CN112540135A - Method for separating and detecting related substances in naphthalene-sensitized vitamin eye drops - Google Patents
Method for separating and detecting related substances in naphthalene-sensitized vitamin eye drops Download PDFInfo
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- 239000000126 substance Substances 0.000 title claims abstract description 73
- 239000003889 eye drop Substances 0.000 title claims abstract description 61
- 229940012356 eye drops Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000011782 vitamin Substances 0.000 title claims abstract description 39
- 229940088594 vitamin Drugs 0.000 title claims abstract description 39
- 229930003231 vitamin Natural products 0.000 title claims abstract description 37
- 235000013343 vitamin Nutrition 0.000 title claims abstract description 37
- 150000003722 vitamin derivatives Chemical class 0.000 title abstract description 11
- 239000012085 test solution Substances 0.000 claims abstract description 63
- 238000010828 elution Methods 0.000 claims abstract description 41
- 238000000926 separation method Methods 0.000 claims abstract description 41
- -1 alcohol compound Chemical group 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 18
- CNIIGCLFLJGOGP-UHFFFAOYSA-N 2-(1-naphthalenylmethyl)-4,5-dihydro-1H-imidazole Chemical compound C=1C=CC2=CC=CC=C2C=1CC1=NCCN1 CNIIGCLFLJGOGP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229960005016 naphazoline Drugs 0.000 claims abstract description 14
- 239000012088 reference solution Substances 0.000 claims abstract description 13
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims description 86
- 239000000243 solution Substances 0.000 claims description 75
- DJDFFEBSKJCGHC-UHFFFAOYSA-N Naphazoline Chemical compound Cl.C=1C=CC2=CC=CC=C2C=1CC1=NCCN1 DJDFFEBSKJCGHC-UHFFFAOYSA-N 0.000 claims description 62
- 229960004760 naphazoline hydrochloride Drugs 0.000 claims description 52
- 238000012360 testing method Methods 0.000 claims description 52
- DBAKFASWICGISY-BTJKTKAUSA-N Chlorpheniramine maleate Chemical compound OC(=O)\C=C/C(O)=O.C=1C=CC=NC=1C(CCN(C)C)C1=CC=C(Cl)C=C1 DBAKFASWICGISY-BTJKTKAUSA-N 0.000 claims description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000011715 vitamin B12 Substances 0.000 claims description 13
- 229940046978 chlorpheniramine maleate Drugs 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- 238000004364 calculation method Methods 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 125000003158 alcohol group Chemical group 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000002798 polar solvent Substances 0.000 claims description 3
- CMWTZPSULFXXJA-UHFFFAOYSA-N Naproxen Natural products C1=C(C(C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-UHFFFAOYSA-N 0.000 abstract description 21
- 229960002009 naproxen Drugs 0.000 abstract description 21
- 239000004480 active ingredient Substances 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 33
- 229930003270 Vitamin B Natural products 0.000 description 28
- 239000011720 vitamin B Substances 0.000 description 28
- 235000019156 vitamin B Nutrition 0.000 description 28
- 238000004128 high performance liquid chromatography Methods 0.000 description 22
- 239000012488 sample solution Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- CMWTZPSULFXXJA-VIFPVBQESA-N naproxen Chemical compound C1=C([C@H](C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-VIFPVBQESA-N 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010051625 Conjunctival hyperaemia Diseases 0.000 description 1
- 206010052140 Eye pruritus Diseases 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 208000003464 asthenopia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960003291 chlorphenamine Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000009658 destructive testing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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Abstract
The invention relates to a method for separating and detecting related substances in naphthalene-sensitive vitamin eye drops, belonging to the field of drug analysis. The separation method comprises injecting the naphazoline into a high performance liquid chromatograph, and separating under the following conditions: the chromatographic column is C8 or C18 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is disodium hydrogen phosphate, and the mobile phase B is an alcohol compound and/or acetonitrile; elution time and volume content of mobile phase B during the elution time were as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45-18%. The separation method can separate related substances corresponding to three effective components in the naphthalene-Min-Wei eye drops. The detection method comprises taking the naphazoline eye drops as a test solution, preparing a reference solution, performing chromatographic detection on the naphazoline eye drops and the reference solution, and calculating the content of each related substance according to the recorded chromatogram. The detection method can quickly and accurately detect the contents of the three active ingredients corresponding to the related substances in the naproxen vitamin eye drops.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and detecting related substances in naphthalene-sensitive vitamin eye drops.
Background
The naphazoline hydrochloride, chlorphenamine maleate and vitamin B are used as the eyedrops12And multiple adjuvants, and is suitable for treating eye fatigue, conjunctival congestion, and eye pruritus.
