CN114264747A - High performance liquid chromatography detection method for empagliflozin intermediate and related substances thereof - Google Patents
High performance liquid chromatography detection method for empagliflozin intermediate and related substances thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 70
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- 229960003345 empagliflozin Drugs 0.000 title claims abstract description 47
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 8
- 239000000243 solution Substances 0.000 claims abstract description 84
- 239000003085 diluting agent Substances 0.000 claims abstract description 51
- 239000007788 liquid Substances 0.000 claims abstract description 40
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 15
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- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- RCSSDHAONHNBCD-UHFFFAOYSA-N (2,3,4,5,6-pentafluorophenyl)silane Chemical group FC1=C(F)C(F)=C([SiH3])C(F)=C1F RCSSDHAONHNBCD-UHFFFAOYSA-N 0.000 claims abstract description 4
- PZKYKDWPZSLPDI-UHFFFAOYSA-N [SiH4].CCCCCCCC Chemical group [SiH4].CCCCCCCC PZKYKDWPZSLPDI-UHFFFAOYSA-N 0.000 claims abstract description 4
- WGZCUXZFISUUPR-UHFFFAOYSA-N acetonitrile;oxolane Chemical compound CC#N.C1CCOC1 WGZCUXZFISUUPR-UHFFFAOYSA-N 0.000 claims abstract description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
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- 238000000926 separation method Methods 0.000 claims description 10
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- 230000006978 adaptation Effects 0.000 claims description 7
- 230000003044 adaptive effect Effects 0.000 claims description 4
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 2
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- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
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- 239000010452 phosphate Substances 0.000 claims description 2
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a high performance liquid chromatography detection method for an empagliflozin intermediate and related substances thereof. The detection method comprises the following steps: a chromatographic column which adopts octane silane bonded silica gel or pentafluoro-phenyl silane bonded silica gel as a filling agent; performing gradient elution by using a phosphoric acid aqueous solution or a phosphate buffer solution with the pH value of 2.5-6.5 as a mobile phase A and an acetonitrile-methanol solution or an acetonitrile-tetrahydrofuran solution as a mobile phase B; preparing a sample solution from a sample containing the Engelliflozin intermediate and related substances thereof by using a diluent, injecting the sample solution into a liquid chromatograph, and detecting and recording a chromatogram. The detection method has the advantages of short analysis time, high sensitivity, good specificity and durability and the like, can simply, quickly, sensitively, accurately and reliably detect the Empagliflozin intermediate A and related substances thereof, and is favorable for effectively monitoring and controlling the product quality.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry analysis, in particular to a high performance liquid chromatography detection method for an empagliflozin intermediate and related substances thereof.
Background
The Empagliflozin (Jardance) is a sodium-glucose cotransporter-2 (SGLT-2) inhibitor drug jointly developed by Boringer Invitrogen and Gift company, can block the reabsorption of glucose in the kidney and excrete excessive glucose to the outside of the body, thereby achieving the effect of reducing the blood sugar level, and the blood sugar reducing effect is independent of beta cell function and insulin resistance.
The key intermediate of the empagliflozin is an empagliflozin intermediate A (CAS number: 915095-94-2), and the molecular formula is as follows: c17H16ClIO2Molecular weight: 414.67, the structural formula is as follows:
empagliflozin intermediate A
According to the patent literatureThe synthetic route of the intermediate A of the canagliflozin is CN101193903B as follows:
usually, the resulting intermediate a of engagliflozin, after the reaction of the above synthetic route, contains related substances such as starting materials, intermediates and degradation products which are not completely reacted, wherein,
the Engelliflozin related substance A1 is a starting material of a synthesis process, and has a molecular formula: c13H7ClFIO, molecular weight: 360.55, the chemical formula is as follows:
the related substance A2 of the empagliflozin is a positional isomer of a starting material of a synthesis process, and has a molecular formula: c13H7ClFIO, molecular weight: 360.55, the chemical formula is as follows:
the empagliflozin related substance A3 is an intermediate produced in the first step of reaction, and has the molecular formula: c17H14ClIO3Molecular weight: 428.65, the chemical formula is as follows:
the related substance A6 of the empagliflozin is a derivative impurity of the related substance A2 in the first step of reaction, and the molecular formula is as follows: c17H14ClIO3Molecular weight: 428.65, the chemical formula is as follows:
the substance A7 related to engagliflozin isThe process impurity of the first step reaction is produced by deiodination of the intermediate A, and the molecular formula is as follows: c17H15ClO3Molecular weight: 302.75, the chemical structural formula is as follows:
the related substance A8 of Empagliflozin is a process impurity of the first step reaction, and is generated by the reaction of a starting material A1 and two molecules of (S) -3-hydroxytetrahydrofuran, and the molecular formula is as follows: c21H21IO5Molecular weight: 480.29, the chemical formula is as follows:
the related substance A5 of the empagliflozin is a derivative impurity of the related substance A7 in the second step of reaction, and the molecular formula is as follows: c17H17ClO2Molecular weight: 288.77, the chemical formula is as follows:
the related substance A4 of the empagliflozin is a derivative impurity of the related substance A6 in the second step of reaction, and the molecular formula is as follows: c17H16ClIO2Molecular weight: 414.67, the chemical formula is as follows:
CN112986456A discloses an analytical detection method for the intermediate of engagliflozin, but the method cannot effectively separate the engagliflozin intermediate a and related substances thereof in the above synthetic route.
