CN112782405A - Detection method for antioxidant degradation product in levofloxacin lactate and sodium chloride injection - Google Patents
Detection method for antioxidant degradation product in levofloxacin lactate and sodium chloride injection Download PDFInfo
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- ONDRRTIAGBDDKP-PPHPATTJSA-N 294662-18-3 Chemical compound CC(O)C(O)=O.C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 ONDRRTIAGBDDKP-PPHPATTJSA-N 0.000 title claims abstract description 55
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 239000008354 sodium chloride injection Substances 0.000 title claims abstract description 35
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 31
- 239000007857 degradation product Substances 0.000 title claims abstract description 27
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 25
- ICKWICRCANNIBI-UHFFFAOYSA-N 2,4-di-tert-butylphenol Chemical compound CC(C)(C)C1=CC=C(O)C(C(C)(C)C)=C1 ICKWICRCANNIBI-UHFFFAOYSA-N 0.000 claims abstract description 84
- 239000000243 solution Substances 0.000 claims abstract description 52
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 48
- WPMYUUITDBHVQZ-UHFFFAOYSA-N 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoic acid Chemical compound CC(C)(C)C1=CC(CCC(O)=O)=CC(C(C)(C)C)=C1O WPMYUUITDBHVQZ-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 38
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 35
- 238000010828 elution Methods 0.000 claims abstract description 7
- 239000012071 phase Substances 0.000 claims abstract description 5
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 54
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 27
- 239000013558 reference substance Substances 0.000 claims description 19
- 239000002904 solvent Substances 0.000 claims description 18
- 239000012085 test solution Substances 0.000 claims description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 8
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 6
- 239000011780 sodium chloride Substances 0.000 claims 3
- 239000000945 filler Substances 0.000 claims 1
- 239000007790 solid phase Substances 0.000 claims 1
- 238000004140 cleaning Methods 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 description 15
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- 239000000047 product Substances 0.000 description 6
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- 238000001802 infusion Methods 0.000 description 5
- JKIJEFPNVSHHEI-UHFFFAOYSA-N Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) Chemical compound CC(C)(C)C1=CC(C(C)(C)C)=CC=C1OP(OC=1C(=CC(=CC=1)C(C)(C)C)C(C)(C)C)OC1=CC=C(C(C)(C)C)C=C1C(C)(C)C JKIJEFPNVSHHEI-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- SSDSCDGVMJFTEQ-UHFFFAOYSA-N octadecyl 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate Chemical compound CCCCCCCCCCCCCCCCCCOC(=O)CCC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 SSDSCDGVMJFTEQ-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 3
- BGYHLZZASRKEJE-UHFFFAOYSA-N [3-[3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoyloxy]-2,2-bis[3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoyloxymethyl]propyl] 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate Chemical compound CC(C)(C)C1=C(O)C(C(C)(C)C)=CC(CCC(=O)OCC(COC(=O)CCC=2C=C(C(O)=C(C=2)C(C)(C)C)C(C)(C)C)(COC(=O)CCC=2C=C(C(O)=C(C=2)C(C)(C)C)C(C)(C)C)COC(=O)CCC=2C=C(C(O)=C(C=2)C(C)(C)C)C(C)(C)C)=C1 BGYHLZZASRKEJE-UHFFFAOYSA-N 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
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- 229960003376 levofloxacin Drugs 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
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- 238000011109 contamination Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000004806 packaging method and process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- WXZMFSXDPGVJKK-UHFFFAOYSA-N pentaerythritol Chemical compound OCC(CO)(CO)CO WXZMFSXDPGVJKK-UHFFFAOYSA-N 0.000 description 1
- 239000002530 phenolic antioxidant Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention provides a method for detecting an antioxidant degradation product in a levofloxacin lactate and sodium chloride injection. The method adopts solid-phase extraction to treat the levofloxacin lactate sodium chloride injection, and adds a step of cleaning the levofloxacin lactate on a solid-phase extraction column by using an acetic acid solution with a specific concentration before elution, so that the interference of the levofloxacin lactate on the detection of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is avoided, and then the detection is carried out by using an ACQUITY UPLC BEHC18 and an acetonitrile-water solution with a volume ratio of 70:30 as a mobile phase at a detection wavelength of 280 nm.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a method for detecting an antioxidant degradation product in a levofloxacin lactate and sodium chloride injection.
