Method for detecting histidine content

Abstract

The invention discloses a histidine content detection method, and relates to the technical field of detection, and the histidine content detection method comprises the following steps: S1, preparing a standard curve solution, a verification standard solution, a test solution and a blank control solution; s2, hydrophilic chromatography taking thiobetaine as a bonding phase is taken as a chromatographic column, gradient elution is carried out on a mobile phase A and a mobile phase B, the mobile phase A is an acetonitrile solution, the mobile phase B is 0.3% formic acid + 20mM ammonium formate solution, and a CAD detector is adopted to detect each sample; and S3, according to an external standard method, calculating a linear regression equation by taking a peak area logarithm value of the standard curve solution as a vertical coordinate y and a histidine concentration logarithm value of the standard curve solution as a horizontal coordinate x, and then calculating the histidine content in the test solution. The method is low in operation difficulty, high in accuracy, good in repeatability and short in consumed time.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for detecting the histidine content. Background Art

[0002] Histidine is an alkaline amino acid, and its molecular structure contains an α-amino group (pKa≈9.2), an α-carboxyl group (pKa≈1.8), and an imidazole side chain (pKa≈6.0), which makes it exhibit excellent buffering ability in the pH range of 5.5 - 7.5. Among them, the imidazole group has a high buffering efficiency between pH 6.0 - 7.0, making it particularly suitable for application scenarios such as protein purification, antibody drug formulations, and cell culture.

[0003] In recent years, with the gradual rise of the biopharmaceutical industry, the application of histidine has become more extensive. However, histidine does not contain a chromophore and cannot be directly detected by an ultraviolet detector, and the sample needs to be derivatized. In the existing detection methods, the histidine sample is first derivatized and then detected by HPLC. Its operation is complex, with low sensitivity, poor repeatability, and long time consumption. The detection of a single sample takes 35 minutes.

[0004] To solve the above problems, the present invention is hereby proposed. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the present invention provides a method for detecting the histidine content, which has low operation difficulty, high accuracy, good repeatability, and short time consumption.

[0006] To achieve the purpose of the present invention, the following scheme is proposed: A method for detecting the histidine content, comprising the following steps: S1. Sample preparation Prepare a dilution solution: Measure 70 ml of acetonitrile and 30 ml of ultrapure water and mix them evenly to obtain it; Prepare mobile phase B: Weigh 1.261 g of ammonium formate, dissolve it with 1000 ml of ultrapure water, add 3 ml of formic acid, mix evenly and filter through a 0.22 μm filter membrane to obtain a 0.3% formic acid + 20 mM ammonium formate solution, with a shelf life of 1 month; Prepare a standard curve solution: Mix 50 μl, 50 μl, 75 μl, 100 μl, 125 μl, and 150 μl of histidine reference substance solutions with 1950 μl, 950 μl, 925 μl, 900 μl, 875 μl, and 850 μl of dilution solution respectively to obtain STD1 - STD6 standard curve solutions; Prepare a calibration standard substance solution: Mix 100 μl of histidine reference substance solution with 900 μl of dilution solution to obtain a calibration standard substance solution; Preparation of test sample solution: Mix 100 μl of the test sample with 900 μl of the diluent to obtain the test sample solution; prepare 2 portions of the test sample solution in parallel, and after mixing, filter through a 0.2 μm PTFE-Q filter membrane; Preparation of blank control solution: Mix 100 μl of ultrapure water with 900 μl of the diluent to obtain the blank control solution.

[0007] S2. Detection Use hydrophilic chromatography with sulfobetaine as the bonded phase as the chromatographic column, and perform gradient elution with mobile phases A and B. Detect each sample using a CAD detector; The chromatographic column is Chromcore HILIC-ZW, with dimensions of 4.6×150 mm, a particle size of 5 μm, a column temperature of 35 °C, a flow rate of 1.5 ml / min, and an injection volume of 20 μl; Mobile phase A is an acetonitrile solution; Mobile phase B is a 0.3% formic acid + 20 mM ammonium formate solution; The atomization chamber temperature of the CAD detector is 35 °C, the acquisition frequency is 2 Hz, and the filtering is 5 s; Inject the standard curve solution once, the calibration standard solution 5 times, the blank control solution once, and the test sample solution once; after injecting the test sample solution 10 times, inject the calibration standard solution once again.

