CN114839293B - Quantitative determination method for genotoxic impurities in calcium dobesilate - Google Patents
Quantitative determination method for genotoxic impurities in calcium dobesilate Download PDFInfo
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- CN114839293B CN114839293B CN202210484138.8A CN202210484138A CN114839293B CN 114839293 B CN114839293 B CN 114839293B CN 202210484138 A CN202210484138 A CN 202210484138A CN 114839293 B CN114839293 B CN 114839293B
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- benzoquinone
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- calcium dobesilate
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- sulfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 75
- QGNBTYAQAPLTMX-UHFFFAOYSA-L calcium dobesilate Chemical compound [Ca+2].OC1=CC=C(O)C(S([O-])(=O)=O)=C1.OC1=CC=C(O)C(S([O-])(=O)=O)=C1 QGNBTYAQAPLTMX-UHFFFAOYSA-L 0.000 title claims abstract description 72
- 229960005438 calcium dobesilate Drugs 0.000 title claims abstract description 72
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 7
- 231100000024 genotoxic Toxicity 0.000 title abstract description 12
- 230000001738 genotoxic effect Effects 0.000 title abstract description 12
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims abstract description 169
- 229940005561 1,4-benzoquinone Drugs 0.000 claims abstract description 93
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 90
- QOIXLGYJPBDQSK-UHFFFAOYSA-N 3,6-dioxocyclohexa-1,4-diene-1-sulfonic acid Chemical compound OS(=O)(=O)C1=CC(=O)C=CC1=O QOIXLGYJPBDQSK-UHFFFAOYSA-N 0.000 claims abstract description 68
- 239000000243 solution Substances 0.000 claims abstract description 64
- 238000001212 derivatisation Methods 0.000 claims abstract description 38
- 239000000523 sample Substances 0.000 claims abstract description 36
- 239000012488 sample solution Substances 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 6
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000013558 reference substance Substances 0.000 claims description 26
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 20
- 239000002904 solvent Substances 0.000 claims description 17
- 239000000337 buffer salt Substances 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical group [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 4
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 4
- 238000010812 external standard method Methods 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 4
- 239000006012 monoammonium phosphate Substances 0.000 claims description 4
- 239000012085 test solution Substances 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 239000008351 acetate buffer Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 abstract description 70
- 229960004337 hydroquinone Drugs 0.000 abstract description 35
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 14
- 239000011575 calcium Substances 0.000 abstract description 14
- 229910052791 calcium Inorganic materials 0.000 abstract description 14
- 238000002360 preparation method Methods 0.000 abstract description 11
- 239000002994 raw material Substances 0.000 abstract description 9
- 238000011002 quantification Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000002378 acidificating effect Effects 0.000 abstract 1
- 239000000945 filler Substances 0.000 abstract 1
- 239000000741 silica gel Substances 0.000 abstract 1
- 229910002027 silica gel Inorganic materials 0.000 abstract 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 238000011084 recovery Methods 0.000 description 11
- 239000012483 derivatization solution Substances 0.000 description 10
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 10
- -1 2-sulfo-1, 4-benzoquinone manganese salt Chemical compound 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000012071 phase Substances 0.000 description 8
- 239000012088 reference solution Substances 0.000 description 8
- 238000007865 diluting Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 150000004057 1,4-benzoquinones Chemical class 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000009616 inductively coupled plasma Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 229910052748 manganese Inorganic materials 0.000 description 3
- 239000011572 manganese Substances 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 238000011111 UV-scan method Methods 0.000 description 1
- GOPYZMJAIPBUGX-UHFFFAOYSA-N [O-2].[O-2].[Mn+4] Chemical group [O-2].[O-2].[Mn+4] GOPYZMJAIPBUGX-UHFFFAOYSA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000010405 reoxidation reaction Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- DGQOCLATAPFASR-UHFFFAOYSA-N tetrahydroxy-1,4-benzoquinone Chemical compound OC1=C(O)C(=O)C(O)=C(O)C1=O DGQOCLATAPFASR-UHFFFAOYSA-N 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a quantitative determination method of genotoxic impurities in calcium dobesilate, which comprises the steps of preparing a derivatization reagent by adopting an acidic acetonitrile solution, ensuring that calcium dobesilate, 2-sulfo-1, 4-benzoquinone, hydroquinone and 1, 4-benzoquinone can exist stably, ensuring that benzoquinone impurities can react completely with the derivatization reagent, and fully dissolving samples and impurities by adopting an acetonitrile aqueous solution. Aiming at the derivatized sample solution, the separation and quantification of the p-benzoquinone impurities are realized by using a chromatographic column with end-capped octadecylsilane bonded silica gel as a filler and using a mixed solution of buffer solution and acetonitrile with pH of 4.5+/-0.5 as a mobile phase. The method is convenient to operate, has good specificity, high sensitivity, good precision and repeatability in detection of the benzoquinone impurities in the sample, can accurately quantitatively analyze the two benzoquinone impurities in the calcium p-hydroxybenzosulfonate, and ensures the safety and quality controllability of the raw material and the preparation of the calcium p-hydroxybenzosulfonate.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for separating and measuring impurities in calcium dobesilate by using pre-column derivatization and adopting a high performance liquid chromatography.
