CN111579704A - Method for detecting content of N, N' -diacetyl-L-cystine in compound amino acid injection - Google Patents
Method for detecting content of N, N' -diacetyl-L-cystine in compound amino acid injection Download PDFInfo
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- YTPQSLLEROSACP-YUMQZZPRSA-N (2R)-2-acetamido-3-[[(2R)-2-acetamido-2-carboxyethyl]disulfanyl]propanoic acid Chemical compound CC(=O)N[C@H](C(O)=O)CSSC[C@@H](C(O)=O)NC(C)=O YTPQSLLEROSACP-YUMQZZPRSA-N 0.000 title claims abstract description 60
- 238000002347 injection Methods 0.000 title claims abstract description 50
- 239000007924 injection Substances 0.000 title claims abstract description 50
- -1 compound amino acid Chemical class 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000012085 test solution Substances 0.000 claims abstract description 44
- 239000013558 reference substance Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 39
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 39
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 39
- 239000008055 phosphate buffer solution Substances 0.000 claims description 33
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 25
- 238000001514 detection method Methods 0.000 claims description 24
- 238000010790 dilution Methods 0.000 claims description 21
- 239000012895 dilution Substances 0.000 claims description 21
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 12
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 claims description 11
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 3
- 239000012088 reference solution Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims 1
- 229910002027 silica gel Inorganic materials 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 12
- 238000000926 separation method Methods 0.000 abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 229910019142 PO4 Inorganic materials 0.000 abstract 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 1
- 239000010452 phosphate Substances 0.000 abstract 1
- 229940090044 injection Drugs 0.000 description 38
- 239000000523 sample Substances 0.000 description 32
- 230000014759 maintenance of location Effects 0.000 description 10
- 239000012488 sample solution Substances 0.000 description 8
- 238000001612 separation test Methods 0.000 description 8
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 229960004308 acetylcysteine Drugs 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000012224 working solution Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 2
- 229940021715 acetylcysteine injection Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004190 ion pair chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
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Abstract
The invention relates to a method for detecting the content of N, N' -diacetyl-L-cystine in a compound amino acid injection by using a high performance liquid chromatography, which comprises the following steps: preparing a mobile phase, preparing a reference substance solution, preparing a test solution, setting chromatographic instrument parameters, detecting the reference substance solution, detecting the test solution and calculating the content of the N, N' -diacetyl-L-cystine in the test solution. The invention adopts a phosphate mobile phase system, can realize the separation of N, N' -diacetyl-L-cystine and adjacent chromatographic peaks on a common C18 chromatographic column, and has the advantages of simple and convenient operation and easy implementation. Compared with an ion-pair mobile phase system, the method has good reproducibility, does not need to use expensive ion-pair reagents, and is more economical.
Description
Technical Field
The invention relates to a method for detecting the content of N, N '-diacetyl-L-cystine in a compound amino acid injection, in particular to a method for detecting the content of N, N' -diacetyl-L-cystine in the compound amino acid injection by using a high performance liquid chromatography.
Background
N, N' -diacetyl-L-cystine is an oxidative degradation impurity of N-acetylcysteine in the compound amino acid injection, and monitoring the content of the impurity in the compound amino acid injection can provide a basis for optimizing process parameters and ensuring the product quality. In acetylcysteine bulk drugs and acetylcysteine single preparations (such as acetylcysteine injection and acetylcysteine inhalation preparations), quantitative detection methods for the impurities have been reported, and ion pair chromatography is mostly adopted. At present, no report of a method for detecting N, N' -diacetyl-L-cystine in a compound amino acid injection is found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using high performance liquid chromatography, which has good reproducibility and is more economical.
According to the technical scheme provided by the invention, the method for detecting the content of N, N' -diacetyl-L-cystine (impurity C) in the compound amino acid injection comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.1-2.5 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of (93-95) to (7-5) to form a mobile phase;
step two, diluting N, N' -diacetyl-L-cystine with water to obtain a solution with the concentration of 0.6-20 mg/L as a reference substance;
step three, adding water into the compound amino acid injection for dilution to be used as a test solution;
step four, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the thickness of 5 mu m, the column temperature is set to be 33-37 ℃, the detection wavelength is set to be 210nm, the flow rate is set to be 0.9-1.1 ml/min, the temperature of a sample injector is set to be 4 ℃, and the sample injection volume is set to be 10 mu L;
step five, detecting the reference solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of an N, N '-diacetyl-L-cystine chromatographic peak, and recording the peak area of the N, N' -diacetyl-L-cystine chromatographic peak;
step six, detecting the test solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, and recording the peak area of the chromatographic peak of the N, N' -diacetyl-L-cystine in the test solution;
and step seven, calculating the content of the N, N' -diacetyl-L-cystine in the test sample according to an external standard method.