[0008] S3. Calculation (1) Judge whether the test is valid according to the following conditions: There should be no chromatographic peaks in the blank control; The relative standard deviation (RSD) of the peak areas of the 5 calibration standard solutions should be ≤2.0%; The relative standard deviation (RSD) of the retention time of histidine should be ≤2.0%; Compare the average value of the detection results of the 5 calibration standard solutions with the theoretical value, and the absolute value of the relative error should not be greater than 3.0%; The absolute value of the relative deviation of the concentration results of the two parallel test sample solutions should be ≤2.0%; relative deviation = (parallel 1 - parallel 2) / (parallel 1 + parallel 2); (2) According to the external standard method, use the logarithm of the peak area of the standard curve solution as the ordinate y, and the logarithm of the histidine concentration of the standard curve solution as the abscissa x to calculate the linear regression equation, and then calculate the content of histidine in the test sample solution.

[0009] The beneficial effects of the present invention are as follows: 1. This detection method does not require sample derivatization, and a single sample detection only takes 13 minutes; the detection method has low operation difficulty, high accuracy, good repeatability, and short time consumption.

[0010] 2. When using a HILIC-Amide type chromatographic column and adjusting the mobile phase gradient, histidine can elute, but the peak is severely tailing, making it impossible to perform chromatographic analysis on histidine. The ChromCore HILIC-ZW chromatographic column can be used for the chromatographic analysis of histidine.

[0011] 3. Under different mobile phase conditions, histidine peaks only when the mobile phase contains ammonium formate and formic acid, and the asymmetry remains stable.

[0012] 4. Since histidine has ultraviolet absorption only at low wavelengths (190 nm - 230 nm) with a maximum absorption wavelength of around 210 nm, it is not suitable to be detected using an ultraviolet detector. Moreover, formic acid has ultraviolet absorption at low wavelengths, and when used as a mobile phase additive, it will cause baseline noise and baseline drift at low wavelengths. The CAD detector is suitable for the detection of histidine. Different CAD parameters and acquisition frequencies have little effect on the noise. Filtering for 5 s can reduce the baseline noise more effectively than 2 s. The temperature of the nebulization chamber can affect the noise and peak area, but both can meet the usage requirements.

[0013] 5. Considering that substances such as proteins and nucleic acids contained in the test sample are likely to precipitate on the chromatographic column, the test sample is diluted in advance with an acetonitrile solution to prevent the precipitation and contamination of the chromatographic column by proteins and nucleic acids. The test sample is diluted 10-fold with different ratios of acetonitrile solution. As a result, the higher the acetonitrile ratio, the smaller the peak area of histidine measured. Through screening experiments, it is finally considered to dilute the sample 10-fold with 70% acetonitrile and simultaneously adjust the initial ratio of acetonitrile in the elution gradient to 65%. Description of the Drawings

[0014] Figure 1 Shows the chromatogram of the negative control; Figure 2 Shows the superimposed chromatogram of STD1 - STD6; Figure 3 Shows the standard curve graph; Figure 4 Shows the chromatogram of the 40% spiked solution; Figure 5 Shows the chromatogram of the 100% spiked solution; Figure 6 Shows the chromatogram of the 150% spiked solution; Figure 7 Shows the superimposed chromatogram of the test sample solution; Figure 8 Shows the chromatogram of histidine for different condition groups; Figure 9 Shows the ultraviolet spectrum graph; Figure 10 Shows the chromatogram of histidine and arginine; Figure 11Chromatograms of H-1, H-2, H-3 and H-4 are shown; Figure 12 Chromatograms of M-1, M-2, M-3 and M-4 are shown. Detailed implementation mode

[0015] Example 1 This example provides a method for detecting the histidine content, which is as follows: Table 1 Instrument and equipment list The reagents involved are as follows: Formic acid: Sinopharm Chemical Reagent Co., Ltd., mass spectrometry grade; Ammonium formate: Sinopharm Chemical Reagent Co., Ltd., mass spectrometry grade; Acetonitrile: manufacturer is Fisher Chemical, HPLC grade.

