Imidazole content detection method

Abstract

The invention discloses a method for detecting the content of imidazole. A mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is a potassium dihydrogen phosphate buffer solution containing sodium heptanesulfonate, the mobile phase B is methanol, three-stage gradient elution is adopted, the proportion of the mobile phase A is 95%-30%-95%, and the total proportion of the mobile phase B and the mobile phase A is 100%. The detection and reagent treatment process is simple, instruments and equipment are conventional, the method is good in specificity, repeatability and sensitivity, and the peak appearance time of a target peak is short.

Description

Technical Field

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

[0002] Imidazole is an important chemical raw material, widely used in the synthesis of pharmaceuticals and pesticides, and as a curing agent for epoxy resins. In the purification of protein biopharmaceuticals, it is often used to elute contaminants and collect the target protein during the purification of histidine-tagged (His-tag) proteins. The elution mechanism is to compete with the imidazole group in the His-tag, thereby eluting contaminants and collecting the high-purity target protein.

[0003] During the production of large-molecule biologics, the imidazole content needs to be detected and controlled to ensure the safety and quality of the production process and product quality. Therefore, a method for detecting the imidazole content in large-molecule biologics is particularly important.

[0004] Currently, the main methods for determining imidazoles include thin-layer chromatography, ultraviolet spectrophotometry, liquid chromatography-mass spectrometry, and high-performance liquid chromatography. However, these methods suffer from low sensitivity, poor reproducibility, high instrument costs, complex operation, and a complex and time-consuming detection process. Specific methods for determining imidazoles in large protein-based biologics are limited. The main method, the limit comparison method for thin-layer chromatography (under clotrimazole in the Chinese Pharmacopoeia), is time-consuming, with sample testing (activation + development) taking approximately one hour, and a detection limit generally around 0.5 mg / mL. Furthermore, thin-layer chromatography often uses organic reagents, resulting in large errors in colorimetric quantification and low reproducibility.

[0005] In the invention CN107525791A, ultraviolet light is used to determine methimazole by UV spectrophotometer. The detection process of the fluorescence spectrum of the sample mixture or the complex color indicator of Cu (II) ions in the mixed solution is complicated, and the operations such as sample processing and solution preparation are complicated.

[0006] In the invention CN108303478A, a high performance liquid chromatography tandem mass spectrometry method is used to detect nitroimidazole residues in royal jelly. The sample processing procedure is complicated and the instrument and equipment cost is high.

[0007] Moreover, when high performance liquid chromatography is currently used to detect imidazole, the separation effect of the imidazole peak is poor and there is a tailing phenomenon. For example, in the literature Liu Na, Xie Zili, Wu Xiaoying, Wang Ye, Liu Qianqian, Xin Jie. Study on the method of determining the content of imidazole in clotrimazole vaginal tablets by HPLC [J]. Guangzhou Chemical Industry, 2017, 45(18): 92-94., it is mentioned that when using HPLC to determine the content of imidazole, the imidazole peak is too early, and the imidazole and solvent peaks cannot be effectively separated, which easily leads to inaccurate determination of the imidazole content; in addition, it is found that the imidazole peak is easy to overlap with the solvent peak and the expected effect cannot be achieved. In addition, the polarity of imidazole is relatively large, and the proportion of methanol needs to be reduced from 70% to 5% to effectively detect the imidazole content. However, the detection time of this method is long, and the gradient elution time required is 47 minutes, while the sample stability time after preparation is only 8 hours. It cannot be effectively detected when facing a large number of samples. Therefore, there is an urgent need for a method for the exclusive determination of imidazole in macromolecular protein preparations with shorter detection time, better separation effect, good repeatability and high sensitivity, especially a method for reducing solvent costs in daily testing and multi-batch continuous testing. Summary of the Invention

[0008] To solve the problems existing in the prior art, the present invention provides a method for detecting the imidazole content in macromolecular protein preparations. The detection and reagent processing process is simple, the instrumentation is conventional, the method is specific, the repeatability and sensitivity are good, and the target peak emergence time is fast.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] The present invention provides a method for detecting imidazole by using high performance liquid chromatography. The mobile phase consists of mobile phases A and B, wherein mobile phase A is a potassium dihydrogen phosphate (KH2PO4) buffer solution containing sodium heptanesulfonate, and mobile phase B is methanol. A three-stage gradient elution is adopted, wherein the proportion of mobile phase A is 95%-30%-95%, and the total proportion of mobile phases B and A is 100%.

