CN114384193B - Method for detecting chiral isomer of peramivir - Google Patents
Method for detecting chiral isomer of peramivir Download PDFInfo
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- CN114384193B CN114384193B CN202011114364.4A CN202011114364A CN114384193B CN 114384193 B CN114384193 B CN 114384193B CN 202011114364 A CN202011114364 A CN 202011114364A CN 114384193 B CN114384193 B CN 114384193B
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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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- 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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- 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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- 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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Abstract
The invention discloses a method for detecting an enantiomer of peramivir, which is characterized by comprising the following chemical names: (1R, 2R,3S, 4S) -3- [ (1R) -1-acetamide-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid with a structural formula as shown in formula I, and detecting by reversed phase high performance liquid chromatography of mobile phase containing copper sulfate and L-isoleucine.
Description
Technical Field
The invention relates to an analytical chemistry detection method, in particular to a method for detecting peramivir enantiomers by adopting a high performance liquid chromatography method with copper sulfate-L-isoleucine as a mobile phase.
Background
The chemical name of the prasu Mi Weisan hydrate is: (1S, 2S,3R, 4R) -3- [ (1S) -1-acetamide-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid trihydrate; molecular formula C 15 H 28 N 4 O 4 ·3H 2 O; molecular weight 382.45; the structural formula is as follows:
peramivir is a novel cyclopentane anti-influenza virus drug, which is another novel influenza virus NA inhibitor developed successfully with Zanamivir (Zanaamivir) and Oseltamivir (Oseltamivir) and marketed 1999. The national food and drug administration approves the new anti-influenza drug peramivir sodium chloride injection on 5 days 4 and 5 in 2013, and the existing clinical test data prove that the novel anti-influenza drug peramivir sodium chloride injection is effective on influenza A and influenza B.
The peramivir enantiomer is (1R, 2R,3S, 4S) -3- [ (1R) -1-acetamide-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid of formula C 15 H 28 N 4 O 4 The molecular weight is 328.41, and the structural formula is shown as follows:
the structural formula of the peramivir can be seen that the peramivir has five chiral centers, and whether the chiral isomer can be effectively detected directly influences the quality and the medication safety of the peramivir, so that the peramivir needs to be detected and controlled in quality.
Reverse-phase high performance liquid chromatography is a common detection method, which adopts a non-polar or relatively weak-polar stationary phase, and uses a stronger-polar solvent as a mobile phase, and is commonly used for separating and detecting non-polar and weaker-polar compounds; the reversed phase high performance liquid chromatography is most widely applied in the modern liquid chromatography, and is about 80% of the application of the whole high performance liquid chromatography according to statistics.
Diastereoisomers can be separated by reverse phase high performance liquid chromatography, but the enantiomers have the same effect as the stationary phase and are therefore difficult to separate by reverse phase high performance liquid chromatography.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for detecting peramivir enantiomer by using a reversed-phase high performance liquid chromatography with copper sulfate-L-isoleucine as a mobile phase.
The technical scheme is as follows: the invention relates to a method for detecting an enantiomer of peramivir, which is characterized by the chemical name: (1R, 2R,3S, 4S) -3- [ (1R) -1-acetamide-2-ethylbutyl ] -4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid, structural formula is shown as formula I, characterized in that the detection is carried out by reversed phase high performance liquid chromatography of mobile phase containing copper sulfate and L-isoleucine.
In some embodiments, the mobile phase comprising copper sulfate, L-isoleucine is a mobile phase comprising a phosphate-copper sulfate-L-isoleucine buffer.
In some embodiments, the mobile phase comprising copper sulfate, L-isoleucine is (phosphate-copper sulfate-L-isoleucine buffer) -acetonitrile is the mobile phase.
