Purity high-grade measuring method for purity standard substance of frog skin peptide

Abstract

The application discloses a purity high-grade measuring method of a standard substance of purity of frog skin peptide, and relates to the technical field of analytical chemistry and standard substance value determination. The application realizes accurate quantification of characteristic amino acids in frog skin peptide by means of isotope dilution-acid hydrolysis-liquid chromatography-tandem mass spectrometry, and then calculates the purity of frog skin peptide, so as to meet the metrology traceability requirement of the standard substance. The application provides high-grade metrology support for quality control, biological activity research and clinical detection of frog skin peptide related products, and significantly improves the accuracy and reliability of the value determination result of the purity of frog skin peptide.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry and standard reference determination technology, and more specifically, to a method for measuring the high purity level of a dermalin purity standard reference. Background Technology

[0002] Bombesin is a fourteen-peptide with important physiological activities, its amino acid sequence being: Glp-Gln-Arg-Leu-Asn-Gln-Trp-Ala-Val-His-Leu-Met-NH2. It is widely used in neuroendocrine research, tumor diagnosis, and the development of therapeutic targets. High-purity, high-accuracy bombesin standards are crucial for ensuring the accuracy and comparability of relevant bioanalytical, pharmacodynamic, and clinical test results.

[0003] Currently, the main methods for determining the purity of peptide standards include mass balance method, amino acid analysis method, and hydrolysis high performance liquid chromatography. However, conventional methods have the following shortcomings: (1) The mass balance method has stringent requirements for the determination of impurities (moisture, volatiles, inorganic impurities, etc.), resulting in a large cumulative uncertainty; (2) Conventional amino acid analysis method uses external standard method for quantification, which is easily affected by sample pretreatment and matrix effect, thus limiting accuracy.

[0004] Therefore, establishing a highly accurate, highly specific method for measuring the purity of dermalin that can be directly traced to SI units (moles) is of great significance for the preparation and determination of high-grade dermalin purity standard materials.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a high-level method for measuring the purity of a standard reference material for dermalin. By using isotope dilution-acid hydrolysis-liquid chromatography-tandem mass spectrometry, the method achieves accurate quantification of characteristic amino acids (valine and leucine) in dermalin, thereby calculating the purity of dermalin and meeting the metrological traceability requirements of the standard reference material.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for measuring the high purity level of a dermal filler purity standard, comprising the following steps: S1: Prepare a mixed internal standard solution of isotopically labeled valine and leucine, and a mixed standard calibration solution of valine and leucine; the mixed standard calibration solution includes a mixed internal standard solution mixed in a specified ratio and an unlabeled amino acid mixed standard solution; the unlabeled amino acid mixed standard solution includes valine and leucine without isotopic labeling. S2: Mix the aqueous solution of the test frog dermal extract with the mixed internal standard solution at a mass ratio of 0.9-1:0.9-1, add hydrochloric acid with a final concentration of 6-7.2 mM, deoxygenate, and hydrolyze at 130℃ for 24-48 h; S3: After the hydrolysate from step S2 is dried, it is reconstituted to obtain the hydrolysate sample to be tested; S4: The hydrolysate and the mixed standard calibration solution were loaded separately for analysis. The peak areas of leucine, valine, and their corresponding isotope labels in the hydrolysate were detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The peak areas of leucine, valine, and their corresponding isotope labels in the mixed standard calibration solution were also detected. The LC conditions were: C18 column, mobile phase of 0.2% formic acid aqueous solution and acetonitrile at a volume ratio of 98:2, isocratic elution, flow rate of 0.3 mL / min; and the mass spectrometry conditions were: leucine cone voltage 45-50 V, collision energy 10-15 V; isotope-labeled leucine cone voltage 45-50 V, collision energy 10-15 V; valine cone voltage 35-40 V, collision energy 10-15 V; and isotope-labeled valine cone voltage 35-40 V / collision energy 10-15 V. S5: The mass fractions of leucine and valine in the sample were calculated using the isotope dilution method, and the purity of frog skin extract was obtained.

