CN116754705A - Method for detecting acetic acid and acetate content - Google Patents
Method for detecting acetic acid and acetate content Download PDFInfo
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 165
- 238000000034 method Methods 0.000 title claims abstract description 61
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 title claims abstract description 39
- 239000003814 drug Substances 0.000 claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 238000010828 elution Methods 0.000 claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 15
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 15
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 15
- 239000007864 aqueous solution Substances 0.000 claims abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 229960000583 acetic acid Drugs 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 51
- 239000002904 solvent Substances 0.000 claims description 39
- 238000007865 diluting Methods 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 19
- 239000012488 sample solution Substances 0.000 claims description 19
- 239000011550 stock solution Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000005303 weighing Methods 0.000 claims description 16
- 239000012362 glacial acetic acid Substances 0.000 claims description 13
- 229940079593 drug Drugs 0.000 claims description 10
- 239000012085 test solution Substances 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 9
- 239000013558 reference substance Substances 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 239000012490 blank solution Substances 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000003643 water by type Substances 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 1
- 238000009210 therapy by ultrasound Methods 0.000 claims 1
- 239000012071 phase Substances 0.000 description 37
- 238000004817 gas chromatography Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 239000013557 residual solvent Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000004737 colorimetric analysis Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 150000004683 dihydrates Chemical class 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- VPPJLAIAVCUEMN-GFCCVEGCSA-N lacosamide Chemical compound COC[C@@H](NC(C)=O)C(=O)NCC1=CC=CC=C1 VPPJLAIAVCUEMN-GFCCVEGCSA-N 0.000 description 2
- 229960002623 lacosamide Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 150000005837 radical ions Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012362 drug development process Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960005112 moxifloxacin hydrochloride Drugs 0.000 description 1
- IDIIJJHBXUESQI-DFIJPDEKSA-N moxifloxacin hydrochloride Chemical compound Cl.COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 IDIIJJHBXUESQI-DFIJPDEKSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002604 ultrasonography Methods 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
- G01N30/89—Inverse chromatography
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention provides a high performance liquid chromatography detection method for acetic acid and acetate in a medicine, which comprises the following steps: the chromatographic column is a reverse chromatographic column, the sodium dihydrogen phosphate aqueous solution and acetonitrile are used as a mobile phase A and/or a mobile phase B for gradient elution, and the method has good detection specificity and high accuracy.
Description
Technical Field
The invention relates to a detection method of a medicine residual solvent, in particular to a high performance liquid chromatography detection method of acetic acid and acetate residual quantity in a medicine.
Background
In order to ensure the safety of the medicine in human body, ICH formulates Q3C impurity: the guidelines for residual solvents suggest acceptable amounts of residual solvents for pharmaceuticals under conditions of human safety. The guiding principle classifies acetic acid into low-toxicity solvents in the process of drug production and purification, and the solvents have little harm to human bodies, but many of the solvents have not been subjected to long-term toxicity or carcinogenic research, so the content limitation is required in GMP or other quality requirements.
Based on this, the acetic acid content in the residual solvent needs to be limited in the drug development process. Acetic acid used in the drug synthesis process may be remained in the form of acetic acid or acetate, so quality detection of acetic acid and acetate is required in the drug synthesis research and development process to limit the content level of acetic acid.
At present, a common detection method of the residual solvent acetic acid of the medicine is a gas chromatography method, for example, a detection method of the residual solvent acetic acid in moxifloxacin hydrochloride published in China patent document CN 104297365A, namely, a gas chromatography method is adopted. However, because acetic acid has stronger polarity and high boiling point, the formed gas chromatography peak shape is poor, and acetic acid is easy to react with gas phase solvents such as DMSO, DMF and the like to generate unknown peaks, which is not beneficial to accurate determination of the acetic acid content.
