CN115389681B - Detection method for dithiothreitol residue in thiolated hyaluronic acid derivative - Google Patents
Detection method for dithiothreitol residue in thiolated hyaluronic acid derivative Download PDFInfo
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Abstract
The invention discloses a detection method of dithiothreitol residue in a thiolated hyaluronic acid derivative, which comprises the following steps: preparing a DTT standard working solution; detecting the DTT standard substance by gel permeation chromatography to obtain a corresponding standard curve; preparing a working solution of a sample to be detected; and detecting the working solution of the sample to be detected by gel permeation chromatography to obtain the residual content of dithiothreitol in the sample. The sample of the invention does not need to be processed, the operation method is simple and easy to implement, and the DTT has a good linear range (the linear correlation coefficient (R) thereof) within the range of 1-20 ppm 2 ) 0.9994), the lowest detection limit can reach 1ppm (signal to noise ratio 3.67), the quantification limit can reach 2.72 ppm (signal to noise ratio 10), the accuracy is high (the recovery rate (R) is between 105.6% and 111.9% (RSD is 3%)), and the requirements in actual detection can be met.
Description
Technical Field
The invention relates to the technical field of dithiothreitol detection, and particularly relates to a detection method of dithiothreitol residues in thiolated hyaluronic acid derivatives.
Background
Hyaluronic Acid (HA for short), also known as "Hyaluronic Acid", is widely distributed in the extracellular matrix of animals and humans, is an important constituent of cell matrices and various tissues, and HAs a variety of important physiological functions. Performing coupling reaction on an amino reagent containing a disulfide bond and a side chain carboxyl group of hyaluronic acid; after the coupling reaction is finished, the disulfide bond can be reduced into free sulfydryl through a simple reduction process, so that the sulfhydrylation hyaluronic acid derivative can be obtained, which is the most common preparation method for the current sulfhydrylation hyaluronic acid derivative; in the above reduction of disulfide bonds to free thiol groups, the reducing agent most widely used at present is dithiothreitol.
Dithiothreitol (DTT) is a small-molecule organic reducing agent with a chemical formula of C 4 H 10 O 2 S 2 . It is a linear molecule in its reduced state and becomes a six-membered cyclic structure containing a disulfide bond after oxidation. DTT can maintain sulfhydryl groups in a reduced state, and is commonly used to reduce disulfide bonds in protein molecules and polypeptides, and more commonly to prevent protein cysteine residues from forming intramolecular and intermolecular disulfide bonds, often as a protein sulfhydryl protecting agent. The introduction of DTT into the body may destroy the protein structure, thereby causing enzyme inactivation and the like, and the residue of DTT may cause potential safety hazard, so that the residual amount of the reducing agent DTT in the sample and the final product in the process needs to be strictly monitored.
According to the current report, when the traditional spectrophotometer method is used for measurement, DTT is easy to be oxidized, response is unstable, sensitivity is low, and detection requirements cannot be met. Still another liquid chromatography-based test method is to derive DTT with a fluorescent probe, monobromodiamine (mBBr), and quantitatively analyze DTT by using reversed-phase liquid chromatography and a fluorescence detector after derivation, which has high sensitivity, but has complex sample pretreatment process and strict requirements on reaction environment, and is not suitable for detecting DTT residue in an industrial production process. Chinese patent CN112526028A discloses a liquid chromatography method for simultaneously and quantitatively analyzing the two coexisting forms of reduction state and oxidation state of dithiothreitol by liquid chromatography tandem mass spectrometry (LC-MS/MS), but this method needs to generate the standard curves of the two substances at the same time, increasing the detection workload. There are also reports of the measurement of DTT content by back titration of cobalt ion, but DTT is easily oxidized and the sensitivity of this method is not high. Chinese patent CN107255660B discloses a method for detecting dithiothreitol in a solution by using a graphene quantum dot electrode, which is simple to operate and high in sensitivity, but is limited to high price of a preparation process thereof and less application in industrial production.
