A method for analyzing the content of a target nitrogen-containing substance in a nitrogen-containing complex

Abstract

The application belongs to the field of chemical analysis, and particularly relates to an analysis method for the content of a target nitrogen-containing substance in a nitrogen-containing compound, which comprises the following steps: S1, respectively determining the protein conversion coefficients of each nitrogen-containing substance in the nitrogen-containing compound; S2, detecting the content of other nitrogen-containing substances except the target nitrogen-containing substance in the nitrogen-containing compound; S3, detecting the total nitrogen content of the nitrogen-containing compound by the Kjeldahl method, and recording the volume of total inorganic acid consumed; and S4, calculating the content of the target nitrogen-containing substance in the nitrogen-containing compound by a preset function formula. The analysis method disclosed by the application is simple in operation, high in spectrum, specificity and accuracy, good in stability, and extremely high in detection efficiency, and is suitable for a wide range of applications, and is a preferred method for detecting the content of a nitrogen-containing substance in a nitrogen-containing compound at present, and is suitable for detecting any target nitrogen-containing component in any nitrogen-containing compound.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis, and specifically relates to an analytical method for the content of target nitrogen-containing substances in nitrogen-containing complexes. Background Technology

[0002] Currently, commonly used protein detection methods mainly include ultraviolet (UV) absorption, the Bradford method (Coomassie Brilliant Blue method), the Lowry method, and the BCA method (dioctannic acid method). UV absorption is simple to operate and fast, but it is easily interfered with by other UV-absorbing substances in the sample and requires a high protein concentration. The Bradford method is easy to operate, develops color rapidly, and has good compatibility with most non-protein substances, but its linear range is narrow, it is easily interfered with by surfactants and strongly alkaline buffers, and there are significant differences in response between different proteins. The Lowry method has high sensitivity, but it is cumbersome and time-consuming, requires precise control of the reaction time, has poor specificity, and is susceptible to interference from various substances. The BCA method has good compatibility, a wide linear range, and strong colorimetric stability, but its reaction requires incubation at high temperatures and is easily interfered with by reducing agents, chelating agents, and other substances. The above methods are all for the detection of total protein or total nitrogenous substances, and they have different detection obstacles, such as cumbersome sample pretreatment process, difficulty in obtaining the standards required for quantification, and easy interference from other proteins or chemicals in the sample in actual detection, thus making it difficult to achieve accurate quantitative detection of specific components.

[0003] The Kjeldahl method, a classic chemical analytical method for determining the total nitrogen content in substances, is widely used in food, agriculture, medicine, environment, and materials science. This method converts organic nitrogen in the sample into inorganic ammonium salts through digestion, distillation, and titration, and then quantitatively analyzes these salts to obtain the total nitrogen content. However, the result obtained by this method is the total nitrogen content. If multiple nitrogen-containing components or other nitrogen-containing impurities are present in the sample, they will significantly interfere with the detection results. More importantly, the Kjeldahl method has an inherent limitation: it cannot distinguish the contribution of different nitrogen-containing components to the total nitrogen. In complex systems containing multiple nitrogen-containing substances, the total nitrogen content alone cannot provide the accurate content of any specific component, which limits its application in complex systems.

[0004] To overcome this limitation, some studies have attempted to combine the Kjeldahl method with other detection methods that can specifically identify a particular component. For example, published patent CN120741699A proposes a "method for detecting the content of collagen and regenerated silk fibroin in a nitrogen-containing complex." This method uses high-performance liquid chromatography (HPLC) to directly determine the content of regenerated silk fibroin, then uses the Kjeldahl method to determine the total nitrogen content of the nitrogen-containing complex, and finally uses the difference method to calculate the collagen content. This method achieves, to some extent, the separate quantification of the two main components in a complex nitrogen-containing system. However, this method still has the following significant shortcomings: (1) Reliance on target standard: This method requires obtaining a pure sample of the target nitrogenous substance (such as silk fibroin) as an HPLC quantitative standard for establishing a standard curve or external standard method for quantification. However, in many practical applications, the target nitrogenous substance may be difficult to separate and purify with high purity, or its standard may be difficult to obtain, expensive, or even not yet commercialized. In the above cases, this method loses its basis for application.

