CN112540136A - Method for separating and detecting components of naphthalene-sensitized vitamin eye drops - Google Patents

Abstract

The invention relates to a method for separating and detecting components of naphthalene-sensitized 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 selected from alcohol compounds and/or acetonitrile; elution time and volume content of mobile phase B during the elution time were as follows: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55 to 30 percent for 10 to 11 min. The separation method can separate three effective components in the naphthalene-Min-Wei eye drop. The detection method comprises the steps of taking the naproxen vitamin eye drops as a test solution, respectively preparing a control solution containing each component of a reference substance in the naproxen vitamin eye drops, respectively carrying out chromatographic detection on the naproxen vitamin eye drops and the control solution, and calculating the content of each component according to a recorded chromatogram. The detection method can simultaneously, rapidly and accurately detect the contents of three effective components in the naproxen vitamin eye drops.

Description

Method for separating and detecting components of naphthalene-sensitized vitamin eye drops

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and detecting components of a naphthylamine vitamin eye drop.

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.

At present, naphazoline hydrochloride, chlorphenamine maleate and vitamin B which are main components of the naphazoline hydrochloride, the naphazoline maleate and the vitamin B are respectively measured by different methods in the 'Chinese pharmacopoeia' 2020 edition₁₂The content of (A) has the disadvantage of being time-consuming and tedious.

Therefore, there is a need to develop a new method for separating and detecting components of the naproxen-vitamin eye drops so as to simultaneously separate and detect three active components in the naproxen-vitamin eye drops.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for separating components of naphthylamine vitamin eye drops, which is simple and convenient, can realize the separation of three effective components in the naphthylamine vitamin eye drops, and has higher separation degree. The invention also aims to provide a method for detecting the components of the naproxen vitamin eye drops, which can quickly and accurately simultaneously detect the content of each effective component in the naproxen vitamin eye drops.

In order to achieve the above object, the present invention provides a method for separating components of an ophthalmic solution of naphazoline, comprising the steps of:

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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min;

the Naminovir eye drop contains vitamin B₁₂Naphazoline hydrochloride and chlorpheniramine maleate.

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 components of the naphthalene-sensitized vitamin eye drops, which comprises the following steps:

taking the naphazoline hydrochloride eye drops as a test solution, and respectively preparing a reference solution of a reference substance containing each component in the naphazoline hydrochloride eye drops;

subjecting the test solution and the control solution to chromatographic detection and recording chromatograms respectively according to the separation conditions in the separation method according to the first aspect of the invention;

calculating the content of each component according to the peak area corresponding to each component in the recorded chromatogram;

the Naminovir eye drop contains vitamin B₁₂Naphazoline hydrochloride and chlorpheniramine maleate.

Preferably, in the control solution, vitamin B₁₂Concentration of controlThe concentration of the naphazoline hydrochloride is 0.05-0.15 mg/mL, the concentration of the naphazoline hydrochloride reference substance is 0.01-0.03 mg/mL, and the concentration of the chlorpheniramine maleate reference substance is 0.1-0.3 mg/mL.

Preferably, the calculation formula of the content of each component is as follows:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

Preferably, the detection wavelength of the components of the naphthylamine vitamin eye drops is 220-300 nm.

Compared with the prior art, the invention has the beneficial effects that:

the method for separating the components of the naphazoline hydrochloride can realize the vitamin B in the naphazoline hydrochloride₁₂The naphazoline hydrochloride and the chlorphenamine maleate are completely separated, 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 the components of the naproxen-vitamin eye drops can quickly and accurately detect naphazoline hydrochloride, chlorpheniramine maleate and vitamin B in the naproxen-vitamin eye drops simultaneously₁₂The contents of the three effective components. 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 a sample solution of example 1 of the present invention at a wavelength of 270 nm;

FIG. 2 is an HPLC chromatogram of a control solution of example 1 of the present invention at a wavelength of 270 nm;

FIG. 3 is an HPLC chromatogram of a sample solution of example 2 of the present invention at a wavelength of 225 nm;

FIG. 4 is an HPLC chromatogram of a control solution of example 2 of the present invention at a wavelength of 225 nm;

FIG. 5 is an HPLC chromatogram of a sample solution of example 3 of the present invention at a wavelength of 290 nm;

FIG. 6 is an HPLC chromatogram of a control solution of example 3 of the present invention at a wavelength of 290 nm.

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 the components of the naproxen eyedrops according to the embodiment of the present invention.