The naproxen eyedrops contain a plurality of related substances (impurities). In order to ensure the safety and quality controllability of the product of the naphthalene-sensitized vitamin eye drops, the content of related substances in the eye drops needs to be detected. At present, the Chinese pharmacopoeia 2020 does not specify a method for detecting related substances. Although the chinese patent CN201911385569.3 provides a method for detecting related substances in naphazoline hydrochloride, it can only detect related substances of naphazoline hydrochloride and chlorphenamine maleate, which are effective components in naphazoline hydrochloride, and fails to detect vitamin B, which is an effective component, simultaneously12The related substances of (1).
Therefore, there is a need to develop a new method for separating and detecting related substances in the naproxen-vitamin eye drops so as to simultaneously separate and detect related substances corresponding to three active ingredients in the naproxen-vitamin eye drops.
Disclosure of Invention
In view of the defects in the prior art, an object of the present invention is to provide a method for separating related substances in naphthylamine vitamin eye drops, which is simple and convenient, can realize effective separation between related substances corresponding to three effective components in the naphthylamine vitamin eye drops, and has high separation degree of the related substances corresponding to each effective component. The invention also aims to provide a method for detecting related substances in the naproxen vitamin eye drops, which can quickly and accurately simultaneously detect the total content of the related substances corresponding to each effective component in the naproxen vitamin eye drops and is convenient for controlling the product quality.
In order to achieve the above object, the present invention provides a method for separating related substances from naphazoline, comprising the following steps:
injecting the naphazoline eye drops into a high performance liquid chromatograph, and separating under the following conditions:
chromatographic conditions are as follows: the chromatographic column is C8 or C18 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is disodium hydrogen phosphate, and the mobile phase B is selected from alcohol compounds and/or acetonitrile;
elution conditions: elution time and volume content of the mobile phase B during the elution time were as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45-18%.
Preferably, the related substances in the naproxen vitamin eye drops comprise vitamin B12Impurities, naphazoline hydrochloride impurities and chlorpheniramine maleate impurities.
Preferably, the column temperature in the separation process is 25-45 ℃.
Preferably, the flow rate of the mobile phase in the separation process is 0.5-1.5 mL/min.
Preferably, the concentration of the mobile phase A is 0.01-2.0 mol/L; the pH value of the mobile phase A is 2.0-5.0.
Preferably, the alcohol compound in the mobile phase B is selected from one or more of methanol, ethanol, isopropanol and ethylene glycol.
Preferably, the sample injection amount of the high performance liquid chromatograph is 10-100 mu L.
The second aspect of the invention provides a method for detecting related substances in naphthylamine vitamin eye drops, which comprises the following steps:
taking the naphazoline eye drops as a test solution, and preparing a test reference solution;
carrying out chromatographic detection on the test solution and the test control solution respectively according to the separation conditions in the separation method according to the first aspect of the invention, and recording chromatograms;
and calculating the content of each related substance according to the peak area of each related substance in the recorded chromatogram.
Preferably, the preparation method of the test article control solution comprises the following steps: the test solution is diluted to a concentration of 1.0% by a polar solvent.
Preferably, in the detection method, the calculation formula of the content of the related substances is as follows:
content of related substance%x/As)×1.0×100％
Wherein, Sigma AxIs the sum of chromatographic peak areas of the related substances in the test solution, AsIs the corresponding chromatographic peak area in the test sample control solution.
Preferably, the related substance is vitamin B12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are respectively as follows: 250 to 370nm, 220 to 300 nm.
Compared with the prior art, the invention has the beneficial effects that:
the method for separating related substances in the naproxen vitamin eye drops can realize the separation of vitamin B in the naproxen vitamin eye drops12The naphazoline hydrochloride and the chlorphenamine maleate completely separate related substances corresponding to the three effective components, the front and rear separation degrees meet the requirement of more than 1.5, the theoretical plate number is calculated to be more than 2000, and the column effect is better. Moreover, the method for detecting related substances in the naproxen vitamin eye drops can quickly and accurately detect naphazoline hydrochloride, chlorphenamine maleate and vitamin B in the naproxen vitamin eye drops simultaneously12The total content of the related substances corresponding to the three active ingredients is convenient for controlling the product quality and ensuring the safety and quality attribute of the naphthalene-Min vitamin eye drops. In addition, the method is simple, convenient and rapid, and has high specificity, accuracy and precision.
Drawings
In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings needed to be used in the embodiment will be briefly described below. It is appreciated that the following drawings depict only certain embodiments of the invention and are therefore not to be considered limiting of its scope.
