Non-derivatization quantitative detection method for amino acid in high-concentration protein preparation

Abstract

The invention discloses a non-derivatization quantitative detection method for amino acid in a high-concentration protein preparation, which comprises the following steps: separating by using a graphitized carbon chromatographic column, adding an ion pair reagent trifluoroacetic acid into a mobile phase, and detecting by using an electric fog detector (CAD); the method comprises the following steps: preparing a plurality of groups of amino acid standard substance solutions with different concentration groups, wherein the concentration range is 1mmol / L-20mmol / L; drawing a quadratic equation standard curve of the amino acid standard product concentration and the peak area as a detection equation for detecting the amino acid concentration; the method comprises the following steps: diluting a test sample by using ultrapure water for multiple times, carrying out ultrafiltration centrifugal treatment, removing protein, introducing the sample, obtaining a corresponding peak area according to the spectrum integral of the test sample, and substituting the peak area into the detection equation to obtain the concentration of amino acid in the test sample. The method has the advantages of high detection precision, high detection efficiency and simple operation, and greatly reduces the adverse effect of high-concentration protein on the chromatographic column.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical detection, and particularly relates to a non-derivatized quantitative detection method for amino acids in high-concentration protein preparations. Background Art

[0002] At present, there are more and more antibody subcutaneous injection biological products on the market. Such biological products are generally high-concentration protein solutions, and high-concentration protein solutions usually add excipients such as arginine and histidine to reduce the viscosity of the protein solution. However, during the ultrafiltration process, high-concentration protein solutions are easily affected by the Donan effect, which affects the concentration of arginine and histidine in the excipients. Therefore, accurate determination of the concentration of arginine and histidine in high-concentration proteins plays a vital role in the ultrafiltration process of high-concentration protein preparations.

[0003] There are many methods for the detection of amino acids. The common methods are as follows: (1) Pre-column derivatization of amino acids.

[0004] Derivatization reagents such as phenyl isothiocyanate, carbamate, 2,4-dinitrofluorobenzene or o-phthalaldehyde are used to react with amino acids in a specific solvent to generate derivatization products with fluorescent or ultraviolet signals. Ultra-high performance liquid chromatography instruments with ultraviolet detectors or fluorescence detectors are used to separate and quantify the amino acids.

[0005] The disadvantages of this method are: the pre-column derivatization reaction is cumbersome, the reagents are toxic to a certain extent, and the commercial derivatization kits are expensive.

[0006] (2) Amino acid analyzer.

[0007] An amino acid analyzer and an ion exchange chromatography column are used for separation, and ninhydrin derivatization is performed after the column to generate a blue-purple substance that can be detected by a spectrophotometer for quantification.

[0008] The disadvantages of this method are that the amino acid analyzer is specifically used to detect amino acids and cannot be used for other tests. The instrument has great limitations and is expensive, making it unsuitable for use by pharmaceutical companies.

[0009] Due to the high polarity of amino acids, they are weakly retained on conventional reversed-phase C18 columns, and most amino acids do not have ultraviolet luminescent groups, making direct detection difficult. Therefore, a common method for determining amino acid concentration is to perform pre-column derivatization on amino acids to increase the hydrophobicity and ultraviolet absorption capacity of amino acids. For example, in the prior art, patent CN115932079A discloses a high-performance liquid chromatography method for detecting free chiral amino acid fingerprints of protein products and its application. In this method, derivatization reagents need to be configured, and the derivatization process is cumbersome and complicated, the pretreatment time is long, and the operation time on the reversed-phase C18 column after being put on the machine is long, and the detection efficiency is low; and because there are many operating steps, a variety of different reagents need to be added during the derivatization process, and light-proof operation is required, which increases the complexity of the operation; in addition, the addition of a variety of different reagents causes poor repeatability of the results.

