CN114137126A - Method for measuring content of L-lactic acid and D-lactic acid in levofloxacin lactate - Google Patents
Method for measuring content of L-lactic acid and D-lactic acid in levofloxacin lactate Download PDFInfo
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- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title claims abstract description 154
- 229930182843 D-Lactic acid Natural products 0.000 title claims abstract description 100
- 229940022769 d- lactic acid Drugs 0.000 title claims abstract description 99
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 title claims abstract description 97
- 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 47
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000012086 standard solution Substances 0.000 claims abstract description 53
- 239000000243 solution Substances 0.000 claims abstract description 41
- 239000000126 substance Substances 0.000 claims abstract description 28
- 229910000365 copper sulfate Inorganic materials 0.000 claims abstract description 18
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000012085 test solution Substances 0.000 claims abstract description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- VVNCNSJFMMFHPL-VKHMYHEASA-N D-penicillamine Chemical compound CC(C)(S)[C@@H](N)C(O)=O VVNCNSJFMMFHPL-VKHMYHEASA-N 0.000 claims description 7
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 2
- 239000002994 raw material Substances 0.000 abstract description 16
- 238000004458 analytical method Methods 0.000 abstract description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 88
- 239000004310 lactic acid Substances 0.000 description 40
- 235000014655 lactic acid Nutrition 0.000 description 37
- 239000012071 phase Substances 0.000 description 22
- 239000000523 sample Substances 0.000 description 15
- 239000003446 ligand Substances 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 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 8
- 229960003376 levofloxacin Drugs 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000000178 monomer Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- -1 lactate ions Chemical class 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 229910001431 copper ion Inorganic materials 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 150000004696 coordination complex Chemical class 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
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- 239000012488 sample solution Substances 0.000 description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000013375 chromatographic separation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000007859 condensation product Substances 0.000 description 1
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- 229910052744 lithium Inorganic materials 0.000 description 1
- GKQWYZBANWAFMQ-HSHFZTNMSA-M lithium;(2r)-2-hydroxypropanoate Chemical compound [Li+].C[C@@H](O)C([O-])=O GKQWYZBANWAFMQ-HSHFZTNMSA-M 0.000 description 1
- GKQWYZBANWAFMQ-UHFFFAOYSA-M lithium;2-hydroxypropanoate Chemical compound [Li+].CC(O)C([O-])=O GKQWYZBANWAFMQ-UHFFFAOYSA-M 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
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- 239000008213 purified water Substances 0.000 description 1
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- 238000001179 sorption measurement Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to a content determination method of L-lactic acid and D-lactic acid in levofloxacin lactate. The determination method comprises the following specific steps: preparing an L-lactic acid standard substance solution, preparing a D-lactic acid standard substance solution, preparing a test solution and performing HPLC (high performance liquid chromatography) determination, wherein the HPLC method adopts a chiral chromatographic column, and the mobile phase is 2-2.5mmol/L copper sulfate solution-isopropanol. The determination method of the invention respectively uses the L-lactic acid standard solution and the D-lactic acid standard solution which are prepared in advance as the standard solutions, adopts an HPLC method, uses a chiral chromatographic column to determine the content of the L-lactic acid and the D-lactic acid in the levofloxacin lactate sample, has the advantages of good specificity, good precision, high accuracy, good repeatability and the like, and can be used for determining the content of the L-lactic acid and the D-lactic acid in the levofloxacin lactate raw material.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a content determination method of L-lactic acid and D-lactic acid in levofloxacin lactate.
Background
In healthy humans, lactic acid exists almost exclusively in the form of L-lactic acid, and L-lactic acid produced or taken in by itself is catabolized by L-lactate dehydrogenase in the liver, and D-lactic acid is hardly metabolized by the human body. The World Health Organization (WHO) advocates the use of L-lactic acid in place of the commonly used D-lactic acid in the food and pharmaceutical industries. WHO stipulates that the amount of D-lactic acid taken by a human body per day should be less than 100mg/kg of body mass, and does not limit the amount of L-lactic acid. The configuration of lactic acid is not described in detail in the Chinese pharmacopoeia 2020 edition, and the main three types of lactic acid auxiliary materials for common medicine in the market are: l-lactic acid, racemic lactic acid and mixed lactic acid (the configuration ratio of L-lactic acid and D-lactic acid is uncertain). In addition, the enterprise research shows that the configuration of lactic acid in the levofloxacin lactate raw material influences the specific rotation degree of the raw material. Based on the above two points, it is necessary to determine the content of L-lactic acid and D-lactic acid in the levofloxacin lactate raw material.
