A detection method for heterobifunctional cross-linking agent

Abstract

The present invention relates to the field of chemical analysis technology, and in particular to a detection method for a heterobifunctional cross-linking agent. The detection method for a heterobifunctional cross-linking agent provided by the present invention includes the following conditions: the mobile phase A in the liquid chromatography detection process is a mixed solution of formic acid and acetonitrile, and the mobile phase B is a mixed solution of formic acid and water; the mobile phase elution mode is gradient elution, and the diluent is water. The fragmentation voltage during mass spectrometry detection is 90-115V, the dry gas flow rate is 10-20L / min, the dry gas temperature is 300-400°C, and the atomizer pressure is (30-40) psi. The detection method for the heterobifunctional cross-linking agent of the present invention can be within the trace concentration of 0.005μg / ml-0.5μg / ml, and the heterobifunctional cross-linking agent response value is in a good linear relationship with the concentration, and the detection limit can reach 5ng / ml.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting a heterobifunctional cross-linking agent. Background Art

[0002] 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (CAS#92921-24-9) is a common water-soluble heterobifunctional crosslinker. The sodium salt contains an amine-reactive hydroxysuccinimide group and a thiol-reactive maleimide group. It is often used to combine amines and thiols to form stable amide bonds and thioether bonds.

[0003] As reported in the paper Versatile RHDV Virus-Like Particles: Incorporation of Antigens by Genetic Modification and Chemical Conjugation (Biotechnology and Bioengineering, 98(5), 968-977, 2007), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt can be used as a cross-linking agent to bind small peptides and proteins to the surface of rabbit hemorrhagic disease virus-like particle vaccine, thereby overcoming the limitations of chimeric virus-like particle vaccine formation and production.

[0004] As reported in the paper Improved performance of collagen scaffolds crosslinked by Traut's reagent and Sulfo-SMCC (Journal of Biomaterials Science, Polymer Edition, 28(7), 629-647, 2017), cross-linking collagen scaffolds with an appropriate amount of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt is an effective and safe method that can be used to modify naturally derived collagen scaffolds and has significant potential use in tissue regeneration.

[0005] However, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt may cause skin irritation and corrosion, severe eye irritation and damage, respiratory irritation, and specific target organ toxicity. Therefore, it is necessary to study the detection method of this heterobifunctional cross-linking agent.

[0006] In the prior art, LC-DAD is generally used to detect 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt. However, the detection limit of LC-DAD is generally 0.5 μg / mL, which makes it difficult to detect 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt at a lower concentration. Summary of the invention

[0007] The present invention is made to solve the above problems, and aims to provide a detection method that can be used to detect low concentrations of heterobifunctional crosslinking agents, especially a method for detecting low concentrations of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt.

[0008] The present invention provides a method for detecting a heterobifunctional cross-linking agent, which is a liquid chromatography-mass spectrometry detection method and has the following characteristics, including:

[0009] The mobile phases in the liquid chromatography detection process are mobile phase A and mobile phase B, mobile phase A is a first mixed solution, mobile phase B is a second mixed solution, the first mixed solution is a mixed solution of formic acid and acetonitrile, and the second mixed solution is a mixed solution of formic acid and water;

[0010] In the process of liquid chromatography detection, the mobile phase is passed through the chromatographic column by gradient elution, and the elution gradient is: the total elution time is divided into N steps for gradient elution in sequence, the proportion of mobile phase A in the first N-1 steps of the gradient gradually increases from (0-10)% to (85-95)%, the proportion of mobile phase B gradually decreases from (90-100)% to (5-15)%, in the Nth step, the proportion of mobile phase A is (85-95)%, the proportion of mobile phase B is (5-15)%, in the Nth step of the gradient, the proportion of mobile phase A gradually decreases from (85-95)% to (0-10)%, the proportion of mobile phase B gradually increases from (5-15)% to (90-100)%, and 2≤N≤10;

[0011] The fragmentation voltage during mass spectrometry detection was 90-115V.

[0012] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, it can also have the following characteristics: wherein, in the first mixed solution, the volume ratio of formic acid to acetonitrile is (0.25-3):(950-1050), and in the second mixed solution, the volume ratio of formic acid to water is (0.25-3):(950-1050).