[0005] (2) Applicable only to specific composition systems: This method is specifically designed for the binary nitrogen-containing complex system of "collagen + regenerated silk fibroin". The detection process, chromatographic conditions and calculation methods are highly dependent on the chemical and physical properties of this system and lack broad applicability. Once the composition of the nitrogen-containing complex changes, such as the appearance of other proteins, peptides or nitrogen-containing polymers, this method cannot be directly extended or adjusted.

[0006] (3) The detection sequence is logically dependent: In this method, the content of the target nitrogenous substance (silk fibroin) must first be determined by HPLC, and then this part must be subtracted from the total nitrogen before the content of the other component (collagen) can be estimated. This means that if the target nitrogenous substance itself is difficult to detect directly in a certain application scenario (e.g., lack of specific chromatographic response, inability to separate from the matrix, lack of suitable detection wavelength, or insolubility), the entire difference scheme will fail. In other words, this method requires that "the target substance can be measured first," rather than flexibly choosing any easily detectable component as the starting point.

[0007] In summary, while existing strategies based on "specific direct determination + total nitrogen subtraction" are effective in specific systems, they generally suffer from rigid dependence on standards, specific system compositions, and direct detection capabilities. Furthermore, conventional total protein and total nitrogen detection methods cannot distinguish specific components or are susceptible to interference, making it difficult for current technologies to achieve universal and specific quantitative detection of any target nitrogen-containing component in nitrogen-containing complexes. Therefore, there is an urgent need to develop a novel subtraction detection method that does not require target analyte standards, is not limited by complex composition, and does not require priority direct determination of the target analyte, in order to overcome the shortcomings of existing technologies and achieve specific, accurate, and universal quantitative detection of any target nitrogen-containing component in nitrogen-containing complexes. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide an analytical method for the content of target nitrogen-containing substances in nitrogen-containing complexes. The method of this invention is simple to operate, has a wide spectrum, high specificity, high accuracy, good stability, and extremely high detection efficiency. It is applicable to a wide range of applications and can be used to detect any target nitrogen-containing component in any nitrogen-containing complex.

[0009] This invention provides an analytical method for the content of a target nitrogen-containing substance in a nitrogen-containing complex, the analytical method comprising the following steps: S1: Determine the protein conversion factor of each nitrogen-containing substance in the nitrogen-containing complex; S2: Detect the content of nitrogen-containing substances other than the target nitrogen-containing substance in the nitrogen-containing complex; S3: The total nitrogen content of the nitrogen-containing complex was determined by the Kjeldahl method, and the total volume of inorganic acid consumed was recorded; S4: Calculate the content of the target nitrogen-containing substance in the nitrogen-containing complex using a preset function formula.

[0010] In some embodiments of the present invention, the function formula is as follows: ; In the formula, C 目 V represents the content of the target nitrogen-containing substance in the nitrogen-containing complex, in mg / g; M represents the mass of the nitrogen-containing complex, in g; V 总 V0 is the total volume of inorganic acid consumed by the nitrogen-containing complex when detected by the Kjeldahl method, in mL; V0 is the volume of inorganic acid consumed by the blank sample when the nitrogen-containing complex is detected by the Kjeldahl method, in mL; n is the number of nitrogen-containing substances in the nitrogen-containing complex; C i F represents the content of the i-th nitrogen-containing substance in the nitrogen-containing complex, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in the nitrogen-containing complex, in units of 1; H+ The concentration of inorganic acid, expressed in mol / L, is used for the detection of nitrogen-containing complexes by the Kjeldahl method; F 目 1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen in g / mmol; n' represents the correction factor for inorganic acids.

[0011] In some embodiments of the present invention, in step S1, the protein conversion factor F is determined in the following manner: (a) For nitrogen-containing substances with well-defined chemical structures and known purity, the molecular formula can be used to calculate F = molecular weight / (number of nitrogen atoms in the molecule × 14); (b) For nitrogen-containing substances with unclear chemical structures or for which pure samples cannot be obtained, nitrogen content is determined by the Kjeldahl method. Specifically, a pure sample of the nitrogen-containing substance of known concentration is taken and its nitrogen content is determined by the Kjeldahl method. F = mass of the nitrogen-containing substance / mass of nitrogen obtained by the Kjeldahl method for the mass of the substance.

[0012] In some embodiments of the present invention, the inorganic acid is hydrochloric acid or sulfuric acid.

[0013] Preferably, when the inorganic acid is hydrochloric acid, n'=1; when the inorganic acid is sulfuric acid, n'=1 / 2.

