CN112986456A - HPLC analysis detection method of empagliflozin intermediate - Google Patents
HPLC analysis detection method of empagliflozin intermediate Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 29
- OBWASQILIWPZMG-QZMOQZSNSA-N empagliflozin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=CC=C(Cl)C(CC=2C=CC(O[C@@H]3COCC3)=CC=2)=C1 OBWASQILIWPZMG-QZMOQZSNSA-N 0.000 title claims description 23
- 229960003345 empagliflozin Drugs 0.000 title claims description 23
- 238000004128 high performance liquid chromatography Methods 0.000 title abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000010828 elution Methods 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 15
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 57
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- 239000012085 test solution Substances 0.000 claims description 16
- 239000003085 diluting agent Substances 0.000 claims description 14
- 239000012046 mixed solvent Substances 0.000 claims description 10
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 9
- 239000005695 Ammonium acetate Substances 0.000 claims description 9
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 235000019257 ammonium acetate Nutrition 0.000 claims description 9
- 229940043376 ammonium acetate Drugs 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- 239000007975 buffered saline Substances 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 2
- 239000000337 buffer salt Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 239000012071 phase Substances 0.000 abstract description 28
- 239000012535 impurity Substances 0.000 abstract description 21
- 239000000126 substance Substances 0.000 abstract description 17
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000006227 byproduct Substances 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 238000003908 quality control method Methods 0.000 abstract description 3
- 239000000872 buffer Substances 0.000 abstract description 2
- 239000012074 organic phase Substances 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 239000000543 intermediate Substances 0.000 description 43
- 238000000926 separation method Methods 0.000 description 10
- 230000014759 maintenance of location Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 102000000070 Sodium-Glucose Transport Proteins Human genes 0.000 description 3
- 108010080361 Sodium-Glucose Transport Proteins Proteins 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- -1 4-chloro-3- (4- (((S) -tetrahydrofuran-3-yl) oxy) benzyl) phenyl Chemical group 0.000 description 2
- DCERHCFNWRGHLK-UHFFFAOYSA-N C[Si](C)C Chemical compound C[Si](C)C DCERHCFNWRGHLK-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000012801 analytical assay Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- VQUPQWGKORWZII-WDPYGAQVSA-N (2r,3r)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2s,3r,4r,5r,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy-2,3-dihydrochromen-4-one Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1C(=O)C2=C(O)C=C(O)C=C2O[C@@H]1C1=CC=C(O)C=C1 VQUPQWGKORWZII-WDPYGAQVSA-N 0.000 description 1
- JYZBRIZBJDEAIX-INIZCTEOSA-N (3S)-3-[4-[(2-chlorophenyl)methyl]phenoxy]oxolane Chemical compound C1COC[C@H]1OC2=CC=C(C=C2)CC3=CC=CC=C3Cl JYZBRIZBJDEAIX-INIZCTEOSA-N 0.000 description 1
- YLUHNGIWRCCQMQ-INIZCTEOSA-N (3s)-3-[4-[(2-chloro-5-iodophenyl)methyl]phenoxy]oxolane Chemical compound ClC1=CC=C(I)C=C1CC(C=C1)=CC=C1O[C@@H]1COCC1 YLUHNGIWRCCQMQ-INIZCTEOSA-N 0.000 description 1
- SSWRXTPXZOULOF-INIZCTEOSA-N 4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenol Chemical compound C1COC[C@H]1OC2=CC=C(C=C2)CC3=C(C=CC(=C3)O)Cl SSWRXTPXZOULOF-INIZCTEOSA-N 0.000 description 1
- VQUPQWGKORWZII-KTLFEHCLSA-N Dihydrokaempferol-3-O-alpha-L-rhamnopyranoside Natural products O([C@@H]1[C@H](c2ccc(O)cc2)Oc2c(c(O)cc(O)c2)C1=O)[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O1 VQUPQWGKORWZII-KTLFEHCLSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- VQUPQWGKORWZII-UHFFFAOYSA-N Neoisoengelitin Natural products OC1C(O)C(O)C(C)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C=C1 VQUPQWGKORWZII-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- CELWCAITJAEQNL-UHFFFAOYSA-N oxan-2-ol Chemical compound OC1CCCCO1 CELWCAITJAEQNL-UHFFFAOYSA-N 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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Abstract
The invention discloses an HPLC (high performance liquid chromatography) analytical detection method of an engagliflozin intermediate, which uses a high performance liquid system, takes octadecylsilane chemically bonded silica as a chromatographic column, and takes a buffer salt-organic phase as a mobile phase for gradient elution, so that the engagliflozin intermediate and related substances thereof can be quantitatively measured, effective monitoring data is provided for the preparation process of the engagliflozin, the generation of by-products and impurities in the reaction is reduced, and the quality control of the engagliflozin is realized; through perfect methodology verification, the method is proved to have strong specificity, high accuracy and good stability.
