Method for determining content of hexyl aminolevulinate hydrochloride bulk drug

Abstract

The invention discloses a method for determining the content of an aminolevulinic acid hexyl ester hydrochloride raw material medicine, and belongs to the technical field of medicines. The method adopts a high performance liquid chromatography method to measure the content of a hexyl aminolevulinate hydrochloride bulk drug, and comprises the following steps: firstly, preparing a test solution and a reference solution for later use; and then setting high performance liquid detection conditions, taking octadecyl bonded silica gel as a filling agent, and taking sodium dihydrogen phosphate buffer solution-acetonitrile as a mobile phase to detect the hexyl aminolevulinate hydrochloride bulk drug. According to the present invention, with the high performance liquid chromatography method, the content of the hexyl ammonovalerate hydrochloride bulk drug can be rapidly, effectively, accurately and reliably separated and detected, such that the product quality of the hexyl ammonovalerate hydrochloride can be easily controlled, and the medication safety of the patient can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a method for determining the content of aminolevulinic acid hexyl hydrochloride raw material medicine. Background Art

[0002] Aminolevulinic acid hexyl hydrochloride, whose chemical name is 5-aminolevulinic acid hexyl hydrochloride, is highly consistent with 5-aminolevulinic acid in its related application fields. As a photosensitizer, it has good applications in photodynamic therapy and the diagnosis and treatment of related tumors. In addition, the lipid solubility and stability of aminolevulinic acid hexyl hydrochloride are higher than those of 5-aminolevulinic acid, and it is easier to penetrate the lipid bilayer of the cell membrane and has better transdermal permeability.

[0003] At present, the main methods for detecting aminolevulinic acid hexyl hydrochloride include high-performance liquid chromatography with ultraviolet detector, fluorescence detector or high-performance liquid chromatography-tandem mass spectrometry technology. First, the ultraviolet absorption of aminolevulinic acid hexyl hydrochloride is weak, which cannot meet the analysis and detection of low-concentration compound samples; secondly, aminolevulinic acid hexyl hydrochloride does not have a fluorescent group, and it needs to be chemically derivatized before fluorescence detection can be used, which is complicated, time-consuming, labor-intensive, and has poor stability; finally, high-performance liquid chromatography-tandem mass spectrometry technology has problems such as expensive related instruments and high reagent requirements, which leads to its inability to be widely used.

[0004] Therefore, developing a stable and reliable content analysis method is of great significance to the production of aminolevulinic acid hexyl hydrochloride raw materials and preparations. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for determining the content of aminolevulinic acid hexyl hydrochloride raw material medicine. The method can quickly, effectively, accurately and reliably separate and detect aminolevulinic acid hexyl hydrochloride, is beneficial to detecting the product quality of aminolevulinic acid hexyl hydrochloride, and improves the safety of patients' medication.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for determining the content of aminolevulinic acid hexyl hydrochloride raw material drug, using high performance liquid chromatography to detect the content of aminolevulinic acid hexyl hydrochloride, comprising the following steps:

[0008] (1) Prepare test sample solution and reference sample solution and set aside;

[0009] (2) Setting HPLC detection conditions;

[0010] (3) Take the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of aminolevulinic acid hexyl ester hydrochloride raw material.

[0011] The chromatographic column uses octadecyl bonded silica gel as a filler, has a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

[0012] The mobile phase consists of 10mmol / L-50mmol / L sodium dihydrogen phosphate buffer solution and acetonitrile, with a volume ratio of 50-70:30-50.

[0013] The detection wavelength of the high performance liquid chromatograph detector is 210 nm.

[0014] The column temperature of the chromatographic column is 30° C. and the flow rate is 1.0 mL / min.

[0015] Preparation of the test solution: take about 20 mg of aminolevulinic acid hexyl ester hydrochloride and place it in a 20 mL volumetric flask, add a solvent to dissolve it, dilute it to the fixed volume, and mix it evenly; preparation of the reference solution: take about 20 mg of aminolevulinic acid hexyl ester hydrochloride and place it in a 20 mL volumetric flask, add a solvent to dissolve it, dilute it to the fixed volume, and mix it evenly.