Background
Levofloxacin lactate is a third-generation quinolone broad-spectrum antibacterial drug, and the injection is clinically used for treating infection caused by sensitive bacteria, and has definite curative effect. Compared with glass bottle packed infusion products, plastic bottle packed infusion products have the characteristics of light weight, convenient transportation, difficult damage of bottle bodies, easy product quality guarantee, convenient bottle cap opening by adopting the easy-to-pull ring design and the like, and are widely used in various medical institutions in China in recent years. In addition, because the light transmission of the plastic bottle body is weaker than that of the glass bottle, the lactic acid levofloxacin product which is sensitive to light and easy to decompose is easier to store, and the product period can be further prolonged.
In order to prevent thermal oxidation degradation reaction, antioxidants are usually added to the polymer materials for infusion packaging to maintain the excellent properties of the polymer materials and delay the oxidation process of the polymer materials. At present, most antioxidants used in infusion materials in China are phenolic antioxidants or phosphorous antioxidants, and the most widely used antioxidants include pentaerythritol tetrakis- [ beta- (3, 5-di-tert-butyl-4-hydroxyphenyl) propionate (antioxidant 1010), tris (2, 4-di-tert-butylphenyl) phosphite (antioxidant 168), n-octadecyl beta- (3, 5-di-tert-butyl-4-hydroxyphenyl) propionate (antioxidant 1076), and the like. However, since the infusion packaging material is in direct contact with the liquid medicine, the antioxidant 1010, the antioxidant 168 or the antioxidant 1076 may be oxidized or hydrolyzed in the liquid medicine to generate degradation products, and the degradation products may migrate into the liquid medicine to cause contamination of the preparation and safety risks. At present, the detection method and migration rule of the antioxidant are researched more, and high performance liquid chromatography is mostly adopted, but the degradation product of the antioxidant of the levofloxacin lactate injection cannot be quantitatively analyzed by the HPLC method at present. Therefore, there is a need to develop a method for detecting the degradation products of the antioxidant, so as to enhance the quality safety control of levofloxacin lactate injection.
Disclosure of Invention
Aiming at the technical problem that antioxidant degradation products possibly contained in levofloxacin lactate injection cannot be detected in the prior art, the invention provides a method for detecting the antioxidant degradation products in levofloxacin lactate and sodium chloride injection.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a detection method for antioxidant degradation products in levofloxacin lactate and sodium chloride injection comprises the following steps of:
preparing a reference substance solution: mixing 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid reference substances, and preparing a reference substance solution by using a solvent;
solid phase extraction: adding 0.001-0.003mol/L acetic acid solution into a solid phase extraction column after the levofloxacin lactate and sodium chloride injection is over-activated to wash the solid phase extraction column, then sequentially eluting with methanol and tetrahydrofuran, collecting eluent, and fixing the volume of a solvent to be used as a test solution;
and (4) taking the reference substance solution and the test solution to enter a high performance liquid chromatograph for detection.
The antioxidant mainly refers to antioxidant 1010, antioxidant 168, antioxidant 1076 and the like, and other degradation products are antioxidants of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid.
Three substances, namely levofloxacin lactate and a degradation product of an antioxidant, namely 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid, may exist in the levofloxacin lactate sodium chloride injection at the same time, and all the three substances are enriched on a solid-phase extraction column when the degradation product of the antioxidant is enriched through the solid-phase extraction column, the levofloxacin lactate enriched on the solid-phase extraction column cannot be cleaned by adopting a conventional elution step, and the levofloxacin lactate influences the detection of the 2, 4-di-tert-butylphenol and the 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid during detection.
According to the method for detecting the antioxidant degradation product in the levofloxacin lactate and sodium chloride injection, the step of washing the solid-phase extraction column by adding the acetic acid solution with a specific concentration before elution is performed, so that levofloxacin lactate on the solid-phase extraction column is sufficiently removed, and two substances of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid cannot be eluted, so that only 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid exist on the solid-phase extraction column during elution, and the purpose of accurate quantitative detection is achieved.
Preferably, the packing of the solid phase extraction column is N-vinylpyrrolidone and a lipophilic divinylbenzene polymer.
Further preferably, the solid phase extraction column is a Subel-Select HLB SPE solid phase extraction column.
