CN112782332A - HPLC (high performance liquid chromatography) detection method for p-aminophenol impurities in acetaminophen medicine - Google Patents
HPLC (high performance liquid chromatography) detection method for p-aminophenol impurities in acetaminophen medicine Download PDFInfo
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- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 title claims abstract description 126
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 239000012535 impurity Substances 0.000 title claims abstract description 48
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 44
- 229960005489 paracetamol Drugs 0.000 title claims abstract description 42
- 239000003814 drug Substances 0.000 title claims abstract description 22
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 82
- 239000000243 solution Substances 0.000 claims abstract description 71
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 41
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 41
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 41
- 239000002904 solvent Substances 0.000 claims abstract description 26
- 239000012085 test solution Substances 0.000 claims abstract description 24
- OQKFGIANPCRSSK-UHFFFAOYSA-N azanium;methanol;acetate Chemical compound [NH4+].OC.CC([O-])=O OQKFGIANPCRSSK-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000012488 sample solution Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 238000012360 testing method Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 41
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 19
- 239000005695 Ammonium acetate Substances 0.000 claims description 19
- 235000019257 ammonium acetate Nutrition 0.000 claims description 19
- 229940043376 ammonium acetate Drugs 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 10
- 238000010812 external standard method Methods 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 5
- 239000013558 reference substance Substances 0.000 claims description 5
- 238000007877 drug screening Methods 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims description 2
- 238000010268 HPLC based assay Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 16
- 239000000523 sample Substances 0.000 abstract description 15
- 230000007547 defect Effects 0.000 abstract description 4
- 238000007865 diluting Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 6
- 239000002245 particle Substances 0.000 description 5
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 5
- 235000019345 sodium thiosulphate Nutrition 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
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- 238000004090 dissolution Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
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- 239000000126 substance Substances 0.000 description 4
- 241000599985 Beijerinckia mobilis Species 0.000 description 3
- 241000218671 Ephedra Species 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- VJBCNMFKFZIXHC-UHFFFAOYSA-N azanium;2-(4-methyl-5-oxo-4-propan-2-yl-1h-imidazol-2-yl)quinoline-3-carboxylate Chemical compound N.N1C(=O)C(C(C)C)(C)N=C1C1=NC2=CC=CC=C2C=C1C(O)=O VJBCNMFKFZIXHC-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940124579 cold medicine Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 230000000202 analgesic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention provides an HPLC detection method for p-aminophenol impurities in acetaminophen medicaments, which comprises the following steps: dissolving the acetaminophen medicament by using an ammonium acetate-methanol solution containing ascorbic acid as a solvent to obtain a test sample solution; and detecting the content of p-aminophenol impurities in the test solution by using a high performance liquid chromatograph with the ammonium acetate-methanol solution as a mobile phase. According to the HPLC detection method provided by the invention, when a sample to be detected is pretreated, ascorbic acid is added into an ammonium acetate-methanol solution serving as a solvent, so that p-aminophenol impurities are stable and cannot be oxidized and degraded, the sample solution is not required to be prepared on site, and the defect that the p-aminophenol impurities are lost in a solution preparation link is overcome.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to an HPLC (high performance liquid chromatography) detection method for p-aminophenol impurities in a p-acetaminophen medicine.
Background
Acetaminophen has antipyretic and analgesic effects, and can be widely used in compound cold medicine, and can be used for treating arthralgia, neuralgia, migraine, cancer pain and postoperative pain. Para-aminophenol is a process and degradation impurity of acetaminophen and therefore can be produced either during production or storage. The acetaminophen has high toxicity and can cause mutation, and the limit of the acetaminophen in the medicine is strict, and the acetaminophen is generally required to be 0.1% less than the labeled amount.
Currently, the detection of p-aminophenol mainly comprises an electrochemical method, a Thin Layer Chromatography (TLC) method and a High Performance Liquid Chromatography (HPLC) method. The electrochemical method has multiple interference factors and poor specificity, and is easy to generate larger measurement errors. The sensitivity of the TLC method is low, and accurate quantitative measurement cannot be performed. The HPLC method has high sensitivity, precision and accuracy, and is the mainstream method adopted at present.
