CN113341019A - Analysis method of edetate in levofloxacin lactate and sodium chloride injection - Google Patents
Analysis method of edetate in levofloxacin lactate and sodium chloride injection Download PDFInfo
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- CN113341019A CN113341019A CN202110641208.1A CN202110641208A CN113341019A CN 113341019 A CN113341019 A CN 113341019A CN 202110641208 A CN202110641208 A CN 202110641208A CN 113341019 A CN113341019 A CN 113341019A
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- edetate
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- sodium chloride
- chloride injection
- levofloxacin lactate
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 229940009662 edetate Drugs 0.000 title claims abstract description 56
- 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 30
- 239000008354 sodium chloride injection Substances 0.000 title claims abstract description 30
- 238000004458 analytical method Methods 0.000 title claims abstract description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000001212 derivatisation Methods 0.000 claims abstract description 48
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 45
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 43
- 230000014759 maintenance of location Effects 0.000 claims abstract description 29
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 26
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 26
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 24
- 229910052742 iron Inorganic materials 0.000 claims abstract description 21
- -1 iron ion Chemical class 0.000 claims abstract description 19
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical class [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 14
- 238000012360 testing method Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 35
- 229940124274 edetate disodium Drugs 0.000 claims description 34
- 229910001431 copper ion Inorganic materials 0.000 claims description 13
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 9
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000010949 copper Substances 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 44
- 239000000523 sample Substances 0.000 description 38
- 229910001447 ferric ion Inorganic materials 0.000 description 24
- 239000012071 phase Substances 0.000 description 21
- 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 19
- 229960003376 levofloxacin Drugs 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 239000011550 stock solution Substances 0.000 description 16
- 238000011084 recovery Methods 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- 238000012790 confirmation Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- UEAVLBXLOZNDHT-UHFFFAOYSA-K calcium;sodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxymethyl)amino]acetate Chemical compound [Na+].[Ca+2].OC(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEAVLBXLOZNDHT-UHFFFAOYSA-K 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- KYGZCKSPAKDVKC-UHFFFAOYSA-N Oxolinic acid Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC2=C1OCO2 KYGZCKSPAKDVKC-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000003306 quinoline derived antiinfective agent Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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 belongs to the field of drug analysis, and particularly relates to an analysis method of edetate in levofloxacin lactate and sodium chloride injection. The analysis method comprises the steps of utilizing an iron ion derivatization reagent to derivatize edetate in the levofloxacin lactate and sodium chloride injection to be tested to obtain a test sample a; determining edetate in the sample a by HPLC method; the HPLC method adopts sodium dihydrogen phosphate solution-acetonitrile as a mobile phase and a C18 column as a chromatographic column. According to the invention, a sample is directly derivatized by iron ions, and rapid screening and determination are realized by optimizing chromatographic conditions and utilizing the characteristic of short iron ion derivatization retention time; and then, the suspicious sample is verified by utilizing the characteristic of long retention time of the copper ion derivative, so that the occurrence of false positive is effectively avoided, and the method is simple, rapid, accurate and good in repeatability.
Description
Technical Field
The invention belongs to the field of drug analysis, and particularly relates to an analysis method of edetate in levofloxacin lactate and sodium chloride injection.
Background
Levofloxacin is a third-generation quinolone antibiotic, is easy to form a complex with metal ions to lose activity, and Fe3+The ions are easy to complex with the levofloxacin to darken (dark brown) the color of the levofloxacin lactate and sodium chloride injection, so that part of production enterprises add edetate (disodium edetate or calcium sodium edetate) into the prescription, the stability of a complex formed by the edetate and metal ions is higher than that of the levofloxacin, the levofloxacin can be prevented from losing activity and darkening the color of the injection, and whether illegal behaviors of producing the levofloxacin without adding the edetate or adding the edetate according to the prescription are examined.