In addition, gas chromatography is suitable for detecting gases or volatile gases and solids, and can not detect acetate, so the currently commonly used method for detecting acetate is a colorimetric method. The method for detecting the content of acetate in the medicine disclosed in the Chinese patent document CN 115046950A comprises the following colorimetric method: the method comprises the steps of establishing a standard curve of absorbance-acid radical ion concentration, obtaining absorbance of a sample to be detected, and calculating the content of acid radical ions in the sample according to the standard curve and the absorbance of the sample to be detected. Although the colorimetric method is simple to operate, the detection accuracy is extremely low, because the colorimetric method is based on the color development of the generated colored compound, the pertinence is strong, the use condition is more strict, the colorimetric method is easily interfered by other components, and the colorimetric method is easily influenced by light, environment and the like.
Therefore, the development of the rapid and highly-universal acetic acid and acetate detection method has important significance in the aspect of quality control of medicines.
Disclosure of Invention
The invention aims to establish a high performance liquid chromatography method capable of accurately detecting the residual quantity of acetic acid and acetate in a medicine, and makes up the defects of the prior art.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, the invention provides a method for detecting acetic acid and acetate content in a drug, which is a high performance liquid chromatography method, wherein a reversed phase chromatographic column is adopted, sodium dihydrogen phosphate aqueous solution and acetonitrile are used as a mobile phase A and/or a mobile phase B, and the elution is performed according to isocratic elution or gradient elution.
In some embodiments, the drug is a laninamide.
In other embodiments, the molar concentration of the sodium dihydrogen phosphate aqueous solution is 0.01 to 0.05M.
Preferably, the molar concentration of the sodium dihydrogen phosphate aqueous solution is 0.02 and M.
In some embodiments, the chromatographic column employed is a pure water resistant phase chromatographic column.
Further, the pure water resistant phase chromatographic column is Waters Atlantis ® T3, its specification is 150 mm ×4.6 mm ×5 μm.
In some embodiments, the column temperature is 20-40 ℃.
Preferably, the column temperature is 25 ℃.
In some embodiments, the elution is performed according to a gradient in the elution process, wherein the initial proportion of the elution gradient is 95% -100% of the phase A, and the final proportion is 50% -100% of the phase B; wherein the mobile phase proportion is the volume percentage of each mobile phase to the total volume of the eluent, and the sum of the volume proportions of mobile phases A and B is 100%.
In some embodiments, the eluent has a flow rate of 0.5 to 1.5 mL/min; preferably, the eluent flow rate is 1.0 mL/min.
In some embodiments, a high performance liquid chromatograph detector ultraviolet detector is employed.
Further, the detection wavelength is 200 nm-280 nm.
Preferably, the detection wavelength is 210 nm.
In some embodiments, one or more of the following steps are included:
(a) Preparing blank solution, reference substance solution and test substance solution (namely medicine solution), wherein:
blank solvent: namely diluting solvent which is pure water or high-proportion water solution;
control solution: dissolving glacial acetic acid with a diluting solvent to dilute to a certain concentration;
test solution: dissolving a sample solution in a diluting solvent to dilute the sample solution to a certain concentration;
(b) Respectively taking 10-100 mu L of the blank solution, the reference substance solution and the sample solution, injecting the blank solution, the reference substance solution and the sample solution into a high performance liquid chromatograph, performing gradient elution according to the following table, and recording a chromatogram;
| time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 7 | 100 | 0 |
| 8 | 30 | 70 |
| 20 | 30 | 70 |
| 21 | 100 | 0 |
| 30 | 100 | 0 |
(c) According to the chromatographic peak area, the contents of the acetic acid and the acetate of the test sample are calculated, and the calculation method is as follows:
in some embodiments, the specific formulation method of the control and test solutions is as follows:
control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; precisely measuring the stock solution 1mL, placing in a 50 mL measuring flask, diluting to scale with solvent, shaking to obtain reference solution;
test solution: taking about 250 mg of the lank amide, precisely weighing, placing into a 25 mL measuring flask, adding a proper amount of solvent, ultrasonically shaking for 10 min to dissolve, diluting to a scale with a diluent, and shaking uniformly to obtain a sample solution;
mobile phase a (i.e. sodium dihydrogen phosphate aqueous solution): weighing 2.3996 g (+ -0.24 g) sodium dihydrogen phosphate (such as dihydrate according to the molar concentration, and converting into sample), dissolving in a reagent bottle containing 1000 mL water, adding 1.0 mL phosphoric acid, mixing, filtering, and ultrasonic treating.