[0008] The present invention has the following beneficial effects: This invention selects leucine (Leu) and valine (Val), which are acid-hydrolyzable in dermalin, as characteristic amino acids. Isotope-labeled forms of these two amino acids are used as internal standards. Under specific hydrolysis conditions with hydrochloric acid, dermalin is completely hydrolyzed into free amino acids. The absolute content of the two characteristic amino acids in the free amino acids is directly determined using isotope dilution mass spectrometry. Based on the molecular weight of dermalin and the stoichiometry of its amino acids, the purity value of dermalin is calculated. This method is independent of traditional chromatographic purity methods, and the measurement results can be directly traced to national amino acid standard materials, and further to the International System of Units (SI). This invention provides high-level metrological support for the quality control, bioactivity research, and clinical testing of dermalin-related products, significantly improving the accuracy and reliability of the determination results. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 Mass spectrum of the mixed standard calibration solution by LC-MS; Figure 2 LC-MS mass spectrum of amino acid samples hydrolyzed from frog dermal extract; Figure 3 The optimized concentration of hydrochloric acid for the purity standard of frog dermalin is shown in the figure. Figure 4 The optimal amount of hydrochloric acid used for hydrolyzing the purity standard substance of frog dermatin is shown in the diagram. Figure 5 An optimized diagram showing the hydrolysis temperature of the purity standard substance for frog dermalin; Figure 6 The diagram shows the optimized hydrolysis time for the purity standard of frog dermalin. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0012] Definition of Bombesin purity standard material: refers to high-purity Bombesin products used as quantitative reference standards in experiments.

[0013] In a first aspect, the present invention provides a method for measuring the high purity of a dermal dermal purity standard, comprising the following steps: S1: preparing a mixed internal standard solution of isotopically labeled valine and leucine; preparing a mixed standard calibration solution of valine and leucine; wherein the mixed standard calibration solution comprises the mixed internal standard solution mixed in a specified proportion and an unlabeled amino acid mixed standard solution; wherein the unlabeled amino acid mixed standard solution comprises valine and leucine without isotopic labeling; S2: Mix the aqueous solution of the analyte to be tested with the mixed internal standard solution at a mass ratio of 0.9-1:0.9-1, add hydrochloric acid with a final concentration of 6-7.2 mM, deoxygenate, and hydrolyze at 130℃ for 24-48 h; S3: After the hydrolysate from step S2 is dried, it is reconstituted to obtain the hydrolysate sample to be tested; S4: The hydrolysate and the mixed standard calibration solution were loaded and detected separately. The peak areas of leucine, valine, and their corresponding isotope labels in the hydrolysate sample were detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The peak areas of leucine, valine, and their corresponding isotope labels in the mixed standard calibration solution were also detected. The LC conditions were: C18 column, mobile phase of 0.2% formic acid aqueous solution and acetonitrile at a volume ratio of 98:2, isocratic elution, flow rate of 0.3 mL / min; the mass spectrometry conditions were: leucine cone voltage 45-50 V, collision energy 10-15 V; isotope-labeled leucine cone voltage 45-50 V, collision energy 10-15 V; valine cone voltage 35-40 V, collision energy 10-15 V; isotope-labeled valine cone voltage 35-40 V / collision energy 10-15 V. S5: The mass fractions of leucine and valine in the sample were calculated using the isotope dilution method, and the purity of frog skin extract was obtained.

[0014] In an optional embodiment, the mixed internal standard solution of isotopically labeled valine and leucine in S1, and the mixed standard calibration solution of valine and leucine, are prepared according to the following method: (1) Target concentration calculation 1) Calculate the mass of this amino acid in 1 mg of frog dermatin. According to the formula: (1); where M represents the mass of the amino acid in 1 mg of dinoflagellin, in μg; M is the relative molecular mass of dinoflagellin, dimensionless; M AA n represents the molecular weight of the amino acid, dimensionless; AA The number of amino acids measured in dinoflagellin is dimensionless. The expression “×1 mg×1000” means: using 1 mg of dinoflagellin as a baseline, and converting the calculation result from mg to µg (1 mg = 1000 µg).

[0015] 2) Calculate the mass of the amino acid contained in 50 μL of frog skin extract solution.

[0016] In this embodiment, 50 µL of frog dermalin solution (containing 1 mg of frog dermalin) was diluted 20-fold before injection for detection. Therefore, the mass of the amino acid in the 50 µL diluted solution was calculated according to formula (2): (2). Wherein, m 50,i The mass of amino acid i in 50 µL of diluted solution is expressed in µg; m i The mass of amino acid i in 1 mg of frog skin extract calculated by formula (1) is in µg; 20 is the dilution factor.