If the Edetate (EDTA) is directly measured, the EDTA is easy to complex with metal ions in a sample and a mobile phase, a plurality of chromatographic peaks are formed, and accurate quantification cannot be realized, so that the derivatization method is considered for measuring the amount of the edetate in the sample number. Logarithm of stability constants (lgKMY) Fe of edetate salts with common metal ions3+Is 24.23, Cu2+Is 18.7 of Ca2+Is 10.69, which shows Fe3+Highest stability, Cu2+Second, Fe3+And Cu2+All can replace Ca2+Ions.
At present, the determination method of edetate in injection has been studied for a long time, and the content of edetate disodium is generally determined by an iron ion reagent by using an HPLC method for analysis, and the analysis time is long. The analysis speed can be accelerated by optimizing chromatographic conditions of an HPLC method and shortening the retention time of peaks, but if the retention time of the iron ion derivative edetate disodium is shortened, a false positive phenomenon is likely to occur, so that the determination is wrong and misjudgment is caused. How to obtain a quick, accurate and effective method for analyzing the edetate is a problem to be solved at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an analysis method of edetate in a levofloxacin lactate and sodium chloride injection, which directly derives the edetate in the levofloxacin lactate and sodium chloride injection to be tested by using iron ions, rapidly measures the content of the edetate by optimizing chromatographic conditions and compressing analysis time, and then further confirms whether the retention time of a derived peak is consistent with that of a derived peak of an edetate disodium standard solution for a suspicious sample by using the edetate in the copper ion derived sample.
The invention provides an analysis method of edetate in levofloxacin lactate and sodium chloride injection, which comprises the following steps:
s1: directly derivatizing edetate in the levofloxacin lactate and sodium chloride injection to be tested by using an iron ion derivatization reagent to obtain a test sample a;
s2: determining edetate in the sample a by HPLC method;
the HPLC method adopts sodium dihydrogen phosphate solution-acetonitrile as a mobile phase and a C18 column as a chromatographic column.
In the technical scheme, the edetate (disodium edetate or calcium sodium edetate) in the levofloxacin lactate and sodium chloride injection is directly derived by utilizing iron ions to form an iron edetate complex (EDTA-Fe)3+) Thereby rapidly determining the edetate content in the levofloxacin lactate and sodium chloride injection, wherein the edetate content is edetate disodium (C)10H14N2Na2O8·2H2O) calculating. The method directly uses iron ions to deriveAnd the method has the advantages that the sample does not need to be extracted, the pH value of a derivatization reagent does not need to be adjusted, the iron ion derivatization and the copper ion derivatization are combined, the false positive is effectively avoided, and the method is simple, rapid, accurate and good in repeatability.
Further, the analysis method further comprises:
s3: and derivatizing the sample to be tested by using a copper ion derivatization reagent to obtain a test sample b, and determining by using the HPLC method to confirm whether the peak retention time of the test sample b is consistent with the derivatization peak of the edetate disodium standard solution.
In the technical scheme, the method is used for further confirming whether the retention time of the peak after derivatization is consistent with the derivatization peak of the standard edetate solution or not by using copper ions to derivatize the edetate in the sample to be detected for the suspicious sample, the analysis method can combine iron ion derivatization and copper ion derivatization, so that false positive is avoided, and the method is simple, rapid and accurate.
Further, the volume ratio of the sodium dihydrogen phosphate solution to the acetonitrile in the sodium dihydrogen phosphate solution-acetonitrile mobile phase is (88-92) to (8-12);
preferably, the volume ratio of the sodium dihydrogen phosphate solution to the acetonitrile in the mobile phase is 90: 10.
In the technical scheme, the consumption of the mobile phase acetonitrile is less, but the analysis time is short, so that the detection speed is kept, and the detection accuracy is also ensured.
Further, the sodium dihydrogen phosphate solution contains 4-6mmol/L tetrabutylammonium bromide, and the pH is adjusted to 4-5 by using phosphoric acid solution.
Preferably, the sodium dihydrogen phosphate solution contains 5mmol/L tetrabutylammonium bromide, and the pH is adjusted to 4.5 with a phosphoric acid solution.