The technical scheme for measuring the acetic acid and the acetate which are the residual solvents of the medicine provided by the invention has the advantages of high accuracy, strong specificity and good repeatability, and can qualitatively and quantitatively detect the acetic acid and the acetate in the medicine, so that the quality of the medicine is ensured, and the safety of the medicine is improved.
Drawings
FIG. 1 shows a gas chromatography detection acetic acid residue pattern
FIG. 2 is a diagram showing a blank solution for detecting acetic acid and acetate content by high performance liquid chromatography
FIG. 3 is a diagram showing the high performance liquid chromatography for detecting acetic acid and acetate content
FIG. 4 is a solution chart of a high performance liquid chromatography detection acetic acid and acetate content measurement reference substance
Detailed Description
The following detailed description of the invention relates to methods for measuring acetic acid and acetate content in a drug by way of example, it being apparent that the examples described are only some, but not all, examples of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Terminology
The medicine is used in preventing, treating and diagnosing diseases, and may be natural chemical matter and its preparation, synthesized compound and its preparation, recombinant protein, antibody, etc. Preferably, some embodiments of the present invention relate to synthetic compounds and pharmaceutical materials and formulations formed therefrom.
The acetate is an acetate ion, and has a chemical formula of CH3COO-, and can exist in a free ion form in a medicine solution, and can also form acetate with other substances to exist in an acetate form.
The pure water resistant chromatographic column disclosed by the invention refers to a chromatographic column with a tolerance effect on pure water or a high-proportion aqueous solution mobile phase, and comprises, but is not limited to, an AQ series, such as a ShimNex HE C18-AQ pure water resistant chromatographic column Shimadzu ODS carbon-eighteen high-purity silica gel reversed-phase liquid column; ODS series such as an Epidet SinoChrom ODS-BP column; RP series such as Waters XTerra RP-18 liquid chromatography columns; t3 chromatographic columns, e.g. Waters Atlantis ® T3, etc.
The "test substance" in the present invention refers to a target drug to be detected.
The invention relates to a test solution, which is prepared from a target drug to be detected.
The inventor performs a large number of experiments in the process of exploring the detection method of acetic acid and acetate in the medicine, and finds that the gas chromatography is adopted to detect the content of the acetic acid in the medicine, the chromatographic peak is seriously trailing, the peak shape is worse, and the accurate detection of the content of the acetic acid in the medicine is not facilitated.
In the process, the inventor unexpectedly discovers that the content of acetic acid and acetate in the medicine can be rapidly and accurately detected by adopting a high performance liquid chromatography method, taking a sodium dihydrogen phosphate aqueous solution and/or acetonitrile mixed solution as a mobile phase and using a reversed phase chromatographic column to detect the acetic acid and the acetate.
In addition, the inventor finds that when the adopted reversed-phase chromatographic column is a pure water-resistant chromatographic column in the experimental process, the detection method has more stable results, good chromatographic peak shape and more accurate results.
In some embodiments, the agents detected by the present invention are amide-based agents, including, but not limited to, lacosamide, and the like.
In some embodiments, the molar concentration of the sodium dihydrogen phosphate aqueous solution employed in the present invention is 0.01 M,0.02 M,0.03 M,0.04 M,0.05M; preferably, the molar concentration of the sodium dihydrogen phosphate aqueous solution is 0.02. 0.02M.