Preferably, in step one, the pH of the sodium dihydrogen phosphate buffer is adjusted and the buffer is filtered through a 0.45 μm filter.
Preferably, in step one, the pH of the sodium dihydrogen phosphate buffer is adjusted to 2.3 with phosphoric acid.
Preferably, in step one, the sodium dihydrogen phosphate buffer and acetonitrile are mixed according to a volume ratio of 94:6 to form a control solution.
Preferably, in the third step, the compound amino acid injection is diluted by 5 times.
Preferably, in step four, the flow rate is set to 1.0ml/min and the column temperature of the column is set to 35 ℃.
Preferably, in the fourth step, C18 chromatographic column with product parameters of 150mm × 4.6mm and 5 μm is selected.
Compared with an ion-pair mobile phase system, the method has good reproducibility, does not need to use expensive ion-pair reagents, and is more economical.
Drawings
FIG. 1 is a chromatogram of a control working solution in example 1.
FIG. 2 is a chromatogram of the test solution in example 1.
FIG. 3 is a chromatogram of the test solution in example 2.
FIG. 4 is a chromatogram of the test solution in example 3.
FIG. 5 is a chromatogram of the test solution in example 4.
FIG. 6 is a chromatogram of the test solution in example 5.
FIG. 7 is a chromatogram of the test solution in example 6.
FIG. 8 is a chromatogram of the test solution in example 7.
FIG. 9 is a chromatogram of the test solution in example 8.
FIG. 10 is a chromatogram of the test solution in example 9.
Detailed Description
The present invention will be further described with reference to the following specific examples.
The column used in the examples below was an octadecylsilane bonded silica C18 column with product parameters of 5 μm, 150 mm. times.4.6 mm.
Example 1
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, accurately weighing 64.11mg of the N, N '-diacetyl-L-cystine reference substance, placing the reference substance in a 100mL measuring flask, dissolving the reference substance with a proper amount of water and diluting the reference substance to the highest scale to obtain a reference substance storage solution of the N, N' -diacetyl-L-cystine; precisely transferring 1ml of N, N' -diacetyl-L-cystine reference substance stock solution into a 50ml measuring flask, adding water for diluting until the highest scale is reached, and shaking up to obtain a reference substance solution;
step three, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step four, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.0ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
step five, detecting the reference solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of an N, N '-diacetyl-L-cystine chromatographic peak, and recording the peak area of the N, N' -diacetyl-L-cystine chromatographic peak;
step six, detecting the test solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, and recording the peak area of the chromatographic peak of the N, N' -diacetyl-L-cystine in the test solution;
and step seven, calculating the content of the N, N' -diacetyl-L-cystine in the test solution according to an external standard method.
And (3) detection results: the retention time of the reference working solution and the test solution is consistent. The separation degree of N, N' -diacetyl-L-cystine from the adjacent chromatographic peak in the test solution was 7.93, as shown in Table 1.
TABLE 1
| Sample (I) | Retention time (min) | Degree of separation |
| Reference working solution | 8.888 | N.A. |
| Test solution | 8.893 | 7.93 |
Example 2
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.1 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.0ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 2.
TABLE 2
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 9.643 | 3.117 | 4.65 |
Example 3
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.5 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.0ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 3.
TABLE 3
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 8.597 | 2.893 | 2.69 |
Example 4
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 93:7 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.0ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 4.
TABLE 4
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 7.184 | 0.461 | 3.17 |
Example 5
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 95:5 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.0ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 5.
TABLE 5
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 11.456 | 0.4 | 11.93 |
Example 6
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 μm, the column temperature is set to be 33 ℃, the detection wavelength is set to be 210nm, the flow rate is set to be 1.0ml/min, the temperature of a sample injector is set to be 4 ℃, and the sample injection volume is set to be 10 μ L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and referring to table 6.
TABLE 6
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 9.203 | 0.395 | 9.59 |
Example 7
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to be 37 ℃, the detection wavelength is set to be 210nm, the flow rate is set to be 1.0ml/min, the temperature of a sample injector is set to be 4 ℃, and the sample injection volume is set to be 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 7.
TABLE 7
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 8.619 | 0.402 | 8.12 |
Example 8
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 0.9ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 8.
TABLE 8
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 9.952 | 0.458 | 8.06 |
Example 9
A detection method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection by using a high performance liquid chromatography comprises the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.3 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of 94:6 to form a mobile phase;
step two, adding water into the compound amino acid injection for dilution to serve as a test solution, wherein the dilution multiple is 5;
step three, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, a C18 chromatographic column with the diameter of 5 mu m, the column temperature is set to 35 ℃, the detection wavelength is set to 210nm, the flow rate is set to 1.1ml/min, the temperature of a sample injector is set to 4 ℃, and the sample injection volume is set to 10 mu L;
and step four, detecting the sample solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of the chromatographic peak of the N, N' -diacetyl-L-cystine and the separation degree of the chromatographic peak and the adjacent chromatographic peak, and showing in table 9.