[0016] The content of hexyl aminolevulinate hydrochloride in the chromatogram of the test solution is in the range of 98.0% to 102.0%.

[0017] The injection volume of the test solution and the reference solution was 10 μL.

[0018] The solvent in the test solution and the reference solution is purified water.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The high performance liquid chromatography method used in the present invention can quickly, effectively, accurately and reliably separate and detect aminolevulinic acid hexyl hydrochloride, which is beneficial to controlling the product quality of aminolevulinic acid hexyl hydrochloride and improving the safety of patients' medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the system suitability spectrum under the chromatographic conditions of the present invention;

[0022] Figure 2 It is the spectrum of the test product under the chromatographic conditions of the present invention;

[0023] Figure 3 It is the spectrum of the reference substance under the chromatographic conditions of the present invention;

[0024] Figure 4 It is the undestructed spectrum of aminolevulinic acid hexyl ester hydrochloride;

[0025] Figure 5 It is the oxidation destruction spectrum of aminolevulinic acid hexyl ester hydrochloride;

[0026] Figure 6 It is the high temperature destruction spectrum of aminolevulinic acid hexyl hydrochloride;

[0027] Figure 7 It is the thermal decomposition destruction spectrum of aminolevulinic acid hexyl hydrochloride;

[0028] Figure 8 It is the light destruction spectrum of aminolevulinic acid hexyl ester hydrochloride;

[0029] Fig. 9 It is the aminolevulinic acid hexyl hydrochloride-acid destruction spectrum;

[0030] Fig.10 It is the alkali destruction spectrum of aminolevulinic acid hexyl hydrochloride;

[0031] Fig.11 This is the standard curve of aminolevulinic acid hexyl ester hydrochloride. DETAILED DESCRIPTION

[0032] The present invention is further illustrated below in conjunction with specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0033] Example 1

[0034] A method for determining the content of aminolevulinic acid hexyl hydrochloride raw material drug, using high performance liquid phase method to determine the content of aminolevulinic acid hexyl hydrochloride, specifically comprising the following steps:

[0035] (1) Prepare a test solution and a reference solution. Preparation of the test solution: take about 20 mg of aminolevulinic acid hexyl hydrochloride and place it in a 20 mL volumetric flask, add purified water to dissolve it, dilute it to the fixed volume, and mix it evenly; Preparation of the reference solution: take about 20 mg of aminolevulinic acid hexyl hydrochloride and place it in a 20 mL volumetric flask, add purified water to dissolve it, dilute it to the fixed volume, and mix it evenly; prepare a mixed solution for sampling to test the test effect of the method of the present invention. Preparation of the mixed solution: take appropriate amounts of aminolevulinic acid hexyl hydrochloride, aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride, dissolve and dilute them with purified water to prepare a mixed solution containing 1 mg / mL aminolevulinic acid hexyl hydrochloride, 5 μg / mL aminolevulinic acid hydrochloride and 1.5 μg / mL aminolevulinic acid ethyl hydrochloride; the solvent in the test solution, the reference solution and the mixed solution is purified water;

[0036] (2) Setting HPLC detection conditions; Detection conditions: The chromatographic column is Waters Xselect HSS T3, with octadecyl bonded silica as the filler, the specification is 250 mm × 4.6 mm, 5 μm, the mobile phase is composed of 10 mmol / L to 50 mmol / L sodium dihydrogen phosphate buffer and acetonitrile, and the volume ratio of sodium dihydrogen phosphate buffer to acetonitrile is 50 to 70:30 to 50; the column temperature is 30 °C, the flow rate is 1.0 mL / min; the detection wavelength of the HPLC detector is 210 nm;

[0037] (3) Accurately measure 10 μL of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram. Adjust the detection sensitivity so that the peak height of the main component peak is full scale, such as Figure 1 As shown. Figure 1 It can be seen that under this chromatographic condition, the main component does not interfere with aminolevulinic acid hydrochloride and aminolevulinic acid ethyl ester, and the blank solvent has no interference, so this method is applicable.

[0038] Example 2

[0039] The detection method of Example 1 was verified from several aspects, including system applicability, destructive test, linear relationship, precision, accuracy, solution stability, etc., which are described in detail below.