[0011] Three-stage gradient elution is an HPLC technique that divides the elution process into three phases: an initial phase in which the mobile phase composition is kept constant to achieve steady-state conditions and initial separation; a gradient phase in which the mobile phase composition is gradually changed to optimize the separation of the components; and a final phase in which the mobile phase is adjusted back to its initial state or a new constant composition to clean the column. This method can improve separation efficiency and quantitative analysis accuracy, making it particularly suitable for analyzing complex samples.

[0012] In some embodiments, the method satisfies one or more of the following:

[0013] 1) The concentration of heptanesulfonic acid is 0.05-0.5% (w / v), preferably 0.1% (w / v);

[0014] 2) The concentration of potassium dihydrogen phosphate in the mobile phase A is 0.01-0.2 M; preferably 0.05 M; and,

[0015] 3) The pH of the mobile phase A is 3-4, preferably 3.5.

[0016] In some embodiments, mobile phase A is an aqueous solution of 0.05 M potassium dihydrogen phosphate and 0.1% (w / v) sodium heptane sulfonate at a pH of 3.5.

[0017] In some embodiments, the method satisfies one or more of the following:

[0018] 1) During gradient elution, the time for the mobile phase ratio to switch from one stage to the next is 0.1 minutes;

[0019] 2) a detection wavelength of 210-215 nm, preferably 214 nm; and,

[0020] 3) The flow rate of the mobile phase is 0.5-5 mL / min, for example, 1.0 mL / min.

[0021] In some embodiments, the mobile phase gradient is: 0-10 min, 95% A; 10-10.1 min, 95%-30% A; 10.1-15 min, 30% A; 15-15.1 min, 30%-95% A; 15.1-25 min, 95% A.

[0022] In some embodiments, the method satisfies one or more of the following:

[0023] 1) The high performance liquid chromatography uses a C18 column with a specification of 250×4.6 mm and 5 μm; preferably, Shimadzu's Shim-pack GIST C18;

[0024] 2) the HPLC injection volume is 10-50 µL, for example 20 µL; and,

[0025] 3) The column temperature of the HPLC column is room temperature, such as 28°C, 30°C and 32°C, preferably 30°C.

[0026] In some embodiments, the imidazole standard is dissolved or diluted using a dissolving or diluting solution.

[0027] In some embodiments, the pH of the dissolving solution or diluent is 7.0-9.0.

[0028] In some embodiments, the dissolving solution or diluent comprises the following components: 1-2 g / L of NaH2PO4•H2O, 5-7 g / L of Na2HPO4, 350-370 g / L of urea, and 7-10 g / L of sodium chloride.

[0029] In some embodiments, 1.2-1.4 g / L of NaH2PO4•H2O, 5.5-6.0 g / L of Na2HPO4, 355-365 g / L of urea, and 8.5-9 g / L of sodium chloride.

[0030] In some embodiments, 1.31 g / L NaH2PO4•H2O, 5.75 g / L Na2HPO4, 60.4 g / L urea, and 8.8 g / L sodium chloride.

[0031] In some embodiments, the method comprises the following steps:

[0032] 1) Use dissolving or diluting solution to dissolve or dilute the imidazole sample;

[0033] 2) injecting the prepared sample into the chromatographic system using an injector; and,

[0034] 3) Analyze and calculate the content of imidazole in the sample.

[0035] In some embodiments, the imidazole detected by HPLC is imidazole in a macromolecular biological preparation.

[0036] In some embodiments, the macromolecular biologic is a recombinant protein injection.

[0037] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0038] The reagents and raw materials used in the present invention are commercially available.

[0039] The present invention provides a promising and advantageous method for detecting imidazole in macromolecular protein preparations using high-performance liquid chromatography. Using a C18 chromatographic column and a UV detector, and utilizing specific chromatographic conditions, the method rapidly isolates the specific imidazole peak without interference from reagents or proteins. The detection and reagent handling procedures are simple, the instrumentation is conventional, the method is highly specific, and the repeatability and sensitivity are excellent, resulting in a rapid peak emission time for the target peak. The amount of imidazole in the sample can be calculated based on a standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the detection spectrum of imidazole chromatography when the concentration of potassium dihydrogen phosphate in mobile phase A is 0.05 M, 0.01 M, and 0.2 M.

