CN108226337B - HPLC method for resolving cinacalcet hydrochloride enantiomer - Google Patents
HPLC method for resolving cinacalcet hydrochloride enantiomer Download PDFInfo
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Abstract
The invention discloses a method for separating, analyzing and measuring cinacalcet hydrochloride enantiomer, which selects silica gel coated with cellulose-tri (4-methylbenzoate) as a chiral stationary phase and a mixed solvent of n-hexane, absolute ethyl alcohol and ethanolamine in a certain proportion as a mobile phase for separating and analyzing the cinacalcet hydrochloride enantiomer.
Description
Technical Field
The invention relates to a high performance liquid chromatography, in particular to a high performance liquid chromatography for resolving cinacalcet hydrochloride enantiomer.
Background
Cinacalcet hydrochloride is a 2 nd generation calcium receptor regulator, and a novel compound with an alkylaniline skeleton in a chemical structure can act on a calcium receptor of a G-protein conjugated receptor. In parathyroid cells, calcium ion receptors are responsible for extracellular calcium ion (Ca)2 +) The concentration is sensitive, and the parathyroid hormone (PTH) secretion is regulated. Cinacalcet hydrochloride binds to calcium ion receptors and enhances extracellular Ca through allosteric effects2+Activating calcium ion receptor (calcium ion receptor isoagonist). Cinacalcet hydrochloride also inhibits the division of PTHProliferation of secreted and parathyroid cells, lowering serum PTH levels when administered orally [1]It can be used for treating secondary hyperparathyroidism caused by chronic kidney disease receiving dialysis and hypercalcemia of parathyroid tumor patients.
Cinacalcet hydrochloride has an asymmetric chiral carbon atom in the molecule, and a pair of enantiomers exist, which are racemic mixtures consisting of two single enantiomers, the R and S enantiomers. Wherein the structural formulas of the S-cinacalcet hydrochloride and the R-cinacalcet hydrochloride are as follows:
r-cinacalcet hydrochloride S-cinacalcet hydrochloride
Because the S enantiomer has no therapeutic activity, the single R enantiomer is clinically used, and the chemical name of the enantiomer is (R) -N- (1- (1-naphthyl) ethyl) -3- (3- (trifluoromethyl) phenyl) -1-propylamine hydrochloride. Due to the difference of cell expression, the cinacalcet hydrochloride enantiomers have significant difference in pharmacokinetics. The effective separation of the two becomes a difficult point in the separation and purification and quality control of the cinacalcet hydrochloride. Therefore, the high performance liquid chromatography method for resolving cinacalcet hydrochloride enantiomer is established, and has important application value. Firstly, a basis is provided for the process improvement of synthesizing, separating and purifying R-cinacalcet hydrochloride; secondly, providing basis for controlling the quality of the R-cinacalcet hydrochloride or the preparation; thirdly, the method is used for discussing the transformation or metabolism rule of the enantiomer in the body of an animal or a human body and provides a basis for clinical reasonable medication.
So far, only a few documents report the resolution of the chiral drug, and Vadde et al (JChromolographia, 2009, 70: 229-. Ravi et al (J Biomed chromatography, 2011, 25(6):674-679) established indirect reversed-phase high performance liquid chromatography and indirect reversed-phase thin layer chromatography of cinacalcet enantiomer by Maefey reagent, and the microwave derivation is needed, so the operation is complicated and the sensitivity is not high. Lougenqiang and the like (chemical research and application, 2015, 27 (6): 882-885) establish a method for splitting cinacalcet enantiomer by using a chiral stationary phase, and on a Chiralpak AD-H chiral column, the mobile phase is n-hexane-isopropanol-triethylamine (90: 10: 0.1) as a mobile phase for splitting, the retention time of the enantiomer is within 20min, but the peak type difference and the number of theoretical plates are low. Zhang Feng et al (journal of Waxi pharmacy, 2014, 29(1): 075-076) on the same chromatographic column, using n-hexane-ethanol-ethanolamine (95: 5: 0.1) as a mobile phase, and analyzing the content of enantiomer in the cinacalcet hydrochloride starting material R-1- (1-naphthyl) ethylamine at a flow rate of 1.0ml/min, but when the method is used for analyzing cinacalcet hydrochloride, the enantiomer cannot be effectively separated and influences the column effect.
In a word, the methods have the problems that the analysis time is long, baseline separation of cinacalcet hydrochloride enantiomers cannot be realized, the sensitivity is low, the operation is complicated, the cost is high and the like.
Disclosure of Invention
The inventor finds that the high performance liquid chromatography can effectively separate the cinacalcet hydrochloride enantiomers. The method has simple and convenient operation and low cost, and can simply, quickly and accurately resolve the cinacalcet hydrochloride enantiomer.
