CN113567565A - Method for detecting glutaraldehyde in amoxicillin - Google Patents
Method for detecting glutaraldehyde in amoxicillin Download PDFInfo
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- CN113567565A CN113567565A CN202010357054.9A CN202010357054A CN113567565A CN 113567565 A CN113567565 A CN 113567565A CN 202010357054 A CN202010357054 A CN 202010357054A CN 113567565 A CN113567565 A CN 113567565A
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- glutaraldehyde
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- amoxicillin
- acetonitrile
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 title claims abstract description 88
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 title claims abstract description 60
- 229960003022 amoxicillin Drugs 0.000 title claims abstract description 60
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 90
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 46
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 131
- 239000007788 liquid Substances 0.000 claims description 68
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 66
- 238000001212 derivatisation Methods 0.000 claims description 60
- 239000003153 chemical reaction reagent Substances 0.000 claims description 50
- 238000006243 chemical reaction Methods 0.000 claims description 40
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 38
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical group NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 20
- 238000007865 diluting Methods 0.000 claims description 20
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- QAUJHIMXYLFORD-GCBPPVMSSA-N n-[(e)-[(5e)-5-[(2,4-dinitrophenyl)hydrazinylidene]pentylidene]amino]-2,4-dinitroaniline Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC=C1N\N=C\CCC\C=N\NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O QAUJHIMXYLFORD-GCBPPVMSSA-N 0.000 description 3
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
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- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-aminopenicillanic acid Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 description 1
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 1
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- MQXQVCLAUDMCEF-CWLIKTDRSA-N amoxicillin trihydrate Chemical compound O.O.O.C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 MQXQVCLAUDMCEF-CWLIKTDRSA-N 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Abstract
The invention relates to a method for determining glutaraldehyde in amoxicillin by high performance liquid chromatography, which uses octadecylsilane chemically bonded silica as a chromatographic column of a stationary phase, and uses water as a mobile phase: and (3) acetonitrile. The method has the advantages of appropriate peak time, good peak type, strong specificity and simple operation, and solves the problem of detecting residual glutaraldehyde in amoxicillin, thereby ensuring controllable quality of amoxicillin raw materials and preparations in the production process.
Description
Technical Field
The invention relates to a method for determining genotoxic impurity glutaraldehyde in amoxicillin by a derivatization method, in particular to a method for determining glutaraldehyde in amoxicillin by a high performance liquid chromatography method.
Background
Amoxicillin (Amoxicillin) is a penicillin with amino side chain, the chemical structure of which is that a hydroxyl group is added on the benzene ring of the side chain of Ampicillin (Ampicillin, AMP), the basic structure mainly playing the role of antibiosis is a beta-lactam ring in 6-amino penicillanic acid, which can be specifically combined with a target spot on the inner membrane of bacteria to inhibit the activity of the bacterial cell wall mucopeptide synthetase, thereby hindering the synthesis of cell wall mucopeptide, leading the cell wall of the bacteria to be defective and leading the thalli to expand and break. Is one of the oral penicillins which are widely applied at present, and the preparations of the oral penicillins comprise capsules, tablets, granules and the like.
The chemical structural formula of amoxicillin is as follows:
the product is white to off-white crystalline powder, slightly soluble in water and methanol, and has a chemical name: (2S,5R,6R) -3, 3-dimethyl-6- [ (R) - (-) -2-amino-2- (4-hydroxyphenyl) acetylamino ] -7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid trihydrate.
The synthesis method of amoxicillin is various and can be roughly divided into a chemical synthesis method and an enzymatic synthesis method, because the enzymatic synthesis method can reduce the reaction steps and the generation of wastes, is beneficial to protecting the environment and reducing the production cost, has excellent product quality and contains few impurities, the enzymatic reaction is a necessary trend, large-scale production is carried out, glutaraldehyde used in the enzyme production process belongs to genotoxic impurities and is also an impurity which is necessary to be researched in the amoxicillin raw material, and there is no literature report about the method for detecting the glutaraldehyde in the amoxicillin.
