CN113960202A - Detection method of 4-dimethylaminopyridine in dapagliflozin intermediate - Google Patents
Detection method of 4-dimethylaminopyridine in dapagliflozin intermediate Download PDFInfo
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- CN113960202A CN113960202A CN202111233205.0A CN202111233205A CN113960202A CN 113960202 A CN113960202 A CN 113960202A CN 202111233205 A CN202111233205 A CN 202111233205A CN 113960202 A CN113960202 A CN 113960202A
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- HYAFETHFCAUJAY-UHFFFAOYSA-N pioglitazone Chemical compound N1=CC(CC)=CC=C1CCOC(C=C1)=CC=C1CC1C(=O)NC(=O)S1 HYAFETHFCAUJAY-UHFFFAOYSA-N 0.000 description 2
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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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- 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
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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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- G01N30/28—Control of physical parameters of the fluid carrier
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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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- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention provides a method for detecting 4-dimethylaminopyridine in a dapagliflozin intermediate, and relates to the technical field of analytical chemistry. The detection method provided by the invention has higher sensitivity and durability, can effectively detect whether the dapagliflozin intermediate contains residual 4-dimethylaminopyridine and the content thereof, effectively controls the quality of a dapagliflozin product and improves the medication safety of medicines.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, and particularly relates to a detection method of 4-dimethylaminopyridine in a dapagliflozin intermediate.
Background
Dapagliflozin is a sodium-glucose cotransporter 2(SGLT2) selective inhibitor. SGLT2 was selectively expressed in the first segment of the proximal tubule of the glomerulus (S1), regulating the reabsorption of most (90%) of the permeate glucose. Inhibition of SGLT2 by dapagliflozin results in the excretion of excess glucose through the urine, thereby improving glycemic control without increasing insulin secretion. In vitro studies have shown that dapagliflozin is effective in selectively inhibiting human (h) SGLT2 relative to closely related selective 1242-1600-folded hSGLT 1. Dapagliflozin has also been shown to selectively inhibit SGLT2 in mice (m) and dogs (d) relative to mGLT1 and dSGLT 1. Clinical research results show that the dapagliflozin used alone or in combination with the drugs such as metformin, pioglitazone, glimepiride and insulin can significantly reduce HbA1c and fasting blood glucose of type 2 diabetes patients, the incidence rate of adverse reactions is similar to that of placebo, and the risk of hypoglycemia is low.
4-Dimethylaminopyridine (DMAP) is a catalyst used in the dapagliflozin synthesis process, is a potential genotoxic impurity, and is reported in the literature, DMAP is toxic when being taken orally or contacted with skin and has irritation to eyes, respiratory system and skin, and in order to better control the quality of dapagliflozin, a measurement method is needed to detect whether 4-dimethylaminopyridine is contained in dapagliflozin, so that the safety of medication is ensured, but no relevant report of a 4-dimethylaminopyridine detection method in a dapagliflozin intermediate is found at present.
Disclosure of Invention
The invention aims to provide a method for detecting 4-dimethylaminopyridine in a dapagliflozin intermediate, which can effectively detect whether the dapagliflozin intermediate contains residual 4-dimethylaminopyridine and the content of the residual 4-dimethylaminopyridine, effectively control the quality of a dapagliflozin product and improve the medication safety of a medicine.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting 4-dimethylaminopyridine in a dapagliflozin intermediate, which comprises the following steps:
dissolving a 4-dimethylaminopyridine reference substance in a water-acetonitrile solution to obtain a reference substance solution;
dissolving the dapagliflozin intermediate to be detected in a water-acetonitrile solution to obtain a test sample solution;
respectively carrying out high performance liquid chromatography detection on the reference solution and the test solution to obtain the peak area of 4-dimethylaminopyridine in the 4-dimethylaminopyridine reference substance and the peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
calculating by adopting a method shown in a formula I to obtain the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
in the formula I, FFlat plateThe calculation method of (A) is shown as a formula II;
Afor supplying toThe peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is shown;
Wfor supplying toThe weight of dapagliflozin intermediate to be tested, mg;
the unit of the content of 4-dimethylaminopyridine is ppm;
in formula II, n is the sample injection frequency of the reference substance, FiThe calculation method is shown in formula III as the correction factor;
in the formula III, WTo iWeight of the ith 4-dimethylaminopyridine control, mg;
Pto pair% pure 4-dimethylaminopyridine controlDegree;
Ato iThe peak area of the ith 4-dimethylaminopyridine control.
