CN109115914B - High performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof - Google Patents
High performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof Download PDFInfo
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- CN109115914B CN109115914B CN201811163936.0A CN201811163936A CN109115914B CN 109115914 B CN109115914 B CN 109115914B CN 201811163936 A CN201811163936 A CN 201811163936A CN 109115914 B CN109115914 B CN 109115914B
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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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention discloses a high performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof, which comprises the following chromatographic conditions: the chromatographic column is a Zorbax SB-phenyl column (4.6 mm. times.250 mm, 5 μm); the mobile phase was acetonitrile containing 10mM (R) -4-phenyl-2-oxazolidinone: 0.02mol/L potassium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) (35: 65); the preparation method of the 0.02mol/L potassium dihydrogen phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate, adding water, dissolving, mixing to desired concentration, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane. The method provided by the invention is a conventional HPLC chromatographic separation method, ramosetron hydrochloride and S-type enantiomer thereof can be effectively separated by only using a common phenyl chromatographic column, and the method has good repeatability and low cost of chromatographic consumables.
Description
Technical Field
The invention belongs to the field of medicine detection, and relates to separation and detection of ramosetron hydrochloride and S-type enantiomer thereof.
Background
Ramosetron hydrochloride (1 hydrochloride) is a strong 5-hydroxytryptamine (5-HT3) receptor antagonist developed by Nippon mountain company, and is mainly used for preventing and treating digestive tract symptoms such as nausea and vomiting caused by anti-malignant tumor drug therapy, and postoperative nausea and vomiting symptoms. 1 has a chiral center at the junction of the indole structure and the carbonyl group. It was reported that (R) -form 1 had 112-fold greater affinity for the 5-HT3 receptor than its enantiomer, 2[ (S) -form ], in mouse maternal nerve cells. Clinically used 1 is the (R) -form, and more drugs are administered clinically as a single enantiomer due to possible differences in the enantiomers in pharmacology, pharmacokinetics and toxicology. Therefore, in the study of pharmaceutical prescription process and the selection of storage conditions, the influence factors of isomer impurities and the quality control method thereof should be fully examined.
In the prior art, an HPLC chiral column separation method and a capillary electrophoresis method are mainly adopted to separate 1 and 2, but the following steps are adopted: the repeatability of the capillary electrophoresis method is poor; HPLC chiral column separation methods require reliance on expensive chiral columns.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a high performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof.
The above purpose of the invention is realized by the following technical scheme:
a high performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof comprises the following chromatographic conditions:
the chromatographic column is a Zorbax SB-phenyl column (4.6 mm. times.250 mm, 5 μm);
the mobile phase was acetonitrile containing 10mM (R) -4-phenyl-2-oxazolidinone: 0.02mol/L potassium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) (35: 65); the preparation method of the 0.02mol/L potassium dihydrogen phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate, adding water, dissolving, mixing to desired concentration, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane.
Preferably, the flow rate is 1.0 ml/min.
Preferably, the detection wavelength is 306 nm.
Preferably, the column temperature is 30 ℃.
Preferably, the sample size is 20. mu.l.
Has the advantages that:
the method provided by the invention is a conventional HPLC chromatographic separation method, ramosetron hydrochloride and S-type enantiomer thereof can be effectively separated by only using a common phenyl chromatographic column, and the method has good repeatability and low cost of chromatographic consumables.
Drawings
FIG. 1 is a chart showing the HPLC separation effect of the mixed control solution of example 1;
FIG. 2 is a chart showing the HPLC separation effect of the mixed control solution of example 2;
FIG. 3 is a chart showing the HPLC separation effect of the mixed control solution of example 3.
Detailed Description
The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.
Example 1:
instrument and reagent
Model LC-20A high performance liquid chromatograph (Shimadzu, japan, equipped with a quaternary pump, an on-line degasser, an autosampler, a DAD detector, an LC solution chemical workstation).