In the authoritative pharmacopoeias at home and abroad such as the China pharmacopoeia 2020 edition, the British pharmacopoeia 2020 edition, the United states pharmacopoeia 43 edition and the like, impurity inspection items are arranged in various theories of acetaminophen bulk drugs and preparations thereof for monitoring the content of acetaminophen impurities, and HPLC methods are adopted. However, because the chemical property of the p-aminophenol impurity is unstable, the p-aminophenol impurity is rapidly oxidized and degraded into other substances in a solution, thereby influencing the measurement result. In order to solve this problem, each pharmacopoeia requires a solution to be prepared on site, but this has an influence on the working efficiency, and particularly when the sample size is large, the examiner must keep the apparatus and continuously perform the solution preparation operation. And the problem cannot be completely solved by 'preparation in situ after use', when a sample is contacted with a solution, oxygen in the solution inevitably reacts with p-aminophenol impurities, even if the sample is injected immediately after the preparation is finished, the p-aminophenol is degraded to a certain degree in the preparation process of the solution, and particularly, the solution is a variety which needs to be subjected to complex pretreatment and has long solution preparation time.
In addition, part of the compound cold medicines in China still do not have examination items for aminophenol impurities, so that the supervision of medicine quality is not facilitated, and an easy-to-operate and accurate determination method is urgently needed.
Disclosure of Invention
In view of the problems of the prior art, the present invention aims to provide an HPLC method suitable for detecting p-aminophenol impurities in acetaminophen type medicines.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for HPLC detection of p-aminophenol impurities in a p-acetaminophen preparation, said method comprising:
dissolving the acetaminophen medicament by using an ammonium acetate-methanol solution containing ascorbic acid as a solvent to obtain a test sample solution; and detecting the content of p-aminophenol impurities in the test solution by using a high performance liquid chromatograph with the ammonium acetate-methanol solution as a mobile phase.
According to the HPLC detection method provided by the invention, when a sample to be detected is pretreated, ascorbic acid is added into an ammonium acetate-methanol solution serving as a solvent, so that p-aminophenol impurities are stable and cannot be oxidized and degraded, the sample solution is not required to be prepared on site, and the defect that the p-aminophenol impurities are lost in a solution preparation link is overcome.
As a preferred embodiment of the present invention, the sample solution contains ascorbic acid at a mass concentration of 0.001 to 0.1mg/mL, and may be, for example, 0.001mg/mL, 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.05mg/mL, 0.06mg/mL, 0.08mg/mL, or 0.1 mg/mL.
Preferably, the concentration of acetaminophen in the sample solution is 1 to 5mg/mL, and may be, for example, 1mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, or 5 mg/mL.
Preferably, the concentration of the p-aminophenol impurity in the sample solution is 1 to 5. mu.g/mL, and may be, for example, 1mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, or 5 mg/mL.
The concentration of the p-aminophenol impurity is 1-5 mu g/mL, which means the concentration of the p-aminophenol impurity obtained through final test, and the dilution multiple of the sample can be determined according to the calibrated or estimated content of the p-aminophenol impurity, so that the concentration of the p-aminophenol impurity obtained through final test is in the range, and if the measured concentration is obviously not in the range, the sample needs to be diluted again for detection.
In the invention, if a chromatographic peak consistent with the retention time of p-aminophenol exists in a chromatogram of a test solution, the content of p-aminophenol is 0.1 percent of the labeled amount of p-acetaminophen calculated by a peak area according to an external standard method.
Preferably, the ammonium acetate-methanol solution is obtained by mixing an ammonium acetate solution and methanol.
Preferably, the volume ratio of the ammonium acetate solution to the methanol is (85-91): 15-9, and may be, for example, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, or the like.
Preferably, the molar concentration of ammonium acetate in the ammonium acetate solution is 0.03-0.07 mol/L, and may be, for example, 0.03mol/L, 0.035mol/L, 0.04mol/L, 0.045mol/L, 0.05mol/L, 0.055mol/L, 0.06mol/L, 0.065mol/L, or 0.07 mol/L.
In a preferred embodiment of the present invention, the HPLC detection method uses a C18 column for detection.
The chromatographic column used in the invention is a C18 chromatographic column, and can separate substances in a sample, so that the separation degree of a p-aminophenol peak, a p-acetaminophen peak and adjacent peaks in a map is higher. The column used in the present invention may be a Zorbax SB-C18 column or a Shim-pack Scepter C18 column, and may have a size of 4.6mm X250 mm and a particle size of about 5 μm.