There are also reports of quantitation of Dithiothreitol (DTT) residual by HPLC, for example: high performance liquid chromatography for measuring dithiothreitol and oxidized dithiothreitol (report of analytical science 2016, 32 537-540) in the medicine, the content of DTT in the metabolite (an active protein) of the engineering bacteria is measured by HPLC, a Waters X-Bridge C18 chromatographic column is adopted, and the mobile phase is 30 (v/v); the reversed-phase high performance liquid chromatography is used for detecting the residual amount of dithiothreitol in the recombinant virus-like particle vaccine stock solution (journal of Chinese biologicals 2015, 28. However, the active protein and the recombinant HPV vaccine both have a definite single molecular weight protein structure, and the thiolated hyaluronic acid derivative related to the application is a macromolecular polysaccharide derivative mixture with dispersibility, and researches show that the two detection methods are difficult to meet the actual requirements of separation and detection of trace DTT residues in the thiolated hyaluronic acid derivative.
In the preparation of the thiolated hyaluronic acid derivative, quantitative detection of dithiothreitol residues becomes a problem to be solved, and a detection method for trace residues of DTT in the thiolated hyaluronic acid derivative does not exist at present, so that limitation is brought to the wide application of the thiolated hyaluronic acid derivative in medicines or medical instruments, and the establishment of an effective detection method for trace residues of DTT in the thiolated hyaluronic acid derivative has important significance.
Disclosure of Invention
The invention aims to provide a detection method for dithiothreitol residues in thiolated hyaluronic acid derivatives, which has high accuracy and can meet actual requirements, and a gel permeation chromatography detection method for dithiothreitol residues.
In order to solve the technical problem, the invention provides a method for detecting dithiothreitol residue in a thiolated hyaluronic acid derivative, which comprises the following steps: step (1), standard substance working solution: precision scaleTaking a certain amount of DTT standard substance, and taking phosphate buffer solution with pH of 3.5-4.5 as a diluent to prepare working solution of the DTT standard substance with not less than 4 concentration gradients and a range of 1-20 ppm; step (2), sample working solution to be detected: precisely weighing a certain amount of thiolated hyaluronic acid derivative, and preparing a thiolated hyaluronic acid derivative solution with the concentration range of 3-20 mg/ml by taking phosphate buffer solution with the pH of 3.5-4.5 as a diluent; step (3) standard curve: sequentially carrying out gel permeation chromatography detection on the standard substance working solution in the step (1) to respectively obtain the peak areas of corresponding dithiothreitol, and carrying out linear regression with the concentration of the standard substance working solution to generate a standard curve, or automatically generating the standard curve by a software system; and (4) determining a sample to be detected: detecting the working solution of the sample to be detected in the step (2) through gel permeation chromatography to obtain the peak area of dithiothreitol of the sample, substituting the peak area into the standard curve in the step (3), and calculating to obtain the residual content of dithiothreitol in the sample to be detected; the gel permeation chromatography detection in the step (2) and the step (4) adopts a Water 2695-2998 liquid chromatograph; the type of the water-phase gel permeation chromatographic column is Ultrahydrogel TM 120,7.8 multiplied by 300mm, and the grain size of the filler is 6 mu m; column temperature: at 30 ℃.
Specifically, the sample injection amount of the gel permeation chromatography is 10-100 mul; the flow rate is 0.5-1.0 ml/min.
Specifically, the mobile phase of the gel permeation chromatography is a mixed solution of phosphate buffer salt with pH of 3.5-4.5 and acetonitrile.
Specifically, the volume ratio of the phosphate buffer solution to the acetonitrile is 85-95.
Specifically, the flow rate is 0.7 ml/min.
Specifically, the sample injection amount is 40 μ l.
In particular, the detector of gel permeation chromatography is a photodiode array detector.
Specifically, the detection wavelength of the gel permeation chromatography is 210nm.
Specifically, the concentration gradients of the standard working solution are 1, 2, 5, 10 and 20ppm respectively.
Specifically, the molecular weight of the thiolated hyaluronic acid derivative is greater than 120 KDa.