The preferable chromatographic column is matched with a specific cleaning solution and an eluent, so that separation of the levofloxacin lactate and the two substances of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid is favorably realized, and accurate quantitative detection of the two substances of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid by a high performance liquid detection method is favorably carried out subsequently.
Preferably, the dosage of the levofloxacin lactate and sodium chloride injection is 10-100mL, and the dosage of the acetic acid solution is 5 mL.
Preferably, the concentration of the acetic acid solution is 0.002 mol/L.
The optimized dosage of the acetic acid solution is favorable for sufficiently eluting the lactic acid levofloxacin enriched on the solid-phase extraction column.
Preferably, the total volume of the methanol and the tetrahydrofuran is 10mL, wherein the volume ratio of the methanol to the tetrahydrofuran is 1:9-9: 1.
Further preferably, the total volume of the methanol and the tetrahydrofuran is 10mL, wherein the volume ratio of the methanol to the tetrahydrofuran is 6: 4.
The preferable dosage of the eluent is favorable for fully eluting the two substances of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid on the solid phase extraction column, thereby being favorable for accurately quantifying the contents of the 2, 4-di-tert-butylphenol and the 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid in the levofloxacin lactate sodium chloride injection.
Preferably, the volume of the solvent is 10mL after collecting the eluent.
Optionally, the activation method of the solid phase extraction column comprises: before the solid phase extraction column is used, the solid phase extraction column is washed by 10mL of methanol and 10mL of water in sequence, and the solid phase extraction column is kept in a wet state in the whole process.
Preferably, the detection conditions of the high performance liquid chromatography are as follows:
a detector: an ultraviolet detector;
detection wavelength: 280 nm;
a chromatographic column: ACQUITY UPLC BEH C18, 2.1 × 100mm, 1.7 μm;
mobile phase: acetonitrile-water solution with volume ratio of 70: 30;
flow rate: 0.38-0.42 mL/min;
the elution mode is isocratic elution.
Preferably, the column temperature is 28-32 ℃ and the injection volume is 3 muL.
More preferably, the flow rate is 0.4mL/min and the column temperature is 30 ℃.
Preferably, the concentration of 2, 4-di-tert-butylphenol in the control solution is 6.7. mu.g/mL, and the concentration of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid in the control solution is 0.8. mu.g/mL.
Through the research and verification of methodologies such as specificity, sensitivity and the like, the high performance liquid detection method provided by the invention is found to be sensitive, accurate and good in reproducibility, can be used for simultaneously and quantitatively detecting two substances, namely antioxidant degradation products 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid, in the levofloxacin lactate sodium chloride injection by a simpler method, is simple, good in separation effect and high in sensitivity, provides reliable guarantee for better controlling the product quality of the levofloxacin lactate sodium chloride injection, and has wide application prospect.
Drawings
FIG. 1 is a high performance liquid chromatogram of a control solution in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of a test sample solution detected by method one in example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of a test sample solution detected by method two in example 1 of the present invention;
FIG. 4 is a high performance liquid chromatogram of a test solution obtained by the third method in example 1 of the present invention;
FIG. 5 is a high performance liquid chromatogram of the labeled solution of the sample detected by method III in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Preparing a reference substance solution: mixing 2, 4-di-tert-butylphenol and a 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid reference substance, and preparing a reference substance solution with the concentration of 6.7 mu g/mL of 2, 4-di-tert-butylphenol and 0.8 mu g/mL of 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid by using a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1).
HPLC detection conditions of two substances of antioxidant degradation products 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid are as follows:
a detector: an ultraviolet detector;
detection wavelength: 280 nm;
a chromatographic column: ACQUITY UPLC BEH C18, 2.1 × 100mm, 1.7 μm;
mobile phase: acetonitrile-water solution with volume ratio of 70: 30;
flow rate: 0.4 mL/min;
column temperature: 30 ℃;
sample introduction amount: 3 mu L of the solution;
the elution mode is isocratic elution.
The prepared reference substance solution is taken and subjected to sample injection detection according to the liquid chromatography conditions, the result is shown in figure 1, the peak time of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is 1.262min, the peak time of 2, 4-di-tert-butylphenol is 2.599min and the degree of separation is high, and the HPLC detection method provided by the invention can realize accurate quantitative detection of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid.
The method comprises the following steps:
in order to eliminate the interference of levofloxacin lactate on 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid, the inventor tries various methods and selects various cleaning liquids to remove the levofloxacin lactate enriched in the solid-phase extraction column.
