A method for quantitative detection of amino acids in high-concentration protein preparations without derivatization

Abstract

The present invention discloses a method for quantitatively detecting amino acids in high-concentration protein preparations without derivatization. The method uses a graphitized carbon chromatographic column for separation, adds the ion-pairing reagent trifluoroacetic acid to the mobile phase, and employs a charged aerosol detector (CAD) for detection. Multiple amino acid standard solutions with varying concentrations ranging from 1 mmol / L to 20 mmol / L are prepared. A quadratic standard curve is plotted, representing the relationship between amino acid standard concentration and peak area, to serve as the detection equation for amino acid concentration. The test sample is diluted multiple times with ultrapure water, then ultrafiltered and centrifuged for protein removal before injection. The corresponding peak area is then integrated from the test sample's chromatographic profile and then substituted into the detection equation to determine the amino acid concentration in the test sample. This method achieves high detection accuracy, high efficiency, and simple operation, while significantly reducing the adverse effects 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] Currently, an increasing number of subcutaneous antibody biologics are appearing on the market. These biologics are generally high-concentration protein solutions, to which excipients such as arginine and histidine are often added to reduce the viscosity of the protein solution. However, during the ultrafiltration process, high-concentration protein solutions are susceptible to the Donan effect, which affects the concentrations of arginine and histidine in the excipients. Therefore, accurately measuring the concentrations of arginine and histidine in high-concentration protein plays a crucial role in the ultrafiltration process of high-concentration protein preparations.

[0003] There are currently many methods for detecting amino acids, among which the most common methods are as follows:

[0004] (1) Pre-column derivatization of amino acids.

[0005] 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 derivatized products with fluorescent or UV signals. An ultra-high performance liquid chromatography instrument equipped with a UV detector or a fluorescence detector is used to separate and quantify the amino acids.

[0006] 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.

[0007] (2) Amino acid analyzer.

[0008] 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.

[0009] 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.

[0010] Due to the high polarity of amino acids, they are poorly retained on conventional reverse-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 the amino acids to increase their hydrophobicity and ultraviolet absorption capacity. 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. This method requires the configuration of derivatization reagents, and the derivatization process is cumbersome and complicated, with a long pre-treatment time. The run time on the reverse-phase C18 column after being put on the machine is long, and the detection efficiency is low. Moreover, due to the large number of operating steps, a variety of different reagents need to be added during the derivatization process, and the operation needs to be kept away from light, which increases the complexity of the operation. In addition, the addition of a variety of different reagents causes poor reproducibility of the results.

[0011] 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

[0012] 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.

[0013] The present invention achieves the above-mentioned object through the following technical solution: a method for quantitative detection of amino acids in high-concentration protein preparations without derivatization, comprising the following steps:

[0014] S1. Equilibrate the chromatographic column with an initial mobile phase, wherein the initial mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile; and the chromatographic column is a Hypercarb graphitized carbon chromatographic column from Thermo Fisher Scientific.

[0015] 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);

[0016] The gradient elution process is as follows:

[0017] From 0 to 20 minutes, the volume fraction of mobile phase A was changed from 100% to 75% at a constant rate, and the volume fraction of mobile phase B was changed from 0% to 25% at a constant rate. The flow rate of the mobile phase was 0.8 to 1.2 mL / min.

[0018] From 20 to 20.1 minutes, the volume fraction of mobile phase A was changed from 75% to 100% at a constant rate, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant rate. The flow rate of the mobile phase was 0.8 to 1.2 mL / min.

[0019] 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%;

[0020] 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;

[0021] S4. Dilute the test solution at least four times with ultrapure water, replace the solution with an ultrafiltration centrifuge tube, and centrifuge. Take the filtrate and inject it into the chromatograph, run the gradient elution process, and obtain a spectrum of the test solution.

[0022] S5. Data Analysis:

[0023] S51. Integrate the N groups of standard curve solution spectra to obtain N peak areas Q, use the concentrations of the N groups of standard curve solutions as the abscissa and the corresponding peak areas Q as the ordinate to obtain peak area-concentration curves of the amino acids, and fit them to obtain a quadratic equation Y;

[0024] S52, integrating the spectrum of the solution to be tested to obtain the amino acid peak area Q';

[0025] S53. Substitute the peak area Q' into the quadratic equation Y to obtain the amino acid detection concentration.

[0026] Furthermore, the volume fraction of trifluoroacetic acid in the trifluoroacetic acid aqueous solution is 0.5%.

[0027] 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.