[0010] Therefore, it is necessary to provide a new non-derivatized quantitative detection method for amino acids in high-concentration protein preparations to solve the above technical problems. Summary of the invention

[0011] The main purpose of the present invention is to provide a non-derivatized quantitative detection method for amino acids in high-concentration protein preparations, which has high detection accuracy, high detection efficiency, simple operation, and greatly reduces the adverse effects on the chromatographic column.

[0012] The present invention achieves the above object through the following technical scheme: a method for quantitatively detecting amino acids in high-concentration protein preparations without derivatization, comprising the following steps: S1. Balancing the chromatographic column with an initial mobile phase, wherein the initial mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile; and the chromatographic column is a Hypercarb graphitized carbon chromatographic column produced by Thermo Fisher Scientific; S2. After the chromatographic column is balanced, a blank solution is injected into the chromatograph, and a gradient elution process is run to obtain a blank solution spectrum; the chromatograph is a high performance liquid chromatograph connected to a charged aerosol detector CAD; The gradient elution process is: From 0 to 20 minutes, the volume fraction of mobile phase A changes from 100% to 75% at a constant rate, and the volume fraction of mobile phase B changes from 0% to 25% at a constant rate. The flow rate of the mobile phase is 0.8-1.2 mL / min. From 20 to 20.1 minutes, the volume fraction of mobile phase A was changed from 75% to 100% at a constant speed, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant speed. The flow rate of the mobile phase was 0.8 to 1.2 mL / min. From 20.1 to 30 min, the volume fraction of mobile phase A was maintained at 100%, and the volume fraction of mobile phase B was maintained at 0%; S3, respectively injecting N groups of amino acid standard curve solutions into the chromatograph, running the gradient elution process, and obtaining N groups of corresponding standard curve solution spectra, where N is an integer greater than or equal to 5; S4, diluting the solution to be tested with ultrapure water at least four times, replacing the liquid with an ultrafiltration centrifuge tube and centrifuging, taking out the filtrate and injecting it into the chromatograph, running the gradient elution process, and obtaining a spectrum of the solution to be tested; S5. Data analysis: S51, integrating N groups of standard curve solution spectra to obtain N peak areas Q, taking the concentration of the N groups of standard curve solutions as the abscissa and the corresponding peak area Q as the ordinate, obtaining the peak area-concentration curve of the amino acid, and fitting to obtain a quadratic equation Y; S52, integrating the spectrum of the solution to be tested to obtain the amino acid peak area Q'; S53. Substitute the peak area Q' into the quadratic equation Y to obtain the amino acid detection concentration.

[0013] Furthermore, the volume fraction of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.5%.

[0014] Furthermore, the N groups of amino acid standard curve solutions have the same components but different concentrations, and the concentration range is 1 mmol / L to 20 mmol / L.

[0015] Furthermore, the amino acids include one or more of histidine, arginine, methionine, lysine, proline or glycine.

[0016] Furthermore, the chromatographic column temperature is 30°C.

[0017] Furthermore, the centrifugal treatment time is at least 5 minutes, and the rotation speed is above 12000 rpm.

[0018] Furthermore, the blank solution is a 0.1 mol / L hydrochloric acid solution.