In the technical scheme, EDTA-Fe can be prolonged by adding tetrabutylammonium bromide3+The peak retention time can be effectively avoided, and EDTA-Fe can be effectively prevented from tailing by controlling the pH value of the mobile phase3+The retention time of the peak and the peak is controlled to be about 5min, and the number of theoretical plates, tailing factors and the peak height can be optimized.
Further, the chromatographic column was InertSustain AQ-C18, the specification was 4.6mm × 250mm, and the particle size was 5 μm.
Further, the chromatographic conditions of the HPLC method are as follows:
mobile phase: 10-100mmol/L sodium dihydrogen phosphate solution-acetonitrile;
flow rate: 1.0 mL/min;
a chromatographic column: InertSustain AQ-C18;
column temperature: 30-45 ℃;
sample introduction amount: 10-20 μ L;
detection wavelength: 250-270 nm;
in the technical scheme, the retention time of the edetate can be prolonged by adding the ion pair reagent into the mobile phase; EDTA-Fe can be effectively improved by controlling the concentration of sodium dihydrogen phosphate3+Peak type and number of theoretical plates.
Preferably, the chromatographic conditions of the HPLC method are as follows:
mobile phase: 50mmol/L sodium dihydrogen phosphate solution-acetonitrile;
flow rate: 1.0 mL/min;
a chromatographic column: InertSustain AQ-C18;
column temperature: 40 ℃;
sample introduction amount: 10-20 μ L;
detection wavelength: 257 nm;
wherein, the sample amount in the step S2 is preferably 10 μ L; the amount of sample injection in step S3 is preferably 20. mu.L.
Further, the iron ion derivatization reagent in step S1 is ferric chloride solution, wherein Fe is3+The concentration is 0.01mol/L, and the volume ratio of the iron ion derivatization reagent to the sample to be detected is (0.1-1.5) to 5;
and/or the copper ion-derivatizing agent in the step S3 is a copper sulfate solution, wherein Cu is2+The concentration is 0.01mol/L, and the volume ratio of the copper ion derivatization reagent to the sample to be detected is (0.1-1.5) to 5. Specifically, the dosage of the copper ion derivatization reagent can be dynamically adjusted according to the edetate concentration in the suspicious sample.
In the technical scheme, the derivatization reagent is directly prepared by pure water, the pH value is not required to be saved, and the method is simple; the accuracy of the assay is improved by the optimal selection of the volume (amount added) of derivatizing agent.
Compared with the prior art, the method has the beneficial effects that:
1. according to the invention, the edetate in the levofloxacin lactate and sodium chloride injection to be tested is directly derivatized by using iron ions, and the content of the edetate is rapidly measured by optimizing chromatographic conditions, utilizing the characteristic of short retention time of the iron ion derivative and compressing analysis time, so that the speed is high, and the method is accurate and reliable;
2. the method directly uses iron ions for derivatization, does not need to extract a sample, does not need to adjust the pH value of a derivatization reagent, is simple and fast, realizes fast screening, and has the time less than 10 min;
3. the method can utilize copper ions to derivatize the edetate in the sample to be detected, utilizes the characteristic of long retention time of the copper ion derivative, and further confirms whether the retention time of the peak after derivatization is consistent with the derivatization peak of the standard solution of edetate disodium, can effectively avoid the occurrence of false positive, and is particularly suitable for the detection and analysis of low-content edetate;
4. the method can combine iron ion derivatization and copper ion derivatization, can avoid the occurrence of false positive, and has the advantages of simplicity, rapidness, accuracy and good repeatability.
Drawings
FIG. 1 shows EDTA-Fe according to the present invention3+A typical chromatogram, wherein A is a chromatogram obtained by derivatizing edetate disodium with ferric chloride; b is a typical chromatogram of levofloxacin lactate and sodium chloride injection (the formula contains edetate disodium) after being subjected to ferric chloride derivatization; c is a typical chromatogram of levofloxacin lactate and sodium chloride injection (without edetate in the prescription) after being derivatized by ferric chloride; d is a typical chromatogram of the levofloxacin lactate and sodium chloride injection without derivatization;
FIG. 2 is a confirmation chart of the present invention, wherein A is a chromatogram of disodium edetate derivatized with copper sulfate; b is a chromatogram of a sample 1 of company E after being derivatized by copper sulfate; and C is a chromatogram of a sample 2 of the company E after being subjected to copper sulfate derivatization.
