A method for purifying digitoxin and determining its purity

Abstract

The present invention provides a method for purifying digitoxin and determining its purity. Using the pure digitoxin obtained by separation and purification with supercritical fluid chromatography technology as a control, the purity of the crude digitoxin is determined by chromatography and nuclear magnetic resonance spectroscopy. The present invention uses supercritical fluid chromatography as a pretreatment means for quantitative nuclear magnetic analysis, reducing the consumption of a large amount of solvents, shortening the sample pretreatment time, avoiding interference with the measurement results such as sample degradation, and improving the efficiency and measurement accuracy of digitoxin purity determination. The present invention provides a reliable and convenient detection method for the organic purity measurement of digitoxin, and has important value in the fields of drug production, quality control and organic chemical purity analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation, purification and quantitative analysis, and particularly relates to a method for purifying digitoxin and determining its purity. Background Art

[0002] Digitoxin, also known as digoxin, is a cardiac glycoside drug mainly used for the treatment of heart failure and certain arrhythmias. Digitoxin has a slow action, a large cumulative effect, and strong toxic and side effects. Its clinical usage rate is lower than that of another cardiac glycoside drug, digoxin, also known as ouabain. As the main impurity of digoxin, digitoxin has been included in the pharmacopoeias of countries and regions such as China, the United States, the United Kingdom, and Europe. The pharmacopoeias of various countries have clear regulations on the content limit of digitoxin in digoxin. For example, the Chinese Pharmacopoeia stipulates that it shall not exceed 2%.

[0003] The content of digitoxin is often determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Most pharmacopoeias of various countries use digitoxin as a reference substance and adopt the external standard method of chromatography for quantitative analysis. However, the national standard substance of digitoxin is missing, and the drug reference substances of various countries are different, resulting in difficulty in ensuring the accuracy of the measurement results of existing methods and introducing technical risks to drug quality and drug control. Existing cardiac glycoside drugs are often purified by an HPLC system. This method can effectively separate and purify different structurally similar impurities, metabolites and degradation products to ensure purity and quality control, but it has the characteristics of large solvent consumption and low purification amount per unit time.

[0004] Quantitative nuclear magnetic resonance technology (qNMR) is a potential reference method for the determination of organic purity. It can achieve the accurate purity determination of most organic compounds by using a small amount of traceable national standard substances as internal standards. A series of existing organic purity determination methods are combined with an HPLC system as a pretreatment means for qNMR to achieve the purity determination of low-purity organic compounds. However, such technologies usually require long-time sample pretreatment to replace conventional solvents with deuterated reagents, or use specific NMR pulse sequences to suppress solvent peaks. However, the above processes may cause sample degradation or signal distortion, resulting in errors in the measurement of organic purity. Summary of the Invention

[0005] In view of the above defects and requirements of the prior art, the present invention aims to provide a method for purifying digitoxin and determining its purity by using supercritical fluid chromatography (SFC), qNMR, and an HPLC system, named SFC-ISC-qNMR.

[0006] To achieve the above object, on the one hand, the present invention provides a method for purifying digitoxin.

[0007] The method for purifying digitoxin provided by the present invention is to perform separation and purification by supercritical fluid chromatography technology.

[0008] The method for purifying digitoxin includes the following steps: completely dissolve digitoxin in an organic solvent, and perform separation and purification on a chromatographic column by supercritical fluid chromatography to obtain pure digitoxin;

[0009] Among them, the mobile phase for the separation and purification is a mixture of supercritical carbon dioxide (phase A) and a modifier (phase B);

[0010] The stationary phase for the separation and purification is normal-phase silica gel (Nucifera SI 5 um 12 nm 10×250 mm);

[0011] The organic solvent is one or two of methanol and dichloromethane;

[0012] The modifier is one or two of methanol and ethanol;

[0013] The elution conditions of the mobile phase are: 0 - 25 min, isocratic elution with 23% B, or 0 - 25 min, isocratic elution with 25% B, or 0 - 25 min, isocratic elution with 24 - 26% B;

[0014] The back pressure of the supercritical fluid chromatograph is 8 - 12 MPa, specifically it can be 10 MPa;

[0015] The flow rate of the mobile phase in the chromatographic column in the supercritical fluid chromatograph is 3 - 10 mL / min, specifically it can be 8 mL / min, and the detection wavelength is 220 nm;

[0016] The injection volume is 10 - 200 μL.