FIG. 1 is an HPLC chromatogram of system suitability solution 1 at a wavelength of 361 nm;
FIG. 2 is an HPLC chromatogram of system suitability solution 2 at a wavelength of 280 nm;
FIG. 3 is an HPLC chromatogram of a test solution of example 1 of the present invention at a wavelength of 361 nm;
FIG. 4 is an HPLC chromatogram of a test sample control solution of example 1 of the present invention at a wavelength of 361 nm;
FIG. 5 is an HPLC chromatogram of a sample solution of example 1 of the present invention at a wavelength of 280 nm;
FIG. 6 is an HPLC chromatogram of a sample control solution of example 1 of the present invention at a wavelength of 280 nm;
FIG. 7 is an HPLC chromatogram of a sample solution of example 1 of the present invention at a wavelength of 262 nm;
FIG. 8 is an HPLC chromatogram of a sample control solution of example 1 of the present invention at a wavelength of 262 nm;
FIG. 9 is an HPLC chromatogram of a sample solution of example 2 of the present invention at a wavelength of 370 nm;
FIG. 10 is an HPLC chromatogram of a test sample control solution of example 2 of the present invention at a wavelength of 370 nm;
FIG. 11 is an HPLC chromatogram of a sample solution of example 2 of the present invention at a wavelength of 220 nm;
FIG. 12 is an HPLC chromatogram of a sample control solution of example 2 of the present invention at a wavelength of 220 nm;
FIG. 13 is an HPLC chromatogram of a sample solution of example 2 of the present invention at a wavelength of 300 nm;
FIG. 14 is an HPLC chromatogram of a test sample control solution of example 2 of the present invention at a wavelength of 300 nm;
FIG. 15 is an HPLC chromatogram of a test solution of example 3 of the present invention at a wavelength of 310 nm;
FIG. 16 is an HPLC chromatogram of a test sample control solution of example 3 of the present invention at a wavelength of 310 nm;
FIG. 17 is an HPLC chromatogram of a test solution of example 3 of the present invention at a wavelength of 260 nm;
FIG. 18 is an HPLC chromatogram of a test sample control solution of example 3 of the present invention at a wavelength of 260 nm;
FIG. 19 is an HPLC chromatogram of a sample solution of example 3 of the present invention at a wavelength of 220 nm;
FIG. 20 is an HPLC chromatogram of a sample control solution of example 3 of the present invention at a wavelength of 220 nm;
in the figure, 1-vitamin B12The peak position of (d); the peak position of 2-naphazoline hydrochloride; the position of the peak of 3-maleichlorophenamin.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It is to be understood that these examples are illustrative only and are not intended to limit the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following is a detailed description of the method for separating and detecting related substances in the naproxen eyedrops according to the embodiment of the present invention.
The invention firstly provides a method for separating related substances in the naproxen vitamin eye drops, and the method can realize the separation of vitamin B in the naproxen vitamin eye drops12The naphazoline hydrochloride and the chlorphenamine maleate corresponding to the three effective components are completely separated, namely the vitamin B is realized12Separating impurities, naphazoline hydrochloride impurities and chlorphenamine maleate impurities.
Specifically, the method for separating related substances in the naphazoline hydrochloride provided by the invention comprises the following steps: injecting the naphazoline eye drops into a high performance liquid chromatograph for separation. Preferably, the sample injection amount of the high performance liquid chromatograph is 10-100 mu L. More preferably, the sample volume of the high performance liquid chromatograph is 50-100 μ L.
Further, the separation conditions of the high performance liquid chromatography in the present invention are as follows:
chromatographic conditions
A chromatographic column: a C8 or C18 chromatography column; preferably, the C8 column is a Waters XBidge C8 column (150 mm. times.4.6 mm, 3.5 μm) and the C18 column is an octadecylsilane bonded silica gel column, Thermo Scientific Acclaim (TM) 120C 18 column (250 mm. times.4.6 mm, 5 μm) or an Endeovsil C18 column (250 mm. times.4.6 mm, 5 μm).
Mobile phase: comprising a mobile phase A and a mobile phase B. Specifically, the mobile phase A is disodium hydrogen phosphate, and the mobile phase B is selected from alcohol compounds and/or acetonitrile. The concentration of the mobile phase A is 0.01-2.0 mol/L, preferably 0.07 mol/L. The pH of the mobile phase A is 2.0-5.0, preferably 3.5, and more preferably, the pH of the mobile phase A is adjusted with phosphoric acid. The mobile phase B is selected from alcohol compounds and/or acetonitrile, preferably selected from one or more of methanol, ethanol, isopropanol and ethylene glycol, and more preferably methanol.
Preferably, the column temperature in the separation process is 25-45 ℃. Further, the column temperature is preferably 30 ℃.
Second, elution conditions
Elution time: preferably not more than 100 min.
And (3) an elution mode: gradient elution.