[0019] Compared with the prior art, the beneficial effect of the non-derivatized quantitative detection method of amino acids in high-concentration protein preparations of the present invention is that: by using a graphitized carbon chromatographic column, combining trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and designing a specific gradient elution process, and using an electric aerosol detector as a detector, the amino acids can have a long retention time on the chromatographic column without derivatization treatment before injection, and the separation of different amino acids is good, and the linear relationship of the external standard method is good. This method dilutes it with ultrapure water at least four times before injection, and centrifuges it with an ultrafiltration centrifuge tube. The retention time of amino acids on the chromatographic column is prolonged without causing protein to form adsorption on the chromatographic column, thereby eliminating the adverse effects of high-concentration protein on the chromatographic column; in addition, this method has higher detection efficiency. Specifically: (1) A graphitized carbon column was used, and trifluoroacetic acid aqueous solution and acetonitrile were used as the mobile phase. Gradient elution was completed within 20 minutes. The sample was injected after 10 minutes of equilibration (i.e., the 20.1-30 minute process in the gradient elution). The separation effect was good, the retention time was moderate, the repeatability was good, and the quantitative accuracy was high. The retention of polar compounds by the graphitized carbon black column was significantly better than that of the reversed phase column. Under the condition of adding the ion pair reagent trifluoroacetic acid as the mobile phase, the ion pair reagent could further prolong the retention time of amino acids, and good retention behavior could be achieved without derivatization. The retention time of amino acids in this method was moderate and the separation was good. (2) Compared with the traditional pre-column derivatization method, this method is simple, efficient, easy to operate, and greatly improves the detection efficiency; (3) The charged aerosol detector is used for detection, and the quantitative limit is 1mmol / L, which can meet the quantitative requirements of amino acid excipients in high-concentration preparations; the charged aerosol detector used is a universal detector that does not require the sample to have ultraviolet absorption and can directly detect underivatized amino acids, greatly shortening the pretreatment process of amino acids; (4) The method of ultrafiltration after dilution is used to remove proteins in the test solution. This is simple and quick, eliminates the adverse effects of high-concentration proteins on the chromatographic column, and extends the service life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a chromatogram of a blank solution in an embodiment of the present invention; Figure 2 The chromatogram of the standard curve SC-1 solution in the embodiment of the present invention; Figure 3 The chromatogram of the standard curve SC-2 solution in the embodiment of the present invention; Figure 4 The chromatogram of the standard curve SC-3 solution in the embodiment of the present invention; Figure 5 The chromatogram of the standard curve SC-4 solution in the embodiment of the present invention; Figure 6 The chromatogram of the standard curve SC-5 solution in the embodiment of the present invention; Figure 7 : is the peak area-concentration relationship curve of His in the embodiment of the present invention; Figure 8 is the peak area-concentration relationship curve of Arg in the embodiment of the present invention; Fig. 9 is a chromatogram of the solution to be tested in an embodiment of the present invention; Fig.10 It is the chromatogram of Comparative Example 1 of the present invention; Fig.11It is the chromatogram of Comparative Example 2 of the present invention; Fig.12 It is the chromatogram of Comparative Example 3 of the present invention; Fig.13 It is the chromatogram of Comparative Example 4 of the present invention; Fig.14 It is the chromatogram of Comparative Example 5 of the present invention; Fig.15 1 is a chromatogram of various types of amino acids in the embodiments of the present invention. DETAILED DESCRIPTION

[0021] Embodiment 1: This embodiment is a method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization, which comprises the following steps: S1. Equilibrate the chromatographic column with an initial mobile phase, wherein the initial mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.5% trifluoroacetic acid aqueous solution, and mobile phase B is acetonitrile; the temperature of the chromatographic column is 30° C.; the chromatographic column is a Hypercarb graphitized carbon chromatographic column from Thermo Fisher Scientific, with a specification of “5 μm, 4.6×150 mm”.

[0022] S2. After the chromatographic column is balanced, a blank solution is injected into the chromatograph and a gradient elution process is run to obtain a blank solution spectrum TP1, such as Figure 1 As shown; the blank solution is a 0.1 mol / L hydrochloric acid solution to prove that the sample solvent has no interfering peaks at the amino acid peak position; the chromatograph is a high performance liquid chromatograph connected to a charged aerosol detector CAD; The gradient elution process is: From 0 to 20 minutes, the volume fraction of mobile phase A changes from 100% to 75% at a constant rate, and the volume fraction of mobile phase B changes from 0% to 25% at a constant rate. The flow rate of the mobile phase is 0.8-1.2 mL / min. From 20 to 20.1 minutes, the volume fraction of mobile phase A was changed from 75% to 100% at a constant speed, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant speed. The flow rate of the mobile phase was 0.8 to 1.2 mL / min. From 20.1 to 30 min, the volume fraction of mobile phase A was maintained at 100%, and the volume fraction of mobile phase B was maintained at 0%.