[0014] In some embodiments of the present invention, in step S1, the nitrogen-containing complex is one or more of biomedical materials, tissue engineering, medical devices, biological agents, pharmaceuticals, cosmetics, food, feed, textile materials, and environmental samples.

[0015] In some embodiments of the present invention, in step S1, the nitrogen-containing composite is a solid, a semi-solid, a liquid, a gel, a powder, a film, a sponge, a fiber, or any combination of the above forms.

[0016] In some embodiments of the present invention, in step S2, the target nitrogen-containing substance is one or more of the following: protein, polypeptide, amino acid, nucleic acid, alkaloid, nitrogen-containing drug, nitrogen-containing polymer material, nitrogen-containing small molecule metabolite, nitrogen-containing inorganic salt, and nitrogen-containing medical aesthetic active ingredient.

[0017] Preferably, the protein is one or more of collagen, whey protein, and silk fibroin; the polypeptide is one or more of glutathione, cyclic peptide, and antimicrobial peptide; the amino acid is one or more of arginine, lysine, glycine, and taurine; the nucleic acid is natural nucleic acid and / or artificial nucleic acid, including but not limited to those derived from animals, plants, fungi, bacteria, and viruses; the alkaloid is one or more of caffeine, nicotine, and berberine; the nitrogen-containing drug is one or more of antibiotics, antitumor drugs, anesthetics, cardiovascular drugs, and central nervous system drugs; the nitrogen-containing polymer material is one or more of chitosan, polylysine, polyarginine, polyacrylamide, and polyethyleneimine; the nitrogen-containing small molecule metabolite is one or more of urea, uric acid, creatinine, and amine compounds; the nitrogen-containing inorganic salt is one or more of ammonium salts, nitrates, and nitrites; and the nitrogen-containing medical aesthetic active ingredients include one or more of hyaluronic acid derivatives, quaternary ammonium salt preservatives, biological dressings, protein gels, and PDRN stock solutions. The analytical method of this invention for the content of target nitrogen-containing substances in nitrogen-containing complexes has excellent broad-spectrum applicability.

[0018] In some embodiments of the present invention, in step S2, the other nitrogen-containing substances are one or more of sodium hyaluronate, chondroitin sulfate, heparin, collagen, gelatin, chitosan, sodium alginate (containing nitrogen impurities), polyvinylpyrrolidone, and polyamino acids.

[0019] In some embodiments of the present invention, in step S2, the detection is obtained by a detection method corresponding to other nitrogen-containing substances in the nitrogen-containing complex besides the target nitrogen-containing substance. The specific detection method includes, but is not limited to: high performance liquid chromatography, gas chromatography, spectrophotometry, enzymatic methods, immunoassay, capillary electrophoresis, and mass spectrometry.

[0020] In some embodiments of the present invention, the nitrogen contribution ratio of the target nitrogen-containing substance to other nitrogen-containing components in the nitrogen-containing complex is ≥1:100.

[0021] In some embodiments of the present invention, the detection limit of the analytical method is ≤0.1 mg / g and the quantitation limit is ≤0.5 mg / g.

[0022] In some embodiments of the present invention, the analytical method has a detection error of ≤5% when there are ≤10 nitrogen-containing components in the nitrogen-containing complex.

[0023] This invention differs from existing technologies that directly detect the target analyte. Instead, it performs subtraction analysis after detecting all interfering substances, determines the total nitrogen content using the Kjeldahl method, and then calculates the corresponding total protein equivalent. A specific method is then used to determine the content of each nitrogen-containing component other than the target nitrogen-containing substance. The nitrogen contribution of the target nitrogen-containing substance is obtained by subtracting the nitrogen contribution of other nitrogen-containing components from the total nitrogen contribution, and then multiplied by the protein conversion factor of the target nitrogen-containing substance. Specifically, this invention establishes a quantitative relationship between the content of other nitrogen-containing substances (excluding the target nitrogen-containing substance) and the corresponding hydrochloric acid consumption volume in the Kjeldahl method by separately determining the protein conversion factor of each nitrogen-containing substance in the nitrogen-containing complex. By establishing a functional relationship, the content of the target nitrogen-containing substance in the nitrogen-containing complex is obtained rapidly and efficiently. Compared with existing technologies, this invention, using the Kjeldahl method, is applicable to various forms of nitrogen-containing complexes, ensuring the integrity and accuracy of sample detection. The only source of interference in this method is other nitrogen-containing substances in the sample besides the target nitrogen-containing substance. By accurately quantifying these interfering substances, the accurate determination of the target nitrogen-containing substance content is achieved.