[0041] Figure 2 The detection spectrum of imidazole chromatography when the concentration of sodium heptane sulfonate in mobile phase A is 0.05% (w / v), 0.1% (w / v), and 0.5% (w / v).

[0042] Figure 3 This is the linear and range chromatographic detection spectrum of imidazole.

[0043] Figure 4 This is the specific chromatographic detection spectrum of imidazole.

[0044] Figure 5 This is the detection spectrum of imidazole in macromolecular protein injection. DETAILED DESCRIPTION

[0045] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0046] The reagents used in the examples are imidazole (Sangon), sodium heptanesulfonate (Sangon), high performance liquid chromatography (UV detector) (Shimadzu), and chromatographic columns (Shimadzu). Other raw materials, unless otherwise specified, are commercially available conventional raw materials. The process methods used in the examples, unless otherwise specified, are conventional methods in the art.

[0047] Example 1 Sample preparation

[0048] 1.1 Preparation of test samples

[0049] 200 μL of a purified macromolecular biological preparation sample (Sox2 protein, prepared according to the method disclosed in patent CN118240054A) was taken into an inner insert tube, and the inner insert tube was placed in a sample bottle for later use.

[0050] 1.2 Preparation of standard samples

[0051] Prepare standard dilution solution: Take 1.31 g of NaH2PO4•H2O, 5.75 g of Na2HPO4, 360.40 g of urea, and 8.80 g of sodium chloride, dissolve them in appropriate amount of ultrapure water, adjust the pH to 7.0-9.0 with NaOH, transfer to a 1000 mL volumetric flask and make up to volume with ultrapure water.

[0052] Prepare imidazole standard stock solution (0.5 mg / mL): Dissolve 131 mg of NaH2PO4•H2O, 575 mg of Na2HPO4, 36,000 mg of urea, 877 mg of sodium chloride, and 50 mg of imidazole in appropriate amount of ultrapure water, adjust the pH to 7.0-9.0 with NaOH, transfer to a 100 mL volumetric flask, and make up to volume with ultrapure water.

[0053] Take the imidazole standard stock solution, and then dilute it with standard diluent to 50 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, 5.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL, 0.3 μg / mL, 0.005 μg / mL, and 0.001 μg / mL, respectively.

[0054] The dilution served as a negative blank.

[0055] Example 2 Sample detection conditions

[0056] The standard sample prepared in Example 1 and the sample to be tested were tested using the following instruments and conditions and the mobile phase gradient shown in Table 3.

[0057] Instruments and conditions

[0058] High performance liquid chromatography: Shimadzu LC-20AT;

[0059] Chromatographic column: Shimadzu Shim-pack GIST C18 (250×4.6 mm, 5 μm);

[0060] Detection wavelength: 214 nm;

[0061] Injection volume: 20 µL;

[0062] Column temperature: 30°C;

[0063] Flow rate: 1.0 mL / min;

[0064] Detection was performed using the mobile phase gradient shown in Table 1 below:

[0065] Table 1 Mobile phase gradient

[0066]

[0067] 2.1 Mobile phase selection

[0068] 1) Take the imidazole standard stock solution and dilute it to 0.005 μg / mL and 0.001 μg / mL respectively with standard diluent.

[0069] 2) Prepare mobile phase:

[0070] Mobile phase A (sodium hexanesulfonate): Dissolve 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium hexanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly and filter.

[0071] Mobile phase A (sodium heptane sulfonate): Dissolve 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium heptane sulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly and filter.

[0072] Mobile phase A (sodium octane sulfonate): Dissolve 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium octane sulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly and filter.

[0073] Mobile phase B (methanol)

[0074] 3) Detect the standards using mobile phase A (sodium hexane sulfonate), mobile phase A (sodium heptane sulfonate), and mobile phase A (sodium octane sulfonate) according to the chromatographic conditions mentioned above and the mobile phase gradient shown in Table 3. Determine the minimum limit of quantification for the samples based on a signal-to-noise ratio of approximately 10.

[0075] The experimental results are shown in Table 2.

[0076] Table 2

[0077]

[0078] Sodium hexanesulfonate, sodium octanesulfonate, and sodium heptanesulfonate were used as mobile phase A for comparison. The results are shown in Table 2. When sodium heptanesulfonate was used as the mobile phase component, the detection sensitivity was higher than that of sodium hexanesulfonate and sodium octanesulfonate, and the quantification limit could be increased from 0.005 μg / mL to 0.001 μg / mL. Sodium heptanesulfonate can better balance the retention time and separation efficiency, thereby improving the peak shape and detection sensitivity.

