Method for detecting related substances in hexyl aminolevulinate hydrochloride for bladder perfusion by using high performance liquid chromatography

Abstract

The invention discloses a method for detecting related substances in hexyl aminolevulinate hydrochloride for bladder perfusion by using high performance liquid chromatography, and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: 1) respectively weighing a proper amount of a reference substance and a proper amount of a test substance, and diluting and dissolving with water to prepare a test substance solution and a reference substance solution; (2) carrying out gradient elution by adopting a GL Intsutin chromatographic column; (3) respectively and precisely measuring the test solution and the reference solution, injecting into a liquid chromatograph, starting to detect and recording a chromatogram; and 4) drawing a standard curve to obtain a standard curve equation, and calculating the contents of aminolevulinic acid hydrochloride and ethyl aminolevulinate hydrochloride in hexyl aminolevulinate hydrochloride for bladder perfusion. The method disclosed by the invention is good in specificity, can be used for directly carrying out trace detection on aminolevulinic acid hydrochloride and ethyl aminolevulinate hydrochloride in hexyl aminolevulinate hydrochloride for bladder perfusion by using a liquid phase, and is good in linearity, reliable in accuracy and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by utilizing high performance liquid chromatography. Background Art

[0002] 5-Aminolevulinicacid hexyl ester hydrochloride for bladder instillation, main component: 5-Aminolevulinicacid hexyl ester hydrochloride, molecular formula: C 11 H 22 ClNO3, molecular weight is 251.76, its chemical structure is as shown below:

[0003] .

[0004] Aminolevulinic acid hexyl hydrochloride and special solvent for bladder instillation are currently the only approved contrast agent drugs in the world to assist in the diagnosis and management of bladder cancer. They can be highly selectively absorbed by tumor tissue first, and through combined use with blue light cystoscopy, the detection rate of bladder cancer in situ can be improved. The combined use of aminolevulinic acid hexyl hydrochloride for bladder instillation and blue light cystoscopy as a method for the management of non-muscle invasive bladder cancer (NMIBC) has been included in the global expert consensus guidelines and the Chinese guidelines for the diagnosis and treatment of urological and men's diseases.

[0005] At present, the drug has not been marketed in China. There is no literature at home or abroad reporting the detection method of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride in its related substances. Summary of the invention

[0006] The technical problem solved by the present invention is to provide a method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography, which can separate multiple starting materials, intermediates and main peaks, fully meet the qualitative and quantitative analysis of various impurities in the product, and effectively control the product quality.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography comprises the following steps:

[0009] 1) Weigh appropriate amounts of reference substance and test substance respectively, dilute and dissolve them with water to prepare test substance solution and reference substance solution;

[0010] 2) Setting the HPLC detection conditions: using a GL Inertsustain column with a size of 4.6 mm × 250 mm and a filler particle size of 5 µm; the mobile phase consists of phase A and phase B, where phase A is phosphate buffer, phase B is acetonitrile, and the solvent is water, with gradient elution;

[0011] 3) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, the detection wavelength of the liquid chromatograph is 210nm, start detection and record the chromatogram;

[0012] 4) Draw a standard curve, obtain the standard curve equation, and calculate the content of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride in aminolevulinic acid hexyl hydrochloride for bladder instillation.

[0013] Preferably, in step 1), aminolevulinic acid hexyl hydrochloride for bladder instillation is taken and water is added to prepare a solution containing 10 mg of aminolevulinic acid hexyl hydrochloride per 1 mL, which is used as the test solution.

[0014] Preferably, in step 1), an appropriate amount of aminolevulinic acid hydrochloride reference substance is taken, accurately weighed, dissolved in water and quantitatively diluted to prepare a solution containing 50 μg of aminolevulinic acid hydrochloride per 1 mL, as the reference substance solution.

