Heparin precursor content detection method and application thereof

Abstract

The invention provides a heparin precursor content detection method and application thereof. According to the method, the sample concentration is linearly correlated with the product A * theta 360 of the nuclear magnetic resonance hydrogen spectrum peak area A of a characteristic hydrogen signal in a 1H-NMR spectrogram and the 360-degree pulse width theta 360. According to the method, the concentration of the heparin precursor can be directly and rapidly tested and obtained in a nuclear magnetic sample without adding an internal standard substance into the nuclear magnetic sample; specifically, according to the method, the concentrations of the heparin precursors in different systems are measured by utilizing the linear relation between the heparin precursor hydrogen spectrum A * theta 360 and the concentration C of the heparin precursors established in different systems (such as D2O, D2O-H2O or D2O-fermentation liquor), and the accuracy of the measurement result is high. According to the method disclosed by the invention, efficient and rapid quantitative detection of the heparin precursor is realized, and complexity of heparin precursor sample treatment and purification steps in a traditional method is avoided, so that the real-time monitoring capability in the production process is improved, the heparin production efficiency and quality control are optimized, and the method has important application value in heparin industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for detecting the content of a heparin precursor and an application thereof, in particular to a method for detecting the content of a heparin precursor in a fermentation broth. Background Art

[0002] Heparin precursor polysaccharides are glycosaminoglycans, consisting of repeating disaccharide units consisting of alternating glucuronic acid (GlcUA) and acetylglucosamine (GlcNAc). They are found in large quantities in the capsules of various bacteria. Currently, heparin precursors are primarily extracted from Escherichia coli K5. Heparin precursors (heparosan) have a polysaccharide backbone structure similar to heparin / heparan sulfate. Appropriate modification can yield non-animal-derived heparin or its analogs. Therefore, heparin precursors are ideal raw materials for the enzymatic synthesis of heparin. The structural formula of heparin precursors is shown below: .

[0003] Heparin precursors have potential applications in ophthalmology, plastic surgery, dermatology, and medical device coatings. Heparin precursors and their derivatives are also promising candidates for high-quality biomaterials such as gels and scaffolds for tissue engineering and as drug delivery vehicles. Heparin precursors can also be used as drug carriers to enhance the targeting of anticancer drugs, offering significant potential in pharmaceutical development. The efficient and cost-effective production of heparin precursors with controllable molecular weights through microbial fermentation holds significant application value and strategic significance. However, during the fermentation process, heparin precursors can contain a large number of small molecules and produce substances such as acetic acid, which can affect the detection of heparin precursor content in the fermentation broth.

[0004] Currently, there are few reports on the detection and quality control methods for heparin precursor content. Heparin precursor content is generally determined using the sulfuric acid-carbazole method, which is suitable for measuring the uronic acid content in acidic polysaccharides. Acidic polysaccharides are hydrolyzed in concentrated sulfuric acid to produce glucuronic acid, which reacts with carbazole in the presence of sulfuric acid to form a purple-red compound with a carbonyl group. This carbonyl-containing purple-red compound has a maximum absorption at 530 nm. Heparin precursor polysaccharides are composed of acetylglucosamine and glucuronic acid, with the glucuronic acid content being 45.56%. By establishing a standard curve, the heparin precursor content in a sample can be determined based on the glucuronic acid content.

[0005] However, the biggest problem with using the sulfuric acid-carbazole method to measure the content of heparin precursors is its poor specificity and susceptibility to interference from impurities in reagents or samples (heparin precursor analogs, residual culture medium). It has the disadvantages of cumbersome operation, harsh reaction reagents and reaction conditions that are unfriendly to personnel, poor reproducibility, many interference factors, poor specificity and low accuracy.

[0006] 1H-NMR technology can effectively detect different chemical structures in a sample. By distinguishing the chemical shifts of different structures, it can help identify heparin precursors and other impurities. Because the intensity of the NMR signal is proportional to the number of hydrogen atoms in the corresponding structure, this technology can be used not only for qualitative analysis but also for quantitative determination.

[0007] In nuclear magnetic resonance (NMR) quantitative experiments, if the molecule to be measured is an electrolyte, the conductivity of the sample will increase as its concentration increases; however, the increase in conductivity will lead to a decrease in the signal-to-noise ratio of the sample, making the relationship between the sample signal and concentration non-linear. To improve accuracy, an internal standard substance (such as benzyl alcohol) of known concentration is usually added, and the concentration of the target substance is estimated by integrating the signal and combining it with the concentration of the internal standard substance. However, heparin-like substances are biological macromolecules, and their 1 The H-NMR signal distribution is broad and complex, making it difficult to find an internal standard substance that does not overlap with its signal. Furthermore, the internal standard substance may interact with heparin or impurities, affecting the quantitative results. Therefore, the use of NMR technology for the quantification of heparin precursors remains challenging.

[0008] Controlling the content of heparin precursors has always been a key and challenging issue in their development, production, and utilization. Current methods for determining and detecting heparin precursor content have numerous shortcomings. Therefore, there is an urgent need to develop methods for controlling and detecting heparin precursor content to ensure the accuracy of content detection results during fermentation preparation and application. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the present invention aims to provide a method for detecting the content of heparin precursors and its application, in particular a method for detecting the content of heparin precursors in fermentation broth and its application. 1 The characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A and 360° pulse width θ in H-NMR spectrum 360 The product A×θ 360 The method can be used to quickly measure the concentration of heparin precursor in the NMR sample without adding an internal standard substance to the NMR sample. Specifically, the method uses the heparin precursor hydrogen spectrum A×θ established in different systems (such as D2O, D2O-H2O or D2O-fermentation broth) 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in different systems was successfully determined with high accuracy. The method of the present invention achieves efficient and rapid quantitative detection of heparin precursor, avoiding the complexity of heparin precursor sample processing and purification steps in traditional methods, thereby improving the real-time monitoring capability of the production process and optimizing the efficiency and quality control of heparin production. The method has important application value in the industrial production of heparin.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for detecting the content of heparin precursor, the detection method comprising: (S1) The 360° pulse width θ of the heparin precursor reference solution and the heparin precursor test solution were measured by nuclear magnetic resonance spectroscopy. 360 and the characteristic hydrogen signal NMR 1H spectrum peak area A, calculate the characteristic hydrogen signal NMR 1H spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 ; (S2) The least squares method was used for linear regression to establish the relationship between the characteristic hydrogen signal H NMR spectrum peak area A and the 360° pulse width θ of the heparin precursor reference solution. 360 The mathematical relationship between the product Y of the heparin precursor reference solution and the concentration X is Y = kX + b; (S3) Calculating the concentration of the heparin precursor in the heparin precursor sample solution according to the mathematical relationship of step (S2); wherein the calculation formula for the concentration of the heparin precursor in the heparin precursor sample solution is:

[0011] In the above formula, A 供试品 is the characteristic hydrogen signal H NMR spectrum peak area of ​​the heparin precursor test solution; θ 360 is the 360° pulse width of the heparin precursor test solution; k is the slope of the linear equation; b is the intercept of the linear equation; and N is the dilution factor of the heparin precursor test sample solution into the heparin precursor test solution.

[0012] According to an embodiment of the present invention, the heparin precursor test sample solution is diluted to become a heparin precursor test solution, and the dilution factor is N, where N is a number greater than or equal to 1; illustratively, N=the ratio of the volume of the heparin precursor test solution to the volume of the heparin precursor test sample solution.

[0013] According to an embodiment of the present invention, the method further comprises the steps of: (S0) preparing a heparin precursor reference substance into a heparin precursor reference substance solution; and preparing a heparin precursor sample to be tested into a heparin precursor test solution.

[0014] According to an embodiment of the present invention, the method is a simple and rapid nuclear magnetic resonance method for detecting the content of heparin precursors, in particular a rapid and quantitative nuclear magnetic resonance method for detecting the content of heparin precursors in fermentation broth. 1 The characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A and 360° pulse width θ in H-NMR spectrum 360 The product A×θ 360A linear correlation is performed, and the content of heparin precursor can be quickly measured directly in the NMR sample without adding an internal standard substance to the NMR sample.

[0015] According to an embodiment of the present invention, in step (S1), the concentration of heparin precursor in the heparin precursor reference solution is 0.25-20.0 mg / mL; preferably 0.75-15.0 mg / mL, for example, 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL or 20.0 mg / mL.

[0016] According to an embodiment of the present invention, in step (S1), the heparin precursor reference solution includes heparin precursor and deuterated D2O; or the heparin precursor reference solution includes heparin precursor, deuterated D2O and water H2O; or the heparin precursor reference solution includes heparin precursor, deuterated D2O and fermentation broth.

[0017] According to an embodiment of the present invention, in step (S1), when the heparin precursor reference solution includes heparin precursor, deuterated water D2O and water H2O, the volume ratio of deuterated water D2O to water H2O is 5-20:95-80, for example, 5:95, 10:90, 15:85 or 20:80.

[0018] According to an embodiment of the present invention, in step (S1), when the heparin precursor reference solution includes heparin precursor, deuterated D2O and fermentation broth, the volume ratio of deuterated D2O to fermentation broth is 5-20:95-80, for example, 5:95, 10:90, 15:85 or 20:80.

[0019] According to an embodiment of the present invention, the fermentation broth comprises a fermentation supernatant.

[0020] According to an embodiment of the present invention, the fermentation broth does not comprise a heparin precursor.

[0021] According to an embodiment of the present invention, the fermentation broth can be prepared by methods known in the art, or can be purchased through commercial channels; illustratively, the fermentation broth can be prepared by the following method: Ecoli glycerol strains that do not express heparin precursors were fermented in a culture medium and the supernatant was collected.

[0022] According to an embodiment of the present invention, illustratively, the fermentation conditions are known in the art, such as the fermentation conditions satisfying the following conditions: incubation at 37°C and 200 rpm in a shaker for 8-12 hours, followed by incubation at 30°C and 200 rpm for 48 hours. More specifically, 200 mL of sterilized culture medium is added with 3 mL of 50% glycerol (sterilized), followed by the addition of 100 μL of Ecoli glycerol bacteria that do not express heparin precursors; the culture is transferred to a shaker and incubated overnight at 37°C and 200 rpm; the next day, the temperature is increased to 30°C, and incubation is continued for 2 days; after fermentation is complete, the fermentation broth is transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a supernatant; the supernatant is placed in boiling water for 10 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant is obtained to obtain the fermentation broth.