[0028] Furthermore, the amino acids include one or more of histidine, arginine, methionine, lysine, proline or glycine.

[0029] Furthermore, the temperature of the chromatographic column is 30°C.

[0030] Furthermore, the centrifugal treatment time is at least 5 minutes, and the rotation speed is above 12000 rpm.

[0031] Furthermore, the blank solution is 0.1 mol / L hydrochloric acid solution.

[0032] 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 charged atomizer 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 external standard method has a good linear relationship. 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 extended while the protein does not 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:

[0033] (1) Using a graphitized carbon column, trifluoroacetic acid aqueous solution and acetonitrile as the mobile phase, gradient elution was completed within 20 minutes, and the sample was injected after 10 minutes of equilibrium (i.e., the 20.1-30 minute process in the gradient elution). The separation effect was good, the retention time was moderate and the repeatability was good, and the quantitative accuracy was high. The graphitized carbon black column had significantly better retention of polar compounds than the reversed-phase column. Under the mobile phase conditions with the addition of the ion-pairing agent trifluoroacetic acid, the ion-pairing agent could further extend the retention time of amino acids, and good retention behavior could be achieved without derivatization. The amino acid retention time in this method was moderate and the separation was good.

[0034] (2) Compared with the traditional pre-column derivatization method, this method is simple, efficient, and easy to operate, which greatly improves the detection efficiency;

[0035] (3) The detection is carried out using a charged aerosol detector with a quantitative limit of 1 mmol / 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 ultraviolet absorption of the sample and can directly detect underivatized amino acids, greatly shortening the pre-treatment process of amino acids.

[0036] (4) The protein in the test solution is removed by ultrafiltration after dilution, which is simple and quick, eliminates the adverse effects of high-concentration protein on the chromatographic column, and extends the service life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a chromatogram of a blank solution in an embodiment of the present invention;

[0038] Figure 2 The chromatogram of the standard curve SC-1 solution in the embodiment of the present invention is shown in FIG.

[0039] Figure 3 The chromatogram of the standard curve SC-2 solution in the embodiment of the present invention is shown;

[0040] Figure 4 This is a chromatogram of the standard curve SC-3 solution in the embodiment of the present invention;

[0041] Figure 5 The chromatogram of the standard curve SC-4 solution in the embodiment of the present invention is shown;

[0042] Figure 6 This is a chromatogram of the standard curve SC-5 solution in the embodiment of the present invention;

[0043] Figure 7 : is the peak area-concentration relationship curve of His in the embodiment of the present invention;

[0044] Figure 8 : is the peak area-concentration relationship curve of Arg in the embodiment of the present invention;

[0045] Figure 9 is a chromatogram of the solution to be tested in an embodiment of the present invention;

[0046] Figure 10 It is the chromatogram of Comparative Example 1 of the present invention;

[0047] Figure 11 It is the chromatogram of Comparative Example 2 of the present invention;

[0048] Figure 12 This is the chromatogram of Comparative Example 3 of the present invention;

[0049] Figure 13 It is the chromatogram of Comparative Example 4 of the present invention;

[0050] Figure 14 This is the chromatogram of Comparative Example 5 of the present invention;

[0051] Figure 15 1 is a chromatogram of various types of amino acids in the examples of the present invention. DETAILED DESCRIPTION

[0052] Example 1:

[0053] This embodiment provides a method for quantitatively detecting amino acids in a high-concentration protein preparation without derivatization, which comprises the following steps:

[0054] 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 chromatographic column temperature 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".

[0055] S2. After the chromatographic column is balanced, inject the blank solution into the chromatograph and run the gradient elution process to obtain the blank solution spectrum TP1, as shown in Figure 1 As shown; the blank solution is 0.1 mol / L hydrochloric acid solution to prove that the sample solvent has no interfering peak at the amino acid peak position; the chromatograph is a high performance liquid chromatograph connected to a charged aerosol detector CAD;

[0056] The gradient elution process is as follows:

[0057] From 0 to 20 minutes, the volume fraction of mobile phase A was changed from 100% to 75% at a constant rate, and the volume fraction of mobile phase B was changed from 0% to 25% at a constant rate. The flow rate of the mobile phase was 0.8 to 1.2 mL / min.

[0058] From 20 to 20.1 minutes, the volume fraction of mobile phase A was changed from 75% to 100% at a constant rate, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant rate. The flow rate of the mobile phase was 0.8 to 1.2 mL / min.

[0059] 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%.

[0060] In this embodiment, the flow rate of the mobile phase is 1.0 mL / min.