Detailed Description
The technical features of the present invention described above and those described in detail below (as an embodiment) can be combined with each other to form a new or preferred technical solution, but the present invention is not limited to these embodiments, and the embodiments also do not limit the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The formulations according to the following examples are all commercially available products and are commercially available, unless otherwise specified.
The invention is described in further detail below with reference to the figures and examples:
example 1: optimization of experimental conditions
1. Instruments and reagents
1.1 Instrument: LC-20AD high performance liquid chromatograph (Shimadzu, Japan), MS205DU electronic balance (METTLER TOLEDO, Switzerland);
1.2 reagent: disodium ethylenediaminetetraacetate reference reagent (manufacturer: Shanghai Shanpu chemical Co., Ltd., lot number: 2020, 4, 12 days, content is calculated as 100%), ferric trichloride and copper sulfate are analytically pure (manufacturer: Xiong science, Ltd.), water is purified water, acetonitrile is chromatographically pure, and other reagents are analytically pure;
1.3 medicine: levofloxacin lactate sodium chloride injection: 15 batches of samples from east Asia pharmaceutical Co., Ltd, E2 batches, and 1 batch of samples from Pueisi pharmaceutical Co., Ltd, Sha, Zhejiang.
2. Chromatographic conditions
Mobile phase: 50mmol/L sodium dihydrogen phosphate solution (containing 5mmol/L tetrabutylammonium bromide, pH adjusted to 4.5 with phosphoric acid solution) -acetonitrile (90: 10); flow rate: 1.0 mL/min; the chromatographic column is InertSustain AQ-C18(4.6mm × 250mm, 5 μm); the column temperature is 40 ℃; the sample volume is 10 mu L; detection wavelength: 257 nm.
3. Solution preparation
Precisely weighing 50.30mg of an ethylenediaminetetraacetic acid disodium reference reagent, placing the ethylenediaminetetraacetic acid disodium reference reagent into a 50mL measuring flask, adding water to dissolve the ethylenediaminetetraacetic acid disodium reference reagent, diluting the ethylenediaminetetraacetic acid disodium reference reagent to a scale, and shaking up the disodium ethylenediaminetetraacetate reference reagent to serve as a disodium edetate standard stock solution (about 1000 mu g/mL);
weighing 0.54g of ferric chloride, adding 200mL of water to dissolve and shake the ferric chloride uniformly to serve as a ferric ion derivatization reagent (containing Fe)3+About 0.01 mol/L);
precisely measuring 5mL of a to-be-detected levofloxacin lactate and sodium chloride injection sample, adding 1mL of a derivatization reagent, adding water to dilute to 10mL, and shaking up to obtain a sample solution a;
4. condition selection
4.1 selection of detection wavelength
EDTA and Fe3+Complex of (EDTA-Fe)3+) The absorption maximum at a wavelength of 257nm is selected so that 257nm is selected as the detection wavelength.
4.2 selection of phosphate buffer salt pH
The sodium dihydrogen phosphate solutions in the mobile phases were adjusted to different pH values, respectively, and analyzed under the chromatographic conditions of the present invention. From Table 1 phosphate buffer salt pH vs EDTA-Fe3+The influence of the peak results show that the pH value of the sodium dihydrogen phosphate solution is changed between 3.0 and 6.0, and the larger the pH value is, the EDTA-Fe is3+The slower the time to peak, the EDTA-Fe in the test sample solution3+The peak, theoretical plate number, tailing factor and peak height all worsened. Secondly, the larger the pH value is, the longer the retention time of the levofloxacin peak is; the retention time of the levofloxacin peak at the pH value of 6.0 is about 20min, and the peak appearance time is late. Therefore, the mobile phase pH is preferably 4.5 in combination.