The chemical name of lactic acid is 2-hydroxypropionic acid, the commercially available lactic acid reagents or pharmaceutic adjuvants are mixtures of 2-hydroxypropionic acid and condensates thereof, and the content of the lactic acid monomer (2-hydroxypropionic acid) in the commercially available adjuvants, reagents and reference products of D-lactic acid or L-lactic acid is lower than the marked content.
The levofloxacin lactate is an ionic compound obtained by salifying lactic acid and levofloxacin, and the lactic acid dissolved in water exists in the form of lactate ions without condensed lactic acid. If the D-lactic acid or L-lithium lactate standard substance is directly prepared into the standard solution to measure the content of D or L-lactic acid in the levofloxacin lactate raw material, the measured content is higher. Therefore, it is necessary to find a method for accurately measuring the content of D-lactic acid or L-lactic acid in levofloxacin lactate.
Disclosure of Invention
The invention aims to provide a method for measuring the content of L-lactic acid and D-lactic acid in levofloxacin lactate, which respectively takes a pre-prepared L-lactic acid standard solution and a pre-prepared D-lactic acid standard solution as standard solutions, adopts an HPLC method, uses a chiral chromatographic column to measure the content of the L-lactic acid and the D-lactic acid in a levofloxacin lactate sample, has the advantages of good specificity, good precision, high accuracy, good repeatability and the like, and can be used for measuring the content of the L-lactic acid and the D-lactic acid in a levofloxacin lactate raw material.
The invention provides a method for measuring the content of L-lactic acid and D-lactic acid in levofloxacin lactate, which comprises the following specific steps:
1) preparing an L-lactic acid standard solution: precisely weighing the L-lithium lactate standard substance, placing the L-lithium lactate standard substance in a volumetric flask, adding water to dissolve the L-lithium lactate standard substance, and shaking up the L-lithium lactate standard substance;
2) preparing a D-lactic acid standard solution: precisely weighing a D-lactic acid standard substance, placing the D-lactic acid standard substance in a conical flask, adding 1mol/L sodium hydroxide solution, carrying out boiling water bath for 4-6min, adding 1-2 drops of phenolphthalein indicator solution, adding 1mol/L hydrochloric acid solution while the solution is hot, neutralizing to neutrality, transferring all the liquid to a volumetric flask, adding water to a constant volume to reach a scale, and shaking up;
3) preparing a test solution: taking a levofloxacin lactate sample, placing the levofloxacin lactate sample in a volumetric flask, adding a mobile phase for dissolving, diluting to a scale, and shaking up;
4) and (3) HPLC determination: respectively taking the L-lactic acid standard solution and the D-lactic acid standard solution as standard solutions, and determining the contents of L-lactic acid and D-lactic acid in the levofloxacin lactate sample by an HPLC method;
wherein, the HPLC method adopts a chiral chromatographic column, and the mobile phase is 2-2.5mmol/L copper sulfate solution-isopropanol.
The L-lithium lactate is an ionic compound and is obtained by salifying L-lactic acid and metal lithium, and no condensed lactic acid exists, so that the content of the L-lactic acid in the levofloxacin lactate can be determined by adopting the L-lithium lactate as a standard substance. Because the corresponding D-lactic acid ionic compound cannot be purchased in the market as a standard product, the hydrolysis method is adopted in the invention, the lactic acid polymer in the D-lactic acid is hydrolyzed by sodium hydroxide to be completely converted into D-lactic acid ions, and then the D-lactic acid ions are used as a standard solution to measure the content of the D-lactic acid in the levofloxacin lactate, so that the error is small, and the accuracy can be improved.
Preferably, the chromatographic column in the technical scheme is a fenoxam 3126(D) -penicillamine chiral chromatographic column with the specification of 250 × 4.6 mm.
In the technical scheme, a Fenomei 3126(D) -penicillamine chiral chromatographic column is adopted, which is a chiral chromatographic column containing a selective chiral ligand D-penicillic acid, and the ligand is adsorbed (hydrophobic effect) on an inverse filler and is complexed with copper ions. The separation mechanism is based on the formation of a reversible diastereomeric metal complex between a chiral selection ligand and a chiral solute ligand (the enantiomeric compound to be detected) by coordination with a metal ion (copper ion). Because the selection ligand is chiral, the stereochemistry of the chiral solute will determine the elution order, the longer the retention time of the enantiomer forming a thermodynamically stable complex with the chiral selection ligand. 3126 the chromatographic column generally uses reverse phase separation mode, and comprises regulating retention time of the separated substance by adding small amount of organic phase, separating lactic acid enantiomer based on exchange equilibrium and stability between ligand (lactic acid) in mobile phase and ligand in stationary phase, and regulating retention time of the substance to be tested by adding organic solvent isopropanol.
