CN111707750A - HPLC detection method for related substances of sodium salicylate - Google Patents
HPLC detection method for related substances of sodium salicylate Download PDFInfo
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- CN111707750A CN111707750A CN202010585699.8A CN202010585699A CN111707750A CN 111707750 A CN111707750 A CN 111707750A CN 202010585699 A CN202010585699 A CN 202010585699A CN 111707750 A CN111707750 A CN 111707750A
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- G—PHYSICS
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- 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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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
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Abstract
The invention provides a method for detecting related substances of sodium salicylate, which adopts a reversed-phase high performance liquid chromatography, uses octadecylsilane chemically bonded silica with polar end groups sealed as a chromatographic column of a stationary phase, and uses a mixed solution of methanol, water and trifluoroacetic acid as a mobile phase; the method can simultaneously separate sodium salicylate and related substances such as 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol, is convenient for controlling the product quality in the production and quality control processes, and has the advantages of low cost, simplicity, easy implementation, high accuracy and precision, and good stability and reproducibility.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an HPLC (high performance liquid chromatography) detection method for sodium salicylate related substances.
Background
Sodium Salicylate (Sodium Salicylate), also known as Sodium ortho-hydroxybenzoate, of the formula: c7H5NaO3Molecular weight: 160.10.
the structural formula is as follows:
sodium salicylate is white flaky crystal or powder, and is one of the early human medicines. The ancient books in China and abroad have been recorded with the treatment of diseases by willow, salicyl and the like. The application of the sodium salicylate ointment, the sodium salicylate tincture and the like has been in history for more than one hundred years, and the sodium salicylate ointment, the sodium salicylate tincture and the like are still sold in the market as old medicines with definite curative effect and very low price. At present, the requirement of various external medicaments such as ointment, tincture, powder and the like prepared by taking the traditional Chinese medicine as a raw material is large. The sodium salicylate has wide application and is an important pharmaceutical chemical raw material. The medicine is mainly used for preparing antipyretic, analgesic and antirheumatic medicines, and is a raw material for producing and synthesizing a plurality of important medicines such as aspirin, sodium acetylsalicylate, sodium methyl salicylate, salicylamide, magnesium salicylate, salicylamine, salsalate and the like.
At present, only a salicylic acid related substance detection method is collected in Chinese pharmacopoeia, the related substance detection method adopts a reversed-phase high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filler, methanol-water-glacial acetic acid (60:40:1) is used as a mobile phase, and the detection wavelength is 270 nm.
Disclosure of Invention
The invention provides an HPLC detection method of sodium salicylate related substances, and conditions of high performance liquid chromatography comprise:
the chromatographic column adopts octadecylsilane chemically bonded silica with polar end groups sealed as a stationary phase;
an ultraviolet detector is adopted, and the detection wavelength is 200-300 nm; the flow rate of the mobile phase is 0.5-2.0 ml/min; the column temperature is 25-35 ℃; the mobile phase adopts a mixed solution of methanol, water and trifluoroacetic acid, the volume ratio of the methanol to the water to the trifluoroacetic acid is (35-45): 55-65): 0.05-0.15, and isocratic elution is carried out.
The HPLC detection method of the sodium salicylate related substance comprises the following conditions of high performance liquid chromatography:
the length of the chromatographic column is 150-250 mm; the detection wavelength is 260-280 nm; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 28-32 ℃; the volume ratio of the mobile phase methanol, water and trifluoroacetic acid is 38:62:0.1 or 40:60: 0.1.
The HPLC detection method of the sodium salicylate related substance has the detection wavelength of 270nm, 271nm or 272 nm.
The HPLC detection method of the sodium salicylate related substance comprises the following steps: taking a sodium salicylate sample, and dissolving and diluting the sodium salicylate sample by using a diluent to prepare a test solution.
According to the HPLC detection method of the sodium salicylate related substances, the diluent is a mixed solution of methanol and water or a mixed solution of methanol, water and trifluoroacetic acid.