Description
Technical Field
The application relates to the technical field of drug analysis, in particular to an HPLC (high performance liquid chromatography) analysis and detection method of an empagliflozin intermediate.
Background
Engagliflozin is a selective sodium-glucose cotransporter (SGLT) inhibitor useful for treating adult type II diabetes. The engeletin limits the reabsorption of most glucose in vivo through the selective inhibition effect on the sodium-glucose cotransporter, and promotes the massive excretion of glucose from urine to achieve the aim of controlling the blood sugar level.
In the process of synthesizing the empagliflozin, the purity of some key intermediates needs to be controlled so as to reduce the occurrence of side reactions and the generation of impurities, and then the yield and the purity of the empagliflozin are improved.
The establishment of an effective separation method for the empagliflozin intermediate has important significance for the preparation of the empagliflozin product.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present application.
The synthesis of engagliflozin involves the following intermediate compounds: the chemical name of the compound is (2S,3R,4S,5R,6R) -2- (4-chloro-3- (4- (((S) -tetrahydrofuran-3-yl) oxy) benzyl) phenyl) -3,4, 5-tri ((trimethyl silicon) oxy) -6- (((trimethyl silicon) oxy) methyl) tetrahydro-2H-pyran-2-ol, the structure of which is shown as the following formula (I),
TMS in the formula (I) is trimethylsilyl.
There are 4 related substances (i.e. impurities) used and producible in the process of synthesizing the compound, which are respectively SM1, M1Z1, M1Z2 and M1Z3, and the structural formulas are respectively:
SM1
(S) -3- (4- (2-chloro-5-iodobenzyl) phenoxy) tetrahydrofuran
M1Z1
(S) -3- (4- ((4,4 ' -dichloro-3 ' - (4- ((R) -tetrahydrofuran-3-yl) oxy) benzyl) - [1,1 ' -biphenyl ] -3-yl) methyl) phenoxy) tetrahydrofuran
M1Z2
(S) -3- (4- (2-chlorobenzyl) phenoxy) tetrahydrofuran
M1Z3
(S) -4-chloro-3- (4- ((tetrahydrofuran-3-yl) oxy) benzyl) phenol.
The method can quantitatively determine the engagliflozin intermediate and related substances thereof, provides effective monitoring data for the preparation process of the engagliflozin, reduces the generation of byproducts and impurities in the reaction, and realizes the quality control of the engagliflozin.
The invention provides an analytical detection method of an empagliflozin intermediate, which comprises the following steps:
(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is one or more mixed solvents selected from acetonitrile, dimethyl sulfoxide, tetrahydrofuran and methanol;
(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; performing gradient elution by adopting a mobile phase A and a mobile phase B as mobile phases, wherein the mobile phase A is a buffered saline solution, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;
(3) and (3) determination: and (2) injecting 5-50 mul of the test solution obtained in the step (1) into a high performance liquid chromatograph, and recording a chromatogram.
