Method for determining content of stabilizer in sample

Abstract

The present disclosure relates to a method for determining the content of a stabilizer in a sample. The method uses a high-performance liquid chromatography method for determination, wherein a chromatographic column comprises a pentafluorophenyl or pentabromophenyl bonded silica gel as a filler. The method is simple and convenient, has a good specificity and high accuracy, and can be used for the determination of both a high-polarity stabilizer and a low-polarity stabilizer.

Description

A method for detecting stabilizer content in a sample

[0001] Citation of relevant applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202410950997.0, filed on July 16, 2024, entitled "A Method for Detecting the Content of Stabilizer in a Sample", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of analytical testing technology, specifically to a method for detecting the content of stabilizers in a sample using high performance liquid chromatography. Background Technology

[0004] Injectable drugs are widely used in clinical practice as a dosage form of pharmaceuticals. To improve the stability of injectable drugs, stabilizers may be added. In some injectable solutions, such as those for radiopharmaceuticals, one or more stabilizers may be added.

[0005] Radiopharmaceuticals exhibit radioactive self-decomposition; particles or rays emitted by radioactive isotopes can directly act on the radiopharmaceutical itself, causing structural changes in active molecules and resulting in the loss of its biological activity, thus affecting the final therapeutic effect. To reduce radioactive decomposition and improve drug stability, multiple stabilizers are typically added to radiopharmaceutical injection solutions. Considering the significant impact of stabilizer content on drug quality, safety, and efficacy, it is necessary to control the stabilizer content.

[0006] Sodium vitamin C and gentic acid are candidate stabilizers for radiation resistance. The Chinese Pharmacopoeia, Part II, uses titration for the detection of sodium vitamin C as an excipient. No detection method for gentic acid was found. However, due to the multiple components and radioactivity of radiopharmaceutical injections, titration is unsuitable for measuring sodium vitamin C content in radiopharmaceutical injections due to drawbacks such as numerous interfering factors, large measurement errors, and high radiation doses.

[0007] When using high-performance liquid chromatography (HPLC) to detect the content of substances, the commonly used column packing material is octadecyl bonded silica gel (C18 column). However, sodium vitamin C is a strongly polar substance, while gentic acid is a weakly polar substance, exhibiting a significant difference in polarity. When using the commonly used C18 column for detection in HPLC, it is difficult to achieve ideal separation of sodium vitamin C and gentic acid, or although they can be separated, there are problems such as long analysis time or poor peak shape, resulting in the method not meeting the detection requirements or having poor stability. Summary of the Invention

[0008] The purpose of this disclosure is to provide a method for simultaneously and accurately detecting the content of two stabilizers with significantly different polarities in a sample. This method is simple, convenient, specific, and accurate.

[0009] This disclosure provides a method for detecting the content of stabilizers in a sample, including detection using high performance liquid chromatography, wherein the chromatographic column comprises pentafluorophenyl or pentabromophenyl bonded silica gel as packing material.

[0010] In some embodiments, the sample contains one or more of a strong polar stabilizer (e.g., sodium vitamin C) and a weak polar stabilizer (e.g., gentianic acid).

[0011] In some embodiments, the sample contains both a strong polar stabilizer (e.g., sodium vitamin C) and a weak polar stabilizer (e.g., gentianic acid).

[0012] In some embodiments, the stabilizer comprises one or more of sodium vitamin C and gentianic acid.

[0013] In some embodiments, the stabilizer contains both sodium vitamin C and gentianic acid.

[0014] In some embodiments, the packing material of the chromatographic column is pentabromophenyl-bonded silica gel, such as pentabromophenyl-bonded porous spherical silica gel.

[0015] In some embodiments, the chromatographic column is a COSMOSIL PBr column.

[0016] In some embodiments, the inner diameter of the chromatographic column is 2 to 8 mm, for example 4 to 6 mm, for example about 4.6 mm.

[0017] In some embodiments, the length of the chromatographic column is 50 to 300 mm, for example, about 250 mm.