[0023] According to an embodiment of the present invention, the fermentation broth can be any fermentation broth known in the art that can produce a heparin precursor; illustratively, the fermentation broth is LB fermentation broth, SOC fermentation broth or TB fermentation broth.

[0024] According to an embodiment of the present invention, the culture medium selected for the LB fermentation broth is LB culture medium (Luria-Bertani Broth).

[0025] According to an embodiment of the present invention, the LB medium includes peptone, yeast, sodium chloride and water.

[0026] According to an embodiment of the present invention, the LB medium comprises the following components at the following concentrations: 5-30 g / L peptone, 5-25 g / L yeast, and 1-10 g / L sodium chloride, and the solvent is water. Exemplarily, the LB medium comprises the following components at the following concentrations: 16 g / L peptone, 10 g / L yeast, and 5 g / L sodium chloride, and the solvent is water.

[0027] According to an embodiment of the present invention, the culture medium selected for the SOC fermentation broth is SOC culture medium (SuperOptimal Broth with Catabolite repression).

[0028] According to an embodiment of the present invention, the SOC medium includes peptone, yeast, sodium chloride, glucose, magnesium chloride, magnesium sulfate and water.

[0029] According to an embodiment of the present invention, the SOC medium comprises the following components at the following concentrations: 5-30 g / L peptone, 1-10 g / L yeast, 5-25 g / L sodium chloride, 10-30 mM glucose, 5-20 mM magnesium chloride, and 5-20 mM magnesium sulfate, and the solvent is water. Exemplarily, the SOC medium comprises the following components at the following concentrations: 16 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, 20 mM glucose, 10 mM magnesium chloride, and 10 mM magnesium sulfate, and the solvent is water.

[0030] According to an embodiment of the present invention, the culture medium selected for the TB fermentation broth is TB culture medium (Terrific Broth).

[0031] According to an embodiment of the present invention, the TB culture medium includes peptone, yeast, glycerol and / or glucose, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0032] According to an embodiment of the present invention, the TB culture medium comprises the following components at the following concentrations: 10-20 g / L peptone, 20-30 g / L yeast, 2-8 g / L glycerol and / or glucose, 1.5-5 g / L potassium dihydrogen phosphate, and 10-20 g / L dipotassium hydrogen phosphate.

[0033] According to an embodiment of the present invention, in step (S1), the concentration of heparin precursor in the heparin precursor test solution is 0.25-20.0 mg / mL; preferably 0.75-15.0 mg / mL.

[0034] According to an embodiment of the present invention, in step (S1), the heparin precursor reference solution and the heparin precursor test solution have the same composition, that is, when the heparin precursor test solution includes fermentation broth, the heparin precursor reference solution also includes fermentation broth, and the composition of the fermentation broth is the same; when the heparin precursor test solution includes water H2O, the heparin precursor reference solution also includes water H2O; when the heparin precursor test solution includes deuterated water D2O, the heparin precursor reference solution also includes deuterated water D2O.

[0035] According to an embodiment of the present invention, in step (S1), the heparin precursor test solution is prepared by the following method: fermenting Ecoli glycerol bacteria expressing a heparin precursor in a culture medium, collecting the supernatant, and preparing the heparin precursor test sample solution; adding deuterated water (D2O) and optionally adding or not adding a diluent to obtain a heparin precursor test solution having a heparin precursor concentration of 0.25-20.0 mg / mL. The diluent is deuterated water (D2O) or a mixed solution of deuterated water (D2O) and water (H2O), or a fermentation broth containing deuterated water (D2O), wherein the volume ratio of deuterated water (D2O) to water (H2O) is preferably 5-20:95-80, for example, 5:95, 10:90, 15:85, or 20:80; and the volume ratio of deuterated water (D2O) to fermentation broth is 5-20:95-80, for example, 5:95, 10:90, 15:85, or 20:80. If the concentration of the heparin precursor test solution is high (>20.0 mg / mL) after adding heavy water D2O, a diluent may be added to further dilute the concentration of the heparin precursor test solution to obtain a heparin precursor test solution with a heparin precursor concentration of 0.25-20.0 mg / mL.

[0036] Illustratively, deuterated D2O is added to the heparin precursor sample solution to obtain a heparin precursor test solution having a heparin precursor concentration of 0.25-20.0 mg / mL; alternatively, deuterated D2O and a mixed solution of deuterated D2O and water H2O are added to the heparin precursor sample solution to obtain a heparin precursor test solution having a heparin precursor concentration of 0.25-20.0 mg / mL; alternatively, deuterated D2O and a fermentation broth containing deuterated D2O are added to the heparin precursor sample solution to obtain a heparin precursor test solution having a heparin precursor concentration of 0.25-20.0 mg / mL.

[0037] According to an embodiment of the present invention, the fermentation conditions of the Ecoli glycerol bacteria expressing the heparin precursor are the same as the fermentation conditions of the Ecoli glycerol bacteria that do not express the heparin precursor. In the present invention, the Ecoli glycerol bacteria that express the heparin precursor and the Ecoli glycerol bacteria that do not express the heparin precursor differ only in that the Ecoli glycerol bacteria that express the heparin precursor contain genes related to expression of the heparin precursor, while the Ecoli glycerol bacteria that do not express the heparin precursor only contain an empty plasmid.

[0038] According to an embodiment of the present invention, factors affecting the nuclear magnetic resonance signal include magnetic field intensity, temperature, concentration, instrument parameters, and chemical environment. When the magnetic field intensity, temperature, concentration, and instrument parameters are consistent, the nuclear magnetic resonance signals under different solution environments may have large differences. Therefore, in the present invention, it is preferred that the composition of the heparin precursor reference solution and the heparin precursor test solution are the same to ensure consistency of the solution conditions of the heparin precursor reference solution and the heparin precursor test solution, thereby obtaining higher result accuracy.

[0039] As a preferred embodiment, when the heparin precursor test solution includes deuterated D2O and fermentation broth, the heparin precursor reference solution also includes deuterated D2O and fermentation broth, and the fermentation broth in the heparin precursor reference solution has the same composition as the fermentation broth in the heparin precursor test solution, and the volume ratio of deuterated D2O to fermentation broth in the heparin precursor reference solution is the same as the volume ratio of deuterated D2O to fermentation broth in the heparin precursor test solution. This can be achieved by preparing a series of heparin precursor reference solutions with a concentration gradient using fermentation broth that does not contain heparin precursor.

[0040] As a preferred embodiment, when the heparin precursor test solution includes deuterated D2O and water H2O, the heparin precursor reference solution also includes deuterated D2O and water H2O, and the volume ratio of deuterated D2O to water H2O in the heparin precursor reference solution is the same as the volume ratio of deuterated D2O to water H2O in the heparin precursor test solution. This can be achieved by dissolving the heparin precursor in deuterated D2O and water H2O to prepare a series of heparin precursor reference solutions with a concentration gradient.

[0041] As a preferred embodiment, when the heparin precursor test solution includes deuterated D2O, the heparin precursor reference solution also includes deuterated D2O. This can be achieved by dissolving the heparin precursor in deuterated D2O and preparing a series of heparin precursor reference solutions with a concentration gradient.

[0042] According to an embodiment of the present invention, in step (S1), the nuclear magnetic resonance spectrometer used in the nuclear magnetic resonance spectroscopy analysis is a pulsed Fourier transform (PFT) spectrometer; illustratively, the nuclear magnetic resonance spectrometer is a Bruker nuclear magnetic resonance spectrometer.

[0043] According to an embodiment of the present invention, for a 360° pulse width θ of a sample 360 , any pulse sequence can be used for detection (preferably the pulse sequence used for one-dimensional spectrum acquisition in nuclear magnetic resonance), because for the same sample, the 360° pulse width θ 360 In order to facilitate the detection, a relatively simple Zg pulse sequence can be selected for detection.

[0044] According to an embodiment of the present invention, the 360° pulse width θ is measured. 360 The detection conditions of the nuclear magnetic resonance spectroscopy analysis method are shown in Table a below: Table a Detection conditions of nuclear magnetic resonance spectroscopy

[0045] According to a preferred embodiment of the present invention, the 360° pulse width θ is measured. 360 The detection conditions of the nuclear magnetic resonance spectroscopy analysis method are shown in Table b below: Table b Detection conditions of nuclear magnetic resonance spectroscopy

[0046] According to an embodiment of the present invention, the 360° pulse width θ 360 The calculation steps include: collecting the hydrogen spectrum of heparin precursor according to the above detection conditions, then performing Fourier transform (EFP), correcting the phase and baseline, calculating the 90° pulse width to obtain the 90° pulse width P1, and multiplying the P1 value by 4 to obtain the 360° pulse width θ 360 The calculation of the 90° pulse width is achieved by inputting the command "pulsecal".

[0047] According to an embodiment of the present invention, the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area can be measured using a ledbppg2s1d pulse sequence.

[0048] According to an embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table C below: Table c Detection conditions of nuclear magnetic resonance spectroscopy

[0049] According to a preferred embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table d below: Table d Detection conditions of nuclear magnetic resonance spectroscopy

[0050] According to an embodiment of the present invention, the Zgcppr pulse sequence can be used to measure the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum.

[0051] According to an embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table e below: Table e Detection conditions of nuclear magnetic resonance spectroscopy

[0052] According to a preferred embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table f below: Table f Detection conditions of nuclear magnetic resonance spectroscopy

[0053] According to an embodiment of the present invention, the Zggpw5 pulse sequence may be used to measure the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum.

[0054] According to an embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table g below: Table g Detection conditions of nuclear magnetic resonance spectroscopy

[0055] According to a preferred embodiment of the present invention, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are shown in Table h below: Table h Detection conditions of nuclear magnetic resonance spectroscopy

[0056] According to the embodiment of the present invention, in the deuterated water D2O and fermentation broth system, the ledbppg2s1d pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor can be used to determine the concentration of heparin precursor in the deuterated water (D2O) and fermentation broth system. This method is suitable for detecting the heparin precursor content in crude heparin precursor during the preparation process (fermentation process).

[0057] According to the embodiment of the present invention, in the deuterated water D2O and water H2O system, the ledbppg2s1d pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360 The linear relationship between the concentration of heparin precursor and C is established, and thus the concentration of heparin precursor in a system of deuterated water (D2O) and water (H2O) can be determined. This method is suitable for detecting the content of heparin precursor in a prepared heparin precursor product.