The volume contents of mobile phase a and mobile phase B in the mobile phase during the elution time are shown in table 1.
TABLE 1 volume content of mobile phase A and mobile phase B in the mobile phase during the elution time
| Elution time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 82 | 18 |
| 25 | 80 | 20 |
| 70 | 55 | 45 |
| 80 | 55 | 45 |
| 85 | 82 | 18 |
Elution flow rate: the flow rate of the mobile phase in the separation process is controlled to be 0.5-1.5 mL/min. Preferably, the flow rate of the mobile phase is 1.0 mL/min.
The invention also provides a method for detecting related substances in the naproxen vitamin eye drops, and the method can be used for quickly and accurately detecting the vitamin B in the naproxen vitamin eye drops at the same time12The total content of related substances corresponding to the three effective components of naphazoline hydrochloride and chlorphenamine maleate can simultaneously realize the vitamin B12Quantitative determination of impurities, naphazoline hydrochloride impurities and chlorphenamine maleate impurities.
Specifically, the method for detecting related substances in the naphazoline hydrochloride eye drops provided by the invention comprises the following steps:
taking the naphazoline eye drops as a test solution, and preparing a test reference solution;
respectively carrying out chromatographic detection on the test solution and the test reference solution according to the separation conditions in the separation method, and recording chromatograms;
and reading peak areas of chromatographic peaks corresponding to the related substances in the recorded chromatogram, and calculating the content of the related substances.
Preferably, the vitamin B in the substances involved in the invention12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are respectively as follows: 250 to 370nm, 220 to 300nm and 220 to 300 nm.
Preferably, the method for preparing the test sample control solution of the present invention comprises: the test solution is diluted with a polar solvent (preferably water) to a concentration of 1.0%. Specifically, 1mL of the test solution is precisely transferred and placed in a 100mL volumetric flask, diluted to the scale with water and shaken up to obtain the test control solution.
Further, in the detection method provided by the invention, a calculation formula of the content of the related substances is as follows:
content of related substance%x/As)×1.0×100％
Wherein, Sigma AxIs the sum of chromatographic peak areas of the related substances in the test solution, AsIs the corresponding chromatographic peak area in the test sample control solution.
Specifically, when the content of naphazoline hydrochloride-related substances (impurities) in the test solution is calculated, Sigma AxIs the sum of chromatographic peak areas of naphazoline hydrochloride impurities in the test solution; a. thesIs the chromatographic peak area of naphazoline hydrochloride in the test sample control solution;
when the content of related substances (impurities) of the chlorpheniramine maleate in the test solution is calculated, Sigma AxIs the sum of the chromatographic peak areas of chlorpheniramine maleate impurities in the test solution; a. thesThe area of the chromatographic peak of the chlorpheniramine maleate in the test sample control solution is shown;
when calculating vitamin B in the test solution12Content of related substances (impurities) Sigma AxAs vitamin B in the test solution12Sum of chromatographic peak areas of impurities; a. thesIs a vitamin in a reference solution of a test sampleElement B12Chromatographic peak area of (a).
The inventor of the invention researches and discovers that naphazoline hydrochloride and vitamin B in a test solution are contained in a high performance liquid chromatography separation12The separation of the corresponding related substances is difficult, and the separation conditions provided by the invention can realize naphazoline hydrochloride, chlorphenamine maleate and vitamin B in the test solution12The three relevant substances corresponding to the three effective components are effectively and completely separated. In order to verify the separation effect, the invention also prepares vitamin B only before the relevant substances are separated and the content is detected12Systematic applicability of control solution 1 (vitamin B)12Concentration of the reference substance is 0.1mg/mL) and a system suitability solution 2 containing only the naphazoline hydrochloride reference substance (concentration of the naphazoline hydrochloride reference substance is 0.02mg/mL), which are respectively injected into a high performance liquid chromatograph, and chromatographic analysis is carried out according to the separation conditions, and a chromatogram is recorded.
Specifically, the method of preparing the system-compatible solution 1 and the system-compatible solution 2 in the present invention is as follows.
Preparation of system applicability solution 1: precisely weighing vitamin B1210mg, placing in a 100mL volumetric flask, adding a proper amount of water to make vitamin B12And (3) completely dissolving, adding 1mL of 1mol/L NaOH solution, shaking up, adding 1mL of 1mol/L HCl solution for neutralization after 10min, then diluting to scale with water, shaking up to obtain a system applicability solution 1, and immediately injecting samples after standing for 60 min.
Preparation of system applicability solution 2: precisely weighing 2mg of naphazoline hydrochloride, placing the naphazoline hydrochloride into a 100mL volumetric flask, adding a proper amount of water to completely dissolve the naphazoline hydrochloride, adding 1mL of 1mol/L NaOH solution, shaking up, adding 1mL of 1mol/L HCl solution for neutralization after 10min, then diluting the solution to a scale with water, shaking up to obtain a system applicability solution 2, and immediately injecting samples after standing for 60 min.