CN106706768B discloses a method for separating and measuring engeletin and related substances, and the method can not effectively separate the engeletin intermediate a and related substances of the synthetic route.
Therefore, in order to track and control the quality of the medicine, it is necessary to establish an effective detection method of the empagliflozin intermediate A and related substances thereof.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a high performance liquid chromatography detection method for the Engelliflozin intermediate A and the related substances thereof, which has the advantages of short analysis time, high sensitivity, good specificity and durability and the like, can simply, quickly, sensitively, accurately and reliably detect the Engelliflozin intermediate A and the related substances thereof, and is favorable for effectively monitoring and controlling the product quality.
The technical scheme provided by the invention is as follows:
a high performance liquid chromatography detection method for an engagliflozin intermediate A and related substances A1-A8 thereof is disclosed, wherein the structural formula of the engagliflozin intermediate A and related substances A1-A8 is as follows:
the detection method of the present invention is characterized in that,
1) chromatographic conditions are as follows:
a chromatographic column which adopts octane silane bonded silica gel or pentafluoro-phenyl silane bonded silica gel as a filling agent; performing gradient elution by using a phosphoric acid aqueous solution or a phosphate buffer solution with the pH value of 2.5-6.5 as a mobile phase A and an acetonitrile-methanol solution or an acetonitrile-tetrahydrofuran solution as a mobile phase B;
2) a detection step:
and respectively injecting the blank solution, the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording the chromatogram.
Furthermore, the chromatographic column used in the invention uses octane silane bonded silica gel or pentafluoro-phenyl silane bonded silica gel as a filler, and the available brands are not limited, such as agilent, Shimadzu, Sammerfei, Watts, YMC and the like; the length of the chromatographic column is 150-250 mm, the particle size of the filler is 3-5 mu m, and the inner diameter of the chromatographic column is 4.6 mm; preferably selected from Thermo Accucore XL C8, 150mm × 4.6mm, 4 μm; phenomenex Kinetex F5, 250X 4.6mm, 5 μm; ACE 3C18-PFP, 150X 4.6mm, 3 μm; agilent ZORBAX RX-C8, 250X 4.6mm, 5 μm.
Further, an aqueous phosphoric acid solution or a phosphate buffer solution having a pH of 2.5 to 3.5 is preferable, and an aqueous phosphoric acid solution having a pH of 3 is more preferable.
Further, the phosphate is selected from potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate, and the concentration of the phosphate buffer is 0.005-0.01 mol/L.
Further, the volume ratio of acetonitrile to methanol or acetonitrile to tetrahydrofuran in the mobile phase B is 100: 0-70: 30; preferably the volume ratio of acetonitrile to methanol is 80:20, or, preferably, the volume ratio of acetonitrile to tetrahydrofuran is 90: 10.
Further, the gradient elution procedure is shown in table 1.
TABLE 1
The preferred gradient elution procedure is shown in table 2.
TABLE 2
Further, the flow rate of the mobile phase is 0.8-1.2 ml/min; preferably 1.0 ml/min.
Further, the temperature of the chromatographic column is 20-40 ℃; preferably 25 to 35 ℃, and more preferably 30 ℃.
Further, the detection wavelength is 210-230 nm; preferably 220 nm.
Further, the diluent is a mixed solution of an organic solvent and purified water; the volume ratio of the organic solvent to the purified water is 40: 60-60: 40, preferably the volume ratio of organic solvent to purified water is 50: 50.
further, the organic solvent is acetonitrile, methanol or a mixed organic solvent of acetonitrile and methanol, wherein the volume ratio of acetonitrile to methanol in the mixed organic solvent is 30-50: 5-15, preferably 40: 10.
furthermore, the concentration of the test solution is 0.3-0.8 mg/ml, preferably 0.5 mg/ml.