[0016] S1. Sample preparation Prepare the diluent: Measure 70 ml of acetonitrile and 30 ml of ultrapure water and mix them evenly; Prepare the standard curve solution, calibration standard solution, test solution, and blank control solution; Table 2 Concentration table of STD1-STD6 standard curve solutions Preparation of calibration standard solution, test solution, and blank control solution: Take 100 μl of histidine reference solution, test sample, and ultrapure water respectively, and add 900 μl of diluent to each, and mix them evenly; Prepare 2 parallel test solutions, and after mixing, filter them with a 0.2 μm PTFE-Q filter membrane.

[0017] S2. Detection Use high performance liquid chromatography (CAD detector) to detect the standard curve solution, calibration standard solution, blank control solution, and test solution: Chromatographic column: Chromcore HILIC-ZW, size 4.6×150 mm, particle size 5 μm, column temperature 35 °C, flow rate 1.5 ml / min, injection volume 20 μl; Mobile phase A is an acetonitrile solution; Mobile phase B is 0.3% formic acid + 20 mM ammonium formate solution; The atomization chamber temperature of the CAD detector is 35 °C, the acquisition frequency is 2 Hz, and the filtering is 5 s; Table 3 Gradient elution condition table Inject the standard curve solution once, the calibration standard solution five times, the blank control solution once, and the test sample solution once; after injecting the test sample solution ten times, inject the calibration standard solution once again.

[0018] S3. Calculation (2) Judge whether the test is valid according to the following conditions: There should be no chromatographic peaks in the blank control; The relative standard deviation (RSD) of the peak areas of the five calibration standard solutions should be ≤ 2.0%; The relative standard deviation (RSD) of the retention time of histidine should be ≤ 2.0%; When comparing the average value of the test results of the five calibration standard solutions with the theoretical value, the absolute value of the relative error should not be greater than 3.0%; The absolute value of the relative deviation of the concentration results of the two parallel test sample solutions should be ≤ 2.0%; relative deviation = (parallel 1 - parallel 2) / (parallel 1 + parallel 2); (2) According to the external standard method, with the logarithm of the peak area of the standard curve solution as the ordinate y and the logarithm of the histidine concentration of the standard curve solution as the abscissa x, calculate the linear regression equation, and then calculate the content of histidine in the test sample solution.

[0019] Experiment 1 Conduct a specificity test on the detection method provided in Example 1.

[0020] Inject the negative control sample according to the experimental procedure of Example 1, as Figure 1 shown, there is no interference at the target peak in the negative control, and the specificity meets the requirements.

[0021] Experiment 2 Conduct a linearity and range test on the detection method provided in Example 1.

[0022] Prepare the standard curve solution according to Table 4: Table 4 Standard curve solution table of STD1 - STD5 Detect according to the detection method of Example 1, and the detection results are as Figure 2 shown, and the original data is shown in Table 5: Table 5 Detection result table of the standard curve solution According to the external standard method, calculate the linear regression equation with the logarithm of the histidine concentration and the corresponding logarithm of the peak area of the standard curve (STD1 - STD6), and the standard curve is as Figure 3As shown, the regression equation is: Log (peak area) = 0.8294 × Log (concentration μg / ml) - 0.6143, and the correlation coefficient R² = 0.9998, indicating good linearity in Example 1.

[0023] According to the verification results of linearity, precision, and accuracy, the method meets the usage requirements when the histidine concentration in the test sample is in the range of 246.75 μg / ml - 1480.50 μg / ml, indicating a wide detection range in Example 1.

[0024] Experiment 3 The repeatability (precision) test was conducted on the detection method provided in Example 1.

[0025] Take the test samples (i.e., the samples to be measured) from the same batch of Example 1, and prepare 6 samples in parallel. Then, conduct the detection and result calculation according to the experimental procedure in Example 1, and record the peak areas of the samples.

[0026] The detection results are as Figure 7 shown, and the original data is shown in Table 6: Table 6 Peak areas corresponding to the test sample solutions The relative standard deviation of the concentrations of 6 test sample solutions is 1.5%, which is less than 3.0%, indicating good repeatability of the method provided in Example 1.