In the present invention, vitamin B can be identified by the chromatogram of the applicability solution 1 (FIG. 1)12The peak position of (d); by means of the chromatogram of the system suitability solution 2 (fig. 2), the peak position of naphazoline hydrochloride can be determined; combining with the test solutionThe chromatogram of (fig. 5) can determine the peak position of the maleyl chlorpheniramine.
Examples
The features and properties of the present invention are described in further detail below with reference to specific examples.
Example 1
Taking the naproxen vitamin eye drops as a test solution, precisely measuring 1.0mL of the test solution, placing the test solution in a 100mL volumetric flask, adding water to dilute the solution to a scale, and shaking the solution uniformly to serve as a test reference solution (the concentration is 1.0%). Precisely measuring the sample solution and the sample control solution by 100 μ L, respectively, injecting into a DAN U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatograms shown in FIGS. 3-8.
Chromatographic conditions are as follows: the chromatographic column is a Thermo Scientific Acclaim (TM) 120C 18 chromatographic column (250 mm. times.4.6 mm, 5 μm); mobile phase a is 0.07mol/L disodium hydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and said mobile phase B is methanol; the column temperature was 25 ℃.
Elution conditions: gradient elution is adopted, and the elution time and the volume content of the mobile phase B in the mobile phase within the elution time are as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45% -18%; the elution flow rate was 1.0 mL/min.
Detection conditions are as follows: vitamin B12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are 361nm, 280nm and 262nm respectively.
The separation degrees (which can be directly read in a spectrogram) between all main peaks and other peaks in the high performance liquid chromatogram are all larger than 1.5, and the theoretical plate numbers (obtained by a conventional calculation method in the field) are all larger than 2000, which indicates that the separation degrees and the column efficiency of each impurity in the naproxen vitamin eye drops provided by the embodiment of the invention are higher.
Reading peak areas of related substance (impurity) chromatographic peaks corresponding to the wavelengths in the high performance liquid chromatogram, and calculating the content of the related substances in the test solution according to the following mode:
content of related substance (impurity)% (∑ a)x/As)×1.0×100％
In the formula: sigma AxIs the sum of chromatographic peak areas of the related substances in the test solution, AsIs the corresponding chromatographic peak area in the test sample control solution;
when calculating the content of naphazoline hydrochloride impurity in the test solutionxIs the sum of chromatographic peak areas of naphazoline hydrochloride impurities in the test solution; a. thesIs the chromatographic peak area of naphazoline hydrochloride in the test sample control solution;
when the content of the chlorphenamine maleate impurities in the test solution is calculated, Sigma AxIs the sum of the chromatographic peak areas of chlorpheniramine maleate impurities in the test solution; a. thesThe area of the chromatographic peak of the chlorpheniramine maleate in the test sample control solution is shown;
when calculating vitamin B in the test solution12When the content of impurities is sigma AxAs vitamin B in the test solution12Sum of chromatographic peak areas of impurities; a. thesIs vitamin B in a reference solution of a test sample12Chromatographic peak area of (a).
Respectively calculating to obtain vitamin B in the naphazoline through the formula120.45 percent of impurity, 0.21 percent of naphazoline hydrochloride impurity and 0.15 percent of chlorphenamine maleate impurity.
Example 2
Taking the naphthylamine vitamin eye drops as a test solution, precisely measuring 1.0mL of the test solution, placing the test solution in a 100mL volumetric flask, adding water to dilute the solution to a scale, and shaking the solution uniformly to serve as a test reference solution (the concentration is 1.0%). Precisely measuring the sample solution and the sample control solution at 50 μ L, respectively, injecting into a DAN U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatograms shown in FIGS. 9-14.
Chromatographic conditions are as follows: the chromatographic column is a Waters Xbridge C8 chromatographic column (150 mm. times.4.6 mm, 3.5 μm); mobile phase a is 1.2mol/L disodium hydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and said mobile phase B is acetonitrile; the column temperature was 30 ℃.
Elution conditions: gradient elution is adopted, and the elution time and the volume content of the mobile phase B in the mobile phase within the elution time are as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45% -18%; the elution flow rate was 0.7 mL/min.
Detection conditions are as follows: vitamin B12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are 370nm, 220nm and 300nm respectively.
The separation degrees (which can be directly read in a spectrogram) between all main peaks and other peaks in the high performance liquid chromatogram are all larger than 1.5, and the theoretical plate numbers (obtained by a conventional calculation method in the field) are all larger than 2000, which indicates that the separation degrees and the column efficiency of each impurity in the naproxen vitamin eye drops provided by the embodiment of the invention are higher.
