CN112611817B - HPLC method for separating naftopidil and enantiomer thereof - Google Patents
HPLC method for separating naftopidil and enantiomer thereof Download PDFInfo
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- CN112611817B CN112611817B CN202011465890.5A CN202011465890A CN112611817B CN 112611817 B CN112611817 B CN 112611817B CN 202011465890 A CN202011465890 A CN 202011465890A CN 112611817 B CN112611817 B CN 112611817B
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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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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8877—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample optical isomers
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Abstract
The invention discloses an HPLC method for separating naftopidil and an enantiomer thereof. Naftopidil has a chiral center, and a pair of enantiomers (R) -naftopidil and (S) -naftopidil exist. The active ingredient used in clinic is (R) -naftopidil. In order to control the purity of (R) -naftopidil, it is necessary to establish an analytical method capable of separating (R) -naftopidil from (S) -naftopidil. At present, chiral chromatographic columns are used for separating the two, so that the cost is high. The HPLC method provided by the invention is based on an inexpensive eighteen carbon chromatographic column as a separation means, effectively separates (R) -naftopidil and (S) -naftopidil, has short separation time, and can greatly reduce the separation cost of naftopidil and enantiomers thereof.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to an HPLC (high performance liquid chromatography) method for separating naftopidil and an enantiomer thereof.
Background
Naftopidil (Naftopidil) is chemically named as 1- (2-methoxy) -4- [3- (1-naphthoxy) -2-hydroxypropyl ] -piperazine, is a novel receptor antagonist which is developed and marketed by the francium and the Asahi Kasei Pharma Kasei, is firstly approved in Japan for marketing in 1999 within 6 months, and is used for treating diseases such as prostatic hyperplasia.
Naftopidil has a chiral center and a pair of enantiomers, and the chemical structures of the naftopidil are respectively shown as follows:
the active ingredient used in clinic is (R) -naftopidil. In order to control the purity of (R) -naftopidil, it is necessary to establish an analytical method capable of separating (R) -naftopidil from (S) -naftopidil.
YInXiang Sun et al established a method for measuring naftopidil enantiomer by chiral high performance liquid chromatography, and used a chromatographic column of AD-H chiral column (Development of a chiral HPLC method for the analysis of naftopidil enantiomers. journal of Chinese Pharmaceutical sciences. 2009). The shisol and the like establish a method for directly splitting the naftopidil enantiomer by a Chiralpak AD-RH chiral column, and the adopted chromatographic column is the Chiralpak AD-RH chiral column (the Chiralpak AD-RH chiral column directly splits the naftopidil enantiomer, and the journal of drug analysis, 2009).
The AD-H chiral column and the ChiralpakAD-RH chiral column used in the documents are both chiral chromatographic columns specially used for separating chiral compounds, the manufacturing cost and the selling price are both high, and the service life of the chiral chromatographic column is much shorter than that of a conventional eighteen-carbon chromatographic column.
The invention is especially proposed in order to develop an HPLC method for separating (R) -naftopidil and (S) -naftopidil with low cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an HPLC method for separating naftopidil and an enantiomer thereof.
The purpose of the invention is realized by the following technical scheme:
an HPLC method for separating (R) -naftopidil and (S) -naftopidil at low cost is characterized in that a proper amount of chiral additive is added into a mobile phase, and the chiral additive is Fmoc-N-methyl-L-isoleucine.
Preferably, the mobile phase A is a 5% methanol aqueous solution containing 0.05 mol/LFmoc-N-methyl-L-isoleucine, the mobile phase B is a mixed organic solvent formed by acetonitrile and methanol according to a volume ratio of 4:3, and the A phase and the B phase are eluted at an isocratic rate of 35:65 according to the volume ratio.
Preferably, the separation column is a Waters Xbridge C18 column (4.6 mm. times.150 mm, 5 μm).
Has the advantages that:
the HPLC method provided by the invention is based on an inexpensive eighteen-carbon chromatographic column as a separation means, effectively separates (R) -naftopidil and (S) -naftopidil, has short separation time, and can greatly reduce the separation cost of naftopidil and enantiomers thereof.
Drawings
FIG. 1 shows the structural formulas of (R) -naftopidil and (S) -naftopidil.
FIG. 2 is an HPLC chromatogram under the conditions of example 1.
FIG. 3 is an HPLC chromatogram under the conditions of example 2.
Detailed Description
The following examples are intended to illustrate the essential technical content of the present invention, and the scope of the present invention should not be limited by the specific general details described in the following examples.
Example 1:
first, instrument and reagent
1. Instrument
Agilent 1260 liquid chromatograph (vacuum degasser, binary pump, autosampler), USA.
A chromatographic column: waters Xbridge C18 column (4.6 mm. times.150 mm, 5 μm).
A Mettler toledo electronic balance (XS 105).
2. Reagent
The purity of the (R) -naftopidil reference substance and the purity of the (S) -naftopidil reference substance are both not lower than 99%, and the structural formula is shown in figure 1.
The chiral additive Fmoc-N-methyl-L-isoleucine was purchased from Meclin reagent with a purity of 98%.
Acetonitrile and methanol are used as chromatographic purity, and water is used as ultrapure water.