Further, the sample injection volume is 5-10 mu l.
Further, solution preparation is performed prior to the detection step:
blank solution: diluent
Impurity reference stock solution: respectively taking a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance, dissolving with acetonitrile, and preparing into impurity reference substance stock solutions with a diluent; the concentration of related substances in the impurity reference substance stock solution is 0.3-0.8 mg/ml, preferably 0.5 mg/ml;
system adaptation solution: taking an reference substance A of the empagliflozin intermediate and the stock solutions of all impurity reference substances, dissolving the reference substance A and the stock solutions with acetonitrile, and preparing a system adaptive solution by using a diluent; the concentration of the empagliflozin intermediate A in the system adaptive solution is 0.3-0.8 mg/ml, preferably 0.5mg/ml, and the concentration of each related substance is 3-8 mu g/ml, preferably 5 mu g/ml;
test solution: dissolving the Engelliflozin intermediate A with acetonitrile, and preparing a test solution with a diluent; the concentration of the empagliflozin intermediate A in the test solution is 0.3-0.8 mg/ml, and preferably 0.5 mg/ml.
The reference substance of the Engelliflozin intermediate A used in the invention has the purity of more than 99 percent after the structure is confirmed; the empagliflozin intermediate a used in the invention is a randomly drawn production batch.
Further, the purity of the empagliflozin intermediate A and the content of related substances A1-A8 thereof are calculated according to a peak area normalization method; the S/N ratio is not less than 10, and 0.03% of the sample concentration is taken as a quantification limit; the S/N ratio is about 3, and 0.01% of the sample concentration is taken as the detection limit. Furthermore, the separation degree between the empagliflozin intermediate A and the related substances A1-A8 is more than 1.5; the quantitative limit of the Engelliflozin intermediate A and the related substances A1-A8 is 0.03%; the detection limit of the Engelliflozin intermediate A and the related substances A1-A8 is 0.01 percent.
The high performance liquid chromatography method for determining the Engelliflozin intermediate A and the related substances thereof optimizes the detection method by comprehensively investigating the influence of a chromatographic column, a mobile phase, a gradient elution program, a flow rate and a column temperature on separation detection, has the advantages of short analysis time (25-35 min), high sensitivity (the limit of quantification is 0.03 percent and the limit of detection is 0.01 percent), specificity (the separation degree of the Engelliflozin intermediate A and the related substances A1-A8 is not less than 1.5) and good durability, and the like, and provides a simple, quick, sensitive, accurate and reliable method for quality tracking and quality control in the synthesis of the Engelliflozin intermediate A.
Drawings
FIG. 1 is a liquid chromatogram of a blank solution examined under the conditions of example 1.
FIG. 2 is a liquid chromatogram of a system adapted solution tested under the conditions of example 1.
FIG. 3 is a liquid chromatogram of a test solution examined under the conditions of example 1.
FIG. 4 is a liquid chromatogram of the quantitative limiting solution examined under the conditions of example 1.
FIG. 5 is a liquid chromatogram of a detection limit solution detected under the conditions of example 1.
FIG. 6 is a liquid chromatogram of the system adapted solution tested under the conditions of example 4.
FIG. 7 is a liquid chromatogram of the system adapted solution tested under the conditions of example 5.
FIG. 8 is a liquid chromatogram of the system adapted solution tested under the conditions of example 6.
FIG. 9 is a liquid chromatogram of a system-adapted solution tested under the conditions of example 7.
FIG. 10 is a liquid chromatogram of a system-adapted solution tested under the conditions of comparative example 1.
FIG. 11 is a liquid chromatogram of a system-adapted solution tested under the conditions of comparative example 2.
FIG. 12 is a liquid chromatogram of a system-adapted solution tested under the conditions of comparative example 2.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
The high performance liquid chromatograph used in the embodiment of the invention is a liquid chromatograph of Agilent company.
The Engelliflozin intermediate A and related substances used in the embodiment of the invention are self-made in the Haixiang pharmaceutical industry and are confirmed by the structure.
Example 1
(1) Chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: chromatographic column using octyl silane bonded silica gel as filler (Thermo Accucore XL C8, 150X 4.6mm, 4 μm)
Flow rate: 1.0ml/min column temperature: 30 deg.C
Detection wavelength: sample size at 220 nm: 5 μ l
Mobile phase A: phosphoric acid water solution, taking purified water, adjusting pH value to 3 with phosphoric acid
Mobile phase B: acetonitrile-methanol (80:20V/V)
Diluent agent: acetonitrile-methanol-purified water (40:10:50V/V/V)
The gradient elution procedure is shown in table 3.
