrometry. According to the method, the volume exclusion chromatography-mass balance method is combined with the acid hydrolysis isotope dilution mass spectrometry to accurately measure the content of the porcine I-type collagen, so that the content measurement precision is improved.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a quantitative detection method for porcine type I collagen. Background Art

[0002] Porcine type I collagen is a natural macromolecular protein compound with a molecular weight of 360KDa to 400KDa. Due to its unique α1, α2 tripeptide chain helical structure, the spatial configuration is complex. Porcine type I collagen is widely used in various fields such as medical care, beauty and skin care, food additives and packaging materials. In all these related application fields, the accurate determination of its content is the basis and important step of application. Therefore, it is urgent to establish a detection technology for accurately determining the content of porcine type I collagen.

[0003] However, due to the special conformation of porcine type I collagen, existing detection methods have problems such as large deviation in measurement results and low accuracy. Summary of the invention

[0004] In order to solve the defects in the prior art, the present invention provides a quantitative detection method for porcine type I collagen. The present invention uses size exclusion chromatography-mass balance method combined with acid hydrolysis isotope dilution mass spectrometry to accurately determine the content of porcine type I collagen and improve the content determination accuracy.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a quantitative detection method for porcine type I collagen, comprising the following steps:

[0007] S1. Determination of the macromolecular purity of porcine type I collagen by size exclusion chromatography;

[0008] S2. Determine the contents of water, ash, anions and volatile organic components in porcine type I collagen;

[0009] S3, based on the data obtained in S1 and S2, the content of porcine type I collagen was obtained by mass balance method;

[0010] S4. Use isotope dilution mass spectrometry to calibrate the porcine type I collagen content obtained by the mass balance method.

[0011] Preferably, the chromatographic conditions of S1 are: mobile phase: 150 mmol / L sodium phosphate buffer and pH=6.8 buffer salt system; detection wavelength: 210 nm; flow rate: 0.4 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase gradient: isocratic elution, 60 min.

[0012] More preferably, the buffer salt system with pH=6.8 is prepared as follows: weigh 24.639 g of anhydrous sodium phosphate, add water to 1000 mL, adjust the pH value to 6.8 with phosphoric acid, and filter with a 0.45 μm filter membrane before use.

[0013] Preferably, the content of anions in S2 is the sum of anions such as fluoride, acetate, chloride, nitrite, bromide, nitrate, sulfate, and phosphate in collagen.

[0014] Preferably, the volatile organic components in S2 include methanol, acetonitrile, dichloromethane, n-hexane, and ethyl hexanoate.

[0015] Preferably, the calculation formula for the content of porcine type I collagen measured by the S3 mass balance method is:

[0016] w=P×(1-ABCD)

[0017] Wherein, W is the content of porcine type I collagen, P is the purity of type I collagen determined by high performance liquid chromatography size exclusion method, A is the moisture content, B is the ash content, C is the volatile component content, and D is the anion component content.

[0018] Preferably, S4 specifically comprises: determining the content of porcine type I collagen by isotope dilution mass spectrometry, and performing statistical analysis on the obtained content data and the porcine type I collagen content data obtained by the mass balance method.

[0019] Preferably, in the process of determining the content of porcine type I collagen by isotope dilution mass spectrometry, the isotope marker is L- 13 C6 phenylalanine, L- 13 C6 Leucine and L- 13 C6 isoleucine.

[0020] Preferably, the process of determining the content of porcine type I collagen by isotope dilution mass spectrometry includes: weighing the mass ratio of amino acids and isotope-labeled amino acids by a balance, and determining the peak area ratio of amino acids and isotope-labeled amino acids by high performance liquid chromatography-tandem mass spectrometry to calculate the content of porcine type I collagen.

[0021] More preferably, the HPLC conditions include: mobile phase A is 10% 100 mM ammonium formate aqueous solution + 90% water (adjusted with formic acid to pH = 4.5), mobile phase B is 10% 100 mM ammonium formate aqueous solution + 90% acetonitrile (adjusted with formic acid to pH = 4.5); column temperature: 30°C; flow rate: 0.5 ml / min.

[0022] More preferably, the mass spectrometry conditions include: using an electrospray ion source, positive ion mode, nebulizer gas temperature: 300°C, nebulizer gas flow rate: 5L / min, sheath gas temperature: 300°C, sheath gas flow rate: 11L / min, nebulizer gas pressure: 45psi, capillary outlet voltage: 4500V.

