CN111426769B - Chiral mobile phase additive HPLC resolution method of nadifloxacin enantiomer - Google Patents
Chiral mobile phase additive HPLC resolution method of nadifloxacin enantiomer Download PDFInfo
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- CN111426769B CN111426769B CN202010320124.3A CN202010320124A CN111426769B CN 111426769 B CN111426769 B CN 111426769B CN 202010320124 A CN202010320124 A CN 202010320124A CN 111426769 B CN111426769 B CN 111426769B
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- 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 provides an HPLC resolution method for a chiral mobile phase additive of an nadifloxacin enantiomer, belonging to the field of pharmaceutical analysis. The chromatographic conditions of the method provided by the invention comprise a chromatographic mobile phase system which is formed by adding a mobile phase into a C18 chromatographic column and a hydroxypropyl-beta-cyclodextrin (HP-beta-CD) chiral mobile phase additive; the method specifically comprises the following steps: the mobile phase is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile with a volume ratio of 80-83; the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution is 5-10mmol/L; flow rate: 1.0mL/min; the chromatographic column is Welch XB-C18,4.6mm is multiplied by 250mm, and 5 mu m is adopted; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 290nm. The method is simple to operate, economical and practical, and has good linearity, precision and sensitivity.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to a chiral mobile phase additive HPLC resolution method of nadifloxacin enantiomer.
Background
At present, the resolution of High Performance Liquid Chromatography (HPLC) on drug enantiomers is generally applied, various technical methods are developed rapidly, and the methods are mainly divided into a pre-column derivatization method, a chiral mobile phase addition method and a chiral stationary phase method. The pre-column derivatization method has complex pretreatment, long derivatization time and poor stability of the derivative; the chiral stationary phase method utilizes the stability difference of diastereomer complexes formed between a drug enantiomer and a stationary phase to realize the resolution of the enantiomer, has the advantage of simple sample processing steps, but no commonly applicable chiral column similar to a C18 column exists so far, isomers with different chemical properties have to adopt different types of chiral columns, and the commercially available chiral chromatographic column is usually expensive. The chiral mobile phase addition method can realize the resolution of the drug enantiomer on a C18 column by taking the chiral additive as the mobile phase, and has simple pretreatment and low price. The chiral mobile phase addition method is mainly divided into a method using copper sulfate-chiral amino acid, beta-cyclodextrin and derivatives thereof as additives, wherein the method using copper sulfate-chiral amino acid as the additives is widely used for resolution of enantiomers of quinolone drugs, such as resolution of drugs such as pazufloxacin, antofloxacin, ofloxacin and the like. The method is suitable for separating compounds with chiral carbon atoms close to chiral coordination sites, but cannot be used for separating drugs with chiral carbon atoms far away from chiral coordination sites. The beta-cyclodextrin and the derivatives thereof show good selectivity when being used for chiral mobile phase enantiomer resolution, and the pretreatment is simple and the analysis is fast.
Nadifloxacin, a third generation quinolone antibacterial drug developed and developed by tsukamur japan, was first marketed in japan in 1993 as a 1% ointment for the treatment of acne. At present, few reports are provided for the resolution method of the enantiomer of the nadifloxacin, and the invention mainly introduces the resolution of the nadifloxacin by using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as a chiral mobile phase additive. The enantiomers of nadifloxacin are shown below.
Disclosure of Invention
The invention aims to provide a chiral mobile phase additive HPLC resolution method of nadifloxacin enantiomer.
In order to achieve the above purpose, the present invention provides a chiral mobile phase additive HPLC resolution method for nadifloxacin enantiomers, wherein the chromatographic conditions of the HPLC resolution method include a chromatographic mobile phase system composed of a C18 chromatographic column and a hydroxypropyl- β -cyclodextrin (HP- β -CD) chiral mobile phase additive added into a mobile phase; the method comprises the following specific steps:
the mobile phase is a mixed solution of potassium dihydrogen phosphate solution and acetonitrile, the concentration of the potassium dihydrogen phosphate solution is 0.05-0.1mol/L, and the pH value of the potassium dihydrogen phosphate solution is adjusted to 5.9-7.5 by potassium hydroxide solution;
the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution is 5-10mmol/L.
Wherein the volume ratio of the potassium dihydrogen phosphate solution to the acetonitrile is 80-83:20-17, preferably 83: 17;
preferably, the concentration of the potassium dihydrogen phosphate solution is 0.05mol/L;
preferably, the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution is 7.5mmol/L;
preferably, the potassium dihydrogen phosphate solution is adjusted to a pH of 6.9 with a potassium hydroxide solution.
Flow rate: 1.0mL/min;
wherein, the C18 chromatographic column is Welch XB-C18,4.6mm is multiplied by 250mm,5 μm;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 290nm.
The nadifloxacin has maximum absorption at 237nm and 290nm, and 290nm is selected as the detection wavelength to avoid low-wavelength noise infection because the mobile phase contains HP-beta-CD.