[0058] According to the embodiment of the present invention, in the deuterated water D2O system, the ledbppg2s1d pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360The linear relationship between the concentration of heparin precursor C and the heparin precursor concentration C can be used to determine the concentration of heparin precursor in a deuterated water D2O system. This method is suitable for detecting the heparin precursor content in a prepared heparin precursor product.

[0059] According to the embodiment of the present invention, in the deuterated water D2O and water H2O system, the zggpw5 pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360 The linear relationship between the concentration of heparin precursor and C is established, and thus the concentration of heparin precursor in a system of deuterated water (D2O) and water (H2O) can be determined. This method is suitable for detecting the content of heparin precursor in a prepared heparin precursor product.

[0060] According to the embodiment of the present invention, in the deuterated water D2O system, the zggpw5 pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360 The linear relationship between the concentration of heparin precursor C and the heparin precursor concentration C can be used to determine the concentration of heparin precursor in a deuterated water D2O system. This method is suitable for detecting the heparin precursor content in a prepared heparin precursor product.

[0061] According to the embodiment of the present invention, in the deuterated water D2O system, the zgcppr pulse sequence is selected in the nuclear magnetic resonance spectroscopy to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A, and the heparin precursor hydrogen spectrum A×θ is established. 360 The linear relationship between the concentration of heparin precursor C and the heparin precursor concentration C can be used to determine the concentration of heparin precursor in a deuterated water D2O system. This method is suitable for detecting the heparin precursor content in a prepared heparin precursor product.

[0062] According to an embodiment of the present invention, by measuring the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A under the conditions of the ledbppg2s1d pulse sequence, the signal interference of the culture medium components and metabolite small molecules in the fermentation broth can be effectively filtered out, greatly improving the spectral quality of the hydrogen spectrum of the heparin precursor in the fermentation broth; and the feasibility of selecting the ledbppg2s1d pulse sequence for measuring the heparin precursor was verified in deuterated water D2O, deuterated water D2O and water H2O system, and deuterated water D2O and fermentation broth system, and the hydrogen spectra of heparin precursor reference solutions of different concentrations were collected and the 360° pulse width θ was measured. 360 The characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It is linearly related to the concentration of heparin precursor.

[0063] According to the embodiment of the present invention, the feasibility of selecting the zggpw5 pulse sequence for determining heparin precursors was verified in deuterated water D2O, deuterated water D2O, and water H2O systems by measuring the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A under the conditions of the zggpw5 pulse sequence. The hydrogen spectra of heparin precursor reference solutions with different concentrations were collected and the 360° pulse width θ was measured. 360 The characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It is linearly related to the concentration of heparin precursor.

[0064] According to the embodiment of the present invention, the feasibility of selecting the zgcppr pulse sequence to measure heparin precursors was verified in a heavy water D2O system by measuring the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A under the conditions of the zgcppr pulse sequence. The hydrogen spectra of heparin precursor reference solutions with different concentrations were collected and the 360° pulse width θ was measured. 360 The characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 It is linearly related to the concentration of heparin precursor.

[0065] According to an embodiment of the present invention, by comparing the detection effects of different pulse sequences zgcppr, zggpw5 and ledbppg2s1d, the results show that when the pulse sequence is set to ledbppg2s1d, it has a better detection effect on heparin precursors in fermentation broth; in contrast, the pulse sequences zgcpprr and zggpw5 are more suitable for the detection of high-purity heparin precursors, and are not suitable for the detection of fermentation broth.

[0066] According to an embodiment of the present invention, by using the heparin precursor hydrogen spectrum A×θ established in different systems (such as deuterated water D2O, deuterated water D2O-water H2O or deuterated water D2O-fermentation broth), 360 The linear relationship between the concentration of heparin precursor and the fermentation broth was established, and the concentration of heparin precursor in different systems was successfully determined. The determination results were highly accurate, with a recovery rate of more than 99%, which met the detection requirements.

[0067] According to an embodiment of the present invention, the characteristic hydrogen signal of the heparin precursor refers to the hydrogen atom signal of the methyl group on the N-acetyl group (NAc) of the heparin precursor.

[0068] In a second aspect, the present invention provides an application of the method for detecting the content of heparin precursor according to the first aspect in the detection of heparin precursor.

[0069] According to an embodiment of the present invention, the method for detecting the content of the heparin precursor is applied to monitoring the fermentation process of the heparin precursor, so as to determine whether the fermentation is completed.

[0070] According to an embodiment of the present invention, the method for detecting the heparin precursor content is applied to the screening of E. coli with high heparin precursor productivity during the transformation of E. coli, thereby obtaining E. coli with high heparin precursor productivity.

[0071] According to an embodiment of the present invention, the method for detecting the content of heparin precursor is applied to monitoring the preparation and purification processes of heparin precursor, so as to determine whether the purity of the prepared heparin precursor meets the requirements of the quality standards.

[0072] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0073] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for detecting the content of a heparin precursor, and in particular, provides a method for detecting the content of a heparin precursor in a fermentation broth using nuclear magnetic resonance spectroscopy. The detection method can be used to detect the content of a crude heparin precursor in a preparation process (fermentation process) and in a finished heparin precursor product. The detection method has the characteristics of mild operating conditions and high sensitivity. The detection method can efficiently and quickly detect the content of a crude heparin precursor in a preparation process and in a finished heparin precursor product, and is convenient for application in large-scale industrial production.

[0074] (2) The method for detecting the content of heparin precursor provided by the present invention is characterized by high efficiency, rapidity, and strong adaptability. The detection method does not require pretreatment of the sample to be tested, nor does it require special chemical modification or labeling of the sample to be tested, and can quickly and quantitatively detect the content of heparin precursor in the sample to be tested. The detection method is based on the hydrogen spectrum signal obtained from the sample to be tested, thereby analyzing and judging the structure of the sample to be tested, and inferring the presence of impurity analogs. The detection method can overcome the signal interference of other substances in the reaction system (such as culture medium components and metabolite small molecules in the fermentation broth) on the reaction results. The detection method is suitable for high-throughput detection, and the detection method can obtain nuclear magnetic resonance spectra of multiple samples in a short period of time. The detection method is simpler, faster, more efficient, and the detection results are more objective, and is suitable for large-scale industrial production.

[0075] (3) In the experiment of determining the content of heparin precursor by the sulfuric acid-carbazole method, the experimental results show that the purified water group and the blank culture medium group in the control experimental group also have absorbance, indicating that there is an error in the determination of heparin precursor by the sulfuric acid-carbazole method. The nuclear magnetic resonance spectroscopy detection method of the heparin precursor of the present invention can effectively overcome the influence of the introduction of reagents such as blank culture medium and carbazole on the detection results during the experiment. The nuclear magnetic resonance spectroscopy detection method of the heparin precursor of the present invention has higher accuracy, better precision, and can provide more intuitive detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The heparin precursor reference solution (concentration of 20 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by using the ledbppg2s1d pulse sequence in Example 2 is 1 H-NMR spectrum.

[0077] Figure 2 The heparin precursor reference solution (solvent is heavy water D2O) obtained by the Zgcppr pulse sequence in Example 3 is 1 The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0078] Figure 3 The heparin precursor reference solution (solvent is heavy water D2O) obtained by the Zgcppr pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0079] Figure 4 The heparin precursor reference solution (solvent: heavy water D2O) obtained by the Zggpw5 pulse sequence in Example 3 is 1 The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0080] Figure 5 The heparin precursor reference solution (solvent: heavy water D2O) obtained by the Zggpw5 pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0081] Figure 6 The heparin precursor reference solution (solvent is heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 3 is 1The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0082] Figure 7 The heparin precursor reference solution (solvent is heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0083] Figure 8 The heparin precursor reference solution (the solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by the Zggpw5 pulse sequence in Example 3 is 1 The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0084] Figure 9 The heparin precursor reference solution (the solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by the Zggpw5 pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0085] Figure 10 The heparin precursor reference solution (the solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 3 is 1 The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0086] Figure 11 The heparin precursor reference solution (the solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0087] Figure 12 The heparin precursor reference solution (concentration of 5 mg / mL, solvent is an aqueous solution containing 10 vol% heavy water D2O) obtained by using the zg pulse sequence in Example 3 is 1 H-NMR spectrum.

[0088] Figure 13The zggpw5 pulse sequence was used to collect the heparin precursor reference solution (concentration of 5 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) in Example 3. 1 H-NMR spectrum.

[0089] Figure 14 The heparin precursor in Example 3 was prepared under different solvents and pulse sequence conditions. 1 Overlay of H-NMR spectra, where Figure 14 a in Example 3 is the heparin precursor reference solution (concentration of 5 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) collected using the ledbppg2s1d pulse sequence. 1 H-NMR spectrum; Figure 14 b is the heparin precursor reference solution (concentration of 20 mg / mL, solvent is deuterated water D2O) collected using the zggpw5 pulse sequence in Example 3 1 H-NMR spectrum; Figure 14 c is the heparin precursor reference solution (concentration of 5 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) collected using the zggpw5 pulse sequence in Example 3 1 H-NMR spectrum.

[0090] Figure 15 The heparin precursor reference solution (the solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by using the ledbppg2s1d pulse sequence in Example 3 is 1 The result of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C in the H-NMR spectrum.

[0091] Figure 16 The heparin precursor reference solution (the solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by using the ledbppg2s1d pulse sequence in Example 3 is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results are plotted against the heparin precursor concentration, C.

[0092] Figure 17 The heparin precursor reference solution (5 mg / mL, solvent: LB fermentation broth containing 10 vol% heavy water D2O) obtained by using the zgcppr pulse sequence in Example 3 is 1 H-NMR spectrum.

[0093] Figure 18The heavy water D2O and 10vol% D2O-90vol% LB fermentation liquid obtained by using the ledbppg2s1d pulse sequence in Example 4 are 1 H-NMR spectrum.

[0094] Figure 19 The heparin precursor reference solution (concentration of 10.3 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 5 was placed at 4°C for 0 h, 24 h, and 48 h. 1 H-NMR spectrum.

[0095] Figure 20 The heparin precursor reference solution (concentration of 10.3 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 5 was placed at room temperature for 0 h, 24 h, and 48 h. 1 H-NMR spectrum.

[0096] Figure 21 The samples 1 to 6 obtained by using the ledbppg2s1d pulse sequence in Example 6 are 1 H-NMR spectrum. DETAILED DESCRIPTION

[0097] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0098] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0099] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0100] All reagents and solvents used in the present invention are commercially available and can be used without further purification. The E. coli Nissle 1917 used in the present invention was obtained from Beijing CESI Biotechnology Co., Ltd. and is a lyophilized powder.