The Gel Permeation Chromatography (GPC) instrument comprises a pump system, an (automatic) sample introduction system, a gel chromatographic column, a detection system and a data acquisition and processing system. Similar to High Performance Liquid Chromatography (HPLC) detection, the GPC detection method has the advantages of accurate quantification, short detection period and the like, and is one of the methods relatively suitable for trace detection. HPLC performs sample separation by means of a chromatographic column filled with very small gel particle packing, which is achieved based on the affinity between the various molecules in the substance to be separated and the gel particle packing. The separation principle of GPC is a size exclusion theory, and porous channels with various sizes are arranged on the surface and inside of a porous filler filled in a gel permeation chromatographic column, so that the separation is realized based on the molecular size difference of various molecules in a substance to be separated.
In order to realize the quantitative detection of DTT, good separation between DTT and the thiolated hyaluronic acid derivative and other possible substances must be realized, and the separation degree needs to be more than or equal to 1.5 according to the general guidelines 0512 of high performance liquid chromatography in pharmacopoeia of the people's republic of China (2020 edition, four). However, the thiolated hyaluronic acid derivative is a derivative of a macromolecular natural polysaccharide, has a certain dispersibility in molecular weight, is a macromolecular mixture with different macromolecular weight, and is difficult to be well separated from DTT in a chromatographic column (such as a C18 silica gel primary chromatographic column) commonly used for HPLC. Therefore, in the present invention, the separation and detection of a trace amount of DTT remaining in the thiolated hyaluronic acid derivative is carried out by using a GPC gel permeation chromatography column.
Researches show that the separation degree of the thiolated hyaluronic acid derivative and DTT is related to various factors such as the components of a mobile phase, the pH value of the mobile phase, the proportion of the mobile phase and the like. The detection method of the invention is obtained by comprehensively considering all factors, and can obtain good analysis results.
The invention has the beneficial effects that:
the method for detecting the trace DTT residue in the thiolated hyaluronic acid derivative provided by the invention has the advantages that the sample does not need to be processed, the operation method is simple and feasible, and the DTT is in the range of 1-20 ppmHas a good linear range (its linear correlation coefficient (R)) in the periphery 2 ) 0.9994), the lowest detection limit can reach 1ppm (signal to noise ratio 3.67), the quantitative limit can reach 2.72 ppm (signal to noise ratio 10), and the accuracy is high (the recovery rate (R) is between 105.6% and 111.9% (RSD is 3%)), so that the requirements in actual detection can be met.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings required for the present invention are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a DTT chromatogram (separation of different organic phases) provided by a detection method according to the invention;
FIG. 2 is a DTT chromatogram (different mobile phase pH) provided by a detection method according to the invention;
FIG. 3 is a DTT chromatogram (different mobile phase ratios) provided by the detection method according to the invention;
FIG. 4 is a DTT standard curve provided by the detection method according to the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The device information in the following examples is as follows: water 2695-2998 liquid chromatograph (PDA detector); aqueous gel permeation chromatography column (Ultrahydrogel) TM 120 7.8 x 300mm, and the particle size of the filler is 6 mu m; column temperature: at 30 ℃.
Example 1 examination of test conditions
1.1 Preparation of DTT test solution
Accurately weighing 0.100g of DTT standard substance, dissolving with pure water, and diluting to 100ml to obtain standard stock solution with concentration of 1000 ppm; and (3) transferring a certain volume of standard stock solution, and diluting the stock solution into standard working solution with the concentration of 1, 2, 5, 10 and 20ppm by using pure water.
1.2 Thiolated hyaluronic acid derivatives
Thiolated hyaluronic acid derivatives (MW 120 kDa, 200 kDa, 500 kDa) in aqueous solution (10 mg/ml) (pH 3.5).
1.3 Determination of detection wavelength
The DTT test solution was scanned at full wavelength on an ultraviolet-visible spectrophotometer. As a result, DTT showed an absorption from about 260nm to a maximum at 200 to 210nm and a marginal absorption at 200nm, and 210nm was selected as a detection wavelength of GPC.
1.4 selection of the Mobile phase
In the mobile phase, the organic and aqueous phases, buffer substances and pH have an influence on the separation and detection of the sample.
The water phase is phosphate buffer solution, methanol and acetonitrile are respectively used as organic phases for separation, the retention time of the thiolated hyaluronic acid is about 9 min (the retention time is slightly reduced along with the increase of molecular weight), and the retention time of DTT is about 37 min. The results show that acetonitrile has a better resolution (see figure 1). Therefore, the subsequent experiments of this example chose acetonitrile as the organic phase.