In the above analytical measurement method, the engletin intermediate is (2S,3R,4S,5R,6R) -2- (4-chloro-3- (4- (((S) -tetrahydrofuran-3-yl) oxy) benzyl) phenyl) -3,4, 5-tris ((trimethylsilyl) oxy) -6- (((trimethylsilyl) oxy) methyl) tetrahydro-2H-pyran-2-ol, and the structure thereof is represented by the following formula (I),
in the above analytical measurement method, the diluent is one or a mixed solvent of two or more selected from acetonitrile, dimethyl sulfoxide, tetrahydrofuran and methanol.
In the above analytical determination method, the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile, optionally, the volume ratio of dimethyl sulfoxide to acetonitrile in the mixed solvent is (10-30): (70-90), for example, 20: 80.
In the above analytical assay method, the column may be selected from the group consisting of Agilent, Welch brand, and others, preferably Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um.
In the above analytical determination method, the buffer salt is selected from one of acetate, phosphate and formate, preferably acetate. The salt may be a sodium, potassium or ammonium salt.
In the above analytical measurement method, the concentration of the buffered saline solution is 4mmol/L to 6 mmol/L; optionally, the pH of the buffered saline solution is 7.3-9.0.
In the above analytical determination method, the buffered saline solution was an ammonium acetate buffer solution having a concentration of 5mmol/L, and the pH was adjusted to 7.5 with aqueous ammonia.
In the above analytical measurement method, the procedure of the gradient elution is:
in the above analytical measurement method, the high performance liquid chromatograph is an Agilent 1260 liquid chromatograph;
a chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um);
the detection wavelength is 225 nm;
the flow rate is 1.0 ml/min;
column temperature: 30 ℃;
sample introduction volume: 10 μ l.
In the above analytical determination method, the mobile phase A was 5mmol/L ammonium acetate buffer solution (pH adjusted to 7.5 with ammonia water); the mobile phase B is acetonitrile; the gradient elution procedure was:
in the above analytical assay method, the preparing a test solution includes: taking appropriate amount of the engagliflozin intermediate, SM1, M1Z1, M1Z2 and M1Z3, adding a diluent to dissolve and quantitatively dilute to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 mu g of SM1, about 20 mu g M1Z1, about 20 mu g M1Z2 and about 20 mu g M1Z3 in each 1ml of solution as a test solution.
In one of the above analytical detection methods, the analytical detection method includes the steps of:
(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80;
(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; the mobile phase adopts a mobile phase A and a mobile phase B to carry out gradient elution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;
here, the mobile phase a was an ammonium acetate buffer solution having a concentration of 5mmol/L and adjusted to pH 7.5 with ammonia water;
the procedure for the gradient elution was:
(3) and (3) determination: and (2) taking 10 mu l of the test solution obtained in the step (1), injecting into a high performance liquid chromatograph, and recording a chromatogram.
The method uses octadecylsilane chemically bonded silica as a chromatographic column and uses a buffer salt-organic phase as a mobile phase for gradient elution, so that the method can quantitatively determine the engagliflozin intermediate and related substances, provide effective monitoring data for the preparation process of the engagliflozin, reduce the generation of byproducts and impurities in the reaction, and realize the quality control of the engagliflozin. Through perfect methodology verification, the method is proved to have strong specificity, high accuracy and good stability.
The methodology of the analytical determination method provided by the invention is verified, and the results are as follows:
additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.
Drawings
The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not to limit the disclosure.
FIG. 1 is an HPLC plot of the Engelliflozin intermediate and related substances from example 1;
FIG. 2 is an HPLC plot of the Engelliflozin intermediate and related substances from example 2;
FIG. 3 is an HPLC plot of the Engelliflozin intermediate and related substances at example 3;
fig. 4 is an HPLC profile of the engagliflozin intermediate and related substances in example 4.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.
The present invention will be explained in more detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit and scope of the present invention.