The molecular structure of the nadifloxacin is different from the structures of the pazufloxacin, the antofloxacin and the ofloxacin, a mother ring of the nadifloxacin is connected with a chiral carbon to form a C-C group, and the mother rings of the pazufloxacin, the antofloxacin and the ofloxacin are connected with the chiral carbon to form a C-O group. The research attempts to resolve the nadifloxacin by using a mobile phase with copper sulfate-chiral amino acid (D-phenylalanine) as a chiral additive, and results are unsuccessful. The HP-beta-CD is used as the mobile phase chiral additive, chiral inclusion substances with different physicochemical properties are formed through the inclusion effect of an internal cavity structure of a cyclodextrin derivative on the nadifloxacin enantiomer, so that different distribution coefficients are generated in a mobile phase and a fixed phase, and the resolution of the enantiomer is realized.
The invention has the beneficial effects that:
the invention provides a simple, convenient and quick method for splitting nadifloxacin enantiomer by an HPLC method of a chiral mobile phase additive, which does not need to perform derivatization on diastereomer, does not need to use an expensive beta-CD chiral stationary phase chromatographic column, only needs to add hydroxypropyl-beta-cyclodextrin into a mobile phase, and has the advantages of simple operation, economy and practicality, and better linearity, precision and sensitivity.
Drawings
FIG. 1 is a chromatogram of example 1 of the present invention
The invention is further described with reference to the following figures and examples.
Detailed Description
Example 1
A chiral mobile phase additive HPLC resolution method of nadifloxacin enantiomer comprises the following chromatographic conditions:
mobile phase: mixed solution of potassium dihydrogen phosphate solution and acetonitrile, wherein the concentration of the potassium dihydrogen phosphate solution is 0.05-0.1mol/L, and the pH value of the potassium dihydrogen phosphate solution is adjusted to 5.9-7.5 by potassium hydroxide solution;
wherein the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution is 5-10mmol/L;
wherein the volume ratio of the potassium dihydrogen phosphate solution to the acetonitrile is 83: 17;
flow rate: 1.0mL/min;
the chromatographic column is Welch XB-C18,4.6mm is multiplied by 250mm, and 5 mu m is adopted;
column temperature: 35 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 290nm.
Example 2
The procedure of example 1 was repeated except that the pH was adjusted to 7.5 with a potassium hydroxide solution.
Example 3
The procedure of example 1 was repeated except that the pH was adjusted to 5.9 with a potassium hydroxide solution.
Example 4
Wherein the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution was 10mmol/L, as in example 1.
Example 5
Wherein the concentration of HP-beta-CD in the potassium dihydrogen phosphate solution was 5mmol/L, as in example 1.
Example 6
Wherein the concentration of the potassium dihydrogen phosphate solution in the mobile phase was 0.1mol/L, as in example 1.
Example 7
Wherein the concentration of the potassium dihydrogen phosphate solution in the mobile phase was 0.05mol/L, as in example 5.
Example 8
Wherein the volume ratio of the potassium dihydrogen phosphate solution to the acetonitrile in the mobile phase is 80: 20, the rest of the same procedure as in example 1.
Example 9
Wherein the volume ratio of potassium dihydrogen phosphate solution to acetonitrile in the mobile phase was 78: 18, the rest of the procedure was the same as in example 1.
The results of the tests of examples 1 to 9 are shown in FIG. 1 and tables 1 to 4.
TABLE 1 Effect of pH of potassium dihydrogen phosphate solution on retention time and degree of separation
TABLE 2 Effect of HP-beta-CD concentration in Potassium dihydrogen phosphate solution on Retention time and separation
TABLE 3 Effect of monopotassium phosphate concentration in the mobile phase on the Retention time and degree of separation
TABLE 4 influence of the volume ratio of potassium dihydrogen phosphate solution and acetonitrile in the mobile phase on the retention time and the degree of separation
As shown in fig. 1, the nadifloxacin can be better resolved under the chromatographic conditions of example 1, the retention time of the two enantiomer peaks is about 31.5min and 34.1min, respectively, the separation degree between the two enantiomer peaks is 2.1, and the requirement that the separation degree between the chromatographic peaks of the substance to be detected in 'chinese pharmacopoeia' 2015 should be greater than 1.5 is met.
As can be seen from table 1, when comparing examples 1, 2 and 3, the pH of the mobile phase varied between 5.9 and 7.5, and the peak time of nadifloxacin increased with increasing pH, and the degree of separation between enantiomers decreased after increasing. Wherein, the pH value of the mobile phase is controlled to be 6.9, and the separation effect is best.
As can be seen from Table 2, the chiral mobile phase additive HP-beta-CD concentration is changed from 5mmol/L to 7.5mmol/L for comparison among examples 1, 4 and 5, the retention time of the enantiomer of the nadifloxacin is obviously shortened, and the separation degree is also slightly improved (more than 2.0); while the reduction of the retention time of the enantiomer of the nadifloxacin from 7.5mmol/L to 10mmol/L and the improvement of the resolution are not obvious, the HP-beta-CD concentration is preferably 7.5mmol/L in view of the cost.