At higher pH of the mobile phase, the DTT becomes broader in peak shape and tends to show superimposed hetero-peaks, probably related to the DTT being easily oxidized at higher pH values. As a result, it was found that the peak shape of DTT was good at a pH of 3.5 to 4.5 in the phosphate buffer (see FIG. 2). Therefore, the subsequent experiments of this example chose phosphate buffer at pH 4.0.
Since the thiolated hyaluronic acid derivative is insoluble in an organic solvent, the volume ratio of the organic solvent cannot be excessively large. The results show that the volume ratio of the phosphate buffer solution to the acetonitrile is 85-95, and that good separation degree can be achieved, especially when the volume ratio is 90. Therefore, the subsequent experiments of this example select a volume ratio of phosphate buffer to acetonitrile of 90.
And (5) inspecting different sample volumes (10-100 mu l) of the DTT test solution. The result shows that when the sample introduction amount is 40 mu l, the response height of the DTT in the detection wavelength band is moderate, and the accuracy of the detection method can be further increased, so that 40 mu l is selected as the detection wavelength of GPC.
Different flow rates of the mobile phase (0.5-1.0 ml/min) were examined. As a result, the flow rate is preferably 0.7 ml/min. Too fast a flow rate will result in too short a retention time for the separated material, and not good enough separation, and too slow a flow rate will result in a broadening of the peak shape of the material and affect the peak shape.
Under the preferable detection conditions (when the sample volume is 40 mul, the flow rate is 0.7 ml/min, and the mobile phase is phosphate buffer solution (pH 4.0) and acetonitrile with a volume ratio of 90 to 10, and the detection wavelength is 210 nm), the chromatogram of DTT is shown in figures 1 and 3 (the separation degree is not less than 14.4), so that the requirement that the separation degree is not less than 1.5 in general pharmacopoeia of the people's republic of China (2020 edition four) 0512 high performance liquid chromatography is met, and the chromatogram conditions are adopted in the subsequent examples.
EXAMPLE 2 creation of Standard Curve
Accurately weighing 0.100g of DTT standard substance, dissolving with preferable mobile phase, and fixing to 100ml to obtain standard stock solution with concentration of 1000 ppm; a volume of the standard stock solution is removed, diluted with the preferred mobile phase to 1, 2, 5, 10, 20ppm of the standard working solution and tested under the preferred test conditions.
The test results are shown in the following table 1:
TABLE 1
As can be seen from the standard curve (FIG. 4), DTT has a good linear range in the range of 1-20 ppm, and its linear correlation coefficient (R) 2 ) 0.9994, satisfies the detection method with respect to the linear correlation coefficient (R) 2 ) Greater than 0.998.
Example 3 detection and quantitation limits
Comparing a signal detected by using a low-concentration DTT standard working solution with a signal detected by a blank sample, calculating a detection limit by using a corresponding concentration when a signal-to-noise ratio is not lower than 3, and calculating a quantitative limit by using a corresponding concentration when the signal-to-noise ratio is not lower than 10; the result shows that the lowest detection limit can reach 1ppm (signal-to-noise ratio is 3.67), and the quantitative limit can reach 2.72 ppm (signal-to-noise ratio is 10).
Example 4 precision
Taking 6 parts of DTT standard working solution (5 ppm) to carry out detection under the preferable detection condition, and calculating relative deviation according to the peak area obtained by each test sample, wherein the results are shown in the following table 2:
TABLE 2
The RSD is 0.94 percent, has good precision and meets the target requirement that the RSD is less than or equal to 2 percent.
Example 5 accuracy
Three DTT standard working solutions (8 ppm, 10ppm and 12 ppm) were added to the above-mentioned thiolated hyaluronic acid derivative aqueous solution in three different concentrations by the sample-addition recovery test method, and each concentration of the standard solution was prepared in triplicate, and the amount of DTT therein was measured using the standard curve in example 2, and the recovery rate was calculated. The test results are given in table 3 below:
TABLE 3
The accuracy is the degree of the measured result of the characterization detection method close to the true value, and is generally characterized by the recovery rate (R), the requirement is between 80 and 120 percent (RSD is not more than 10 percent), and the test result meets the requirement.