The invention aims to provide a high performance liquid chromatography method for simply and rapidly splitting cinacalcet hydrochloride enantiomer, thereby realizing effective separation and accurate determination of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride.
Specifically, in one embodiment of the invention, a method for resolving cinacalcet hydrochloride enantiomer by high performance liquid chromatography is provided, which comprises adopting a fiber chiral column and using a mixed solvent of n-hexane-absolute ethyl alcohol-ethanolamine as a mobile phase.
In one embodiment provided by the invention, the fiber-like chiral column is preferably a cellulose-tris (4-methylbenzoate) chiral column (Chiralcel OJ-H, 250mm × 4.6mm, 5 μm).
In one embodiment of the invention, the volume ratio of the n-hexane-absolute ethyl alcohol-ethanolamine mixed solvent as a mobile phase is 60: 40: 0.08-80: 20: 0.12, more preferably 65: 35: 0.08-75: 25: 0.12, and most preferably 70: 30: 0.1. The flow rate is preferably 0.4-0.6ml/min, most preferably 0.5 ml/min.
The cinacalcet hydrochloride enantiomer is completely resolved in a short time with considerable difficulty, and the prior art has many reports that complete separation is difficult to realize within 15min by adopting various chiral separation columns; furthermore, the present invention preferably finds, through a large number of experiments, that under the conditions of the separation column used in the present invention, if the mobile phase composition and the ratio are not within the selected range, effective separation is difficult, which fully indicates that the selection of the present invention has obvious specificity.
In one embodiment of the present invention, there is provided a method for the resolution and quantification of cinacalcet hydrochloride enantiomers comprising the steps of:
(1) taking a proper amount of cinacalcet hydrochloride sample, dissolving with a mobile phase, and quantitatively diluting to prepare a test sample solution containing 0.2mg of cinacalcet hydrochloride per 1 ml; precisely measuring 1ml of the test solution, placing in a 100ml measuring flask, adding mobile phase to dilute to scale, and shaking up to obtain a control solution.
(2) Setting the flow rate of the mobile phase to be 0.4-0.6ml/min, preferably 0.5ml/min, the detection wavelength to be 222 +/-2 nm, preferably 222nm, the column temperature: 20-30 ℃, preferably 25 ℃;
(3) and (3) injecting 20 mu l of the sample solution obtained in the step (1) into a liquid chromatograph, and recording a chromatogram.
Here, the liquid chromatograph is a high performance liquid chromatograph, and a Sammerfei U3000 high performance liquid chromatograph can be selected;
wherein, the chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
detection wavelength: 222 nm;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
The volume ratio of the n-hexane-absolute ethyl alcohol-ethanolamine mixed solvent to the mobile phase is 60: 40: 0.08-80: 20: 0.12, more preferably 65: 35: 0.08-75: 25: 0.12, and most preferably 70: 30: 0.1. The flow rate is preferably 0.4-0.6ml/min, most preferably 0.5 ml/min.
Under the selection conditions of the mobile phase and the separation column, the overall separation effect of the invention is particularly better through the selection of the flow rate, the column temperature and the composition proportion of the mobile phase.
The beneficial effects of the invention compared with the prior art comprise:
(1) the cinacalcet hydrochloride enantiomer can be effectively resolved by adopting a cellulose-tri (4-methylbenzoate) chiral column (Chiralcel OJ-H, 250mm multiplied by 4.6mm, 5 mu m);
(2) selecting a mobile phase dissolving sample, wherein the volume ratio of the mixed solvent of n-hexane-absolute ethyl alcohol-ethanolamine as the mobile phase is 60: 40: 0.08-80: 20: 0.12, so that the stability of the solution is ensured; the sample injection volume is 20 mul, the column temperature is 25 ℃, and the symmetry of chromatographic peaks is improved. The method solves the resolution problem of the cinacalcet hydrochloride enantiomer, and has important significance in various aspects such as drug metabolism research, chiral impurities, purity control and the like.
(3) Selecting a mobile phase dissolving sample, wherein the volume ratio of a mixed solvent of n-hexane-absolute ethyl alcohol-ethanolamine as the mobile phase is 70: 30: 0.1, under the preferred combination of conditions, achieves optimal separation of the cinacalcet hydrochloride enantiomers in as short a time as possible (within 10 min), can be used for accurate analysis of the product, and can be prepared in high yield and purity.
Drawings
FIG. 1, example 1 chromatographic conditions high performance liquid chromatogram of R-cinacalcet hydrochloride.
FIG. 2, high performance liquid chromatogram of chromatographic conditions S-cinacalcet hydrochloride of example 1.
FIG. 3, high performance liquid chromatogram of cinacalcet hydrochloride sample under chromatographic conditions of example 1.