Preferably, the volume ratio of water to acetonitrile in the water-acetonitrile solution is 1: 9.
Preferably, the conditions for the high performance liquid chromatography detection include:
column temperature: 25-35 ℃;
mobile phase: the mobile phase A is a phosphoric acid aqueous solution with the mass concentration of 0.16-0.24%, and the mobile phase B is acetonitrile;
gradient elution;
flow rate: 0.9 to 1.1 mL/min-1；
Detection wavelength: 280 nm.
Preferably, the conditions of the gradient elution are
Preferably, the concentration of the control solution is 1.4-1.6 mug/mL; the concentration of the test solution is 9-11 mg/mL.
Preferably, the injection volume of the reference solution and the sample solution is 10 μ L when the high performance liquid chromatography is performed.
Preferably, the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is 0.0045-1.5 mu g/mL.
Preferably, the detection limit concentration of the high performance liquid chromatography is 0.0045 mu g/mL.
Preferably, the limit concentration of the high performance liquid chromatography detection is 0.015 mu g/mL.
The invention provides a method for detecting 4-dimethylaminopyridine in a dapagliflozin intermediate, which has higher sensitivity and durability, can effectively detect whether the dapagliflozin intermediate contains residual 4-dimethylaminopyridine and the content of the residual 4-dimethylaminopyridine, can effectively control the quality of a dapagliflozin product and improves the medication safety of medicines.
Drawings
FIG. 1 is a typical HPLC chromatogram of a solvent in the specificity verification example;
FIG. 2 is a typical HPLC chromatogram of a control solution in the specificity verification example;
FIG. 3 is a typical HPLC chromatogram of a sample solution in the specificity verification example;
FIG. 4 is a typical HPLC chromatogram of a sample-spiking solution in the specificity verification example;
FIG. 5 is a typical HPLC chromatogram of a limiting solution;
FIG. 6 is a typical HPLC chromatogram of a detection limit solution;
FIG. 7 is a typical HPLC chromatogram of the UV absorption maximum of a control solution.
Detailed Description
The invention provides a method for detecting 4-dimethylaminopyridine in a dapagliflozin intermediate, which comprises the following steps:
dissolving a 4-dimethylaminopyridine reference substance in a water-acetonitrile solution to obtain a reference substance solution;
dissolving the dapagliflozin intermediate to be detected in a water-acetonitrile solution to obtain a test sample solution;
respectively carrying out high performance liquid chromatography detection on the reference solution and the test solution to obtain the peak area of 4-dimethylaminopyridine in the 4-dimethylaminopyridine reference substance and the peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
calculating by adopting a method shown in a formula I to obtain the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
in the formula I, FFlat plateThe calculation method of (A) is shown as a formula II;
Afor supplying toThe peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is shown;
Wfor supplying toThe weight of dapagliflozin intermediate to be tested, mg;
the unit of the content of 4-dimethylaminopyridine is ppm;
in the formula II, FiThe calculation method is shown in formula III as the correction factor;
in the formula III, WTo iWeight of the ith 4-dimethylaminopyridine control, mg;
Pto pair% purity of 4-dimethylaminopyridine control;
Ato iThe peak area of the ith 4-dimethylaminopyridine control.
In the invention, the dilution multiple of the test sample refers to the total dilution multiple of a test sample solution for high performance liquid chromatography detection, which is prepared from a 4-dimethylaminopyridine reference substance; the reference substance dilution factor refers to the total dilution factor of a reference substance solution for high performance liquid chromatography detection, which is prepared from a 4-dimethylaminopyridine reference substance.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
The invention dissolves 4-dimethylaminopyridine reference substance in water-acetonitrile solution to obtain reference substance solution. In the present invention, the purity of the 4-dimethylaminopyridine reference is preferably 95 wt% or more. In the present invention, the volume ratio of water to acetonitrile in the water-acetonitrile solution is preferably 1: 9. The method adopts the water-acetonitrile solution to completely dissolve the 4-dimethylaminopyridine.