The invention firstly provides a method for separating components of the naphazoline hydrochloride eye drops, and the method can realize the vitamin B in the naphazoline hydrochloride eye drops₁₂Naphazoline hydrochloride and chlorpheniramine maleate and complete separation of three effective components.

Specifically, the method for separating the components of the naphazoline hydrochloride eye drops 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 XBridge C8 column (150 mm. times.4.6 mm, 3.5 μm), 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), an Endeovsil C18 column (250 mm. times.4.6 mm, 5 μm) or a Dionex Acclaim 120C 18 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, ethylene glycol and acetonitrile, more preferably methanol.

Preferably, the column temperature in the separation process is 25-45 ℃. Further, the column temperature is preferably 30 to 40 ℃.

Second, elution conditions

Elution time: preferably not more than 20 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 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 the components of the naphazoline, and the method can quickly and accurately detect the vitamin B in the naphazoline at the same time₁₂The contents of the three effective components of naphazoline hydrochloride and chlorphenamine maleate can simultaneously realize the vitamin B₁₂Quantitative determination of naphazoline hydrochloride and chlorphenamine maleate.

Specifically, the method for detecting the components of the naphazoline hydrochloride eye drops comprises the following steps:

taking the naphazoline hydrochloride eye drops as a test solution, and respectively preparing a reference solution of a reference substance containing each component in the naphazoline hydrochloride eye drops;

respectively carrying out chromatographic detection on the test solution and the control solution according to the separation conditions in the separation method, and recording chromatograms;

and reading the peak area of the chromatographic peak corresponding to each component in the recorded chromatogram, and calculating the content of each component.

Preferably, the detection wavelength of the components of the naproxen-vitamin eye drops is 220-300 nm.

Preferably, in the control solution of the present invention, vitamin B₁₂The concentration of the reference substance is 0.05-0.15 mg/mL, the concentration of the naphazoline hydrochloride reference substance is 0.01-0.03 mg/mL, and the concentration of the chlorphenamine maleate reference substance is 0.1-0.3 mg/mL. The preparation method of the control solution comprises the following steps: precisely weighing vitamin B₁₂And appropriate amount of reference substances of naphazoline hydrochloride and chlorphenamine maleate, and water is added to dilute the reference substances to the required concentration.

Further, in the detection method provided by the invention, the calculation formula of the content of each component is as follows:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

In particular, when computing a testWhen the relative labeled content of naphazoline hydrochloride in the product solution is A_{For supplying to}Is the chromatographic peak area of naphazoline hydrochloride in the test solution, A_{To pair}Is the chromatographic peak area, m, of naphazoline hydrochloride in control solution_{To pair}As the concentration of naphazoline hydrochloride in the control solution;

when calculating the relative labeled content of chlorphenamine maleate in the test solution, A_{For supplying to}Is the chromatographic peak area of chlorphenamine maleate in the test solution, A_{To pair}Is the chromatographic peak area, m, of chlorpheniramine maleate in the control solution_{To pair}The concentration of chlorpheniramine maleate in the control solution;

when calculating vitamin B in the test solution₁₂In relative labeled amounts of (A)_{For supplying to}As vitamin B in the test solution₁₂Chromatographic peak area of (A)_{To pair}As vitamin B in a control solution₁₂Chromatographic peak area of, m_{To pair}As vitamin B in a control solution₁₂And (4) concentration.

The inventor of the invention researches and discovers that the prior art can not realize the simultaneous realization of naphazoline hydrochloride, chlorpheniramine maleate and vitamin B in a test solution during the separation and detection of high performance liquid chromatography₁₂The separation and detection of the invention can realize the separation of naphazoline hydrochloride, chlorphenamine maleate and vitamin B in the test solution by adopting the separation conditions provided by the invention₁₂The three effective components are effectively and completely separated and detected.

Examples

The features and properties of the present invention are described in further detail below with reference to specific examples.

Example 1

The naphazoline eye drops are used as a test solution. Precisely weighing vitamin B₁₂Appropriate amount of reference substances of naphazoline hydrochloride and chlorphenamine maleate are added with water to prepare vitamin B with the concentration of 0.1mg/mL₁₂Control, naphazoline hydrochloride control 0.02mg/mL, and chlorphenamine maleate control 0.2 mg/mL.

Respectively sucking 20 μ L of test solution and control solution, injecting into a DAON U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatogram shown in FIG. 1-FIG. 2.