The ramosetron hydrochloride reference substance is purchased from China food and drug testing research institute, and the purity is not lower than 99 percent; the S enantiomer control was purchased from TLC pharmachem, inc, with a purity of not less than 99%.
Acetonitrile is chromatographically pure, potassium dihydrogen phosphate is superior grade pure, water is ultrapure water, and other reagents are analytically pure.
Second, method and results
2.1 chromatographic conditions
The chromatographic column is a Zorbax SB-phenyl column (4.6 mm. times.250 mm, 5 μm);
the mobile phase was acetonitrile containing 10mM (R) -4-phenyl-2-oxazolidinone: 0.02mol/L potassium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) (35: 65); the preparation method of the 0.02mol/L potassium dihydrogen phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate according to the formula amount, adding water to the formula volume, dissolving, mixing, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane;
the flow rate is 1.0 ml/min; the detection wavelength is 306 nm; the column temperature is 30 ℃; the amount of sample was 20. mu.l.
2.2 preparation of the solution
Ramosetron hydrochloride control solution: accurately weighing ramosetron hydrochloride reference substance, dissolving the ramosetron hydrochloride reference substance by using 35% acetonitrile aqueous solution and fixing the volume to 100 mu g/ml; s enantiomer control solution: precisely weighing an S-type enantiomer reference substance, dissolving the S-type enantiomer reference substance by using 35% acetonitrile water solution to be constant volume to 100 mu g/ml;
mixing the reference solution: accurately weighing ramosetron hydrochloride reference substances and S-type enantiomer reference substances respectively, dissolving with 35% acetonitrile water solution, and fixing the volume to respective concentration of 100 mu g/ml;
2.3 separation Effect
And (3) taking the ramosetron hydrochloride reference substance solution, the S-type enantiomer reference substance solution, the mixed reference substance solution and a blank solvent, respectively injecting samples according to the chromatographic conditions, measuring, and recording a chromatogram (shown in figure 1). The results show that ramosetron hydrochloride has a separation degree of more than 2.0 from the S enantiomer and a theoretical plate number of more than 10000 under the chromatographic conditions.
Example 2:
instrument and reagent
Model LC-20A high performance liquid chromatograph (Shimadzu, japan, equipped with a quaternary pump, an on-line degasser, an autosampler, a DAD detector, an LC solution chemical workstation).
The ramosetron hydrochloride reference substance is purchased from China food and drug testing research institute, and the purity is not lower than 99 percent; the S enantiomer control was purchased from TLC pharmachem, inc, with a purity of not less than 99%.
Acetonitrile is chromatographically pure, potassium dihydrogen phosphate is superior grade pure, water is ultrapure water, and other reagents are analytically pure.
Second, method and results
2.1 chromatographic conditions
The chromatographic column is a Zorbax SB-phenyl column (4.6 mm. times.250 mm, 5 μm);
the mobile phase was acetonitrile containing 10mM (2S,3R) -3-amino-2-hydroxy-4-phenylbutyric acid: 0.02mol/L potassium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) (35: 65); the preparation method of the 0.02mol/L potassium dihydrogen phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate according to the formula amount, adding water to the formula volume, dissolving, mixing, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane;
the flow rate is 1.0 ml/min; the detection wavelength is 306 nm; the column temperature is 30 ℃; the amount of sample was 20. mu.l.
2.2 preparation of the solution
Ramosetron hydrochloride control solution: accurately weighing ramosetron hydrochloride reference substance, dissolving the ramosetron hydrochloride reference substance by using 35% acetonitrile aqueous solution and fixing the volume to 100 mu g/ml; s enantiomer control solution: precisely weighing an S-type enantiomer reference substance, dissolving the S-type enantiomer reference substance by using 35% acetonitrile water solution to be constant volume to 100 mu g/ml;
mixing the reference solution: accurately weighing ramosetron hydrochloride reference substances and S-type enantiomer reference substances respectively, dissolving with 35% acetonitrile water solution, and fixing the volume to respective concentration of 100 mu g/ml;
2.3 separation Effect
And (3) taking the ramosetron hydrochloride reference substance solution, the S-type enantiomer reference substance solution, the mixed reference substance solution and a blank solvent, respectively injecting samples according to the chromatographic conditions, measuring, and recording a chromatogram (shown in figure 2). The results show that ramosetron hydrochloride has a separation degree of more than 2.0 from the S enantiomer and a theoretical plate number of more than 10000 under the chromatographic conditions.