[0018] In some embodiments, the particle size of the chromatographic column packing material is 2 to 8 μm, preferably 3 to 6 μm, more preferably 4 to 6 μm, for example about 5 μm.

[0019] In some embodiments, the chromatographic column has the following specifications: COSMOSIL PBr, 4.6 mm × 250 mm, 5 μm.

[0020] In some embodiments, the mobile phase used in the high-performance liquid chromatography method is a mixture of mobile phase A and mobile phase B.

[0021] In some implementations, mobile phase A does not include buffer salt.

[0022] In some embodiments, mobile phase A is an aqueous solution containing an acid. In some embodiments, the acid is trifluoroacetic acid, formic acid, acetic acid, or phosphoric acid.

[0023] In some embodiments, mobile phase A is selected from aqueous trifluoroacetic acid or aqueous formic acid.

[0024] In some embodiments, mobile phase A is an aqueous solution of trifluoroacetic acid. In some embodiments, the concentration of the aqueous trifluoroacetic acid solution is 0.01 to 0.5% (v / v), preferably 0.02 to 0.2% (v / v), for example 0.03% to 0.1% (v / v), for example about 0.03% (v / v), about 0.05% (v / v), about 0.07% (v / v), or about 0.1% (v / v).

[0025] In some embodiments, mobile phase A is an aqueous formic acid solution. In some embodiments, the concentration of the aqueous formic acid solution is 0.01 to 0.5% (v / v), preferably 0.02 to 0.2% (v / v), for example 0.03% to 0.1% (v / v), for example about 0.03% (v / v), about 0.05% (v / v), about 0.07% (v / v), or about 0.1% (v / v).

[0026] In some embodiments, the pH of mobile phase A is 1.5 to 5.0, preferably 1.5 to 4.0, more preferably 2.0 to 3.0, for example about 2.0.

[0027] In some embodiments, mobile phase B is selected from organic solvents. In some embodiments, the organic solvent is selected from one or more of acetonitrile, methanol, and tetrahydrofuran, preferably acetonitrile.

[0028] In some implementations, the flow rate is 0.8 to 1.5 ml / min, for example 1.0 to 1.4 ml / min or 1.2 to 1.4 ml / min, and even more for example about 1.3 ml / min.

[0029] In some implementations, the detection wavelength is 200–300 nm, preferably 205–270 nm, more preferably 230–270 nm, and even more preferably 235–245 nm, for example, about 240 nm.

[0030] In some implementations, the detection wavelength is 200–300 nm, preferably 205–270 nm, more preferably 205–250 nm, and even more preferably 205–245 nm.

[0031] In some implementations, the detection wavelength is approximately 210 nm.

[0032] In some embodiments, the column temperature is 20–40°C, preferably 30–40°C, for example, about 35°C.

[0033] In some implementations, the sample injection volume is 2 to 30 μl, for example 2 to 20 μl, or even 2 μl, 5 μl, 10 μl, or 20 μl.

[0034] In some implementations, the high-performance liquid chromatography method employs gradient elution.

[0035] In some embodiments, the gradient elution procedure is as follows: the initial gradient is 90%–100% of the mobile phase volume of mobile phase A; the second gradient is 40%–60% (e.g., 48%–52%) of mobile phase A volume of mobile phase A, varying from 90%–100%; the third gradient is 40%–60% (e.g., 48%–52%) of mobile phase A volume of mobile phase A, varying from 40%–60% (e.g., 48%–52%) to 90%–100%; and the final gradient is 90%–100% of mobile phase A volume of mobile phase A. The elution time for the initial gradient is 5–8 min, the elution time for the second gradient is 6–8 min, the elution time for the third gradient is 4–6 min, the elution time for the fourth gradient is 0.5–3 min, and the elution time for the final gradient is 4–8 min.