EXAMPLE 6 durability
Taking 5 parts of DTT standard working solution, under the condition of other unchanged chromatographic conditions, changing the column temperature by +/-1 ℃ and the flow rate by +/-5%, respectively carrying out sample injection detection, recording and analyzing peak areas, and obtaining a representative result shown in the following table 4 (the concentration of the DTT standard working solution is 5 ppm):
TABLE 4
The RSD is 3.2 percent, the durability is good, and the target requirement that the RSD is less than or equal to 5 percent is met.
Example 7 application of the method
The residual amount of trace DTT in the three thiolated hyaluronic acid derivative solutions of example 1 was measured according to the chromatographic conditions determined in example 1, and a standard curve was established according to example 2, and the results of the measurement showed that the residual amount of DTT in the two thiolated hyaluronic acid derivatives (MW 120 KDa, 200 KDa) solutions was < the limit of detection (1 ppm), and the residual amount of DTT in the one thiolated hyaluronic acid derivative (MW 500 KDa) solution was 4.2 ppm.
According to ICH Q3AR2: impurities in New crude drug substance the definition threshold value of impurities contained in the drug with the maximum daily dose is 0.1% or 1mg, and the DTT residual quantity limit of the three thiol-modified hyaluronic acid derivative solutions is 10ppm based on the effective components (10 mg/ml), which meets the specification.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for detecting dithiothreitol residues in thiolated hyaluronic acid derivatives is characterized by comprising the following steps:
step (1), standard substance working solution: precisely weighing a certain amount of DTT standard substance, and taking phosphate buffer solution with pH of 3.5-4.5 as a diluent to prepare working solution of the DTT standard substance with not less than 4 concentration gradients and a range of 1-20 ppm;
step (2) standard curve: sequentially carrying out gel permeation chromatography detection on the standard substance working solution in the step (1) to respectively obtain the peak areas of corresponding dithiothreitol, and carrying out linear regression with the concentration of the standard substance working solution to generate a standard curve, or automatically generating the standard curve by a software system;
step (3), working solution of a sample to be detected: precisely weighing a certain amount of thiolated hyaluronic acid derivative, and preparing a thiolated hyaluronic acid derivative solution with the concentration range of 3-20 mg/ml by taking a phosphate buffer solution with the pH of 3.5-4.5 as a diluent;
and (4) determining a sample to be detected: detecting the working solution of the sample to be detected in the step (3) through gel permeation chromatography to obtain the peak area of dithiothreitol of the sample, substituting the peak area into the standard curve in the step (2), and calculating to obtain the residual content of dithiothreitol in the sample to be detected;
the gel permeation chromatography detection in the step (2) and the step (4) adopts a Water 2695-2998 liquid chromatograph; the type of the water-phase gel permeation chromatographic column is Ultrahydrogel TM 120,7.8 × 300mm, and the particle size of the filler is 6 μm; column temperature: 30 ℃; the mobile phase of the gel permeation chromatography is a mixed solution of phosphate buffer salt with pH of 3.5-4.5 and acetonitrile; the volume ratio of the phosphate buffer solution to the acetonitrile is 85-95; the detector of the gel permeation chromatography is a photodiode array detector, and the detection wavelength of the gel permeation chromatography is 210nm.
2. The detection method according to claim 1, wherein the sample size of the gel permeation chromatography is 10 to 100 μ l; the flow rate is 0.5-1.0 ml/min.
3. The method of claim 2, wherein the flow rate is 0.7 ml/min.
4. The detection method according to claim 2, wherein the sample size is 40 μ l.
5. The detection method according to claim 1, wherein the concentration gradients of the standard working solution are 1, 2, 5, 10 and 20ppm, respectively.
6. The detection method according to claim 1, wherein the thiolated hyaluronic acid derivative has a molecular weight of greater than 120 kDa.
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| US20090111133A1 (en) * | 2007-10-31 | 2009-04-30 | Abbott Laboratories | Gel Filtration Standard |
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| JPH1010112A (en) * | 1996-06-24 | 1998-01-16 | Daicel Chem Ind Ltd | Method for determining polysaccharide derivative in low-molecular organic compound |
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