Second, method and results
1. Chromatographic conditions
A chromatographic column: waters Xbridge C18 chromatography column (4.6 mm. times.150 mm, 5 μm);
mobile phase a phase: 5% methanol aqueous solution containing 0.05 mol/LFmoc-N-methyl-L-isoleucine (preparation method: first, methanol and water are mixed uniformly according to volume ratio of 5:95, then chiral additive is added to prepare the required concentration);
mobile phase B phase: a mixed organic solvent formed by acetonitrile and methanol according to the volume ratio of 4: 3;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 35: 65;
flow rate: 1.0 mL/min;
detection wavelength: 283 nm;
column temperature: 30 ℃;
sample introduction amount: 10 μ L.
2. Solution preparation
Mixing the reference solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to serve as a solvent, and preparing a mixed reference substance solution containing 0.6mg/mL of (R) -naftopidil reference substance and 0.6mg/mL of (S) -naftopidil reference substance.
(R) -naftopidil control solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to be used as a solvent, and preparing a solution containing 0.1mg/mL of (R) -naftopidil reference substance.
(S) -naftopidil control solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to be used as a solvent, and preparing a solution containing 0.1mg/mL of (S) -naftopidil reference substance.
3. Analysis of sample introduction
Respectively and precisely measuring 10 mu L of mixed reference substance solution, (R) -naftopidil reference substance solution and (S) -naftopidil reference substance solution, injecting into a liquid chromatograph, analyzing according to the chromatographic conditions, and recording a chromatogram. The results are shown in fig. 2, under the chromatographic conditions, (R) -naftopidil and (S) -naftopidil can achieve baseline separation.
Example 2: comparative example, mobile phase without addition of chiral additive
Instrument and reagent
1. Instrument for measuring the position of a moving object
Agilent 1260 liquid chromatograph (vacuum degasser, binary pump, autosampler), USA.
A chromatographic column: waters Xbridge C18 column (4.6 mm. times.150 mm, 5 μm).
Mettler toledo electronic balance (XS 105).
2. Reagent
The purity of the (R) -naftopidil reference substance and the purity of the (S) -naftopidil reference substance are both not lower than 99%, and the structural formula is shown in figure 1.
Acetonitrile and methanol are used as chromatographic purity, and water is used as ultrapure water.
Second, method and results
1. Chromatographic conditions
And (3) chromatographic column: waters Xbridge C18 chromatography column (4.6 mm. times.150 mm, 5 μm);
mobile phase a phase: 5% methanol water solution (preparation method: methanol and water are uniformly mixed according to the volume ratio of 5: 95);
mobile phase B phase: a mixed organic solvent formed by acetonitrile and methanol according to the volume ratio of 4: 3;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 35: 65;
flow rate: 1.0 mL/min;
detection wavelength: 283 nm;
column temperature: 30 ℃;
sample introduction amount: 10 μ L.
2. Solution preparation
Mixing the reference solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to serve as a solvent, and preparing a mixed reference substance solution containing 0.6mg/mL of (R) -naftopidil reference substance and 0.6mg/mL of (S) -naftopidil reference substance.
(R) -naftopidil control solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to serve as a solvent, and preparing a solution containing 0.1mg/mL (R) -naftopidil reference substance.
(S) -naftopidil control solution: taking a proper amount of mobile phase A and B, mixing the two phases according to the volume ratio of 35:65 to be used as a solvent, and preparing a solution containing 0.1mg/mL of (S) -naftopidil reference substance.
3. Analysis of sample introduction
Respectively and precisely measuring 10 mu L of mixed reference substance solution, (R) -naftopidil reference substance solution and (S) -naftopidil reference substance solution, injecting into a liquid chromatograph, analyzing according to the chromatographic conditions, and recording a chromatogram. The results are shown in fig. 3, under the chromatographic conditions, (R) -naftopidil and (S) -naftopidil elute peaks together and cannot be separated effectively.
In conclusion, the method of the invention is based on the cheap eighteen carbon chromatographic columns as the separation means, effectively separates (R) -naftopidil and (S) -naftopidil, has short separation time, and can greatly reduce the separation cost of naftopidil and enantiomers thereof.
The above-described embodiments are intended to embody the essential technical content of the present invention, and the scope of the present invention should not be limited by the specific general details described in the above-described embodiments.
Claims (1)
1. An HPLC method for separating (R) -naftopidil and (S) -naftopidil is characterized in that the chromatographic conditions are as follows:
a chromatographic column: waters Xbridge C18, specification 4.6mm × 150mm, 5 μm;
mobile phase a phase: the 5% methanol water solution containing 0.05 mol/LFmoc-N-methyl-L-isoleucine is prepared by the following steps: uniformly mixing methanol and water according to the volume ratio of 5:95, and then adding Fmoc-N-methyl-L-isoleucine to prepare the required concentration;
mobile phase B phase: a mixed organic solvent formed by acetonitrile and methanol according to the volume ratio of 4: 3;
elution mode and ratio: the phase A and the phase B are eluted at equal degrees according to the volume ratio of 35: 65;
flow rate: 1.0 mL/min;
detection wavelength: 283 nm;
column temperature: at 30 ℃.
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