Preferably, the concentration of the copper sulfate solution in the mobile phase in the technical scheme is 2-2.5 mmol/L.
Preferably, in the above technical scheme, the volume ratio of the copper sulfate solution to the isopropanol in the mobile phase is 92.5-93.5: 6.5-7.5.
Since lactic acid peak time is fast and levofloxacin peak very late, an appropriate mobile phase needs to be found to shorten the analysis time. The invention adopts the Fennomei 3126(D) -penicillamine chiral chromatographic column to need copper sulfate solution as the mobile phase, the concentration of the copper sulfate is generally controlled at 1-3mmol/L, and Cu is increased2+The sample retention time can be shortened by adding organic modifier such as isopropanol, but the proportion of isopropanol needs to be controlled within 15%.
Preferably, the volume ratio of the copper sulfate solution to the isopropanol in the mobile phase in the technical scheme is 93: 7.
Preferably, the chromatographic conditions in the above technical solution further include a column temperature of 40 ℃, a flow rate of 1.0mL/min, a sample volume of 10 μ L, and a detection wavelength of 254 nm.
The invention also provides the application of the content determination method in the content determination of L-lactic acid and D-lactic acid in levofloxacin lactate raw materials and preparations.
Compared with the prior art, the method has the beneficial effects that:
1. the method takes the pre-prepared L-lactic acid standard solution and the D-lactic acid standard solution (obtained by hydrolyzing the D-lactic acid) as the standard solutions respectively, can determine the L-lactic acid content and the D-lactic acid content in the levofloxacin lactate, and has small error and high accuracy;
2. the HPLC method takes a Fennom 3126(D) -penicillamine chiral chromatographic column as an adsorption chromatographic column, and a reversible diastereomer metal complex is formed between a chiral selection ligand and a chiral solute ligand through coordination with copper ions in a mobile phase, and because the retention time with the chiral selection ligand is longer, the retention time of L-lactic acid and D-lactic acid can be adjusted and separated by adding a small amount of organic phase, so that the purpose of respectively measuring the contents of the L-lactic acid and the D-lactic acid is achieved;
3. the method has the advantages of linear relation, high precision, high accuracy, low detection limit and the like, and can be used for measuring the content of L-lactic acid and D-lactic acid in the levofloxacin lactate raw material.
Drawings
FIG. 1 is a chromatogram of each solution of the present invention, wherein A is a chromatogram of a mixed solution of an L-lactic acid standard solution and a D-lactic acid standard solution, B is a chromatogram of a levofloxacin lactate solution, C is a chromatogram of an L-lactic acid standard solution, and D is a chromatogram of a D-lactic acid standard solution;
FIG. 2 is a typical chromatogram of a levofloxacin solution;
FIG. 3 is a chromatogram of a mixed solution of L-lithium lactate and D-lactic acid.
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:
1. instruments and reagents
1.1 Instrument: LC-20AD high performance liquid chromatograph (Shimadzu, Japan), MS205DU electronic balance (METTLER TOLEDO, Switzerland);
1.2 reagent: l lithium lactate standard (manufacturer: ALDRICH, lot: MKCK3402, L-lactic acid content 89.1%); d lactic acid standard (manufacturer: Michelin company, batch number: C10190971, D-lactic acid content 90%); the water is purified water; the isopropanol is chromatographically pure; the other reagents are analytically pure;
1.3 medicine: levofloxacin lactate raw material 1, manufacturing enterprise: xinchang pharmaceutical factory, Zhejiang pharmaceutical products, Ltd, lot number: 219C 225-190901; levofloxacin lactate raw material 2, manufacturing enterprise: shangyu Jingxin pharmaceutical Co., Ltd, batch number: DK 24-1903021-VI; levofloxacin lactate raw material 3, manufacturing enterprise: jiangxi geodetic drug limited liability company, batch number: 180806-2.
2. Chromatographic conditions
Mobile phase: 2.5mmol/L copper sulfate solution-isopropanol (93: 7);
flow rate: 1.0 mL/min;
a chromatographic column: feminomei 3126(D) -penicillamine chiral chromatography columns (250 × 4.6 mm);
column temperature: 40 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 254 nm.
