CN114264734A - Detection method of aminolevulinic acid hydrochloride related substances - Google Patents
Detection method of aminolevulinic acid hydrochloride related substances Download PDFInfo
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- CN114264734A CN114264734A CN202111360171.1A CN202111360171A CN114264734A CN 114264734 A CN114264734 A CN 114264734A CN 202111360171 A CN202111360171 A CN 202111360171A CN 114264734 A CN114264734 A CN 114264734A
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- aminolevulinic acid
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- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 229960000781 aminolevulinic acid hydrochloride Drugs 0.000 title claims abstract description 30
- 239000000126 substance Substances 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 239000012535 impurity Substances 0.000 claims abstract description 39
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims abstract description 8
- 239000000523 sample Substances 0.000 claims abstract description 7
- 239000012488 sample solution Substances 0.000 claims abstract description 7
- 238000007865 diluting Methods 0.000 claims abstract description 6
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 3
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 25
- 238000012937 correction Methods 0.000 claims description 14
- 239000012085 test solution Substances 0.000 claims description 14
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 claims description 13
- 239000002253 acid Substances 0.000 claims description 13
- HRQDCDQDOPSGBR-UHFFFAOYSA-M sodium;octane-1-sulfonate Chemical compound [Na+].CCCCCCCCS([O-])(=O)=O HRQDCDQDOPSGBR-UHFFFAOYSA-M 0.000 claims description 11
- 239000013558 reference substance Substances 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 7
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 5
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 4
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000004364 calculation method Methods 0.000 claims description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- XFXIJSRNFKHZFW-UHFFFAOYSA-N [Na].CCCCCCCC Chemical compound [Na].CCCCCCCC XFXIJSRNFKHZFW-UHFFFAOYSA-N 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
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- 239000012071 phase Substances 0.000 description 11
- 229960002749 aminolevulinic acid Drugs 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
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- 238000005303 weighing Methods 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010034972 Photosensitivity reaction Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
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- QGQXAMBOYWULFX-LZWSPWQCSA-N 2-morpholin-4-ylethyl (e)-6-(4,6-dihydroxy-7-methyl-3-oxo-1h-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound OC=1C=2C(=O)OCC=2C(C)=C(O)C=1C\C=C(/C)CCC(=O)OCCN1CCOCC1 QGQXAMBOYWULFX-LZWSPWQCSA-N 0.000 description 1
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- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for detecting related substances of aminolevulinic acid hydrochloride, which is a reversed-phase high-performance liquid chromatography and comprises the steps of preparing an octane sodium sulfonate buffer solution with the pH value of 2.0 +/-0.2, further preparing an octane sodium sulfonate buffer solution with acetonitrile accounting for 10-20% (v/v) -acetonitrile serving as a mobile phase, preparing a sample to be detected with the aminolevulinic acid hydrochloride as a main component into a sample solution with the concentration of 10mg/ml by using the mobile phase, putting 1ml of the sample solution into a 20ml measuring flask, diluting the sample solution to a scale by using the mobile phase, shaking up, and then preparing a reference solution for detection operation. The detection method of the aminolevulinic acid hydrochloride related substances has good specificity, has high detection sensitivity on the related substances, can realize complete separation of impurity peaks and main peaks, has more detectable substance types, and can identify 4 impurities generated in the degradation process of the aminolevulinic acid hydrochloride, wherein the two impurities are new substances which have not been identified before.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting aminolevulinic acid hydrochloride related substances.
Background
Aminolevulinic acid hydrochloride(Aminolevulinic Acid Hydrochloride) with the chemical name of 5-amino-4-ketovalerate and the molecular formula: c5H9NO3HCl, molecular weight: 167.59. the metabolism of aminolevulinic acid (ALA) is the first step in the biochemical pathway for heme synthesis in humans. ALA is not a photosensitizer by itself, but a metabolic precursor of the photosensitizer protoporphyrin ix (ppix). ALA synthesis is typically regulated by intracellular heme levels through ALA synthetase feedback inhibition. Exogenous ALA circumvents this regulation, resulting in accumulation of PpIX. Photosensitivity is produced by conversion and accumulation of ALA to PpIX upon dermal application of aminolevulinic acid hydrochloride. Upon exposure to light of a certain wavelength and energy, the accumulated PpIX produces a photodynamic effect, a light and oxygen dependent cytotoxic effect: PpIX absorbs light and assumes an excited state, which subsequently undergoes electron transfer with oxygen molecules to generate highly reactive singlet oxygen and free radicals. The photosensitivity of the applied ALA postfocal light together with the specific wavelength and energy of the light is the basis of ALA photodynamic therapy.
Aminolevulinic acid hydrochloride is unstable in alkaline solution and under illumination conditions, and can be slowly degraded at high temperature to generate impurities. In order to ensure the safety and effectiveness of the product, impurities need to be controlled, so that the detection of related substances is an important index for the quality control of the product.