TABLE 9
| Sample (I) | Retention time (min) | Peak area | Degree of separation |
| Test solution | 8.085 | 0.374 | 7.62 |
Linearity of the method of the invention
The linear relationship was measured according to the chromatographic conditions of example 1, and control solutions were prepared at concentrations of 0.6413 μ g/mL, 1.2826 μ g/mL, 3.2065 μ g/mL, 6.4130 μ g/mL, 9.6195 μ g/mL, 12.8260 μ g/mL and 19.2390 μ g/mL, respectively, and linear regression was performed using peak areas and corresponding concentrations, and the results showed that, when the concentration of N, N '-diacetyl-L-cystine in the control solution was 0.6413-19.2390 mg/L, which corresponds to the concentration of N, N' -diacetyl-L-cystine in the test sample of 3.2065-96.195 mg/L, regression was performed using peak areas and corresponding concentrations, and the regression equation was y 7.7039x +0.3249, and r was 0.9993.
Accuracy and precision of the method of the invention
The accuracy and precision of the method of the present invention were tested according to the chromatographic conditions of example 1 by adding N, N '-diacetyl-L-cystine to samples that did not contain N, N' -diacetyl-L-cystine, respectively, to design 3 different concentrations, preparing 3 test solutions for each concentration, and evaluating the results of the measurements of 9 samples, as shown in table 10.
As can be seen from table 10, when the concentration of N, N '-diacetyl-L-cystine in the test solution is 6.09 to 15.60mg/L, which corresponds to the concentration of N, N' -diacetyl-L-cystine in the test solution of 30.5 to 78.0mg/L, the average recovery rate is 98.4%, and the RSD (relative standard deviation) is 2.1%.
Quantitative limits of the method of the invention
The quantitative limit of the detection method of the present invention was tested according to the chromatographic conditions of example 1, and the results showed that the quantitative limit of the detection method of the present invention was 16mg/L of N, N' -diacetyl-L-cystine in the test sample under the condition that the signal-to-noise ratio was not less than 10.
Claims (7)
1. A method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection is characterized by comprising the following steps:
firstly, weighing sodium dihydrogen phosphate dihydrate, adding water to prepare a sodium dihydrogen phosphate buffer solution with the concentration of 20mM, then adjusting the pH value of the sodium dihydrogen phosphate buffer solution to 2.1-2.5 by using phosphoric acid, and finally mixing the sodium dihydrogen phosphate buffer solution and acetonitrile according to the volume ratio of (93-95) to (7-5) to form a mobile phase;
step two, diluting N, N' -diacetyl-L-cystine with water to obtain a solution with the concentration of 0.6-20 mg/L as a reference substance;
step three, adding water into the compound amino acid injection for dilution to be used as a test solution;
fourthly, the parameters of a chromatographic column product of the high performance liquid chromatograph are 150mm multiplied by 4.6mm, the octadecyl silane bonded silica gel C18 chromatographic column with the thickness of 5 mu m, the column temperature is set to be 33-37 ℃, the detection wavelength is set to be 210nm, the flow rate is set to be 0.9-1.1 ml/min, the temperature of a sample injector is set to be 4 ℃, and the sample injection volume is set to be 10 mu L;
step five, detecting the reference solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, recording the position of an N, N '-diacetyl-L-cystine chromatographic peak, and recording the peak area of the N, N' -diacetyl-L-cystine chromatographic peak;
step six, detecting the test solution by using a high performance liquid chromatograph, outputting a corresponding chromatogram, and recording the peak area of the chromatographic peak of the N, N' -diacetyl-L-cystine in the test solution;
and step seven, calculating the content of the N, N' -diacetyl-L-cystine in the test sample according to an external standard method.
2. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in step one, the pH of the sodium dihydrogen phosphate buffer is adjusted and the buffer is filtered through a 0.45 μm filter.
3. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in step one, the pH of the sodium dihydrogen phosphate buffer is adjusted to 2.3 with phosphoric acid.
4. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in the first step, sodium dihydrogen phosphate buffer and acetonitrile are mixed according to a volume ratio of 94:6 to form a mobile phase.
5. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in the third step, the compound amino acid injection has 5 times of dilution.
6. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in step four, the flow rate was set to 1.0ml/min and the column temperature of the column was set to 35 ℃.
7. The method for detecting the content of N, N' -diacetyl-L-cystine in compound amino acid injection according to claim 1, which is characterized in that: in the fourth step, C18 chromatographic column with product parameters of 150mm × 4.6mm and 5 μm is selected.
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