[0036] In some implementations, the gradient elution procedure is as follows:

[0037] The inventors of this disclosure discovered that highly polar stabilizers (such as sodium vitamin C) have very weak retention times on C18 columns, and these short retention times make them highly susceptible to interference from other substances. Therefore, it is desirable to extend the retention time of highly polar stabilizers (such as sodium vitamin C). On the other hand, weakly polar stabilizers (such as gentianic acid) have longer retention times on C18 columns. Therefore, using a C18 column cannot simultaneously and effectively detect both highly polar stabilizers (such as sodium vitamin C) and weakly polar stabilizers (such as gentianic acid).

[0038] Through extensive experimental investigation, the inventors of this disclosure unexpectedly discovered that when using chromatographic columns containing pentafluorophenyl or pentabromophenyl bonded silica as packing material, the retention time of strongly polar stabilizers (such as sodium vitamin C) can be prolonged, while the retention time of weakly polar stabilizers (such as gentianic acid) can be shortened.

[0039] In particular, when the detection conditions are set as described in any of the preceding descriptions, the retention time of a strong polar stabilizer (e.g., sodium vitamin C) can be significantly prolonged, and the retention time of a weak polar stabilizer (e.g., gentianic acid) on the chromatographic column can be shortened, thus enabling the simultaneous and accurate detection of the contents of both strong polar stabilizers (e.g., sodium vitamin C) and weak polar stabilizers (e.g., gentianic acid).

[0040] In some implementations, the sample is an injection solution.

[0041] In some embodiments, the sample is an injection containing a compound of formula I:

[0042] A represents a nuclide.

[0043] In some embodiments, A in the compound of formula I is a non-radioactive nuclide, for example... 69 Ga、 175 Lu et al.

[0044] In some embodiments, A in compound I is 175 Lu. This compound is referred to as compound A below.

[0045] In some embodiments, A in the compound of formula I is a radionuclide, for example... 68 Ga、 177 Lu、 225 Ac、 64 Cu or 211 At.

[0046] In some embodiments, A in compound I is 177 Lu. This compound is referred to as compound B below.

[0047] In some implementations, the sample is an injection containing compound A, which is referred to as compound A injection.

[0048] In some embodiments, the sample is an injection containing compound B, which is referred to as compound B injection.

[0049] The inventors have discovered that when the detection method of this disclosure described in any of the preceding claims is used to detect stabilizers in an injection solution, other components in the injection solution do not interfere with the detection in the system.

[0050] For example, the detection method provided in this disclosure can accurately and rapidly detect the content of gentic acid and / or sodium vitamin C in injections containing compound I. When the sample is an injection containing compound I, sodium vitamin C, and gentic acid, the detection method of this disclosure can simultaneously detect the content of sodium vitamin C and gentic acid, with a resolution greater than 1.5. Moreover, this detection method is simple, convenient, specific, and accurate. In particular, this detection method can accurately detect the content of stabilizer in compound A / B injections, thus playing an important guiding role in the development and optimization of the formulation process of compound A / B injections.

[0051] This disclosure also provides the use of the detection method described above in the detection of samples.

[0052] In some embodiments, the sample contains one or more of a strongly polar substance (e.g., sodium vitamin C) and a weakly polar substance (e.g., gentianic acid).

[0053] In some embodiments, the sample contains both a highly polar substance (e.g., sodium vitamin C) and a weakly polar substance (e.g., gentianic acid).

[0054] In some embodiments, the sample contains one or more of sodium vitamin C and gentianic acid.

[0055] In some embodiments, the sample contains both sodium vitamin C and gentic acid.

[0056] All technical features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive technical features and / or steps.

[0057] Unless otherwise specified, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±20%, preferably ±10%, more preferably ±5%, and more preferably ±2% of the stated numerical value. Attached Figure Description

[0058] Figure 1 is a chromatogram of the blank solution (water) of Example 1.

[0059] Figure 2 is a chromatogram of the sodium vitamin C reference solution from Example 1.

[0060] Figure 3 is a chromatogram of the gentian acid reference solution from Example 1.

[0061] Figure 4 is a chromatogram of the system suitability solution from Example 1.

[0062] Figure 5 is a chromatogram of the test solution from Example 1.