[0017] The temperature of the chromatographic column is maintained at 32 - 35 °C.

[0018] Another aspect of the present invention provides a method for determining the purity of digitoxin.

[0019] The method for determining the purity of digitoxin provided by the present invention includes the following steps: collect and dry the pure digitoxin fraction and displace it into a deuterated reagent containing an internal standard to obtain a calibration solution, precisely measure the crude digitoxin and the internal standard and mix them to prepare a test solution, use quantitative nuclear magnetic resonance spectroscopy proton nuclear magnetic resonance to measure the characteristic hydrogen nuclear integral areas of digitoxin and the internal standard in the calibration solution, use liquid chromatography to measure the liquid phase peak areas of digitoxin and the internal standard in the calibration solution and the test solution, and calculate the accurate purity of digitoxin in the unknown concentration crude product through a formula.

[0020] Among them, the drying method is any one of nitrogen blowing and vacuum drying;

[0021] The internal standard substance is the ethyl paraben national standard substance;

[0022] The masses of the crude digitalis and the internal standard substance are 0.1 - 10 mg, and the volumes of the calibration solution and the test solution are 0.5 - 1 mL;

[0023] The mass ratio of digitoxin pure product to the internal standard substance in the calibration solution should be in the range of 0.46 - 46

[0024] The mass ratio of crude digitoxin to the internal standard substance in the test solution should be in the range of 0.46 - 46;

[0025] The deuterated reagent can be selected from at least one of deuterated methanol, deuterated DMSO, and deuterated dichloromethane.

[0026] The formula is:

[0027]

[0028] Wherein, is the accurate purity of digitoxin in the crude product to be measured, is the liquid chromatography peak area of digitoxin in the crude product to be measured, is the liquid chromatography peak area of the internal standard substance mixed with the crude product to be measured, is the liquid chromatography peak area of digitoxin pure product in the calibration solution, is the liquid chromatography peak area of the internal standard substance mixed with digitoxin pure product in the calibration solution, is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of digitoxin, is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of the internal standard substance, is the molar mass of digitoxin, is the molar mass of the internal standard substance, is the accurately weighed mass of digitoxin, is the accurately weighed mass of the internal standard substance, is the integral area corresponding to the nuclear magnetic resonance signal of digitoxin, is the integral area corresponding to the nuclear magnetic resonance signal of the internal standard substance, The purity of the internal standard substance.

[0029] The application of the above digitoxin purification and purity determination method in digoxin quality control also belongs to the protection scope of the present invention.

[0030] The digitoxin purification and purity determination method provided by the present invention, which combines supercritical fluid chromatography, quantitative nuclear magnetic resonance technology, and high performance liquid chromatography analysis, has the following beneficial effects:

[0031] (1) The supercritical fluid chromatography purification method provided by the present invention can quickly and efficiently obtain high-purity digitoxin, which can be used for industrial scale-up production;

[0032] (2) By using supercritical fluid chromatography as a pretreatment means for quantitative nuclear magnetic resonance analysis, the present invention reduces the consumption of a large amount of solvents, shortens the sample pretreatment time, avoids interference with the measurement results such as sample degradation, and can improve the efficiency and measurement accuracy of digitoxin purity determination.

[0033] (3) The present invention provides a reliable and convenient detection method for measuring the organic purity of digitoxin, which has important value in the fields of drug production, quality control, and organic chemical purity analysis. Description of the Drawings

[0034] Figure 1 It is the chromatogram and fraction collection interval of supercritical fluid chromatography for the separation and purification of digitoxin under the gradient mobile phase conditions with methanol as the modifier in Example 1 of the present invention.

[0035] Figure 2 It is the HPLC purity analysis chart of the samples at the front, middle, and rear sections of the main component chromatographic peak under the gradient mobile phase conditions with methanol as the modifier in Example 1 of the present invention.