[0023] Preferably, the formula for determining the content of porcine type I collagen by isotope dilution mass spectrometry is:

[0024]

[0025] Wherein, C—is the mass fraction of porcine type I collagen (unit: g / g);

[0026] M—is the mass of the porcine type I collagen sample used for hydrolysis (in g);

[0027] R1—is the peak area ratio of the amino acid in the high-standard solution to the labeled amino acid;

[0028] R2—is the peak area ratio of the amino acid in the low-standard solution to the labeled amino acid;

[0029] P—is the purity of amino acid (unit: g / g);

[0030] m 标 — is the mass of the labeled amino acid in the hydrolyzed sample (unit: g);

[0031] R 样 — is the peak area ratio of the amino acid after sample hydrolysis to the labeled amino acid;

[0032] I1—is the mass ratio of amino acid to labeled amino acid in high standard solution;

[0033] I2—is the mass ratio of amino acid in low-standard solution to labeled amino acid;

[0034] P H — is the hydrolysis efficiency;

[0035] R AA — is the amino acid purity proportional factor (unit: g / g);

[0036] M col — is the molecular weight of collagen;

[0037] N AA — is the number of amino acids in collagen;

[0038] M AA — is the molecular weight of the amino acid.

[0039] Preferably, the correction method in S4 is selected from any one or more of the Shapiro-Wilk method, the Dixon method, the Grubbs method, the F test, the independent sample t test, and the combined normality test.

[0040] The beneficial effects of the present invention are:

[0041] The present invention adopts size exclusion chromatography-mass balance method combined with acid hydrolysis isotope dilution mass spectrometry, firstly performs a preliminary determination of the content of porcine type I collagen by size exclusion chromatography-mass balance method, then corrects the measured content by acid hydrolysis isotope dilution mass spectrometry, and obtains the final content of porcine type I collagen. This method can greatly improve the determination accuracy and precision of the content of such macromolecular protein compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The chromatogram of the determination of the macromolecular purity of porcine type I collagen by size exclusion chromatography in Example 2;

[0043] Figure 2 The total ion current diagram of phenylalanine, leucine, isoleucine and marker in Example 2;

[0044] Figure 3 This is the QQ diagram in the combined normality test in Example 2. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0046] Example 1

[0047] This embodiment provides a quantitative detection method for porcine type I collagen, the steps are as follows:

[0048] (1) Determination of the purity (organic content) of porcine type I collagen macromolecules by size exclusion chromatography

[0049] The determination method is as follows: Take an appropriate amount of this product, dissolve it with 0.01mol / L hydrochloric acid solution and dilute it to make a solution containing 1.0mg per 1mL as the test solution. The dissolution process is to refrigerate and stand at 4℃ for more than 12h, wait for the collagen to dissolve naturally, and do not vortex or ultrasonicate the protein solution during the dissolution process to avoid destroying the collagen peptide chain structure and causing purity determination errors. Take the test sample and inject it into the liquid chromatograph, and calculate it according to the peak area normalization method.

[0050] The chromatographic conditions were as follows:

[0051] 1) Preparation of mobile phase: 150mmol / L sodium phosphate buffer, pH=6.8 buffer salt system is used as mobile phase; wherein the pH=6.8 buffer salt system is prepared by weighing 24.639g of anhydrous sodium phosphate, adding water to 1000mL, adjusting the pH value to 6.8 with phosphoric acid, and filtering with a 0.45μm filter membrane before use.

[0052] 2) Chromatographic column: Agilent AdvanceBio SEC 300A 2.7 μm 7.8×300 mm.

[0053] 3) Detection wavelength: 210nm.

[0054] 4) Flow rate: 0.4 mL / min.

[0055] 5) Column temperature: 30°C.

[0056] 6) Injection volume: 2 μL.

[0057] 7) Mobile phase gradient: isocratic elution, 60 min.

[0058] (2) Determination of moisture content of porcine type I collagen

[0059] Take 200 mg of the porcine type I collagen sample and spread it flat in a flat weighing bottle that has been dried to constant weight. Since collagen is relatively loose, the sample height should not exceed 10 mm and be accurately weighed; open the bottle cap and dry it in an oven at 100-105°C for 5 hours, cover the bottle cap, move it to a dryer, cool it for 30 minutes, and accurately weigh it; then dry it at the above temperature for 1 hour, cool it for 30 minutes, and weigh it until the difference between two consecutive weighings does not exceed 5 mg. Calculate the water content (%) in the sample based on the lost weight.