3. Solution preparation
3.1 preparation of L-lactic acid standard solution: 0.5639g of L-lithium lactate standard substance is precisely weighed, placed in a 50mL volumetric flask, dissolved and diluted to the scale mark by adding water, and shaken up to be used as an L-lactic acid standard substance solution (about 10 mg/mL).
3.2D-lactic acid standard solution preparation: 1.1401g of D-lactic acid standard substance is precisely weighed, the D-lactic acid standard substance is placed in a 250mL conical flask, 25mL of 1mol/L sodium hydroxide solution is added, boiling water bath is carried out for 5min, 1 drop of phenolphthalein indicator solution is added, 1mol/L hydrochloric acid solution (about 13.2mL) is added while the solution is hot to neutralize to neutrality (red color disappears), all the liquid is transferred to a 100mL volumetric flask, the conical flask is washed by water for several times, all the washing liquid is transferred to a measuring flask, water is added to fix the volume to a scale, and the solution is shaken up to be used as D-lactic acid standard substance solution (about 10 mg/mL).
3.3 preparation of test solution: taking a levofloxacin lactate sample of about 13mg, placing the sample into a 10mL volumetric flask (the sample amount and the volumetric flask volume can be scaled up and scaled up), adding a mobile phase for dissolving, diluting to a scale, shaking up, and preparing a solution containing lactic acid (the concentration of two enantiomers is the sum) of about 250 mu g/mL to serve as a test solution.
Example 1: optimization of experimental conditions
1. Selection of chromatographic separation conditions
Research shows that lactic acid peak time is relatively fast, and levofloxacin peak time is very late, so that an appropriate mobile phase needs to be found to shorten analysis time. The Fennomei 3126(D) -penicillamine chiral chromatographic column needs copper sulfate solution as mobile phase, the concentration of copper sulfate is controlled at 1-3mmol/L, in this example by increasing Cu2+The sample retention time can be shortened by adding organic modifier such as isopropanol, but the proportion of isopropanol needs to be controlled within 15%.
This example measures levofloxacin lactate solution by adjusting the ratio of copper sulfate and isopropanol at different concentrations as mobile phase to select appropriate analysis time, and the results are shown in table 1.
TABLE 1 Effect of copper sulfate concentration and Isopropanol ratio on Retention time and degree of separation
The results show that the L-lactic acid peak is earlier than the D-lactic acid, and the retention time of the lactic acid and the levofloxacin can be shortened and the separation degree of lactic acid enantiomer can be properly increased by increasing the ratio of copper sulfate concentration to isopropanol. Therefore, a volume ratio of 2.5mmol/L copper sulfate solution to isopropanol of 93: 7 is preferred as the mobile phase. Wherein, the mixed solution of the L-lactic acid standard solution and the D-lactic acid standard solution, the lactic acid levofloxacin solution, the L-lactic acid standard solution and the D-lactic acid standard solution are shown in figure 1A, B, C, D, and the typical chromatogram of the levofloxacin solution is shown in figure 2, which indicates that the separation degree of the flow is good compared with that of the L-lactic acid, the D-lactic acid and the levofloxacin.
2. Selection of preparation of lactic acid Standard solution
Since lithium D-lactate cannot be obtained, only standard solutions can be prepared using D-lactic acid, but commercially available D-lactic acid is a mixture of D-lactic acid monomers and condensates thereof; in order to prove that the content of the lactic acid monomer in the D-lactic acid standard product is lower than the identification content, a mixed solution is prepared: directly taking a mixed solution containing 1mg/mL of each of L-lithium lactate and D-lactic acid prepared by adding a mobile phase into an L-lithium lactate standard substance and a D-lactic acid standard substance, wherein the D-lactic acid standard substance is not subjected to cracking, and a chromatogram is shown in figure 3 after HPLC (high performance liquid chromatography) measurement, and as can be seen from figure 3, the peak area of the D-lactic acid is only about 60% of the peak area of the L-lithium lactate with the same concentration, which indicates that the content of lactic acid monomers in the D-lactic acid standard substance is only about 60%.
In this example, a sodium hydroxide solution is used for hydrolysis, a lactic acid condensation product is converted into a lactic acid monomer, and the lactic acid monomer is neutralized with hydrochloric acid to prepare a D-lactic acid standard solution, and the liquid phase analysis result is substantially consistent with the peak area of L-lithium lactate at the same concentration, as shown in fig. 1A, which illustrates that a method for preparing the D-lactic acid standard solution by a sodium hydroxide hydrolysis method is feasible.