In some embodiments, the pure water resistant phase chromatographic column employed in the present invention; preferably, the chromatographic column is Waters Atlantis ® T3, its specification is 150 mm ×4.6 mm ×5 μm.
In some embodiments, the column temperature is 20 ℃,25 ℃,30 ℃,35 ℃,40 ℃; preferably, the column temperature is 25 ℃.
In some embodiments, the elution is performed according to a gradient with an elution gradient starting at 95%, 96%, 97%, 98%, 99%, 100% of phase a and ending at a final ratio in the range of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of phase B; wherein the mobile phase proportion is the volume percentage of each mobile phase to the total volume of the eluent, and the sum of the volume proportions of mobile phases A and B is 100%.
In some embodiments, the eluent has a flow rate of 0.5 mL/min, 0.6 mL/min, 0.7 mL/min, 0.8 mL/min, 0.9 mL/min, 1.0 mL/min, 1.1 mL/min, 1.2 mL/min, 1.3 mL/min, 1.4 mL/min, 1.5 mL/min; preferably, the eluent flow rate is 1.0 mL/min.
In some embodiments, the high performance liquid chromatograph detector employed is an ultraviolet detector.
In some embodiments, the detection wavelengths are 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm; preferably, the detection wavelength is 210 nm.
In some embodiments, one or more of the following steps are included:
(a) Preparing blank solution, reference substance solution and test substance solution (medicine solution), wherein:
blank solvent: namely diluting solvent which is pure water or high-proportion water solution;
control solution: dissolving glacial acetic acid with a diluting solvent to dilute to a certain concentration;
test solution: dissolving a sample solution in a diluting solvent to dilute the sample solution to a certain concentration;
(b) The blank solution, the reference substance solution and the sample solution are respectively injected into the high-performance liquid chromatograph, the injection volume can be 10 mu L, 20 mu L, 30 mu L, 40 mu L, 50 mu L, 60 mu L, 70 mu L, 80 mu L, 90 mu L and 100 mu L. Gradient elution was performed according to table 1 and chromatograms were recorded;
TABLE 1 gradient elution procedure table for mobile phases
| Time (min) | Mobile phase a (%) | Mobile phase (B) |
| 0 | 100 | 0 |
| 7 | 100 | 0 |
| 8 | 30 | 70 |
| 20 | 30 | 70 |
| 21 | 100 | 0 |
| 30 | 100 | 0 |
(c) According to the chromatographic peak area, the contents of the acetic acid and the acetate of the test sample are calculated, and the calculation method is as follows:
the methodology for determining acetic acid and acetate content by high performance liquid chromatography is demonstrated by specific examples. The materials and reagents used in the examples were all commercially available unless otherwise specified.
EXAMPLE 1 detection of acetic acid residue by gas chromatography
Chromatographic conditions
Chromatographic column: wonda Cap wax (0.25 mm X30 m X0.25 mu m)
Detector temperature: 250 DEG C
Sample inlet temperature: 220 DEG C
Heating program: the initial column temperature was 80℃and was raised to 200℃at a rate of 10℃per minute for 5 min.
Flow rate: 1.0 mL/min
Split ratio: 10:1
Sample injection amount: 1. mu L
Blank solvent: methanol
Control solution: weighing glacial acetic acid about 10 mg, precisely weighing, placing into 20 mL measuring flask, diluting with methanol to scale, and shaking.
The solution 1 [ mu ] L is measured, injected into a gas chromatograph, and a chromatogram is recorded, as shown in FIG. 1.
As shown in FIG. 1, the glacial acetic acid detected by the gas chromatography shows a peak at 3.969 min, a tailing factor at 4.151, and a peak shape is poor due to severe tailing, so that the detection accuracy of the acetic acid content is low.