Background
Calcium dobesilate is used as a capillary protective medicine for long-term clinical application, has a structure containing a p-diphenol hydroxyl group structure, is easily oxidized once being contacted with water or oxygen in air to generate 2-sulfo-1, 4-benzoquinone impurities, even if a sample is placed in a temperature-control sample injector at 4 ℃, the 2-sulfo-1, 4-benzoquinone in the solution is still rapidly increased, so that the result of the sample exceeds the limit, and the fact that the overrun factor is unqualified by the sample itself or is caused by the preparation process cannot be judged. The impurity has a genotoxic structure and needs to be controlled, and the impurity is a novel compound which is not reported at home and abroad and is self-made by the synthesis department of the company, and the specific preparation process is as follows:
Concentrated sulfuric acid (29.4 g) is slowly added into cyclohexane (100 g) solution of hydroquinone (20 g) under the protection of nitrogen and mechanical stirring at room temperature (20-25 ℃), the mixture is heated to 70-80 ℃ for reaction for 0.5 hours after the mixture is completely added, the mixture is cooled to room temperature, cyclohexane is separated, acetonitrile (200 mL) is added for dissolving solid, after the solid is dissolved, manganese dioxide (80 g) is added at the internal temperature of less than 30 ℃, and the mixture is stirred at room temperature for reaction for 12 hours. After the reaction, the reaction solution was poured into ice countless ethanol (400 mL,2 v/v), the manganese dioxide residue was removed by filtration, the obtained filtrate was added to ice water (800 mL,4v/v, based on the volume of the reaction solution), and the obtained filtrate was filtered with a 0.45 μm filter membrane, and the obtained filtrate was lyophilized to obtain 2-sulfonic acid-1, 4-benzoquinone yellow powdery solid. The chemical structure of the material is determined by nuclear magnetic resonance hydrogen spectrum, high-resolution mass spectrum and inductively coupled plasma measurement, and detailed maps are shown in figures 1-3.
In addition, the starting material hydroquinone of calcium dobesilate, like calcium dobesilate in structure, is itself easily oxidized to produce 1, 4-benzoquinone, and the component also has a genotoxic structure to be controlled. By inquiring the database The Carcinogenic Potency Database, the TD 50 data of 1, 4-benzoquinone is 5.07mg/Kg/d; the impurity limit is calculated according to ICH M7 guidelines, the TTC (toxicology control threshold) of 1, 4-benzoquinone is 5.07 mug/d, the daily maximum dose of the product is 1.5g/d, and therefore, the limit of 1, 4-benzoquinone is formulated to be 3.38ppm; 2-sulfo-1, 4-benzoquinone is similar in structure to 1, 4-benzoquinone and has the same warning structure, so that the limit is 3.38ppm.
Therefore, to ensure the safety of the drug, the above two impurities must be controlled. However, two genotoxic impurities of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone belong to trace components and are not easy to detect; in addition, calcium dobesilate and a trace amount of impurity hydroquinone are easy to oxidize due to the nature of self groups, and 2-sulfonic acid group-1, 4-benzoquinone and 1, 4-benzoquinone have strong oxidability, so that four components cannot coexist in a solution.
Firstly, for quantification of 1, 4-benzoquinone, as the oxidation reduction reaction of 1, 4-benzoquinone and calcium dobesilate cannot coexist in a solution state, after 1, 4-benzoquinone is quantitatively added into a raw material sample solution, the 1, 4-benzoquinone does not show a peak in a system applicability solution, and 2-sulfonic acid group-1, 4-benzoquinone and hydroquinone are obviously increased (as shown in figure 4), so that the 1, 4-benzoquinone rapidly and completely reacts with calcium dobesilate in the solution state to generate 2-sulfonic acid group-1, 4-benzoquinone and hydroquinone, thus not only the quantification of 1, 4-benzoquinone can not be realized, but also the quantification of 2-sulfonic acid group-1, 4-benzoquinone can be interfered.
In addition, for quantification of 2-sulfo-1, 4-benzoquinone, the presence of 1, 4-benzoquinone, the 2-sulfo-1, 4-benzoquinone measured in calcium dobesilate solution, cannot be used to determine whether the component source is calcium dobesilate itself or a reaction product in the solution preparation process. Meanwhile, after the calcium dobesilate solution is prepared into a solution, the 2-sulfo-1, 4-benzoquinone is continuously increased, the impurities are still increased after the solution is continuously placed, which indicates that the calcium dobesilate as a main component is unstable in a solution state and is continuously oxidized to generate the 2-sulfo-1, 4-benzoquinone, and the difficulty of quantifying the impurities is further increased.
In addition, the four structural formulas of calcium dobesilate, 2-sulfo-1, 4-benzoquinone, hydroquinone and 1, 4-benzoquinone are similar, the polarities are large, the liquid phase in reverse phase is kept weak, the peak-out time is short, meanwhile, the sample liquid is required to be prepared into high concentration due to the low impurity limit, the quantitative requirement can be met, the peak width of the calcium dobesilate peak in the liquid chromatogram is large, the impurities to be detected are contained in the main peak of the calcium dobesilate, and the impurities to be detected are difficult to separate (as shown in fig. 5).