The HPLC detection method of the sodium salicylate related substances comprises 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol.
The invention aims to obtain a detection method capable of simultaneously separating sodium salicylate and related substances thereof, namely 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol.
In an embodiment of the present application, the related substances comprise 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol. The invention aims to solve the separation between the main component of the sodium salicylate and each impurity so as to establish a set of complete sodium salicylate related substance control standard, and the method has the advantages of simple operation, high precision, good stability and good reproducibility.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a typical HPLC chromatogram of the detection method of the embodiment of the present invention, wherein 5.974 is 4-hydroxybenzoic acid, 9.952 is phenol, 14.477 is 4-hydroxyisophthalic acid, 23.181 is sodium salicylate;
FIG. 2 is a HPLC chromatogram of the detection method of comparative example 1 according to the present invention;
FIG. 3 is a HPLC chromatogram of the detection method of comparative example 2 of the present invention;
FIG. 4 is a HPLC chromatogram of the detection method of comparative example 3 according to the present invention;
FIG. 5 is an HPLC chromatogram of the detection method of comparative example 4 of the present invention.
Detailed Description
The above-mentioned contents are further explained by way of examples, but it should not be construed that the scope of the present invention is limited to the following examples. In the present application, the embodiments and features of the embodiments may be arbitrarily combined with each other without conflict.
Example 1
Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: phenomenex SynergiTM4μm Hydro-RP
250×4.6mm；
Flow rate: 1.0 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-trifluoroacetic acid (40:60:0.1) isocratic elution.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
trifluoroacetic acid: TEDIA, HPLC grade.
The implementation steps are as follows:
solution preparation: precisely weighing about 5mg of each of a 4-hydroxybenzoic acid reference substance, a 4-hydroxyisophthalic acid reference substance and a phenol reference substance, placing the reference substances in a 10ml measuring flask, dissolving and diluting the reference substances to scales by using a mobile phase, shaking up to obtain a reference stock solution, precisely weighing 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to scales by using the mobile phase, and shaking up to obtain an impurity reference substance solution; precisely weighing about 0.1g of sodium salicylate sample, placing the sodium salicylate sample in a 10ml measuring flask, adding an impurity reference substance solution to dissolve and dilute the sodium salicylate sample to a scale, and shaking up to obtain a test sample solution.
Precisely measuring 20 μ l of the test solution, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in table 1 and fig. 1, and the degrees of separation between each impurity and between sodium salicylate and the adjacent impurity are both greater than 1.5, indicating that the method is good in specificity.
TABLE 1 chromatographic Peak location and resolution test results
| Name of Compound | Retention time (minutes) | Degree of separation | Number of theoretical plate |
| 4-hydroxybenzoic acid | 5.974 | — | 9580.51 |
| Phenol and its preparation | 9.952 | 14.27 | 17235.88 |
| 4-Hydroxyisophthalic acid | 14.477 | 11.59 | 15610.15 |
| Salicylic acid sodium salt | 23.181 | 11.62 | 8277.75 |
Example 2
Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: phenomenex Synergi of different batchesTM4μm Hydro-RP
250×4.6mm；
Flow rate: 0.9-1.1 ml/min;
detection wavelength: 270 nm;
column temperature: 28-32 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-trifluoroacetic acid (40:60:0.1) or methanol-water-trifluoroacetic acid (38:62:0.1) isocratically.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
trifluoroacetic acid: TEDIA, HPLC grade.
The implementation steps are as follows:
the solution formulation was as described in example 1.
On the basis of the established chromatographic conditions, the column temperature and the flow rate are changed, chromatographic columns of different batches are replaced, 20 mu l of the test solution is precisely measured and injected into a liquid chromatograph, and the chromatogram is recorded. The results are shown in Table 2.