[0017] 3) Calculate the concentration of this amino acid in the frog dermatin solution. Based on a 50 µL dilution solution, the concentration of this amino acid in the protein solution is calculated according to formula (3): (3). In equation (3): c i The concentration of amino acid i in the frog dermatin solution is expressed in µg / g; m 50,i The mass of amino acid i in the 50 µL diluted solution calculated by formula (2) is in µg; 0.05 g is the mass of the 50 µL diluted solution (based on a solution density of ≈1 g / mL, 50 µL ≈ 0.05 g).

[0018] The concentrations of valine, leucine, and their internal standard were calculated according to the above steps as follows: valine: 72.76 ug / g; leucine: 162.94 ug / g; valine marker: 76.03 ug / g; leucine marker: 164.50 ug / g.

[0019] (2) Preparation of amino acid mixed standard working solution Accurately weigh 722 μg of valine and 1620 μg of leucine, place them in a preparation container, add 9.9938 g of 0.1 mol / L hydrochloric acid solution, and shake thoroughly to obtain the amino acid mixed standard working solution.

[0020] (3) Preparation of isotope-labeled internal standard working solution Accurately weigh 754 μg of valine label and 1644 μg of leucine label, place them in another preparation container, add 9.9902 g of 0.1 mol / L hydrochloric acid solution, and shake thoroughly to obtain the isotope-labeled amino acid mixed internal standard working solution.

[0021] Before performing the analysis, accurately weigh 50 mg of amino acid mixed internal standard solution and 50 mg of isotope-labeled internal standard working solution using a 1 / 1,000,000 analytical balance, add 1 ml of 0.1 M hydrochloric acid aqueous solution, and mix thoroughly to prepare a mixed standard calibration solution (here, it is used as the test solution for the external standard method).

[0022] This invention employs an internal standard method for detecting the purity of dermalin. Leucine (Leu) and valine (Val), which are acid-hydrolyzable in dermalin, are selected as characteristic amino acids. Isotope-labeled forms of these two amino acids are used as internal standards. Under specific hydrolysis conditions using hydrochloric acid, dermalin is completely hydrolyzed into free amino acids. The absolute content of the two characteristic amino acids in the free amino acids is directly determined using isotope dilution mass spectrometry. The mass fractions of leucine (Leu) and valine (Val) in the sample are calculated. Based on the molecular structure of dermalin (which is known to contain specific molar amounts of leucine and valine), and according to the molecular weight of dermalin and the stoichiometry of its amino acids, the purity value of dermalin is calculated.

[0023] This method is independent of traditional chromatographic purity methods, and the measurement results can be directly traced to national amino acid standard materials, and further traced to the International System of Units (SI). This invention provides high-level metrological support for the quality control, bioactivity research, and clinical testing of frog dermalin-related products, significantly improving the accuracy and reliability of the determination results.

[0024] Leucine and valine were selected as the characteristic amino acids because, compared to other amino acids in frog dermalin, these two amino acids exhibit extremely high stability under high-concentration acid hydrolysis conditions.

[0025] Increasing the final concentration of hydrochloric acid during acid hydrolysis leads to a decrease in the measured purity of frog dermalin, resulting in inaccurate test results. Setting a specific final concentration of hydrochloric acid balances the efficiency of frog dermalin hydrolysis with ease of operation, ensuring the accuracy of sample purity testing.

[0026] Excessively high hydrolysis temperatures can lead to a decrease in the purity of frog dermalin, possibly due to thermal degradation of amino acids caused by high temperatures. Insufficient hydrolysis time will result in incomplete hydrolysis.

[0027] In a preferred embodiment of the present invention, the isotopically labeled valine is selected from any of the following: deuterated valine, carbon-13 monolabeled valine, and carbon-13 and nitrogen-15 double-labeled valine. Carbon-13 monolabeled valine may be labeled with all carbons (M+5), or it may be labeled with only some carbons.

[0028] In a preferred embodiment of the present invention, the isotopically labeled valine is selected from valine-U- 13 C5, 15 N(M+6).

[0029] In a preferred embodiment of the present invention, the isotopically labeled leucine is selected from any of the following: deuterated labeling, carbon-13 single labeling, and carbon-13 and nitrogen-15 double labeling.

[0030] In a preferred embodiment of the present invention, the isotopically labeled leucine is: 1,2- 13 C2-Leucine (M+2) is labeled with both the carboxyl carbon and the α-carbon.