In view of the above, there is a need to develop a method capable of effectively separating and accurately detecting four components of calcium dobesilate, 2-sulfo-1, 4-benzoquinone, hydroquinone and 1, 4-benzoquinone.
Disclosure of Invention
The invention aims to provide a quantitative determination method for impurities in calcium dobesilate, which can effectively separate and accurately detect two components of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in the calcium dobesilate so as to ensure the safety of raw materials and preparations of the calcium dobesilate.
The invention relates to a quantitative determination method of impurities in calcium dobesilate, wherein the impurities are 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone,
Separating and measuring by liquid chromatography, derivatizing the sample phase by a derivatization reagent, and adding a solvent to prepare a sample solution; the solvent is an acetonitrile aqueous solution with the concentration of 10-80%, and the derivatization reagent is prepared by the following method: taking 2, 4-dinitrophenylhydrazine, adding 0.4-0.6 ml of phosphoric acid solution with the volume fraction of 80-90%, and then adding acetonitrile, and uniformly mixing to obtain a solution with the concentration of 0.1-10 mg/ml of 2, 4-dinitrophenylhydrazine, namely the derivatization reagent.
In a specific example, the quantitative determination method of impurities in calcium dobesilate disclosed by the invention is characterized in that the impurities are 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone, and the separation determination is carried out by adopting liquid chromatography under the following chromatographic conditions:
chromatographic column: a capped octadecylsilane chemically bonded silica chromatographic column;
The detection wavelength is as follows: 300 nm-400 nm;
Flow rate: 0.8-1.2 ml/min;
Column temperature: 0-40 ℃;
Mobile phase: taking a mixed solution of buffer salt solution-acetonitrile with pH of 4.5+/-0.5 as a mobile phase, wherein the volume ratio of the buffer salt solution to the acetonitrile is 80:20-35:65;
2-sulfo-1, 4-benzoquinone with 1, 4-benzoquinone control solution: preparing a mixed solution of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone by adding a solvent, adding 1-10 ml of derivatization reagent, and adding the solvent to prepare a reference substance solution of 0.1-0.2 mug/ml after derivatization for 1-24 hours;
Test solution: adding 1-10 ml of derivatization reagent into a sample to be tested, derivatizing for 1-24 h, and adding a solvent to prepare 0.03-0.05 g/ml of sample solution;
The solvent is an acetonitrile aqueous solution with the concentration of 10-80%, and the derivatization reagent is prepared by the following method: taking 2, 4-dinitrophenylhydrazine, adding 0.4-0.6 ml of phosphoric acid solution with the volume fraction of 80-90%, and then adding acetonitrile, and uniformly mixing to obtain a solution with the concentration of 0.1-10 mg/ml of 2, 4-dinitrophenylhydrazine, namely the derivatization reagent.
The column according to the invention may be chosen from the end-capped octadecylsilane chemically bonded silica columns commonly used in the art, such as the trade mark Phenomenex Gemini C 18 column (specification 4.6 mm. Times.250 mm,5 μm).
The buffer salt solution with the pH of 4.5+/-0.5 in the mobile phase is phosphate buffer solution, acetate buffer solution or triethylamine buffer solution, and is preferably monoammonium phosphate buffer solution. These buffer salt solutions may be formulated according to conventional methods in the art or purchased directly.
The molar amount of 2, 4-dinitrophenylhydrazine according to the invention ensures that it can be more than 2 times the molar total amount of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone, preferably 2 to 5 times the molar total amount of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone.
In some embodiments, the present invention is derivatized for 5 to 6 hours, more preferably 4.5 to 5.5 hours;
In some embodiments, the volume ratio of mobile phase buffer salt solution to acetonitrile described in the present invention is (45-55): (47.5-52.5), preferably 50:50.
In some embodiments, the detection wavelength described herein is 378 nm.+ -. 5nm.
In some embodiments, the flow rates described herein are in the range of 0.8 to 1.2ml/min.
In some embodiments, the column temperature described herein is 25 ℃ ± 5 ℃.
In some embodiments, the solvent described herein is a 20% to 35% acetonitrile in water.
The method of the invention further comprises the steps of taking 5-20 mu l of each of the sample solution and the reference solution, respectively injecting into a high performance liquid chromatograph, recording the chromatograms, and calculating the amounts of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in the sample according to an external standard method.
According to the invention, through pre-column derivatization treatment, on one hand, impurities 2-sulfo-1, 4-benzoquinone, 1, 4-benzoquinone and 2, 4-dinitrophenylhydrazine can be reacted (as shown in the following formula), and the reaction of 1, 4-benzoquinone and 2, 4-dinitrophenylhydrazine no longer reacts with calcium dobesilate to interfere the quantification of 2-sulfo-1, 4-benzoquinone; meanwhile, different phenylhydrazone compounds are generated from the 2-sulfo-1, 4-benzoquinone and the 2, 4-dinitrophenylhydrazine in the sample, so that simultaneous quantitative detection of the target components 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone can be realized. On the other hand, the main component calcium dobesilate and the interference component hydroquinone in the derivatization treatment process not only participate in the reaction, but also can inhibit the self-oxidation of the calcium dobesilate and the hydroquinone, maintain the stability of the calcium dobesilate and the hydroquinone, and ensure that the calcium dobesilate and the hydroquinone do not generate oxidation reaction in the solution, thereby realizing the quantitative detection of the calcium dobesilate and the hydroquinone. Through derivatization treatment, the polarity of phenylhydrazone compounds obtained by the reaction of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone is increased, the phenylhydrazone compounds are maintained and enhanced in a reverse phase system, and calcium dobesilate and hydroquinone do not participate in the reaction, and the polarity is not changed, so that the four components can be effectively separated.