Preferably, the detector used in the HPLC detection method is an ultraviolet detector and/or a diode array detector.
Preferably, the detection wavelength of the detector may be 230nm, 231nm, 232nm or the like, preferably 231 nm.
Preferably, the HPLC detection method adopts an external standard method to detect the content of p-aminophenol impurities.
Preferably, the concentration of p-aminophenol in the control solution used in the external standard method is 2 to 2.5. mu.g/mL, for example, 2. mu.g/mL, 2.1. mu.g/mL, 2.2. mu.g/mL, 2.3. mu.g/mL, 2.4. mu.g/m, or 2.5. mu.g/mL, and preferably 2.5. mu.g/mL.
As a preferable technical scheme of the invention, the detection time of the HPLC detection method is 8-15 min, for example, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15 min.
As a preferred technical scheme of the invention, the HPLC detection method comprises the following steps:
(1) mixing an ammonium acetate solution with the molar concentration of 0.03-0.07 mol/L and methanol according to the volume ratio of (85-91) to (15-9) to obtain an ammonium acetate-methanol solution;
adding ascorbic acid into the ammonium acetate-methanol solution, and then dissolving the acetaminophen medicine to obtain a test solution, wherein the test solution contains ascorbic acid with the mass concentration of 0.001-0.1 mg/mL, the concentration of acetaminophen is 1-5 mg/mL, and the concentration of acetaminophen impurities is 1-5 mu g/mL;
preparing a reference substance solution, wherein the concentration of p-aminophenol in the reference substance solution is 2.5 mu g/mL;
(2) and detecting the content of p-aminophenol impurities in the test solution by using a high performance liquid chromatograph under the detection wavelength of 231nm by taking the ammonium acetate-methanol solution as a mobile phase.
Illustratively, the HPLC detection method of the present invention can be performed using the following steps:
(1) solution preparation:
solvent: adding ascorbic acid into 0.05mol/L ammonium acetate solution-methanol (volume ratio is 88:12), dissolving and diluting, and diluting to reach volume of 1000mL, wherein the concentration of the ascorbic acid is 0.01 mg/mL;
mobile phase: taking 0.05mol/L ammonium acetate solution-methanol (volume ratio is 88:12) as a mobile phase;
test solution: taking a proper amount of a sample to be measured, precisely weighing, adding a solvent for dissolution and quantitatively diluting to prepare a solution containing about 2.5mg of acetaminophen in every 1mL, and filtering;
control solution: taking a proper amount of p-aminophenol reference substances, precisely weighing, adding a mobile phase for dissolving, and quantitatively diluting to prepare a solution containing 2.5 mu g of p-aminophenol in 1 mL;
sensitivity solution: precisely measuring 1mL of the reference solution, placing the reference solution in a 10mL measuring flask, diluting the reference solution to a scale with the test solution, and shaking up;
(2) and (3) computer detection:
precisely measuring a test solution and a reference solution, respectively injecting into a liquid chromatograph, and recording a chromatogram until the peak appearance of the acetaminophen peak is complete;
the detection column is a C18 chromatographic column, the column temperature is 30 ℃, and the detection wavelength is 231 nm; the sample injection volume is 10 mu L;
and (4) after the spectrogram is recorded, calculating the content of the p-aminophenol according to an external standard method.
In a second aspect, the present invention also provides a use of the HPLC detection method of the first aspect in drug screening or drug quality control.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
in order to solve the problem of oxidative degradation of the aminophenol impurities, the method provided by the invention uses a solvent with an antioxidant effect, the ascorbic acid can realize a protection effect under a very low concentration, and is superior to other common antioxidants, the stability of the aminophenol impurities can be protected within 12-24 hours, a sample solution is not required to be prepared in situ, and the defect that the aminophenol impurities are lost in a solution preparation link is eliminated;
meanwhile, within the concentration range limited by the invention, ascorbic acid does not generate any interference to the determination process, the base line of the chromatogram can be kept smooth, and the chromatogram acquired under the set wavelength does not generate an interference chromatographic peak after the solvent peak, so that the method is also suitable for detecting other related substances in the medicine while detecting p-aminophenol impurities.