[0013] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0014] Among them, the gradient elution time is 12 minutes, and the elution gradient is: the total elution time is divided into 2 steps for gradient elution in sequence, the elution time of the first step is 0-8 minutes, the proportion of mobile phase A from 0 to 2 minutes is 5%, the proportion of mobile phase B is 95%, from 2 to 8 minutes, the proportion of mobile phase A gradually increases to 90%, and the proportion of mobile phase B gradually decreases to 10%, the elution time of the second step is 8-12 minutes, from 8 to 10 minutes, the proportion of mobile phase A is 90%, and the proportion of mobile phase B is 10%, from 10 to 10.1 minutes, the proportion of mobile phase A gradually decreases to 5%, the proportion of mobile phase B gradually increases to 95%, from 10.1 to 12 minutes, the proportion of mobile phase A is maintained at 5%, and the proportion of mobile phase B is maintained at 95%.

[0015] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0016] Among them, the negative ion mode under the SIM detection mode was used in the liquid chromatography-mass spectrometry detection process.

[0017] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0018] The mobile phase flow rate in the liquid chromatography detection process is 0.1-1.0 mL / min; the column temperature of the chromatographic column is 25-35°C.

[0019] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0020] The gradient elution time is 10 to 20 minutes.

[0021] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0022] Among them, the liquid chromatography column is a Phenomenex Kinetex C18 chromatography column.

[0023] In the detection method of the heterobifunctional cross-linking agent provided by the present invention, the following characteristics may also be present:

[0024] The flow rate of the drying gas during the mass spectrometry detection process is 10 to 20 L / min, the temperature of the drying gas during the mass spectrometry detection process is 300 to 400° C., and the pressure of the nebulizer during the mass spectrometry detection process is (30 to 40) psi.

[0025] Functions and Effects of the Invention

[0026] According to the detection method of the heterobifunctional cross-linking agent involved in the present invention, because liquid chromatography-mass spectrometry is used, and a suitable elution gradient and mass spectrometry fragmentation voltage are selected, the detection method of the heterobifunctional cross-linking agent of the present invention can show a good linear relationship between the response value of the heterobifunctional cross-linking agent and the concentration within the trace concentration range of 0.005μg / mL-0.5μg / mL, and the quantitative limit can reach 5ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a liquid phase mass spectrum of the heterobifunctional cross-linking agent sample in Example 1 of the present invention;

[0028] Figure 2 is the SPC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the SCAN mode positive ion mode;

[0029] Figure 3 is a TIC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the positive ion mode of the SCAN mode;

[0030] Figure 4 is the SPC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the negative ion mode of the SCAN mode;

[0031] Figure 5 is a TIC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the negative ion mode of the SCAN mode;

[0032] Figure 6 is the SPC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the negative ion mode of the SIM mode;

[0033] Figure 7 is a TIC graph of the heterobifunctional cross-linking agent sample in Example 2 of the present invention in the negative ion mode of the SIM mode;

[0034] Figure 8 is a liquid phase mass spectrum of the heterobifunctional cross-linking agent sample in Example 3 of the present invention at a fragmentation voltage of 70V;

[0035] Fig. 9 is a liquid phase mass spectrum of the heterobifunctional cross-linking agent sample in Example 3 of the present invention at a fragmentation voltage of 90V;

[0036] Fig.10 is a liquid phase mass spectrum of the heterobifunctional cross-linking agent sample in Example 3 of the present invention at a fragmentation voltage of 110V;

[0037] Fig.11 is a liquid phase mass spectrum of the heterobifunctional cross-linking agent sample with a concentration of 0.005 μg / mL in Example 4 of the present invention; and

[0038] Fig.12 It is a linear equation diagram in Example 5 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below in conjunction with embodiments and drawings.

[0040] In the following examples, unless otherwise specified, the mobile phases used were all commercially available analytically pure products.

[0041] In the following examples, unless otherwise specified, the heterobifunctional cross-linking agent samples used are all 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt, CAS number: 92921-24-9, a commercially available product.

[0042] In the following examples, unless otherwise specified, the water used was deionized water.