EXAMPLE 2 method for measuring acetic acid and acetate content by high Performance liquid chromatography-specificity
Chromatographic conditions
Chromatographic column: waters Atlantis ® T3（150 mm×4.6 mm×5 µm）
Flow rate: 1.0 mL/min
Column temperature: 25 DEG C
Detection wavelength: 210 nm (nm)
Sample injection amount: 100. mu L
Mobile phase
Mobile phase a:0.02 M sodium dihydrogen phosphate aqueous solution
Weighing 2.3996 g (+ -0.24 g) sodium dihydrogen phosphate (such as dihydrate according to the molar concentration, converting into sample, dissolving in 1000 mL reagent bottle containing 1000 mL water, adding 1.0 mL phosphoric acid, mixing, suction filtering, and ultrasound.
Mobile phase B: acetonitrile
Solvent: i.e. diluting the solvent, mobile phase A
Run time: 30 min
Gradient elution is as follows table 2:
TABLE 2 gradient elution procedure table for mobile phases
| Time (min) | Mobile phase a (%) | Mobile phase (B) |
| 0 | 100 | 0 |
| 7 | 100 | 0 |
| 8 | 30 | 70 |
| 20 | 30 | 70 |
| 21 | 100 | 0 |
| 30 | 100 | 0 |
Solution preparation
Blank solvent: mobile phase a.
Control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; the stock solution 1mL was precisely measured, placed in a 50 mL measuring flask, diluted to scale with solvent, and shaken well to serve as a control solution. Two portions were prepared in parallel.
Test solution: taking about 250 mg of the lank amide, precisely weighing, placing into a 25 mL measuring flask, adding a proper amount of solvent, ultrasonically shaking for 10 min to dissolve, diluting to a scale with a diluent, and shaking uniformly to obtain a sample solution. Two parts are prepared by the same method.
Precisely measuring 100 mu L of blank solvent, 100 mu L of reference substance solution and 100 mu L of sample solution, respectively injecting into a high performance liquid chromatograph, running according to the chromatographic conditions and the mobile phase, and recording a chromatogram. The patterns are shown in figures 2 to 4.
Results: as can be seen from fig. 2 to 4, the blank solvent does not interfere with the measurement of acetic acid content; the target peak retention time was about 4.3 minutes, the peak appearance time was relatively good, and the peak shape was good.
Example 3 method for measuring acetic acid and acetate content by high Performance liquid chromatography-precision of sample injection
Control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; the stock solution 1mL was precisely measured, placed in a 50 mL measuring flask, diluted to scale with solvent, and shaken well to serve as a control solution. According to the chromatographic conditions, the detection method and the operation method of the example 2, 6 needles were continuously injected, the chromatograms were recorded, and the precision of the instrument was examined. The results are shown in Table 3 below.
TABLE 3 summary of proprietary results
Conclusion: and the continuous sample injection is carried out by 6 needles, the RSD of the peak area is 0.23%, and the instrument sample injection precision is good.
EXAMPLE 4 method for measuring acetic acid and acetate content by high Performance liquid chromatography-solution stability
Control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; the stock solution 1mL was precisely measured, placed in a 50 mL measuring flask, diluted to scale with solvent, and shaken well to serve as a control solution. After being placed under the room temperature condition 0 h,2 h,4 h,8 h,12 h,24 h, 100 mu L of the sample is measured, the sample is injected into a liquid chromatograph, and the sample is operated according to the chromatographic conditions and the operation method of the example 2, and a chromatogram is recorded. The results are shown in Table 4 below.
TABLE 4 stability results summary table
Conclusion: the control solution was placed in 24 h at room temperature with a peak area RSD of 0.26% and good stability.
Example 5 method for measuring acetic acid and acetate content by high Performance liquid chromatography-sensitivity
Precisely measuring control solution 1mL, placing in a 50 mL measuring flask, adding solvent to dilute to scale, shaking uniformly, and taking as quantitative limiting solution. Taking a quantitative limit solution 3 mL, placing the quantitative limit solution into a 10 mL measuring flask, adding a solvent to dilute the quantitative limit solution to a scale, and shaking the mixture uniformly to serve as a detection limit solution. Precisely measuring 100 mu L of each solution, injecting the solution into a liquid chromatograph, operating according to the chromatographic conditions and the operation method of the embodiment 2, and recording a chromatogram. The results are shown in Table 5 below.