[0031] In a preferred embodiment of the present invention, the formula for calculating the mass fractions of leucine and valine in a sample using the isotope dilution method is as follows: (4), where M is the relative molecular mass of dinoflagellin, dimensionless (or it can be g / mol); m x·AA The mass of the amino acids measured in the hydrolyzed sample of frog dermatin is expressed in mg; n AAM represents the number of amino acids measured in frog dermalin, dimensionless; AA is the relative molecular mass of the amino acid being tested, dimensionless (can also be g / mol); m is the sample weight of dermalin, mg; take the arithmetic mean of the results of independently calculated purity of dermalin for leucine and valine.

[0032] Where, m x AA Calculated using the following formula: (5) m x AA : Mass of the amino acids measured in the protein hydrolysate sample, mg; I x AA Peak area of ​​the amino acid being measured in the hydrolyzed sample, mg; I 'S AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the hydrolyzed sample; m STD AA Mass of the amino acid being tested in the mixed standard calibration solution, in units of m. x·AA The units are the same, for example, mg; m IS AA Mass of the amino acid isotope internal standard in the mixed standard calibration solution, in units of m. x·AA The units are the same, for example, mg; I IS AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the mixed standard calibration solution; I STD AA : Peak area of ​​the amino acid being measured in the mixed standard calibration solution; m 'IS AA Mass of the internal standard of the amino acid isotope being measured in the hydrolyzed sample, in units of m. x·AA The units are the same, for example, mg; In a preferred embodiment of the present invention, the chromatographic column used in the liquid chromatography is a KINETEX C18 column with dimensions of 2.1 mm × 150 mm and a diameter of 2.6 μm.

[0033] In a preferred embodiment of this invention, the mass spectrometry conditions for leucine are: cone voltage 50 V, collision energy 15 V; for isotopically labeled leucine: cone voltage 47 V, collision energy 14 V; for valine: cone voltage 40 V, collision energy 15 V; and for isotopically labeled valine: cone voltage 40 V, collision energy 15 V. The advantage is that an optimal balance point is found for each target analyte, thereby maximizing detection sensitivity and selectivity. Using other cone voltages and collision energies will affect the response intensity of the mass spectrometry peaks.

[0034] In a preferred embodiment of the present invention, in step S2, the aqueous solution of the analyte dermatin and the mixed internal standard solution are mixed at a mass ratio of 0.9-1:0.9-1, and hydrochloric acid with a final concentration of 6 mM is added. A final concentration of 6 mM hydrochloric acid is the optimal hydrolysis concentration, providing the best detection effect for analyte dermatin purity.

[0035] Step S1, the preparation of the mixed internal standard solution of isotopically labeled valine and leucine includes: mixing the isotopic label of valine and the isotopic label of leucine in hydrochloric acid solution according to a preset ratio; The mixed standard calibration solution includes a mixed internal standard solution mixed in a mass ratio of 0.9-1.1:0.9-1.1 and an unlabeled amino acid mixed standard solution; the unlabeled amino acid mixed standard solution includes valine and leucine without isotopic labeling, and in the unlabeled amino acid mixed standard solution, valine and leucine are mixed in the following ratio: 722 μg±10 μg: 1620 μg±10 μg.

[0036] In a preferred embodiment of the present invention, the resolution in step S3 uses a 0.1 mol / L hydrochloric acid solution, and the resolution solution is filtered through a filter membrane.

[0037] During reconstitution, vortex to fully dissolve the solution, filter through a 0.20 μm filter membrane, and transfer the filtrate to a sample vial for LC-MS / MS analysis.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] Example 1 This embodiment provides a method for measuring the high purity of frog dermalin purity standard material. The specific experimental instruments and reagents are as follows: Experimental apparatus: Analytical balance AL-104 (Mettler, Switzerland), analytical balance XS-105 (Mettler, Switzerland), nitrogen evaporator EFAA-DC24-RT (Shanghai Anpu Experimental Technology Co., Ltd.), oxyhydrogen flame ampoule sealing machine OKFKJ-2 (Hunan Walker Energy Technology Co., Ltd.), electric heating drying oven 101-OBS (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), liquid chromatography-mass spectrometry (LC-MS) system AB Triple Quad 4500 (AB SCIEX, USA). Experimental reagents and standard substances: Acetonitrile (chromatographic grade, Merck GmbH, Germany), hydrochloric acid (chromatographic grade, Shanghai Anpu Experimental Technology Co., Ltd.), valine GBW09236 (purity 98.8%–100%) (National Institute of Metrology, China), leucine 09237 (purity 99.3%–101%) (National Institute of Metrology, China), valine marker (L-Valine (¹³C5, 99%; ... 5 N, 99%) (Cambridge Isotope Laboratory, USA), leucine marker (L-Leucine (1,2-¹³C2, 99%) (Cambridge Isotope Laboratory, USA).