[0036] Figure 3 It is the chromatogram and fraction collection interval of supercritical fluid chromatography for the separation and purification of digitoxin under the isocratic mobile phase conditions with ethanol as the modifier in Example 2 of the present invention.

[0037] Figure 4 It is the HPLC purity analysis chart of the samples at the front, middle, and rear sections of the main component chromatographic peak under the isocratic mobile phase conditions with ethanol as the modifier in Example 2 of the present invention.

[0038] Figure 5 It is the chromatogram and fraction collection interval of liquid chromatography for the separation and purification of digitoxin in Comparative Example 2.

[0039] Figure 6 It is the HPLC purity analysis chart of the middle section sample of the main component chromatographic peak collected by liquid chromatography purification in Comparative Example 2.

[0040] Figure 7 It is the integration interval used in the quantitative nuclear magnetic resonance experiment for the calibration solution in Example 3 of the present invention.

[0041] Figure 8 It is the first integration method used in the quantitative nuclear magnetic resonance value determination experiment of digitoxin crude product in Comparative Example 3.

[0042] Figure 9 It is the second integration method used in the quantitative nuclear magnetic resonance value determination experiment of digitoxin crude product in Comparative Example 3.

[0043] Figure 10 It is the liquid chromatogram of the crude digitoxin to be measured. Specific embodiments

[0044] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0045] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0046] Example 1

[0047] This example provides a method for purifying digitoxin using methanol as a modifier. The method includes the following steps:

[0048] Weigh 60 mg of crude digitoxin and dissolve it with an equal proportion mixture solution of 1 mL of methanol and dichloromethane to prepare a mother liquor with a concentration of 60 mg / mL.

[0049] In this example, a supercritical fluid chromatograph from Jiangsu Hanbang Technology Co., Ltd., model SFC-LAB20, is selected. The chromatographic column specifications are Nucifera SI 5 um 12 nm 10×250 mm. The flow rate of the mobile phase system is 8 mL / min, the back pressure is set at 10 MPa, and the column temperature is 35°C. The injection volume is 20 μL, and the detection wavelength is 220 nm. Phase A of the SFC mobile phase is supercritical CO2, and phase B is methanol. The mobile phase conditions are isocratic elution with 23%B from 0 to 25 min; isocratic elution with 25%B from 0 to 25 min; gradient elution with 24 - 26%B from 0 to 25 min.

[0050] Figure 1 It is the chromatogram and fraction collection interval of supercritical fluid chromatography for the separation and purification of digitoxin under the gradient elution mobile phase conditions with 24% - 26% methanol as the modifier. Collect the effluent under the flat-top time period of the chromatographic peak and dry it under nitrogen assistance to obtain high-purity digitoxin.

[0051] The liquid-phase purity analysis of the samples at the front, middle, and rear segments of the main component chromatographic peak under three mobile phase conditions was carried out using a high-performance liquid chromatograph. In this example, an LC-20AD liquid chromatograph from Shimadzu Corporation was selected, with a chromatographic column specification of Waters XBridge BEH C18 5 μm 4.6 mm × 250 mm, a column temperature of 25 °C, an injection volume of 20 μL, a PDA detector, a starting wavelength of 190 nm, an ending wavelength of 800 nm, and an analysis wavelength of 220 nm. The mobile phase was a binary high-pressure gradient, with phase A being H2O and phase B being acetonitrile. The mobile phase gradient is shown in Table 1.

[0052]

[0053] Under the three mobile phase conditions, the liquid-phase purity of the samples collected at the front, middle, and rear segments of the main component chromatographic peak in the supercritical fluid chromatogram exceeded 99.5% (Table 2).

[0054]

[0055] Figure 2 It is an HPLC purity analysis chart of the samples at the front, middle, and rear segments of the main component chromatographic peak under the gradient elution mobile phase condition with 24% - 26% methanol as the modifier. It can be seen that the liquid-phase purity of the samples collected at the front, middle, and rear segments of the main component chromatographic peak in the supercritical fluid chromatogram exceeded 99.4%. Among them, the liquid-phase purity of digitoxin in the effluent during the flat-top time period was the highest, reaching 99.85%.