TABLE 1 phosphate buffer salt pH vs EDTA-Fe3+Influence of the Peak
4.3 selection of ion versus reagent concentration
In the present invention refer toKeeping the acetonitrile proportion, the phosphate concentration and the pH unchanged according to the chromatographic conditions, and investigating the change of the tetrabutylammonium bromide concentration to the standard solution EDTA-Fe3+Influence of peak measurement. From Table 2 mobile phase tetrabutylammonium bromide concentration vs EDTA-Fe3+The effect of the peaks can be seen in the change of tetrabutylammonium bromide concentration from 0mmol/L to 5mmol/L EDTA-Fe3+The peak retention time is obviously prolonged, and the number of theoretical plates is greatly improved. Therefore, the final preferred tetrabutylammonium bromide concentration is 5mmol/L in combination.
TABLE 2 mobile phase tetrabutylammonium bromide concentration vs EDTA-Fe3+Influence of the Peak
| ρTetrabutylammonium bromide(mmol/L) | Retention time (min) | Number of theoretical plates | Tailing factor |
| 5 | 5.071 | 10670 | 1.197 |
| 2.5 | 4.393 | 8896 | 1.181 |
| 0 | 3.251 | 7249 | 1.187 |
4.4 selection of phosphate concentration
In the present invention, the influence of the change in the concentration of sodium dihydrogenphosphate on the measurement was examined while maintaining the acetonitrile ratio, the tetrabutylammonium bromide concentration in the mobile phase and the pH with reference to the above-mentioned chromatographic conditions. From Table 3 mobile phase sodium dihydrogen phosphate concentration vs EDTA-Fe3+As a result of the influence of the peaks, it can be seen that EDTA-Fe is present at a higher concentration of sodium dihydrogen phosphate3+The shorter the peak retention time and the smaller the concentration of sodium dihydrogen phosphate, EDTA-Fe in the test and standard solutions3+The peak retention time is slightly deviated, and the levofloxacin peak is in contact with EDTA-Fe3+There was some overlap of peaks, interfering with the assay. Therefore, the final preferred concentration of sodium dihydrogen phosphate is 50 mmol/L.
TABLE 3 mobile phase sodium dihydrogen phosphate concentration vs EDTA-Fe3+Influence of the Peak
4.5 selection of acetonitrile ratio and column temperature
In the present invention, the influence of the acetonitrile ratio and the change in the column temperature on the measurement was examined while keeping the conditions of the aqueous phase constant. From Table 4 mobile phase acetonitrile ratio versus EDTA-Fe3+The influence of the peaks and the levofloxacin peak shows that the temperature has little influence on the retention time, and the acetonitrile is reduced to 5 percent although EDTA-Fe3+The peak retention time was delayed by approximately 2min, but the levofloxacin peak retention time was extended to about 24min, which was about 3 times the levofloxacin peak retention time at 10% acetonitrile analysis. Therefore, the acetonitrile volume ratio is selected to be 10% and the column temperature is selected to be 40 ℃.
TABLE 4 mobile phase acetonitrile ratio vs EDTA-Fe3+Effect of Peak and levofloxacin Peak
4.6 selection of chromatographic separation conditions
EDTA-Fe3+The polarity is greater and the mobile phase is hardly retained in the C18 column without the addition of ion pairing reagents. Optionally, the retention time can be extended by adding an ion-pairing agent to the mobile phase. The EDTA-Fe can be well prolonged by adopting the chromatographic conditions3+The peak retention time is 5min, the peak retention time of levofloxacin is about 7min, and EDTA-Fe3+The peak and the levofloxacin peak are completely separated, the whole analysis time is less than 10min, and EDTA-Fe is shown in a chromatogram chart 13+A typical chromatogram can also show that EDTA-Fe is present under the chromatographic separation conditions3+The peak and levofloxacin peak are completely separated, and therefore, the chromatographic conditions are considered together as preferred conditions.