[0079] 2.2 Selection of different KH2PO4 concentrations in the mobile phase

[0080] 1) Take the imidazole standard stock solution and dilute it to 20 μg / mL with standard diluent.

[0081] 2) Prepare mobile phase:

[0082] Mobile phase A (0.05 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium hexanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0083] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 1.36 g potassium dihydrogen phosphate and 1.0 g sodium heptanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0084] Mobile phase A (0.2 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 27.2 g of potassium dihydrogen phosphate and 1.0 g of sodium heptanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0085] Mobile phase B (methanol).

[0086] 3) Detect the standards using three different mobile phases according to the chromatographic conditions, using the instrumentation and conditions mentioned above and the mobile phase gradients shown in Table 3. Determine the optimal conditions based on the top brightness peak shape.

[0087] The results are as follows Figure 1 As shown, 0.05 M KH2PO4, 0.01 M KH2PO4, and 0.2 M KH2PO4 were used as mobile phase A for comparison. The results are shown in Figure 1 The results showed that using 0.05 M KH2PO4 as the mobile phase component resulted in better detection of the target peak compared to 0.01 M KH2PO4 and 0.2 M KH2PO4. The phosphate concentration in 0.01 M KH2PO4 was too low, preventing detection of the target peak. The phosphate concentration in 0.2 M KH2PO4 was too high, affecting the baseline and causing the pump pressure to drop to zero, preventing proper detection. 0.05 M KH2PO4 achieved a better balance between retention time and separation efficiency, resulting in better peak shape and detection sensitivity. In subsequent experiments, 0.05 M KH2PO4 and 0.1% (w / v) sodium heptanesulfonate in water (pH 3.5) were selected as mobile phase A.

[0088] 2.3 Selection of different heptanesulfonic acid concentrations in the mobile phase

[0089] 1) Prepare the standard solution as in Example 2.

[0090] 2) Prepare mobile phase:

[0091] Mobile phase A (0.05M KH2PO4 0.05% (w / v) sodium heptanesulfonate (pH 3)): Dissolve 6.8 g of potassium dihydrogen phosphate and 0.5 g of sodium heptanesulfonate in water. Adjust the pH to 3 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0092] Mobile phase A (0.05M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium heptanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0093] Mobile phase A (0.05 M KH2PO4 0.5% (w / v) sodium heptanesulfonate (pH 4)): Dissolve 6.8 g of potassium dihydrogen phosphate and 5.0 g of sodium heptanesulfonate in water. Adjust the pH to 4 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0094] Mobile phase B (methanol)

[0095] 3) Based on the chromatographic conditions, use three different mobile phases A, and detect the standards using the instruments and conditions mentioned above and the mobile phase gradients shown in Table 3. Determine the optimal conditions.

[0096] The results are as follows Figure 2 As shown in FIG, 0.05% (w / v) sodium heptane sulfonate, 0.1% (w / v) sodium heptane sulfonate, and 0.5% (w / v) sodium heptane sulfonate were used as mobile phase A for comparison. The results are shown in FIG. Figure 2 The results showed that using 0.1% (w / v) sodium heptane sulfonate as the mobile phase component resulted in better detection of the target peak compared to 0.05% (w / v) and 0.5% (w / v) sodium heptane sulfonate. The 0.05% (w / v) sodium heptane sulfonate peak eluted earlier, closer to the solvent peak, which interfered with the target peak. The 0.5% (w / v) sodium heptane sulfonate peak eluted later. 0.1% (w / v) sodium heptane sulfonate achieved a better balance between retention time and separation efficiency, resulting in better peak shape and detection sensitivity. In subsequent experiments, 0.05 M KH2PO4 and 0.1% (w / v) sodium heptane sulfonate in water (pH 3.5) were selected as mobile phase A.

[0097] 2.4 Selection of different pH values in mobile phase

[0098] 1) Prepare samples and standards as in Example 1.