[0040] Prepare a mixed internal standard working solution of isotopically labeled valine and leucine, and a mixed standard calibration solution of valine and leucine: (1) Target concentration calculation 1) Calculate the mass of the amino acid in 1 mg. According to the formula: (1); where M represents the mass of amino acid i in 1 mg of dermatan, in μg; M represents the relative molecular mass of dermatan, dimensionless; M AA n represents the relative molecular mass of the amino acid, dimensionless; AA The number of amino acids measured in dinoflagellin is dimensionless. The expression “×1 mg×1000” means: using 1 mg of dinoflagellin as a baseline, and converting the calculation result from mg to µg (1 mg = 1000 µg).

[0041] 2) Calculate the mass of the amino acid contained in 50 μL of frog skin extract solution.

[0042] In this embodiment, 50 µL of frog dermalin solution (containing 1 mg of frog dermalin) was diluted 20-fold before injection for detection. Therefore, the mass of the amino acid in the 50 µL diluted solution was calculated according to formula (2): (2). Wherein, m 50,i The mass of amino acid i in 50 µL of diluted solution is expressed in µg; m iThe mass of amino acid i in 1 mg of frog skin extract calculated by formula (1) is in µg; 20 is the dilution factor.

[0043] 3) Calculate the concentration of the amino acid in the frog skin extract solution.

[0044] Based on a 50 µL dilution solution, the concentration of this amino acid in the protein solution is calculated according to formula (3): (3). In equation (3): c i The concentration of amino acid i in the frog dermatin solution is expressed in µg / g; m 50,i The mass of amino acid i in the 50 µL diluted solution calculated by formula (2) is in µg; 0.05 g is the mass of the 50 µL diluted solution (based on a solution density of ≈1 g / mL, 50 µL ≈ 0.05 g).

[0045] The concentrations of valine, leucine, and their internal standard in the unlabeled mixed standard working solution of valine and leucine were calculated according to the above formula as follows: valine: 72.76 ug / g; leucine: 162.94 ug / g; valine label in the isotope-labeled mixed internal standard working solution: 76.03 ug / g; leucine label: 164.50 ug / g.

[0046] (2) Preparation of amino acid mixed standard working solution Accurately weigh 722 μg of valine and 1620 μg of leucine, place them in a preparation container, add 9.9938 g of 0.1 mol / L hydrochloric acid solution, and shake thoroughly to obtain the amino acid mixed standard working solution.

[0047] (3) Preparation of isotope-labeled internal standard working solution Accurately weigh 754 μg of valine label and 1644 μg of leucine label, place them in another preparation container, add 9.9902 g of 0.1 mol / L hydrochloric acid solution, and shake thoroughly to obtain the isotope-labeled amino acid mixed internal standard working solution.

[0048] Purity testing methods 1. Sample pretreatment of frog dermal extract Approximately 1 mg of the purity standard of dinoflagellin was accurately weighed using a 1 / 1,000,000 analytical balance and dissolved in purified water to prepare a 1 mg / mL dinoflagellin aqueous solution. 50 mg of this dinoflagellin aqueous solution and 50 mg of a mixed internal standard solution of isotopically labeled amino acids (containing Val and Leu isotope labels at concentrations of 75.47 μg / g and 164.56 μg / g, respectively) were accurately transferred into a brown ampoule. 1 mL of 6 mol / L hydrochloric acid solution was added, and the mixture was shaken to mix. After purging with nitrogen for 30 seconds to remove oxygen, the ampoule was sealed and hydrolyzed in an oven at 130°C for 24 hours. After hydrolysis, the ampoule was dried under nitrogen, reconstituted with 1 mL of 0.1 M hydrochloric acid aqueous solution, filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a sample vial for LC-MS / MS analysis.

[0049] Before performing the analysis, accurately weigh 50 mg of amino acid mixed internal standard solution and 50 mg of isotope-labeled internal standard working solution using a 1 / 1,000,000 analytical balance, add 1 ml of 0.1 M hydrochloric acid aqueous solution, mix thoroughly to prepare a calibration solution (here used as the test solution for the external standard method).

[0050] The sample solution to be tested, obtained by hydrolyzing the mixture of frog dermal extract and isotopically labeled amino acid mixed internal standard solution with hydrochloric acid, and the aforementioned calibration solution were loaded separately for liquid chromatography-mass spectrometry detection.