The method can successfully quantify two benzoquinone impurities, calcium dobesilate and hydroquinone have larger polarity because the two benzoquinone impurities do not participate in derivatization reaction, and can realize effective separation after the peaks are formed at the peak positions of the solvent.
The invention has the beneficial effects that:
(1) According to the invention, impurities 2-sulfo-1, 4-benzoquinone, 1, 4-benzoquinone and 2, 4-dinitrophenylhydrazine react to form different phenylhydrazones with increased polarities, meanwhile, oxidation of calcium oxybenzene sulfonate and hydroquinone is inhibited, calcium oxybenzene sulfonate, 2-sulfo-1, 4-benzoquinone, hydroquinone and 1, 4-benzoquinone in the solution can be kept stable, the polarities are different, and separation of four components can be realized.
(2) The derivatization reaction of the invention can be completed in 5 hours at room temperature, the reaction condition is mild, and the time is controllable.
(3) According to the invention, after derivatization treatment, acetonitrile-water is used for dissolving the sample solution, so that the sample can be completely dissolved, the complete extraction of target impurities is ensured, the reoxidation of calcium dobesilate in the sample is avoided, and the stability of the solution is ensured.
(4) The method is convenient to operate, has good specificity, high sensitivity, precision and accuracy after verification, and can accurately quantitatively analyze 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone which are genotoxic impurities of calcium p-hydroxybenzosulfonate, thereby ensuring the quality controllability of calcium p-hydroxybenzosulfonate raw materials and preparations.
Drawings
FIG. 1 is a diagram showing the nuclear magnetic resonance hydrogen spectrum (1H-NMR) of 2-sulfonic acid-1, 4-benzoquinone manganese salt as a genotoxic impurity.
FIG. 2 is a High Resolution Mass Spectrum (HRMS) of the genotoxic impurity 2-sulfonic acid-1, 4-benzoquinone manganese salt.
FIG. 3 is an inductively coupled plasma direct reading (ICP) diagram of the genotoxic impurity 2-sulfonic acid-1, 4-benzoquinone manganese salt.
FIG. 4 is a graph showing the comparison of the localization of calcium dobesilate test solutions and genotoxic impurities.
FIG. 5 shows the peak pattern of benzoquinone impurities and calcium dobesilate.
FIG. 6 is a graph showing the solution of a control for measuring impurities of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in a calcium dobesilate raw material of example 1.
FIG. 7 is a blank solvent pattern for determining impurities of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in calcium dobesilate starting material of example 1.
FIG. 8 is a graph showing a UV full-wavelength scan contrast of calcium dobesilate, hydroquinone, 2-sulfo-1, 4-benzoquinone derivatives, and 1, 4-benzoquinone derivatives of example 1.
FIG. 9 is a graph showing the comparison of the response of calcium dobesilate at various wavelengths of detection in the calcium dobesilate starting material of example 1.
FIG. 10 is a graph showing the response of hydroquinone in the hydroquinone positioning solution of example 1 at different detection wavelengths.
FIG. 11 is a chart showing the localization of calcium dobesilate, hydroquinone, 2-sulfo-1, 4-benzoquinone derivatives and 1, 4-benzoquinone derivatives of example 1.
FIG. 12 is a chart showing the sample solution for measuring 1 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone impurities in calcium dobesilate as a raw material of example 1.
FIG. 13 is a chart showing the measurement of a sample solution of 1 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone impurity in a calcium dobesilate starting material of example 1.
FIG. 14 is a graph of a blank adjuvant for determining impurities of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in calcium dobesilate tablets of example 2.
FIG. 15 is a graph showing the impurity measurement test sample solution of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in calcium dobesilate tablet of example 2.
Detailed Description
The examples are provided for further illustration only and are not intended to limit the invention, the technical scope of which is defined by the claims. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The derivatizing reagent 2, 4-dinitrophenylhydrazine used in the examples below was HPLC grade; the calcium dobesilate raw material manufacturer is Hubei Guangchen pharmaceutical industry Co., ltd; the 1, 4-benzoquinone reference substance is a USP reference substance, and the 2-sulfo-1, 4-benzoquinone manganese salt reference substance is self-made by the company.
The apparatus used in the following examples: thermo U-3000 high performance liquid chromatograph, chromeleon chromatography workstation (Themo Co.), XSE105DU analytical electronic balance (METTLER TOLEDO Switzerland).
Example 1 validation of the method for determining the benzoquinone impurity 1, 4-benzoquinone and 2-sulfo-1, 4-benzoquinone in calcium dobesilate
Chromatographic column: phenomenex Gemini C 18 chromatography column (specification 4.6 mm. Times.250 mm,5 μm);
mobile phase: 20mmol/L monoammonium phosphate solution-acetonitrile (50:50);
Detection wavelength: 378nm;
Flow rate: 1.0mL/min;
Column temperature: 25 ℃.