TABLE 3
(2) Solution preparation
Impurity reference stock solution: taking about 25mg of a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance respectively, precisely weighing, respectively placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up to obtain the product.
System adaptation solution: precisely weighing about 50mg of the reference substance A of the empagliflozin intermediate, placing the reference substance A into a 100ml volumetric flask, precisely adding 1ml of the impurity reference substance storage solution into the 100ml volumetric flask respectively, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up to obtain the empagliflozin intermediate.
Test solution: taking about 25mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
Engagliflozin intermediate control stock solutions: precisely weighing about 25mg of reference substance of the Engelliflozin intermediate A, placing the reference substance in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up to obtain the product.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the reference substance storage solution of the Empagliflozin intermediate in volumetric flasks of 1ml to 100ml respectively, adding a diluent to dilute to the scales, shaking up, precisely measuring 3ml of the solution in volumetric flasks of 100ml, adding the diluent to dilute to the scales, and shaking up to obtain the product.
Detection limiting solution: precisely measuring 3ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml volumetric flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.
(3) Measurement of
And respectively injecting a blank solution (namely, a diluent), a system adaptability solution, a sample solution, a quantitative limiting solution and a detection limiting solution into a high performance liquid chromatograph for detection, and recording the chromatogram.
Chromatograms of blank solution (i.e., diluent), system adaptation solution, test sample solution, quantitation limit solution, and detection limit solution are shown in fig. 1-5, respectively.
FIG. 1 shows that the blank is not interfering with the detection; FIG. 2 shows that the separation degree between the Empagliflozin intermediate A and each related substance is good and is more than 1.5; FIG. 3 shows that the related substance A3 and the related substance A5 are detected in the test solution, and other related substances are not detected; FIG. 4 shows that the quantitative limits of the Empagliflozin intermediate A and each related substance are both 0.03%; fig. 5 shows that the detection limit of the empagliflozin intermediate a and each related substance is 0.01%. The maps and the data show that the detection method has good specificity and high detection sensitivity.
Example 2
(1) Chromatographic conditions
The flow rate, column temperature, mobile phase ratio, mobile phase pH, wavelength and sample amount were varied within certain ranges as shown in tables 4 and 5, and other chromatographic conditions were the same as in example 1.
(2) Solution preparation
Impurity reference stock solution: taking a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance, precisely weighing, respectively placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up to obtain the product;
system adaptation solution: precisely weighing about 50mg of the reference substance A of the empagliflozin intermediate, placing the reference substance A into a 100ml volumetric flask, precisely adding 1ml of the impurity reference substance storage solution into the 100ml volumetric flask respectively, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up to obtain the empagliflozin intermediate.
Test solution: taking about 25mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
(3) Measurement of
And respectively injecting the blank solution (namely, the diluent), the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording the chromatogram.
The wavelength and the sample amount were varied within a certain range, and the appropriate wavelength and sample amount were selected, and the results of the system-adapted solution are shown in Table 4.
TABLE 4 chromatographic conditions for investigation of wavelength and sample size
| Wavelength, sample size selection | 210nm，5μl | 220nm，5μl | 230nm，5μl | 220nm，10μl |
| Related substance A1 (%) | 0.906 | 0.915 | 0.770 | 0.940 |
| Related substance A2 (%) | 0.729 | 0.826 | 0.899 | 0.854 |
| Related substance A3 (%) | 0.661 | 0.947 | 0.883 | 0.977 |
| Related substance A4 (%) | 1.110 | 1.010 | 0.713 | 1.045 |
| Related substance A5 (%) | 0.878 | 0.992 | 0.659 | 1.022 |
| Related substance A6 (%) | 0.722 | 1.024 | 0.695 | 1.053 |
| Related substance A7 (%) | 0.760 | 0.924 | 0.475 | 0.948 |
| Related substance A8 (%) | 0.616 | 1.013 | 0.988 | 1.041 |
| Intermediate A (%) | 93.617 | 92.348 | 93.918 | 92.120 |
The result shows that when the wavelength is 210-230 nm, the detection result of the system adaptive solution (containing about 1% of related substances A1-A8) is relatively accurate, and the optimal detection result is 220 nm; the results of the tests were consistent when the amount of the sample was 5. mu.l to 10. mu.l, but the main peak heights were compared, preferably 5. mu.l and 955.
The chromatographic parameters of flow rate, column temperature, mobile phase proportion and mobile phase pH are changed within a certain range, single factor investigation is carried out, the specific chromatographic conditions are shown in table 5, and the results of the test solution are shown in table 6.