[0015] Preferably, in the step 2), the gradient elution conditions are: 0-5 min, the volume percentage of mobile phase A is 85%, and the volume percentage of mobile phase B is 15%; 5-10 min, the volume content of mobile phase A changes from 85% to 80%, and the volume content of mobile phase B changes from 15% to 20%; 10-22 min, the volume content of mobile phase A changes from 80% to 65%, and the volume content of mobile phase B changes from 20% to 35%; 22-35 min, the volume content of mobile phase A changes from 65% to 50%, and the volume content of mobile phase B changes from 35% to 50%; 35-35.1 min, the volume content of mobile phase A changes from 50% to 85%, and the volume content of mobile phase B changes from 50% to 15%; 35.1-40 min, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%.

[0016] Preferably, in step 2), the preparation process of phosphate buffer is as follows: 2.16 g of sodium octane sulfonate and 3.12 g of sodium dihydrogen phosphate dihydrate are dissolved in water and diluted to 1000 mL, and the pH value is adjusted to 3.2 with phosphoric acid.

[0017] Preferably, in step 2), the flow rate of the mobile phase is 1 mL / min and the column temperature is 45°C.

[0018] Preferably, in step 3), the injection volume is 30 μL.

[0019] Preferably, in the step 4), a linear regression equation is made according to the chromatogram with the peak area A as the ordinate and the concentration C as the abscissa. The linear equation of aminolevulinic acid hydrochloride is A=0.0218C+0.0123, and the linear equation of aminolevulinic acid ethyl hydrochloride is A=0.0142C-0.0042.

[0020] Preferably, the linear concentration range of aminolevulinic acid hydrochloride is 2.488-74.64µg / mL, and the limit of quantification is 2.488µg / mL; the linear concentration range of aminolevulinic acid ethyl hydrochloride is 3.605-21.63µg / mL, and the limit of quantification is 3.605µg / mL.

[0021] Preferably, the detection limit of aminolevulinic acid hydrochloride is 2.488µg / mL; the detection limit of aminolevulinic acid ethyl hydrochloride is 3.605µg / mL

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0023] 1) The method of the present invention can separate impurities from the main peak, fully satisfying the qualitative and quantitative detection and analysis of relevant impurities in the product, so that the product quality can be effectively controlled;

[0024] 2) The present invention provides a method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by using high performance liquid chromatography. The separation degree between the aminolevulinic acid hydrochloride chromatographic peak and the aminolevulinic acid ethyl hydrochloride chromatographic peak is high, and there is no interference between them. The aminolevulinic acid hydrochloride and the aminolevulinic acid ethyl hydrochloride can be accurately detected at the same time. The method is simple to operate, easy to control, and has low detection cost. It also has good linear relationship, specificity, precision, stability, and repeatability, high sample recovery rate, and accurate and reliable detection results. It provides an effective detection method for monitoring the content of aminolevulinic acid and aminolevulinic acid ethyl hydrochloride in the aminolevulinic acid hexyl hydrochloride drug for bladder instillation, and further ensures the product quality of aminolevulinic acid hexyl hydrochloride for bladder instillation and the medication safety of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the HPLC chromatogram of the system suitability solution in Example 1;

[0026] Figure 2 It is the linear curve diagram of aminolevulinic acid hydrochloride of the present invention;

[0027] Figure 3 It is the linear curve diagram of ethyl aminolevulinate hydrochloride of the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further illustrated below in conjunction with specific examples. The examples are implemented under the premise of the technical solution of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] In the following examples, the source of the test sample aminolevulinic acid hexyl hydrochloride for bladder instillation used was Guangdong Xinghao Pharmaceutical Co., Ltd., and the specification of each vial was 85 mg calculated as aminolevulinic acid hexyl hydrochloride; all reagents used were analytically pure.

[0030] The structural formula of the related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation is:

[0031] Aminolevulinic acid hydrochloride: ;

[0032] Ethyl aminolevulinate hydrochloride: .