[0060] (3) Determination of ash content of porcine type I collagen

[0061] Accurately weigh 1000 mg of porcine type I collagen test sample, place it in a crucible that has been cleaned in advance and burned in a muffle furnace until constant weight, accurately weigh it, burn it at 700-800℃ until it is completely incinerated, move it to a dryer and cool it to room temperature, then accurately weigh it; then burn it at 700-800℃ until constant weight, repeat the above steps, and calculate the ash content based on the residual mass after burning.

[0062] (4) Determination of anions in porcine type Ⅰ collagen

[0063] Accurately weigh 10 mg of porcine type I collagen sample, dilute with 10 mL of deionized water, vortex and sonicate for 30 minutes until the collagen is completely dissolved in water, and filter the fluoride ion, acetate, chloride ion, nitrite, bromide ion, nitrate, sulfate, and phosphate in the collagen to be tested with a 0.45 μm filter membrane. The sum of the measured anions is the total amount of anions in porcine type I collagen. The determination conditions are as follows:

[0064] ICS-2100 ion chromatograph (DIONEX, USA);

[0065] Chromatographic column: Dionex IonPacTMAS11-HC (4×250 mm);

[0066] Guard column: Dionex IonPacTMAG11-HC (4×50 mm);

[0067] Column temperature: 30°C;

[0068] Injection volume: 50 μL;

[0069] Flow rate: 1 mL / min;

[0070] Current: 75mA;

[0071] Gradient elution: 0-8min: 15mM KOH→9-25min: 25mM KOH→26-35min: 15mM KOH.

[0072] (5) Determination of volatile organic components in porcine type Ⅰ collagen

[0073] The volatile organic components that may exist in collagen are determined by headspace sampling-gas chromatography-mass spectrometry, which are mainly residual solvents in the collagen synthesis process, such as methanol, acetonitrile, dichloromethane, n-hexane, ethyl hexanoate, etc. The total amount of the measured compounds is the volatile organic component content in porcine type I collagen. The determination steps are as follows:

[0074] 1) Headspace experimental conditions:

[0075] Sample loop volume: 1mL;

[0076] Equilibrium temperature: 70℃;

[0077] Quantitative loop temperature: 90°C;

[0078] Transmission line temperature: 100°C;

[0079] Equilibration time: 30 min;

[0080] Headspace bottle pressure 138kPa;

[0081] Pressurization time: 0.20min;

[0082] Sample ring equilibration time: 0.05min;

[0083] Injection time: 1.0min.

[0084] 2) Gas chromatography-mass spectrometry experimental conditions:

[0085] Chromatographic column: DB-624 60m×320μm×1.8μm;

[0086] Carrier gas: helium, purity ≥99.999%, flow rate: 1.0mL / min;

[0087] Inlet temperature: 230°C;

[0088] Injection mode: split injection, split ratio: 5:1;

[0089] Heating program: 40℃ for 2 min, then increase the temperature to 150℃ at a rate of 5℃ / min, then increase the temperature to 230℃ at a rate of 20℃ / min and hold for 10 min;

[0090] Ion source temperature: 230°C;

[0091] GC-MS interface temperature: 230°C;

[0092] Selected ion monitoring: Select one quantitative ion and two qualitative ions for each compound. Since there are many volatile components to be tested, in order to avoid cross-interference, the volatile components to be tested are divided into three groups. All ions to be tested in each group are tested separately in the order of peak elution and in different time periods.

[0093] (6) The content of porcine type I collagen was measured by mass balance method

[0094] The content of the sample by the mass balance method can be defined as the value of the percentage of moisture, ash, volatile components and anions removed from the result of the main component determination. After the purity (organic matter) of collagen was determined by size exclusion chromatography, the moisture, ash, anions and volatile components of porcine type I collagen were determined respectively, and the content of porcine type I collagen was calculated by the following formula.

[0095] w=P×(1-ABCD)(1)

[0096] Wherein, W is the content of porcine type I collagen, P is the purity of type I collagen determined by high performance liquid chromatography size exclusion method, A is the moisture content, B is the ash content, C is the volatile component content, and D is the anion component content.

[0097] The porcine type Ⅰ collagen content was initially obtained by the mass balance method in 5 determination steps, and then calibrated by acid hydrolysis isotope dilution mass spectrometry.