As can be seen from Table 3, the potassium dihydrogen phosphate concentration had a small effect on the retention time of the enantiomer of nadifloxacin. Comparative examples 6, 7, HP-beta-CD had a greater effect on retention time than potassium dihydrogen phosphate. When the concentration of the potassium dihydrogen phosphate is 0.05-0.1mol/L, the separation degree is more than 1.5. From the viewpoint of cost, the concentration of potassium dihydrogen phosphate is preferably 0.05mol/L.
As is clear from Table 4, in comparison among examples 1, 8 and 9, the higher the acetonitrile ratio, the faster the peak time of nadifloxacin appeared, and the degree of separation became worse. And when the volume ratio of the potassium dihydrogen phosphate solution to the acetonitrile is 80-83.
The methodology of the resolution method disclosed in the present invention was examined, specifically as follows.
Naofloxacin reference (Source: china institute for testing food and drug, batch No. 130462-201001, content: 97.0%; racemate)
Accurately weighing 10.31mg of the nadifloxacin reference substance, putting the nadifloxacin reference substance into a 10mL measuring flask, adding acetonitrile to dissolve and dilute the nadifloxacin reference substance to a scale, and shaking up to obtain the nadifloxacin reference substance solution.
The methodology was examined linearly according to the chromatographic conditions of example 1. The norfloxacin reference substance solution is injected with 0.1, 0.5, 2, 5, 10 and 15 mu L of sample respectively. The peak area of the single enrofloxacin enantiomer is used as the ordinate (y), the mass (x) of the single enrofloxacin enantiomer is used as the abscissa for linear regression, and the result shows that the mass of the single enrofloxacin enantiomer is between 0.05 and 7.50 mu g and has a good linear relation with the corresponding peak area. The regression equation for enantiomer 1 is: y =3.139 × 10 6 x+1.352×10 4 ，R 2 =0.99999; the enantiomer 2 regression equation is: y =3.151 × 10 6 x+2.302×10 4 ，R 2 ＝0.99998。
Taking the norfloxacin reference solution, carrying out precision measurement by continuously feeding 6 times (10 mu L) according to the chromatographic conditions of the example 1, and respectively calculating the RSD of each enantiomer peak area of the norfloxacin to be 0.12% and 0.17%, which indicates that the method has good precision.
And (3) taking the control solution to dilute the control solution into solutions containing about 1 mu g/mL and 3 mu g/mL of the nadifloxacin (the concentrations of the single enantiomers are 0.5 mu g/mL and 1.5 mu g/mL respectively), and respectively determining, wherein the signal-to-noise ratios of the two enantiomers are both greater than 3 when the concentration of the nadifloxacin is 1 mu g/mL, and the signal-to-noise ratios of the two enantiomers are both greater than 10 when the concentration of the nadifloxacin is 3 mu g/mL. The detection limit and the quantification limit of the single enantiomer of nadifloxacin may be set at 0.5. Mu.g/mL and 1.5. Mu.g/mL.
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 (4)
1. An HPLC resolution method of chiral mobile phase additive of nadifloxacin enantiomer is characterized in that chromatographic conditions of the HPLC resolution method comprise a chromatographic mobile phase system which is formed by adding a mobile phase into a chromatographic column which is C18 and a chiral mobile phase additive which is hydroxypropyl-beta-cyclodextrin (HP-beta-CD); the method specifically comprises the following steps:
the mobile phase is a mixed solution of a potassium dihydrogen phosphate solution and acetonitrile, the concentration of the potassium dihydrogen phosphate solution is 0.05mol/L, and the pH value of the potassium dihydrogen phosphate solution is adjusted to 6.9 by using a potassium hydroxide solution;
the concentration of the chiral mobile phase additive HP-beta-CD in the potassium dihydrogen phosphate solution is 7.5mmol/L;
the chromatographic column is Welch XB-C18,4.6mm is multiplied by 250mm, and 5 mu m is particularly adopted;
the flow rate of the mobile phase is 1.0mL/min; the column temperature was 35 ℃;
the volume of the potassium dihydrogen phosphate solution and the acetonitrile in the mobile phase is 80-83: 20-17.
2. A process for the chiral mobile phase additive HPLC resolution of nadifloxacin enantiomers as claimed in claim 1 wherein the volume of potassium dihydrogen phosphate solution and acetonitrile in said mobile phase is 83: 17.
3. A process for the chiral mobile phase additive HPLC resolution of nadifloxacin enantiomer according to claim 1, wherein the sample is administered in an amount of 10 μ L.
4. The process for HPLC resolution of an enantiomer of nadifloxacin with chiral mobile phase additive according to claim 1, wherein the detection wavelength is 290nm.
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