[0033] Example 1

[0034] (1) Testing conditions

[0035] Instrument: High performance liquid chromatograph Agilent 1260;

[0036] Detection wavelength: 210nm;

[0037] Chromatographic column: GL Inertsustain, size 4.6mm×250mm, filler particle size 5µm;

[0038] Mobile phase A: phosphate buffer (take 2.16 g of sodium octane sulfonate and 3.12 g of sodium dihydrogen phosphate dihydrate, dissolve in water and dilute to 1000 mL, and adjust the pH to 3.2 with phosphoric acid);

[0039] Mobile phase B: acetonitrile;

[0040] Gradient elution program: 0~5min, the volume percentage of mobile phase A is 85%, and the volume percentage of mobile phase B is 15%; 5~10min, the volume content of mobile phase A changes from 85% to 80%, and the volume content of mobile phase B changes from 15% to 20%; 10~22min, the volume content of mobile phase A changes from 80% to 65%, and the volume content of mobile phase B changes from 20% to 35%; 22~35min, the volume content of mobile phase A changes from 65% to 50%, and the volume content of mobile phase B changes from 35% to 50%; 35~35.1min, the volume content of mobile phase A changes from 50% to 85%, and the volume content of mobile phase B changes from 50% to 15%; 35.1~40min, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%;

[0041] Sample concentration: 10 mg / mL;

[0042] Injection volume: 30 μL;

[0043] Flow rate: 1 mL / min;

[0044] Column temperature: 45°C;

[0045] Solvent: Water.

[0046] (2) Testing steps

[0047] System suitability solution: Take appropriate amount of aminolevulinic acid hexyl hydrochloride, aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances, weigh accurately, dissolve and dilute with water to make a mixed solution containing 10 mg of aminolevulinic acid hexyl hydrochloride, 50 μg of aminolevulinic acid hydrochloride and 15 μg of aminolevulinic acid ethyl hydrochloride per 1 mL.

[0048] Test solution: Take an appropriate amount of aminolevulinic acid hexyl hydrochloride for bladder instillation, add water to prepare a solution containing 10 mg of aminolevulinic acid hexyl hydrochloride per 1 mL as the test solution.

[0049] Reference solution: Take an appropriate amount of aminolevulinic acid hydrochloride reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 50 μg of aminolevulinic acid hydrochloride per 1 mL.

[0050] The diluent is water.

[0051] Accurately measure 30 μL of system suitability solution, reference solution, and test solution, respectively, inject into liquid chromatograph, and record chromatogram. In the chromatogram of system suitability solution, the theoretical plate number calculated based on the aminolevulinic acid hydrochloride peak is not less than 3000, and the separation between the aminolevulinic acid hydrochloride peak and the aminolevulinic acid ethyl hydrochloride peak is greater than 1.5. The retention time of related substances is: aminolevulinic acid hydrochloride is 5.6min, and aminolevulinic acid ethyl hydrochloride is 15.6min.

[0052] Figure 1 This is the chromatogram of the system suitability solution at a wavelength of 210 nm. Aminolevulinic acid hydrochloride and ethyl aminolevulinic acid hydrochloride are eluted in sequence, and the separation is good.

[0053] Example 2

[0054] 1. Specificity test

[0055] Blank solvent: water;

[0056] Ethyl aminolevulinate hydrochloride fixative solution: Take an appropriate amount of ethyl aminolevulinate hydrochloride reference substance, weigh it accurately, and dissolve and dilute it in water to make a solution containing 150 μg of ethyl aminolevulinate hydrochloride per 1 mL.

[0057] Aminolevulinic acid hydrochloride localization solution: Take an appropriate amount of aminolevulinic acid hydrochloride reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing 50 μg of aminolevulinic acid hydrochloride per 1 mL.

[0058] System suitability solution: Take appropriate amount of aminolevulinic acid hexyl hydrochloride, aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances, weigh accurately, dissolve and dilute with water to make a mixed solution containing 10 mg of aminolevulinic acid hexyl hydrochloride, 50 μg of aminolevulinic acid hydrochloride and 15 μg of aminolevulinic acid ethyl hydrochloride per 1 mL.

[0059] Take 50 μL of the blank solvent, ethyl aminolevulinate hydrochloride positioning solution, aminolevulinic acid hydrochloride positioning solution, and system suitability solution, respectively, and operate according to the above chromatographic conditions. The results are shown in Table 1.