[0098] (7) Acid hydrolysis isotope dilution mass spectrometry

[0099] Isotope dilution mass spectrometry (IDMS) is a method of obtaining the absolute amount of an element in a sample by accurate mass spectrometry measurement of isotope abundance and accurate weighing of the added diluent. The present invention hydrolyzes porcine type I collagen into three amino acids: phenylalanine, leucine and isoleucine, and adds their isotope labeling reagents: L- 13 C6 phenylalanine, L- 13 C6 leucine, L- 13 C6 isoleucine, the mass ratio of amino acids and isotope-labeled amino acids is measured by a balance, and the peak area ratio of amino acids and isotope-labeled amino acids is measured by high performance liquid chromatography-tandem mass spectrometry to calculate the final porcine type I collagen content. The experimental steps are as follows:

[0100] S1. Prepare 10 μg / g standard solutions of phenylalanine, leucine and isoleucine with 0.01 mol / L dilute hydrochloric acid solution. Similarly, prepare 10 μg / g standard solutions of phenylalanine, leucine and isoleucine with 0.01 mol / L dilute hydrochloric acid solution. μg / g L- 13 C6 phenylalanine, L- 13 C6 leucine, L- 13 C6 isoleucine isotope labeling solution;

[0101] S2. Weigh about 5 mg of porcine type I collagen sample using a balance with a minimum division of 0.01 mg, dissolve it in 100 mL of 0.01 mol / L hydrochloric acid, accurately weigh its mass, and prepare a 0.05 mg / g porcine type I collagen mother solution;

[0102] S3, take 100 μL collagen mother solution and add it into 5 mL ampoule bottle, calculate the theoretical amino acid mass of phenylalanine, leucine and isoleucine contained in 100 μL mother solution according to the theoretical amino acid sequence of porcine type I collagen, and then add the corresponding L- 13 C6 phenylalanine, L- 13 C6 leucine, L- 13 C6 isoleucine marker solution, ensuring the mass ratio of amino acid and isotope-labeled amino acid is 0.9:1 and 1.1:1;

[0103] S4. Place the ampoule in a vacuum freeze dryer and freeze dry for 1 hour. After all the water in the sample is removed, take it out, add 1500μL 8mol / L hydrochloric acid, pass nitrogen to deoxygenate, seal it, and hydrolyze it in a (150.0±0.5)℃ oven for 5 hours. After hydrolysis, blow dry with nitrogen, re-dissolve it with 1mL 0.1% formic acid water-acetonitrile mixed solution (50%-50%), filter it through a 0.22μm filter membrane, and then measure it on the machine;

[0104] S5. Chromatographic conditions: mobile phase A is 10% 100 mM ammonium formate aqueous solution + 90% water (adjusted with formic acid to pH = 4.5), mobile phase B is 10% 100 mM ammonium formate aqueous solution + 90% acetonitrile (adjusted with formic acid to pH = 4.5); chromatographic column: HILIC-Z, 2.7 μm, 2.1 mm×150 mm; column temperature: 30°C; flow rate: 0.5 ml / min, the mobile phase elution gradient is shown in Table 1.

[0105] Table 1. Mobile phase elution gradient

[0106] Time / min A％ B％ 0 0 100 10 15 85 11 40 60 15 40 60 15.1 0 100 25 0 100

[0107] S6. Mass spectrometry conditions: The mass spectrometer was operated with an electrospray ion source, positive ion mode, nebulizer gas (N2) temperature: 300°C, nebulizer gas flow rate: 5 L / min, sheath gas temperature: 300°C, sheath gas flow rate: 11 L / min, nebulizer gas pressure: 45 psi, capillary outlet voltage: 4500 V. The optimized mass spectrometry parameters in multiple reaction monitoring mode are shown in Table 2.