4.7 selection of derivatization conditions
Precisely measuring 2mL of edetate disodium standard stock solution (about 1000 mu g/mL), respectively adding 0.25-2 mL of derivatization reagent, adding water to dilute to 10mL, shaking up, analyzing according to the chromatographic conditions, and investigating EDTA-Fe by different volume of derivatization reagents3+Influence of the peak area. Derivatization reagent volumes vs EDTA-Fe from Table 53+The influence of the peak and the levofloxacin peak can be seen, when 0.5mL of EDTA-Fe is added in the derivatization reagent3+The peak area was already 96% of 1mL, while EDTA-Fe at 1mL and 1.5mL3+The peak area was substantially unchanged. Since it is sufficient to add 1mL of the derivatizing agent depending on the mass of disodium edetate in the solution to 2000. mu.g, it is preferable that the volume of the derivatizing agent is 1 mL.
TABLE 5 volume of derivatizing agent versus EDTA-Fe3+Effect of Peak and levofloxacin Peak
5. Methodology investigation
5.1 Linear relationship investigation
Precisely measuring 1mL of the disodium edetate standard stock solution (about 1000 mu g/mL), putting the disodium edetate standard stock solution into a 50mL measuring flask, adding water to dilute the disodium edetate standard stock solution to a scale, and shaking up to obtain a second disodium edetate standard stock solution (about 20 mu g/mL). Respectively taking 2mL, 1.5mL, 1mL and 0.5mL of the edetate disodium standard stock solution with the concentration of about 1000 mu g/mL and two 5mL, 2mL and 1mL of the edetate disodium standard stock solution with the concentration of about 20 mu g/mL into a 10mL measuring flask, adding 1mL of derivatization reagent, adding water to dilute to 10mL, shaking up to prepare the edetate disodium standard curve series solution (corresponding to the concentration of the edetate disodium contained is about 200, 150, 100, 50, 10, 4 and 2 mu g/mL).
According to the chromatographic conditions, 10 mu L of edetate disodium standard curve series solution is taken for sample injection and chromatogram is recorded. With EDTA-Fe3+The peak area is the ordinate (y), the linear regression is carried out by taking the edetate disodium concentration (x) as the abscissa, and the result shows that the edetate disodium concentration is between 2 and 200 mu g/mL and EDTA-Fe3+The peak areas are in good linear relation, and the regression equation is as follows: y is 1.417 × 104x+9.71×102，R2＝0.99998。
5.2 precision
Precisely measuring 1mL of edetate disodium standard stock solution (about 1000 mu g/mL), placing in a 10mL measuring flask, adding 1mL of derivatization reagent, adding water to dilute to 10mL, shaking up, continuously feeding samples for 6 times (10 mu L) according to the chromatographic strip, and recording the chromatogram. Calculation of EDTA-Fe3+The RSD of the peak areas (1427924, 1432473, 1432811, 1432186, 1432774 and 1431269) is 0.35%, which indicates that the method has good precision.
5.3 repeatability
6 portions of sample solution to be tested are prepared from samples (batch number: 2019101913, specification: 100 mL: 0.2g of levofloxacin) of east Asia pharmaceutical Co., Ltd, Jiangxi, Biebi group according to the preparation method of the solution, the analysis is carried out according to the chromatographic conditions, and the chromatogram is recorded. Calculation of EDTA-Fe in 6 samples3+The RSD of the peak area (657226, 660738, 664585, 661920, 654754, 661488) is 0.54%, which indicates that the method has good repeatability.
5.4 stability
Accurately measuring edetic acid1mL of disodium standard stock solution (about 1000 mu g/mL) is put into a 10mL measuring flask, 1mL of derivatization reagent is added, water is added for dilution to 10mL, the mixture is shaken up and is measured according to the chromatographic conditions at 0h, 2h, 4h, 6h, 8h, 10h and 12h respectively, and EDTA-Fe at each time is calculated3+RSD of the peak area (1414008, 1426801, 1426861, 1427951, 1427742, 1427092, 1426761) was 0.4%. Description of EDTA-Fe3+It was still stable within 12 h.