[0063] Figure 6 is a linear and range working curve of vitamin C sodium in Example 3.

[0064] Figure 7 is a graph showing the linearity and range of gentian acid in Example 3.

[0065] Figure 8 is a chromatogram of the test solution of Comparative Example 1.

[0066] Figure 9 shows the chromatogram of the test solution of Comparative Example 2. Detailed Implementation

[0067] The embodiments of this disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain this disclosure, and should not be construed as limiting this disclosure. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0068] Preparation of Solution Example 1

[0069] Prepare a blank solution: Use water as the blank solution.

[0070] Preparation of sodium vitamin C reference solution: Based on the theoretical content of sodium vitamin C in the sample, weigh about 30 mg of sodium vitamin C reference standard, dissolve it in water, and make up to 10 ml to prepare a 3.0 mg / ml reference solution.

[0071] Preparation of gentianic acid reference solution: Based on the theoretical content of gentianic acid in the sample, weigh about 10 mg of gentianic acid reference standard, dissolve it in water, and make up to 25 ml to prepare a 0.4 mg / ml reference solution.

[0072] Prepare the system suitability solution (also known as the mixed reference solution of sodium vitamin C and gentianic acid): Transfer 1 ml of sodium vitamin C reference solution and 0.1 ml of gentianic acid reference solution to the same 10 ml volumetric flask, dilute to volume with water, and shake well.

[0073] Preparation of test solution: Transfer 0.5 ml of compound A injection solution, dilute with water and bring the volume to 50 ml, shake well, and use as test solution.

[0074] Prepare a solution free of sodium vitamin C and gentianic acid: Transfer 0.5 ml of Compound A solution free of sodium vitamin C and gentianic acid (compared to Compound A injection, it does not contain sodium vitamin C and gentianic acid), dilute with water to a final volume of 50 ml, and shake well.

[0075] Example 1

[0076] Take 20 μl each of blank solution, sodium vitamin C reference solution, gentic acid reference solution, system suitability solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of sodium vitamin C and gentic acid in the test solution by peak area using the external standard method.

[0077] Chromatographic conditions

[0078] Column: COSMOSIL PBr, 4.6 mm × 250 mm, 5 μm;

[0079] Mobile phase A: 0.05% (v / v) aqueous trifluoroacetic acid, pH approximately 2.0;

[0080] Mobile phase B: Acetonitrile;

[0081] Flow rate: 1.3 ml / min;

[0082] Detection wavelength: 240nm;

[0083] Column temperature: 35℃;

[0084] Injection volume: 20 μl;

[0085] Gradient elution is used, and the procedure is as follows:

[0086] Test results

[0087] The detection results are shown in Figures 1-5. This method can simultaneously detect the content of the stabilizer sodium vitamin C and gentianic acid in compound A injection solution, with a resolution greater than 1.5.

[0088] Example 2 Specificity

[0089] Refer to the chromatographic conditions of Example 1. Measure blank solution, solution without sodium vitamin C and gentianic acid, sodium vitamin C reference solution, gentianic acid reference solution, system suitability solution, and test solution, and inject them into the liquid chromatograph separately. Record the chromatograms. The results are shown in the table below.

[0090] Table 1. Specificity

[0091] Experimental results show that there is no interference at the retention times of gentic acid and sodium vitamin C in the chromatograms of the blank solution and the solution without sodium vitamin C and gentic acid; the system suitability solution meets the requirement that the resolution between sodium vitamin C and gentic acid is greater than 1.5; the retention times of gentic acid and sodium vitamin C in the test solution are consistent with the retention times in their respective reference solutions; indicating that the high performance liquid chromatography method provided in this disclosure for detecting the content of sodium vitamin C and gentic acid in compound A injection has good specificity.

[0092] Example 3: Linearity and Range

[0093] Solution preparation

[0094] Linear solution of sodium vitamin C: Weigh an appropriate amount of sodium vitamin C reference standard, dissolve it in ultrapure water and dilute it quantitatively to prepare a series of solutions with concentrations of approximately 450.0 μg / ml, 300.0 μg / ml, 225.0 μg / ml, 150.0 μg / ml, 75.0 μg / ml and 30.0 μg / ml.