[0108] Table 2. Mass spectrometry parameters of each amino acid

[0109]

[0110]

[0111] S7. The calculation formula for determining the content of porcine type I collagen by acid hydrolysis isotope dilution mass spectrometry is as follows:

[0112]

[0113] C—is the mass fraction of porcine type I collagen (unit: g / g);

[0114] M—is the mass of the porcine type I collagen sample used for hydrolysis (in g);

[0115] R1—is the peak area ratio of the amino acid in the high-standard solution to the labeled amino acid;

[0116] R2—is the peak area ratio of the amino acid in the low-standard solution to the labeled amino acid;

[0117] P—is the purity of amino acid (unit: g / g);

[0118] m 标 — is the mass of the labeled amino acid in the hydrolyzed sample (unit: g);

[0119] R 样 — is the peak area ratio of the amino acid after sample hydrolysis to the labeled amino acid;

[0120] I1—is the mass ratio of amino acid to labeled amino acid in high standard solution;

[0121] I2—is the mass ratio of amino acid in low-standard solution to labeled amino acid;

[0122] P H — is the hydrolysis efficiency;

[0123] R AA — is the amino acid purity proportional factor (unit: g / g);

[0124] M col — is the molecular weight of collagen;

[0125] N AA — is the number of amino acids in collagen;

[0126] M AA — is the molecular weight of the amino acid.

[0127] (8) Statistical analysis of the data on the content of porcine type I collagen measured by the mass balance method and the isotope dilution mass spectrometry method. The values ​​obtained by the two methods were subjected to the following statistical analysis in turn to determine whether the data of the two methods were consistent.

[0128] Ⅰ. Use the Shapiro-Wilk (3≤n≤50) method to test the normal distribution of the fixed value data of mass balance method and isotope dilution mass spectrometry;

[0129] II. Perform outlier tests on the data of mass balance method and isotope dilution mass spectrometry using Dixon method and Grubbs method respectively to determine whether the two methods contain outliers;

[0130] III. The F test method was used to test the homogeneity of variance of the two groups of fixed value results of mass balance method and isotope dilution mass spectrometry to observe whether the variance between groups is significantly different from the variance within groups and whether they have the same precision;

[0131] IV. Use independent sample t test to test the consistency of data between the two value determination methods and observe whether there is significant difference in the data;

[0132] V. Combined normality test: After combining the data from the two determination methods, perform a normality test again to observe whether the data obey the normal distribution. If the data obey the normal distribution, it is considered that the data obtained by the mass balance method and the isotope dilution mass spectrometry are consistent and conform to the normal distribution, and can be used as the final content value of porcine type I collagen.

[0133] Example 2

[0134] In this example, a batch of porcine type I collagen product was taken and the porcine type I collagen content was determined according to the detection method described in Example 1, and the steps were as follows:

[0135] (1) Determination of the purity (organic content) of porcine type I collagen macromolecules by size exclusion chromatography

[0136] According to the determination method and experimental conditions of (1) in Example 1, 5 parallel samples were taken and each sample was analyzed 3 times to obtain the purity of porcine type I collagen. The results are shown in Table 3 and the chromatogram is shown in Figure 1 (The above picture is a typical chromatogram of the purity analysis of porcine type I collagen, and the below picture is a partial enlarged picture of the above picture).

[0137] Table 3. Purity of porcine type Ⅰ collagen (%)

[0138]

[0139]

[0140] (2) Determination of moisture content of porcine type I collagen

[0141] According to the determination method and experimental conditions of (2) in Example 1, the water content of porcine type I collagen was determined twice in parallel, as shown in Table 4. The average water content of the two times was 13.1%.

[0142] Table 4. Moisture content of pig type Ⅰ collagen purity standard material determined by drying method

[0143] Baking dish 1 Baking dish 2 Baking dish / mg 50553.8 49257.4 Sample (20mg) / bottle 256.2 260.6 Drying (105℃) 4h / mg 50777.5 49482.8 Re-drying (105℃) 2h / mg 50777.4 49482.6 Baking dish + sample / mg 50810.0 49518.0 Water mass / mg 32.6 35.2 Water content % 12.7 13.5

[0144] (3) Determination of ash content of porcine type I collagen

[0145] According to the determination method and experimental conditions of (3) in Example 1, the ash content of porcine type I collagen was determined twice in parallel. The results are shown in Table 5. The average ash content of the two times was 0.16%.

[0146] Table 5. Ash content of pig type Ⅰ collagen purity standard material determined by burning method

[0147] Crucible 1 Crucible 2 Crucible constant weight / mg 36388.1 37591.2 Drying sample mass / mg 1000.7 1000.6 Burn at 500℃ for 2h, burn at 750℃ for 4h 36389.6 37592.7 750℃ for another 2h 36389.8 37592.6 Ash mass / mg 1.7 1.4 Ash content / % 0.170 0.140

[0148] (4) Determination of anions in porcine type Ⅰ collagen

[0149] According to the determination method and experimental conditions of (4) in Example 1, four parallel determinations were performed to obtain the content of anions in pig type I collagen. The experimental results are shown in Table 6. The anion content in the sample was 0.091%.