Drawings
FIG. 1 is a HPLC analysis chart obtained when 0.001mg/mL ascorbic acid was contained in the test solution in example 1.
FIG. 2 is a HPLC analysis chart obtained when the test solution in example 6 contains 1mg/mL of ascorbic acid.
FIG. 3 is a HPLC analysis chart obtained when 0.1mg/mL ascorbic acid was contained in the test solution in example 7.
FIG. 4 is a HPLC detection pattern obtained when 0.01mg/mL sodium thiosulfate was contained in the test solution in comparative example 2.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
In the following examples, unless otherwise specified, reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.
Example 1
The embodiment provides an HPLC detection method for p-aminophenol impurities in an acetaminophen medicament, which specifically includes the following steps:
(1) solution preparation:
a. solvent: mixing ammonium acetate solution with the molar concentration of 0.05mol/L and methanol, wherein the volume ratio of the ammonium acetate solution to the methanol is 88:12, adding 1mg of ascorbic acid, dissolving and diluting to 1000mL to obtain a solvent with the ascorbic acid concentration of 0.001 mg/mL;
b. mobile phase: mixing ammonium acetate solution with the molar concentration of 0.05mol/L and methanol according to the volume ratio of 88: 12;
c. test solution: 1.2637g of paracetamol, ephedra and American dry suspension (with the calibrated content of paracetamol being 0.080g/g) is taken as a sample to be detected, 10mL of solvent is added for dissolution, and quantitative dilution is carried out to prepare a solution containing 2.5mg of paracetamol in each 1mL, and filtration is carried out;
d. control solution: a control solution containing 2.5. mu.g of p-aminophenol per 1mL was prepared by dissolving 25.03mg of p-aminophenol control in 10mL of a solvent (i.e., an ammonium acetate-methanol solution containing ascorbic acid) and quantitatively diluting.
(2) And (3) computer detection: detecting with high performance liquid chromatograph (Waters corporation, USA, e2695-2998), with ultraviolet detector wavelength of 231 nm;
the injection volume is 10 μ L, and the chromatographic column is Zorbax SB-C18(4.6mm × 250mm, particle size 5 μm);
the obtained detection spectrum is shown in figure 1, and the peak of p-aminophenol appears about 4min, and the obtained spectrum has a smooth baseline and no interference peak, so that the obtained detection concentration is more accurate.
Example 2
The embodiment provides an HPLC detection method for p-aminophenol impurities in an acetaminophen medicament, which specifically includes the following steps:
(1) solution preparation:
a. solvent: mixing ammonium acetate solution with the molar concentration of 0.07mol/L and methanol according to the volume ratio of 85:15, adding ascorbic acid for dissolving and diluting to 1000mL to obtain a solvent with the ascorbic acid concentration of 0.01 mg/mL;
b. mobile phase: mixing ammonium acetate solution with the molar concentration of 0.07mol/L and methanol according to the volume ratio of 85: 15;
c. test solution: 1.2637g of paracetamol, ephedra and American dry suspension (with the calibrated content of paracetamol being 0.080g/g) is taken as a sample to be detected, 10mL of solvent is added for dissolution, and quantitative dilution is carried out to prepare a solution containing 2.5mg of paracetamol in each 1mL, and filtration is carried out;
d. control solution: a control solution containing 2.5. mu.g of p-aminophenol per 1mL was prepared by dissolving 25.03mg of p-aminophenol control in 10mL of a solvent (i.e., an ammonium acetate-methanol solution containing ascorbic acid) and quantitatively diluting.
(2) And (3) computer detection: detecting with high performance liquid chromatograph (Waters corporation, USA, e2695-2998), with ultraviolet detector wavelength of 231 nm;
the injection volume was 10. mu.L, and the column was Zorbax SB-C18(4.6 mm. times.250 mm, particle size 5 μm).