[0099] 2) Prepare mobile phase:

[0100] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3)): Dissolve 1.36 g of potassium dihydrogen phosphate and 1.0 g of sodium heptanesulfonate in water. Adjust the pH to 3 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0101] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 1.36 g potassium dihydrogen phosphate and 1.0 g sodium heptanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0102] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 4)): Dissolve 1.36 g potassium dihydrogen phosphate and 1.0 g sodium heptanesulfonate in water. Adjust the pH to 4 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0103] Mobile phase B (methanol)

[0104] 3) Based on the chromatographic conditions, use three different mobile phases A and test using the instruments and conditions mentioned above. Determine the optimal conditions based on the stability of the sample test results.

[0105] The results are shown in Table 3. Comparison was made using pH 3, pH 3.5, and pH 4 as mobile phase A. As shown in the table, when pH 3.5 was used as the mobile phase component, the results of the test samples were smaller and more stable. In subsequent experiments, 0.05 M KH2PO4, 0.1% (w / v) sodium heptane sulfonate aqueous solution (pH 3.5) was selected as mobile phase A.

[0106] Table 3 Comparison results of mobile phases

[0107]

[0108] 2.5 Selection of different flow rates in instruments and conditions

[0109] 1) Prepare samples and standards as in Example 1.

[0110] 2) Prepare mobile phase:

[0111] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 1.36 g potassium dihydrogen phosphate and 1.0 g sodium heptanesulfonate in water. Adjust the pH to 3.5 with phosphoric acid and add water to a final volume of 1000 mL. Mix thoroughly, filter, and set aside.

[0112] Mobile phase B (methanol)

[0113] 3) Based on the chromatographic conditions and the instrumentation and testing conditions mentioned above, conduct experiments at different flow rates (0.8 mL / min, 1.0 mL / min, and 1.2 mL / min). Determine the optimal flow rate based on the stability of the sample test results.

[0114] The results are shown in Table 4. Different flow rates (0.8 mL / min, 1.0 mL / min, and 1.2 mL / min) were used for comparison. As shown in the table, when 1.0 mL / min was used as the flow rate, the results of the test samples were smaller and more stable. The flow rate of 1.0 mL / min was selected for subsequent experiments.

[0115] Table 4 Flow rate comparison results

[0116]

[0117] 2.6 Selection of different column temperatures in instruments and conditions

[0118] 1) Prepare samples and standards as in Example 1.

[0119] 2) Prepare mobile phase:

[0120] Mobile phase A (0.01 M KH2PO4 0.1% (w / v) sodium heptanesulfonate (pH 3.5)): Dissolve 1.36 g of potassium dihydrogen phosphate and 1.0 g of sodium heptanesulfonate in appropriate amount of water, adjust the pH to 3.5 with phosphoric acid, and add water to a final volume of 1000 mL. Mix thoroughly and filter.

[0121] Mobile phase B (methanol)

[0122] 3) Based on the chromatographic conditions, perform experiments using different column temperatures (28°C, 30°C, and 32°C) using the instrumentation and conditions mentioned above. Determine the optimal conditions based on the stability of the sample test results.

[0123] The results are shown in Table 5. Different column temperatures (28°C, 30°C, and 32°C) were used for comparison. As shown in the table, when the column temperature was 30°C, the results of the test samples were smaller and more stable. The column temperature of 30°C was selected for subsequent experiments.

[0124] Table 5 Column temperature comparison results

[0125]

[0126] Example 3

[0127] 3.1 Linearity and range

[0128] The detection method screened in Example 2 was used to detect different imidazole standard concentrations (50 μg / mL, 10 μg / mL, 5.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL, blank).

[0129] The linear regression equation is y=61741x+1807.3, where R 2=0.999. The results showed that the linear relationship was good within the concentration range of 0.5 mg / L-30 mg / L.

[0130] 3.2 Reproducibility of the detection method

[0131] The standard samples (30 μg / mL, 20 μg / mL, 10 μg / mL) prepared in Example 1 were tested using the method of Example 2. The results are shown in Table 6:

[0132] Table 6 Reproducibility of samples

[0133]

[0134] As can be seen from Table 6, the recovery rate of each sample concentration is close to 100%, and the highest RSD does not exceed 0.06.

[0135] 3.3 Limit of quantification of the detection method

[0136] The standard sample (0.001 μg / mL) prepared in Example 1 was tested using the method described in Example 2. The results are shown in Table 7 below:

[0137] Table 7

[0138]

[0139] As can be seen from Table 7, the detection limit of quantification is 0.001 μg / mL, with high sensitivity and good repeatability.