TABLE 5 sensitivity results summary table
| Name of the name | Concentration (mg/mL) | Peak area | Equivalent to the sample solution | S/N |
| Quantitative limiting solution | 0.001 | 2.58 | 0.01% | 19.25 |
| Detection limiting solution | 0.0003 | 0.81 | 0.003% | 6.14 |
Conclusion: the concentration of the quantitative limit solution is 0.001 mg/mL, which is equivalent to 0.01% of the test sample solution, the concentration of the detection limit solution is 0.0003 mg/mL, which is equivalent to 0.003% of the test sample solution, and the detection limit requirement is met.
EXAMPLE 6 method for measuring acetic acid and acetate content by high Performance liquid chromatography-Linear
The control stock solution is precisely measured and properly prepared into linear solutions of 2%,10%,50%,80%,100% and 120% respectively. Precisely measuring 100 mu L of each solution, injecting the solution into a liquid chromatograph, operating according to the chromatographic conditions and the operation method of the embodiment 2, and recording a chromatogram. The results are shown in Table 6.
TABLE 6 Linear results summary table
Conclusion: glacial acetic acid has good linear relation in the range of 1.0 [ mu ] g/mL-60.043 [ mu ] g/mL.
EXAMPLE 7 method for measuring acetic acid and acetate content by high Performance liquid chromatography-repeatability
Control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; the stock solution 1mL was precisely measured, placed in a 50 mL measuring flask, diluted to scale with solvent, and shaken well to serve as a control solution. Two portions were prepared in parallel.
Test solution: taking about 250 mg of the lank amide, precisely weighing, placing into a 25 mL measuring flask, adding a proper amount of solvent, ultrasonically shaking for 10 min to dissolve, diluting to a scale with a diluent, and shaking uniformly to obtain a sample solution. Six parts were prepared in the same manner.
Precisely measuring 100 mu L of each of the control solution and the sample solution, injecting into a liquid chromatograph, operating according to the chromatographic conditions and the operation method of the embodiment 2, and recording a chromatogram. The results are shown in Table 7 below.
Table 7: repeated results summary table
Conclusion: the results of the repeated measurement of 6 samples show no obvious difference and good repeatability.
EXAMPLE 8 method for measuring acetic acid and acetate content by high Performance liquid chromatography-recovery rate
Control solution: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; the stock solution 1mL was precisely measured, placed in a 50 mL measuring flask, diluted to scale with solvent, and shaken well to serve as a control solution. Two portions were prepared in parallel.
Test solution: about 250 percent mg percent of the lacosamide is taken, precisely weighed, placed in a 25 mL measuring flask, added with a proper amount of solvent, ultrasonically shaken for 10 minutes to dissolve, respectively added with a control stock solution 0.4 mL,0.5 mL,0.6 mL, diluted to scale with a diluent, and shaken uniformly to obtain 80 percent, 100 percent and 120 percent of sample solution. Each concentration was formulated in triplicate.
Precisely measuring 100 mu L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, operating according to the chromatographic conditions and the operation method of the embodiment 2, and recording the chromatograms. The results are shown in Table 8 below.
Table 8: recovery results summary table
Conclusion: the method has the advantages that the average recovery rate is 99.7% within the range of 80% -120%, the RSD is 0.39%, and the method recovery rate is good.
EXAMPLE 9 method for measuring acetic acid and acetate content by high Performance liquid chromatography-durability
The control solution and the test solution were sampled under the conditions of flow rate.+ -. 0.2 mL/min, wavelength.+ -. 5 nm and organic phase proportion.+ -. 5%, and the sample was run according to the chromatographic conditions and running method of example 2, with the results shown in Table 9 below.