Example 3: comparative examples
Instrument and reagent
Model LC-20A high performance liquid chromatograph (Shimadzu, japan, equipped with a quaternary pump, an on-line degasser, an autosampler, a DAD detector, an LC solution chemical workstation).
The ramosetron hydrochloride reference substance is purchased from China food and drug testing research institute, and the purity is not lower than 99 percent; the S enantiomer control was purchased from TLC pharmachem, inc, with a purity of not less than 99%.
Acetonitrile is chromatographically pure, potassium dihydrogen phosphate is superior grade pure, water is ultrapure water, and other reagents are analytically pure.
Second, method and results
2.1 chromatographic conditions
The chromatographic column is a Zorbax SB-phenyl column (4.6 mm. times.250 mm, 5 μm);
the mobile phase is acetonitrile: 0.02mol/L potassium dihydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) (35: 65); the preparation method of the 0.02mol/L potassium dihydrogen phosphate buffer solution comprises the following steps: weighing potassium dihydrogen phosphate according to the formula amount, adding water to the formula volume, dissolving, mixing, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane;
the flow rate is 1.0 ml/min; the detection wavelength is 306 nm; the column temperature is 30 ℃; the amount of sample was 20. mu.l.
2.2 preparation of the solution
Ramosetron hydrochloride control solution: accurately weighing ramosetron hydrochloride reference substance, dissolving the ramosetron hydrochloride reference substance by using 35% acetonitrile aqueous solution and fixing the volume to 100 mu g/ml; s enantiomer control solution: precisely weighing an S-type enantiomer reference substance, dissolving the S-type enantiomer reference substance by using 35% acetonitrile water solution to be constant volume to 100 mu g/ml;
mixing the reference solution: accurately weighing ramosetron hydrochloride reference substances and S-type enantiomer reference substances respectively, dissolving with 35% acetonitrile water solution, and fixing the volume to respective concentration of 100 mu g/ml;
2.3 separation Effect
And (3) taking the ramosetron hydrochloride reference substance solution, the S-type enantiomer reference substance solution, the mixed reference substance solution and a blank solvent, respectively injecting samples according to the chromatographic conditions, measuring, and recording a chromatogram (see figure 3). The results show that ramosetron hydrochloride cannot be separated from the S enantiomer under the chromatographic conditions, and the two are eluted together.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (1)
1. A high performance liquid chromatography method for separating ramosetron hydrochloride and S-type enantiomer thereof is characterized in that the chromatographic conditions are as follows:
the chromatographic column is a Zorbax SB-phenyl column with the specification of 4.6mm multiplied by 250mm and 5 mu m;
the mobile phase is a mixture of 10mM (R) -4-phenyl-2-oxazolidinone in acetonitrile and 0.02mol/L potassium dihydrogen phosphate buffer at a volume ratio of 35: 65; wherein, 0.02mol/L potassium dihydrogen phosphate buffer solution is adjusted to pH 3.0 by phosphoric acid, and the specific preparation method comprises the following steps: weighing potassium dihydrogen phosphate, adding water, dissolving, mixing to desired concentration, adjusting pH to 3.0 with phosphoric acid, and filtering with 0.45 μm filter membrane;
the flow rate is 1.0 mL/min;
the detection wavelength is 306 nm;
the column temperature is 30 ℃;
the sample size is 20 muL.
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Citations (7)
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