[0023] In this embodiment, the flow rate of the mobile phase is 1.0 mL / min.

[0024] S3, standard curve solution injection: S31. Prepare 100 mmol / L arginine Arg stock solution and 100 mmol / L histidine His stock solution, and dissolve them in 0.1 mol / L hydrochloric acid solution.

[0025] S32, preparing five groups of arginine Arg standard curve solutions and five groups of histidine His standard curve solutions: (1) Take 200 μL of 100 mmol / L Arg stock solution and 100 mmol / L His stock solution, add 600 μL of 0.1 mol / L hydrochloric acid solution and mix well to obtain 20 mmol / L Arg solution and 20 mmol / L His solution, respectively, which are recorded as SC-5 solution; (2) Take 500 μL of SC-5 solution and add 500 μL of 0.1 mol / L hydrochloric acid solution to obtain 10 mmol / L Arg solution and 10 mmol / L His solution, respectively, which are recorded as SC-4 solution; (3) Take 500 μL of SC-4 solution and add 500 μL of 0.1 mol / L hydrochloric acid solution to obtain 5 mmol / L Arg solution and 5 mmol / L His solution, respectively, which are recorded as SC-3 solution; (4) Take 400 μL of the solution in SC-3 and add 600 μL of 0.1 mol / L hydrochloric acid solution to obtain 2 mmol / L Arg solution and 2 mmol / L His solution, respectively, which are recorded as SC-2 solution; (5) Take 500 μL of SC-2 solution and add 500 μL of 0.1 mol / L hydrochloric acid solution to obtain 1 mmol / L Arg solution and 1 mmol / L His solution, respectively, which are recorded as SC-1 solution; The SC-1 solution, SC-2 solution, SC-3 solution, SC-4 solution and SC-5 solution constitute five sets of arginine Arg standard curve solutions and five sets of histidine His standard curve solutions.

[0026] S33, respectively inject five groups of arginine Arg standard curve solutions and five groups of histidine His standard curve solutions into the chromatograph, run the gradient elution process, and obtain five groups of corresponding standard curve solution spectra TP2, such as Figure 2-Figure 6 shown.

[0027] S4. Injection of the solution to be tested: S41, dilute the test solution four times with ultrapure water, change the solution in a 10KD ultrafiltration centrifuge tube at 12000rpm and centrifuge for 5min, remove the filtrate and load the sample; the protein concentration in the test solution in this embodiment is as high as 170mg / mL, and the theoretical concentrations of arginine and histidine are 60mmol / L and 20mmol / L respectively; S42, injecting the pre-treated solution to be tested into the chromatograph, running the gradient elution process, and obtaining the spectrum TP4 of the solution to be tested.

[0028] S5. Data analysis: S51, integrating the five sets of Arg standard curve solution spectra TP2 to obtain N peak areas Q1, using the concentrations of the five sets of Arg standard curve solutions as the abscissa and the corresponding peak areas Q1 as the ordinate, fitting to obtain the peak area-concentration curve of arginine Arg and its equation Y1; the peak area-concentration curve of arginine Arg is as follows Figure 7 As shown, equation Y1 is: y = -3.05 × 10 6 x 2 +2.31×10 8 x +1.72×10 8 ; Equation absolute coefficient R 2 =0.9994.

[0029] S52, integrating the five groups of His standard curve solution spectra TP3 to obtain N peak areas Q2, using the concentrations of the five groups of His standard curve solutions as the abscissa and the corresponding peak areas Q2 as the ordinate, fitting to obtain the peak area-concentration curve of histidine His and its equation Y2; the peak area-concentration curve of histidine His is as follows Figure 8 As shown, equation Y2 is: y = -3.01 × 10 6 x 2 +2.37×10 8 x +1.89×10 8 ; Equation absolute coefficient R 2 =0.9992.