[0040] 1) System suitability determination:

[0041] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 1 mg of aminolevulinic acid hexyl hydrochloride per 1 mL, which is used as the reference substance solution.

[0042] Take appropriate amounts of aminolevulinic acid hydrochloride and aminolevulinic acid ethyl hydrochloride reference substances respectively, weigh them accurately, dissolve them in solvent and dilute them to make separate fixed-frame solutions containing about 0.5 mg of aminolevulinic acid hydrochloride per 1 mL and separate fixed-frame solutions containing about 0.15 mg of aminolevulinic acid ethyl hydrochloride per 1 mL.

[0043] Accurately weigh an appropriate amount of the test sample into a volumetric flask, add appropriate amounts of aminolevulinic acid hydrochloride and ethyl aminolevulinic acid hydrochloride into the same volumetric flask, dissolve and dilute with a solvent to make a mixed solution containing approximately 1 mg of ethyl aminolevulinic acid hydrochloride, approximately 5 μg of aminolevulinic acid hydrochloride, and approximately 1.5 μg of ethyl aminolevulinic acid hydrochloride per 1 mL as the system suitability solution.

[0044] 10 μL of blank solvent, system suitability solution, impurity location solution and control solution were injected into the HPLC, and the chromatogram was recorded to examine the separation of each component. The results are shown in Table 1. As can be seen from Table 1, at a detection wavelength of 210 nm, the peak order of the mixed solution is aminolevulinic acid hydrochloride, ethyl aminolevulinic acid hydrochloride, hexyl aminolevulinic acid hydrochloride and impurities can be effectively separated, and the solvent peak does not interfere with the determination of hexyl aminolevulinic acid hydrochloride.

[0045] Table 1 System suitability results

[0046]

[0047] 2) Destructive testing

[0048] Undamaged solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, dissolve it with solvent and dilute it to the scale, shake well, and use it as the solution before destruction.

[0049] Acid destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add 1 mL of 0.5 mol / L hydrochloric acid solution, destroy it at room temperature for 30 minutes, add 1 mL of 0.5 mol / L sodium hydroxide solution to neutralize it, dissolve it with a solvent and dilute it to the scale, shake it well, and use it as the acid destruction solution.

[0050] Alkali destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add 1 mL of 0.1 mol / L sodium hydroxide solution, and immediately add 1 mL of 0.1 mol / L hydrochloric acid solution to neutralize it. Dissolve and dilute to the scale with solvent, shake well, and use this as the alkali destruction solution.

[0051] Oxidative destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add 1 mL of 30% hydrogen peroxide, destroy it in an oven at 60°C for 6 hours, dissolve it in a solvent and dilute it to the scale, shake it well, and use it as the oxidative destruction solution.

[0052] High temperature destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, place it in a 60°C oven for destruction for 6 hours, cool to room temperature, then dissolve and dilute to the scale with a solvent, shake well, and use it as the high temperature destruction solution.

[0053] Pyrolysis destruction solution: Take about 20 mg of this product, place it in a 20 mL volumetric flask, add 5 mL of solvent to dissolve, then destroy it in a 60°C oven for 6 hours, cool to room temperature, then dissolve and dilute to the scale with solvent, shake well, and use it as the pyrolysis destruction solution.

[0054] Light-damaged solution: Take the undamaged solution and place it under ultraviolet wavelengths of 254nm and 365nm for 6 hours to be used as the light-damaged solution.

[0055] Take 10 μL of each of the above test solutions and inject them into the chromatograph, record the chromatogram, and analyze the content changes. For the test solution under various destruction conditions, the diode array was used for peak purity or spectral similarity determination. The results are shown in Table 2. It can be seen from Table 2 that under various destruction conditions, the degradation products produced by the test sample can be effectively separated from the main peak, the degradation products do not interfere with the determination of aminolevulinic acid hexyl hydrochloride, the reduction in the destruction of the main peak is basically consistent with the decrease in its content, the purity of the main peak is not less than 990, and the material is conserved.