[0061] S3. Standard curve solution injection:

[0062] 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.

[0063] S32, preparing five groups of arginine Arg standard curve solutions and five groups of histidine His standard curve solutions:

[0064] (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;

[0065] (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;

[0066] (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;

[0067] (4) Take 400 μL of the SC-3 solution 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;

[0068] (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;

[0069] The SC-1 solution, SC-2 solution, SC-3 solution, SC-4 solution and SC-5 solution constitute five groups of arginine Arg standard curve solutions and five groups of histidine His standard curve solutions.

[0070] S33, respectively injecting five groups of arginine Arg standard curve solutions and five groups of histidine His standard curve solutions into the chromatograph, running the gradient elution process, and obtaining five groups of corresponding standard curve solution spectra TP2, as shown in FIG. Figure 2-Figure 6 shown.

[0071] S4. Injection of the test solution:

[0072] S41. Dilute the test solution four-fold with ultrapure water, replace the solution with centrifuge at 12,000 rpm for 5 minutes in a 10KD ultrafiltration centrifuge tube, and remove the filtrate for sample loading. In this embodiment, the protein concentration in the test solution is as high as 170 mg / mL, and the theoretical concentrations of arginine and histidine are 60 mmol / L and 20 mmol / L, respectively.

[0073] 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.

[0074] S5. Data Analysis:

[0075] S51. Integrate the five sets of Arg standard curve solution spectra TP2 to obtain N peak areas Q1. Use the concentrations of the five sets of Arg standard curve solutions as the abscissa and the corresponding peak areas Q1 as the ordinate 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:

[0076] y = -3.05 × 106 x 2 +2.31×10 8 x +1.72×10 8 ;

[0077] Equation absolute coefficient R 2 =0.9994.

[0078] S52, integrating the five sets of His standard curve solution spectra TP3 to obtain N peak areas Q2, using the concentrations of the five sets 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:

[0079] y = -3.01 × 10 6 x 2 +2.37×10 8 x +1.89×10 8 ;

[0080] Equation absolute coefficient R 2 =0.9992.

[0081] 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: Figure 9 As shown;

[0082] S54. Substitute peak area Q3 into equation Y1 to obtain the arginine concentration. Substitute peak area Q4 into equation Y2 to obtain the histidine concentration, where the His concentration is 16.8 mmol / L and the Arg concentration is 60.3 mmol / L. A comparison of the test results with the theoretical concentrations is shown in Table 1.

[0083] Table 1

[0084]

[0085] To verify the effectiveness of the detection method described in this example, the accuracy of the detection results of the test solution was verified. The recovery rates of His and Arg are shown in Table 2.

[0086] Table 2

[0087]

[0088] The results shown in Table 2 show that within the His concentration range of 1.0 to 20.0 mmol / L, the recovery rate was lowest at 1.0 mmol / L, at 86%, while the recovery rates at other concentrations ranged from 98% to 105%, indicating high recovery rates. Within the Arg concentration range of 1.0 to 20.0 mmol / L, the recovery rate was lowest at 1.0 mmol / L, at 84%, while the recovery rates at other concentrations ranged from 97% to 105%, indicating high recovery rates. Therefore, controlling the sample injection concentration between 2.0 and 20.0 mmol / L provides a high degree of accuracy for this method.

[0089] In order to verify the repeatability of the detection method described in this example, the retention times of His and Arg in 5 test samples were tested, as shown in Table 3.

[0090] Table 3

[0091]

[0092] From the results shown in Table 3, it can be seen that the retention times of His and Arg in the five samples are stable and the RSDs are small, indicating that this detection method has good repeatability.

[0093] 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 the following factors: the use of a graphitized carbon column as a 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 aerosol detector. The combined effect of these 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 achieving rapid, effective, and reliable detection of amino acid concentrations in high-concentration protein preparations.

[0094] 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 efficacy, this embodiment designed comparative examples 1 to 5. The specific methods of the five comparative examples are as follows.

[0095] Comparative Example 1:

[0096] The graphitized carbon chromatographic column was equilibrated with the initial mobile phase, wherein the mobile phase A was water and the mobile phase B was acetonitrile, and the column temperature was 30°C. After the column was equilibrated, a blank solution was injected into the high performance liquid chromatograph, and the charged aerosol detector was used for detection, and the same gradient elution process as in Example 1 was run. Then, the 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 the spectrum of the test solution as shown in FIG. Figure 10 shown.