1.1 Specificity test
(1) Investigation of interference of blank solvent
Because the concentration of the derivatization reagent is higher, impurities in the derivatization reagent can generate peaks at the front and rear positions of the component to be detected, and the detection of the sample is interfered. It is therefore provided that the degree of separation between the impurities of the target component and the derivatizing agent should be greater than 2.0.
Taking a proper amount of each of the 1, 4-benzoquinone reference substance and the 2-sulfo-1, 4-benzoquinone manganese salt reference substance, adding 50% acetonitrile for dissolving, and preparing a solution containing 0.14mg of each of the 1, 4-benzoquinone and the 2-sulfo-1, 4-benzoquinone in each 1ml, and taking the solution as a reference substance stock solution.
About 0.10g of 2, 4-dinitrophenylhydrazine and 85% (volume fraction) of phosphoric acid were taken and diluted to a scale with acetonitrile in a 100ml measuring flask to obtain a derivatization solution.
1Ml of reference stock solution is precisely measured respectively, the solution is placed in a 10ml measuring flask, 6ml of derivatization solution is added, water is added for dilution to a scale, shaking is carried out, and the reaction is carried out for 5 hours at room temperature. Accurately measuring 20 μl, and analyzing according to the chromatographic conditions, wherein the result shows that: the 2, 4-dinitrophenylhydrazine, the 2-sulfo-1, 4-benzoquinone-derived product, and the 1, 4-benzoquinone-derived product peak in this order, wherein the degree of separation between the 2-sulfo-1, 4-benzoquinone-derived product and the fore-and-aft impurities is 4.4 and the degree of separation between the 4.2,1,4-benzoquinone-derived product and the fore-and-aft impurities is 4.0 and 4.8, giving a baseline separation (see FIGS. 6 and 7).
The results show that the derivatization reagent has no interference to the detection of the sample, and the two components to be detected can be completely separated in the reversed phase chromatography due to the difference of the polarities of the derivatives of the 1, 4-benzoquinone and the 2-sulfo-1, 4-benzoquinone.
(2) Investigation of interference of known components
In addition, in order to examine the interference of calcium oxybenzene sulfonate and hydroquinone on determination of genotoxic impurities, calcium oxybenzene sulfonate and hydroquinone need to be injected under the same chromatographic condition, and whether peaks appear near two components to be detected or not is observed. However, as is clear from the UV scan of calcium oxybenzene sulfonate, hydroquinone, 2-sulfo-1, 4-benzoquinone derivative and 1, 4-benzoquinone derivative (see FIG. 8), the difference in UV absorption of the four components is large, calcium oxybenzene sulfonate and hydroquinone have maximum absorption at 300nm, and 1, 4-benzoquinone derivative and 2-sulfo-1, 4-benzoquinone derivative have maximum absorption at 378nm, so that when 378nm is selected as the detection wavelength, the chromatograms of calcium oxybenzene sulfonate and hydroquinone hardly respond, and the peak positions of calcium oxybenzene sulfonate and hydroquinone cannot be observed (see FIGS. 9 to 10).
Comparing the chromatograms of the detection wavelengths with the maximum ultraviolet response of the sample solution, the hydroquinone positioning solution and the reference solution (see figure 11), wherein the detection wavelengths of the three solutions are respectively 300nm, 300nm and 378nm, the preparation method of the sample solution is the same as 1.5, the hydroquinone positioning solution is 0.1mg/ml, the preparation method of the reference solution is the same as 1.5, and as a result, calcium oxybenzene sulfonate and impurity hydroquinone are both found out at the positions before the peak of the derivatization reagent 2, 4-dinitrophenylhydrazine component, and the impurity 1, 4-benzoquinone reference to be detected and the 2-sulfo-1, 4-benzoquinone 2, 4-dinitrophenylhydrazine component are found out at the positions after the peak of the 2, 4-dinitrophenylhydrazine component, so that the main component and hydroquinone in the sample can not interfere with the detection of the impurity to be detected;
In summary, it is known that since 1, 4-benzoquinone and 2-sulfo-1, 4-benzoquinone react with derivatizing agents to form stable and less polar derivatizing compounds, calcium oxybenzene sulfonate and hydroquinone do not participate in derivatization reaction, and the polarity is unchanged, thereby eliminating interference of known components in a sample on detection.
1.2 Linear and Range test
Precisely weighing 1, 4-benzoquinone reference substance and 2-sulfo-1, 4-benzoquinone manganese salt reference substance, placing into a measuring flask, and adding 50% acetonitrile for dissolving and diluting to obtain a series of mixed standard solutions with concentration. 1ml of each of the above-mentioned materials is precisely measured, placed in a 10ml measuring flask, 6ml of the derivatization solution is added, 25% acetonitrile is added to dilute to a scale, and the mixture is shaken uniformly to react for 5 hours at room temperature. 20. Mu.l of each sample was precisely measured and analyzed under the above-mentioned chromatographic conditions, the quantitative limit concentration of each component was used as the lowest concentration, 0.0004% of the concentration of the sample solution (preparation method same as 1.5) was used as the highest concentration of each component, the concentration was used as the abscissa, and the peak area was used as the ordinate, and the results were shown in Table 1. The results show that the correlation coefficient (R) of the regression line of each component standard curve is more than 0.990, and meets the requirements.