Example 2: methodology investigation
1. Investigation of linear relationships
1mL of L-lactic acid and D-lactic acid standard solution with the concentration of about 10mg/mL is precisely measured and placed in a 10mL volumetric flask and a 25mL volumetric flask, and a mobile phase is added to dilute the solution to a scale, so as to prepare a mixed lactic acid standard solution 1 with the concentration of about 1000 mu g/mL of L-lactic acid and D-lactic acid and a mixed lactic acid standard solution 2 with the concentration of about 400 mu g/mL. 2mL, 1.5mL, 1mL, 0.5mL and 0.2mL of the mixed lactic acid standard solution (the concentrations of L-lactic acid and D-lactic acid are about 1000. mu.g/mL respectively) are precisely measured and placed in a 10mL measuring flask, a mobile phase is added to dilute the solution to a scale, and the solution is shaken up to prepare 3-7 mixed lactic acid standard solutions containing the concentrations of L-lactic acid and D-lactic acid of about 200. mu.g/mL, 150. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL and 20. mu.g/mL respectively.
And (3) respectively taking 2-7 of the mixed lactic acid standard solution, and determining according to the 2. chromatographic conditions. Linear regression was performed with the peak area of each lactic acid enantiomer as the ordinate (y) and the lactic acid concentration (x) as the abscissa.
The results show that: the concentration of the L-lactic acid is between 20 and 400 mu g/mL and has a good linear relation with the peak area, and the regression equation is as follows: 4880.16x-6891.12, r20.9999; the concentration of the D-lactic acid is in a good linear relation with the peak area between 20 and 400 mu g/mL, and the regression equation is as follows: 4884.12x-6142.29, r20.9998. The regression equations of the two lactic acid enantiomers are approximately consistent in linearity and intercept, further illustrating the feasibility of the preparation method of the D-lactic acid standard solution by the sodium hydroxide hydrolysis method.
2. Precision degree
Taking a mixed lactic acid standard solution 5 with the concentration of L-lactic acid and D-lactic acid of about 100 mu g/mL, continuously injecting samples for 6 times (10 mu L) according to the 2-chromatographic condition, and recording a chromatogram. The RSD calculated for the L-lactic acid areas (473301, 466690, 464941, 472580, 480716, 470730, 471493) was 1.2% and the RSD calculated for the D-lactic acid areas (482040, 483128, 485038, 491062, 486403, 483029) was 0.7%, indicating that the method of the present invention is highly accurate.
3. Repeatability of
The levofloxacin lactate raw material (the manufacturing enterprise: Xinchang pharmaceutical factory, Zhejiang medicine, Ltd., lot number: 219C225-190901) was taken and 6 parts of test solution was prepared according to "3.3 parts of test solution preparation". Measured according to the '2. chromatographic conditions' and the chromatogram recorded. The content of lactic acid enantiomer in 6 samples was calculated, and the results are shown in Table 2, and the RSD of the L-lactic acid and D-lactic acid contents is 0.7% and 0.9%, respectively, which shows that the method of the present invention has good repeatability.
TABLE 2 repeatability test results table
4. Quantitative limit and detection limit
Taking a proper amount of the mixed lactic acid standard solution 5, and diluting the mixed lactic acid standard solution with a mobile phase into mixed lactic acid standard solutions 8 and 9 with the concentrations of L-lactic acid and D-lactic acid of about 0.6 and 2 mu g/mL respectively. Respectively according to the determination of 2. chromatographic conditions, the signal to noise ratios of the L-lactic acid peak and the D-lactic acid peak are both more than 3 when the concentration is 0.6 mug/mL, and the signal to noise ratios of the L-lactic acid peak and the D-lactic acid peak are both more than 10 when the concentration is 2 mug/mL. The detection limit and the quantification limit of the L-lactic acid and the D-lactic acid in the method of the invention can be 0.6 mug/mL and 2 mug/mL.