[0140] 3.4 Effect of proteins in macromolecular biological preparations on imidazole detection

[0141] To determine the effect of proteins in biological preparations on imidazole detection, Sox2 protein samples prepared in different purification stages in Example 1 were taken (Sox2 sample-1, Sox2 sample-2, Sox2 sample-3, Sox2 sample-4, and Sox2 sample-5 had the same protein content but different imidazole contents). The method of Example 2 was used for detection, and the results were as follows: Figure 4 As shown in the figure, imidazole detection has good specificity, is not interfered by reagents and proteins, and has a fast peak elution time.

[0142] 3.5 Stability of sample preparation

[0143] The test samples prepared in Example 1 were placed for 0, 2, 6, 12, 24, and 48 hours, respectively, and tested using the method of Example 2. The results are shown in Table 8:

[0144] Table 8

[0145]

[0146] As can be seen from Table 8, the RSD of the main peak purity of the samples at each time point was ≤2%. The test solution remained basically stable within 48 hours at room temperature.

[0147] Example 4 Detection of test samples

[0148] The detection method of Example 2 was used to detect the sample to be tested in Example 1.

[0149] The results are as follows Figure 5 As shown, the concentration of imidazole in the sample to be tested was calculated according to the linear regression equation in Example 3 to be 13 μg / mL.

Claims

1. A method for detecting imidazole using high performance liquid chromatography, characterized in that, The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is potassium dihydrogen phosphate buffer containing sodium heptanesulfonate, and mobile phase B is methanol. A three-stage gradient elution is used. The proportion of mobile phase A is 95%-30%-95%, and the total proportion of mobile phase B and A is 100%.

2. The method according to claim 1, wherein The method satisfies one or more of the following: 1) The concentration of heptanesulfonic acid is 0.05-0.5% (w / v), preferably 0.1% (w / v); 2) The concentration of potassium dihydrogen phosphate in the mobile phase A is 0.01-0.2 M; preferably 0.05 M; and, 3) The pH of the mobile phase A is 3-4, preferably 3.

5.

3. The method according to claim 2, wherein Mobile phase A was an aqueous solution of 0.05 M potassium dihydrogen phosphate and 0.1% (w / v) sodium heptane sulfonate at a pH of 3.

5.

4. The method according to any one of claims 1 to 3, wherein The method satisfies one or more of the following: 1) During gradient elution, the time for the mobile phase ratio to switch from one stage to the next is 0.1 minutes; 2) a detection wavelength of 210-215 nm, preferably 214 nm; and, 3) The flow rate of the mobile phase is 0.5-5 mL / min, for example, 1.0 mL / min.

5. The method according to claim 4, wherein The mobile phase gradient was: 0-10 min, 95% A; 10-10.1 min, 95%-30% A; 10.1-15min, 30%A; 15-15.1min, 30%-95%A; 15.1-25min, 95%A.

6. The method according to any one of claims 1 to 5, wherein: The method satisfies one or more of the following: 1) The high performance liquid chromatography uses a C18 column with a specification of 250×4.6 mm and 5 μm; preferably, Shimadzu's Shim-pack GIST C18; 2) the HPLC injection volume is 10-50 µL, for example 20 µL; and, 3) The column temperature of the HPLC column is room temperature, such as 28°C, 30°C and 32°C, preferably 30°C.

7. The method according to any one of claims 1 to 6, wherein: The standard imidazole is dissolved or diluted using a dissolving solution or diluent; Preferably, the pH of the dissolving solution or diluent is 7.0-9.

0.

8. The method according to claim 7, wherein The dissolving solution or diluent comprises the following components: 1-2 g / L of NaH2PO4•H2O, 5-7 g / L of Na2HPO4, 350-370 g / L of urea and 7-10 g / L of sodium chloride; Preferably, the dissolving solution or diluent comprises the following components: 1.2-1.4 g / L of NaH2PO4•H2O, 5.5-6.0 g / L of Na2HPO4, 355-365 g / L of urea and 8.5-9 g / L of sodium chloride.

9. The method according to any one of claims 1 to 8, wherein The method comprises the following steps: 1) Use dissolving or diluting solution to dissolve or dilute the imidazole sample; 2) injecting the prepared sample into the chromatographic system using an injector; and, 3) Analyze and calculate the content of imidazole in the sample.

10. The method according to any one of claims 1 to 9, wherein The imidazole detected by high performance liquid chromatography is the imidazole in the macromolecular biological preparation; the macromolecular biological preparation is, for example, a recombinant protein injection.

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