FIG. 4, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 2.
FIG. 5, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 3.
FIG. 6, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 4.
FIG. 7, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 5.
FIG. 8, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 6.
FIG. 9, high performance liquid chromatogram of cinacalcet hydrochloride under chromatographic conditions of example 7.
FIG. 10, high performance liquid chromatogram of cinacalcet hydrochloride under the chromatographic conditions of comparative example 1.
FIG. 11, high performance liquid chromatogram of cinacalcet hydrochloride under the chromatographic conditions of comparative example 2.
FIG. 12, high performance liquid chromatogram of cinacalcet hydrochloride under the chromatographic conditions of comparative example 3.
Detailed Description
The present invention will be explained below by referring to specific examples and drawings, but the present invention is not limited thereto.
Example 1
Apparatus and conditions
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine 70: 30: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
R-cinacalcet hydrochloride: accurately weighing appropriate amount of R-cinacalcet hydrochloride reference substance, dissolving with mobile phase, diluting with mobile phase to obtain solution containing about 2 μ g per 1ml, precisely measuring 20 μ l, injecting into chromatograph, recording chromatogram, wherein the chromatogram of R-cinacalcet hydrochloride is shown in figure 1, and the peak is about 9.9 min.
S-cinacalcet hydrochloride solution: accurately weighing appropriate amount of S-cinacalcet hydrochloride reference substance, dissolving with mobile phase, diluting with mobile phase to obtain solution containing 10 μ g per 1ml, and making chromatogram of S-cinacalcet hydrochloride as shown in figure 2, with peak at about 9.4 min.
Test solution: a proper amount of cinacalcet hydrochloride sample is accurately weighed, dissolved by a mobile phase and diluted into a solution containing about 0.2mg per 1ml by the mobile phase. The chromatogram of the cinacalcet hydrochloride sample is shown in figure 3, wherein the peak 1 is S-cinacalcet hydrochloride, the peak is about 9.4min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 9.9min, the separation degree of the two is more than 1.5, and the symmetry is good. Under the chromatographic condition, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride are completely separated, and the isomer content can be accurately quantified by adopting an external standard method or a self-contrast method.
Example 2
Apparatus and conditions
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine ═ 65: 35: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 4, the peak 1 in the figure is S-cinacalcet hydrochloride, the peak is about 9.4min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 10.0min, the separation degree of the two reaches more than 1.5, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Example 3
Apparatus and conditions
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine 75: 25: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 5, the peak 1 in the figure is S-cinacalcet hydrochloride, the peak is about 9.9min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 10.5min, the separation degree of the two is more than 1.5 except that the retention time is slightly changed, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Example 4
Apparatus and conditions
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine 70: 30: 0.08;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving and quantitatively diluting with a mobile phase to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 6, the peak 1 in the figure is S-cinacalcet hydrochloride, the peak is about 9.9min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 10.5min, the separation degree of the two is more than 1.5 except that the retention time is slightly changed, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Example 5
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine 70: 30: 0.12;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.5 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing about 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording the chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 7, the peak 1 in the figure is S-cinacalcet hydrochloride, the peak is about 9.8min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 10.4min, and the separation degree reaches more than 1.5 except for slight change of retention time, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Example 6
Apparatus and conditions
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine ═ 70: 30: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.4 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 8, the peak 1 in the figure is S-cinacalcet hydrochloride, the peak is about 11.8min, the peak 2 is R-cinacalcet hydrochloride, the peak is about 12.4min, the separation degree of the two is more than 1.5 except that the retention time is slightly changed, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Example 7
A Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine ═ 70: 30: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 0.6 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking appropriate amount of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride (or raceme) respectively, dissolving with mobile phase and quantitatively diluting to prepare system applicability solution containing 10 mug of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mug, injecting into a liquid chromatograph, recording chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 9, in the figure, the No. 1 peak is S-cinacalcet hydrochloride, the peak is about 7.8min, the No. 2 peak is R-cinacalcet hydrochloride, the peak is about 8.2min, the two have slight change of retention time, the separation degree reaches more than 1.5, and the symmetry is good. Under the chromatographic conditions, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride can be completely separated.
Comparative example 1
Agilent 1200 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine ═ 95: 5: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 1.0 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 10, the peak of S-cinacalcet hydrochloride is about 5.9min, the peak of R-cinacalcet hydrochloride is about 6.1min, and the two peaks are not completely separated and appear inverted peaks. Under the chromatographic conditions, the two cannot be effectively separated, and the isomer content of the two cannot be accurately quantified.