[0097] Comparative Example 1 used a mobile phase without the ion-pairing agent trifluoroacetic acid, performed separation on a graphitized carbon column, detected using a charged aerosol detector, and injected a mixed solution of histidine and arginine. The experimental conditions are shown in Table 4.

[0098] Table 4

[0099]

[0100] The detection spectrum of comparative example 1 is as follows Figure 10 As shown in the experiment, it was found that histidine and arginine could not be well separated. Figure 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.

[0101] Comparative Example 2:

[0102] The graphitized carbon chromatographic column was equilibrated with the initial mobile phase, wherein the mobile phase A was 0.5% trifluoroacetic acid aqueous solution and the mobile phase B was acetonitrile, and the column temperature was 30°C. After the column was equilibrated, a blank solution was injected into the high performance liquid chromatograph, and the elution was detected by an ultraviolet detector at 254 nm. The same gradient elution process as in Example 1 was run. Then, the mixed solution of histidine and arginine to be tested was injected, and the elution was detected by an ultraviolet detector at 254 nm. The same gradient elution process was run to obtain the spectrum of the solution to be tested as shown in FIG. Figure 11 shown.

[0103] Comparative Example 2 used a mobile phase containing trifluoroacetic acid, separated on a graphitized carbon column, detected using a UV detector at 254 nm, and injected a mixed solution of histidine and arginine. The experimental conditions are shown in Table 5.

[0104] Table 5

[0105]

[0106] The detection spectrum of comparative example 2 is as follows Figure 11 As shown, from Figure 11 It 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.

[0107] Comparative Example 3:

[0108] The reverse phase C18 column was equilibrated with the initial mobile phase, wherein the mobile phase A was 0.5% trifluoroacetic acid aqueous solution and the mobile phase B was acetonitrile, and the column temperature was 30°C. After the column was equilibrated, a blank solution was injected into the HPLC, and the HPLC was used for detection by a charged aerosol detector. The same gradient elution process as in Example 1 was run. The histidine and arginine mixed solution to be tested was then injected, and the charged aerosol detector was used for detection. The same gradient elution process was run. The chromatogram of the test solution was obtained as shown in FIG. Figure 12 shown.

[0109] Comparative Example 3 used a mobile phase containing trifluoroacetic acid, performed separation on a reversed-phase C18 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.

[0110] Table 6

[0111]

[0112] The detection spectrum of comparative example 3 is as follows Figure 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. Figure 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 cannot achieve quantitative detection of amino acids.

[0113] Comparative Example 4:

[0114] The graphitized carbon chromatographic column was equilibrated with the initial mobile phase, wherein mobile phase A was 5% acetonitrile, 1% methanol, and 0.08% trifluoroacetic acid aqueous solution; mobile phase B was 60% acetonitrile, 1% methanol, and 0.08% trifluoroacetic acid aqueous solution; the column temperature was 30°C; after the column was equilibrated, a blank solution was injected into a high performance liquid chromatograph, and the chromatographic analysis was performed using a charged aerosol detector. The same gradient elution process as in Example 1 was then performed. The histidine and arginine mixed solution to be tested was then injected, and the chromatogram of the test solution was obtained using a charged aerosol detector. Figure 13 shown.

[0115] 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, and detection was performed using a charged aerosol detector. A mixed solution of histidine and arginine was injected. The experimental conditions are shown in Table 7.

[0116] Table 7

[0117]

[0118] The detection spectrum of Comparative Example 4 is as follows Figure 13 As shown, during the experiment, it was found 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 and improving the peak shape of non-derivatized amino acids. In Example 1, no methanol and acetonitrile were added to the mobile phase A, and only trifluoroacetic acid aqueous solution was added, and the solubility reached 0.5%, which can effectively enhance the retention time and improve the peak shape of non-derivatized amino acids.

[0119] Comparative Example 5:

[0120] The graphitized carbon column was equilibrated with the initial mobile phase. Mobile phase A was 0.5% trifluoroacetic acid in water. Mobile phase B was acetonitrile. The column temperature was 30°C. After the column was equilibrated, a blank solution was injected into the HPLC, and the column was detected using a charged aerosol detector. The following gradient elution process was performed:

[0121] 0.0-5.0 min: 100% A, 0% B;

[0122] 5.0-25.0 min: 85% A, 15% B;

[0123] 30.1-40.0 min: 100%, 0% B;

[0124] Then the mixed solution of histidine and arginine to be tested was injected and detected by charged aerosol detector. The above gradient elution process was run to obtain the spectrum of the solution to be tested as shown in the figure. Figure 14 shown.

[0125] 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.