In the invention, the concentration of the control solution is preferably 1.4-1.6 mug/mL, and more preferably 1.5 mug/mL.
The method comprises the step of dissolving a dapagliflozin intermediate to be detected in a water-acetonitrile solution to obtain a test sample solution. In the invention, the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is preferably 0.0045-1.5 mu g/mL. In the present invention, the volume ratio of water to acetonitrile in the water-acetonitrile solution is preferably 1: 9.
In the invention, the concentration of the test solution is preferably 9-11 mg/mL, and more preferably 10 mg/mL.
After a reference solution and a test solution are obtained, the high performance liquid chromatography detection is respectively carried out on the reference solution and the test solution to obtain the peak area of 4-dimethylaminopyridine in the 4-dimethylaminopyridine reference and the peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected.
In the present invention, the injection volumes of the control solution and the test solution are preferably 10 μ L.
In the present invention, the conditions for the high performance liquid chromatography detection preferably include:
column temperature: 25-35 ℃, more preferably 30 ℃;
mobile phase: the mobile phase A is a phosphoric acid aqueous solution with the mass concentration of 0.16-0.24%, and is more preferably 0.2%; the mobile phase B is acetonitrile; gradient elution;
flow rate: 0.9 to 1.1 mL/min-1More preferably 1.0 mL/min-1；
Detection wavelength: 280 nm.
In the present invention, the conditions of the gradient elution are shown in table 1:
TABLE 1 conditions of gradient elution
According to the method, the impurities in the dapagliflozin intermediate and the dapagliflozin intermediate can be eluted by adopting gradient elution and selection of a mobile phase, so that the interference of the rest impurities on the determination of the 4-dimethylaminopyridine is avoided, and the specificity of the method is ensured.
In the invention, the chromatographic column for high performance liquid chromatography detection preferably comprises an octadecylsilane chemically bonded silica reverse phase chromatographic column, and particularly preferably contains caochlorin CAPCELL PAK C18; the inner diameter of the chromatographic column is preferably 4.6mm, the length of the chromatographic column is preferably 250mm, and the pore diameter of sieve plates at two ends of the chromatographic column is preferably 5 mu m.
In the present invention, the detector for high performance liquid chromatography detection is preferably an ultraviolet detector. In the present invention, a typical HPLC chromatogram of the ultraviolet maximum absorption of the control solution is shown in FIG. 7, and from FIG. 7, the detection wavelength of the HPLC detection can be determined to be 280 nm.
After the peak area of 4-dimethylaminopyridine in the 4-dimethylaminopyridine reference substance and the peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected are obtained, the content of the 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is calculated by adopting the method shown in the formula I.
In the specific embodiment of the invention, the detection limit concentration of the high performance liquid chromatography is 0.0045 mu g/mL, and the quantification limit concentration is 0.015 mu g/mL.
The invention proves the sensitivity and durability of the detection method through methodology verification, can effectively detect whether the dapagliflozin intermediate contains residual 4-dimethylaminopyridine and the content thereof, effectively controls the quality of dapagliflozin products through the method, and improves the medication safety of medicines.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials, reagents, instruments and HPLC chromatographic conditions used in the following examples and verification examples were:
1. instrument and HPLC chromatographic conditions:
thermo Ultimate 3000 high performance liquid chromatograph (quaternary pump, degasser unit, diode array detector, column oven, autosampler, system monitor and Chromeleon7 workstation) (Thermo Fisher, usa); MSA6.6S-0CE type millionths of a day (Sartorius, Germany); one hundred thousand analytical balances (METTLER TOLEDO, USA) type AB 265-S.
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica gel as filler (4.6 mm × 250mm, 5 μm in major Cao CAPCELL PAK C18) for chromatographic column; the mobile phase A is phosphoric acid aqueous solution with the mass concentration of 0.2%, the mobile phase B is acetonitrile, and gradient elution is carried out according to the table 1; the flow rate was 1.0mL per minute; an ultraviolet detector with the detection wavelength of 280 nm; the column temperature is 30 ℃; the injection volume is 10. mu.L.
TABLE 1 conditions of gradient elution
2. Material
4-dimethylaminopyridine control (source: aladine, batch number: F2019075); dapagliflozin intermediate (available from Shandong Shanglu anti-medicine, Inc., lot numbers 2008001, 20170402, 20170403, and 20170404); acetonitrile (source: ThermoFisher, batch No. 20025052); phosphoric acid (source: Michael, lot: C11206512).