Chromatographic conditions are as follows: the column was a Dionex Acclaim 120C 18 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 40 ℃.

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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min; the elution flow rate was 1.0 mL/min.

Detection conditions are as follows: vitamin B₁₂And the detection wavelengths of naphazoline hydrochloride and chlorpheniramine maleate are 270 nm.

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 the spectral peaks in the naphazel provided by the embodiment of the invention are higher.

Reading the peak area of the chromatographic peak corresponding to each component in the recorded chromatogram, and calculating the content of each component in the test solution according to the following formula:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

Respectively calculating to obtain vitamin B in the naphazoline through the formula₁₂The contents of naphazoline hydrochloride and chlorphenamine maleate are respectively 100.1%, 100.2% and 99.8%.

Example 2

The naphazoline eye drops are used as a test solution. Precisely weighing vitamin B₁₂Appropriate amount of reference substances of naphazoline hydrochloride and chlorphenamine maleate are added with water to prepare the vitamin B with the concentration of 0.05mg/mL₁₂Control, naphazoline hydrochloride control 0.01mg/mL, and chlorphenamine maleate control 0.1 mg/mL.

Respectively sucking sample solution and control solution 40 μ L, injecting into a DAON U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatogram shown in fig. 3-4.

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 0.55mol/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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min; the elution flow rate was 1.0 mL/min.

Detection conditions are as follows: vitamin B₁₂The detection wavelengths of naphazoline hydrochloride and chlorpheniramine maleate are 225 nm.

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 the spectral peaks in the naphazel provided by the embodiment of the invention are higher.

Reading the peak area of the chromatographic peak corresponding to each component in the recorded chromatogram, and calculating the content of each component in the test solution according to the following formula:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

Respectively calculating to obtain vitamin B in the naphazoline through the formula₁₂The contents of naphazoline hydrochloride and chlorpheniramine maleate are respectively 98.6%, 100.1% and 99.5%.

Example 3

The naphazoline eye drops are used as a test solution. Precisely weighing vitamin B₁₂Appropriate amount of reference substances of naphazoline hydrochloride and chlorphenamine maleate are added with water to prepare the vitamin B with the concentration of 0.15mg/mL₁₂Control, naphazoline hydrochloride control 0.03mg/mL, and chlorphenamine maleate control 0.3 mg/mL.

Respectively sucking sample solution and control solution 50 μ L, injecting into a DAON U3000 high performance liquid chromatograph, and separating under the following conditions to obtain high performance liquid chromatogram shown in FIGS. 5-6.

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 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 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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min; the elution flow rate was 1.0 mL/min.

Detection conditions are as follows: vitamin B₁₂And the detection wavelengths of the naphazoline hydrochloride and the chlorpheniramine maleate are 290 nm.

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 the spectral peaks in the naphazel provided by the embodiment of the invention are higher.

Reading the peak area of the chromatographic peak corresponding to each component in the recorded chromatogram, and calculating the content of each component in the test solution according to the following formula:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

Respectively calculating to obtain vitamin B in the naphazoline through the formula₁₂The contents of naphazoline hydrochloride and chlorpheniramine maleate are respectively 99.9%, 97.1% and 100.2%.

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 column was a Dionex Acclaim 120C 18 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 40 ℃.

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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min; the elution flow rate was 1.0 mL/min.

Detection conditions are as follows: vitamin B₁₂And the detection wavelength of naphazoline hydrochloride and chlorphenamine maleate is 270 nm.

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.

20 mu L of the test sample solution is sucked and injected into a high performance liquid chromatograph, and simultaneously, a photodiode array detector is adopted to scan the UV wavelength of 200-400 nm of each sample solution, and 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 the naphazoline hydrochloride and the chlorpheniramine maleate are all more than 0.98, which shows that the three main peaks do not contain other degradation impurity peaks, and shows that the method has good specificity and no interference when the three main components of the naphazoline hydrochloride and chlorpheniramine maleate eye drops are measured.

Test example 2

The method for detecting the components of the naphazoline hydrochloride eye drops is used for carrying out 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 column was a Dionex Acclaim 120C 18 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 40 ℃.

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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55-30% for 10-11 min; the elution flow rate was 1.0 mL/min.

Detection conditions are as follows: vitamin B₁₂And the detection wavelengths of naphazoline hydrochloride and chlorpheniramine maleate are 270 nm.

Test solution: taking the naphazoline hydrochloride eye drops as a test solution.