[0027] Experiment 4 The intermediate precision test was conducted on the detection method provided in Example 1.

[0028] Experimenters A and B prepared standard curve solutions, calibration standard solutions, blank control solutions, and 6 parallel test sample solutions respectively according to the operation method in Example 1 for testing. The results are shown in Table 7: Table 7 Relative standard deviation values of the concentrations of test sample solutions The relative standard deviation of the histidine determination results in 12 test sample solutions among different experimenters is 1.6%, which meets the requirement that the relative standard deviation is less than 3%, indicating good intermediate precision, stability, and reliability of the method provided in Example 1.

[0029] Experiment 5 The accuracy test was conducted on the method provided in Example 1.

[0030] The theoretical content of histidine in the test sample solution in Example 1 is 940 μg / ml. Prepare a histidine spiked solution accurately, and add it to the test sample to prepare three spiked samples with high, medium, and low levels of 40%, 100%, and 150% respectively.

[0031] Detection and calculation were carried out according to the method in Example 1. The detection results of the three spiked samples were respectively Figure 4 , Figure 5 , Figure 6 . The original data is shown in Table 8, and the calculation results are shown in Table 9: Table 8 Peak areas corresponding to different spiked solutions Table 9 Recovery rates corresponding to different spiked solutions The spiked recovery rates were between 95% and 105%. The detection results were stable and had good repeatability, indicating that the method provided in Example 1 had high accuracy.

[0032] Experiment 6 The durability test was carried out on the method provided in Example 1.

[0033] According to the method in Example 1, the test conditions were changed according to Table 10, and the samples were detected. The results of Condition 1 and Condition 2 are shown in Table 11: Table 10 Comparison table of test conditions Table 11 Results table of Condition 1 and Condition 2 The ratios of the histidine contents of the test samples measured under different durability conditions to the histidine contents of the test samples under the corresponding normal conditions were between 99.0% and 101.5%, meeting the requirement that the ratios of the histidine contents of the test samples under different chromatographic conditions to the histidine contents of the test samples under normal conditions are between 98% and 102%.

[0034] Experiment 7 The screening experiment of chromatographic conditions was carried out as follows: An AcuuCore-150-Amide-HILIC (4.6×100mm, 2.6μm) chromatographic column was used, and gradient elution was carried out respectively under different gradient elution conditions. The column temperature was 35°C, the flow rate was 1 ml / min, the atomization chamber temperature of the CAD detector was 35°C, the acquisition frequency was 5 Hz, and the filtering was 3.6 s.

[0035] Table 12 Gradient elution condition 4 Table 13 Gradient elution condition 5 Table 14 Gradient elution condition 6 Table 15 Gradient elution condition 7 Among them, mobile phase A is acetonitrile, mobile phase B is 20 mM ammonium acetate with pH 3.5, and mobile phase D is 20 mM ammonium formate + 0.3% formic acid solution. The chromatograms of histidine in different condition groups are as Figure 8 shown.

[0036] The conclusion is that when using a HILIC-Amide type chromatographic column and adjusting the mobile phase gradient, histidine can elute, but the peak tailing is severe, and chromatographic analysis of histidine cannot be carried out.

[0037] Experiment 8 Using Nanodrop for ultraviolet spectrum scanning, the spectrograms of 40 mM ammonium formate, 40 mM ammonium formate + 1.2% formic acid, and 0.987 mg / ml histidine are as Figure 9 shown. Since histidine only has ultraviolet absorption at low wavelengths (190 nm - 230 nm) and the maximum absorption wavelength is around 210 nm, it is not suitable to use an ultraviolet detector for detection. Moreover, formic acid has ultraviolet absorption at low wavelengths, and when used as a mobile phase additive, it will cause baseline noise and baseline drift at low wavelengths.

[0038] Experiment 9 The mobile phase screening experiment is as follows: Using a ChromCore HILIC-ZW chromatographic column, mobile phase A is acetonitrile, mobile phase B is ultrapure water, mobile phase C is 40 mM ammonium formate solution, and mobile phase D is 40 mM ammonium formate + 1.2% formic acid solution. Gradient elution is carried out according to the mobile phase gradient in Table 16, with a column temperature of 35 °C, a flow rate of 1.5 ml / min, the atomization chamber temperature of the detector CAD being 35 °C, a collection frequency of 2 Hz, filtering for 5 s, and setting the chromatographic column to equilibrate for 3 min.