[0030] S53, integrating the spectrum TP4 of the solution to be tested to obtain the peak area Q3 corresponding to arginine Arg and the peak area Q4 corresponding to histidine His; the spectrum TP4 of the solution to be tested is as follows: Fig. 9 As shown; S54, substituting the peak area Q3 into equation Y1, the arginine detection concentration was obtained; substituting the peak area Q4 into equation Y2, the histidine detection concentration was obtained, where the His concentration was 16.8 mmol / L and the Arg concentration was 60.3 mmol / L. The comparison between the detection results and the theoretical concentrations is shown in Table 1.

[0031] Table 1

[0032] In order to verify the effectiveness of the detection method described in this example, the accuracy of the detection results of the test solution is verified. The recovery rates of His and Arg are shown in Table 2.

[0033] Table 2

[0034] From the results shown in Table 2, it can be seen that: within the concentration range of 1.0~20.0mmol / L for His, the recovery rate at 1.0mmol / L is the lowest, which is 86%, and the recovery rates at other concentration points are between 98% and 105%, with a high recovery rate; within the concentration range of 1.0~20.0mmol / L for Arg, the recovery rate at 1.0mmol / L is the lowest, which is 84%, and the recovery rates at other concentration points are between 97% and 105%, with a high recovery rate. Therefore, if the injection concentration of the test sample is controlled between 2.0~20.0mmol / L, this method has a high accuracy.

[0035] In order to verify the repeatability of the detection method described in this example, the retention times of His and Arg in 5 samples to be tested were tested, as shown in Table 3.

[0036] Table 3

[0037] From the results shown in Table 3, it can be seen that the retention times of His and Arg in the 5 samples are stable and the RSD is small, indicating that this detection method has good repeatability.

[0038] The non-derivatized quantitative detection method for amino acids in high-concentration protein preparations described in this embodiment is mainly based on the combined effect of factors such as the selection of a graphitized carbon chromatographic column for the chromatographic column, the use of 0.5% trifluoroacetic acid water as the mobile phase, the design of a specific gradient elution program, and the use of a chromatograph with an charged atomizer detector. The combined effect of the above factors ensures that even if the solution to be detected is not subjected to amino acid derivatization treatment, it can have a good retention time on the chromatographic column selected in this embodiment, can quickly produce peaks on the spectrum, and different types of amino acids can also be well separated, thereby quickly, effectively and reliably realizing the detection of amino acid concentrations in high-concentration protein preparations.

[0039] In order to verify that the selection of the chromatographic column, the design of the mobile phase and elution program, and the selection of the detector in this embodiment must work together to achieve the above technical effects, this embodiment designed comparative examples 1 to 5, and the specific methods of the five comparative examples are as follows.

[0040] Comparative Example 1: The graphitized carbon chromatographic column was balanced with the initial mobile phase, wherein the mobile phase A was water and the mobile phase B was acetonitrile, and the temperature of the chromatographic column was 30°C; after the chromatographic column was balanced, a blank solution was injected into a high performance liquid chromatograph, and a charged aerosol detector was used for detection, and the same gradient elution process as in Example 1 was run; then, a mixed solution of histidine and arginine to be tested was injected, and the charged aerosol detector was used for detection, and the same gradient elution process was run to obtain a spectrum of the solution to be tested as shown in FIG. Fig.10 shown.

[0041] Comparative Example 1 uses a mobile phase without ion-pairing agent trifluoroacetic acid, performs separation on a graphitized carbon chromatographic column, uses a charged aerosol detector for detection, and injects a mixed solution of histidine and arginine. The experimental conditions are shown in Table 4.