[0095] Gentian acid linear solution: Weigh an appropriate amount of gentian acid reference standard, dissolve it in ultrapure water and quantitatively dilute it to prepare a series of solutions with concentrations of approximately 8.0 μg / ml, 4.0 μg / ml, 3.0 μg / ml, 2.0 μg / ml, 1.0 μg / ml and 0.4 μg / ml.

[0096] Detection methods

[0097] Following the chromatographic conditions of Example 1, 20 μl of each of the linear solutions of different concentrations was injected into the liquid chromatograph, and the chromatograms were recorded. A linear regression equation was plotted with concentration on the x-axis and peak area on the y-axis. The results are shown in Table 2. The chromatograms of linearity and range for sodium vitamin C and gentian acid are shown in Figures 6 and 7, respectively.

[0098] Table 2. Linearity and Range

[0099] The experimental results showed that sodium vitamin C had a linear correlation coefficient of 0.9992 in the concentration range of 30.0-450 μg / ml, which met the quantitative requirement of a correlation coefficient of not less than 0.999, indicating a good linear relationship between peak area and concentration. Similarly, gentic acid had a linear correlation coefficient of 0.9999 in the concentration range of 0.4-8.0 μg / ml, which also met the quantitative requirement of a correlation coefficient of not less than 0.999, indicating a good linear relationship between peak area and concentration.

[0100] Example 4 System Precision

[0101] Following the chromatographic conditions of Example 1, 20 μl of the system suitability solution was injected into the liquid chromatograph, and the injection was repeated six times consecutively. The chromatograms were recorded. The results are shown in Tables 3 and 4.

[0102] Table 3. Precision of the Vitamin C Sodium Assay System

[0103] Table 4. Precision of the gentian acid determination system

[0104] Experimental results show that, with six consecutive injections of the system suitability solution (a mixed standard solution of sodium vitamin C and gentianic acid), the relative standard deviation (%RSD) of the retention time and peak area of ​​the main peak both meet the requirement of not exceeding 2.0%. This indicates that the high-performance liquid chromatography method provided in this disclosure for determining the content of sodium vitamin C and gentianic acid in samples has good system precision.

[0105] Example 5 Precision (Repeatability)

[0106] Following the chromatographic conditions of Example 1, six test solutions were prepared, and 20 μl of each solution was injected into the liquid chromatograph. The chromatograms were recorded. The results are shown in Tables 5 and 6.

[0107] Table 5. Repeatability results of sodium vitamin C

[0108] Table 6. Repeatability results of gentianic acid

[0109] The experimental results showed that the %RSD of the retention time of sodium vitamin C and gentianic acid chromatographic peaks did not exceed 2.0%, and the %RSD of the peak area did not exceed 5.0%, both of which met the acceptable standards, indicating that the determination had good repeatability.

[0110] Example 6 Accuracy

[0111] Prepare the spiking and recovery solution containing sodium vitamin C and gentic acid: Transfer 0.5 ml of the blank control solution of compound A injection (i.e., the solution that does not contain compound A compared to the compound A injection, also known as the base solution), 0.5 ml of sodium vitamin C reference solution, and 50 μl of gentic acid reference solution to the same 50 ml volumetric flask, add water to the mark, and shake well. Prepare 6 parallel solutions.

[0112] Following the chromatographic conditions of Example 1, 20 μl of each of the six recovered solutions were injected into the high-performance liquid chromatograph, and the chromatograms were recorded. The results are shown in Tables 7 and 8.

[0113] Table 7. Accuracy - Sodium Vitamin C

[0114] Table 8. Accuracy - Gentian Acid

[0115] Experimental results showed that the average recoveries of sodium vitamin C and gentianic acid in the six spiking solutions determined by this method were between 90% and 108%, indicating good accuracy.