Reading peak areas of related substance (impurity) chromatographic peaks corresponding to the wavelengths in the high performance liquid chromatogram, and calculating the content of the related substances in the test solution according to the following mode:
content of related substance (impurity)% (∑ a)x/As)×1.0×100％
In the formula: sigma AxIs the sum of chromatographic peak areas of the related substances in the test solution, AsIs the corresponding peak area in the test sample control solution;
when calculating the content of naphazoline hydrochloride impurity in the test solutionxIs the sum of chromatographic peak areas of naphazoline hydrochloride impurities in the test solution; a. thesIs the chromatographic peak area of naphazoline hydrochloride in the test sample control solution;
when the content of the chlorphenamine maleate impurities in the test solution is calculated, Sigma AxIs the sum of the chromatographic peak areas of chlorpheniramine maleate impurities in the test solution; a. thesThe area of the chromatographic peak of the chlorpheniramine maleate in the test sample control solution is shown;
when calculating vitamin B in the test solution12When the content of impurities is sigma AxAs vitamin B in the test solution12Sum of chromatographic peak areas of impurities; a. thesIs vitamin B in a reference solution of a test sample12Chromatographic peak area of (a).
By the above formulas respectivelyCalculating to obtain vitamin B in the naphazoline120.42 percent of impurity, 0.22 percent of naphazoline hydrochloride impurity and 0.17 percent of chlorphenamine maleate impurity.
Example 3
Taking the naphthylamine vitamin eye drops as a test solution, precisely measuring 1.0mL of the test solution, placing the test solution in a 100mL volumetric flask, adding water to dilute the solution to a scale, and shaking the solution uniformly to serve as a test reference solution (the concentration is 1.0%). Precisely measuring the sample solution and the sample control solution by 100 μ L, respectively, injecting into a Dyan U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatograms shown in FIGS. 15-20.
Chromatographic conditions are as follows: the chromatographic column is an Endeovsil C18 chromatographic column (250 mm. times.4.6 mm, 5 μm); mobile phase a is 0.5mol/L disodium hydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and said mobile phase B is methanol; the column temperature was 45 ℃.
Elution conditions: gradient elution is adopted, and the elution time and the volume content of the mobile phase B in the mobile phase within the elution time are as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45% -18%; the elution flow rate was 1.0 mL/min.
Detection conditions are as follows: vitamin B12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are 310nm, 260nm and 220nm respectively.
The separation degrees (which can be directly read in a spectrogram) between all main peaks and other peaks in the high performance liquid chromatogram are all larger than 1.5, and the theoretical plate numbers (obtained by a conventional calculation method in the field) are all larger than 2000, which indicates that the separation degrees and the column efficiency of each impurity in the naproxen vitamin eye drops provided by the embodiment of the invention are higher.
Reading peak areas of related substance (impurity) chromatographic peaks corresponding to the wavelengths in the high performance liquid chromatogram, and calculating the content of the related substances in the test solution according to the following mode:
content of related substance (impurity)% (∑ a)x/As)×1.0×100％
In the formula: sigma AxFor the substance concerned in the test solutionSum of chromatographic peak areas, AsIs the corresponding peak area in the test sample control solution;
when calculating the content of naphazoline hydrochloride impurity in the test solutionxIs the sum of chromatographic peak areas of naphazoline hydrochloride impurities in the test solution; a. thesIs the chromatographic peak area of naphazoline hydrochloride in the test sample control solution;
when the content of the chlorphenamine maleate impurities in the test solution is calculated, Sigma AxIs the sum of the chromatographic peak areas of chlorpheniramine maleate impurities in the test solution; a. thesThe area of the chromatographic peak of the chlorpheniramine maleate in the test sample control solution is shown;
when calculating vitamin B in the test solution12When the content of impurities is sigma AxAs vitamin B in the test solution12Sum of chromatographic peak areas of impurities; a. thesIs vitamin B in a reference solution of a test sample12Chromatographic peak area of (a).
Respectively calculating to obtain vitamin B in the naphazoline through the formula120.47 percent of impurity, 0.25 percent of naphazoline hydrochloride impurity and 0.18 percent of chlorphenamine maleate impurity.
Test example 1
And carrying out a destructive degradation test on the naphthalene-sensitized vitamin eye drops, and checking the purity of a main peak by using a photodiode array detector to verify whether the specificity of the method meets the requirement.
The instrument comprises the following steps: daian U3000 high performance liquid chromatograph
Chromatographic conditions are as follows: the chromatographic column is a Thermo Scientific Acclaim (TM) 120C 18 chromatographic column (250 mm. times.4.6 mm, 5 μm); mobile phase a is 0.07mol/L disodium hydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and said mobile phase B is methanol; the column temperature was 30 ℃.