5.5 limit of quantitation and detection
Taking a proper amount of the second standard edetate product stock solution (about 20 mu g/mL), respectively placing the appropriate amount of the second standard edetate product stock solution into 10mL measuring bottles, respectively adding 1mL of derivatization reagent, adding water to dilute the solution to 10mL, shaking the solution uniformly to prepare solutions which are about 0.4 mu g/mL and 1.2 mu g/mL of edetate disodium, respectively measuring according to the chromatographic conditions, and determining the EDTA-Fe when the concentration of the edetate disodium is 0.4 mu g/mL3+The peak signal-to-noise ratios are all larger than 3, and the EDTA-Fe is added when the concentration of the edetate disodium is 1.2 mu g/mL3+The peak signal-to-noise ratios are all greater than 10. When the sample to be tested is sampled in 5mL and the dilution volume is 10mL, the detection limit and the quantification limit of the method can be 0.8 mu g/mL and 2.4 mu g/mL.
5.6 accuracy
5mL of Zhejiang Sha Proesi pharmaceutical industry Co., Ltd (lot number: 190917-2A1, specification: 250 mL: levofloxacin 0.5g, no edetate used in the prescription) was taken, placed in a 10mL measuring flask (9 parts), 1.5mL, 1.0mL and 0.5mL of edetate disodium standard stock solution (about 1000. mu.g/mL), 1.0mL and 0.5mL (each concentration being 3 parts) were added, each derivatization reagent was added, water was added to dilute to the scale, shaken, and the concentration and recovery rate of edetate disodium in the recovered sample were determined according to the above chromatographic conditions, respectively. The recovery results are shown in Table 6, and it can be seen from Table 6 that the recovery is 100.26% -101.02%, the average recovery is 100.7%, and the recovery RSD is 0.28%, indicating that the method has good accuracy.
TABLE 6 recovery results table
Example 2: sample assay
Preparing a sample solution to be tested by taking a sample to be tested according to the method of '3. solution preparation' in the example 1, analyzing according to '2. chromatographic conditions', recording a chromatogram, and calculating the amount of the edetate disodium in the sample according to a standard regression curve.
The formula of the company E does not add edetate, and 2 batches of edetate, sample 1 and sample 2, respectively have the concentration of 16.5 mu g/mL and 17.9 mu g/mL in terms of edetate disodium, are detected.
No edetate was detected from samples of Zhejiang Sha Precission pharmaceutical Co.
The addition amount of the edetate disodium in the prescription of the Jiangxi east Asia pharmaceutical Co., Ltd, of the Bibi group, is 100 mu g/mL, 15 batches of samples detect the edetate disodium, and the average value is 96.3 mu g/mL.
Example 3: sample validation
1. Instrumentation and chromatographic conditions
The apparatus and liquid phase conditions were the same as those of "1. apparatus and reagent" and "2. chromatographic conditions" in example 1 except that the amount of the sample was adjusted to 20. mu.l.
2. Solution preparation
Copper ion derivatization reagent: weighing copper sulfate 0.25g, adding water 100mL to dissolve and shake uniformly as copper sulfate derivatization reagent (containing Cu)2+About 0.01 mol/L).
Precisely measuring 5mL of a to-be-detected levofloxacin lactate and sodium chloride injection sample, adding 1mL of a copper ion derivatization reagent, adding water to dilute to 10mL, and shaking up to obtain a sample solution b to be detected.
Precisely measuring 1mL of the edetate disodium standard stock solution (about 1000 mu g/mL), placing the edetate disodium standard stock solution into a 10mL measuring flask, adding water to dilute the edetate disodium standard stock solution to a scale mark, and shaking up the solution. Precisely measuring 1mL of the solution, adding 1mL of copper sulfate derivatization reagent, adding water to dilute to 10mL, and shaking up to prepare a standard solution which is about 10 mu g/mL of edetate disodium.
3. Confirmation assay
Two batches of samples from company E were taken for measurement, and test sample solution b and edetate disodium standard solution were prepared as described above and measured as described above with the instrument and chromatographic conditions. As can be seen from the confirmation map of fig. 2, the retention time of the peak of edetate disodium in the two batches of samples of company E is 13.931min for sample 1 and 13.915min for sample 2, which is consistent with the retention time of 13.968min for the standard edetate disodium solution, and it can be confirmed that both batches of samples contain edetate.