Example 1
Preparation of a reference solution: an appropriate amount of 4-dimethylaminopyridine control was precisely weighed, dissolved in a water-acetonitrile solution (the volume ratio of water to acetonitrile was 1:9), and diluted to a solution containing 1.5. mu.g of 4-dimethylaminopyridine control per 1mL as a control solution.
Preparing a test solution: taking a proper amount of dapagliflozin intermediate, precisely weighing, adding water-acetonitrile solution (the volume ratio of water to acetonitrile is 1:9) to dissolve, and quantitatively diluting to prepare solution containing 10mg of dapagliflozin intermediate per 1mL as test solution.
Respectively and precisely measuring 10 mu L of each solution, injecting the solution into a high performance liquid chromatograph, recording the peak area, and calculating the content of 4-dimethylaminopyridine in the dapagliflozin intermediate by adopting a method shown in a formula I, wherein the result is shown in a table 2.
TABLE 2 determination of 4-dimethylaminopyridine in dapagliflozin intermediates
| Sample batch number | Content (wt.) |
| 2008001 | Not detected out |
| 20170402 | Not detected out |
| 20170403 | Not detected out |
| 20170404 | Not detected out |
Verification example methodology verification
1 specificity
Solvent: water-acetonitrile solution, the volume ratio of water to acetonitrile is 1: 9.
Reference stock solution: precisely weighing 1.5mg of 4-dimethylaminopyridine as a reference substance, placing the reference substance into a 10mL volumetric flask, adding a solvent to dissolve the reference substance, fixing the volume to a scale, and shaking up to obtain a reference substance stock solution;
control solution: precisely measuring 0.2mL of the reference stock solution, placing the reference stock solution in a 20mL volumetric flask, adding a solvent for dilution, fixing the volume to a scale, and shaking up to obtain a reference solution.
Test solution: and (3) precisely weighing 100mg of dapagliflozin intermediate 2008001 batches, placing the dapagliflozin intermediate into a 10mL volumetric flask, adding a solvent to dissolve the dapagliflozin intermediate, fixing the volume to a scale, and shaking up to obtain a test solution.
Adding a standard test solution: and (3) taking 100mg of dapagliflozin intermediate, precisely weighing, placing in a 10mL volumetric flask, adding the reference substance solution to dissolve, fixing the volume to a scale, and shaking up to obtain a solution added with the standard test substance.
Precisely measuring the solvent, the reference solution, the sample solution and the sample standard solution by 10 μ L, respectively, injecting into a high performance liquid chromatograph, and recording chromatogram, wherein the results are shown in Table 3; typical spectra are shown in FIGS. 1-4.
As can be seen from the graphs of FIGS. 1 to 4, the solvent and the sample solution have no interference to the detection of the 4-dimethylaminopyridine, and the separation degree of the 4-dimethylaminopyridine peak and the main peak in the chromatogram of the sample solution meets the requirement.
TABLE 3 determination methodology of 4-dimethylaminopyridine content in dapagliflozin intermediates-specificity results
| Name (R) | Retention time (min) | Peak area | Degree of separation |
| Blank solvent | / | / | / |
| Control solution | 2.613 | 2.4466 | / |
| Test solution | / | / | / |
| Adding standard sample solution | 2.610 | 2.4026 | / |
2 limit of quantification and detection
The limit of detection (LOD) and limit of quantitation (LOQ) are determined according to a signal-to-noise ratio method. The 4-dimethylaminopyridine reference stock solution with known concentration is diluted, the concentration with S/N ≈ 10 is taken as the limit concentration for quantification, and the concentration with S/N ≈ 3 is taken as the limit concentration for detection.
Precisely measuring each quantitative limiting solution and detection limiting solution by 10 μ L according to the above chromatographic conditions, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in Table 4, and typical spectra are shown in FIGS. 5-6. As can be seen from table 4, the limit of detection S/N is 6.9>3, the concentration is 0.0045 μ g/mL, which is less than 20% of the limit concentration of 1.5 μ g/mL; the minimum S/N in the six-needle quantitative limit is 10.1, which is more than 10, the concentration is 0.015 mu g/mL and is less than 30 percent of the concentration limit of 1.5 mu g/mL, the continuous six-needle RSD in the quantitative limit is 4.68 percent and is less than 6.0 percent, and the method has high sensitivity.