In some embodiments, the method of detecting an enantiomer of peramivir comprises the steps of:
a. preparing a sample solution;
b. the test sample solution is detected by reversed-phase high performance liquid chromatography with phosphate-copper sulfate-L-isoleucine buffer solution as a mobile phase, and specific detection conditions are as follows:
the column temperature of the chromatographic column is 20-50 ℃, preferably 25-40 ℃, most preferably 35 ℃,
the filler of the chromatographic column stationary phase is octadecylsilane chemically bonded silica gel,
the temperature of the differential refractometer is 20-50 ℃, preferably 25-40 ℃, most preferably 35 ℃,
mobile phase: the phosphate-copper sulfate-L-isoleucine buffer solution-acetonitrile is used as a mobile phase, the sample injection amount is 5-100. Mu.l, the sample injection amount is preferably 10-50. Mu.l, and most preferably 10. Mu.l, 20. Mu.l or 30. Mu.l.
In some embodiments, the phosphate salt of step b may be selected from the group consisting of sodium phosphate, disodium hydrogen phosphate, sodium hexametaphosphate, monoammonium phosphate, magnesium phosphate, barium phosphate, ammonium phosphate, calcium phosphate, potassium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, magnesium hydrogen phosphate, and preferably ammonium dihydrogen phosphate.
In some embodiments, the peramivir enantiomer is detected using a method wherein the phosphate salt of step b is monoammonium phosphate at a molar concentration in the buffer of 1-50mmol/L, preferably 5-15mmol/L, more preferably 10mmol/L.
In some embodiments, the molar concentration of copper sulfate in the buffer of step b is 1-50mmol/L, preferably 3-15mmol/L, more preferably 10mmol/L, of the peramivir enantiomer detection method.
In some embodiments, the molar concentration of L-isoleucine in the buffer of step b is 1-50mmol/L, preferably 5-15mmol/L, more preferably 10mmol/L, of the peramivir enantiomer detection method.
In some embodiments, the phosphate-copper sulfate-L-isoleucine buffer pH of step b is 3.0-5.0, preferably 3.0-4.0, more preferably 3.5, of the peramivir enantiomer detection method.
In some embodiments, the volume ratio of peramivir enantiomer detection method, step b (phosphate-copper sulfate-L-isoleucine buffer) to acetonitrile is selected from the group consisting of 10-50:1, preferably 10-20:1, more preferably 10-11: 1. 11-12: 1. 12-13: 1. 13-14: 1. 14-15: 1. 15-16: 1. 16-17: 1. 17-18: 1. 18-19: 1. 19-20:1, most preferably 94:6.
In some embodiments, the test solution concentration of step a is 0.1-100mg/mL, preferably 0.1-10mg/mL,5-10mg/mL, of the peramivir enantiomer detection method.
In some embodiments, the method of detecting the peramivir isomer, the chromatographic column is Waters Xselect CSH C.
The beneficial effects are that: (1) The method for detecting the peramivir enantiomer by using the reversed-phase high performance liquid chromatography with the copper sulfate-L-isoleucine as the mobile phase can effectively separate enantiomer peaks in the API; (2) The method optimizes the concentration of the copper sulfate-L-isoleucine and the operation parameters of the liquid chromatograph in the detection process, so that the detection time is short, and meanwhile, the specificity, the accuracy and the sensitivity of the detection are ensured.
Drawings
FIG. 1 is a blank solvent spectrum;
FIG. 2 is a graph of a mixed solution;
FIG. 3 is a graph of the palatami Wei Dingwei solution;
FIG. 4 is a graph of the peramivir enantiomer localization solution;
fig. 5 is a graph of a lapachomide Wei Lingmin degree solution chromatogram.
Detailed Description
Example 1
Example peramivir and its enantiomer form complexes with copper ions and L-isoleucine, respectively, and are detected by reverse-phase high performance liquid chromatography.