[0060] Table 1 Specificity test results

[0061]

[0062] As shown in Table 1, the blank solvent has no interference, and the peaks of the known impurity positioning solution and the system suitability solution are consistent.

[0063] 2. Limit of Quantitation and Limit of Detection Test

[0064] Take the aminolevulinic acid ethyl ester positioning solution and the aminolevulinic acid hydrochloride positioning solution under the item "1 Specificity Test", and gradually dilute them until the signal-to-noise ratio (S / N) is 10, and the amount at this time is calculated as the quantitative limit; when the signal-to-noise ratio (S / N) is 3, the amount at this time is calculated as the detection limit. The results are shown in Table 2.

[0065] Table 2 Quantitation limit test data

[0066]

[0067] Table 3 Detection limit test data

[0068]

[0069] It can be seen from Tables 2 and 3 that the quantification limits of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl ester measured by this method were 2.488µg / mL and 3.605µg / mL, respectively, equivalent to 0.025% and 0.036% of the test sample concentration; the RSD values ​​of the peak areas of the six quantification limit solutions were 8.30% and 9.55%, both less than 10%, meeting the verification requirements.

[0070] 3. Linearity and range test

[0071] Linear mother solution: Take appropriate amount of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances, weigh accurately, dissolve and dilute with water to make a mixed solution containing 500 μg of aminolevulinic acid hydrochloride and 150 μg of aminolevulinic acid ethyl hydrochloride per 1 mL, as the linear mother solution. The linear mother solution was used to prepare the linear solutions in Table 4 below, and the results are shown in Tables 5 and 6.

[0072] Table 4 Linear master solution preparation table

[0073]

[0074] Table 5 Linearity and range test data of aminolevulinic acid hydrochloride

[0075]

[0076] As shown in Table 5, the standard curve equation of aminolevulinic acid hydrochloride is y=0.0218x+0.0123, the correlation coefficient r=0.9998 (>0.998), and 20% of the peak area is greater than the intercept at 100% concentration, so the linear relationship is good in the range of 2.488µg / mL~74.64µg / mL.

[0077] Table 6 Linearity and range test data of ethyl aminolevulinate hydrochloride

[0078]

[0079] It can be seen from Table 6 that the standard curve equation of aminolevulinic acid ethyl ester is y=0.0142x-0.0042, the correlation coefficient r=0.9998 (>0.998), and 20% of the peak area is greater than the intercept at 100% concentration, so the linear relationship is good in the range of 3.605µg / mL~21.63µg / mL.

[0080] Adopt linear standard curve y=ax+b, by comparing the slope (a) of aminolevulinic acid hydrochloride and the slope (a) of ethyl aminolevulinic acid hydrochloride, the correction factor of ethyl aminolevulinic acid hydrochloride is obtained. According to the data in "3 linearity and range" test, the correction factor of ethyl aminolevulinic acid hydrochloride is calculated, and the result is shown in Table 7.

[0081] Table 7 Correction factor results

[0082]

[0083] As can be seen from Table 7, the correction factor of ethyl aminolevulinate hydrochloride is 1.44.

[0084] 4. Stability test

[0085] System suitability solution: Take appropriate amount of aminolevulinic acid hexyl hydrochloride, aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances, weigh accurately, dissolve and dilute with water to make a mixed solution containing 10 mg of aminolevulinic acid hexyl hydrochloride, 50 μg of aminolevulinic acid hydrochloride and 15 μg of aminolevulinic acid ethyl hydrochloride per 1 mL.

[0086] Test solution: Take 5 vials of this product, add appropriate amount of water to dissolve aminolevulinic acid hexyl hydrochloride, place in a 50mL volumetric flask, wash the container with water several times, add the washing liquid to the volumetric flask, dilute with water to the scale, and shake well. (Prepare 6 portions in parallel)

[0087] Reference solution: Take an appropriate amount of aminolevulinic acid hydrochloride reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 50 μg per 1 mL.

[0088] Take 50 μL of the system suitability solution, test solution, and reference solution, respectively, and operate according to the above chromatographic conditions. The results are shown in Tables 8 to 10.