[0101] In the present invention, the term "content of heparin precursor" unless otherwise defined refers to the concentration of heparin precursor, specifically the mass concentration of heparin precursor.

[0102] In the present invention, the term "360° pulse width" refers to "360° pulse width" unless otherwise defined.

[0103] In this application, the "characteristic hydrogen signal" of a heparin precursor refers to the signal of methyl hydrogen atoms in the NAc of the heparin precursor. Here, A represents the peak area of ​​the H NMR spectrum of the characteristic hydrogen signal of the heparin precursor. Unless otherwise specified, D2O contains 0.002% (w / v) TSP.

[0104] The present invention uses the following abbreviations: mg / mL represents milligrams per milliliter; r represents the linear correlation coefficient, and the larger the r, the better the quantitative ability; R 2 represents the square of the linear correlation coefficient (i.e. r×r), R 2 The larger the value, the better the quantitative ability; RSD stands for relative standard deviation; M stands for mole / liter (mol / L); mM stands for millimole / liter (mmol / L); TSP stands for 2,2,3,3-D4-3-(trimethylsilyl)propionic acid sodium salt; δ stands for the chemical shift value of the atom, in ppm; δ H Represents the chemical shift value of H atoms, in ppm; δ C Represents the chemical shift value of C atom, in ppm; 13 C-NMR ( 13 C-nuclear magnetic resonance) stands for nuclear magnetic resonance carbon spectroscopy; 1 H-NMR ( 1 H-nuclear magnetic resonance) represents the hydrogen nuclear magnetic resonance spectrum; NAc represents the structure of a nitrogen atom connected to an acetyl group (-(CO)-CH3).

[0105] The LB medium used in the following examples (by concentration) contains: 16 g / L peptone, 5 g / L yeast, and 10 g / L sodium chloride, with water as the solvent. This is a culture medium for Escherichia coli and is used to prepare heparin precursors.

[0106] The LB medium used in the following examples was prepared as follows: 16 g of peptone, 10 g of sodium chloride, and 5 g of yeast powder were weighed into a beaker, added to 1 L of ultrapure water, and after complete dissolution, the mixture was divided into shake flasks, each containing 50 mL. After sealing, the flasks were placed in an autoclave for sterilization at 121°C for 30 min.

[0107] Detection conditions of nuclear magnetic resonance spectroscopy: Table 1 The 360° pulse width θ measured using the Zg pulse sequence 360 Detection conditions of nuclear magnetic resonance spectroscopy

[0108] Table 2 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum using the Zgcppr pulse sequence

[0109] Table 3 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum using the Zggpw5 pulse sequence

[0110] Table 4 Detection conditions of the nuclear magnetic resonance spectroscopy method for measuring the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum using the LEDBPPG2S1D pulse sequence

[0111] Example 1 Preparation of Heparin Precursor Reference Substance Heparin precursor was prepared by the same method as that disclosed in Example 1 of Chinese patent document CN119595804A, and used as a reference substance in the study of the method for detecting the content of heparin precursor in the present application.

[0112] Example 2 Detection Method of Heparin Precursor Content 1. Instruments and analytical conditions The NMR spectrometer (Bruker AVIII 600 MHz) was equipped with a QCI ultracold probe and the software version was Topspin 3.0.

[0113] 2. Preparation of reference solution and test solution Diluent: LB fermentation broth containing 10 vol% heavy water D2O (10 vol% D2O-90 vol% LB fermentation broth).

[0114] LB broth refers to a fermentation broth that does not contain heparin precursors. It is prepared by adding 3 mL of 50% glycerol (sterilized) to 200 mL of sterilized LB medium, followed by 100 μL of Ecoli glycerol strains that do not express heparin precursors. The culture is transferred to a shaker and incubated overnight at 37°C and 200 rpm. The next day, the temperature is increased to 30°C and incubated for another two days. After fermentation is complete, the broth is transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant. The supernatant is then placed in boiling water for 10 minutes and centrifuged at 10,000 rpm for 10 minutes. The supernatant is then collected to obtain the LB broth.

[0115] LB fermentation broth containing 10 vol% deuterated D2O: Place 1 mL of deuterated D2O (containing 0.002% (W / V) TSP) in a 10 mL volumetric flask, add the LB fermentation broth prepared above, sonicate and dilute to the mark, and shake well to obtain a 10 vol% D2O-90 vol% LB fermentation broth.

[0116] Preparation of a heparin precursor reference solution (solvent: 10 vol% D2O-90 vol% LB fermentation broth): Accurately weigh 400 mg of the heparin precursor prepared in Example 1 and place it in a 10 mL volumetric flask. Add 10 vol% D2O-90 vol% LB fermentation broth, sonicate, and dilute to volume. Shake well to obtain a 40.0 mg / mL heparin precursor stock solution. Based on this heparin precursor stock solution, heparin precursor reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.5 mg / mL, 5.0 mg / mL, 2.5 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.625 mg / mL, and 0.25 mg / mL were prepared.

[0117] Preparation of heparin precursor test solution: Accurately measure 9 mL of the liquid sample to be tested containing the heparin precursor and 1 mL of heavy water D2O, mix well, and optionally add or not add 10 vol% D2O-90 vol% LB fermentation broth to obtain the heparin precursor test solution.

[0118] The heparin precursor-containing liquid sample to be tested is a liquid sample obtained during the fermentation process of the heparin precursor. Specifically, it is prepared as follows: 200 mL of sterilized LB medium is added with 3 mL of 50% glycerol (sterilized), followed by the addition of 100 μL of Ecoli glycerol bacteria expressing the heparin precursor. The culture is transferred to a shaker and incubated overnight at 37°C and 200 rpm. The next day, the temperature is increased to 30°C, and incubation is continued for two days. Samples obtained at any time during this two-day incubation period constitute the heparin precursor-containing liquid sample to be tested.

[0119] 3. Experimental process and results Take 0.6 mL of the above-prepared gradient concentration heparin precursor reference solution and heparin precursor test solution, transfer them into NMR tubes, sonicate for 5 minutes, and perform 360° pulse width θ in NMR spectrometer. 360 Determination and 1 Acquisition of H-NMR spectra.

[0120] The heparin precursor reference solution was tested using the test conditions (Zg pulse sequence) as shown in Table 1 above. 1 The H-NMR spectrum is collected, Fourier transformed (efp), and phase and baseline corrected. Then, the command "pulsecal" is entered to calculate the 90° pulse width. After the calculation is completed, the software will give the calculated 90° pulse width P1 for the sample. Multiplying this P1 value by 4 gives the 360° pulse width θ. 360 .

[0121] The heparin precursor reference solution was tested using the detection conditions (ledbppg2s1d pulse sequence) as shown in Table 4 above. 1 H-NMR spectra were collected. 1 After the H-NMR spectrum is acquired, Fourier transform (EFP) and phase and baseline corrections are performed. The hydrogen signal of the methyl group on the N-acetyl group (NAc) of the heparin precursor is integrated over a range of 2.01-2.09 ppm. The integrated result is derived as the characteristic hydrogen signal H NMR spectrum peak area A.

[0122] Each heparin precursor control solution 1 The peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) in the H-NMR spectrum and the corresponding 360° pulse width θ 360 Multiply to establish the characteristic hydrogen signal NMR hydrogen spectrum peak area A and 360° pulse width θ 360 The mathematical relationship between the product and the reference substance concentration is Y=kX+b, and the results are shown in Table 5.

[0123] Table 5 Results of A value detection by ledbppg2s1d pulse sequence (LB fermentation broth)

[0124] Take 0.6 mL of the heparin precursor test solution and transfer it into an NMR tube. After ultrasonication for 5 minutes, measure its 360° pulse width θ according to the above method. 360 and 1 H-NMR spectrum, detection 1 The peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) in the H-NMR spectrum is used to calculate the peak area A of the characteristic hydrogen signal NMR spectrum and the 360° pulse width θ. 360 The product of .

[0125] Using the mathematical relationship Y=2.76×10 7 X+1.16×10 7 Calculate the concentration of heparin precursor in the heparin precursor sample solution according to the following formula: 肝素前体 .

[0126] The formula for calculating the concentration of heparin precursor in the heparin precursor test sample is:

[0127] In the above formula, A 供试品 is the peak area of ​​methyl hydrogen atoms in NAc of the H NMR spectrum of the heparin precursor test solution; θ 360 is the 360° pulse width of the heparin precursor test solution; k is the slope of the linear equation; b is the intercept of the linear equation; and N is the dilution factor of the heparin precursor test solution, that is, the ratio of the volume of the heparin precursor test solution to the volume of the heparin precursor sample solution.

[0128] Figure 1 The heparin precursor reference solution (concentration of 20 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by using the ledbppg2s1d pulse sequence in Example 2 is 1 H-NMR spectrum. Figure 1 It can be seen that the use of the LEDBPPG2S1D pulse sequence for nuclear magnetic resonance spectroscopy analysis can effectively filter out the signal interference of culture medium components and metabolite small molecules in the fermentation broth, greatly improving the spectral quality of the hydrogen spectrum of heparin precursor in the fermentation broth, thereby obtaining accurate test results of the heparin precursor content in the fermentation broth.

[0129] Example 3 Optimization experiment of nuclear magnetic resonance conditions The purpose of this example is to investigate the effects of different solvents and NMR pulse sequences on the detection results, thereby screening suitable detection conditions for rapid and quantitative detection of heparin precursor in mixtures such as fermentation broth containing heparin precursor and heparin precursor purification intermediates.

[0130] (1) Investigate the effects of various NMR pulse sequences on the detection results under D2O conditions Heparin precursor test samples are often heparin precursor fermentation broth, heparin precursor process intermediates, or heparin precursor crude products, which contain a large amount of aqueous solvent. This example mainly examines the effects of various NMR pulse sequences on the test results in the presence of D2O, i.e., without the influence of other solvent impurities, as well as the relationship between concentration C and peak area A, A×θ 360 The degree of linear correlation.

[0131] 1. Instruments and analytical conditions: The NMR spectrometer (Bruker AVIII 600 MHz) was equipped with a QCI ultracold probe and the software version was Topspin 3.0.