[0024] This invention eliminates the need for target analyte standards, solving the problem of insufficient applicability of existing analytical methods when the target analyte is difficult to purify or the standard is unavailable. It has broad applicability, suitable for the detection of any target nitrogen-containing component in any nitrogen-containing complex, and has high accuracy with an error value controllable within 5%.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method of the present invention is simple to operate, has a wide range of applications, high specificity, high accuracy, good stability, and extremely high detection efficiency. It is applicable to the detection of any target nitrogen-containing component in any nitrogen-containing complex.

[0026] (2) This invention establishes a quantitative relationship between the content of nitrogenous substances other than the target nitrogenous substance and the corresponding hydrochloric acid consumption volume in the Kjeldahl nitrogen determination method by separately determining the protein conversion coefficient of each nitrogenous substance in the nitrogenous complex. By establishing a functional relationship, the content of the target nitrogenous substance in the nitrogenous complex is obtained quickly and efficiently. Compared with the prior art, this invention, through the Kjeldahl nitrogen determination method, is applicable to nitrogenous complexes of various forms, ensuring the integrity and accuracy of sample detection. The only source of interference in this method is other nitrogenous substances in the sample besides the target nitrogenous substance. By accurately quantifying and detecting the above-mentioned interfering substances, the accurate determination of the content of the target nitrogenous substance is achieved. Detailed Implementation

[0027] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0028] In the description of the specific embodiments of this invention, all reagents are readily available from commercial companies. It should be noted that the Kjeldahl method is an existing method for determining nitrogen content. Specific steps can be found in GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food". The protein conversion factors for silk fibroin, sodium hyaluronate, collagen, chitosan, metformin hydrochloride, polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), and deoxyribonucleotides (DNA) mentioned in the following embodiments are calculated according to their corresponding methods. The determination of sodium hyaluronate content using the method in Appendix C (carbazole method) of standard YY / T 0962-2021, the determination of glucosamine content using the hydrolysis-HPLC method, the determination of collagen content using the hydroxyproline assay method, the determination of PVP content using high-performance liquid chromatography, and the determination of DNA content using the Qubit dsDNA fluorescence quantitative method are all performed according to their corresponding methods. The above determination and calculation methods are well known to those skilled in the art, and this invention will not further limit their disclosure. In the following specific embodiments, the inorganic acid used is hydrochloric acid, with a concentration of C. H+ All values ​​are 0.05021 mol / L. In the function formula, n=2 and n'=1.

[0029] Biomedical Materials: Determination of Silk Fibroin Content in Silk Fibroin / Sodium Hyaluronate Composite Gel In Examples 1-3 below, the protein conversion factor for silk fibroin was calculated based on its amino acid composition, and the protein conversion factor for sodium hyaluronate was calculated using its chemical formula C. 14 H 20 NNaO 11 The corresponding molecular weight was calculated.

[0030] Example 1 An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: 10g of 18.85mg / g silk fibroin hydrogel and 20g of 24.76mg / g sodium hyaluronate gel were mixed to obtain nitrogen-containing complex sample 1, and the mass M of nitrogen-containing complex sample 1 was recorded. S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 1 was determined. The protein conversion factor of silk fibroin was 5.41 and the protein conversion factor of sodium hyaluronate was 28.664. S2: The content of sodium hyaluronate was determined by the method in Appendix C (carbazole method) of standard YY / T 0962-2021, and the result was C=16.45mg / g; S3: The protein content of nitrogen-containing complex sample 1 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 1 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, namely silk fibroin, in nitrogen-containing complex sample 1. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, silk fibroin, in nitrogen-containing complex sample 1, in mg / g; M represents the mass of nitrogen-containing complex sample 1, in g; V 总 V0 is the total volume of inorganic acid consumed by nitrogen-containing complex sample 1 when analyzed by the Kjeldahl method, in mL; V0 is the volume of inorganic acid consumed by the blank sample when nitrogen-containing complex sample 1 is analyzed by the Kjeldahl method, in mL; n is the number of nitrogen-containing substances in the nitrogen-containing complex; C i F represents the sodium hyaluronate content in nitrogen-containing complex sample 1, in mg / g. i C represents the protein conversion factor for sodium hyaluronate in nitrogen-containing complex sample 1, in units of 1; H+ The inorganic acid concentration for nitrogen-containing complex sample 1 when analyzed by the Kjeldahl method is expressed in mol / L; F 目1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the specific data are shown in Table 1.