Table 1 results of linear range investigation
| Component (A) | Linear equation | Range of |
| 1, 4-Benzoquinone | A= 3.3349C-0.0234 a correlation coefficient (R) of 0.9998 | 0.0274μg/ml～0.1646μg/ml |
| 2-Sulfo-1, 4-benzoquinone | A= 1.1451C-0.0019 correlation coefficient (R) is 0.9993 | 0.0348μg/ml～0.1669μg/ml |
1.3 Limit of detection and limit of quantification test
Taking proper amounts of a1, 4-benzoquinone reference substance and a 2-sulfo-1, 4-benzoquinone manganese salt reference substance, gradually and quantitatively diluting with a 50% acetonitrile solution, precisely measuring 1ml, placing into a 10ml measuring flask, adding 6ml of a derivatization solution, reacting for 5 hours at room temperature, diluting to a scale with 25% acetonitrile, shaking uniformly, carrying out sample injection analysis according to the chromatographic conditions, taking the corresponding concentration of each impurity with a signal-to-noise ratio of about 10:1 as a quantitative limit concentration, taking the corresponding concentration with a signal-to-noise ratio of about 3:1 as a detection limit concentration, and respectively obtaining the following quantitative limits of the 1, 4-benzoquinone and the sulfo-1, 4-benzoquinone: 0.024. Mu.g/ml, 0.031. Mu.g/ml, detection limits of 0.007. Mu.g/ml and 0.010. Mu.g/ml, respectively, far below the impurity limit concentration of 0.135. Mu.g/ml.
1.4 Solution stability test
The sample solution to be tested (the preparation method is the same as 1.5) and the reference solution (the preparation method is the same as 1.5), 20 mul of the sample solution is precisely measured after the sample solution is placed for 0h, 2h, 4h, 6h and 8h respectively, and sample injection analysis is carried out according to the chromatographic conditions, and the result shows that the peak area does not obviously change when the reference solution is placed for 8h with the sample solution, the ratio is in the range of 0.8-1.2 when the reference solution is compared with the sample solution, and the reference solution is stable when the reference solution is placed for 8h with the sample solution. The result can show that after the derivatization reagent is added, the reference substance and the 1, 4-benzoquinone reference substance and the 2-sulfo-1, 4-benzoquinone in the test sample solution are both stably present in the solution in the form of derivatives; particularly, after the derivatization reagent is added into the calcium dobesilate in the test solution, the main component is inhibited from being oxidized, the 2-sulfo-1, 4-benzoquinone impurity is not increased any more, and each component can exist independently and stably in the solution.
1.5 Accuracy test
Taking a proper amount of each of the 1, 4-benzoquinone reference substance and the 2-sulfo-1, 4-benzoquinone manganese salt reference substance, adding 50% acetonitrile solution for dissolving and quantitatively diluting to prepare a solution containing about 1.4 mug of each 1, 4-benzoquinone and 2-sulfo-1, 4-benzoquinone in each 1ml, and taking the solution as a reference substance stock solution. Taking about 400mg of a sample (batch number: 20190701) and 12 parts, precisely weighing, placing in a 10ml measuring flask, respectively adding 6ml of a derivatization solution, respectively precisely adding 0ml, 0.8ml, 1.0ml and 1.2ml (3 parts each) of the stock solution, reacting for 5 hours at room temperature, adding water, dissolving and diluting to a scale, and shaking uniformly to obtain a sample solution; and precisely measuring 1ml of the reference substance stock solution respectively, adding 6ml of the derivatization solution into a 10ml measuring flask, reacting for 5 hours at room temperature, adding water to dilute to a scale, and shaking uniformly to obtain the reference substance solution. Precisely measuring 20 μl of each of the control solution and the sample solution, analyzing by sample injection under the above chromatographic conditions, and calculating the recovery rates of 1, 4-benzoquinone and 2-sulfo-1, 4-benzoquinone in the sample solution with low, medium and high impurity concentration levels, wherein the average recovery rates of the low, medium and high concentration levels of 1, 4-benzoquinone are respectively: 107.7%, 106.0%, 103.4%, average recovery (n=9) 105.7%, RSD (n=9) 2.0%; the average recovery rate of the low, medium and high concentration levels of the 2-sulfo-1, 4-benzoquinone is respectively as follows: 99.0%, 94.2%, 92.1%, average recovery (n=9) of 95.1%, RSD (n=9) of 6.0%. The results show that: 1, 4-benzoquinone and 2-sulfo-1, 4-benzoquinone can exist in a test sample solution independently and stably, and can still be detected accurately after being quantitatively added into the test sample, as shown in figure 12, the method has good accuracy, and the specific analysis is as follows:
(1) After the addition of the derivatizing agent, the 1, 4-benzoquinone no longer reacts with the calcium dobesilate, but a stable 1, 4-benzoquinone derivative is formed; therefore, after the 1, 4-benzoquinone is quantitatively added into the sample solution, the impurity can still be detected, and the recovery rate meets the requirement.