Elution conditions: gradient elution is adopted, and the elution time and the volume content of the mobile phase B in the mobile phase within the elution time are as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45% -18%; the elution flow rate was 1.0 mL/min.
Detection conditions are as follows: vitamin preparationB12And the detection wavelengths of the naphazoline hydrochloride and the chlorphenamine maleate impurities are 361nm, 280nm and 262nm respectively.
Photo-destructive testing: taking a proper amount of the naphazoline hydrochloride eye drops, respectively placing in a light instrument, and standing for 10 days under the condition that the illumination intensity is (4500 +/-500) lx. Preparing blank auxiliary material solution by the same method.
High temperature failure test: heating a proper amount of the naphazoline hydrochloride eye drops in a 105 ℃ oven for 4 h. Preparing blank auxiliary material solution by the same method.
Acid destruction test: taking a proper amount of the naproxen vitamin eye drops, adding 0.01mol/L hydrochloric acid solution for dissolving, carrying out water bath for 2h, and then neutralizing with 0.01mol/L sodium hydroxide solution. Preparing blank auxiliary material solution by the same method.
Alkali destruction test: taking a proper amount of the naproxen vitamin eye drops, adding 0.01mol/L sodium hydroxide solution for dissolving, carrying out water bath for 2h, and then neutralizing with 0.01mol/L hydrochloric acid solution. Preparing blank auxiliary material solution by the same method.
Oxidative destruction test: taking a proper amount of the naphazoline hydrochloride eye drops, adding 30% hydrogen peroxide solution for dissolving, and carrying out water bath for 2 h. Preparing blank auxiliary material solution by the same method.
Sucking 10 μ L of the test sample solution, injecting into a high performance liquid chromatograph, and simultaneously scanning each sample solution with UV wavelength of 200-400 nm by using a photodiode array detector, wherein the test results are shown in Table 2.
TABLE 2 degradation destruction test results of Naphthaline eye drops
As can be seen from Table 2, vitamin B was found by purity analysis using a diode array detector scanning three main peaks at full wavelength12The peak purities of naphazoline hydrochloride and chlorphenamine maleate are all more than 0.98, which indicates that the three main peaks do not contain other degradation impurity peaks, the degradation recovery rate obtained by calculation is more than 98%, which indicates that each degradation impurity is basically detected by the method under the destruction condition, and the method has good specificity and is free of interference.
Test example 2
The impurity determination of the naphazoline hydrochloride eye drops is carried out with a repeatability test to investigate the precision of the method.
The instrument comprises the following steps: daian U3000 high performance liquid chromatograph
Chromatographic conditions are as follows: the chromatographic column is a Thermo Scientific Acclaim (TM) 120C 18 chromatographic column (250 mm. times.4.6 mm, 5 μm); mobile phase a is 0.07mol/L disodium hydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid), and said mobile phase B is methanol; the column temperature was 30 ℃.
Elution conditions: gradient elution is adopted, and the elution time and the volume content of the mobile phase B in the mobile phase within the elution time are as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45% -18%; the elution flow rate was 1.0 mL/min.
Detection conditions are as follows: vitamin B12The detection wavelengths of naphazoline hydrochloride and chlorphenamine maleate impurities are 361nm, 280nm and 262nm respectively.
Test solution: the naphazoline eye drops (different from the eye drops in the examples in terms of source) are taken as the test solution.
Test article control solution: precisely transferring 1mL of the test solution, placing the test solution in a 100mL volumetric flask, adding water to dilute the test solution to a scale, and shaking up the test solution to obtain a test control solution.
And (3) detection: precisely absorbing the sample solution and the sample control solution by 50 μ L respectively, repeatedly injecting into high performance liquid chromatograph for 6 times, and measuring the total impurity content corresponding to each effective component in the sample solution. As can be seen from the high performance liquid chromatogram, the separation degrees (directly readable in the chromatogram) are both more than 1.5, and the theoretical plate numbers (obtained by the conventional calculation method in the field) are both more than 2000, which indicates that the separation degrees and the column efficiency of each impurity in the naphazoline eye drops provided by the embodiment of the invention are higher. The contents of impurities corresponding to the three effective components in the naproxen vitamin eye drops calculated according to the formula of the invention are shown in table 3.