[0042] Table 4

[0043] The detection spectrum of comparative example 1 is as follows Fig.10 As shown in the experiment, it was found that histidine and arginine could not be well separated. Fig.10 It can be seen that the peak shapes of histidine and arginine are poor, and quantitative detection of histidine and arginine cannot be achieved, indicating that trifluoroacetic acid in the mobile phase plays an important role in enhancing the retention time of non-derivatized amino acids and improving the peak shape.

[0044] Comparative Example 2: The graphitized carbon chromatographic column was balanced with the initial mobile phase, wherein the mobile phase A was 0.5% trifluoroacetic acid aqueous solution, the mobile phase B was acetonitrile, and the chromatographic column temperature was 30°C; after the chromatographic column was balanced, a blank solution was injected into a high performance liquid chromatograph, and a UV detector was used for detection at 254 nm, and the same gradient elution process as in Example 1 was run; then a mixed solution of histidine and arginine to be tested was injected, and a UV detector was used for detection at 254 nm, and the same gradient elution process was run to obtain a spectrum of the solution to be tested as shown in FIG. Fig.11 shown.

[0045] Comparative Example 2 used a mobile phase containing trifluoroacetic acid, separated on a graphitized carbon column, detected using an ultraviolet detector at 254 nm, and injected a mixed solution of histidine and arginine. The experimental conditions are shown in Table 5.

[0046] Table 5

[0047] The detection spectrum of comparative example 2 is as follows Fig.11 As shown, from Fig.11It can be seen that the peak response value is very low and the peak shape is abnormal, indicating that the characteristic absorption of amino acids at ultraviolet 254nm is not obvious and there are other interferences. It is not suitable for direct detection of non-derivatized amino acids, and thus cannot achieve quantitative detection of amino acids.

[0048] Comparative Example 3: The reverse phase C18 chromatographic column was equilibrated with the initial mobile phase, wherein the mobile phase A was 0.5% trifluoroacetic acid aqueous solution, the mobile phase B was acetonitrile, and the chromatographic column temperature was 30°C; after the chromatographic column was equilibrated, a blank solution was injected into a high performance liquid chromatograph, and a charged aerosol detector was used for detection, and the same gradient elution process as in Example 1 was run; then, a mixed solution of histidine and arginine to be tested was injected, and the charged aerosol detector was used for detection, and the same gradient elution process was run to obtain a spectrum of the solution to be tested as shown in FIG. Fig.12 shown.

[0049] Comparative Example 3 used a mobile phase containing trifluoroacetic acid, performed separation on a reverse phase C18 chromatographic column, used a charged aerosol detector for detection, and injected a mixed solution of histidine and arginine. The experimental conditions are shown in Table 6.

[0050] Table 6

[0051] The detection spectrum of Comparative Example 3 is as follows Fig.12 As shown. Chromatographic columns generally have a non-retention volume, which is called "dead volume" in the industry, and the corresponding time is called "dead time". During the experiment, it was found that amino acids were not retained. Fig.12 It can be seen that the peak appears at the "dead time" and there is no sign of separation of the two amino acids, indicating that the reversed-phase C18 column is not suitable for the separation of non-derivatized amino acids, and thus the quantitative detection of amino acids cannot be achieved.

[0052] Comparative Example 4: The graphitized carbon chromatographic column was balanced with the initial mobile phase, wherein the mobile phase A was 5% acetonitrile, 1% methanol, and 0.08% trifluoroacetic acid aqueous solution; the mobile phase B was 60% acetonitrile, 1% methanol, and 0.08% trifluoroacetic acid aqueous solution; the chromatographic column temperature was 30°C; after the chromatographic column was balanced, a blank solution was injected into a high performance liquid chromatograph, and a charged aerosol detector was used for detection, and the same gradient elution process as in Example 1 was run; then a mixed solution of histidine and arginine to be tested was injected, and a charged aerosol detector was used for detection, and the same gradient elution process was run to obtain a spectrum of the solution to be tested as shown in FIG. Fig.13 shown.