Disclosure of Invention
In order to strictly control the product quality, a method capable of detecting impurities of glutaraldehyde in amoxicillin is established through research. The method can detect and accurately quantify the glutaraldehyde in the amoxicillin, has the advantages of short analysis time, high separation degree, good peak pattern, simple operation, energy consumption saving and the like, and solves the problem of detecting the glutaraldehyde in the amoxicillin, thereby ensuring the controllable quality of the amoxicillin raw material and the preparation thereof in the production process.
The purpose of the invention is realized as follows: a method for detecting glutaraldehyde in amoxicillin is characterized by comprising the following steps:
weighing amoxicillin, placing the amoxicillin in a measuring flask, adding a derivatization reagent and a perchloric acid solution respectively, performing derivatization reaction, diluting with an acetonitrile solution, fixing the volume to obtain a test sample solution, injecting the test sample solution into a high performance liquid chromatograph by taking water-acetonitrile as a mobile phase, detecting, and calculating the main peak area so as to determine the content of glutaraldehyde.
Further, the detection method of glutaraldehyde in amoxicillin of the invention is optimized as follows:
the mobile phase consists of water and acetonitrile with the volume ratio of 25-35: 75-65; preferably, the mobile phase composition is water to acetonitrile in a volume ratio of 35:65, 30:70, 25: 75.
The column temperature of the high performance liquid chromatograph is 25-35 ℃, preferably 30 ℃.
The flow rate of the mobile phase is 1.0-1.4mL/min, preferably 1.0, 1.2 and 1.4 mL/min.
The ultraviolet detection wavelength in the high performance liquid chromatograph is 360 nm.
The concentration of the amoxicillin in the test solution is 15-25mg/mL, preferably 20 mg/mL.
The derivatization reagent is 2, 4-dinitrophenylhydrazine solution, and the addition amount of the derivatization reagent is that the derivatization reagent accounts for 20-40% of the volume ratio of the test solution, preferably 20%, 30% and 40%.
The perchloric acid solution is 30% of perchloric acid solution by volume ratio, and the addition amount of the perchloric acid solution is 5% -20% of perchloric acid solution in the volume ratio of the perchloric acid solution to the test sample solution.
The temperature of derivatization is room temperature to 60 ℃, preferably room temperature, 40 ℃ and 60 ℃.
The derivatization time is 10-40min, preferably 10min, 20min, 30min and 40 min.
The dilution with acetonitrile solution is 50% acetonitrile solution by volume ratio.
The chromatographic column used in the high performance liquid chromatograph is an octadecylsilane bonded silica chromatographic column, such as CAPCELL PAK C18 SG 120S 5 μm 4.6X 250mm, Agilent ZORBAX SB-Aq 4.6X 250mm 5 μm, Waters Symmetry C184.6X 250mm 5 μm, Agilent Eclipse Plus C184.6X 150mm 3.5 μm.
The calculation method for calculating the area of the main peak so as to determine the content of the glutaraldehyde is an external standard method, and specifically comprises the following steps:
the calculation method is that the peak area is calculated according to an external standard method, and the reaction principle is that glutaraldehyde and a derivatization reagent 2, 4-dinitrophenylhydrazine in amoxicillin are derivatized to generate glutaraldehyde 2, 4-dinitrophenylhydrazone for quantification.
The calculation formula is as follows:
wherein: cRThe concentration of glutaraldehyde in the control solution; cXThe concentration is the concentration of the test solution;
ARthe peak area of the main peak of a derivative (glutaraldehyde 2, 4-dinitrophenylhydrazone) in a reference substance solution is shown;
AXis the peak area of the main peak of the derivative (glutaraldehyde 2, 4-dinitrophenylhydrazone) in the test solution.
The invention has the beneficial effects that:
(1) in the method, perchloric acid and a derivatization reagent are added separately, so that the technical problem of reduction of the stability of the derivatization reagent caused by directly adding perchloric acid into the derivatization reagent is effectively solved, and the stability of the derivatization reagent is greatly improved.
(2) The invention directly adds the derivatization reagent and perchloric acid into the sample, ensures the complete reaction of the derivatization reagent and the glutaraldehyde in the sample, and can compete the reaction of the amoxicillin and the glutaraldehyde by a direct adding mode because the amoxicillin can react with the glutaraldehyde.
(3) The method has the advantages of short analysis time, high separation degree, good peak pattern, accurate quantification, simple operation, energy consumption saving and the like, thereby ensuring the controllable quality of the amoxicillin raw material and the preparation thereof in the production process.