Table 9: durability results summary table
| Detection conditions | Content (%) |
| Normal conditions | 0.02 |
| Flow rate is 0.8 mL/min | 0.02 |
| Flow rate 1.2 mL/min | 0.02 |
| Wavelength 205 nm | 0.02 |
| Wavelength 215 nm | 0.02 |
| Organic comparative example 65 | 0.02 |
| Organic comparative example 75 | 0.02 |
Conclusion: minor changes in chromatographic conditions did not affect glacial acetic acid content detection.
From examples 2 to 9, the method for measuring the contents of acetic acid and acetate by using the high performance liquid chromatography has the advantages of strong specificity, good sample injection precision, high sensitivity, good stability, good linear relation, good recovery rate, good repeatability and strong durability. Therefore, the method is suitable for detecting the content of acetic acid and acetate in the medicine.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.
Claims (15)
1. A method for detecting the contents of acetic acid and acetate in a medicine is characterized in that the method is a high performance liquid chromatography method, and adopts a reversed phase chromatographic column, and adopts sodium dihydrogen phosphate aqueous solution and acetonitrile as a mobile phase A and/or a mobile phase B, and the elution is performed according to isocratic elution or gradient elution.
2. The method of claim 1, wherein the drug is a laninamide.
3. The method according to claim 1 or 2, wherein the molar concentration of the sodium dihydrogen phosphate aqueous solution is 0.01-0.05M.
4. A method according to claim 3, wherein the molar concentration of the sodium dihydrogen phosphate aqueous solution is 0.02, M.
5. The method of claim 4, wherein the reverse phase chromatography column is a pure water resistant phase chromatography column.
6. The method according to claim 5, wherein the pure water resistant phase chromatographic column is Waters atlantis t3 with a specification of 150 mm x 4.6 mm x 5 μm.
7. The method of claim 6, wherein the column temperature is 20-40 ℃.
8. The method of claim 7, wherein the column temperature is 25 ℃.
9. The method of claim 8, wherein the elution is performed according to a gradient, the initial proportion of the elution gradient is 95% -100% of the phase A, the final proportion is 50% -100% of the phase B, the proportion of the mobile phases is the volume percentage of each mobile phase in the total volume of the eluent, and the sum of the volume proportions of the mobile phases A and B is 100%.
10. The method of claim 9, wherein the eluent has a flow rate of 0.5 to 1.5 mL/min.
11. The method of claim 10, wherein the eluent flow rate is 1.0 mL/min.
12. The method of claim 11, wherein the detection wavelength is 200 nm to 280 nm using an ultraviolet detector.
13. The method of claim 12, wherein the detection wavelength is 210 nm.
14. The method of claim 13, comprising one or more of the following steps: step (1): preparing blank solution, reference substance solution and test sample solution; step (2): injecting 10-100 mu L of the solution prepared in the step (1) into a high performance liquid chromatograph, performing gradient elution according to the following table, and recording a chromatogram; step (3): calculating the content of acetic acid and acetate in the sample according to the chromatographic peak area
。
15. The method of claim 14, wherein the mobile phase a, the control and the test are formulated as follows:
preparing a reference substance: taking glacial acetic acid 250 mg, precisely weighing, placing in a 100 mL measuring flask, diluting to scale with solvent, shaking uniformly, and taking as stock solution; precisely measuring 1mL of stock solution, placing in a 50 mL measuring flask, diluting to scale with solvent, shaking to obtain reference solution;
test solution: accurately weighing the laninamide 250 mg, placing the obtained mixture into a 25 mL measuring flask, adding a proper amount of solvent, ultrasonically shaking the mixture for 10 minutes to dissolve the mixture, diluting the mixture to a scale with a diluent, and shaking the mixture uniformly to obtain a sample solution;
mobile phase a: weighing 2.3996 g sodium dihydrogen phosphate, dissolving in 1000 mL water in a reagent bottle, adding 1.0 mL phosphoric acid, mixing, filtering, and performing ultrasonic treatment.
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