[0132] 2. Preparation of reference solution Preparation of a heparin precursor reference solution (solvent: deuterated water, D2O): 100 mg of the heparin precursor prepared in Example 1 was added to 2 mL of D2O (containing 0.002% (w / v) TSP). After complete dissolution, a 50 mg / mL heparin precursor stock solution was prepared. Based on this heparin precursor stock solution, heparin precursor reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.0 mg / mL, 5.0 mg / mL, 2.5 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.5 mg / mL, and 0.25 mg / mL were prepared.

[0133] 3. Experimental process and results The 360° pulse width θ of the heparin precursor reference solution was measured according to the method in Example 2. 360 (Use Zg pulse sequence for measurement) and select Zgcppr pulse sequence, Zggpw5 pulse sequence, ledbppg2s1d pulse sequence to collect the heparin precursor control solution 1 The H-NMR spectrum is used to determine the peak area A of the characteristic hydrogen signal of the heparin precursor. The product of the peak area of ​​the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width is A×θ 360 A linear regression equation was drawn for the concentration (C, mg / ml) of the heparin precursor reference solution, and a linear regression equation was drawn for the characteristic hydrogen signal H NMR spectrum peak area A versus the concentration (C, mg / ml). Under different pulse sequence conditions, the characteristic hydrogen signal H NMR spectrum peak area A, 360° pulse width θ corresponding to each sample 360 The linear regression equations are shown in Table 6, Table 7 and Table 8 respectively.

[0134] Table 6 Results of Zgcppr pulse sequence detection of A value (D2O)

[0135] The heparin precursor reference solution acquired by Zgcppr pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 2 As shown, the heparin precursor control solution obtained by Zgcppr pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 3 As shown. Figure 2 and Figure 3 It can be seen from the comparison that the heparin precursor obtained by Zgcppr pulse sequence 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=8.49×10 7 X+1.31×10 7 , R 2 = 0.998; Zgcppr pulse sequence acquisition of heparin precursor 1 The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of A versus concentration C is Y = 1.63 × 10 6 X+1.83×10 6 , R 2 =0.986.

[0136] Table 7 Results of Zggpw5 pulse sequence detection of A value (D2O)

[0137] The heparin precursor reference solution acquired by Zggpw5 pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 4 As shown, the heparin precursor control solution obtained by Zggpw5 pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 5 As shown. Figure 4 and Figure 5 The comparison shows that the heparin precursor obtained by Zggpw5 pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=1.50×10 9 X-1.06×10 8 , R 2 =0.999; the heparin precursor obtained by Zggpw5 pulse sequence 1The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of A versus concentration C is Y = 2.88 × 10 7 X+2.31×10 7 , R 2 =0.991.

[0138] Table 8 Results of A value detection by ledbppg2s1d pulse sequence (D2O)

[0139] The heparin precursor control solution obtained by the ledbppg2s1d pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 6 As shown, the heparin precursor control solution obtained by the ledbppg2s1d pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 7 As shown. Figure 6 and Figure 7 The comparison shows that the heparin precursor obtained by the ledbppg2s1d pulse sequence 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=8.49×10 7 X+1.31×10 7 , R 2 = 0.998; the heparin precursor obtained by the ledbppg2s1d pulse sequence 1 The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of A versus concentration C is Y = 1.63 × 10 6 X+1.83×10 6 , R 2 =0.986.

[0140] From the above test results, it can be seen that Figure 2 、 Figure 4 and Figure 6 By comparison, the heparin precursor reference solution obtained by the zgcppr, zggpw5, ledbppg2s1d pulse sequence in D2O is1 In the H-NMR spectrum, the linear relationship between the peak area A of the characteristic hydrogen signal and the heparin precursor concentration C is relatively poor. Figure 3 、 Figure 5 and Figure 7 By comparison, the heparin precursor reference solution obtained by the zgcppr, zggpw5 and ledbppg2s1d pulse sequences in D2O 1 H-NMR spectrum, the product of the peak area of ​​the characteristic hydrogen signal and the 360° pulse width A×θ 360 It has a good linear relationship with the concentration C of heparin precursor, R 2 Greater than or equal to 0.998.

[0141] (2) Investigate the effects of various NMR pulse sequences on the test results under the conditions of 10 vol% D2O-90 vol% H2O Heparin precursor test samples are often heparin precursor fermentation broth, heparin precursor process intermediates, or heparin precursor crude products, which contain a large amount of aqueous solvent. This example mainly examines the effects of various NMR pulse sequences on the test results in the presence of H2O, as well as the relationship between concentration C and peak area A, A×θ 360 The degree of linear correlation.

[0142] The specific choice of conducting the experiment under the conditions of 10 vol% D2O-90 vol% H2O is because the ultimate goal of the method developed in this study is to detect the concentration of heparin precursor in the fermentation broth. When conducting nuclear magnetic resonance experiments, the simplest sample preparation method is to add 10 vol% D2O to the fermentation broth containing heparin precursor, rather than freeze-drying the fermentation broth containing heparin precursor and then re-dissolving it with 100% D2O.

[0143] 1. Instruments and analytical conditions: The NMR spectrometer (Bruker AVIII 600 MHz) was equipped with a QCI ultracold probe and the software version was Topspin 3.0.

[0144] 2. Preparation of reference solution Preparation of 10 vol% D2O-90 vol% H2O: Take 1 mL of D2O (containing 0.002% (W / V) TSP) in a 15 mL centrifuge tube, add 9 mL of H2O, and mix to obtain 10 vol% D2O-90 vol% H2O.

[0145] Preparation of a heparin precursor reference solution (solvent: aqueous solution containing 10 vol% deuterated D2O): 100 mg of the heparin precursor prepared in Example 1 was added to 2 mL of 10 vol% D2O-90 vol% H2O. After complete dissolution, a 50 mg / mL heparin precursor stock solution was prepared. Based on this heparin precursor stock solution, heparin precursor reference solutions with concentrations of 20.0 mg / mL, 15.0 mg / mL, 10.0 mg / mL, 7.5 mg / mL, 5.0 mg / mL, 3.75 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL were prepared.

[0146] 3. Experimental process and results The 360° pulse width θ of the heparin precursor reference solution was measured according to the method in Example 2. 360 .

[0147] Because the water peak signal was too strong under the conditions of 10 vol% D2O-90 vol% H2O, the zgcppr pulse sequence could not effectively suppress the water peak signal, resulting in a poor signal. Therefore, only the zggpw5 pulse sequence and the ledbppg2s1d pulse sequence were used to collect the heparin precursor reference solution. 1 The H-NMR spectrum is used to determine the peak area A of the characteristic hydrogen signal of the heparin precursor. The product of the peak area of ​​the characteristic hydrogen signal of the H-NMR spectrum and the 360° pulse width is A×θ 360 A linear regression equation was drawn for the concentration (C, mg / ml) of the heparin precursor reference solution, and a linear regression equation was drawn for the peak area A versus the concentration (C, mg / ml). Under different pulse sequence conditions, the characteristic hydrogen signal H NMR spectrum peak area A, 360° pulse width θ corresponding to each sample 360 The linear regression equations are shown in Tables 9 and 10, respectively.

[0148] Table 9 Results of Zggpw5 pulse sequence detection of A values ​​(10 vol% D2O-90 vol% H2O)

[0149] The heparin precursor reference solution acquired by Zggpw5 pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 8 As shown, the heparin precursor control solution obtained by Zggpw5 pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 9 As shown. Figure 8 and Figure 9 The comparison shows that the heparin precursor obtained by Zggpw5 pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=1.94×10 9 X-7.39×10 8 , R 2 =0.999; the heparin precursor obtained by Zggpw5 pulse sequence 1 The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of A versus concentration C is Y = 3.74 × 10 7 X+1.88×10 7 , R 2 =0.992.

[0150] Table 10 Results of A value detection by ledbppg2s1d pulse sequence (10 vol% D2O-90 vol% H2O)

[0151] The heparin precursor control solution obtained by the ledbppg2s1d pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 10 As shown, the heparin precursor control solution obtained by the ledbppg2s1d pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 11 As shown. Figure 10 and Figure 11 The comparison shows that the heparin precursor obtained by the ledbppg2s1d pulse sequence 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=6.95×10 7 X-4.29×10 7 , R 2 = 0.999; heparin precursor obtained by ledbppg2s1d pulse sequence1 The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of the peak area A to the concentration C is Y = 1.34 × 10 6 X+2.32×10 5 , R 2 =0.997.

[0152] From the above test results, it can be seen that Figure 8 and Figure 10 By comparison, the heparin precursors obtained by the zggpw5 pulse sequence and the ledbppg2s1d pulse sequence under the conditions of 10 vol% D2O-90 vol% H2O are 1 In the H-NMR spectrum, the linear relationship between the peak area A of the characteristic hydrogen signal and the concentration C of the heparin precursor is relatively poor. The peak area A of the characteristic hydrogen signal increases more and more slowly with the increase of the concentration C of the heparin precursor. Figure 9 and Figure 11 By comparison, the heparin precursor obtained by zggpw5 and ledbppg2s1d pulse sequences under 10 vol% D2O-90 vol% H2O conditions is 1 H-NMR spectrum, the product of the peak area of ​​the characteristic hydrogen signal and the 360° pulse width A×θ 360 It has a good linear relationship with the heparin precursor concentration C, R 2 Greater than or equal to 0.999.

[0153] This test also investigated the heparin precursor reference solution (solvent: 10 vol% D2O-90 vol% H2O) acquired using the zg pulse sequence. 1 H-NMR spectrum, test results are as follows Figure 12 As shown. Figure 12 It can be seen from the figure that under the conditions of 10 vol% D2O-90 vol% H2O, the water peak signal is too strong, so the zg pulse sequence can only obtain the water signal, and the characteristic hydrogen signal of the heparin precursor cannot be obtained.

[0154] (3) Investigate the effects of various NMR pulse sequences on the detection results under 10 vol% D2O-90 vol% LB fermentation broth conditions Heparin precursor fermentation broth is fermented by various bacteria, producing a large amount of small molecules such as acetic acid. This example introduces 10 vol% D2O-90 vol% LB fermentation broth as a solvent. The main purpose is to investigate the effects of various NMR pulse sequences on the detection results in the presence of H2O and a large amount of acetic acid and other impurities, as well as the relationship between concentration C and peak area A, A×θ 360 The degree of linear correlation.

[0155] 1. Instruments and analytical conditions: The NMR spectrometer (Bruker AVIII 600 MHz) was equipped with a QCI ultracold probe and the software version was Topspin 3.0.