[0116] Example 7 Durability

[0117] Take 20 μl of the mixed reference solution of sodium vitamin C and gentianic acid, and 20 μl of the test solution, inject them into the liquid chromatograph under various chromatographic conditions, record the chromatograms, and the results are shown in Table 9.

[0118] Table 9. Durability *Note: In Table 9, when the chromatographic conditions listed include flow rate, column temperature, mobile phase B ratio, or acid concentration in mobile phase A, it means that all other conditions are the same as those in Example 1, with the only difference being the specified flow rate, column temperature, mobile phase B ratio, or acid concentration in mobile phase A. For example, the column "Flow rate 1.2 ml / min" indicates that the chromatographic conditions used in this column are the same as those in Example 1, except that the flow rate is changed to 1.2 ml / min. Similarly, the column "Mobile phase B ratio (48%)" indicates that the chromatographic conditions used in this column are the same as those in Example 1, except that the mobile phase B ratio is set to 48% for both 13 min and 18 min in the gradient elution program; similarly, the column "Mobile phase B ratio (52%)" indicates that the chromatographic conditions used in this column are the same as those in Example 1, except that the mobile phase B ratio is set to 52% for both 13 min and 18 min in the gradient elution program.

[0119] Experimental results show that, under varying flow rates, column temperatures, gradient ratios of mobile phase B, and trifluoroacetic acid concentrations in mobile phase A, the resolution between the target peak and adjacent chromatographic peaks is greater than 1.5 under all conditions, indicating that the method has good robustness.

[0120] Comparative Example 1

[0121] The test solution was detected under the chromatographic conditions shown below. The results showed that the retention time of sodium vitamin C was too short, only 3.253 min. The separation between sodium vitamin C and adjacent impurity peaks was poor, and the detection error was large. The results are shown in Figure 8.

[0122] Chromatographic conditions

[0123] Column: Inertsil ODS-3, 4.6 mm × 250 mm, 5 μm;

[0124] Mobile phase A: 0.01M sodium acetate buffer solution (pH adjusted to 3.6 with glacial acetic acid);

[0125] Mobile phase B: Acetonitrile;

[0126] Flow rate: 1.0 ml / min;

[0127] Detection wavelength: 245nm;

[0128] Column temperature: 25℃;

[0129] Injection volume: 20 μl;

[0130] Gradient elution is used, and the procedure is as follows:

[0131] Comparative Example 2

[0132] The test solution was detected under the chromatographic conditions shown below. The results showed that the retention time of sodium vitamin C was too short, only 2.621 min. The separation between sodium vitamin C and adjacent impurity peaks was poor, and the detection error was large. The results are shown in Figure 9.

[0133] Chromatographic conditions

[0134] Column: Agilent Infinity Lab Poroshell 120EC-C18, 4.6mm × 150mm, 4μm;

[0135] Mobile phase A: 0.05% formic acid solution;

[0136] Mobile phase B: Acetonitrile;

[0137] Flow rate: 1.0 ml / min;

[0138] Detection wavelength: 245nm;

[0139] Column temperature: 25℃;

[0140] Injection volume: 20 μl;

[0141] Gradient elution is used, and the procedure is as follows:

[0142] Although embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present disclosure. All content disclosed in the specification, including the abstract, and all disclosed methods and steps, can be combined arbitrarily unless these features and / or steps are mutually exclusive combinations. Each technical feature disclosed in the specification, including the abstract, can be replaced by a technical feature that achieves the same, equivalent, or similar purpose, unless otherwise stated. Therefore, unless otherwise stated, each technical feature disclosed in this disclosure is merely one example of an equivalent or similar technical feature in a general series. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this disclosure.

Claims

1. A method for detecting the content of stabilizer in a sample, comprising detecting using a high performance liquid chromatography method, characterized in that, The chromatographic column comprises pentafluorophenyl or pentabromophenyl bonded silica gel as the filler.

2. The method of claim 1, wherein, The stabilizer comprises one or more of a strong polar stabilizer and a weak polar stabilizer; Preferably, the strong polar stabilizer is sodium ascorbate; the weak polar stabilizer is gentisic acid.