[0089] Table 8 System suitability solution-stability test results

[0090]

[0091] Table 9 Reference solution - stability test results

[0092]

[0093] Table 10 Test solution - stability test results

[0094]

[0095] It can be seen from Tables 8 to 10 that within 65.5 hours, the RSDs of the peak area and retention time of aminolevulinic acid hydrochloride and ethyl aminolevulinic acid hydrochloride in the system suitability solution were both less than 2.0%, which met the requirements; within 62 hours, the RSDs of the peak area and retention time of aminolevulinic acid hydrochloride in the reference solution were both less than 2.0%, which met the requirements; within 46 hours, the RSD of the aminolevulinic acid hydrochloride content in the test solution was less than 30%, which met the requirements.

[0096] 5. Precision test

[0097] System suitability solution: Take appropriate amount of aminolevulinic acid hexyl hydrochloride, aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances, weigh accurately, dissolve and dilute with water to make a mixed solution containing 10 mg of aminolevulinic acid hexyl hydrochloride, 50 μg of aminolevulinic acid hydrochloride and 15 μg of aminolevulinic acid ethyl hydrochloride per 1 mL.

[0098] Take 50 μL of the system suitability solution and operate under the above chromatographic conditions. Inject the system suitability solution 6 times continuously and calculate the RSD value of each impurity peak area and retention time. The results are shown in Table 11.

[0099] Table 11 System suitability solution - precision test results

[0100]

[0101] As can be seen from Table 11, the system suitability solution was injected 6 times continuously, and the RSD values ​​of the peak area and retention time of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl ester hydrochloride were both less than 2.0%, which met the verification requirements, indicating that the detection method of the present invention has good precision.

[0102] 6. Accuracy test

[0103] Test solution: Take 5 vials of this product, add appropriate amount of water to dissolve aminolevulinic acid hexyl hydrochloride, place in a 50mL volumetric flask, wash the container with water several times, add the washing liquid into the volumetric flask, dilute with water to the scale, and shake well.

[0104] Impurity mixed solution: Take about 9 mg of aminolevulinic acid hydrochloride and 3 mg of aminolevulinic acid ethyl hydrochloride respectively, accurately weigh, place in a 20 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0105] Impurity control solution: Accurately measure 1.0 mL of impurity mixed solution, place in a 10 mL volumetric flask, add water to dissolve and dilute to the scale.

[0106] Take 50 μL of the test solution, impurity mixed solution, and impurity control solution, and operate according to the above chromatographic conditions. Use the external standard method to calculate the content of known impurities in the test solution. The content of each component obtained after deducting the known impurity content in the test solution is the measured value, and the measured value divided by the added value is the recovery rate of each component. The results are shown in Table 12.

[0107] Table 12 Accuracy test results

[0108]

[0109] As shown in Table 12, the recovery rate of aminolevulinic acid hydrochloride is 100.7%, and the RSD value is 1.08%; the recovery rate of aminolevulinic acid ethyl hydrochloride is 111.9%, and the RSD value is 2.50%; both meet the test requirements, indicating that the detection method of the invention has good accuracy.

[0110] 7. Durability test

[0111] Test solution: Take 5 vials of this product, add appropriate amount of water to dissolve aminolevulinic acid hexyl hydrochloride, place in a 50mL volumetric flask, wash the container with water several times, add the washing liquid to the volumetric flask, dilute with water to the scale, and shake well. (Prepare 6 portions in parallel)

[0112] Reference solution: Take an appropriate amount of aminolevulinic acid hydrochloride reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 50 μg per 1 mL.

[0113] Take 50 μL of the test solution and reference solution respectively and operate according to the above chromatographic conditions. The results are shown in Table 13.

[0114] Table 13 Durability test results

[0115]

[0116] It can be seen from Table 13 that fine-tuning of each chromatographic condition has no significant effect on the sample detection results, and the detection method of the present invention has good durability.