The method comprises the following steps:
50mL of levofloxacin lactate and sodium chloride injection passes through an activated solid-phase extraction column (Suel-Select HLB SPE, 6mL), is sequentially eluted by 6mL of methanol and 4mL of tetrahydrofuran, eluent is collected, volume is increased to 10mL by using a solvent (the volume ratio of the tetrahydrofuran to the methanol is 1: 1), and the solution is filtered to be used as a test solution. Detection was performed according to the above high performance liquid chromatography conditions, and a chromatogram was recorded, as shown in FIG. 2. As can be seen from the figure, obvious bulges exist at about 1.4min and 3.6min, and the detection of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is seriously interfered.
The second method comprises the following steps:
50mL of levofloxacin lactate and sodium chloride injection is filtered through an activated solid-phase extraction column (Suel-Select HLB SPE, 6mL), then 50mL of water is added for cleaning, then 6mL of methanol and 4mL of tetrahydrofuran are sequentially used for eluting, eluent is collected, the volume is adjusted to 10mL by using a solvent (the volume ratio of the tetrahydrofuran to the methanol is 1: 1), and the solution is filtered to be used as a sample solution. Detection was performed according to the above high performance liquid chromatography conditions, and a chromatogram was recorded, as shown in FIG. 3. As can be seen from the figure, obvious swelling appears between 1.4 and 3.5min, and the detection of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is seriously interfered.
The third method comprises the following steps:
the inventor unexpectedly finds that the interference of levofloxacin lactate on the detection of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid can be effectively eliminated by using 0.001-0.003mol/L acetic acid solution as a cleaning agent.
Taking 50mL of levofloxacin lactate and sodium chloride injection, passing through an activated solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, diluting to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1), and filtering to obtain a sample solution.
Taking 50mL of levofloxacin lactate sodium chloride injection, precisely measuring 1mL of the prepared reference solution, adding the reference solution into the levofloxacin lactate sodium chloride injection, uniformly mixing, passing through an activated solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, diluting the volume to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1), filtering, and taking the volume as a sample to add a standard solution.
Taking the prepared test solution and sample adding standard solution, detecting according to the above high performance liquid chromatography conditions, and recording chromatogram, as shown in fig. 4-5. Wherein, fig. 4 is a chromatogram of the test solution, fig. 5 is a chromatogram of the sample-added solution, and as can be seen from fig. 4, levofloxacin lactate, 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid and 2, 4-di-tert-butylphenol are not detected in the test solution. As can be seen from FIG. 5, in the sample-added standard solution, the peak-off time of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is 1.256min, the peak-off time of 2, 4-di-tert-butylphenol is 2.533min, the separation degree is high, and levofloxacin lactate does not generate an interference peak.
The recovery rates of 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid were calculated, and the results are shown in Table 1.
TABLE 1 recovery rate confirmation results
| Composition (I) | Peak area of control | Area of sample normalized peak | The recovery rate is high |
| 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid | 3699 | 3953 | 106.87 |
| 2, 4-di-tert-butylphenol detection | 31463 | 31046 | 98.67 |
Therefore, it was determined that the preparation method of the test sample solution was: taking 50mL of levofloxacin lactate and sodium chloride injection, passing through a solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, diluting to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1), and filtering to obtain a sample solution.
Example 2
Preparing a reference substance solution: mixing 2, 4-di-tert-butylphenol and a 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid reference substance, and preparing a reference substance solution with the concentration of 6.7 mu g/mL of 2, 4-di-tert-butylphenol and 0.8 mu g/mL of 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid by using a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1).
Preparing a test solution: taking 50mL of levofloxacin lactate and sodium chloride injection, passing through a solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, diluting to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1), and filtering to obtain a sample solution.
Preparing a sample labeling solution: taking 50mL of levofloxacin lactate and sodium chloride injection, precisely measuring 1mL of the prepared reference solution, adding the reference solution into the levofloxacin lactate and sodium chloride injection, uniformly mixing, passing through a solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, diluting to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1), and filtering to obtain a sample solution.
And (3) verification of methodology:
2.1 specificity
Detecting the reference solution, the sample solution and the sample labeling solution according to the high performance liquid chromatography conditions, and recording chromatograms, wherein fig. 1 is a chromatogram of the reference solution, fig. 4 is a chromatogram of the sample solution, and fig. 5 is a chromatogram of the sample labeling solution, as shown in fig. 1 and fig. 4-5.