Table 5 chromatographic conditions for examination of durability
TABLE 6 measurement results
The result shows that when the chromatographic parameters are changed within a certain range, the separation degree of the Engelliflozin intermediate A and each related substance is not less than 1.5, the detection results of the Engelliflozin intermediate A and each related substance are basically stable, and the method has good durability.
Example 3
(1) Chromatographic conditions
Same as example 1
(2) Solution preparation
60% linear solution: taking about 15mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
80% linear solution: taking about 20mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
100% linear solution: taking about 25mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
120% linear solution: taking about 30mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
160% linear solution: taking about 40mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding 0.5ml of acetonitrile for dissolving, adding a diluent for diluting to a scale, and shaking up.
Quantitative limiting solution: precisely measuring 1ml to 100ml of 100% linear solution in a volumetric flask, adding a diluent to dilute the solution to a scale, shaking up, precisely measuring 3ml of the solution in the volumetric flask of 100ml, adding the diluent to dilute the solution to the scale, and shaking up to obtain the product.
(3) Measurement of
The blank solution (i.e., diluent), the quantitative limiting solution and each linear solution were injected into a high performance liquid chromatograph in sequence for detection, and the chromatogram was recorded, and the linear results are shown in table 7.
TABLE 7 Linear results
| Factors for investigation | 0.03％ | 60％ | 80％ | 100％ | 120％ | 160％ |
| Concentration (μ g/ml) | 0.15 | 300.2 | 402.4 | 501.2 | 605.2 | 800.4 |
| Peak area | 1.984 | 3609.104 | 4786.131 | 5896.250 | 7108.533 | 9345.529 |
The results show that the linearity curve y is 11.6578x +54.3088, R is 0.99991, and the sample concentration is good at 0.15 μ g/ml to 800.4 μ g/ml.
Example 4
(1) Chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: chromatographic column using octyl silane bonded silica gel as filler (Thermo Accucore XL C8, 150X 4.6mm, 4 μm)
Flow rate: 1.0ml/min column temperature: 20 deg.C
Detection wavelength: sample size at 220 nm: 5 μ l
Mobile phase A: phosphate buffer solution, 0.005mol/L KH2PO4Solution, pH value adjusted to 2.5 with phosphoric acid
Mobile phase B: acetonitrile
Diluent agent: acetonitrile-purified water (40:60V/V)
The gradient elution procedure is shown in table 8.
TABLE 8
(2) Solution preparation
The same as in example 2.
(3) Measurement of
And respectively injecting the blank solution (namely, the diluent), the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram, wherein the system adaptability chromatogram is shown in figure 6. The result shows that the separation degree of the empagliflozin intermediate A and related substances is not less than 1.5, and the detection results of the empagliflozin intermediate A and the related substances are basically stable.
Example 5
(1) Chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: column using pentafluoro-phenylsilane-bonded silica gel as filler (Phenomenex Kinetex F5, 250X 4.6mm, 5 μm)
Flow rate: 1.0ml/min column temperature: 25 deg.C
Detection wavelength: sample size at 220 nm: 5 μ l
Mobile phase A: phosphate buffer solution, 0.005mol/L NaH2PO4Solution, pH adjusted to 3.5 with phosphoric acid
Mobile phase B: acetonitrile
Diluent agent: acetonitrile-purified water (60:40V/V)
The gradient elution procedure is shown in table 9.
TABLE 9
(2) Solution preparation
The same as in example 2.
(3) Measurement of
And respectively injecting the blank solution (namely, the diluent), the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram, wherein the system adaptability chromatogram is shown in figure 7. The result shows that the separation degree of the empagliflozin intermediate A and related substances is not less than 1.5, and the detection results of the empagliflozin intermediate A and the related substances are basically stable.
Example 6
(1) Chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: chromatography column using pentafluorosilanyl bonded silica gel as filler (ACE 3C18-PFP, 150X 4.6mm, 3 μm)
Flow rate: 1.0ml/min column temperature: 30 deg.C
Detection wavelength: sample size at 220 nm: 5 μ l
Mobile phase A: phosphate buffer solution, 0.01mol/L (NH)4)2HPO4Solution, pH value adjusted to 6.5 with phosphoric acid
Mobile phase B: acetonitrile-methanol (70:30V/V)
Diluent agent: acetonitrile-purified water (50:50V/V)
The gradient elution procedure is shown in Table 10.
(2) Solution preparation
The same as in example 2.