The experimental results are as follows:
the column temperature and flow rate are slightly adjusted and the chromatographic column is replaced, the separation degree between the main peak and the impurities and between the impurities is more than 1.5, and the method has good durability.
TABLE 2 test results of the separation degree of sodium salicylate-related substances under different conditions
Comparative example 1
(1) Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: phenomenex
C18
250×4.6mm；
Flow rate: 1.0 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-glacial acetic acid (60:40:1) isocratic elution.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
glacial acetic acid: national chemical group chemical agents limited, AR grade.
The implementation steps are as follows:
solution preparation: precisely weighing about 5mg of each of a 4-hydroxybenzoic acid reference substance, a 4-hydroxyisophthalic acid reference substance and a phenol reference substance, placing the reference substances in a 10ml measuring flask, dissolving and diluting the reference substances to scales by using a mobile phase, shaking up to obtain a reference stock solution, precisely weighing 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to scales by using the mobile phase, and shaking up to obtain an impurity reference substance solution; precisely weighing about 0.1g of sodium salicylate sample, placing the sodium salicylate sample in a 10ml measuring flask, adding an impurity reference substance solution to dissolve and dilute the sodium salicylate sample to a scale, and shaking up to obtain a test sample solution.
Precisely measuring 20 μ l of test solution, injecting into a liquid chromatograph, recording chromatogram, and obtaining experimental results shown in figure 2, wherein the main component of sodium salicylate has serious peak tailing, sodium salicylate and related substances have poor peak shape, and the content measurement result is inaccurate.
Comparative example 2
Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: merck
5μm 250×4.6mm；
Flow rate: 0.6 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-glacial acetic acid (60:40:1) isocratic elution.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
glacial acetic acid: national chemical group chemical agents limited, AR grade.
The implementation steps are as follows:
solution preparation: precisely weighing about 5mg of each of a 4-hydroxybenzoic acid reference substance, a 4-hydroxyisophthalic acid reference substance and a phenol reference substance, placing the reference substances in a 10ml measuring flask, dissolving and diluting the reference substances to scales by using a mobile phase, shaking up to obtain a reference stock solution, precisely weighing 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to scales by using the mobile phase, and shaking up to obtain an impurity reference substance solution; precisely weighing about 0.1g of sodium salicylate sample, placing the sodium salicylate sample in a 10ml measuring flask, adding an impurity reference substance solution to dissolve and dilute the sodium salicylate sample to a scale, and shaking up to obtain a test sample solution.
Precisely measuring 20 μ l of the test solution, injecting into a liquid chromatograph, recording chromatogram, and obtaining experimental results shown in figure 3, wherein the sodium salicylate main component chromatogram peak has poor shape and the impurity chromatogram peaks have overlapping and can not be used for measuring the content of each component.
Comparative example 3
(1) Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: phenomenex SynergiTM4μm Hydro-RP
250×4.6mm；
Flow rate: 0.6 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-glacial acetic acid (60:40:1) isocratic elution.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
trifluoroacetic acid: TEDIA, HPLC grade.
The implementation steps are as follows:
solution preparation: precisely weighing about 5mg of each of a 4-hydroxybenzoic acid reference substance, a 4-hydroxyisophthalic acid reference substance and a phenol reference substance, placing the reference substances in a 10ml measuring flask, dissolving and diluting the reference substances to scales by using a mobile phase, shaking up to obtain a reference stock solution, precisely weighing 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to scales by using the mobile phase, and shaking up to obtain an impurity reference substance solution; precisely weighing about 0.1g of sodium salicylate sample, placing the sodium salicylate sample in a 10ml measuring flask, adding an impurity reference substance solution to dissolve and dilute the sodium salicylate sample to a scale, and shaking up to obtain a test sample solution.
Precisely measuring 20 μ l of test solution, injecting into a liquid chromatograph, recording chromatogram, and obtaining experimental results shown in figure 4, wherein the main component of sodium salicylate has chromatographic peak front extension, peak shape difference, and impurity chromatographic peaks are overlapped and can not be used for measuring the content of each component.