As can be seen from FIG. 1, the peak-off time of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is 1.262min, the peak-off time of 2, 4-di-tert-butylphenol is 2.599min, and the degree of separation is high. The sample solution has no chromatographic peaks of levofloxacin lactate, 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid and 2, 4-di-tert-butylphenol. In the sample adding standard solution, the peak-out time of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is 1.256min, the peak-out time of 2, 4-di-tert-butylphenol is 2.533min, and no chromatographic peak of levofloxacin exists. The method is proved to have good specificity.
2.2 repeatability
Taking 6 parts of levofloxacin lactate and sodium chloride injection, precisely measuring 50mL, preparing 6 parts of test solution according to the preparation method of the test solution, and detecting according to the high performance liquid chromatography conditions, wherein the results are shown in Table 2.
TABLE 2 results of the repeatability tests
The test result shows that 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid and 2, 4-di-tert-butylphenol are not detected in 6 parts of test solution, and the method has good repeatability.
2.3 accuracy
Taking 6 parts of levofloxacin lactate and sodium chloride injection, precisely measuring 50mL, and preparing 6 parts of test solution according to the preparation method of the test solution. Precisely measuring 1mL of the reference solution, adding into 6 10mL volumetric flasks, and diluting to constant volume with the test solution to obtain recovery solution.
The recovery rate was calculated by detecting the 6 recovery rate solutions according to the conditions of the high performance liquid chromatography, and recording chromatograms, and the results are shown in tables 3 to 4.
TABLE 33 results of recovery test of 5-di-tert-butyl-4-hydroxyphenylpropionic acid
TABLE 42 results of recovery test of 4-di-tert-butylphenol
The test result shows that the average recovery rate of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid in 6 parts of test solution is 103.58%, the RSD is 1.86%, the average recovery rate of 2, 4-di-tert-butylphenol is 99.52%, and the RSD is 0.54%, which indicates that the method has good accuracy.
2.4 Linear Range
Precisely measuring a proper amount of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid and 2, 4-di-tert-butylphenol reference substances, uniformly mixing, and preparing a series of linear solutions with a certain volume by using a solvent. Precisely measuring 3 μ L, detecting by sample injection according to the above high performance liquid chromatography conditions, recording chromatogram, and measuring peak area, the results are shown in tables 5-6.
TABLE 53 Linear test results of 5-di-tert-butyl-4-hydroxyphenylpropionic acid
TABLE 62, 4-Di-tert-butylphenol Linear test results
The above results show that 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid is excellent in linearity at a concentration of 0.078 to 117.718. mu.g/ml, and 2, 4-di-tert-butylphenol is excellent in linearity at a concentration of 0.099 to 118.681. mu.g/ml.
2.5 detection and quantitation limits
The control solutions prepared above were diluted to determine the limit of quantitation (S/N. gtoreq.10) and the limit of detection (S/N. gtoreq.3), respectively, and the results are shown in Table 7.
TABLE 7 detection limit and quantitation limit test results
Test results show that the detection limit concentrations of the 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid and the 2, 4-di-tert-butylphenol are 0.031 mug/mL and 0.040 mug/mL respectively; the concentration of the limit of quantification is 0.078 mug/mL and 0.099 mug/mL respectively, the limit of quantification solution is injected repeatedly for 6 times, the RSD of 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid is only 1.34 percent, and the RSD of 2, 4-di-tert-butylphenol is only 2.47 percent, which shows that the method has high sensitivity.
2.6 durability
Taking the prepared reference solution, detecting according to the high performance liquid chromatography conditions, wherein the chromatographic conditions respectively examine the influence of the separation degree under the conditions of flow rate +/-0.2 mL/min and column temperature +/-2 ℃, and the results are shown in tables 8-9.
Table 8 flow rate durability test results
TABLE 9 column temperature durability test results
Test results show that the detection conditions are changed within a certain range (flow rate +/-0.2 mL/min, column temperature +/-2 ℃), 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid and 2, 4-di-tert-butylphenol are still higher, and the method has good durability.
Example 3
Confirmation of recovery ratio of washing method:
preparing a reference substance solution: mixing 2, 4-di-tert-butylphenol and a 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid reference substance, and preparing a reference substance solution with the concentration of 6.7 mu g/mL of 2, 4-di-tert-butylphenol and 0.8 mu g/mL of 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid by using a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1).