Drawings
FIG. 1 shows a high performance liquid chromatogram of example 1 of the present invention;
FIG. 2 shows a high performance liquid chromatogram of example 2 of the present invention;
FIG. 3 shows a high performance liquid chromatogram of example 3 of the present invention;
FIG. 4 shows a high performance liquid chromatogram of example 4 of the present invention;
FIG. 5 shows a high performance liquid chromatogram of example 5 of the present invention;
FIG. 6 shows a high performance liquid chromatogram of example 6 of the present invention;
FIG. 7 shows a high performance liquid chromatogram of example 7 of the present invention;
FIG. 8 shows a high performance liquid chromatogram of example 8 of the present invention;
FIG. 9 shows a high performance liquid chromatogram of example 9 of the present invention;
FIG. 10 shows a high performance liquid chromatogram of example 10 of the present invention;
FIG. 11 is a high performance liquid chromatogram of example 11 of the present invention;
FIG. 12 is a high performance liquid chromatogram of example 12 of the present invention;
FIG. 13 shows a high performance liquid chromatogram of example 13 of the present invention;
FIG. 14 shows a high performance liquid chromatogram of example 14 of the present invention;
FIG. 15 shows a high performance liquid chromatogram of example 15 of the present invention;
FIG. 16 is a high performance liquid chromatogram of example 16 of the present invention;
FIG. 17 is a high performance liquid chromatogram of example 17 of the present invention;
FIG. 18 shows a high performance liquid chromatogram of example 18 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to examples and drawings, but the following examples should not be construed as limiting the scope of the present invention.
The solution described in the embodiments of the present invention preferably refers to an aqueous solution.
Example 1
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6X 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: acetonitrile (water) is 30:70(v/v) as a mobile phase; the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 30min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into a high performance liquid chromatograph, sampling with a sample volume of 10 μ L to obtain a high performance liquid chromatogram, and recording the chromatogram, which is shown in figure 1. The peaks of the retention time at 7.128min and 7.973min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 7.973 min.
Example 2
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing for 30min at 40 ℃ in a water bath to room temperature, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into a high performance liquid chromatograph, sampling with a sample volume of 10 μ L to obtain a high performance liquid chromatogram, and recording the chromatogram, which is shown in figure 2. The peaks of the retention time at 7.144min and 7.994min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 7.994 min.
Example 3
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing for 30min to room temperature in a water bath at 60 ℃, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into a high performance liquid chromatograph, sampling with a sample volume of 10 μ L to obtain a high performance liquid chromatogram, and recording the chromatogram, which is shown in figure 3. The peaks at 7.146min and 7.995min are the peaks of the product generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the peak of the product generated by the reaction of glutaraldehyde and a derivatization reagent has short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 7.995 min.
Example 4
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 10min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 4. The peaks of the retention time at 7.150min and 8.001min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.001 min.
Example 5
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 5. The peaks of the retention time at 7.145min and 7.993min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 7.993 min.
Example 6
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 40min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 6. The peaks of the retention time at 7.159min and 8.013min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.013 min.
Example 7
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 2mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 7. The peaks of the retention time at 7.183min and 8.043min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.043 min.
Example 8
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 8. The peaks of the retention time at 7.189min and 8.048min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.048 min.
Example 9
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 54.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 4mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 9. The peaks of the retention time at 7.184min and 8.042min in the figure are both the product peak generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peak generated by the reaction of glutaraldehyde and a derivatization reagent has short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.042 min.
Example 10
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 35:65
The experimental steps are as follows: water-acetonitrile 35:65(v/v) is used as a mobile phase; the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin in a 10mL measuring flask, precisely adding 1.0mL of glutaraldehyde solution (2 mu g/mL), adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin liquid;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 10. The peaks of the retention time at 10.016min and 11.537min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 11.537 min.
Example 11
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin in a 10mL measuring flask, precisely adding 1.0mL of glutaraldehyde solution (2 mu g/mL), adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin liquid;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 11. The peaks of the retention time at 7.190min and 8.049min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.049 min.