[0150] Table 6. Anion content in samples (%)

[0151]

[0152] (5) Determination of volatile organic components in porcine type Ⅰ collagen

[0153] The measurement was carried out according to the measurement method and experimental conditions in Example 1 (5). The experimental results are shown in Table 7.

[0154] Table 7. Detection limits and test results of volatile components to be tested

[0155]

[0156]

[0157] (6) Analysis of the data of porcine type I collagen quality balance method

[0158] The test results of moisture content, ash content, anion content and residual solvent content obtained from the above tests are summarized as shown in Table 8.

[0159] Table 8. Quantitative results of each impurity component

[0160] Impurity ingredients content% Moisture 13.1 Ash 0.16 Anions 0.091 Volatile organic compounds Not detected

[0161] According to formula (1) in Example 1, the purity value of porcine type I collagen determined by HPLC-SIC was deducted from moisture, ash, anions and volatile organic residues to obtain the mass balance method value result, as shown in Table 9.

[0162] Table 9. Results of the mass balance of pig type Ⅰ collagen standard material (g / g)

[0163]

[0164] The porcine type Ⅰ collagen content initially obtained by the mass balance method was further calibrated by acid hydrolysis isotope dilution mass spectrometry.

[0165] (7) Acid hydrolysis isotope dilution mass spectrometry

[0166] Experimental conditions Based on the determination method and experimental conditions in Example 1 (7), 5 parallel samples were taken and each sample was analyzed 3 times as the determination results of isotope dilution mass spectrometry, as shown in Table 10. Figure 2 Total ion current of phenylalanine, leucine, isoleucine and markers.

[0167] Table 10. Isotope dilution mass spectrometry results (g / g)

[0168]

[0169]

[0170] (8) Statistical analysis of data from two methods

[0171] 1) The Shapiro-Wilk (3≤n≤50) method was used to test the normal distribution of the fixed value data of the mass balance method and isotope dilution mass spectrometry. The results are shown in Tables 11 and 12.

[0172] ①Mass balance method

[0173] Table 11. Normal distribution test of mass balance method

[0174]

[0175] ②Acid hydrolysis isotope dilution mass spectrometry

[0176] Table 12. Normal distribution test of acid hydrolysis isotope dilution mass spectrometry

[0177]

[0178] The skewness values ​​of mass balance method and isotope dilution mass spectrometry are both between -1 and 1, and the kurtosis values ​​are both between -3 and 3, both of which satisfy the normal distribution.

[0179] 2) Dixon and Grubbs outlier tests

[0180] The data of mass balance method and isotope dilution mass spectrometry were tested for outliers using Dixon method and Grubbs method, respectively, and the results are shown in Tables 13 and 14. The fixed value results of the two methods were tested by Grubbs and Dixon outlier test methods, and no outliers were found.

[0181] Table 13. Mass balance method outlier test

[0182]

[0183] Table 14. Isotope dilution mass spectrometry outlier test

[0184]

[0185] 3) Variance Homogeneity Test

[0186] The F test method was used to test the homogeneity of variance of the two groups of determination results of the mass balance method and the isotope dilution mass spectrometry method, as shown in Table 15. Observe whether there is a significant difference between the variance between the groups and the variance within the groups, and judge by F that the determination results of the two methods have homogeneity of variance and the same precision.

[0187] Table 15 Variance homogeneity test of two fixed value methods

[0188]

[0189] 4) Independent samples t-test

[0190] The consistency of the two valuation methods was tested using an independent sample t test, as shown in Table 16. Since the significant difference was 0.01 < 0.05, the original hypothesis of equal variance was not established, and the assumption of unequal variance was accepted, P = 0.233 > 0.05, and the result showed that there was no significant difference.

[0191] Table 16. Inter-group data consistency test

[0192]

[0193]

[0194] 5) Combined normality test

[0195] The data of the two value determination methods were combined for normality test, and the results are shown in Table 17. The skewness value is 0.523, between -1 and 1, and the kurtosis is 3.602, which is close to the kurtosis value of normal distribution 3. By checking the QQ graph, it is further confirmed that the expected standard is basically distributed above and below the straight line, which can be considered to obey the normal distribution. The QQ graph is shown in Figure 3 .