Until now, the detection method of related substances of aminolevulinic acid hydrochloride is only recorded in the United states pharmacopoeia, and the details are described in the following comparative examples. The method has the following defects: (1) the aminolevulinic acid hydrochloride has large polarity and cannot be reserved in an inverse phase system, so that a main peak appears at the dead volume of a chromatographic column, the main peak cannot be effectively separated from a solvent peak, and a chromatographic peak has a shoulder phenomenon for a high-concentration sample solution. (2) The detected number of the impurities is small, only two main impurities, namely 2, 5-dipropionic acid pyrazine (impurity A) and 3- (4' -carbonylvaleric acid) -2, 5-dipropionic acid pyrazine (impurity B), can be identified, and the rest impurities can not be detected or positioned, even the impurities which are not identified exist.
Therefore, it is necessary to optimize the method for detecting aminolevulinic acid hydrochloride-related substances so as to obtain a method with higher sensitivity and more types of detected substances.
Disclosure of Invention
The invention aims to provide a method for detecting aminolevulinic acid hydrochloride related substances with higher sensitivity.
The purpose of the invention is realized by the following technical scheme:
the invention provides a detection method of aminolevulinic acid hydrochloride related substances, which is a reversed-phase high performance liquid chromatography and comprises the following steps:
(1) solution preparation:
A) preparing an octane sodium sulfonate buffer solution with the pH value of 2.0 +/-0.2, and further preparing an octane sodium sulfonate buffer solution with acetonitrile accounting for 10-20% (v/v) -acetonitrile as a mobile phase;
B) preparing a sample to be tested with aminolevulinic acid hydrochloride as a main component into a test solution with the concentration of 10mg/ml by using the mobile phase;
C) placing 1ml of the test solution into a 20ml measuring flask, diluting the test solution to a scale with the mobile phase, shaking up, placing 5ml of the test solution into a 50ml measuring flask, diluting the test solution to a scale with the mobile phase, and shaking up to serve as a control solution;
D) taking 2, 5-pyrazine dipropionate (hereinafter referred to as impurity A) as a reference substance, carrying out ultrasonic treatment on the reference substance for 30 seconds by using a 50% acetonitrile aqueous solution, and then adding the mobile phase for dissolving to prepare a reference substance solution with the concentration of 20 mu g/ml;
(2) chromatographic conditions are as follows: the chromatographic column filler is octadecylsilane chemically bonded silica; the detection wavelength is ultraviolet wavelength; the column temperature is 25-30 ℃; the flow rate is 0.5ml/min to 1.0 ml/min;
(3) measurement and calculation:
A) respectively injecting 20 mul of the test solution, the control solution and the control solution into a liquid chromatograph for the same method determination to obtain a chromatogram of the test solution, a chromatogram of the control solution and a chromatogram of the control solution;
B) respectively calculating the content of each related substance in the chromatogram of the sample solution by using the impurity A as a reference substance and an external standard method with a correction factor;
C) the correction factors for each relevant substance are as follows:
the correction factor for impurity a is 1;
the correction factor for 3- (4' -carbonylvaleric acid) -2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity B) was 1.8; the correction factor for 3-methyl-2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity C) was 1.2;
the correction factor of 3-ethyl-2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity D) is 1.3;
calculating the correction factor of the unknown related substance i according to Ai/At multiplied by 0.5%, wherein Ai is the peak area of the unknown related substance i; at is the major peak area in the control solution chromatogram.
Furthermore, in the octane sodium sulfonate buffer solution, the concentration of the alkane sodium sulfonate is 15 mM-30 mM; the concentration of the phosphate is 15mM to 70 mM.
Preferably, in the sodium octane sulfonate buffer, the concentration of sodium octane sulfonate is 20mM, and the concentration of phosphate is 40 mM.
Further, the phosphate is at least one selected from the group consisting of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and diammonium hydrogen phosphate.
Further, acetonitrile was 15% in the mobile phase.
Further, the detection wavelength is 204 nm-206 nm.
Further, the flow rate was 1.0 ml/min.
The invention has the outstanding effects that:
the detection method of the aminolevulinic acid hydrochloride related substances has good specificity, has high sensitivity for detecting each related substance, can realize complete separation of an impurity peak and a main peak, has more detected substance types, and can identify 4 impurities generated in the degradation process of the aminolevulinic acid hydrochloride, wherein the impurity C and the impurity D are new substances which have not been identified before.
In conclusion, the method for detecting impurities in the production of aminolevulinic acid hydrochloride can greatly improve the quality control level of products, and has great significance for industrial progress.