[0031] Table 1

[0032] Example 2 An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: 15g of 18.85mg / g silk fibroin hydrogel and 15g of 24.76mg / g sodium hyaluronate gel were mixed to obtain nitrogen-containing complex sample 2, and the mass M of nitrogen-containing complex sample 2 was recorded. S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 2 was determined. The protein conversion factor F of silk fibroin was 5.41, and the protein conversion factor F of sodium hyaluronate was 28.664. S2: The content of sodium hyaluronate was determined by the method in Appendix C (carbazole method) of standard YY / T 0962-2021, and the result was C=12.32mg / g; S3: The protein content of nitrogen-containing complex sample 2 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 2 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, namely silk fibroin, in nitrogen-containing complex sample 2. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, silk fibroin, in nitrogen-containing complex sample 2, in mg / g; M represents the mass of nitrogen-containing complex sample 2, in g; V 总 V0 is the total volume of inorganic acid consumed by nitrogen-containing complex sample 2 during Kjeldahl nitrogen determination, in mL; V0 is the volume of inorganic acid consumed by the blank sample during Kjeldahl nitrogen determination of nitrogen-containing complex sample 2, in mL; n is the number of nitrogen-containing substances in the nitrogen-containing complex; C i F represents the sodium hyaluronate content in nitrogen-containing complex sample 2, in mg / g. i C represents the protein conversion factor for sodium hyaluronate in nitrogen-containing complex sample 2, in units of 1; H+ The inorganic acid concentration, expressed in mol / L, is used in the Kjeldahl nitrogen determination of nitrogen-containing complex sample 2. 目1 represents the protein conversion factor for silk fibroin; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the specific data are shown in Table 2.

[0033] Table 2

[0034] Example 3 An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: 20g of 18.85mg / g silk fibroin hydrogel and 10g of 24.76mg / g sodium hyaluronate gel were mixed to obtain nitrogen-containing complex sample 3, and the mass M of nitrogen-containing complex sample 3 was recorded. S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 3 was determined. The protein conversion factor F of silk fibroin was 5.41, and the protein conversion factor F of sodium hyaluronate was 28.664. S2: The content of sodium hyaluronate was determined by the method in Appendix C (carbazole method) of standard YY / T 0962-2021, and the result was C=8.24mg / g; S3: The protein content of nitrogen-containing complex sample 3 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 3 were recorded. 总 ; S4: Substitute into the following function formula to calculate the silk fibroin content C in nitrogen-containing complex sample 3. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, silk fibroin, in nitrogen-containing complex sample 3, in mg / g; M represents the mass of nitrogen-containing complex sample 3, in g; V 总 V0 is the total volume of inorganic acid consumed by nitrogen-containing complex sample 3 when analyzed by the Kjeldahl method, in mL; V0 is the volume of inorganic acid consumed by the blank sample when nitrogen-containing complex sample 1 is analyzed by the Kjeldahl method, in mL; n is the number of nitrogen-containing substances in the nitrogen-containing complex; C i F represents the sodium hyaluronate content in nitrogen-containing complex sample 3, in mg / g. i C represents the protein conversion factor for sodium hyaluronate in nitrogen-containing complex sample 3, in units of 1; H+ The inorganic acid concentration for nitrogen-containing complex sample 1 when analyzed by the Kjeldahl method is expressed in mol / L; F 目1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the specific data are shown in Table 3.

[0035] Table 3

[0036] According to the theoretical concentration = total mass of silk fibroin / total mass of nitrogen-containing complex M, where the total mass of silk fibroin = total mass of silk fibroin hydrogel * concentration of silk fibroin hydrogel; error = (measured concentration C) / (total mass of silk fibroin hydrogel). 目 -Theoretical concentration) / theoretical concentration * 100%, calculate the theoretical concentration and error of Examples 1-3, and the results are shown in Table 4: Table 4

[0037] As can be seen from the test results of Examples 1-3 in Tables 1-3 and Table 4, the analytical method for the content of silk fibroin in nitrogen-containing complexes disclosed in this application can achieve accurate measurement of silk fibroin content. The measurement results of Examples 1, 2 and 3 all have an error within ±3%.