Preparing a test solution: taking 50mL of levofloxacin lactate and sodium chloride injection, passing through an activated solid-phase extraction column (Suel-Select HLB SPE, 6mL), adding 5mL of 0.002mol/L acetic acid solution, sequentially eluting with 6mL of methanol and 4mL of tetrahydrofuran, collecting eluent, and diluting to 10mL with a solvent (tetrahydrofuran and methanol in a volume ratio of 1: 1) to obtain a stock solution for later use.
Precisely measuring 1mL of the prepared reference solution, adding into a 10mL volumetric flask, diluting to constant volume with the stock solution, and filtering to obtain a sample solution.
Taking the reference solution and the sample solution, performing sample injection detection according to the high performance liquid chromatography conditions, recording peak areas, and calculating the recovery rate, wherein the results are shown in Table 10.
TABLE 10 confirmation of recovery rate
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A detection method for antioxidant degradation products in levofloxacin lactate and sodium chloride injection is characterized in that the detection method is a solid phase extraction-high phase liquid chromatography combined method and comprises the following steps:
preparing a reference substance solution: mixing 2, 4-di-tert-butylphenol and 3, 5-di-tert-butyl-4-hydroxy phenylpropionic acid reference substances, and preparing a reference substance solution by using a solvent;
solid phase extraction: adding 0.001-0.003mol/L acetic acid solution into a solid phase extraction column after the levofloxacin lactate and sodium chloride injection is over-activated to wash the solid phase extraction column, then sequentially eluting with methanol and tetrahydrofuran, collecting eluent, and fixing the volume of a solvent to be used as a test solution;
and (4) taking the reference substance solution and the test solution to enter a high performance liquid chromatograph for detection.
2. The method for detecting the degradation products of the antioxidant in the injection of levofloxacin lactate and sodium chloride as claimed in claim 1, wherein the filler of the solid phase extraction column is N-vinylpyrrolidone and lipophilic divinylbenzene polymer.
3. The method for detecting the degradation product of the antioxidant in the levofloxacin lactate and sodium chloride injection as claimed in claim 2, wherein the solid phase extraction column is a Suel-Select HLB SPE solid phase extraction column.
4. The method for detecting the degradation product of the antioxidant in the injection of levofloxacin lactate and sodium chloride as claimed in claim 1, wherein the dosage of the injection of levofloxacin lactate and sodium chloride is 10-100mL, and the dosage of the acetic acid solution is 5 mL.
5. The method for detecting the degradation products of the antioxidant in the levofloxacin lactate and sodium chloride injection as claimed in claim 4, wherein the total volume of the methanol and the tetrahydrofuran is 10mL, and the volume ratio of the methanol to the tetrahydrofuran is 1:9-9: 1; and/or
After collecting the eluent, the volume of the solvent is 10 mL.
6. The method for detecting the degradation products of the antioxidant in levofloxacin lactate and sodium chloride injection according to claim 1, wherein the concentration of the acetic acid solution is 0.002 mol/L.
7. The method for detecting the antioxidant degradation product in the levofloxacin lactate and sodium chloride injection as claimed in claim 1, wherein the detection conditions of the high performance liquid chromatography are as follows:
a detector: an ultraviolet detector;
detection wavelength: 280 nm;
a chromatographic column: ACQUITY UPLC BEH C18, 2.1 × 100mm, 1.7 μm;
mobile phase: acetonitrile-water solution with volume ratio of 70: 30;
flow rate: 0.38-0.42 mL/min;
the elution mode is isocratic elution.
8. The method for detecting the degradation products of the antioxidant in the levofloxacin lactate and sodium chloride injection as claimed in claim 7, wherein the column temperature is 28-32 ℃ and the injection volume is 3 μ L.
9. The method of detecting degradation products of antioxidants in levofloxacin lactate sodium chloride injection of claim 8, wherein the flow rate is 0.4mL/min and the column temperature is 30 ℃.
10. The method for detecting the degradation product of the antioxidant in the levofloxacin lactate and sodium chloride injection as claimed in claim 1, wherein the concentration of 2, 4-di-tert-butylphenol and the concentration of 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid in the control solution are respectively 6.7 μ g/mL and 0.8 μ g/mL.
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