(2) After the derivatization reagent is added, the main component in the sample solution can be inhibited from oxidizing itself and reacting with 1, 4-benzoquinone, and meanwhile, 2-sulfo-1, 4-benzoquinone reacts with the derivatization reagent to generate stable 2-sulfo-1, 4-benzoquinone derivative, so that after the sample solution is added, the 2-sulfo-1, 4-benzoquinone is quantitatively added, and the impurity recovery rate meets the requirement.
1.6 Precision test
The results of the recovery of 1, 4-benzoquinone by another tester, using a different instrument and a different chromatographic column, under the accuracy term, combined with the results under the accuracy term, were found to have a RSD (n=18) of 4.2; recovery of 2-sulfo-1, 4-benzoquinone resulted in RSD (n=18) of 9.4. The method has good precision.
1.7 Durability test
The change of derivatization time + -10%, concentration change of derivatization reagent + -10%, change of detection wavelength + -5 nm, change of column temperature + -5 ℃, change of flow velocity relative value + -20%, change of amount of monoammonium phosphate + -10%, change of amount of acetonitrile + -5% and change of instrument chromatographic behavior when measured by using two different batch number chromatographic columns are examined respectively (reference substances are added into the test sample solution according to a limited amount standard, and recovery rates of 1, 4-benzoquinone and 2-sulfonic-1, 4-benzoquinone are examined). The results show that under each condition, the recovery rates of the 1, 4-benzoquinone and the 2-sulfonic acid group-1, 4-benzoquinone are all in the range of 80-120%. The result shows that the method has good durability.
1.8 Sample measurement
Taking proper amounts of 2-sulfo-1, 4-benzoquinone manganese salt reference substance and 1, 4-benzoquinone reference substance, adding 50% acetonitrile to dissolve, preparing 1.4 mug mixed solution containing 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in each 1ml, precisely weighing 1ml, placing into a 10ml measuring flask, adding 6ml of derivatization solution, adding water to dilute to scale, shaking, and reacting for 5 hours at room temperature to obtain reference substance solution. Taking a proper amount of a sample of calcium dobesilate raw material, placing the sample into a 10ml measuring flask, adding 6ml of a derivatization solution, reacting for 5 hours at room temperature, adding water for dissolving and diluting to a scale, and shaking uniformly to obtain a sample solution. 20. Mu.l of each sample was measured precisely and analyzed by the above-mentioned chromatographic conditions. The amounts of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in each sample were calculated according to the external standard method. As a result, none of the 1, 4-benzoquinone in the three batches 20190701, 20190702 and 20191101 was detected, and the content of 2-sulfo-1, 4-benzoquinone was less than 3.38ppm, which was in accordance with the regulations. See fig. 13.
Because the 1, 4-benzoquinone and the 2-sulfonic acid group-1, 4-benzoquinone belong to trace impurities, when the hydroquinone and the calcium dobesilate are oxidized in a trace amount, the test result of the 1, 4-benzoquinone and the 2-sulfonic acid group-1, 4-benzoquinone in the sample is out of limit, and the sample is judged to be unqualified. Therefore, it is necessary to ensure that calcium dobesilate, hydroquinone and calcium dobesilate exist stably in the solution, and according to the solution stability in example 1, the detected amounts of 1, 4-benzoquinone and 2-sulfonic acid-1, 4-benzoquinone in the raw material sample solution do not change significantly with the prolonged period of time of the solution, which indicates that the hydroquinone and calcium dobesilate remain stable and are not oxidized after the sample is derivatized.
Example 2 determination of the content of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in calcium dobesilate tablets according to the invention.
Taking proper amounts of 2-sulfo-1, 4-benzoquinone manganese salt reference substance and 1, 4-benzoquinone reference substance, adding 50% acetonitrile to dissolve, preparing 1.4 mug mixed solution containing 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in each 1ml, precisely weighing 1ml, placing into a 10ml measuring flask, adding 6ml of derivatization solution, adding water to dilute to scale, shaking, and reacting for 5 hours at room temperature to obtain reference substance solution. Taking a proper amount of a sample of calcium dobesilate tablet, placing the sample into a 10ml measuring flask, adding 6ml of a derivatization solution, reacting for 5 hours at room temperature, adding water for dissolving and diluting to a scale, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution. And taking a proper amount of blank auxiliary materials of the calcium dobesilate tablet, and preparing the blank auxiliary materials by the same method to obtain a blank auxiliary material solution. 20. Mu.l of each sample was measured precisely and analyzed by the above-mentioned chromatographic conditions. The amounts of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in each sample were calculated according to the external standard method. As a result, the blank auxiliary materials have no interference to measurement, and the 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in three batches of samples 191101S, 191102S and 191103S are not detected and meet the regulations. See fig. 14 and 15.