In conclusion, the method is simple and feasible, can be used for determining and confirming the edetate in the levofloxacin lactate and sodium chloride injection, and is rapid, accurate, reliable and good in repeatability.
Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.
Claims (10)
1. An analysis method of edetate in levofloxacin lactate and sodium chloride injection is characterized by comprising the following steps:
s1: derivatizing edetate in the levofloxacin lactate and sodium chloride injection to be tested by using an iron ion derivatization reagent to obtain a test sample a;
s2: determining edetate in the sample a by HPLC method;
the HPLC method adopts sodium dihydrogen phosphate solution-acetonitrile as a mobile phase and a C18 column as a chromatographic column.
2. The method for analyzing edetate contained in levofloxacin lactate sodium chloride injection as claimed in claim 1, further comprising:
s3: and derivatizing the sample to be tested by using a copper ion derivatization reagent to obtain a test sample b, and determining by using the HPLC method to confirm whether the peak retention time of the test sample b is consistent with the derivatization peak of the edetate disodium standard solution.
3. The method for analyzing edetate in levofloxacin lactate sodium chloride injection as claimed in claim 1, wherein the volume ratio of sodium dihydrogen phosphate solution to acetonitrile in step S2 is (88-92) to (8-12).
4. The method for analyzing edetate in levofloxacin lactate sodium chloride injection as claimed in claim 3, wherein the volume ratio of the sodium dihydrogen phosphate solution to the acetonitrile is 90: 10.
5. The method for analyzing edetate in levofloxacin lactate sodium chloride injection as claimed in claim 3 or 4, wherein the sodium dihydrogen phosphate solution contains 4-6mmol/L tetrabutylammonium bromide, and the pH is adjusted to 4-5 with phosphoric acid solution.
6. The method of claim 5, wherein the sodium dihydrogen phosphate solution contains 5mmol/L tetrabutylammonium bromide, and the pH is adjusted to 4.5 with phosphoric acid solution.
7. The method for analyzing edetate in levofloxacin lactate sodium chloride injection according to claim 1, wherein the size of the chromatographic column in step S2 is 4.6mm x 250mm, and the particle size is 5 μm.
8. The method for analyzing edetate in levofloxacin lactate and sodium chloride injection according to claim 1 or 2, wherein the chromatographic conditions of the HPLC method are as follows:
mobile phase: 10-100mmol/L sodium dihydrogen phosphate solution-acetonitrile;
flow rate: 1.0 mL/min;
a chromatographic column: InertSustain AQ-C18;
column temperature: 30-45 ℃;
sample introduction amount: 10-20 μ L;
detection wavelength: 250-270 nm.
9. The method for analyzing the edetate in the levofloxacin lactate and sodium chloride injection as claimed in claim 8, wherein the chromatographic conditions of the HPLC method are as follows:
mobile phase: 50mmol/L sodium dihydrogen phosphate solution-acetonitrile;
flow rate: 1.0 mL/min;
a chromatographic column: InertSustain AQ-C18;
column temperature: 40 ℃;
sample introduction amount: 10-20 μ L;
detection wavelength: 257 nm;
wherein, the sample amount in the step S2 is preferably 10 μ L; the amount of sample injection in step S3 is preferably 20. mu.L.
10. The method for analyzing edetate in levofloxacin lactate sodium chloride injection as claimed in claim 1 or 2, wherein the iron ion-derivatizing agent in step S1 is ferric chloride solution, wherein Fe is3+The concentration is 0.01mol/L, and the volume ratio of the iron ion derivatization reagent to the sample to be detected is (0.1-1.5) to 5;
and/or the copper ion-derivatizing agent in the step S3 is a copper sulfate solution, wherein Cu is2+The concentration is 0.01mol/L, and the volume ratio of the copper ion derivatization reagent to the sample to be detected is (0.1-1.5) to 5.
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