Example 3
The embodiment provides an HPLC detection method for p-aminophenol impurities in an acetaminophen medicament, which specifically includes the following steps:
(1) solution preparation:
a. solvent: mixing ammonium acetate solution with the molar concentration of 0.03mol/L and methanol according to the volume ratio of 91:9, adding ascorbic acid for dissolving and diluting to 1000mL to obtain a solvent with the ascorbic acid concentration of 0.01 mg/mL;
b. mobile phase: mixing ammonium acetate solution with the molar concentration of 0.03mol/L and methanol according to the volume ratio of 91: 9;
c. test solution: 1.2637g of paracetamol, ephedra and American dry suspension (with the calibrated content of paracetamol being 0.080g/g) is taken as a sample to be detected, 10mL of solvent is added for dissolution, and quantitative dilution is carried out to prepare a solution containing 2.5mg of paracetamol in each 1mL, and filtration is carried out;
d. control solution: a control solution containing 2. mu.g of p-aminophenol per 1mL is prepared by dissolving 20mg of p-aminophenol control in 10mL of a solvent (i.e., an ammonium acetate-methanol solution containing ascorbic acid) and quantitatively diluting.
(2) And (3) computer detection: detecting with high performance liquid chromatograph (Waters corporation, USA, e2695-2998), with ultraviolet detector wavelength of 231 nm;
the injection volume was 10. mu.L, and the column was Shim-pack Scepter C18(4.6 mm. times.250 mm, particle size 5 μm).
Example 4
The difference from example 1 is that in this example, the concentration of ascorbic acid in the solvent is 0.01 mg/mL; the rest steps and parameters are consistent with those of the embodiment 1;
example 5
The difference from example 1 is that in this example, the concentration of ascorbic acid in the solvent is 0.005 mg/mL; the rest steps and parameters are consistent with those of the embodiment 1;
example 6
The difference from example 1 is that in this example, the concentration of ascorbic acid in the solvent is 1 mg/mL; the rest steps and parameters are consistent with those of the embodiment 1;
example 7
The difference from example 1 is that in this example, the concentration of ascorbic acid in the solvent is 0.1 mg/mL; the rest steps and parameters are consistent with those of the embodiment 1;
example 8
The difference from example 4 is that in this example, the column used was Shim-pack Scepter C18(4.6 mm. times.250 mm, particle size 5 μm); the rest steps and parameters are consistent with those of the embodiment 1;
comparative example 1
The difference from example 4 is that in this example, ascorbic acid was not added; the rest steps and parameters are consistent with those of the embodiment 1;
comparative example 2
The difference from example 4 is that in this example, ascorbic acid is replaced by sodium thiosulfate; the rest steps and parameters are consistent with those of the embodiment 1;
evaluation of the method
In the present invention, the same sample was tested in the examples and the comparative examples, and the peak areas of p-aminophenol in the detection spectra obtained in examples 1, 4 and 8 were shown in table 1, wherein the peak areas were measured at 0, 1, 2, 3, 6, 12, 18 and 24 hours after the preparation of the sample solution;
TABLE 1
As can be seen from the above table, the sample solutions provided in examples 1, 4 and 8 were all stable (less than 2% change) within 12 hours, whereas example 1 was 9.4% within 24 hours, and both example 2 and example 3 were stable within 24 hours;
comparative example 1 no ascorbic acid was added and tested immediately after compounding, showing a 10.7% loss of p-aminophenol compared to the test solution of example 1;
in addition, the maps obtained in example 6, example 7 and comparative example 2 are shown in fig. 2 to 4;
in example 6 (as shown in fig. 2), when ascorbic acid is contained in 1mg/mL, an interference peak appears on the right side of aminophenol, which affects the integral, and it means that the addition amount of ascorbic acid in the test solution needs to be paid attention to, and is not easy to be too high, otherwise, an interference peak appears to further affect the experimental result; in example 7 (as shown in FIG. 3), when 0.1mg/mL of ascorbic acid is contained, no interference exists after the solvent peak; in comparative example 2 (FIG. 4), when the sample solution contained 0.01mg/mL of sodium thiosulfate, an interference chromatographic peak appeared in the vicinity of the p-aminophenol peak.