[0051] 2. Liquid Chromatography-Mass Spectrometry Detection Conditions Chromatographic conditions: Chromatographic column: KINETEX C18 column (2.1 mm × 150 mm, 2.6 μm); mobile phase: phase A is 0.2% formic acid aqueous solution, phase B is acetonitrile; elution mode: isocratic elution, A:B = 98:2 (v / v), hold for 5 min; flow rate: 0.3 mL / min; injection volume: 3 μL.

[0052] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion mode, multiple reaction monitoring (MRM); Leucine: cone voltage 50 V, collision energy 15 V; isotope-labeled leucine: cone voltage 47 V, collision energy 14 V; Valine: cone voltage 40 V, collision energy 15 V; isotope-labeled valine: cone voltage 40 V, collision energy 15 V. Ion-to-mass-charge ratio: Valine: 118.10→72.10 (Phe) and 124.20→77.10 (labeled Val); Leucine: 132.10→86.2 (Leu) and 134.10→87.19 (labeled Leu); 3. Process three parallel samples of frog dermal extract according to step 1, and perform LC-MS / MS analysis under the conditions of step 2. Record the peak areas of the analyte amino acid isotope internal standards in leucine, valine, and their hydrolysate samples, the peak areas of the analyte amino acid isotope internal standards in the calibration solution, and the peak areas of the analyte amino acid in the calibration solution. Calculate the mass fraction of leucine in the frog dermal extract aqueous solution using isotope dilution mass spectrometry. Substitute the above data into the formula below to calculate the purity values ​​of frog dermal extract based on leucine and valine, respectively, and take the arithmetic mean as the final result.

[0053] The formulas for calculating the mass fractions of leucine and valine in a sample using the isotope dilution method are as follows: The purity of frog skin extract is calculated using the following formula: (4), where M is the relative molecular mass of dinoflagellin, which is dimensionless; m x·AA The mass of the amino acids measured in the hydrolyzed sample of frog dermatin is expressed in mg; n AA M represents the number of amino acids measured in frog dermalin, dimensionless; AA is the relative molecular mass of the amino acid being tested, dimensionless; m is the sample weight of leucine, mg; the arithmetic mean of the results of independently calculated purity of leucine and valine is taken. Among them, mx AA is calculated using the following formula: ; m x AA : Mass of the amino acids measured in the protein hydrolysate sample, mg; I x AA Peak area of ​​the amino acid being measured in the hydrolyzed sample, mg; I' S AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the hydrolyzed sample; m STD AA Mass of the amino acid being tested in the mixed standard calibration solution, mg; m IS AA : The mass of the internal standard of the amino acid isotope being measured in the calibration solution, in mg; I IS AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the mixed standard calibration solution; I STD AA : Peak area of ​​the amino acid being measured in the mixed standard calibration solution; m' IS AA : The mass of the internal standard of the amino acid isotope being tested in the hydrolyzed sample, in mg.

[0054] The relative standard deviation (RSD) of the six parallel samples was less than 0.5%, as shown in Table 1 below, indicating good method precision and demonstrating the high accuracy and metrological traceability of this method.

[0055] Table 1. Determination of purity (%) of dinoflagellin by AA-LC-IDMS / MS method

[0056] The LC-MS chromatogram of the quantitative amino acid standard solution (i.e., the mixed standard calibration solution) prepared from valine GBW 09236 and leucine GBW 09237 is shown in the figure. Figure 1 As shown, the LC-MS chromatogram of the isotopically labeled amino acid mixed internal standard solution is referenced. Figure 2 As shown.

[0057] Experimental Example 1 This experimental example focuses on optimizing the hydrolysis conditions. To ensure the complete and stable hydrolysis of the frog dermalin purity standard into free amino acids while avoiding amino acid degradation, four key parameters—hydrochloric acid concentration, hydrochloric acid dosage, hydrolysis temperature, and hydrolysis time—were systematically optimized. The peak area ratio of each analyte amino acid (leucine, valine) to its corresponding isotope-labeled amino acid in the mass spectrometer was used as the evaluation index. The optimal hydrolysis conditions were defined as those when the peak area ratio reached its maximum or approached a stable plateau. The frog dermalin source and chromatographic-mass spectrometric conditions were the same as in Example 1.