Test instrument for experiments
High performance liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector.
Example 1
Chromatographic conditions
A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)
The detection wavelength is 225nm
The flow rate was 1.0ml/min
Column temperature: 30 deg.C
Sample introduction volume: 10 μ l
Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;
experimental procedure
Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.
The result is shown in figure 1, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and degrees of separation of the empagliflozin intermediate and related materials in figure 1 are shown in the following table:
| name (R) | Retention time | Degree of separation |
| M1Z3 | 4.203 | 14.782 |
| M1Z2 | 7.586 | 11.245 |
| SM1 | 10.309 | 21.673 |
| M1Z1 | 14.979 | 6.384 |
| Empagliflozin intermediate 1 | 25.245 | 4.660 |
The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:
| YPLJ-M1Z2 | 0.09％ |
| YPLJ-SM1 | 0.149％ |
| unknown impurity (RRT:0.68min) | 0.183％ |
| Unknown impurity (RRT:0.71min) | 0.369％ |
| Unknown impurity (RRT:0.92min) | 1.192％ |
| Total miscellaneous% | 1.982％ |
| Number of impurities | 5 |
Example 2
Chromatographic conditions
A chromatographic column: c18(Welch Ultimate C184.6X 100mm, 3 μm)
The detection wavelength is 225nm
The flow rate was 1.0ml/min
Column temperature: 30 deg.C
Sample introduction volume: 10 μ l
Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;
experimental procedure
Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.
The result is shown in figure 2, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in figure 2 are shown in the following table:
| name (R) | Retention time | Degree of separation |
| M1Z3 | 4.364 | / |
| M1Z2 | 7.931 | 23.363 |
| SM1 | 10.739 | 16.988 |
| M1Z1 | 15.438 | 29.219 |
| Empagliflozin intermediate 1 | 26.724 | 4.900 |
The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:
| YPLJ-M1Z2 | 0.105％ |
| YPLJ-SM1 | 0.184％ |
| unknown impurity (RRT:0.68min) | 0.175％ |
| Unknown impurity (RRT:0.71min) | 0.376％ |
| Unknown impurity (RRT:0.92min) | 1.261％ |
| Total miscellaneous% | 2.102％ |
| Number of impurities | 5 |
Example 3
Chromatographic conditions
A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)
The detection wavelength is 225nm
The flow rate was 1.0ml/min
Column temperature: 30 deg.C
Sample introduction volume: 10 μ l
Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.3 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;
experimental procedure
Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.
The result is shown in figure 3, and the effective separation of the empagliflozin intermediate and related substances can be realized under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in figure 3 are shown in the following table:
| name (R) | Retention time | Degree of separation |
| M1Z3 | 4.081 | / |
| M1Z2 | 7.399 | 16.072 |
| SM1 | 10.067 | 12.363 |
| M1Z1 | 14.682 | 23.931 |
| Empagliflozin intermediate 1 | 25.107 | 3.420 |
The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:
| YPLJ-M1Z2 | 0.091％ |
| YPLJ-SM1 | 0.163％ |
| unknown impurity (RRT:0.68min) | 0.219％ |
| Unknown impurity (RRT:0.71min) | 0.373％ |
| Unknown impurity (RRT:0.92min) | 1.412％ |
| Total miscellaneous% | 2.257％ |
| Number of impurities | 5 |
Example 4
Chromatographic conditions
A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)
The detection wavelength is 225nm
The flow rate was 1.0ml/min
Column temperature: 35 deg.C
Sample introduction volume: 10 μ l
Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;
experimental procedure
Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.