[0056] Table 2 Material balance investigation results

[0057] condition Relative content (less damaged) Separation Peak purity Unbroken —— —— 997 acid 90.41％ 30.9 1000 Alkali 92.00％ 30.5 999 Oxidation 99.46％ —— 1000 high temperature 99.17％ —— 999 Pyrolysis 96.97％ —— 999 illumination 92.69％ 20.1 999

[0058] 3) Linear relationship

[0059] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, weigh it accurately, dissolve it in a solvent and dilute it to make a solution containing about 10 mg of aminolevulinic acid hexyl hydrochloride per 1 mL as the linear stock solution. Prepare the linear solution according to Table 3.

[0060] Table 3 Linear solution preparation

[0061]

[0062]

[0063] Accurately measure 10 μL of the above solution and inject it into a high performance liquid chromatograph, record the chromatogram, determine the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Table 4 below.

[0064] Table 4 Linear relationship test results

[0065]

[0066] As shown in Tables 3 and 4, aminolevulinic acid hexyl hydrochloride has a good linear relationship with its peak area within the corresponding concentration range (r>0.999).

[0067] 4) Injection precision

[0068] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, dissolve and dilute with a solvent to make a solution containing about 1 mg of aminolevulinic acid hexyl hydrochloride per 1 mL, take 10 μL and inject into the liquid chromatograph, inject 6 times continuously, and record the peak area. The results are shown in Table 5. It can be seen from Table 5 that after 6 consecutive injections, the peak area RSD of aminolevulinic acid hexyl hydrochloride is ≤2.0%, and the injection precision is good.

[0069] Table 5 Injection precision results

[0070] Injection precision Peak area 1 430.682 2 433.022 3 430.777 4 431.484 5 432.274 6 431.520 RSD 0.21％

[0071] 5) Solution stability test

[0072] The stability tests of the reference solution and the test solution were performed separately.

[0073] Stability of reference solution: Take an appropriate amount of aminolevulinic acid hexyl ester hydrochloride reference substance, dilute it with a solvent to make a solution containing about 1 mg of aminolevulinic acid hexyl ester hydrochloride per 1 mL, inject it at different times, and investigate its intraday stability. The results are shown in Table 6. As can be seen from Table 6, the aminolevulinic acid hexyl ester hydrochloride reference substance has good solution stability within 26.5 hours (RSD≤2.0%).

[0074] Table 6 Stability results of reference solution

[0075] Time (hours) Peak area 0 436.452 1 436.785 2 436.002 4 436.532 5 435.318 8.5 437.649 10 436.823 14 436.759 18 428.594 20 414.747 26.5 415.966 RSD 1.98％

[0076] Stability of test solution: The product was prepared into a test solution containing 1 mg / mL of aminolevulinic acid hexyl hydrochloride according to the proposed method, and the samples were injected at different times to investigate its intraday stability. The results are shown in Table 7. As can be seen from Table 7, the test solution stability was good within 20 hours (RSD≤2.0%).

[0077] Table 7 Stability results of test solution

[0078] Time (hours) Peak area 0 394.049 1 392.370 2 394.684 4 393.588 5 393.722 8.5 394.000 10 393.982 14 392.427 18 387.041 20 371.909 RSD 1.79％

[0079] 6) Repeatability test

[0080] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, weigh it accurately, dissolve it in a solvent, and then dilute it to make a solution containing 1 mg of aminolevulinic acid hexyl hydrochloride per 1 mL. As the reference substance solution, prepare two copies in parallel.

[0081] Take an appropriate amount of the test sample, weigh it accurately, dissolve it in a solvent, and then dilute it to make a solution containing 1 mg of aminolevulinic acid hexyl hydrochloride per 1 mL. Prepare 6 copies in parallel.

[0082] According to the proposed method, 10 μL of the reference solution and the test solution were injected into the liquid chromatograph and the chromatogram was recorded. The first reference solution was injected with 3 needles, the second reference solution was injected with 2 needles, and the test solution was repeated for 6 times, with two needles for each. The results are shown in Table 8. As can be seen from Table 8, the RSD of the 6 repeatability sample test results is ≤2.0%, and the repeatability is good.