Comparative example 4
Experimental apparatus and chromatographic conditions:
the instrument comprises the following steps: the Shimadzu liquid chromatograph LC-20AT is provided with an ultraviolet detector;
a chromatographic column: agilent ZORBAX Eclipse XDB-C185 μm 150X 4.6 mm;
flow rate: 1.0 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu l of the mixture;
mobile phase: methanol-water-trifluoroacetic acid (40:60:0.1) isocratic elution.
Experimental reagent:
methanol: beijing Bailingwei science and technology Co., Ltd., HPLC grade;
trifluoroacetic acid: TEDIA, HPLC grade.
The implementation steps are as follows:
solution preparation: precisely weighing about 5mg of each of a 4-hydroxybenzoic acid reference substance, a 4-hydroxyisophthalic acid reference substance and a phenol reference substance, placing the reference substances in a 10ml measuring flask, dissolving and diluting the reference substances to scales by using a mobile phase, shaking up to obtain a reference stock solution, precisely weighing 1.0ml of the reference stock solution, placing the reference stock solution in a 100ml measuring flask, diluting the reference stock solution to scales by using the mobile phase, and shaking up to obtain an impurity reference substance solution; precisely weighing about 0.1g of sodium salicylate sample, placing the sodium salicylate sample in a 10ml measuring flask, adding an impurity reference substance solution to dissolve and dilute the sodium salicylate sample to a scale, and shaking up to obtain a test sample solution.
Precisely measuring 20 μ l of sample solution, injecting into liquid chromatograph, recording chromatogram, and showing in figure 5 that the main component of sodium salicylate has chromatographic peak front extension, peak shape difference, and impurity chromatographic peaks overlap, and can not be used for measuring the content of each component.
Claims (6)
1. An HPLC detection method of sodium salicylate related substances is characterized in that high performance liquid chromatography is adopted for detection, and the conditions of the high performance liquid chromatography comprise:
the chromatographic column adopts octadecylsilane chemically bonded silica with polar end groups sealed as a stationary phase;
an ultraviolet detector is adopted, and the detection wavelength is 200-300 nm; the flow rate of the mobile phase is 0.5-2.0 ml/min; the column temperature is 25-35 ℃; the mobile phase adopts a mixed solution of methanol, water and trifluoroacetic acid, the volume ratio of the methanol to the water to the trifluoroacetic acid is 35-45: 55-65: 0.05-0.15, and isocratic elution is carried out.
2. The HPLC detection method of sodium salicylate related substance according to claim 1, wherein the conditions of high performance liquid chromatography include:
the length of the chromatographic column is 150-250 mm; the detection wavelength is 260-280 nm; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 28-32 ℃; the volume ratio of the mobile phase methanol, water and trifluoroacetic acid is 38:62:0.1 or 40:60: 0.1.
3. The HPLC detection method for sodium salicylate-related substances according to claim 1 or 2, wherein the detection wavelength is 270nm, 271nm or 272 nm.
4. The HPLC detection method for sodium salicylate-related substances according to claim 1 or 2, wherein the sample solution preparation: taking a sodium salicylate sample, and dissolving and diluting the sodium salicylate sample by using a diluent to prepare a test solution.
5. The HPLC detecting method for sodium salicylate-related substances according to claim 1 or 2, wherein the diluent is a mixed solution of methanol and water or a mixed solution of methanol, water and trifluoroacetic acid.
6. The HPLC detecting method for sodium salicylate related substance according to claim 1 or 2, wherein said related substance comprises 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and phenol.
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| CN202010585699.8A CN111707750A (en) | 2020-06-24 | 2020-06-24 | HPLC detection method for related substances of sodium salicylate |
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| CN202010585699.8A CN111707750A (en) | 2020-06-24 | 2020-06-24 | HPLC detection method for related substances of sodium salicylate |
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Application publication date: 20200925 |