The result is shown in figure 4, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in fig. 4 are shown in the following table:
| name (R) | Retention time | Degree of separation |
| M1Z3 | 4.267 | / |
| M1Z2 | 7.744 | 21.325 |
| SM1 | 10.468 | 15.583 |
| M1Z1 | 15.109 | 27.106 |
| Empagliflozin intermediate 1 | 23.446 | 4.225 |
The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:
| YPLJ-M1Z2 | 0.088％ |
| YPLJ-SM1 | 0.151％ |
| unknown impurity (RRT:0.68min) | 0.191％ |
| Unknown impurity (RRT:0.71min) | 0.385％ |
| Unknown impurity (RRT:0.92min) | 1.793％ |
| Total miscellaneous% | 2.757％ |
| Number of impurities | 5 |
The present application describes embodiments, but the description is illustrative rather than limiting and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein.
Claims (9)
1. An analytical detection method of an empagliflozin intermediate comprises the following steps:
(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution;
(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; performing gradient elution by adopting a mobile phase A and a mobile phase B as mobile phases, wherein the mobile phase A is a buffered saline solution, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;
(3) and (3) determination: and (2) injecting 5-50 mul of the test solution obtained in the step (1) into a high performance liquid chromatograph, and recording a chromatogram.
2. The analytical detection method according to claim 1, wherein the diluent is one or a mixed solvent of two or more selected from acetonitrile, dimethylsulfoxide, tetrahydrofuran and methanol;
optionally, the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile, and optionally, the volume ratio of the dimethyl sulfoxide to the acetonitrile in the mixed solvent is (10-30) to (70-90).
3. The analytical detection method according to claim 1, wherein the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80.
4. The assay of claim 1, wherein the buffer salt is selected from one of acetate, phosphate and formate, optionally acetate;
optionally, the concentration of the buffered saline solution is 4mmol/L to 6 mmol/L; optionally, the pH of the buffered saline solution is 7.3-9.0.
5. The analytical detection method according to claim 4, wherein the buffered saline solution is an ammonium acetate buffer solution having a concentration of 5mmol/L and the pH is adjusted to 7.5 with aqueous ammonia.
6. The analytical detection method according to any one of claims 1 to 5, wherein the gradient elution is performed by:
7. the analytical detection method according to any one of claims 1 to 5, wherein the high performance liquid chromatograph is an Agilent 1260 liquid chromatograph;
a chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um);
the detection wavelength is 225 nm;
the flow rate is 1.0 ml/min;
column temperature: 30 ℃;
sample introduction volume: 10 μ l.
8. The assay detection method of any one of claims 1 to 5, wherein said preparing a test solution comprises: taking appropriate amount of the engagliflozin intermediate, SM1, M1Z1, M1Z2 and M1Z3, adding a diluent to dissolve and quantitatively dilute to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 mu g of SM1, about 20 mu g M1Z1, about 20 mu g M1Z2 and about 20 mu g M1Z3 in each 1ml of solution as a test solution.
9. An analytical detection method of an empagliflozin intermediate, comprising the following steps:
(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80;
(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; the mobile phase adopts a mobile phase A and a mobile phase B to carry out gradient elution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;
here, the mobile phase a was an ammonium acetate buffer solution having a concentration of 5mmol/L and adjusted to pH 7.5 with ammonia water;
the procedure for the gradient elution was:
(3) and (3) determination: and (2) taking 10 mu l of the test solution obtained in the step (1), injecting into a high performance liquid chromatograph, and recording a chromatogram.
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| CN106706769A (en) * | 2015-11-17 | 2017-05-24 | 重庆医药工业研究院有限责任公司 | Separation and determination method of empagliflozin and optical isomers thereof |
| CN106706768A (en) * | 2015-11-17 | 2017-05-24 | 重庆医药工业研究院有限责任公司 | Method for measuring Jardiance and related substances of Jardiance through separation |
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| CN106706769A (en) * | 2015-11-17 | 2017-05-24 | 重庆医药工业研究院有限责任公司 | Separation and determination method of empagliflozin and optical isomers thereof |
| CN106706768A (en) * | 2015-11-17 | 2017-05-24 | 重庆医药工业研究院有限责任公司 | Method for measuring Jardiance and related substances of Jardiance through separation |
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