[0038] Tissue Engineering: Determination of Collagen Content in Collagen / Chitosan Composite Membranes

[0039] Example 4 In Example 4 below, the protein conversion factor of collagen was calculated based on the amino acid composition of collagen, and the protein conversion factor of chitosan was calculated based on a degree of deacetylation of 80%. An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: A collagen / chitosan composite membrane was taken as nitrogen-containing complex sample 4, in which the theoretical content of collagen was 50% and the theoretical content of chitosan was 50%. S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 4 was determined. The protein conversion factor F of collagen was 5.630 and the protein conversion factor F of chitosan was 6.12. S2: The content of glucosamine was determined by hydrolysis-HPLC and then converted into the content of chitosan. The result was C=51.0mg / 100mg composite membrane. S3: The protein content of nitrogen-containing complex sample 4 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 4 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, namely collagen, in nitrogen-containing complex sample 4.目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, collagen, in nitrogen-containing complex sample 4, in mg / g; M represents the mass of nitrogen-containing complex sample 4, in g; V 总 V0 is the total volume of inorganic acid consumed by the nitrogen-containing complex sample 4 during Kjeldahl nitrogen determination, in mL; V0 is the volume of inorganic acid consumed by the blank sample during the Kjeldahl nitrogen determination of nitrogen-containing complex sample 4, in mL; n represents the number of nitrogen-containing substances present in nitrogen-containing complex sample 4; C i F represents the content of the i-th nitrogen-containing substance in nitrogen-containing complex sample 4, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in nitrogen-containing complex sample 4, in units of 1; H+ The inorganic acid concentration for nitrogen-containing complex sample 4, measured by the Kjeldahl method, is expressed in mol / L; F 目 1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the collagen content was calculated to be 49.6 mg / 100mg composite membrane, while the theoretical content was 50.0 mg / 100mg. The recovery rate was calculated to be 99.2%, with an error value of -0.8%.

[0040] Two protein composite systems: Determination of silk fibroin content in silk fibroin / collagen composite hydrogels

[0041] Example 5 The following comparative example is Example 4 disclosed in patent CN120741699A; the protein conversion factor of silk fibroin is calculated based on the amino acid composition, and the protein conversion factor of collagen is calculated based on the amino acid composition of collagen. An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: Take a 1:1 mixture of silk fibroin hydrogel and collagen hydrogel as nitrogen-containing complex sample 5, in which the theoretical concentration of silk fibroin is 10.00 mg / g and the theoretical concentration of collagen is 10.00 mg / g. S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 5 was determined. The protein conversion factor of silk fibroin was 5.41, and the protein conversion factor F of collagen was 5.630. S2: The collagen content (the hydroxyproline content in collagen is about 12%) was determined by the hydroxyproline assay, and the result was C = 9.76 mg / g; S3: The protein content of nitrogen-containing complex sample 5 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 5 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, namely silk fibroin, in nitrogen-containing complex sample 5. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, silk fibroin, in nitrogen-containing complex sample 5, in mg / g; M represents the mass of nitrogen-containing complex sample 5, in g; V 总 V0 is the total volume of inorganic acid consumed by nitrogen-containing complex sample 5 during Kjeldahl nitrogen determination, in mL; V0 is the volume of inorganic acid consumed by the blank sample during Kjeldahl nitrogen determination of nitrogen-containing complex sample 5, in mL; n is the number of nitrogen-containing substances present in nitrogen-containing complex sample 5; C i F represents the content of the i-th nitrogen-containing substance in the nitrogen-containing complex, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in nitrogen-containing complex sample 5, in units of 1; H+ The inorganic acid concentration, expressed in mol / L, is used in the Kjeldahl nitrogen determination of nitrogen-containing complex sample 5. 目 1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the results are shown in Tables 5 and 6.

[0042] Table 5

[0043] Table 6

[0044] A comparison of Tables 5 and 6 shows that the analytical method of the present invention does not require silk fibroin standards, has a shorter detection time, and the results are consistent with the comparative example (difference <0.5%), proving the accuracy of the analytical method of the present invention. However, the analytical method of the present invention does not require silk fibroin standards, which has a significant advantage when standards are difficult to obtain.