[0043] <Example 1>

[0044] A detection method for heterobifunctional cross-linking agent

[0045] This embodiment provides a method for determining the concentration of a heterobifunctional cross-linking agent by liquid chromatography-mass spectrometry. The specific parameters of the liquid chromatography-mass spectrometry provided in this embodiment are shown in Table 1.

[0046] Table 1 Liquid chromatography-mass spectrometry parameters

[0047]

[0048]

[0049] According to the liquid chromatography-mass spectrometry parameters recorded in Table 1, the 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt sample was tested, and the sample concentration was 0.05 μg / mL. The test results are as follows: Figure 1 shown.

[0050] like Figure 1 As shown, the spectrum obtained by the detection method provided in this embodiment has a good main peak shape.

[0051] <Example 2>

[0052] Mode Filtering

[0053] In this example, the mode was screened on the basis of Example 1. In this example, except for the mode, fragmentation voltage and sample concentration, the other conditions are the same as those recorded in Table 1 of Example 1. In this example, the fragmentation voltage is 70V, the sample concentration is 1.0mg / mL, and the detection mode and the corresponding spectra are shown in Tables 2 and 3. Figure 2-Figure 7 shown.

[0054] Table 2 Screening of patterns

[0055] Serial number Sample concentration (mg / mL) Detection Mode Positive and negative ion modes SPC chart / TIC chart 1 1.0 SCAN just Figure 2 / Figure 3 2 1.0 SCAN burden Figure 4 / Figure 5 3 1.0 SIM burden Figure 6 / Figure 7

[0056] like Figure 2-5 As shown in Figure 2, the substance has a strong response in negative ion mode, and the main molecular ion peak M / Z is 413. Figure 3-7 As shown in the figure, compared with the SCAN mode, the substance obtained a better mass spectrum peak shape and lower background interference in the SIM mode.

[0057] <Example 3>

[0058] Screening of fragmentation voltage

[0059] In this example, the value of the fragmentation voltage is screened on the basis of Example 1. In this example, other conditions except the fragmentation voltage and the sample concentration are the same as those described in Example 1. In this example, the sample concentration used is 1.0 mg / mL.

[0060] In this embodiment, three fragmentation voltage values ​​were selected, and their corresponding test results are shown in Table 3 and Table 4. Figure 8-10 shown.

[0061] Table 3 Screening of fragmentation voltage

[0062] Serial number Fragmentation voltage(V) Peak area Atlas 1 70 36245212 Figure 8 2 90 38776732 Fig. 9 3 110 39729480 Fig.10

[0063] As shown in Table 3 and Figure 8-10 As shown, the higher the fragmentation voltage, the larger the peak area, that is, the ion transmission efficiency and response intensity increase with the increase of the fragmentation voltage. Finally, when the fragmentation voltage is 110V, the mass spectrum response of the substance is the strongest and the peak area is the largest.

[0064] <Example 4>

[0065] Detection limit determination

[0066] This example determines the detection limit of the liquid chromatography-mass spectrometry method provided in Example 1.

[0067] like Fig.11As shown, when the absolute concentration of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt solution is determined to be 5 ng / mL, the mass spectrometry response of the substance is still good and the detection limit can be reduced.

[0068] <Example 5>

[0069] Linear range determination

[0070] This example performs a linear range determination on the liquid chromatography-mass spectrometry method provided in Example 1. Specifically, the following steps are included:

[0071] Step 1, prepare a stock solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimidyl ester sodium salt: accurately weigh 20 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimidyl ester sodium salt into a 20 mL volumetric flask, dissolve it in water and dilute to the mark, and shake well to obtain;

[0072] Step 2, preparing 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt linear solutions: prepare the following three groups of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt linear solutions respectively:

[0073] L1 (0.5 μg / mL): Accurately pipette 1 mL of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt stock solution into a 100 mL volumetric flask, dissolve it in water and dilute to the mark, mix well, then accurately pipette 1 mL of the solution from the above volumetric flask into a 20 mL volumetric flask and shake well.

[0074] L2 (0.05 μg / mL): Accurately pipette 1 mL of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt L1 solution into a 10 mL volumetric flask, dissolve it in water and dilute to the mark, and shake well to obtain.