Peramivir and its enantiomer coordination principle:
the analytical steps of the peramivir enantiomer detection are specifically as follows:
chromatographic column: waters Xselect CSH C18 4.6X105 mm 3.5 μm;
mobile phase: 10mmol/L monoammonium phosphate, 10mmol/L copper sulfate pentahydrate, 10mmol/L L-isoleucine buffer (pH adjusted to 3.5 with 50% sodium hydroxide solution) -acetonitrile=94:6;
flow rate: 1.0ml/min;
column temperature: 35 ℃;
differential refractive detector temperature: 35 ℃;
sample injection amount: 20 μl;
solvent: a mobile phase;
peramivir positioning solution: 10mg/ml;
isomer localization solution: 0.5mg/ml;
mixing solution: the peramivir concentration is 10mg/ml and the enantiomer concentration is 0.5mg/ml;
sensitivity solution: 10 μg/ml;
sample injection procedure: the blank solvent, the peramivir positioning solution, the peramivir sensitivity solution, the isomer positioning solution and the mixed solution were measured with precision and 20 μl each, and were injected into a liquid chromatograph, and chromatograms were recorded as shown in fig. 1-5.
The enantiomer content was calculated according to the external standard method.
Results:
TABLE 1 liquid phase data for peramivir and peramivir
Claims (11)
1. A detection method for separating peramivir and peramivir enantiomers, said isomers having chemical names: (1R, 2R,3S, 4S) -3- [ (1R) -1-acetamide-2-ethylbutyl ] -4-guanidino-2-hydroxycyclopentyl-1-carboxylic acid, structural formula is shown as formula I, characterized in that the detection is carried out by adopting a reversed phase high performance liquid chromatography method using phosphate-copper sulfate-L-isoleucine buffer solution/acetonitrile as a mobile phase,
wherein, the filler of the chromatographic column stationary phase is octadecylsilane chemically bonded silica gel; the molar concentration of the copper sulfate in the buffer solution is 1-50mmol/L; the molar concentration of L-isoleucine in the buffer solution is 1-50mmol/L; the pH value of the buffer solution is 3.0-5.0; the volume ratio of the buffer solution to the acetonitrile is 10-50:1.
2. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 1, characterized by comprising the steps of:
a. preparing a sample solution;
b. the test sample solution is detected by reversed-phase high performance liquid chromatography with phosphate-copper sulfate-L-isoleucine buffer solution as a mobile phase, and specific detection conditions are as follows:
the column temperature of the chromatographic column is 20-50 ℃;
the temperature of the differential refraction detector is 20-50 ℃;
mobile phase: phosphate-copper sulfate-L-isoleucine buffer solution/acetonitrile is used as a mobile phase, and the sample injection amount is 5-100 μl.
3. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, characterized in that said column temperature of the chromatographic column is 25-40 ℃; the temperature of the differential refraction detector is 25-40 ℃; the sample injection amount is 10-50 μl.
4. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, wherein said phosphate in step b is selected from the group consisting of sodium phosphate, disodium hydrogen phosphate, sodium hexametaphosphate, ammonium dihydrogen phosphate, magnesium phosphate, barium phosphate, ammonium phosphate, calcium phosphate, potassium phosphate, ammonium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, magnesium hydrogen phosphate.
5. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 4 wherein said phosphate in step b is monoammonium phosphate having a molar concentration of 1-50mmol/L in the buffer.
6. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 5 characterized in that the molar concentration of monoammonium phosphate in the buffer is 5-15mmol/L.
7. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, characterized in that the molar concentration of copper sulfate in the buffer solution in step b is 3-15mmol/L.
8. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, characterized in that the molar concentration of L-isoleucine in the buffer of step b is 5-15mmol/L.
9. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, characterized in that the pH of said phosphate-copper sulfate-L-isoleucine buffer solution of step b is 3.0-4.0.
10. The method for the separation of peramivir and peramivir enantiomers according to claim 2, characterized in that the volume ratio of (phosphate-copper sulfate-L-isoleucine buffer) to acetonitrile in step b is 10-20:1.
11. The method for detecting the separation of peramivir and peramivir enantiomers according to claim 2, wherein said chromatographic column is Waters Xselect CSH C18.
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