[0045] Pharmaceutical field: Determination of metformin hydrochloride content in nitrogen-containing drugs / PVP complexes

[0046] Example 6 In Example 6 below, the protein conversion factor of metformin hydrochloride was calculated based on a molecular weight of 165.62 containing 5 nitrogen atoms, and the protein conversion factor of PVP was calculated based on a molecular weight of 111.14 for the monomer vinylpyrrolidone containing 1 nitrogen atom. An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: A mixed solution of metformin hydrochloride and nitrogen-containing excipient PVP was prepared as nitrogen-containing complex sample 6, wherein the theoretical concentration of metformin hydrochloride was 5.00 mg / mL and the theoretical concentration of PVP was 10.00 mg / mL.

[0047] S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 6 was determined. The protein conversion factor F of metformin hydrochloride was 2.37, and the protein conversion factor F of PVP was 7.14. S2: The content of PVP was determined by high performance liquid chromatography, and the result was C = 9.92 mg / mL; S3: The protein content of nitrogen-containing complex sample 6 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 6 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, namely metformin hydrochloride, in nitrogen-containing complex sample 6. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, metformin hydrochloride, in nitrogen-containing complex sample 6, in mg / g; M represents the mass of nitrogen-containing complex sample 6, in g; V 总 V0 is the total volume of inorganic acid consumed by the nitrogen-containing complex sample 6 during Kjeldahl nitrogen determination, in mL; V0 is the volume of inorganic acid consumed by the blank sample during the Kjeldahl nitrogen determination of nitrogen-containing complex sample 6, in mL; n is the number of nitrogen-containing substances present in nitrogen-containing complex sample 6; C i F represents the content of the i-th nitrogen-containing substance in nitrogen-containing complex sample 6, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in nitrogen-containing complex sample 6, in units of 1; H+ The inorganic acid concentration, expressed in mol / L, is used in the Kjeldahl nitrogen determination of nitrogen-containing complex sample 6. 目 1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the calculated content of metformin hydrochloride was 4.96 mg / mL, the theoretical content was 5.00 mg / mL, and the recovery rate was 99.2%, with an error value of -0.8%.

[0048] Biopharmaceutical field: Determination of protein content in nucleic acid / protein complexes

[0049] Example 7 An analytical method for determining the content of a target nitrogen-containing substance in a nitrogen-containing complex, the method comprising the following steps: A mixed solution of BSA and DNA was prepared as nitrogen-containing complex sample 7, with a theoretical concentration of BSA of 1.00 mg / mL and a theoretical concentration of DNA of 1.50 mg / mL.

[0050] S1: The protein conversion factor of each nitrogen-containing substance in nitrogen-containing complex sample 7 was determined. The protein conversion factor F of BSA was 6.25 and the protein conversion factor F of DNA was 6.956. S2: The DNA content was determined by Qubit dsDNA fluorescence quantitative method, and the result was C = 1.47 mg / mL; S3: The protein content of nitrogen-containing complex sample 7 was determined by the Kjeldahl method. The volume of inorganic acid consumed (V0) of the blank sample and the total volume of inorganic acid consumed (V) of nitrogen-containing complex sample 7 were recorded. 总 ; S4: Substitute into the following function formula to calculate the content C of the target nitrogen-containing substance, BSA, in nitrogen-containing complex sample 7. 目 : ; In the formula, C 目 V represents the content of the target nitrogen-containing substance, BSA, in nitrogen-containing complex sample 7, in mg / g; M represents the mass of nitrogen-containing complex sample 7, in g; V 总 V0 is the total volume of inorganic acid consumed by the nitrogen-containing complex sample 7 during Kjeldahl nitrogen determination, in mL; V0 is the volume of inorganic acid consumed by the blank sample during the Kjeldahl nitrogen determination of nitrogen-containing complex sample 7, in mL; n is the number of nitrogen-containing substances present in nitrogen-containing complex sample 7; C i F represents the content of the i-th nitrogen-containing substance in nitrogen-containing complex sample 7, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in nitrogen-containing complex sample 7, in units of 1; H+ The inorganic acid concentration for nitrogen-containing complex sample 7 when analyzed by the Kjeldahl method is expressed in mol / L; F 目1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen, in g / mmol; n' represents the correction factor for inorganic acids. Three parallel experiments were conducted, and the calculated BSA content was 0.98 mg / mL, the theoretical content was 1.00 mg / mL, and the recovery rate was 98.0%, with an error value of -2.0%.