[0075] L3 (0.005 μg / mL): Accurately pipette 1 mL of sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfonyl succinimidyl ester L2 solution into a 10 mL volumetric flask, dissolve it in water and dilute to the mark, and shake well to obtain.

[0076] Step 3, using the liquid chromatography-mass spectrometry method provided in Example 1 to respectively detect the L1, L2 and L3 solutions, the detection results are shown in Table 4.

[0077] Table 4 Linearity test data

[0078]

[0079]

[0080] Step 4, according to the data in Table 4, the peak area and concentration of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt at each linear concentration are plotted to obtain a linear curve A=aC+b; wherein A is the peak area of ​​the main peak at each linear concentration, and C is the solution concentration of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt. The linear graph finally obtained is as follows: Fig.12 shown.

[0081] like Fig.12 As shown, the linear equation obtained is A = 206590C + 1618.4, and the linear correlation coefficient R 2 =0.9994. Therefore, the liquid chromatography-mass spectrometry method provided in Example 1 can show a good linear relationship between the response value and concentration of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt within the trace concentration linear range of 0.005 μg / mL-0.5 μg / mL.

[0082] Functions and Effects of the Embodiments

[0083] According to the detection method of the heterobifunctional cross-linking agent involved in the above-mentioned embodiment, because liquid chromatography-mass spectrometry technology is adopted, and a suitable elution gradient and mass spectrometry fragmentation voltage are selected, the detection method of the heterobifunctional cross-linking agent provided in Example 1 can show a good linear relationship between the mass spectrometry response value and the concentration of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt in the trace concentration range of 0.005μg / mL-0.5μg / mL, and the quantitative limit can reach 5ng / mL, which provides a good analytical detection means for this type of substance.

[0084] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention.

Claims

1. A method for detecting a heterobifunctional crosslinking agent, wherein the heterobifunctional crosslinking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt, characterized in that: include: A, the mobile phases in the liquid chromatography detection process are mobile phase A and mobile phase B, the mobile phase A is a first mixed solution, the mobile phase B is a second mixed solution, the first mixed solution is a mixed solution of formic acid and acetonitrile, and the second mixed solution is a mixed solution of formic acid and water; In the first mixed solution, the volume ratio of formic acid to acetonitrile is (0.25-3):(950-1050), and in the second mixed solution, the volume ratio of formic acid to water is (0.25-3):(950-1050); B. During the liquid chromatography detection process, the mobile phase is passed through the chromatographic column by gradient elution, the gradient elution time is 12 minutes, and the elution gradient is: the total elution time is divided into 2 steps for gradient elution, the first step elution time is 0 to 8 minutes, 0 to 2 minutes, the proportion of mobile phase A is 5%, the proportion of mobile phase B is 95%, 2 to 8 minutes, the proportion of mobile phase A gradually increases to 90%, and the proportion of mobile phase B gradually decreases to 10%, the second step elution time is 8 to 12 minutes, 8 to 10 minutes, the proportion of mobile phase A is 90%, the proportion of mobile phase B is 10%, 10 to 10.1 minutes, the proportion of mobile phase A gradually decreases to 5%, the proportion of mobile phase B gradually increases to 95%, 10.1 to 12 minutes, the proportion of mobile phase A is maintained at 5%, and the proportion of mobile phase B is maintained at 95%; as well as C, the fragmentation voltage during mass spectrometry detection is 90-115 V; The liquid chromatography column was a Phenomenex Kinetex C18 column.

2. The detection method according to claim 1, characterized in that: in, The negative ion mode under SIM detection mode was used during the liquid chromatography-mass spectrometry detection process.

3. The detection method according to claim 1, characterized in that: in, The flow rate of the mobile phase during the liquid chromatography detection process is 0.1-1.0 mL / min; the column temperature of the chromatographic column is 25-35°C.

4. The detection method according to claim 1, characterized in that: in, The gradient elution time is 10 to 20 minutes.

5. The detection method according to claim 1, characterized in that: in, The flow rate of the drying gas during the mass spectrometry detection process is 10 to 20 L / min; the temperature of the drying gas during the mass spectrometry detection process is 300 to 400°C.

6. The detection method according to claim 1, characterized in that: in, The nebulizer pressure during mass spectrometry detection was (30-40) psi.

Images