Claims (15)
1. The quantitative determination method of impurities in calcium dobesilate is characterized in that the impurities are 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone, liquid chromatography is adopted for separation determination, a sample is derivatized by a derivatization reagent, and then a solvent is added to prepare a sample solution; the solvent is an acetonitrile aqueous solution with the concentration of 10-80%, and the derivatization reagent is prepared by the following method: taking 2, 4-dinitrophenylhydrazine, adding 0.4-0.6 ml of phosphoric acid solution with the volume fraction of 80-90%, adding acetonitrile, and uniformly mixing to obtain a solution with the concentration of 0.1-10 mg/ml of 2, 4-dinitrophenylhydrazine, namely a derivatization reagent, wherein the liquid chromatography conditions are as follows:
chromatographic column: a capped octadecylsilane chemically bonded silica chromatographic column;
The detection wavelength is as follows: 300 nm-400 nm;
Flow rate: 0.8-1.2 ml/min;
Column temperature: 0-40 ℃;
mobile phase: taking a mixed solution of buffer salt solution-acetonitrile with pH of 4.5+/-0.5 as a mobile phase, wherein the volume ratio of the buffer salt solution to the acetonitrile is (80:20) - (35:65);
2-sulfo-1, 4-benzoquinone with 1, 4-benzoquinone control solution: preparing a mixed solution of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone by adding a solvent, adding 1-10 ml of a derivatization reagent, derivatizing for 1-24 h, and adding the solvent to prepare a reference substance solution of 0.1-0.2 mug/ml;
test solution: taking a sample, adding 1-10 ml of derivatization reagent for derivatization for 1-24 hours, and adding a solvent to prepare 0.03-0.05 g/ml of sample solution;
the solvent is an acetonitrile aqueous solution with the concentration of 10-80%; the derivatization reagent is prepared by the following method: taking 2, 4-dinitrophenylhydrazine, adding 0.4-0.6 ml of phosphoric acid solution with the volume fraction of 80-90%, and then adding acetonitrile, and uniformly mixing to obtain a solution with the concentration of 0.1-10 mg/ml of 2, 4-dinitrophenylhydrazine, namely the derivatization reagent.
2. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the solvent is a 20% -35% acetonitrile aqueous solution.
3. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the chromatographic column is Phenomenex Gemini C 18 chromatographic columns with a specification of 4.6mm×250mm,5 μm.
4. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the buffer salt solution with a ph of 4.5±0.5 in the mobile phase is a phosphate buffer solution, an acetate buffer solution or a triethylamine buffer solution.
5. The method for quantitatively determining impurities in calcium dobesilate according to claim 3, wherein the buffer salt solution with pH of 4.5+ -0.5 in the mobile phase is monoammonium phosphate buffer solution.
6. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the molar amount of 2, 4-dinitrophenylhydrazine is more than 2 times the molar total amount of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone.
7. The method for quantitatively determining impurities in calcium dobesilate according to claim 6, wherein the molar amount of 2, 4-dinitrophenylhydrazine is 2 to 5 times the molar total amount of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone.
8. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the derivatization is performed for 5 to 6 hours.
9. The method for quantitatively determining impurities in calcium dobesilate according to claim 8, wherein the derivatization is performed for 4.5 to 5.5 hours.
10. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the volume ratio of the mobile phase buffer salt solution to acetonitrile is (45-55): (47.5-52.5).
11. The method for quantitatively determining impurities in calcium dobesilate according to claim 10, wherein the volume ratio of the mobile phase buffer salt solution to acetonitrile is 50:50.
12. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the detection wavelength is 378nm + -5 nm.
13. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the flow rate is 0.8-1.2 ml/min.
14. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein the column temperature is 25 ℃ ± 5 ℃.
15. The method for quantitatively determining impurities in calcium dobesilate according to claim 1, wherein 5 μl to 20 μl of each of the sample solution and the control solution is taken, the high performance liquid chromatograph is injected, the chromatogram is recorded, and the amounts of 2-sulfo-1, 4-benzoquinone and 1, 4-benzoquinone in the sample are calculated according to an external standard method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1748028A1 (en) * | 1990-05-21 | 1992-07-15 | Киевский Государственный Университет Им.Т.Г.Шевченко | Method of determination of 1,2-naphthaquinone |
| CN112129846A (en) * | 2020-08-14 | 2020-12-25 | 广东人人康药业有限公司 | High performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1748028A1 (en) * | 1990-05-21 | 1992-07-15 | Киевский Государственный Университет Им.Т.Г.Шевченко | Method of determination of 1,2-naphthaquinone |
| CN112129846A (en) * | 2020-08-14 | 2020-12-25 | 广东人人康药业有限公司 | High performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone and application thereof |
| CN112444575A (en) * | 2020-08-14 | 2021-03-05 | 广东人人康药业有限公司 | High performance liquid chromatography for efficiently separating and detecting related substances in hydroquinone and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| HPLC-UV detection for analysis of p-benzoquinone dioxime and p-nitrosophenol, and chromatographic fingerprint applied in quality control of industrial p-benzoquinone dioxime;Lian, HZ 等;JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES;20060131;第29卷(第4期);全文 * |
| RP-HPLC法测定羟苯磺酸钙的有关物质;安宁;吴建敏;张启明;;中国药品标准;20100428(第02期);全文 * |
| RP-HPLC测定羟苯磺酸钙含量及有关物质;于梦;刘文乔;高瑞芳;王忠元;赵怀清;;中国实验方剂学杂志;20130905(第17期);全文 * |
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