(3) Measurement of
And respectively injecting the blank solution (namely, the diluent), the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram, wherein the system adaptability chromatogram is shown in figure 8. The result shows that the separation degree of the empagliflozin intermediate A and related substances is not less than 1.5, and the detection results of the empagliflozin intermediate A and the related substances are basically stable.
Example 7
(1) Chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: chromatography column using octyl silane bonded silica gel as filler (Agilent ZORBAX RX-C8, 250X 4.6mm, 5 μm)
Flow rate: 1.0ml/min column temperature: 40 deg.C
Detection wavelength: sample size at 220 nm: 5 μ l
Mobile phase A: phosphate buffer solution, 0.01mol/L NH4H2PO4Adjusting the pH value of the solution to 4.5 by using phosphoric acid,
mobile phase B: acetonitrile-tetrahydrofuran (90:10V/V)
Diluent agent: methanol-purified water (50:50V/V)
The gradient elution procedure is shown in Table 11.
TABLE 11
(2) Solution preparation
The same as in example 2.
(3) Measurement of
And respectively injecting the blank solution (namely, the diluent), the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording a chromatogram, wherein the system adaptability chromatogram is shown in figure 9. The result shows that the separation degree of the empagliflozin intermediate A and related substances is not less than 1.5, and the detection results of the empagliflozin intermediate A and the related substances are basically stable.
Comparative example 1
In this experiment, the detection method of example 1 of the reference CN112986456A was repeated, and as a comparative experiment of the present application, the specific experimental conditions were as follows:
(1) chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector
A chromatographic column: column packed with octadecylsilane chemically bonded silica (Agilent ZORBAX Eclipse Plus C18, 100X 4.6mm, 3.5 μm)
Flow rate: 1.0ml/min column temperature: 30 deg.C
Detection wavelength: sample size at 225 nm: 10 μ l
Mobile phase A: 0.005mol/L ammonium acetate solution, and adjusting pH to 7.5 with ammonia water
Mobile phase B: acetonitrile
Diluent agent: dimethyl sulfoxide-acetonitrile (20:80V/V)
The gradient elution procedure is shown in table 12.
TABLE 12
(2) Solution preparation
Impurity reference stock solution: taking about 25mg of a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance respectively, precisely weighing, placing in 50ml volumetric flasks respectively, adding a diluent to dissolve and dilute to scale, and shaking uniformly to obtain the product.
System adaptation solution: precisely weighing about 50mg of the reference substance A of the empagliflozin intermediate, placing the reference substance A into a 50ml volumetric flask, precisely adding 1ml of the impurity reference substance stock solution into the 50ml volumetric flask, adding a diluent to dilute to a scale, and shaking up to obtain the empagliflozin intermediate.
Test solution: taking about 50mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding a diluent to dissolve and dilute to a scale, and shaking up.
(3) Measurement of
Respectively injecting blank solution (i.e. diluent), system adaptability solution and test solution into a high performance liquid chromatograph for detection, and recording chromatogram, wherein the system adaptability chromatogram is shown in figure 10.
The results in fig. 10 show that, by the detection method of the reference CN112986456A, the related substance a6 and the related substance A3 are not separated, and the empagliflozin intermediate a tails off, which interferes with the detection of the related substance a4, and the empagliflozin intermediate a and the related substance a1-a8 cannot be separated and detected effectively.
Comparative example 2
In this experiment, the detection method of example 1 of the comparative document CN106706768B was repeated, and as a comparative experiment of the present application, the specific experimental conditions are as follows:
(1) chromatographic conditions
Liquid chromatograph: agilent 1260 liquid chromatograph
A chromatographic column: column packed with octyl silane-bonded silica gel (Agilent ZORBAX Rx-C8, 250X 4.6mm, 5 μ M) or column packed with phenylsilane-bonded silica gel (general-M PH 250X 4.6mm, 5 μ M)
Flow rate: 1.0ml/min column temperature: 25 deg.C
Detection wavelength: 224nm sample size: 20 μ l
Mobile phase A: phosphoric acid water solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid,
mobile phase B: methanol-acetonitrile (50:50V/V)
Diluent agent: mobile phase
The gradient elution procedure is shown in Table 13.
(2) Solution preparation
Impurity reference stock solution: taking about 20mg of a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance respectively, precisely weighing, placing in 50ml volumetric flasks respectively, adding a diluent to dissolve and dilute to a scale, and shaking uniformly to obtain the product;
system adaptation solution: precisely weighing about 40mg of reference substance A of the empagliflozin intermediate, placing the reference substance A into a 50ml volumetric flask, precisely adding 1ml of the impurity reference substance storage solution into the 50ml volumetric flask respectively, adding a diluent to dilute to a scale, and shaking up to obtain the empagliflozin intermediate.