[0156] 2. Preparation of reference solution LB fermentation broth: LB fermentation broth (fermentation broth without heparin precursor) was prepared using the method of Example 1.

[0157] 10 vol% D2O-90 vol% LB fermentation broth: Add 9 mL of the above LB fermentation broth and 1 mL of D2O (containing 0.002% (w / v) TSP) to a 15 mL centrifuge tube and mix well to obtain 10 vol% D2O-90 vol% LB fermentation broth.

[0158] Preparation of a heparin precursor reference solution (the solvent is LB fermentation broth containing 10 vol% heavy water (D2O)): 4 mg of the heparin precursor prepared in Example 1 was added to 1 mL of 10 vol% D2O-90 vol% LB fermentation broth. After complete dissolution, a heparin precursor reference solution with a concentration of 4 mg / mL was prepared.

[0159] 3. Experimental process and results The 360° pulse width θ of the heparin precursor reference solution was measured according to the method in Example 2. 360 .

[0160] Since the water peak signal was too strong under the conditions of 10 vol% D2O-90 vol% LB fermentation broth, the zgcppr pulse sequence could not effectively suppress the water peak signal, resulting in poor signal acquisition. Therefore, only the zggpw5 pulse sequence and the ledbppg2s1d pulse sequence were used to acquire the heparin precursor reference solution. 1 H-NMR spectrum, measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A of the heparin precursor.

[0161] The zggpw5 pulse sequence was used to collect the heparin precursor reference solution. 1 H-NMR spectrum, test results are as follows Figure 13 As shown. Figure 13 The observation and analysis showed that the heparin precursor reference solution (solvent is 10vol% D2O-90vol% LB fermentation broth) 1The H-NMR spectrum exhibits a strong signal peak near a chemical shift of 1.98 ppm. This signal peak is sharp and has a narrow half-width (FWHM). This overly strong signal is presumably derived from small molecules in the LB fermentation broth, such as glycerol and acetic acid. The abundant methyl acetate hydrogen signal peak at a chemical shift of 1.98 ppm buries the characteristic hydrogen signal peak of the heparin precursor (near a chemical shift of 2.04 ppm), making it impossible to accurately measure the peak area of ​​the characteristic hydrogen signal of the heparin precursor in the H-NMR spectrum. Consequently, the zggpw5 pulse sequence cannot be used to detect the content of the heparin precursor in mixtures such as fermentation broth.

[0162] The heparin precursor solution was collected using the ledbppg2s1d pulse sequence. 1 H-NMR spectrum, test results are as follows Figure 14 As shown in a in . Figure 14 The figure also shows the heparin precursor reference solution (concentration 20 mg / mL, solvent is deuterated water D2O) acquired using the zggpw5 pulse sequence. 1 H-NMR spectrum (such as Figure 14 b); and the heparin precursor reference solution (concentration of 5 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) collected using the zggpw5 pulse sequence 1 H-NMR spectrum (such as Figure 14 c). By Figure 14 From the observation and analysis, it can be seen that the majority of small molecule signals in the hydrogen spectrum (green) of the heparin precursor reference solution (solvent is 10 vol% D2O-90 vol% LB fermentation broth) collected using the ledbppg2s1d pulse sequence are filtered out, and the characteristic hydrogen signals of the heparin precursor can be clearly observed. The chemical shifts of these characteristic hydrogen signals are consistent with the chemical shifts of the characteristic hydrogen signals of the heparin precursor in D2O (see Figure 14 a and b in ), indicating Figure 14 The characteristic hydrogen signal (chemical shift 2.04 ppm) remaining in image a is the characteristic hydrogen signal of heparin precursor in LB fermentation broth. Although some other signals remain, they do not overlap with the characteristic hydrogen signal of heparin precursor and do not affect the quantification of heparin precursor. Therefore, the LEDBPPG2S1D pulse sequence can be used to determine the concentration of heparin precursor in fermentation broth.

[0163] Through Figure 14 The comparative analysis shows that the heparin precursor reference solution (the solvent is LB fermentation broth containing 10 vol% heavy water D2O) can collect characteristic hydrogen signal peaks (such as Figure 14 a in the figure), but it is not possible to collect the characteristic hydrogen signal peak under the zggpw5 pulse sequence conditions (such as Figure 14c in the figure), while the zggpw5 pulse sequence can be used to collect characteristic hydrogen signal peaks (such as Figure 14 b) in the above example.

[0164] The heparin precursor control solution obtained by the ledbppg2s1d pulse sequence 1 The results of plotting the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A against the heparin precursor concentration C are shown in the figure below. Figure 15 As shown, the heparin precursor control solution obtained by the ledbppg2s1d pulse sequence is 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 The results of plotting the heparin precursor concentration C are shown in Figure 2. Figure 16 As shown. Figure 15 and Figure 16 The comparison shows that the heparin precursor obtained by the ledbppg2s1d pulse sequence 1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 There is a good linear relationship between the A×θ and the heparin precursor concentration C. 360 The linear regression equation (ten points) for concentration C is Y=2.76×10 7 X+1.16×10 7 , R 2 = 0.999; heparin precursor obtained by ledbppg2s1d pulse sequence 1 The linear relationship between the peak area A of the characteristic hydrogen signal of the H-NMR spectrum and the heparin precursor concentration C is relatively poor. The linear regression equation (ten points) of A versus concentration C is Y = 4.33 × 10 5 X+5.21×10 5 , R 2 =0.998.

[0165] This test also examined the heparin precursor reference solution (solvent: 10 vol% D2O-90 vol% LB fermentation broth) obtained by using the zgcppr pulse sequence. 1 H-NMR spectrum, test results are as follows Figure 17 As shown. Figure 17 It can be seen that under the conditions of 10 vol% D2O-90 vol% LB fermentation broth, the water peak signal is too strong, and the zgcppr pulse sequence can only obtain the water signal, and the characteristic hydrogen signal of the heparin precursor cannot be obtained.

[0166] In summary, under D2O-fermentation conditions, the heparin precursor was obtained by using the ledbppg2s1d pulse sequence.1 The characteristic hydrogen signal of the H-NMR spectrum is the product of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum and the 360° pulse width A×θ 360 It is linearly related to the heparin precursor concentration C; at the same time, since the ledbppg2s1d sequence is added with a diffusion sequence, the small molecule signals in the heparin precursor sample to be tested can be filtered out, which can suppress the water peak signal on the one hand and filter out the signals of other small molecules on the other hand, thereby increasing the quality of the sample spectrum.

[0167] The heparin precursor was obtained by using the ledbppg2s1d pulse sequence under D2O and D2O-H2O conditions. 1 The results of H-NMR spectrum are consistent with those of zgcppr pulse sequence and zggpw5 pulse sequence. The linear relationship between the peak area A of characteristic hydrogen signal nuclear magnetic resonance spectrum and the concentration C of heparin precursor is relatively poor, and with the increase of concentration, the growth of the peak area A of characteristic hydrogen signal nuclear magnetic resonance spectrum becomes slow. The product of the peak area of ​​characteristic hydrogen signal nuclear magnetic resonance spectrum and 360° pulse width A×θ 360 It has a good linear relationship with the concentration C of heparin precursor, R 2 Greater than or equal to 0.998, it can be used for the quantitative detection of heparin precursors in fermentation broth and preparation intermediates.

[0168] The study of Example 3 shows that the present invention has successfully developed a method for direct and rapid quantitative testing of heparin precursor content based on nuclear magnetic resonance (NMR) technology; the detection method is to establish a relationship between the sample concentration C and its 1 The characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A and 360° pulse width θ in H-NMR spectrum 360 The product A×θ 360 The linear relationship between A×θ and θ was found to be consistent with that between A×θ and θ. 360 R of the linear regression equation for concentration C 2 (square of linear correlation coefficient) are higher than the R of the linear regression equation of the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A and concentration C 2 , indicating that A×θ 360 The linear regression equation for concentration C has better quantitative ability.

[0169] The present invention significantly eliminates the interference of small molecular impurities (such as glycerol, acetic acid, etc.) in the fermentation broth on the heparin precursor signal by introducing the LEDBPPG2S1D pulse sequence in the NMR experiment, enhances the clarity and accuracy of the identification of the characteristic hydrogen signal, and successfully establishes the product A×θ of the peak area of ​​the nuclear magnetic resonance hydrogen spectrum of the characteristic hydrogen signal and the 360° pulse width. 360 The linear relationship between the concentration of heparin precursor and the HPLC indicated that the method was suitable for the quantitative analysis of heparin precursor.

[0170] The results of experiments (1), (2) and (3) show that in D2O, the zg pulse sequence, zgcppr pulse sequence, zggpw5 pulse sequence and ledbppg2s1d pulse sequence can all observe the characteristic hydrogen signal of the heparin precursor; under D2O-H2O conditions, the zg pulse sequence cannot observe the characteristic hydrogen signal of the heparin precursor, the zgcppr pulse sequence can observe the characteristic hydrogen signal of the heparin precursor, but the signal is poor, the zggpw5 pulse sequence and ledbppg2s1d pulse sequence can better observe the characteristic hydrogen signal of the heparin precursor; under D2O-fermentation broth conditions, due to the excessively strong signals of water molecules and small molecules such as acetic acid and glycerol, the zg pulse sequence, zgcppr pulse sequence and zggpw5 pulse sequence cannot observe the characteristic hydrogen signal of the heparin precursor, the ledbppg2s1d pulse sequence can better observe the characteristic hydrogen signal of the heparin precursor.

[0171] Example 4 Investigation of the specificity of the heparin precursor content determination method The purpose of this example is to verify that the method for determining the content of heparin precursor of the present invention is not interfered with by blank solution or culture medium, so as to prove that the specificity of the method for determining the content of heparin precursor is good.

[0172] 1. Instruments and analytical conditions Same as Example 2.

[0173] 2. Solution preparation Blank solution 1: heavy water D2O (containing 0.002% (W / V) TSP).

[0174] Blank solution 2: 10 vol% D2O-90 vol% LB fermentation broth, prepared in the same manner as in Example 2.

[0175] 3. Experimental process Take 0.6 mL of blank solution 1 and blank solution 2 respectively, transfer them into NMR tube, ultrasonicate for 5 min, and collect the NMR of blank solution 1 and blank solution 2 according to the pulse sequence of Table 4 of Example 2. 1 H-NMR spectrum.