[0196] Table 17. Normal distribution test of two fixed value methods

[0197] N Minimum Maximum Mean Standard Deviation Skewness Kurtosis Statistics Statistics Statistics Statistics Statistics Statistics Standard error Statistics Standard error 30 0.8537 0.8724 0.862720 0.0032547 0.523 0.427 3.602 0.833

[0198] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A quantitative detection method for porcine type I collagen, characterized in that: The following steps are involved: S1. Determination of the macromolecular purity of porcine type I collagen by size exclusion chromatography; S2. Determine the contents of water, ash, anions and volatile organic components in porcine type I collagen; S3, based on the data obtained in S1 and S2, the content of porcine type I collagen was obtained by mass balance method; S4. Use isotope dilution mass spectrometry to calibrate the porcine type I collagen content measured by the mass balance method.

2. The quantitative detection method according to claim 1, characterized in that: The chromatographic conditions of S1 are as follows: mobile phase: 150 mmol / L sodium phosphate buffer and pH=6.8 buffer salt system; detection wavelength: 210 nm; flow rate: 0.4 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase gradient: isocratic elution, 60 min.

3. The quantitative detection method according to claim 1, characterized in that The volatile organic components in S2 include methanol, acetonitrile, dichloromethane, n-hexane, and ethyl hexanoate.

4. The quantitative detection method according to claim 1, characterized in that: The calculation formula for the content of porcine type I collagen measured by the S3 mass balance method is: Wherein, W is the content of porcine type I collagen, P is the purity of type I collagen determined by high performance liquid chromatography size exclusion method, A is the moisture content, B is the ash content, C is the volatile component content, and D is the anion component content.

5. The quantitative detection method according to claim 1, characterized in that: The S4 specifically includes: using isotope dilution mass spectrometry to determine the content of porcine type I collagen, and performing statistical analysis on the obtained content data and the porcine type I collagen content data obtained by the mass balance method.

6. The quantitative detection method according to claim 1 or 5, characterized in that: The process of determining the content of porcine type I collagen by isotope dilution mass spectrometry includes weighing the mass ratio of amino acids and isotope-labeled amino acids by a balance, and determining the peak area ratio of amino acids and isotope-labeled amino acids by high performance liquid chromatography-tandem mass spectrometry to calculate the content of porcine type I collagen.

7. The quantitative detection method according to claim 6, characterized in that: In the process of determining the content of porcine type I collagen by isotope dilution mass spectrometry, the isotope marker is L- 13 C6 phenylalanine, L- 13 C6 Leucine and L- 13 C6 isoleucine.

8. The quantitative detection method according to claim 6, characterized in that The HPLC conditions include: mobile phase A is 10% 100 mM ammonium formate aqueous solution + 90% water (adjusted with formic acid to pH=4.5), mobile phase B is 10% 100 mM ammonium formate aqueous solution + 90% acetonitrile (adjusted with formic acid to pH=4.5); column temperature: 30°C; flow rate: 0.5 ml / min.

9. The quantitative detection method according to claim 6, characterized in that: The mass spectrometry conditions include: using an electrospray ion source, positive ion mode, nebulizer gas temperature: 300°C, nebulizer gas flow rate: 5L / min, sheath gas temperature: 300°C, sheath gas flow rate: 11L / min, nebulizer gas pressure: 45psi, capillary outlet voltage: 4500V.

10. The quantitative detection method according to claim 6, characterized in that: The formula for determining the content of porcine type I collagen by isotope dilution mass spectrometry is: in, C — is the mass fraction of porcine type I collagen (unit: g / g); M — is the mass of the porcine type I collagen sample used for hydrolysis (in g); R 1—is the peak area ratio of the amino acid in the high-standard solution to the labeled amino acid; R 2—is the peak area ratio of the amino acid in the low-standard solution to the labeled amino acid; P — is the purity of amino acid (unit: g / g); m 标 — is the mass of the labeled amino acid in the hydrolyzed sample (unit: g); R 样 — is the peak area ratio of the amino acid after sample hydrolysis to the labeled amino acid; I 1—is the mass ratio of amino acid to labeled amino acid in high standard solution; I 2—is the mass ratio of amino acid in low-standard solution to labeled amino acid; P H — is the hydrolysis efficiency; R AA — is the amino acid purity proportional factor (unit: g / g); M col — is the molecular weight of collagen; N AA — is the number of amino acids in collagen; M AA — is the molecular weight of the amino acid.

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