Comparative example 2
Agilent 1200 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: 87.5 parts of n-hexane, anhydrous ethanol, ethanolamine: 12.5: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 1.0 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording a chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 11, the peak of S-cinacalcet hydrochloride is about 5.6min, the peak of R-cinacalcet hydrochloride is about 5.9min, two peaks are not completely separated, and the peak shape symmetry is poor. Under the chromatographic conditions, the two cannot be effectively separated, and the isomer content of the two cannot be accurately quantified.
Comparative example 3
Agilent 1200 high performance liquid chromatograph;
a chromatographic column: OJ-H (250 mm. times.4.6 mm, 5 μm) cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine ═ 70: 30: 0.1;
detection wavelength: 222 nm;
column temperature: 25 ℃;
flow rate: 1.0 ml/min;
sample introduction volume: 20 μ l.
Experimental procedure
Taking a proper amount of R-cinacalcet hydrochloride sample and S-cinacalcet hydrochloride (or raceme) respectively, dissolving by using a mobile phase and quantitatively diluting to prepare a system applicability solution containing 10 mu g of R-cinacalcet hydrochloride and S-cinacalcet hydrochloride respectively in each 1ml, precisely measuring 20 mu l, injecting into a liquid chromatograph, recording the chromatogram, wherein the chromatogram of the system applicability solution is shown in figure 12, the peak of the S-cinacalcet hydrochloride in the figure is about 4.7min, the peak of the R-cinacalcet hydrochloride is about 5.1min, and the resolution is only 0.95. Under the chromatographic condition, R-cinacalcet hydrochloride and S-cinacalcet hydrochloride are only primarily separated, but the separation is not good enough, and the isomers are difficult to accurately quantify.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. An HPLC method for resolving cinacalcet hydrochloride enantiomer is characterized in that a cellulose derivative is used as a stationary phase, a mixed solvent of n-hexane, absolute ethyl alcohol and ethanolamine is used as a mobile phase, wherein the cellulose derivative is cellulose-tris (4-methylbenzoate), the volume ratio of n-hexane-absolute ethyl alcohol-ethanolamine in the mixed solvent is 60: 40: 0.08-80: 20: 0.12, and the flow rate of the mobile phase is 0.4-0.6 ml/min.
2. An HPLC method for resolving cinacalcet hydrochloride enantiomer is characterized in that a cellulose derivative is used as a stationary phase, a mixed solvent of n-hexane, absolute ethyl alcohol and ethanolamine is used as a mobile phase, wherein the cellulose derivative is cellulose-tris (4-methylbenzoate), the volume ratio of n-hexane-absolute ethyl alcohol-ethanolamine in the mixed solvent is 65: 35: 0.08-75: 25: 0.12, and the flow rate of the mobile phase is 0.4-0.6 ml/min.
3. An HPLC method for resolving cinacalcet hydrochloride enantiomer is characterized in that a cellulose derivative is used as a stationary phase, a mixed solvent of n-hexane, absolute ethyl alcohol and ethanolamine is used as a mobile phase, wherein the cellulose derivative is cellulose-tris (4-methylbenzoate), the volume ratio of n-hexane-absolute ethyl alcohol-ethanolamine in the mixed solvent is 70: 30: 0.1, 65: 35: 0.1, 75: 25: 0.1, 70: 30: 0.08 or 70: 30: 0.12, and the flow rate of the mobile phase is 0.4-0.6 ml/min.
4. A method according to any one of claims 1 to 3, comprising the steps of:
(1) taking a proper amount of cinacalcet hydrochloride sample, and dissolving the sample by using a mobile phase to obtain 1ml of sample solution containing 0.2mg of cinacalcet hydrochloride;
(2) setting the flow velocity of a mobile phase to be 0.4-0.6ml/min, the detection wavelength to be 222 +/-2 nm, and the column temperature: 20 to 30 ℃.
5. The method according to claim 4, wherein the flow rate of the mobile phase in the step (2) is set to be 0.5ml/min, the detection wavelength is 222nm, and the column temperature is as follows: at 25 ℃.
6. The method of claim 1, wherein the splitting method comprises: a Saimeifei U3000 high performance liquid chromatograph;
a chromatographic column: OJ-H, 250mm × 4.6mm, 5 μm, cellulose-tris (4-methylbenzoate) chiral column;
volume ratio of mobile phase: n-hexane, anhydrous ethanol, ethanolamine 70: 30: 0.1;
detection wavelength: 222 nm;
flow rate: 0.5 ml/min;
sample introduction volume: 20 mu l of the mixture;
the experimental steps are as follows:
taking a proper amount of cinacalcet hydrochloride sample, dissolving with a mobile phase, and quantitatively diluting to prepare a test sample solution containing 0.2mg of cinacalcet hydrochloride per 1 ml; precisely measuring 1ml of the test solution, placing in a 100ml measuring flask, adding mobile phase to dilute to scale, and shaking up to obtain a control solution.
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