[0176] 4. Acceptable standards Blank solution 1 and blank solution 2 1 The H-NMR spectrum showed no signal peak at 2.04 ppm for the N-acetylmethyl group.

[0177] 5. Experimental results Figure 18The blank solution 1 (deuterated water D2O) and blank solution 2 (10 vol% D2O-90 vol% LB fermentation broth) obtained by using the ledbppg2s1d pulse sequence in Example 4 are 1 H-NMR spectrum. Figure 18 It can be seen that blank solution 1 and blank solution 2 do not show obvious signal peaks at the chemical shift value of 2.04 ppm, that is, blank solution 1 and blank solution 2 do not have peaks at 2.04 ppm (characteristic hydrogen chemical shift of NAc methyl), indicating that blank solution 1 and blank solution 2 have no interference at the signal position of heparin precursor N-acetyl, proving that the method has good specificity.

[0178] Example 5 Investigation of Solution Stability of Heparin Precursor Detection Method The purpose of this example is to determine the extent to which the test results of the heparin precursor reference solution used in the assay method of the present invention are not affected after being left for a period of time under certain conditions.

[0179] The storage temperature and storage time of the prepared heparin precursor reference solution were investigated.

[0180] Acceptance criteria: RSD of peak area of ​​all samples ≤ 1.0%.

[0181] 1. Instruments and analytical conditions Same as Example 2.

[0182] 2. Preparation of reference solution A heparin precursor reference solution with a concentration of 10.3 mg / mL was prepared using the method of Example 2 (the solvent was 10 vol% D2O-90 vol% LB fermentation broth).

[0183] 3. Experimental process and results Take 0.6 mL of the prepared heparin precursor reference solution and transfer it to a nuclear magnetic resonance tube to prepare a test sample. Prepare 6 test samples in parallel. The 6 test samples prepared above were evenly divided into 2 groups (3 samples in each group). The 2 groups of samples were placed at room temperature and 4 ° C, respectively. One test sample was taken from each group at 0 h, 24 h, and 48 h for nuclear magnetic resonance detection. The same method as in Example 2 was used to perform 360° pulse width θ in the nuclear magnetic resonance spectrometer. 360 Determination and 1 H-NMR spectrum acquisition, record each sample 1 The peak area A of the NAc methyl hydrogen signal (characteristic hydrogen signal) in the H-NMR spectrum is used to investigate θ 360 The test results of the product of A are shown in Table 11.

[0184] Table 11 Results of investigation on storage temperature and storage time of heparin precursor reference solution

[0185] Figure 19 The heparin precursor reference solution (concentration of 10.3 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 5 was placed at 4°C for 0 h, 24 h, and 48 h. 1 H-NMR spectrum. Figure 20 The heparin precursor reference solution (concentration of 10.3 mg / mL, solvent is LB fermentation broth containing 10 vol% heavy water D2O) obtained by the ledbppg2s1d pulse sequence in Example 5 was placed at room temperature for 0 h, 24 h, and 48 h. 1 H-NMR spectrum. The above experiments and results show that compared with the 0h at room temperature, the θ 360 The product value of A is RSD≤1.0%; compared with the solution placed at 4℃ for 0h, the θ of each heparin precursor reference solution is 360 The product value of A is RSD≤1.0%. It can be seen that the heparin precursor reference solution is stable at room temperature and 4°C within 48 hours, indicating that the heparin precursor reference solution of the present invention can be effectively and accurately used for testing the sample content within 48 hours.

[0186] Example 6 Experiment on the Repeatability of the Detection Method of Heparin Precursor Content This example is to determine the reproducibility of multiple measurement results of the same batch of samples by the same operator.

[0187] 1. Instruments and analytical conditions Same as Example 2.

[0188] 2. Preparation of reference solution and test solution A heparin precursor reference solution with a concentration of 9.6 mg / mL was prepared using the same method as in Example 2 and divided into 6 portions in parallel to serve as heparin precursor test solutions.

[0189] 3. Experimental process and results Take 0.6 mL of the above heparin precursor test solution, transfer it into the NMR tube, and after ultrasonication for 5 minutes, measure the θ 360 and collect its 1 H-NMR spectrum, according to the mathematical relationship obtained in Example 2, Y=2.76×10 7 X+1.16×10 7Calculate the content of heparin precursor in each heparin precursor test solution. The RSD of the heparin precursor content determination results in the six heparin precursor test solutions should be ≤1.0%. The repeatability results are shown in Table 12.

[0190] Table 12 Results of the repeatability study of the heparin precursor content detection method

[0191] Figure 21 The samples 1 to 6 obtained by using the ledbppg2s1d pulse sequence in Example 6 are 1 H-NMR spectrum. Through the test and results, it can be seen that the RSD of the concentration test result of the heparin precursor test solution is 0.35%, which is far less than the acceptable limit of 1.0%. It can be seen that the analytical repeatability of the method of the present invention meets the acceptance standard, proving that the method has good repeatability.

[0192] Example 7 Recovery Test of Heparin Precursor Content Detection Method The same method as in Example 2 was used to prepare heparin precursor reference solutions with concentrations of 0.5 mg / mL, 3.0 mg / mL, and 6.0 mg / mL (the solvent was 10 vol% D2O-90 vol% LB fermentation broth) as heparin precursor test solutions; 0.6 mL of each heparin precursor test solution was selected and transferred to an NMR tube. After sonication for 5 minutes, the 360° pulse width θ of the heparin precursor test solution was measured using the same method as in Example 2. 360 , collect the heparin precursor test solution 1 H-NMR spectrum, measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A of the heparin precursor.

[0193] Table 13 Results of the recovery test of the heparin precursor content detection method

[0194] The above test results indicate that the heparin precursor content detection method of the present invention can accurately determine the concentration of heparin precursor in the test sample. The detection method has good sensitivity within the linear range, and the detection results are reliable, with a recovery rate greater than 99%, which is high and meets the detection requirements.

[0195] When the present invention is actually measured in fermentation broth, the quantitative result of heparin precursor is highly accurate, with a recovery rate exceeding 99%, which proves the reliability of the detection method of the present invention in complex samples.

[0196] Example 8 Detecting the Concentration of Heparin Precursor Using the Heparin Precursor Detection Method The heparin precursor test sample 1 was prepared by the following method: 1 mL of heavy water (D2O) (containing 0.002% (W / V) TSP) was placed in a 10 mL volumetric flask, and the fermentation broth was added. The mixture was ultrasonically diluted to the mark and shaken to obtain the heparin precursor sample 1. The fermentation broth was prepared according to the following method: Transfer colonies from the cultured plates to shake flasks containing synthetic glucose medium and incubate at 37°C, 200 rpm, in a shaker for 6-10 hours. Inoculate 100 mL of synthetic glucose medium with the resulting primary seed solution at a 3% (v / v) inoculum. Incubate at 37°C, 200 rpm, in a shaker for 6-10 hours to obtain the secondary seed solution. Inoculate the secondary seed solution into a fermenter at a 4% (v / v) inoculum. Ferment at 37°C, 450 rpm, and aerate 3-4 L / min for 24 hours. Harvest the culture and centrifuge to obtain the fermentation broth.

[0197] Heparin precursor test sample 2 was prepared by the following method: 1 mL of heavy water (D2O) (containing 0.002% (W / V) TSP) was placed in a 10 mL volumetric flask, and the crude heparin precursor extract was added. The mixture was ultrasonically diluted to the mark and shaken to obtain the heparin precursor sample 2. The crude heparin precursor extract was prepared according to the following method: The fermentation broth prepared above was placed into a 2.0 L centrifuge cup and centrifuged at 3800 rpm for 60 minutes. After centrifugation, if the supernatant is still turbid, repeat the centrifugation 1-2 times until the supernatant is relatively clear. The resulting fermentation broth was centrifuged at 3800 rpm for 60 minutes. If the supernatant is still turbid, repeat the centrifugation 1-2 times until the supernatant is clear. Filter the supernatant using a 0.45 μm filter to remove insoluble particles to protect the ultrafiltration membrane in the next step. The supernatant from the previous step was ultrafiltered (with a molecular weight cutoff of 1000 Da). Slowly add 2.4 L of purified water while ultrafiltration is continued until the volume is concentrated to 800 mL. The ultrafiltration was then terminated to obtain the crude heparin precursor extract.

[0198] The heparin precursor test sample 1 and the heparin precursor test sample 2 obtained above were used as heparin precursor test solutions; 0.6 mL of the heparin precursor test solution was taken respectively, transferred to a nuclear magnetic resonance tube, and after ultrasonication for 5 minutes, the 360° pulse width θ of the heparin precursor test solution was measured using the same method as in Example 2. 360 , collect the heparin precursor test solution 1 H-NMR spectrum, the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A of the heparin precursor was measured, and the mathematical relationship Y=2.76×10 7 X+1.16×10 7Calculate the content of heparin precursor in the heparin precursor test solution (i.e., heparin precursor test sample 1 and heparin precursor test sample 2).

[0199] Table 14 Test results of heparin precursor concentration using heparin precursor detection method

[0200] As can be seen from Table 14, the heparin precursor content is relatively high in heparin precursor test sample 1 and heparin precursor test sample 2. The above detection method has good sensitivity within the linear range and the detection results are reliable.

[0201] Example 9 Linearity test of heparin precursor detection method The purpose of this embodiment is to determine the linear relationship between the measurement result of the test solution and the heparin precursor concentration in the detection method of the present invention within a preset range.

[0202] The concentrations of the selected heparin precursor reference solutions were 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL. Samples of each heparin precursor reference solution were injected and analyzed, with the concentration of the heparin precursor reference solution as the abscissa. The product of the characteristic hydrogen signal H NMR peak area and the 360° pulse width, A×θ, was calculated. 360 Draw a standard curve on the vertical axis and calculate the concentration of the heparin precursor reference solution and A×θ 360 linear relationship.

[0203] 1. Instruments and analytical conditions Same as Example 2.

[0204] 2. Solution preparation Same as Example 2.

[0205] 3. Experimental process and results 0.6 mL of each heparin precursor reference solution (the solvent is LB fermentation broth containing 10 vol% D2O) was selected in sequence and transferred to an NMR tube. After ultrasonication for 5 min, the 360° pulse width θ of the heparin precursor reference solution was measured using the same method as in Example 2. 360 , collect the heparin precursor reference solution 1 H-NMR spectrum, measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A of the heparin precursor.