[0039] Table 16 Mobile phase gradient Using the mixed solution of mobile phase B, mobile phase C, and mobile phase D as the mobile phase, by adjusting the fixed ratio between mobile phase B, mobile phase C, and mobile phase D, the ammonium formate and formic acid concentrations of the mobile phase are adjusted, as shown in detail in Table 17. Inject 10 μl of 10 mM histidine solution for each condition, record the chromatogram, and the results are shown in Table 18.

[0040] Table 17 Mobile phase concentrations under different conditions Table 18 Detection results under different conditions Conclusion: Under different mobile phase conditions, histidine peaks only form when the mobile phase contains ammonium formate and formic acid, and the asymmetry degree remains stable.

[0041] Experiment 10 The specific experiment for optimizing the detector parameters is as follows: Using Condition 8 in Table 17, the condition optimization is carried out with different CAD acquisition parameters. The acquisition parameters are shown in Table 19, and the detection results are shown in Table 20.

[0042] Table 19 CAD acquisition parameters Table 20 Detection results with different CAD acquisition parameters Conclusion: For different CAD parameters, the acquisition frequency has little impact on the noise, and filtering for 5 s can reduce the baseline noise more than filtering for 2 s. The temperature of the nebulization chamber can affect the noise and peak area, but both can meet the usage requirements.

[0043] Experiment 11 The specific experiment for optimizing the mobile phase gradient is as follows: Using Condition 8 in Table 17, the elution gradient is adjusted as shown in Table 21, and the results are shown in Table 22.

[0044] Table 21 Changes in the gradient of mobile phase A Table 22 Results with different elution gradients Conclusion: Under different elution gradients, the asymmetry of histidine remains stable, and all conditions can meet the usage requirements. Among them, the peak areas corresponding to Gradient 5 - Gradient 8 are relatively higher.

[0045] Experiment 12 To make the detection specificity of histidine stronger, arginine is used as an interference to develop a histidine detection method.

[0046] Using a ChromCore HILIC-ZW chromatographic column, with acetonitrile as mobile phase A and 0.3% formic acid + 20 mM ammonium formate solution as mobile phase B, gradient elution is carried out according to Table 23, the column temperature is 35 °C, the flow rate is 1.5 ml / min, the temperature of the nebulization chamber of the detector CAD is 35 °C, the acquisition frequency is 2 Hz, and the filtering time is 5 s.

[0047] Table 23 Elution gradient 1 A: Inject 10 μl of 10 mM arginine hydrochloride solution; B: Inject 10 μl of 10 mM histidine solution; C: Take 1000 μl of 10 mM histidine solution and 500 μl of 10 mM arginine hydrochloride solution, mix them evenly to obtain a histidine and arginine mixed solution, and inject 2 μl.

[0048] The results are as Figure 10 shown, and the two target peaks are baseline-separated with high responses.

[0049] A method for screening sample treatment was carried out using samples with complex components, aiming to make the method applicable not only to histidine detection but also to accurately detect and analyze the content of histidine in preparation samples.

[0050] Experiment 13 Dilute the test sample with acetonitrile solution. The specific screening experiments are as follows: Use a ChromCore HILIC-ZW chromatographic column, with acetonitrile as mobile phase A and 0.3% formic acid + 20 mM ammonium formate solution as mobile phase B. Perform gradient elution according to Table 24, with a column temperature of 35 °C, a flow rate of 1.5 ml / min, a detector CAD, an atomization chamber temperature of 35 °C, a collection frequency of 2 Hz, and a filtering time of 5 s.

[0051] Table 24 Elution gradient 2 Sample A: Contains components such as recombinant protein, liposome finished product, saponin, thio-oligonucleotide, histidine, polysorbate 80, sucrose, etc., covering almost all types of conventional components in biological preparations.

[0052] Take 100 μl of Sample A, add 900 μl of acetonitrile, 90% acetonitrile, 80% acetonitrile, and 70% acetonitrile respectively to dilute 10 times, mix well and filter through a 0.2 μm PTFE-Q filter membrane to obtain M-1, M-2, M-3, and M-4.