3. The method according to claim 1 or 2, characterized in that, The filler of the chromatographic column is pentabromophenyl bonded silica gel.

4. The method according to any one of claims 1-3, wherein the high performance liquid chromatography method uses a mobile phase which is a mixture of mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing an acid, preferably trifluoroacetic acid, formic acid, acetic acid or phosphoric acid, and the mobile phase B is an organic solvent, preferably one or more selected from acetonitrile, methanol and tetrahydrofuran, more preferably acetonitrile.

5. The method according to any one of claims 1-4, characterized in that, The mobile phase A is an aqueous solution of trifluoroacetic acid, preferably at a concentration of 0.01-0.5% (v / v), more preferably 0.02-0.2% (v / v), for example 0.03-0.1% (v / v), for example 0.03% (v / v), 0.05% (v / v), 0.07% (v / v) or 0.1% (v / v); or, The mobile phase A is an aqueous solution of formic acid, preferably at a concentration of 0.01-0.5% (v / v), more preferably 0.02-0.2% (v / v), for example 0.03-0.1% (v / v), for example 0.03% (v / v), 0.05% (v / v), 0.07% (v / v) or 0.1% (v / v).

6. The method according to any one of claims 1-5, characterized in that, The pH of the mobile phase A is 1.5-5.0, preferably 1.5-4.0, more preferably 2.0-3.0, for example 2.

0.

7. The method according to any one of claims 1 to 6, characterized in that, The high performance liquid chromatography method uses a gradient elution, preferably a gradient elution program as follows: an initial gradient of 90-100% (v / v) mobile phase A, a second gradient of 90-100% (v / v) to 40-60% (v / v) mobile phase A, a third gradient of 40-60% (v / v) mobile phase A, a fourth gradient of 40-60% (v / v) to 90-100% (v / v) mobile phase A, and a final gradient of 90-100% (v / v) mobile phase A; the elution time of the initial gradient is 5-8 min, the elution time of the second gradient is 6-8 min, the elution time of the third gradient is 4-6 min, the elution time of the fourth gradient is 0.5-3 min, and the elution time of the final gradient is 4-8 min. More preferably, the following gradient elution procedure is employed:

8. The method according to any one of claims 1-7, characterized in that, The method satisfies one or more of the following conditions: (1) the internal diameter of the chromatographic column is 2-8 mm, for example 4-6 mm, for example 4.6 mm; (2) the length of the chromatographic column is 50-300 mm, for example 250 mm; (3) the particle size of the filler of the chromatographic column is 2-8 μm, preferably 3-6 μm, more preferably 4-6 μm, for example 5 μm; (4) the chromatographic column is a COSMOSIL PBr chromatographic column, preferably having the following specifications: COSMOSIL PBr, 4.6 mm x 250 mm, 5 μm; (5) in the high performance liquid chromatography method, the flow rate is 0.8-1.5 ml / min, for example 1.3 ml / min; (6) in the high performance liquid chromatography method, the detection wavelength is 200-300 nm, preferably 205-270 nm, more preferably 230-270 nm or 205-250 nm, further preferably 235-245 nm or 205-245 nm, for example 240 nm or 210 nm; (7) in the high performance liquid chromatography method, the column temperature is 20-40 °C, preferably 30-40 °C, for example 35 °C.

9. The method according to any one of claims 1-8, characterized in that, The sample is an injection solution, for example an injection solution containing a compound of formula I: wherein A is a nuclide.

10. The method of claim 9, wherein A is a non-radioactive nuclide, for example is 69 Ga or 175 Lu.

11. The method of claim 9, wherein A is a radionuclide, such as 68 Ga, 177 Lu, 225 Ac, 64 Cu or 211 At.

12. Use of the method according to any one of claims 1-11 for detecting a sample.

13. Use according to claim 12, wherein the sample contains one or more of a strongly polar substance and a weakly polar substance; Preferably, the strongly polar substance is sodium ascorbate; the weakly polar substance is gentisic acid.

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