The concentrations of p-aminophenol measured at 12 hours in examples 1 to 8 and comparative examples 1 to 2 are shown in Table 2 below;
TABLE 2
As shown in the table above, the concentration of p-aminophenol detected in 12 hours by the method provided by the invention is more than 2 mug/mL, which indicates that p-aminophenol impurities in the solution are more stable and have smaller change rate; and as can be seen from the comparison between the example 4 and the comparative example 2, the antioxidant in the invention adopts ascorbic acid, and the antioxidant effect is better; in addition, when the sample solution contains 1mg/mL of sodium thiosulfate, the p-aminophenol concentration after being placed for 24 hours is tested, and the p-aminophenol impurity is still degraded by 4.7 percent; when the sample solution contains 0.005mg/mL of ascorbic acid, the concentration of the ascorbic acid is 200 times lower than that of sodium thiosulfate, the aminophenol impurity is still stable after the sample solution is placed for 24 hours, the change rate is less than 0.8 percent,
in contrast, as can be seen from comparison between example 4 and example 6, when the concentration of ascorbic acid in the sample solution is higher, the impurity concentration of the sample obtained by detection is higher, on one hand, the p-aminophenol impurity is more stable, and on the other hand, the result is larger due to the occurrence of an error in the statistical process due to the occurrence of a hetero-peak in the detection map.
In conclusion, the HPLC detection method for the p-aminophenol impurities in the acetaminophen medicament provided by the invention can protect the p-aminophenol impurities to be stable within 12-24 hours, the sample solution is not required to be prepared immediately after use, and the defect that the p-aminophenol impurities are lost in the solution preparation link is overcome.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. An HPLC detection method for p-aminophenol impurities in an acetaminophen medicament, which is characterized by comprising the following steps:
dissolving the acetaminophen medicament by using an ammonium acetate-methanol solution containing ascorbic acid as a solvent to obtain a test sample solution;
and detecting the content of p-aminophenol impurities in the test solution by using a high performance liquid chromatograph with the ammonium acetate-methanol solution as a mobile phase.
2. An HPLC detection method according to claim 1, wherein the sample solution contains ascorbic acid at a mass concentration of 0.001-0.1 mg/mL;
preferably, the concentration of the acetaminophen in the test solution is 1-5 mg/mL;
preferably, the concentration of p-aminophenol impurities in the test solution is 1 to 5 [ mu ] g/mL.
3. An HPLC detection method according to claim 1 or 2, wherein the ammonium acetate-methanol solution is obtained by mixing an ammonium acetate solution and methanol;
preferably, the volume ratio of the ammonium acetate solution to the methanol is (85-91): 15-9;
preferably, the molar concentration of ammonium acetate in the ammonium acetate solution is 0.03-0.07 mol/L.
4. An HPLC detection method according to any one of claims 1 to 3, wherein the HPLC detection method uses a C18 column for detection.
5. An HPLC detection method according to any one of claims 1 to 4, wherein the detector used in the HPLC detection method is an ultraviolet detector and/or a diode array detector;
preferably, the detection wavelength of the detector is 230-232 nm, preferably 231 nm.
6. An HPLC detection method according to any one of claims 1 to 5, wherein the HPLC detection method adopts an external standard method to detect the content of p-aminophenol impurities.
7. An HPLC detection method according to claim 6, wherein the concentration of p-aminophenol in the control solution used in the external standard method is 2-2.5 μ g/mL, preferably 2.5 μ g/mL.
8. An HPLC detection method according to any one of claims 1 to 7, wherein the detection column temperature of the HPLC detection method is 28 to 32 ℃, preferably 30 ℃;
preferably, the detection time of the HPLC detection method is 8-15 min.
9. An HPLC detection method according to any one of claims 1 to 8, wherein the HPLC detection method comprises the steps of:
(1) mixing an ammonium acetate solution with the molar concentration of 0.03-0.07 mol/L and methanol according to the volume ratio of (85-91) to (15-9) to obtain an ammonium acetate-methanol solution;
adding ascorbic acid into the ammonium acetate-methanol solution, and then dissolving the acetaminophen medicine to obtain a test solution, wherein the test solution contains ascorbic acid with the mass concentration of 0.001-0.1 mg/mL, the concentration of acetaminophen is 1-5 mg/mL, and the concentration of acetaminophen impurities is 1-5 mu g/mL;
preparing a reference substance solution, wherein the concentration of p-aminophenol in the reference substance solution is 2.5 mu g/mL;
(2) and detecting the content of p-aminophenol impurities in the test solution by using a high performance liquid chromatograph under the detection wavelength of 231nm by taking the ammonium acetate-methanol solution as a mobile phase.
10. Use of an HPLC assay method according to any one of claims 1 to 9 for drug screening or drug quality control.
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