[0117] 8. Repeatability test

[0118] Test solution: Take 5 vials of this product, add appropriate amount of water to dissolve aminolevulinic acid hexyl hydrochloride, place in a 50mL volumetric flask, wash the container with water several times, add the washing liquid to the volumetric flask, dilute with water to the scale, and shake well. (Prepare 6 portions in parallel)

[0119] Reference solution: Take an appropriate amount of aminolevulinic acid hydrochloride reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 50 μg of aminolevulinic acid hydrochloride per 1 mL. Take 50 μL of the test solution and reference solution respectively, and operate according to the above chromatographic conditions. The results are shown in Table 14.

[0120] Table 14 Repeatability test results

[0121]

[0122] It can be seen from Table 14 that in the 6 samples, the impurities are all within the limit requirements, and there is no significant difference in the number of impurities and the content of each impurity, which meets the verification requirements.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography, characterized in that: The following steps are involved: 1) Weigh appropriate amounts of reference substance and test substance respectively, dilute and dissolve them with water to prepare test substance solution and reference substance solution; 2) Setting the HPLC detection conditions: using a GL Inertsustain column with a size of 4.6 mm × 250 mm and a filler particle size of 5 µm; the mobile phase consists of phase A and phase B, where phase A is phosphate buffer, phase B is acetonitrile, and the solvent is water, with gradient elution; 3) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, the detection wavelength of the liquid chromatograph is 210nm, start detection and record the chromatogram; 4) Draw a standard curve, obtain the standard curve equation, and calculate the content of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride in aminolevulinic acid hexyl hydrochloride for bladder instillation.

2. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 1), aminolevulinic acid hexyl hydrochloride for bladder instillation is taken and water is added to prepare a solution containing 10 mg of aminolevulinic acid hexyl hydrochloride per 1 mL, which is used as the test solution.

3. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 1), an appropriate amount of aminolevulinic acid hydrochloride reference substance is taken, accurately weighed, dissolved in water, and quantitatively diluted to prepare a solution containing 50 μg of aminolevulinic acid hydrochloride per 1 mL, as the reference substance solution.

4. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 2), the gradient elution conditions are: 0-5 min, the volume percentage of mobile phase A is 85%, and the volume percentage of mobile phase B is 15%; 5-10 min, the volume content of mobile phase A changes from 85% to 80%, and the volume content of mobile phase B changes from 15% to 20%; 10-22 min, the volume content of mobile phase A changes from 80% to 65%, and the volume content of mobile phase B changes from 20% to 35%; 22-35 min, the volume content of mobile phase A changes from 65% to 50%, and the volume content of mobile phase B changes from 35% to 50%; 35-35.1 min, the volume content of mobile phase A changes from 50% to 85%, and the volume content of mobile phase B changes from 50% to 15%; 35.1-40 min, the volume content of mobile phase A is 85%, and the volume content of mobile phase B is 15%.

5. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 2), the preparation process of the phosphate buffer is as follows: 2.16 g of sodium octane sulfonate and 3.12 g of sodium dihydrogen phosphate dihydrate are dissolved in water and diluted to 1000 mL, and the pH value is adjusted to 3.2 with phosphoric acid.

6. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 2), the flow rate of the mobile phase is 1 mL / min and the column temperature is 45°C.

7. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 3), the injection volume is 30 μL.

8. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: In the step 4), a linear regression equation is made according to the chromatogram with the peak area A as the ordinate and the concentration C as the abscissa. The linear equation of aminolevulinic acid hydrochloride is A=0.0218C+0.0123, and the linear equation of aminolevulinic acid ethyl hydrochloride is A=0.0142C-0.0042.

9. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: The linear concentration range of aminolevulinic acid hydrochloride is 2.488~74.64µg / mL, and the limit of quantification is 2.488µg / mL; the linear concentration range of aminolevulinic acid ethyl hydrochloride is 3.605~21.63µg / mL, and the limit of quantification is 3.605µg / mL.

10. The method for detecting related substances in aminolevulinic acid hexyl hydrochloride for bladder instillation by high performance liquid chromatography according to claim 1, characterized in that: The detection limit of aminolevulinic acid hydrochloride is 2.488µg / mL; the detection limit of aminolevulinic acid ethyl hydrochloride is 3.605µg / mL.

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