[0126] Table 8

[0127]

[0128] The detection spectrum of Comparative Example 5 is as follows Figure 14 As shown, during the experiment, it was found that histidine and arginine were well separated. Although simultaneous quantitative detection of multiple amino acids was possible, the peak appeared only at about 16 minutes, which was a long time. Compared with the peak appearance at about 11 minutes in Example 1, the detection efficiency was reduced. Therefore, the gradient elution procedure in Example 1 can achieve higher detection efficiency.

[0129] This example also tested the concentration of various amino acids using the non-derivatized quantitative detection method for amino acids in high-concentration protein preparations described in this example. The test results are as follows: Figure 15 As shown. Figure 15 As can be seen from the figure, 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.

[0130] The advantages of this embodiment are:

[0131] First, graphitized carbon columns can enhance the retention of amino acids. If the mobile phase, gradient, and detector remain unchanged but the column is replaced with a reversed-phase column, amino acids will not be retained on reversed-phase columns due to their strong polarity, resulting in no retention and no separation of amino acids.

[0132] Secondly, the ion-pairing agent trifluoroacetic acid can further enhance the retention of amino acids. If the column, gradient, and detector remain unchanged and trifluoroacetic acid is not added to the mobile phase, the retention time of amino acids will be shortened and the resolution will be poor due to the strong polarity of amino acids.

[0133] Finally, electrospray detection can directly detect compounds that do not absorb UV light. If the column, mobile phase, and gradient remain unchanged but the detector is replaced with a UV detector, amino acids will not respond to the UV detector and cannot be detected.

[0134] In this embodiment, a graphitized carbon chromatographic column is used for separation. The ion-pairing reagent trifluoroacetic acid is added to the mobile phase to increase the retention time of arginine and histidine on the chromatographic column, and due to the difference in polarity, there is a good separation degree; an charged atomization detector CAD is used, and the detection signal is sensitive; five groups of standard solutions of arginine and histidine with different concentrations are prepared, and the concentration range is between 1mmol / L and 20mmol / L; a quadratic equation standard curve of the amino acid standard concentration and the peak area is drawn as a detection equation for detecting the amino acid concentration, and then the corresponding peak area is obtained according to the chromatogram integration of the test sample, and the peak area is then substituted into the above detection equation to obtain the detection concentration of the corresponding amino acid type in the test sample; in this embodiment, the test sample is diluted four times with ultrapure water, centrifuged in a 10KD ultrafiltration centrifuge tube, the protein is removed, and the filtrate is taken 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.

[0135] 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 inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for quantitative detection of amino acids in high-concentration protein preparations without derivatization, characterized by: The following steps are involved: S1. Equilibrate the chromatographic column with an initial mobile phase, wherein the initial mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is a trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile; the chromatographic column is a Hypercarb graphitized carbon chromatographic column from Thermo Fisher Scientific; the chromatographic column temperature is 30° C.; and the amount of trifluoroacetic acid added to the trifluoroacetic acid aqueous solution is 0.5% by volume; 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 as follows: From 0 to 20 minutes, the volume fraction of mobile phase A was changed from 100% to 75% at a constant rate, and the volume fraction of mobile phase B was changed from 0% to 25% at a constant rate. The flow rate of the mobile phase was 0.8 to 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 rate, and the volume fraction of mobile phase B was changed from 25% to 0% at a constant rate. 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. Dilute the test solution at least four times with ultrapure water, replace the solution with an ultrafiltration centrifuge tube, and centrifuge. The filtrate is injected into a chromatograph and the gradient elution process is run to obtain a spectrum of the test solution. The centrifugation time is at least 5 minutes and the speed is at least 12,000 rpm. S5. Data Analysis: S51. Integrate the N groups of standard curve solution spectra to obtain N peak areas Q, use the concentrations of the N groups of standard curve solutions as the abscissa and the corresponding peak areas Q as the ordinate to obtain peak area-concentration curves of the amino acids, and fit them 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 quantitative detection of amino acids in a high-concentration protein preparation without derivatization according to claim 1, wherein: The N groups of amino acid standard curve solutions have the same composition but different concentrations, and the concentration range is 1 mmol / L to 20 mmol / L.

3. The method for quantitative detection of amino acids in a high-concentration protein preparation without derivatization according to claim 1, wherein: The amino acids include one or more of histidine, arginine, methionine, lysine, proline or glycine.

4. The method for quantitative detection of amino acids in a high-concentration protein preparation without derivatization according to claim 1, wherein: The blank solution is 0.1 mol / L hydrochloric acid solution.

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