TABLE 4 determination methodology of 4-dimethylaminopyridine content in dapagliflozin intermediate-results of quantitative limit and detection limit
3 durability
Solvent: water-acetonitrile solution, the volume ratio of water to acetonitrile is 1: 9.
Reference stock solution: see the reference stock solution in specificity.
Control solution: precisely measuring 0.2mL of the reference stock solution, placing the reference stock solution in a 200mL volumetric flask, adding a solvent for dilution, fixing the volume to a scale, and shaking up to obtain a reference solution.
Test solution: and (3) taking 100mg of dapagliflozin intermediate, precisely weighing, placing in a 10mL volumetric flask, adding the reference substance solution to dissolve, fixing the volume to a scale, and shaking up to obtain a test solution.
The flow rate (+ -0.1 mL/min), the column temperature (+ -5 ℃) and the mobile phase A phosphoric acid mass concentration (+ -0.04%) were respectively changed, 10. mu.L of each of the solvent, the reference solution and the sample solution was precisely measured, and the solutions were injected into a high performance liquid chromatograph, and the chromatogram was recorded, and the results are shown in Table 5. As can be seen from Table 5, the solvent and the test article solution do not interfere with the detection of 4-dimethylaminopyridine under each durability condition; compared with the content of 4-dimethylaminopyridine measured under normal conditions, the relative deviation is less than +/-5%, and the method has good durability.
TABLE 5 determination methodology of 4-dimethylaminopyridine content in dapagliflozin intermediates-durability results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A detection method of 4-dimethylaminopyridine in dapagliflozin intermediate comprises the following steps:
dissolving a 4-dimethylaminopyridine reference substance in a water-acetonitrile solution to obtain a reference substance solution;
dissolving the dapagliflozin intermediate to be detected in a water-acetonitrile solution to obtain a test sample solution;
respectively carrying out high performance liquid chromatography detection on the reference solution and the test solution to obtain the peak area of 4-dimethylaminopyridine in the 4-dimethylaminopyridine reference substance and the peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
calculating by adopting a method shown in a formula I to obtain the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected;
in the formula I, FFlat plateThe calculation method of (A) is shown as a formula II;
Afor supplying toThe peak area of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is shown;
Wfor supplying toThe weight of dapagliflozin intermediate to be tested, mg;
the unit of the content of 4-dimethylaminopyridine is ppm;
in formula II, n is the sample injection frequency of the reference substance, FiThe calculation method is shown in formula III as the correction factor;
in the formula III, WTo iWeight of the ith 4-dimethylaminopyridine control, mg;
Pto pair% purity of 4-dimethylaminopyridine control;
Ato iThe peak area of the ith 4-dimethylaminopyridine control.
2. The detection method according to claim 1, wherein the volume ratio of water to acetonitrile in the water-acetonitrile solution is 1: 9.
3. The detection method according to claim 1, wherein the conditions of the high performance liquid chromatography detection comprise:
column temperature: 25-35 ℃;
mobile phase: the mobile phase A is a phosphoric acid aqueous solution with the mass concentration of 0.16-0.24%, and the mobile phase B is acetonitrile;
gradient elution;
flow rate: 0.9 to 1.1 mL/min-1；
Detection wavelength: 280 nm.
4. The detection method according to claim 3, wherein the condition of the gradient elution is
5. The detection method according to claim 1, wherein the concentration of the control solution is 1.4 to 1.6 μ g/mL; the concentration of the test solution is 9-11 mg/mL.
6. The detection method according to claim 1 or 5, wherein the sample injection volume of the control solution and the sample solution is 10 μ L when the HPLC detection is performed.
7. The detection method according to claim 1, wherein the content of 4-dimethylaminopyridine in the dapagliflozin intermediate to be detected is 0.0045-1.5 μ g/mL.
8. The detection method according to claim 1, wherein the detection limit concentration of the high performance liquid chromatography is 0.0045 μ g/mL.
9. The detection method according to claim 1, wherein the limit concentration of quantitation of the high performance liquid chromatography detection is 0.015 μ g/mL.
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