Test solution: taking about 40mg of the Engelliflozin intermediate A, precisely weighing, placing in a 50ml volumetric flask, adding a diluent to dissolve and dilute to a scale, and shaking up.
(3) Measurement of
The blank solution (i.e., diluent), the system-adapted solution and the test solution were injected into a high performance liquid chromatograph for detection, and chromatograms were recorded, and a system-adapted map using a C8 column is shown in fig. 11, and a system-adapted map using a phenyl column is shown in fig. 12.
The results of FIGS. 11 and 12 show that the blank baseline was noisy and the analysis time was long with the detection method of reference CN 106706768B; when a C8 column is used, the Engelliflozin intermediate A and the related substance A4, the related substance A2 and the related substance A3, and the related substance A1 and the related substance A5 are not separated; when a phenyl column is used, the Engelliflozin intermediate A and the related substance A4 are not separated, and the related substance A2 and the related substance A5 are not separated; neither of these methods is effective in isolating and detecting the empagliflozin intermediate a and its related substances a 1-A8.
While particular embodiments of the present invention have been described, it is to be understood that the invention is not limited to the precise embodiments described above, and that various changes and/or modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention.
Claims (10)
1. A high performance liquid chromatography detection method for an engagliflozin intermediate A and related substances A1-A8 thereof is disclosed, wherein the structural formula of the engagliflozin intermediate A and related substances A1-A8 is as follows:
the method of detection is characterized in that,
1) chromatographic conditions are as follows:
a chromatographic column which adopts octane silane bonded silica gel or pentafluoro-phenyl silane bonded silica gel as a filling agent; performing gradient elution by using a phosphoric acid aqueous solution or a phosphate buffer solution with the pH value of 2.5-6.5 as a mobile phase A and an acetonitrile-methanol solution or an acetonitrile-tetrahydrofuran solution as a mobile phase B;
2) a detection step:
and respectively injecting the blank solution, the system adaptability solution and the test solution into a high performance liquid chromatograph for detection, and recording the chromatogram.
2. The detection method according to claim 1, wherein the column length of the chromatographic column is 150 to 250mm, the particle size of the filler is 3 to 5 μm, and the inner diameter of the column is 4.6 mm; preferably selected from Thermo Accucore XL C8, 150mm × 4.6mm, 4 μm; phenomenex Kinetex F5, 250X 4.6mm, 5 μm; ACE 3C18-PFP, 150X 4.6mm, 3 μm; agilent ZORBAX RX-C8, 250X 4.6mm, 5 μm.
3. The detection method according to claim 1, wherein the mobile phase A is a phosphoric acid aqueous solution or a phosphate buffer solution with a pH value of 2.5-3.5, preferably a phosphoric acid aqueous solution with a pH value of 3; the phosphate is selected from potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate; the concentration of the phosphate buffer solution is 0.005-0.01 mol/L.
4. The detection method according to claim 1, wherein the volume ratio of acetonitrile to methanol or acetonitrile to tetrahydrofuran in the mobile phase B is 100: 0-70: 30; preferably the volume ratio of acetonitrile to methanol is 80:20, or, preferably, the volume ratio of acetonitrile to tetrahydrofuran is 90: 10.
5. The detection method according to claim 1, wherein the gradient elution is as follows:
preferably, the gradient elution is as follows:
6. the detection method according to claim 1, wherein the flow rate of the mobile phase is 0.8 to 1.2ml/min, preferably 1.0 ml/min; the temperature of the chromatographic column is 20-40 ℃, preferably 25-35 ℃, and more preferably 30 ℃; the detection wavelength is 210-230 nm, preferably 220 nm; the concentration of the test solution is 0.3-0.8 mg/ml, preferably 0.5 mg/ml; the injection volume is 5-10 mul.
7. The detection method according to claim 1, wherein the diluent is a mixed solution of an organic solvent and purified water; the volume ratio of the organic solvent to the purified water is 40: 60-60: 40, preferably the volume ratio of organic solvent to purified water is 50: 50.
8. the detection method according to claim 7, wherein the organic solvent is acetonitrile, methanol, or a mixed organic solvent of acetonitrile and methanol, wherein the volume ratio of acetonitrile to methanol in the mixed organic solvent is 30-50: 5-15, preferably 40: 10.