[0058] (1) Optimization of hydrochloric acid concentration Mix 50 mg each of the frog dermal extract aqueous solution and the internal standard solution in brown ampoules, and add 1.0 mL of hydrochloric acid solutions with concentrations of 2 mol / L, 4 mol / L, 6 mol / L, and 8 mol / L, respectively. Hydrolyze at 130℃ for 24 h. Determine the purity of leucine and valine in the hydrolysate. Figure 3 The results showed that the concentration increased slightly from 2M to 4M, reaching its maximum at 6M, and the purity decreased slightly when further increased to 8 mol / L. Therefore, 6 mol / L was selected as the optimal concentration for hydrolyzing hydrochloric acid.

[0059] (2) Optimization of hydrochloric acid dosage With a fixed hydrochloric acid concentration of 6 mol / L, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of 6 mol / L hydrochloric acid solution were added, respectively, and the solutions were hydrolyzed at 130℃ for 24 h. The purity values ​​of leucine and valine in the sample solutions were determined.

[0060] Figure 4 The results showed that the purity value increased with increasing addition, reaching 0.72 at both 1.0 ml and 1.2 ml, indicating that 1.0 ml was sufficient to obtain the highest average yield. However, the subsequent nitrogen drying time was significantly prolonged. Considering both hydrolysis efficiency and ease of operation, 1.0 mL was selected as the optimal amount of hydrochloric acid.

[0061] (3) Optimization of hydrolysis temperature The hydrochloric acid concentration was fixed at 6 mol / L, and the volume was 1.0 mL. The hydrolysate was carried out at 110℃, 130℃, and 150℃ for 24 h.

[0062] Figure 5 The results showed that the purity of dinoflagellin increased with increasing temperature, reaching a maximum at 130℃ with good repeatability. At 150℃, the purity decreased, possibly due to thermal degradation of amino acids caused by high temperatures. Therefore, 130℃ was selected as the optimal hydrolysis temperature.

[0063] (4) Optimization of hydrolysis time With a fixed hydrochloric acid concentration of 6 mol / L, a dosage of 1.0 mL, and a hydrolysis temperature of 130℃, the hydrolysis times were investigated at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h.

[0064] Figure 6 The results showed that the purity value increased rapidly over time from 0 to 4 hours, indicating that the hydrolysis reaction was not complete. At 8 hours, the ratio reached its maximum value and entered a stable plateau period. Therefore, 24 hours was selected as the optimal hydrolysis time to ensure complete hydrolysis and the highest efficiency.

[0065] The optimized hydrochloric acid hydrolysis conditions were determined as follows: hydrochloric acid concentration 6 mol / L, hydrochloric acid volume 1.0 mL, hydrolysis temperature 130℃, and hydrolysis time 24 hours. These conditions were used for all subsequent quantitative analyses using isotope dilution mass spectrometry.

[0066] Experimental Example 2 Three samples were randomly selected from the hydrolyzed frog dermal extract samples, and the concentration was accurately determined by liquid chromatography-amino acid hydrolysis isotope dilution mass spectrometry. Each sample was measured in parallel three times, and the intra- and inter-group precision was calculated. The results are shown in Table 2. The results show that the relative deviation of the intra- and inter-group purity determination results of frog dermal extract samples does not exceed 1.0%, indicating that the method has good repeatability and precision.

[0067] Table 2 Repeatability and precision of AA-LC-IDMS / MS method for the determination of frog dermatin

[0068] In summary, the purity detection method provided by this invention operates independently of traditional chromatographic purity methods. Measurement results can be directly traced back to national amino acid standard materials, and further to the International System of Units (SI). This invention provides high-level metrological support for the quality control, bioactivity research, and clinical testing of frog dermalin-related products, significantly improving the accuracy and reliability of the determination results.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for measuring the high purity of a standard reference material for frog dermalin, characterized in that, It includes the following steps: S1: Prepare a mixed internal standard solution of isotopically labeled valine and leucine, and prepare a mixed standard calibration solution of valine and leucine. The mixed standard calibration solution includes the mixed internal standard solution and the unlabeled amino acid mixed standard solution mixed in a specified ratio; The unlabeled amino acid standard solution includes valine and leucine without isotopic labeling; S2: Mix the aqueous solution of the analyte to be tested with the mixed internal standard solution at a mass ratio of 0.9-1:0.9-1, add hydrochloric acid with a final concentration of 6-7.2 mM, deoxygenate, and hydrolyze at 130℃ for 24-48 h; S3: After the hydrolysate from step S2 is dried, it is reconstituted to obtain the hydrolysate sample to be tested; S4: The hydrolysate and the mixed standard calibration solution were loaded and detected separately. The peak areas of leucine, valine, and their corresponding isotope labels in the hydrolysate sample were detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The peak areas of leucine, valine, and their corresponding isotope labels in the mixed standard calibration solution were also detected. The LC conditions were: C18 column, mobile phase of 0.2% formic acid aqueous solution and acetonitrile at a volume ratio of 98:2, isocratic elution, flow rate of 0.3 mL / min; the mass spectrometry conditions were: leucine cone voltage 45-50 V, collision energy 10-15 V; isotope-labeled leucine cone voltage 45-50 V, collision energy 10-15 V; valine cone voltage 35-40 V, collision energy 10-15 V; isotope-labeled valine cone voltage 35-40 V / collision energy 10-15 V. S5: The mass fractions of leucine and valine in the sample were calculated using the isotope dilution method, and the purity of frog skin extract was obtained.

2. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, The isotopically labeled valine is selected from any of the following: deuterated valine, carbon-13 monolabeled valine, and carbon-13 and nitrogen-15 double-labeled valine.

3. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 2, characterized in that, Isotopically labeled valine is selected from valine-U- 13 C5, 15 N.

4. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, The isotopically labeled leucine is selected from any of the following: deuterated labeling, carbon-13 single labeling, and carbon-13 and nitrogen-15 double labeling.

5. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 4, characterized in that, The isotopically labeled leucine is: 1,2- 13 C2-Leucine.

6. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, The formulas for calculating the mass fractions of leucine and valine in a sample using the isotope dilution method are as follows: The purity of frog skin extract is calculated using the following formula: Where M is the relative molecular mass of taurine; m x·AA n represents the mass of the amino acids being measured in the hydrolyzed sample of frog dermatin; AA M represents the number of amino acids measured in frog dermalin, dimensionless; AA m is the relative molecular mass of the amino acid being tested; m is the sample mass of frog dermatin, and the unit is the same as m. x·AA Same units; take the arithmetic mean of the results of independently calculated purity of leucine and valine amino acids; Among them, mx AA is calculated using the following formula: ； m x AA : The mass of the amino acid being measured in the hydrolyzed sample; I x AA : Peak area of ​​the amino acid being measured in the hydrolyzed sample; I' S AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the hydrolyzed sample; m STD AA Mass of the amino acid being tested in the mixed standard calibration solution, in units of m. x·AA Same unit; m IS AA Mass of the amino acid isotope internal standard in the mixed standard calibration solution, in units of m. x·AA Same units; I IS AA : Peak area of ​​the internal standard of the amino acid isotope being measured in the mixed standard calibration solution; I STD AA : Peak area of ​​the amino acid being measured in the mixed standard calibration solution; m' IS AA Mass of the internal standard of the amino acid isotope being measured in the hydrolyzed sample, in units of m. x·AA The units are the same.

7. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, The liquid chromatography column used was a KINETEX C18 column, with dimensions of 2.1 mm × 150 mm and a diameter of 2.6 μm.

8. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, For mass spectrometry detection, the mass spectrometry conditions for leucine were: cone voltage 50 V, collision energy 15 V; for isotopically labeled leucine, the mass spectrometry conditions were: cone voltage 47 V, collision energy 14 V; for valine, the mass spectrometry conditions were: cone voltage 40 V, collision energy 15 V; and for isotopically labeled valine, the mass spectrometry conditions were: cone voltage 40 V, collision energy 15 V.

9. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, Step S2: Mix the aqueous solution of the analyte to be tested with the mixed internal standard solution at a mass ratio of 0.9-1:0.9-1, and add hydrochloric acid with a final concentration of 6 mM. Step S1, the preparation of the mixed internal standard solution of isotopically labeled valine and leucine includes: mixing the isotopically labeled valine and the isotopically labeled leucine in hydrochloric acid solution at a ratio of 754 μg ± 10 μg: 1644 ± 10 μg. The mixed standard calibration solution includes the mixed internal standard solution and an unlabeled amino acid mixed standard solution, which are mixed in a mass ratio of 0.9-1.1:0.9-1.

1. The unlabeled amino acid mixed standard solution includes valine and leucine without isotopic labeling. In the unlabeled amino acid mixed standard solution, valine and leucine are mixed in the following ratio: 722 μg±10 μg: 1620 μg±10 μg.

10. The method for measuring the high purity level of the frog dermalin purity standard material according to claim 1, characterized in that, The reconstitution in step S3 uses a 0.1 mol / L hydrochloric acid solution, and the reconstituted test solution is filtered through a filter membrane.

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