Drawings
FIG. 1 is a chromatogram of example 1 of the present invention;
FIG. 2 is a chromatogram of example 2 of the present invention;
FIG. 3 is a chromatogram of example 3 of the present invention;
FIG. 4 is a chromatogram of example 4 of the present invention;
FIG. 5 is a chromatogram of example 5 of the present invention;
FIG. 6 is a chromatogram of example 6 of the present invention;
FIG. 7 is a chromatogram of example 7 of the present invention;
FIG. 8 is a chromatogram of example 8 of the present invention;
FIG. 9 is a chromatogram of example 9 of the present invention;
FIG. 10 is a chromatogram of example 10 of the present invention;
FIG. 11 is a chromatogram of example 11 of the present invention;
FIG. 12 is a chromatogram of a comparative example of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Examples 1 to 10
(1) Experimental equipment and reagent
The instrument comprises the following steps: agilent HPLC 1260Method screening system
(2) Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as the packing of chromatographic column, Agilent Zorbax C184.6X 250mm 5 μm. The following Table 1 shows the chromatographic conditions of examples 1 to 10.
Table 1: chromatographic conditions of examples 1 to 10
Note: mM is an abbreviation for concentration unit mmol/L, 1mM ═ 1 × 103 mol/L.
(3) Sample preparation: preparing a sodium octane sulfonate buffer solution and a mobile phase according to the mixture ratio in the table 2, further weighing a proper amount of the aminolevulinic acid hydrochloride raw material medicine, and preparing the solution with the concentration of 10mg/ml by using the mobile phase.
(4) The experimental results are as follows:
chromatograms obtained by performing chromatographic separation on examples 1 to 10 are shown in the attached drawings 1 to 10 in sequence, and the main component is well separated from the impurity A, the impurity B, the impurity C and the impurity D, wherein the impurity C and the impurity D are new substances which have not been identified before.
As shown in table 2 below, the detection method disclosed in the present invention identifies that the 4 impurities generated during the degradation process of aminolevulinic acid hydrochloride are respectively: impurity A: pyrazine 2, 5-dipropionate;
impurity B: 3- (4' -carbonylvaleric acid) -2, 5-dipropionic acid pyrazine;
impurity C: pyrazine 3-methyl-2, 5-dipropionate;
impurity D: pyrazine 3-ethyl-2, 5-dipropionate.
TABLE 24 impurities that can be identified by the detection methods disclosed in the present invention
Example 11
(1) Experimental equipment and reagent
The instrument comprises the following steps: same as examples 1 to 10
(2) Chromatographic conditions are as follows:
a chromatographic column: same as examples 1 to 10
Flow rate: 1.0ml/min
Detection wavelength: 205nm
Column temperature: 30 deg.C
Sample introduction amount: 20 μ L
Mobile phase: sodium octane sulfonate buffer acetonitrile 86:14(v/v) (sodium octane sulfonate buffer was prepared by dissolving sodium octane sulfonate 4.75g (20mM) and ammonium dihydrogen phosphate 7.59g (66mM) in 980ml of water, adjusting the pH to 1.90 with phosphoric acid, and diluting with water to 1000 ml).
(3) Sample preparation: weighing appropriate amount of aminolevulinic acid hydrochloride preparation containing adjuvants and adjuvants degradation impurities (p-hydroxybenzoic acid), and making into 10mg/ml solution with mobile phase.
(4) The experimental results are as follows:
example 11 was chromatographed, and the resulting chromatogram is shown in FIG. 11.
Further, example 11 was tested for DOE robustness by statistical software Minitab 17 (ID: 30034556135249531400), with the DOE parameters designed as follows:
factor (d):
the salt ion pair concentration is 86 mM-90 mM (namely containing 21.5 mM-22.5 mM of octane sodium sulfonate and 64.5 mM-67.5 mM of ammonium dihydrogen phosphate);
the pH value of the buffer solution is 1.85-1.95;
the organic phase proportion is 13.5% -14.5%;
the flow rate is 0.95 ml/min-1.05 ml/min;
the column temperature is between 28 and 32 ℃;
responding: r1 (degree of separation of main peak from impurity D); r2 (degree of separation of impurity E from the main peak); r3 (degree of separation of impurity F from impurity E); a main peak tailing factor; number of principal peak theoretical plates.
The DOE model: five factor two level (3 center points, three repetitions of center point)
DOE robustness test results: referring to table 3, the results show that, within the DOE parameters, R1>1.0, R2>3.0, R3>2.0, Tailing <3.0, and plant >3000, indicating that the method still has good robustness for substance detection related to adjuvant-containing aminolevulinic acid hydrochloride preparations within the above parameters.
Table 3: DOE robustness test results
Comparative example: detection method for United states pharmacopoeia inclusion
(1) Experimental equipment and reagent
The instrument comprises the following steps: waters UPLC
(2) Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as filler of chromatographic column, Waters BEH Shield RP 182.1 × 100mm 1.7 μm
Flow rate: 0.4ml/min
Detection wavelength: 210nm
Sample introduction amount: 5 μ L
Solution A: 2M sulfuric acid to adjust the pH of the water to 2.2
Diluent agent: methanol and solution A (1:3) (v/v)
Elution procedure: see table 4.