[0053] Comparative Example 4 used the following mobile phases: A: 5% acetonitrile, 1% methanol, 0.08% trifluoroacetic acid aqueous solution; B: 60% acetonitrile, 1% methanol, 0.08% trifluoroacetic acid aqueous solution, gradient elution was performed on a graphitized carbon column, detection was performed using a charged aerosol detector, and a mixed solution of histidine and arginine was injected. The experimental conditions are shown in Table 7.

[0054] Table 7

[0055] The detection spectrum of Comparative Example 4 is as follows Fig.13 As shown, it was found during the experiment that histidine and arginine were weakly retained and could not be separated, and thus quantitative detection of histidine and arginine could not be achieved; this indicates that the concentration of trifluoroacetic acid and the proportion of organic solvent in the mobile phase play an important role in enhancing the retention time of non-derivatized amino acids and improving the peak shape. In the mobile phase A of Example 1, methanol and acetonitrile were not added, only trifluoroacetic acid aqueous solution was added, and the solubility reached 0.5%, which can effectively enhance the retention time of non-derivatized amino acids and improve the peak shape.

[0056] Comparative Example 5: The graphitized carbon column was balanced with the initial mobile phase, where mobile phase A was 0.5% trifluoroacetic acid aqueous solution; mobile phase B was acetonitrile; the column temperature was 30°C; after the column was balanced, the blank solution was injected into the HPLC, and the charged aerosol detector was used for detection, and the following gradient elution process was run: 0.0~5.0min: 100%A, 0%B; 5.0-25.0min: 85%A, 15%B; 30.1-40.0min: 100%, 0%B; Then, the mixed solution of histidine and arginine to be tested is injected, detected by charged aerosol detector, and the above-mentioned gradient elution process is run to obtain the spectrum of the solution to be tested as shown in Fig.14 shown.

[0057] Comparative Example 5 used the same chromatographic column, mobile phase and detector as in Example 1, injected a mixed solution of histidine and arginine, and investigated different gradient elutions. The experimental conditions are shown in Table 8.

[0058] Table 8

[0059] The detection spectrum of Comparative Example 5 is as follows Fig.14As shown, it was found that histidine and arginine were well separated during the experiment. Although simultaneous quantitative detection of multiple amino acids could be achieved, the peak appeared only at about 16 minutes, and the peak time was relatively long. Compared with the peak appearance at about 11 minutes in Example 1, the detection efficiency was reduced. Therefore, the gradient elution program in Example 1 can achieve higher detection efficiency.

[0060] This example also tested the concentration of various amino acids according to the non-derivatized quantitative detection method for amino acids in high-concentration protein preparations described in this example. The test results are as follows: Fig.15 As shown. Fig.15 It can be seen that proline, lysine, and methionine can also achieve good separation from arginine and histidine. Therefore, this method is also feasible and effective for other types of amino acids.

[0061] The advantages of this embodiment are: First, graphitized carbon columns can enhance the retention of amino acids. If the mobile phase, gradient and detector remain unchanged and the column is replaced with a reversed-phase column, the amino acids will not be retained and separated because of their strong polarity.

[0062] Secondly, the ion pair reagent trifluoroacetic acid can further increase the retention behavior of amino acids. If the chromatographic column, gradient and detector are unchanged and trifluoroacetic acid is not added to the mobile phase, the retention time of amino acids will be shortened and the separation will be poor due to the strong polarity of amino acids.

[0063] Finally, electrospray detection can directly detect compounds without UV absorption. If the chromatographic column, mobile phase and gradient remain unchanged and the detector is replaced with a UV detector, amino acids will have no response signal in the UV detector and cannot be detected.