9. the assay of claim 1, wherein the step of assaying is preceded by a solution formulation:
blank solution: diluent
Impurity reference stock solution: respectively taking a related substance A1 reference substance, a related substance A2 reference substance, a related substance A3 reference substance, a related substance A4 reference substance, a related substance A5 reference substance, a related substance A6 reference substance, a related substance A7 reference substance and a related substance A8 reference substance, dissolving with acetonitrile, and preparing into impurity reference substance stock solutions with a diluent;
system adaptation solution: taking an reference substance A of the empagliflozin intermediate and the stock solutions of all impurity reference substances, dissolving the reference substance A and the stock solutions with acetonitrile, and preparing a system adaptive solution by using a diluent;
test solution: taking the Engelliflozin intermediate A, dissolving the Engelliflozin intermediate A with acetonitrile, and preparing a test solution by using a diluent.
10. The detection method according to claim 1, wherein the purity of the empagliflozin intermediate A and the content of the related substance A1-A8 thereof are calculated according to a peak area normalization method; the separation degree between the Engelliflozin intermediate A and the related substances A1-A8 is more than 1.5; the quantitative limit of the Engelliflozin intermediate A and the related substances A1-A8 is 0.03%; the detection limit of the Engelliflozin intermediate A and the related substances A1-A8 is 0.01 percent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114280181A (en) * | 2021-12-23 | 2022-04-05 | 浙江海翔川南药业有限公司 | Detection method of rosuvastatin intermediate and related substances thereof |
| CN115015439A (en) * | 2022-06-30 | 2022-09-06 | 瑞玞生物医学(深圳)有限公司 | Separation detection method for antioxidant in composite solution |
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| CN115407001A (en) * | 2022-11-03 | 2022-11-29 | 山东省食品药品检验研究院 | Analysis method of related substances of dapagliflozin |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170319539A1 (en) * | 2014-10-01 | 2017-11-09 | Mylan Laboratories Ltd. | Amorphous Empagliflozin |
| CN109374782A (en) * | 2018-12-21 | 2019-02-22 | 安徽联创生物医药股份有限公司 | A method of with the related substance of the net bulk pharmaceutical chemicals of HPLC separation determination Yi Palie |
| CN111398457A (en) * | 2020-04-01 | 2020-07-10 | 上海应用技术大学 | Method for detecting substances related to Eagliflozin influence factors under degradation condition |
| CN111983054A (en) * | 2020-07-28 | 2020-11-24 | 安徽联创生物医药股份有限公司 | Method for separating and measuring related substances of empagliflozin intermediate by using HPLC (high performance liquid chromatography) |
-
2021
- 2021-12-27 CN CN202111615625.5A patent/CN114264747A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170319539A1 (en) * | 2014-10-01 | 2017-11-09 | Mylan Laboratories Ltd. | Amorphous Empagliflozin |
| CN109374782A (en) * | 2018-12-21 | 2019-02-22 | 安徽联创生物医药股份有限公司 | A method of with the related substance of the net bulk pharmaceutical chemicals of HPLC separation determination Yi Palie |
| CN111398457A (en) * | 2020-04-01 | 2020-07-10 | 上海应用技术大学 | Method for detecting substances related to Eagliflozin influence factors under degradation condition |
| CN111983054A (en) * | 2020-07-28 | 2020-11-24 | 安徽联创生物医药股份有限公司 | Method for separating and measuring related substances of empagliflozin intermediate by using HPLC (high performance liquid chromatography) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114280181A (en) * | 2021-12-23 | 2022-04-05 | 浙江海翔川南药业有限公司 | Detection method of rosuvastatin intermediate and related substances thereof |
| CN115015439A (en) * | 2022-06-30 | 2022-09-06 | 瑞玞生物医学(深圳)有限公司 | Separation detection method for antioxidant in composite solution |
| CN115015439B (en) * | 2022-06-30 | 2023-11-03 | 瑞玞生物医学(深圳)有限公司 | Separation detection method for antioxidant in composite solution |
| CN115219630A (en) * | 2022-07-25 | 2022-10-21 | 广西壮族自治区食品药品检验所 | Method for determining related substances of berberine trimethoprim capsule |
| CN115219630B (en) * | 2022-07-25 | 2023-11-17 | 广西壮族自治区食品药品检验所 | Method for measuring related substances of berberine trimethoprim capsule |
| CN115407001A (en) * | 2022-11-03 | 2022-11-29 | 山东省食品药品检验研究院 | Analysis method of related substances of dapagliflozin |
| CN117147736A (en) * | 2023-10-27 | 2023-12-01 | 山东百诺医药股份有限公司 | Detection method of D-glucose-delta-lactone related substance |
| CN117147736B (en) * | 2023-10-27 | 2024-01-02 | 山东百诺医药股份有限公司 | Detection method of D-glucose-delta-lactone related substance |
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