Table 4: elution procedure of comparative example
| Time min | Solution A% | Methanol% |
| 0 | 95 | 5 |
| 2 | 95 | 5 |
| 6 | 60 | 40 |
| 8 | 60 | 40 |
| 9 | 95 | 5 |
| 23 | 95 | 5 |
(3) Preparing a sample solution: a proper amount of aminolevulinic acid hydrochloride is weighed and prepared into solutions with the concentration of 40mg/ml and 40 mu g/ml by using a diluent.
(4) Preparing a reference substance solution: respectively weighing appropriate amount of aminolevulinic acid hydrochloride, impurity A and impurity B, and preparing into 0.04mg/ml solution with diluent.
(5) The experimental results are as follows:
calculating the impurity A and the impurity B by respective reference substances according to an external standard method; calculating other single unknown impurities which peak before the impurity A by taking the aminolevulinic acid hydrochloride as a reference substance according to an external standard method; the other single unknown impurities which peak after the related impurity A are calculated by an external standard method by taking the related impurity A as a reference.
The chromatogram obtained is shown in FIG. 12, where the aminolevulinic acid hydrochloride had a peak pattern that shouldered at a concentration of 40mg/ml and peaked at the dead volume position and could not be effectively separated from the solvent peak.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative and that various changes or modifications may be made without departing from the principles and spirit of the invention. The scope of the invention is therefore defined by the appended claims.
Claims (7)
1. A method for detecting related substances of aminolevulinic acid hydrochloride is a reversed-phase high performance liquid chromatography, and is characterized by comprising the following steps:
(1) solution preparation:
A) preparing sodium octane sulfonate buffer solution with the pH value of 2.0 +/-0.2, and further preparing sodium octane sulfonate buffer solution with acetonitrile accounting for 10-20% (v/v) -acetonitrile as a mobile phase;
B) preparing a sample to be tested with aminolevulinic acid hydrochloride as a main component into a test sample solution with the concentration of 10mg/ml by using the mobile phase;
C) placing 1ml of the test solution into a 20ml measuring flask, diluting the test solution to a scale with the mobile phase, shaking up, placing 5ml of the test solution into a 50ml measuring flask, diluting the test solution to a scale with the mobile phase, and shaking up to serve as a control solution;
D) taking 2, 5-dipropionate pyrazine (hereinafter referred to as impurity A) as a reference substance, carrying out ultrasonic treatment for 30 seconds by using 50% acetonitrile aqueous solution, and then adding the mobile phase for dissolving to prepare a reference substance solution with the concentration of 20 mu g/ml;
(2) chromatographic conditions are as follows: the chromatographic column filler is octadecylsilane chemically bonded silica; the detection wavelength is ultraviolet wavelength; the column temperature is 25-30 ℃; the flow rate is 0.5ml/min to 1.0 ml/min;
(3) measurement and calculation:
A) respectively injecting 20 μ l of each of the test solution, the control solution and the control solution into a liquid chromatograph for determination by the same method to obtain a chromatogram of the test solution, a chromatogram of the control solution and a chromatogram of the control solution;
B) respectively calculating the content of each related substance in the chromatogram of the test solution by using the impurity A as a reference substance and an external standard method with a correction factor;
C) the correction factors for each relevant substance are as follows:
the correction factor for impurity a is 1;
the correction factor for 3- (4' -carbonylvaleric acid) -2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity B) was 1.8;
the correction factor for 3-methyl-2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity C) was 1.2;
the correction factor of 3-ethyl-2, 5-dipropionic acid pyrazine (hereinafter referred to as impurity D) is 1.3;
calculating the correction factor of the unknown related substance i according to Ai/At multiplied by 0.5%, wherein Ai is the peak area of the unknown related substance i; at is the major peak area in the control solution chromatogram.
2. The detection method according to claim 1, wherein the concentration of sodium alkane sulfonate in the sodium octane sulfonate buffer solution is 15 mM-30 mM; the concentration of the phosphate is 15 mM-70 mM.
3. The method according to claim 2, wherein the concentration of sodium octane sulfonate is 20mM and the concentration of phosphate is 40mM in the sodium octane sulfonate buffer.
4. The detection method according to claim 2 or 3, wherein the phosphate is at least one selected from the group consisting of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and diammonium hydrogen phosphate.
5. The detection method according to claim 1, wherein the acetonitrile in the mobile phase is 15%.
6. The detection method according to claim 1, wherein the detection wavelength is 204nm to 206 nm.
7. The method of claim 1, wherein the flow rate is 1.0 ml/min.
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