Example 12
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 25:75
The experimental steps are as follows: water-acetonitrile 25:75(v/v) is used as a mobile phase; the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin in a 10mL measuring flask, precisely adding 1.0mL of glutaraldehyde solution (2 mu g/mL), adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin liquid;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 12. The peaks of the retention time at 5.392min and 5.884min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 5.884 min.
Example 13
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.0mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin in a 10mL measuring flask, precisely adding 1.0mL of glutaraldehyde solution (2 mu g/mL), adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin liquid;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 13. The peaks of the retention time at 8.617min and 9.645min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 9.645 min.
Example 14
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.4 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.4mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin in a 10mL measuring flask, precisely adding 1.0mL of glutaraldehyde solution (2 mu g/mL), adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin liquid;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 14. The peaks of the retention time at 6.161min and 6.894min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 6.894 min.
Example 15
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; agilent ZORBAX SB-Aq 4.6X 250mm 5 μm (octadecylsilane chemically bonded silica as filler) was used as a column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 15. The peaks of the retention time at 5.671min and 6.050min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 6.050 min.
Example 16
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; a Waters Symmetry C184.6X 250mm 5 μm (octadecylsilane bonded silica gel as filler) was used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 16. The peaks of the retention time at 7.211min and 8.131min in the figure are the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peaks generated by the reaction of glutaraldehyde and a derivatization reagent have short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 8.131 min.
Example 17
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; agilent Eclipse Plus C184.6X 150mm 3.5 μm (octadecylsilane bonded silica as packing) was used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; putting 1.0mL of glutaraldehyde solution (2 mu g/mL) into a 10mL measuring flask, adding 3mL of 2, 4-dinitrophenylhydrazine solution and 1mL of perchloric acid solution with the volume ratio of 30%, shaking up, standing at room temperature for 20min, diluting with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the product;
injecting the solution to be measured into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 17. The peaks of the retention time of 4.476min and 5.069min in the figure are both the product peak generated by the reaction of glutaraldehyde and a derivatization reagent, and it can be seen from the figure that the product peak generated by the reaction of glutaraldehyde and a derivatization reagent has short analysis time, good peak shape and high separation degree, wherein the main peak area refers to the peak area of 5.069 min.
Example 18
Experimental apparatus and conditions:
agilent 1260 type high performance liquid chromatography system and workstation; automatic sample introduction; CAPCELL PAK C18 SG 120S 5 μm 4.6mm × 250mm (the filler is octadecylsilane chemically bonded silica) is used as a chromatographic column.
Detection conditions are as follows:
ultraviolet detection wavelength: 360 nm; column temperature: 30 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 mu L of the solution; mobile phase: water-acetonitrile 30:70
The experimental steps are as follows: water-acetonitrile is used as a mobile phase at a ratio of 30:70 (v/v); the column temperature is 30 ℃, the flow rate is 1.2mL/min, and the detection wavelength is 360 nm; weighing about 200mg of amoxicillin, placing the amoxicillin into a 10mL measuring flask, adding 3mL2, 4-dinitrophenylhydrazine solution and 1mL perchloric acid solution with the volume ratio of 30%, shaking up, placing the mixture at room temperature for 20min, diluting the mixture with acetonitrile solution with the volume ratio of 50%, and fixing the volume to scale to obtain the amoxicillin-containing liquid;
injecting the solution to be detected into high performance liquid chromatograph, sampling volume of 10 μ L to obtain high performance liquid chromatogram, and recording chromatogram, as shown in figure 18, wherein the detection result of the sample is not detected.
Table 1 comparison of results of examples 1, 2 and 3
As can be seen from Table 1, amoxicillin and glutaraldehyde both react completely at room temperature, 40 deg.C, and 60 deg.C.
Table 2 comparison of results of examples 4, 5 and 6
As can be seen from Table 2, when the derivatization time is 10min, 20min and 40min, the amoxicillin and glutaraldehyde can completely react within 10 min.
Table 3 comparison of results of examples 7, 8 and 9
As shown in Table 3, when the volumes of the derivatization reagents were 2mL, 3mL, and 4mL, both amoxicillin and glutaraldehyde were reacted completely.
TABLE 4 comparison of results of examples 10, 11 and 12
As can be seen from table 4, the mobile phase with the volume ratio of water to acetonitrile of 35:65, 30:70, and 25:75 is used, and the glutaraldehyde derivatization peaks have better separation.