[0064] The present embodiment uses a graphitized carbon chromatographic column for separation, and adds an ion pair reagent trifluoroacetic acid to the mobile phase, which can increase the retention time of arginine and histidine on the chromatographic column, and has a good separation degree due to the difference in polarity; an electric aerosol detector CAD is used, and the detection signal is sensitive; five groups of standard solutions of amino acids and arginine with different concentrations are prepared, and the concentration range is between 1mmol / L and 20mmol / L; a quadratic equation standard curve of amino acid standard concentration and peak area is drawn as a detection equation for detecting amino acid concentration, and then the corresponding peak area is obtained according to the spectrum integration of the test sample, and then the peak area is brought into the above detection equation to obtain the detection concentration of the corresponding amino acid type in the test sample; the present embodiment dilutes the test sample four times with ultrapure water, centrifuges in a 10KD ultrafiltration centrifuge tube, removes protein, and takes out the filtrate for on-machine detection, which can eliminate the attachment of protein to the chromatographic column, reduce the adverse effects on the chromatographic column, and thus ensure the service life of the chromatographic column.

[0065] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for quantitatively detecting amino acids in high-concentration protein preparations without derivatization, characterized in that: The following steps are involved: S1. Balancing the chromatographic column with an initial mobile phase, wherein the initial mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile; and the chromatographic column is a Hypercarb graphitized carbon chromatographic column produced by Thermo Fisher Scientific; S2. After the chromatographic column is balanced, a blank solution is injected into the chromatograph, and a gradient elution process is run to obtain a blank solution spectrum; the chromatograph is a high performance liquid chromatograph connected to a charged aerosol detector CAD; The gradient elution process is: From 0 to 20 minutes, the volume fraction of mobile phase A changes from 100% to 75% at a constant rate, and the volume fraction of mobile phase B changes from 0% to 25% at a constant rate. The flow rate of the mobile phase is 0.8-1.2 mL / min. From 20 to 20.1 minutes, the volume fraction of mobile phase A was changed from 75% to 100% at a constant speed, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant speed. The flow rate of the mobile phase was 0.8 to 1.2 mL / min. From 20.1 to 30 min, the volume fraction of mobile phase A was maintained at 100%, and the volume fraction of mobile phase B was maintained at 0%; S3, respectively injecting N groups of amino acid standard curve solutions into the chromatograph, running the gradient elution process, and obtaining N groups of corresponding standard curve solution spectra, where N is an integer greater than or equal to 5; S4, diluting the solution to be tested with ultrapure water at least four times, replacing the solution with an ultrafiltration centrifuge tube and centrifuging, taking out the filtrate and injecting it into the chromatograph, running the gradient elution process, and obtaining a spectrum of the solution to be tested; S5. Data analysis: S51, integrating the N groups of standard curve solution spectra to obtain N peak areas Q, taking the concentrations of the N groups of standard curve solutions as the abscissa and the corresponding peak areas Q as the ordinate, obtaining the peak area-concentration curves of the amino acids, and fitting to obtain a quadratic equation Y; S52, integrating the spectrum of the solution to be tested to obtain the amino acid peak area Q'; S53. Substitute the peak area Q' into the quadratic equation Y to obtain the amino acid detection concentration.

2. The method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization according to claim 1, characterized in that: The amount of trifluoroacetic acid added to the trifluoroacetic acid aqueous solution is 0.5% by volume.

3. The method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization according to claim 1, characterized in that: The N groups of amino acid standard curve solutions have the same components but different concentrations, and the concentration range is 1 mmol / L to 20 mmol / L.

4. The method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization according to claim 1, characterized in that: The amino acids include one or more of histidine, arginine, methionine, lysine, proline or glycine.

5. The method for quantitative detection of amino acids in high-concentration protein preparations without derivatization according to claim 1, characterized in that: The temperature of the chromatographic column is 30°C.

6. The method for quantitatively detecting amino acids in high-concentration protein preparations without derivatization according to claim 1, characterized in that: The centrifugal treatment time is at least 5 minutes, and the rotation speed is above 12000 rpm.

7. The method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization according to claim 1, characterized in that: The blank solution is 0.1 mol / L hydrochloric acid solution.

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