5 degree of accuracy
Precisely measuring 5mL of L-lactic acid and D-lactic acid standard solution with the concentration of about 10mg/mL, placing the L-lactic acid and D-lactic acid standard solution into a 100mL measuring flask, adding a mobile phase to dilute to a scale, and preparing mixed lactic acid standard solutions with the concentrations of about 500 mu g/mL of L-lactic acid and D-lactic acid. A test sample solution (sample weighing 65.98mg) in the repeatability measurement is precisely measured to be 5mL, the test sample solution is placed into a 10mL measuring bottle (9 parts), mixed lactic acid standard solutions with the concentration of L-lactic acid and D-lactic acid being about 500 mu g/mL are respectively added to be 0.5mL, 1.0mL and 1.5mL (3 parts of each concentration), then mobile phases are respectively added to be diluted to a scale, the mixture is shaken evenly, and the concentration and the recovery rate of the lactic acid enantiomer in a recovered sample are respectively calculated according to the measurement of 2-chromatographic conditions, and are shown in a table 3 and a table 4.
From the results, it is found that the recovery rate of L-lactic acid is 98.78% -101.49%, the average recovery rate is 100.1%, and the recovery rate RSD is 0.9%; the recovery rate of the D-lactic acid is 99.32-101.92%, the average recovery rate is 100.1%, and the recovery rate RSD is 1.0%, which shows that the method has good accuracy.
TABLE 3L-lactic acid recovery results Table
TABLE 4D-lactic acid recovery results Table
Example 3: sample assay
Taking levofloxacin lactate raw materials of 3 production enterprises as samples, respectively weighing about 13mg, placing the samples in a 100mL measuring flask, adding a mobile phase for dissolving, diluting to a scale, and shaking up. Then, analysis was performed under "2. chromatographic conditions", and the chromatogram was recorded. The content of lactic acid enantiomer in 3 samples was calculated by regression equation, and the results are shown in table 5, which shows that the method of the present invention can accurately measure the content of L-lactic acid and D-lactic acid in levofloxacin lactate raw material.
TABLE 5 table of sample measurement results
In conclusion, the determination method provided by the invention respectively uses the L-lactic acid standard solution and the D-lactic acid standard solution (obtained by hydrolyzing D-lactic acid) which are prepared in advance as standard solutions, adopts an HPLC method, and uses a chiral chromatographic column to determine the contents of L-lactic acid and D-lactic acid in the levofloxacin lactate raw material, has the advantages of good specificity, good precision, high accuracy, good repeatability and the like, and can be used for determining the contents of L-lactic acid and D-lactic acid in the levofloxacin lactate raw material.
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. A method for measuring the content of L-lactic acid and D-lactic acid in levofloxacin lactate is characterized by comprising the following specific steps:
1) preparing an L-lactic acid standard solution: precisely weighing the L-lithium lactate standard substance, placing the L-lithium lactate standard substance in a volumetric flask, adding water to dissolve the L-lithium lactate standard substance, and shaking up the L-lithium lactate standard substance;
2) preparing a D-lactic acid standard solution: precisely weighing a D-lactic acid standard substance, placing the D-lactic acid standard substance in a conical flask, adding 1mol/L sodium hydroxide solution, carrying out boiling water bath for 4-6min, adding 1-2 drops of phenolphthalein indicator solution, adding 1mol/L hydrochloric acid solution while the solution is hot, neutralizing to neutrality, transferring all the liquid to a volumetric flask, adding water to a constant volume to reach a scale, and shaking up;
3) preparing a test solution: taking a levofloxacin lactate sample, placing the levofloxacin lactate sample in a volumetric flask, adding a mobile phase for dissolving, diluting to a scale, and shaking up;
4) and (3) HPLC determination: respectively taking the L-lactic acid standard solution and the D-lactic acid standard solution as standard solutions, and determining the contents of L-lactic acid and D-lactic acid in the levofloxacin lactate sample by an HPLC method;
wherein, the HPLC method adopts a chiral chromatographic column, and the mobile phase is 2-2.5mmol/L copper sulfate solution-isopropanol.
2. The method for measuring the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the chromatographic column is a femormet 3126(D) -penicillamine chiral chromatographic column with a specification of 250X 4.6 mm.
3. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the concentration of the copper sulfate solution in the mobile phase is 2-2.5 mmol/L.
4. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the volume ratio of the copper sulfate solution to the isopropanol in the mobile phase is 92.5-93.5: 6.5-7.5.
5. The method for measuring the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 4, wherein the volume ratio of the copper sulfate solution to the isopropanol in the mobile phase is 93: 7.
6. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the column temperature is 40 ℃.
7. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the flow rate is 1.0 mL/min.
8. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the sample amount is 10 μ L.
9. The method for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate according to claim 1, wherein the detection wavelength is 254 nm.
10. Use of the method of any one of claims 1-9 for determining the content of L-lactic acid and D-lactic acid in levofloxacin lactate.
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