TABLE 5 comparison of results of examples 11, 13 and 14
As can be seen from Table 5, the flow rates used were between 1.0 and 1.4mL/min, with better separation between the glutaraldehyde derivatization peaks.
From examples 15 to 17, it can be seen that glutaraldehyde in the sample can be detected by using octadecylsilane chemically bonded silica chromatographic columns of different types, and that the separation degree is good, the peak appearance time is appropriate, and the peak appearance is good.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for detecting glutaraldehyde in amoxicillin is characterized by comprising the following steps:
weighing amoxicillin, placing the amoxicillin in a measuring flask, adding a derivatization reagent and a perchloric acid solution respectively, performing derivatization reaction, diluting with an acetonitrile solution, fixing the volume to obtain a test sample solution, injecting the test sample solution into a high performance liquid chromatograph by taking water-acetonitrile as a mobile phase, detecting, and calculating the main peak area so as to determine the content of glutaraldehyde.
2. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the mobile phase consists of water and acetonitrile with the volume ratio of 25-35: 75-65; preferably, the mobile phase composition is water to acetonitrile in a volume ratio of 35:65, 30:70, 25: 75.
3. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the column temperature of the high performance liquid chromatograph is 25-35 ℃, preferably 30 ℃.
4. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the flow rate of the mobile phase is 1.0-1.4mL/min, preferably 1.0, 1.2 and 1.4 mL/min;
the ultraviolet detection wavelength in the high performance liquid chromatograph is 360 nm.
5. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the concentration of the amoxicillin in the test solution is 15-25mg/mL, preferably 20 mg/mL.
6. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the derivatization reagent is 2, 4-dinitrophenylhydrazine solution, and the addition amount of the derivatization reagent is that the derivatization reagent accounts for 20-40% of the volume ratio of the test solution, preferably 20%, 30% and 40%;
the perchloric acid solution is 30% of perchloric acid solution by volume ratio, and the addition amount of the perchloric acid solution is 5% -20% of perchloric acid solution in the volume ratio of the perchloric acid solution to the test sample solution.
7. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the temperature of derivatization is room temperature to 60 ℃, preferably room temperature, 40 ℃ and 60 ℃.
8. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the derivatization time is 10-40min, preferably 10min, 20min, 30min and 40 min.
9. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the dilution with acetonitrile solution is 50% acetonitrile solution by volume ratio.
10. The method for detecting glutaraldehyde in amoxicillin according to claim 1, characterized in that:
the chromatographic column used in the high performance liquid chromatograph is an octadecylsilane bonded silica chromatographic column, such as CAPCELL PAK C18 SG 120S 5 μm 4.6X 250mm, Agilent ZORBAX SB-Aq 4.6X 250mm 5 μm, Waters Symmetry C184.6X 250mm 5 μm, Agilent Eclipse Plus C184.6X 150mm 3.5 μm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070213333A1 (en) * | 2003-08-14 | 2007-09-13 | Asahi Kasei Phama Corporation | Substituted arylalkanoic acid derivatives and use thereof |
| CN108362803A (en) * | 2018-01-24 | 2018-08-03 | 广东医科大学 | A kind of method of glutaraldehyde content in measurement material |
| CN108918707A (en) * | 2018-07-06 | 2018-11-30 | 无锡市政公用环境检测研究院有限公司 | A kind of method that liquid chromatograph mass spectrography measures glutaraldehyde content in water |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070213333A1 (en) * | 2003-08-14 | 2007-09-13 | Asahi Kasei Phama Corporation | Substituted arylalkanoic acid derivatives and use thereof |
| CN108362803A (en) * | 2018-01-24 | 2018-08-03 | 广东医科大学 | A kind of method of glutaraldehyde content in measurement material |
| CN108918707A (en) * | 2018-07-06 | 2018-11-30 | 无锡市政公用环境检测研究院有限公司 | A kind of method that liquid chromatograph mass spectrography measures glutaraldehyde content in water |
Non-Patent Citations (2)
| Title |
|---|
| "中国药典 2015版三部", 中国医药科技出版社, pages: 73 * |
| 夏南 等: "抗人T 淋巴细胞猪免疫球蛋白中戊二醛残留量的检测", 《中国生物制品学杂志》, vol. 24, no. 8 * |
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