[0206] The product of the characteristic hydrogen signal H NMR spectrum peak area and the 360° pulse width A×θ 360A linear regression equation was constructed for the concentration of the heparin precursor reference solution (C, mg / ml): 10 concentration gradients of 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL, and 20.0 mg / mL were selected to construct the linear regression equation A, specifically Y=2.76×10 7 X+1.16×10 7 , R 2 =0.999; 7 concentration gradients of 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, and 15 mg / mL were selected to construct the linear regression equation B, specifically Y=3×10 7 X+1×10 7 , R 2 =0.999; 5 concentration gradients of 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 7.5 mg / mL, and 15 mg / mL were selected to construct the linear regression equation C, specifically: Y=3×10 7 X+1×10 7 , R 2 =0.9999; 5 concentration gradients of 1.5 mg / mL, 2.5 mg / mL, 5.0 mg / mL, 10 mg / mL, and 20 mg / mL were selected to construct the linear regression equation D, specifically: Y=2.81×10 7 X+5.46×10 6 , R 2 =0.999.

[0207] From the above tests and results, it can be seen that the concentration of heparin precursor reference solution is in the range of 0.25mg / mL-20.0mg / mL, and the concentration of heparin precursor C is related to A×θ 360 There is a good linear relationship, R 2 is greater than or equal to 0.999, indicating that the detection method of the present invention has high quantitative ability.

[0208] The characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area A was used to make a linear regression equation for the concentration of the heparin precursor reference solution (C, mg / ml): 10 concentration gradients of 0.25 mg / mL, 0.625 mg / mL, 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 15 mg / mL and 20.0 mg / mL were selected to construct the linear regression equation E, specifically: Y = 4.33 × 10 5 X+5.21×10 5 , R2 =0.998; 7 concentration gradients of 0.75 mg / mL, 1.5 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, and 15 mg / mL were selected to construct the linear regression equation F, specifically: Y=4.45×10 5 X+5.04×10 5 , R 2 =0.996.

[0209] The above tests and results show that the linear relationship between the concentration C of the heparin precursor reference solution and the peak area A of the characteristic hydrogen signal H NMR spectrum is relatively poor when the concentration of the heparin precursor reference solution is in the range of 0.25 mg / mL to 20.0 mg / mL. 2 It is less than 0.999, indicating that the quantitative ability of this detection method is relatively poor.

[0210] Comparative Example 1 Detection of heparin precursor content in samples by sulfuric acid-carbazole method The heparin precursor content in the sample was detected using the sulfuric acid-carbazole method as in Comparative Example 1 disclosed in Chinese patent document CN119595804A. The experimental results show that both the purified water group and the blank culture medium group in the control experimental group have absorbance, indicating that the sulfate-carbazole method will naturally cause errors in the determination of heparin precursor.

[0211] In summary, the method for detecting the content of heparin precursor provided by the present invention can effectively overcome the influence of the introduction of blank culture medium and reagents such as carbazole on the detection results during the experiment. The method for detecting the content of heparin precursor provided by the present invention has higher accuracy, good precision, and can provide more intuitive detection results.

[0212] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for detecting the content of heparin precursor, characterized in that: The detection method comprises: (S1) The 360° pulse width θ of the heparin precursor reference solution and the heparin precursor test solution were measured by nuclear magnetic resonance spectroscopy. 360 and the characteristic hydrogen signal NMR 1H spectrum peak area A, calculate the characteristic hydrogen signal NMR 1H spectrum peak area A and 360° pulse width θ 360 The product A×θ 360 ; (S2) The least squares method was used for linear regression to establish the relationship between the characteristic hydrogen signal H NMR spectrum peak area A and the 360° pulse width θ of the heparin precursor reference solution. 360 The mathematical relationship between the product Y of the heparin precursor reference solution and the concentration X is Y = kX + b; (S3) Calculating the concentration of the heparin precursor in the heparin precursor sample solution according to the mathematical relationship of step (S2); wherein the calculation formula for the concentration of the heparin precursor in the heparin precursor sample solution is: In the above formula, A 供试品 is the characteristic hydrogen signal H NMR spectrum peak area of ​​the heparin precursor test solution; θ 360 is the 360° pulse width of the heparin precursor test solution; k is the slope of the linear equation; b is the intercept of the linear equation; and N is the dilution factor of the heparin precursor test sample solution into the heparin precursor test solution.

2. The detection method according to claim 1, wherein In step (S1), the concentration of heparin precursor in the heparin precursor reference solution is 0.25-20.0 mg / mL; in step (S1), the concentration of heparin precursor in the heparin precursor test solution is 0.25-20.0 mg / mL.

3. The detection method according to claim 1, wherein In step (S1), the heparin precursor reference solution includes heparin precursor and deuterated D2O; or the heparin precursor reference solution includes heparin precursor, deuterated D2O and water H2O; or the heparin precursor reference solution includes heparin precursor, deuterated D2O and fermentation broth; when the heparin precursor reference solution includes heparin precursor, deuterated D2O and water H2O, the volume ratio of deuterated D2O to water H2O is 5-20:95-80; when the heparin precursor reference solution includes heparin precursor, deuterated D2O and fermentation broth, the volume ratio of deuterated D2O to fermentation broth is 5-20:95-80.

4. The detection method according to claim 3, characterized in that The fermentation broth does not include a heparin precursor; and / or, the fermentation broth is LB fermentation broth, SOC fermentation broth or TB fermentation broth.

5. The detection method according to claim 1, wherein In step (S1), the heparin precursor reference solution and the heparin precursor test solution have the same composition, that is, when the heparin precursor test solution includes fermentation broth, the heparin precursor reference solution also includes fermentation broth, and the composition of the fermentation broth is the same; when the heparin precursor test solution includes water H2O, the heparin precursor reference solution also includes water H2O; when the heparin precursor test solution includes deuterated water D2O, the heparin precursor reference solution also includes deuterated water D2O.

6. The detection method according to claim 1, characterized in that In step (S1), the heparin precursor test solution is prepared by the following method: fermenting Ecoli glycerol bacteria expressing heparin precursor in a culture medium, collecting the supernatant, and preparing the heparin precursor test sample solution; adding deuterated water (D2O) and optionally adding or not adding a diluent to obtain a heparin precursor test solution with a heparin precursor concentration of 0.25-20.0 mg / mL; wherein the diluent is deuterated water (D2O) or a mixed solution of deuterated water (D2O) and water (H2O), or a fermentation broth containing deuterated water (D2O), wherein the volume ratio of deuterated water (D2O) to water (H2O) is 5-20:95-80; and the volume ratio of deuterated water (D2O) to fermentation broth is 5-20:95-80.

7. The detection method according to claim 1, characterized in that The NMR instrument used for NMR spectroscopy analysis was a pulsed Fourier transform spectrometer; Determine the 360° pulse width θ 360 The detection conditions of the nuclear magnetic resonance spectroscopy analysis method are as follows: NMR frequency of 600 MHz, probe of CPQCI, accumulation of 1-8 times, temperature of 298 K, relaxation delay of 1-6 s, spectrum width of 15 ppm, window function of 0.3 Hz, pulse sequence of Zg, number of blank scans of 0 times, number of sampling points of 64k, 90° pulse width of 1 μs, Alternatively, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are as follows: pulse sequence: ledbppg2s1d; NMR frequency: 600 MHz; probe: CPQCI; accumulation: 8-32 times; temperature: 298 K; relaxation delay: 2-12 s; number of empty scans: 4 times; spectrum width: 18 ppm; window function: 0.3 Hz; diffusion time: 0.03 s; gradient duration: 3 ms; gradient strength: 100%; acquisition requirement: no rotation; Alternatively, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are as follows: pulse sequence Zgcppr; NMR frequency 600 MHz; probe CPQCI; accumulation 8-24 times; temperature 298K; relaxation delay 6-20s; spectral width 18ppm; window function 0.3Hz; pre-saturation power 71.8dB; acquisition requirement is non-rotation; Alternatively, the detection conditions of the nuclear magnetic resonance spectroscopy method for determining the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum are: pulse sequence is Zggpw5; NMR frequency is 600 MHz; probe is CPQCI; accumulation is 32-128 times; temperature is 298K; relaxation delay is 6-20s; spectral width is 18ppm; window function is 0.3Hz; double water peak suppression delay is 0.0002s; acquisition requirement is non-rotation.

8. The detection method according to claim 7, characterized in that In the deuterated water D2O and fermentation broth system, the ledbppg2s1d pulse sequence was selected to measure the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum and establish the hydrogen spectrum A×θ of the heparin precursor. 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in the deuterated water D2O and fermentation broth system was determined; Alternatively, in the system of heavy water D2O and water H2O, the ledbppg2s1d pulse sequence is selected to measure the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum to establish the hydrogen spectrum A×θ of the heparin precursor 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in the deuterated water D2O and water H2O system was determined; Alternatively, in a heavy water D2O system, the ledbppg2s1d pulse sequence is selected to determine the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum to establish the heparin precursor hydrogen spectrum A×θ 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in heavy water D2O system was determined; Alternatively, in the deuterated water (D2O) and water (H2O) system, the zggpw5 pulse sequence was selected to determine the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance (NMR) proton spectrum and to establish the proton spectrum of heparin precursor A×θ. 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in the deuterated water D2O and water H2O system was determined; Alternatively, in a heavy water D2O system, the zggpw5 pulse sequence is selected to determine the peak area of ​​the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum and establish the heparin precursor hydrogen spectrum A×θ 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in heavy water D2O system was determined; Alternatively, in a heavy water D2O system, the zgcppr pulse sequence is selected to measure the characteristic hydrogen signal nuclear magnetic resonance hydrogen spectrum peak area and establish the heparin precursor hydrogen spectrum A×θ 360 The linear relationship between the concentration of heparin precursor C and the concentration of heparin precursor in the deuterated water D2O system was determined.

9. Use of the method for detecting the content of heparin precursor according to any one of claims 1 to 8 in the detection of heparin precursor.

10. The use according to claim 9, characterized in that The method for detecting the content of the heparin precursor is applied to monitoring the fermentation process of the heparin precursor, thereby determining whether the fermentation is complete; or, The heparin precursor content detection method is applied to the screening of E. coli with high heparin precursor productivity during the transformation of E. coli, thereby obtaining E. coli with high heparin precursor productivity; or, The method for detecting the content of heparin precursor is applied to monitoring the preparation process and purification process of heparin precursor, so as to determine whether the purity of the prepared heparin precursor meets the requirements of the quality standard.

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