[0053] Take 6.3 ml of 10 mM histidine solution, add 3.7 ml of ultrapure water, mix well, take 100 μl, add 900 μl of acetonitrile, 90% acetonitrile, 80% acetonitrile, and 70% acetonitrile respectively to dilute 10 times, mix well and filter through a 0.2 μm PTFE-Q filter membrane to obtain H-1, H-2, H-3, and H-4.

[0054] Inject 20 μl of each test sample solution.

[0055] The chromatograms of H-1, H-2, H-3, and H-4 are as Figure 11 shown, and the chromatograms of M-1, M-2, M-3, and M-4 are as Figure 12 shown. Treat the histidine solution and Sample A with different concentrations of acetonitrile. As the proportion of acetonitrile increases, histidine in the histidine solution will precipitate, resulting in a decrease in peak area. Histidine in the test sample solution will precipitate together with other substances in the test sample (such as sucrose), resulting in an even smaller peak area.

[0056] Since the peak areas of M-4 and H-4 are basically the same, it is considered to dilute the sample 10 times with 70% acetonitrile, mix well and filter through a 0.2 μm PTFE-Q membrane to obtain the sample. At the same time, adjust the chromatographic conditions to change the initial proportion of acetonitrile in the elution gradient to 65%.

[0057] The above embodiments are only used to illustrate the technical idea and characteristics of the present invention, and do not represent the only or limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the histidine content, characterized in that, It includes the following steps: S1. Sample preparation Prepare standard curve solution, calibration standard solution, test solution, and blank control solution; S2. Detection Use hydrophilic chromatography with sulfobetaine as the bonding phase as the chromatographic column, and perform gradient elution with mobile phases A and B. Mobile phase A is acetonitrile solution, and mobile phase B is 0.3% formic acid + 20 mM ammonium formate solution. Detect each sample using a CAD detector; S3. Calculation According to the external standard method, use the logarithm of the peak area of the standard curve solution as the ordinate y, and the logarithm of the histidine concentration of the standard curve solution as the abscissa x to calculate the linear regression equation, and then calculate the content of histidine in the test solution.

2. The detection method of histidine content according to claim 1, characterized in that, In step S1, mix 50 μl, 50 μl, 75 μl, 100 μl, 125 μl, 150 μl of histidine reference solution with 1950 μl, 950 μl, 925 μl, 900 μl, 875 μl, 850 μl of diluent respectively to obtain STD1 - STD6 standard curve solutions. The diluent is 70% acetonitrile solution.

3. The detection method of histidine content according to claim 1, wherein In step S1, mix 100 μl of histidine reference solution with 900 μl of diluent to obtain the calibration standard solution. The diluent is 70% acetonitrile solution.

4. The detection method of histidine content according to claim 1, wherein In step S1, mix 100 μl of the test sample with 900 μl of diluent to obtain the test solution. The diluent is 70% acetonitrile solution; Prepare 6 parallel test solutions, and filter them through a 0.2 μm PTFE - Q filter membrane after mixing.

5. The detection method of histidine content according to claim 1, wherein In step S1, mix 100 μl of ultrapure water with 900 μl of diluent to obtain the blank control solution. The diluent is 70% acetonitrile solution.

6. The detection method of histidine content according to claim 1, characterized in that, In step S2, the chromatographic column is Chromcore HILIC - ZW, with dimensions of 4.6×150 mm, particle size of 5 μm, column temperature of 35°C, flow rate of 1.5 ml / min, and injection volume of 20 μl.

7. The detection method of histidine content according to claim 1, wherein In step S2, the gradient elution conditions are as follows: 。 8. The detection method of histidine content according to claim 1, wherein In step S2, when using a CAD detector for detection, the nebulizer temperature is 35°C, the acquisition frequency is 2 Hz, and the filtering is 5 s.

9. The detection method of histidine content according to claim 1, characterized in that In step S2, inject the standard curve solution once, the calibration standard solution 5 times, the blank control solution once, and the test solution once; After injecting the test solution 10 times, inject the calibration standard solution once again.

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