[0051] The above implementation examples are shown in Table 7: Table 7

[0052] As shown in Table 7, the analytical method of the present invention is applicable to a variety of nitrogen-containing complexes, and none of them require target standard, thus exhibiting good broad-spectrum applicability.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An analytical method for the content of a target nitrogen-containing substance in a nitrogen-containing complex, characterized in that, The analytical method includes the following steps: S1: Determine the protein conversion factor of each nitrogen-containing substance in the nitrogen-containing complex; S2: Detect the content of nitrogen-containing substances other than the target nitrogen-containing substance in the nitrogen-containing complex; S3: The total nitrogen content of the nitrogen-containing complex was determined by the Kjeldahl method, and the total volume of inorganic acid consumed was recorded; S4: Calculate the content of the target nitrogen-containing substance in the nitrogen-containing complex using a preset function formula.

2. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, The formula for the function is as follows: ； In the formula, C 目 V represents the content of the target nitrogen-containing substance in the nitrogen-containing complex, in mg / g; M represents the mass of the nitrogen-containing complex, in g; V 总 V0 is the total volume of inorganic acid consumed by the nitrogen-containing complex when detected by the Kjeldahl method, in mL; V0 is the volume of inorganic acid consumed by the blank sample when the nitrogen-containing complex is detected by the Kjeldahl method, in mL; n is the number of nitrogen-containing substances in the nitrogen-containing complex; C i F represents the content of the i-th nitrogen-containing substance in the nitrogen-containing complex, in mg / g; i C is the protein conversion factor for the i-th nitrogen-containing substance in the nitrogen-containing complex, in units of 1; H+ The concentration of inorganic acid, expressed in mol / L, is used for the detection of nitrogen-containing complexes by the Kjeldahl method; F 目 1 represents the protein conversion factor for the target nitrogenous substance; 0.014 represents the millimolecular mass of nitrogen in g / mmol; n' represents the correction factor for inorganic acids.

3. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, In step S1, the protein conversion factor F is determined in the following way: (a) For nitrogen-containing substances with well-defined chemical structures and known purity, the molecular formula can be used to calculate F = molecular weight / (number of nitrogen atoms in the molecule × 14); (b) For nitrogen-containing substances with unclear chemical structures or where pure samples cannot be obtained, the nitrogen content is determined by the Kjeldahl method. Specifically, a known concentration of the nitrogen-containing substance is taken and its nitrogen content is determined by the Kjeldahl method. F = mass of the nitrogen-containing substance / mass of nitrogen obtained by the Kjeldahl method from the mass of the substance.

4. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, In step S1, the nitrogen-containing complex is one or more of the following: biomedical materials, tissue engineering, medical devices, biological agents, pharmaceuticals, cosmetics, food, feed, textile materials, and environmental samples.

5. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, In step S2, the target nitrogen-containing substance is one or more of the following: protein, polypeptide, amino acid, nucleic acid, alkaloid, nitrogen-containing drug, nitrogen-containing polymer material, nitrogen-containing small molecule metabolite, nitrogen-containing inorganic salt, and nitrogen-containing medical aesthetic active ingredient.

6. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, In step S2, the other nitrogen-containing substances are one or more of the following: sodium hyaluronate, chondroitin sulfate, heparin, collagen, gelatin, chitosan, sodium alginate (containing nitrogen impurities), polyvinylpyrrolidone, and polyamino acids.

7. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 6, characterized in that, In step S2, the detection is obtained by the detection method corresponding to other nitrogen-containing substances in the nitrogen-containing complex besides the target nitrogen-containing substance. The specific detection method includes, but is not limited to: high performance liquid chromatography, gas chromatography, spectrophotometry, enzymatic method, immunoassay, capillary electrophoresis, and mass spectrometry.

8. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 1, characterized in that, The inorganic acid is hydrochloric acid or sulfuric acid.

9. The analytical method for the content of target nitrogen-containing substances in a nitrogen-containing complex according to claim 8, characterized in that, When the inorganic acid is hydrochloric acid, n'=1; when the inorganic acid is sulfuric acid, n'=1 / 2.

10. The analytical method for the content of a target nitrogen-containing substance in a nitrogen-containing complex according to claim 1, characterized in that, The analytical method has a detection error of ≤5% for the target nitrogen-containing substance when there are ≤10 nitrogen-containing components in the nitrogen-containing complex.

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