[0083] Table 8 Repeatability results

[0084] Serial number content(%) 1 98.8 2 100.7 3 99.4 4 98.8 5 100.2 6 99.8 RSD 0.77％

[0085] 7) Accuracy test

[0086] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, weigh it accurately, dissolve it in a solvent, and then dilute it to make a solution containing 1 mg of aminolevulinic acid hexyl hydrochloride per 1 mL. As the reference substance solution, prepare two copies in parallel.

[0087] Take an appropriate amount of aminolevulinic acid hexyl hydrochloride reference substance, weigh it accurately, dissolve it in a solvent and dilute it to prepare recovery solutions containing 0.5 mg, 1 mg, and 1.5 mg of aminolevulinic acid hexyl hydrochloride per 1 mL.

[0088] Prepare 50%, 100% and 150% solutions according to Table 9, and prepare 3 copies of each concentration.

[0089] Table 9 Accuracy solution preparation

[0090]

[0091] 10 μL of the above solution was accurately weighed and injected into a high performance liquid chromatograph, the chromatogram was recorded, the peak area was determined, and the recovery rate and RSD were calculated. The results are shown in Table 10. As can be seen from Table 10, the recovery rate and average recovery rate of aminolevulinic acid hexyl hydrochloride were both within the range of 98% to 101%, RSD ≤ 2.0%, and the recovery rate was good.

[0092] Table 10 Accuracy results

[0093]

[0094] 8) Intermediate precision

[0095] Different operators used different instruments at different times to perform content checks on the same batch of samples according to the repeatability test method, and the results are shown in Table 11. As can be seen from Table 11, the intermediate precision of the content check method for this product is good.

[0096] Table 11 Intermediate precision test results

[0097]

[0098]

[0099] 9) Durability test

[0100] The durability of the aminolevulinic acid hexyl ester method was mainly verified from five aspects: different mobile phase pH, different mobile phase ratio, different column temperature, different flow rate and different chromatographic column.

[0101] The experiment was carried out according to the conditions in Table 12, and the results are shown in Table 13. It can be seen from Table 13 that when the mobile phase pH, mobile phase ratio, flow rate, column temperature undergo slight changes and when the same type of chromatographic column is replaced, the detection results of aminolevulinic acid hexyl hydrochloride content are not significantly affected, which meets the requirements, indicating that the chromatographic conditions have good durability.

[0102] Table 12 Durability conditions

[0103] parameter Scope of change Mobile phase pH ±0.1 Mobile phase ratio ±1％ Column temperature Column temperature ±2℃ Flow rate Flow rate ±0.05mL / min Different columns Octadecyl bonded silica as filler

[0104] Table 13 Durability results

[0105]

[0106] 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 measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug, characterized in that: The method of detecting the content of aminolevulinic acid hexyl hydrochloride by high performance liquid chromatography comprises the following steps: (1) Prepare test sample solution and reference sample solution and set aside; (2) Setting HPLC detection conditions; (3) Take the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of aminolevulinic acid hexyl ester hydrochloride raw material.

2. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The chromatographic column uses octadecyl bonded silica gel as a filler, has a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

3. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The mobile phase consists of 10mmol / L-50mmol / L sodium dihydrogen phosphate buffer and acetonitrile, with a volume ratio of 50-70:30-50.

4. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatograph detector is 210 nm.

5. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The column temperature of the chromatographic column is 30° C. and the flow rate is 1.0 mL / min.

6. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: Preparation of test solution: take about 20 mg of aminolevulinic acid hexyl hydrochloride and place it in a 20 mL volumetric flask, add solvent to dissolve, dilute to volume, and mix well; Preparation of reference solution: take about 20 mg of aminolevulinic acid hexyl hydrochloride reference substance and place it in a 20 mL volumetric flask, add solvent to dissolve, dilute to volume, and mix well.

7. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The content of hexyl aminolevulinate hydrochloride in the chromatogram of the test solution is in the range of 98.0% to 102.0%.

8. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The injection volume of the test solution and the reference solution was 10 μL.

9. The method for measuring the content of aminolevulinic acid hexyl hydrochloride raw material drug according to claim 1, characterized in that: The solvent in the test solution and the reference solution is purified water.

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