TABLE 3 results of the precision test of the method of the present invention
| Number of |
1 | 2 | 3 | 4 | 5 | 6 | RSD％ |
| Vitamin B12Impurity (%) | 0.32 | 0.30 | 0.35 | 0.30 | 0.33 | 0.28 | 0.080 |
| Naphazoline hydrochloride impurity (%) | 0.15 | 0.13 | 0.11 | 0.14 | 0.12 | 0.16 | 0.14 |
| Chlorpheniramine maleate impurity (%) | 0.10 | 0.09 | 0.08 | 0.10 | 0.12 | 0.11 | 0.14 |
As can be seen from Table 3, the Relative Standard Deviation (RSD) of the impurity content corresponding to each effective component obtained by measurement is less than 0.2%, which shows that the method provided by the invention has better result precision.
In conclusion, the method for separating and detecting related substances in the naphthalene-sensitized vitamin eye drops provided by the invention is simple and convenient, the separation degree of each component is higher, the total content of impurities corresponding to each effective component in the naphthalene-sensitized vitamin eye drops can be rapidly and accurately measured simultaneously, the quality of products can be conveniently controlled, and the safety and quality attributes of the eye drops are ensured. In addition, the method provided by the invention has high specificity and good accuracy and precision.
The present invention is not limited to the above-described embodiments, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and such modifications and improvements are also considered to be within the scope of the present invention. Those not described in detail in this specification are within the skill of the art.
Claims (11)
1. A method for separating related substances in naphthylamine vitamin eye drops is characterized by comprising the following steps:
injecting the naphazoline eye drops into a high performance liquid chromatograph, and separating under the following conditions:
chromatographic conditions are as follows: the chromatographic column is C8 or C18 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is disodium hydrogen phosphate, and the mobile phase B is selected from alcohol compounds and/or acetonitrile;
elution conditions: elution time and volume content of the mobile phase B during the elution time were as follows: 0-25 min, 18% -20%; 20-45% of 25-70 min; 45 percent of the total amount of the raw materials for 70 to 80 min; 80-85 min, 45-18%.
2. The separation method as claimed in claim 1, wherein the related substances in the Naphthaline eyedrops comprise vitamin B12Impurities, naphazoline hydrochloride impurities and chlorpheniramine maleate impurities.
3. The separation method according to claim 1, wherein the column temperature during the separation is 25 to 45 ℃.
4. The separation method according to claim 1, wherein the flow rate of the mobile phase during the separation process is 0.5-1.5 mL/min.
5. The separation method according to claim 1, wherein the concentration of the mobile phase A is 0.01-2.0 mol/L; the pH value of the mobile phase A is 2.0-5.0.
6. The separation method according to claim 1, wherein the alcohol compound in the mobile phase B is selected from one or more of methanol, ethanol, isopropanol and ethylene glycol.
7. The separation method according to any one of claims 1 to 6, wherein the amount of the sample to be introduced into the high performance liquid chromatograph is 10 to 100. mu.L.
8. A method for detecting related substances in naphthylamine vitamin eye drops is characterized by comprising the following steps:
taking the naphazoline eye drops as a test solution, and preparing a test reference solution;
performing chromatographic detection on the test solution and the test control solution respectively according to the separation conditions in the separation method according to any one of claims 1 to 7, and recording chromatograms;
and calculating the content of each related substance according to the peak area of each related substance in the recorded chromatogram.
9. The detection method according to claim 8, wherein the preparation method of the test article control solution comprises: the test solution is diluted to a concentration of 1.0% by a polar solvent.
10. The detection method according to claim 8, wherein the calculation formula of the content of the related substances in the detection method is as follows:
content of related substance%x/As)×1.0×100％
Wherein, Sigma AxIs the sum of chromatographic peak areas of the related substances in the test solution, AsIs the corresponding peak area in the test sample control solution.
11. The method according to any one of claims 8 to 10, wherein the substance of interest contains vitamin B12The detection wavelengths of the impurities, the naphazoline hydrochloride impurities and the chlorphenamine maleate impurities are respectively as follows: 250 to 370nm, 220 to 300 nm.
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| CN114646705A (en) * | 2022-03-22 | 2022-06-21 | 武汉绿合医药科技有限公司 | Method for detecting content of related substances in diquafosol sodium eye drops |
| CN118641658A (en) * | 2024-06-12 | 2024-09-13 | 连云港市食品药品检验检测中心 | Test methods for identification and content determination of two chemical drugs in nasal medical devices |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114646705A (en) * | 2022-03-22 | 2022-06-21 | 武汉绿合医药科技有限公司 | Method for detecting content of related substances in diquafosol sodium eye drops |
| CN118641658A (en) * | 2024-06-12 | 2024-09-13 | 连云港市食品药品检验检测中心 | Test methods for identification and content determination of two chemical drugs in nasal medical devices |
| CN118641658B (en) * | 2024-06-12 | 2025-03-28 | 连云港市食品药品检验检测中心 | Test methods for identification and content determination of two chemical drugs in nasal medical devices |
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