Control solution: precisely weighing vitamin B₁₂Appropriate amount of reference substances of naphazoline hydrochloride and chlorphenamine maleate are added with water to prepare vitamin B with the concentration of 0.1mg/mL₁₂Control, naphazoline hydrochloride control 0.02mg/mL, and chlorphenamine maleate control 0.2 mg/mL.

And (3) detection: precisely absorbing sample solution and control solution 20 μ L respectively, repeatedly injecting into high performance liquid chromatograph for 6 times, and reading vitamin B in chromatogram of sample solution₁₂Chromatographic peak areas for naphazoline hydrochloride and chlorpheniramine maleate (A)_{For supplying to}) Vitamin B in the chromatogram of the control solution₁₂Chromatographic peak areas for naphazoline hydrochloride and chlorpheniramine maleate (A)_{To pair}). The separation degree (which can be directly read in a spectrogram) between all main peaks and other peaks in the high performance liquid chromatogram is more than 1.5, and the theoretical plate number (obtained by a conventional calculation method in the field) is more than 2000. The contents of the three active ingredients in the naproxen vitamin eye drops calculated according to the external standard method formula are shown in table 3.

TABLE 3 results of the precision test of the method of the present invention

Number of measurements	1	2	3	4	5	6	RSD％
								Vitamin B12(％)	100.2	100.4	100.2	100.1	100.5	100.3	0.15
Naphazoline hydrochloride (%)	99.8	99.1	99.7	99.6	99.4	99.2	0.28
								Chlorpheniramine maleate (%)	101.3	101.1	100.5	100.8	101.5	101.2	0.36

As can be seen from Table 3, the Relative Standard Deviation (RSD) of the content of each effective component is less than 0.5%, which indicates that the method provided by the invention has better result precision.

In conclusion, the method for separating and detecting the components of the naphthalene-sensitized vitamin eye drops is simple and convenient, the separation degree of each component is more than 1.5, the theoretical plate number is more than 2000, the chromatographic condition stability is good, and the content of each effective component in the naphthalene-sensitized vitamin eye drops can be rapidly and accurately measured simultaneously. 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 (10)

1. A method for separating components of 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: 30% for 0-3 min; 30-55% for 3-5 min; 55% in 5-10 min; 55 to 30 percent for 10 to 11 min.

The Naminovir eye drop contains vitamin B₁₂Naphazoline hydrochloride and chlorpheniramine maleate.

2. The separation method according to claim1, wherein the column temperature during the separation is 25 to 45 ℃.

3. The separation method according to claim1, wherein the flow rate of the mobile phase during the separation process is 0.5-1.5 mL/min.

4. The separation method according to claim1, 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.

5. The separation method according to claim1, wherein the alcohol compound in the mobile phase B is selected from one or more of methanol, ethanol, isopropanol and ethylene glycol.

6. The separation method according to any one of claims 1 to 5, wherein the amount of the sample to be introduced into the HPLC is 10 to 100. mu.L.

7. A method for detecting components of naphthylamine vitamin eye drops is characterized by comprising the following steps:

taking the naphazoline hydrochloride eye drops as a test solution, and respectively preparing a reference solution of a reference substance containing each component in the naphazoline hydrochloride eye drops;

subjecting said test solution and said control solution to chromatographic detection and recording chromatograms according to the separation conditions in the separation method according to any one of claims 1 to 6;

calculating the content of each component according to the peak area corresponding to each component in the recorded chromatogram;

the Naminovir eye drop contains vitamin B₁₂Naphazoline hydrochloride and chlorpheniramine maleate.

8. The detection method according to claim 7, characterized in thatIn the control solution, vitamin B₁₂The concentration of the reference substance is 0.05-0.15 mg/mL, the concentration of the naphazoline hydrochloride reference substance is 0.01-0.03 mg/mL, and the concentration of the chlorphenamine maleate reference substance is 0.1-0.3 mg/mL.

9. The detection method according to claim 7, wherein the calculation formula of the content of each component is as follows:

wherein w is the relative labeled content of a certain component in the naphazoline, A_{For supplying to}Is the chromatographic peak area of the component in the test solution, A_{To pair}As the chromatographic peak area of the component in the control solution, m_{For supplying to}Is the labeled concentration of a certain component in the test solution, m_{To pair}The concentration of this component in the control solution.

10. The method according to any one of claims 7 to 9, wherein the wavelength of detection of the naproxen-vitamin eye drop component is 220 to 300 nm.

Images