[0056] To ensure the accuracy of the digitoxin purity determination result, the pure digitoxin fraction collected in the flat-top region of the chromatographic peak in Example 1 was dried using the nitrogen blowing method to ensure the signal-to-noise ratio of the subsequent qNMR detection. Taking the gradient elution condition of 24 - 26% as an example, the fraction collected in the flat-top section contained approximately 800 μL of residual methanol. The sample was completely dried using the nitrogen blowing method under a water bath at 30 °C, and the total time for collection and drying was approximately 15 minutes. Compared with the digitoxin purification method based on HPLC in Comparative Example 1, the purification efficiency was increased by 6 times, and the purification rate was increased from 98.43% to 99.85%.

[0057] Comparative Example 1

[0058] This comparative example provides a digitoxin purification method using methanol as a modifier, and the method includes the following steps:

[0059] Weigh 60 mg of digitoxin crude product and dissolve it using an equal-volume mixed solution of 1 mL of methanol and dichloromethane to prepare a mother liquor with a concentration of 60 mg / mL.

[0060] In this example, a supercritical fluid chromatograph from Jiangsu Hanbang Technology Co., Ltd., model SFC-LAB20, was selected. The chromatographic column had a specification of Nucifera SI 5 um 12 nm 10×250 mm. The flow rate of the mobile phase system was 4 mL / min, the back pressure was set at 10 MPa, and the column temperature was 35°C. The injection volumes were 10 μL and 20 μL respectively, and the detection wavelength was 220 nm. Phase A of the SFC mobile phase was supercritical CO2, and phase B was methanol. The detailed mobile phase gradient is shown in Table 3.

[0061]

[0062] High performance liquid chromatography was used to analyze the liquid phase purity of the middle section of the main component chromatographic peak. An LC-20AD liquid chromatograph from Shimadzu Corporation was selected. The chromatographic column had a specification of Waters XBridge BEH C18 5 μm 4.6 mm ×250 mm, and the column temperature was 25°C. The injection volume was 20 μL, and a PDA detector was used. The starting wavelength was 190 nm, and the ending wavelength was 800 nm. The mobile phase was a binary high-pressure gradient. Phase A was H2O, and phase B was acetonitrile. The mobile phase gradient was the same as Table 1 in Example 1. The experimental results showed that the liquid phase purities of digitoxin in the effluent during the flat peak period at the two injection volumes were 99.68% and 99.52% respectively, both lower than the purification rate using 24%-26% methanol as the modifier for gradient elution in Example 1.

[0063] Example 2

[0064] This example provides a method for purifying digitoxin using ethanol as a modifier, which includes the following steps:

[0065] Weigh 60 mg of crude digitoxin and dissolve it using an equal proportion mixed solution of 1 mL of methanol and dichloromethane to prepare a mother liquor with a concentration of 60 mg / mL.

[0066] In this example, a supercritical fluid chromatograph from Jiangsu Hanbang Technology Co., Ltd., model SFC-LAB20, was selected. The chromatographic column had a specification of Nucifera SI 5 um 12 nm 10×250 mm. The flow rate of the mobile phase system was 8 mL / min, the back pressure was set at 10 MPa, and the column temperature was 35°C. The injection volume was 20 μL, and the detection wavelength was 220 nm. Phase A of the SFC mobile phase was supercritical CO2, and phase B was ethanol. The mobile phase conditions were isocratic elution with 23%B from 0 - 25 min; isocratic elution with 25%B from 0 - 25 min; gradient elution with 24 - 26%B from 0 - 25 min.

[0067] Figure 3Chromatogram and fraction collection interval of supercritical fluid chromatography for the separation and purification of digitoxin under the isocratic mobile phase condition with 25% ethanol as the modifier. The effluent during the flat-top period of the chromatographic peak was collected and dried under nitrogen assistance to obtain high-purity digitoxin.

[0068] The high-performance liquid chromatograph was used to analyze the liquid-phase purity of the samples at the front, middle, and rear segments of the main component chromatographic peaks under three mobile phase conditions. In this example, an LC-20AD liquid chromatograph from Shimadzu Corporation was selected, the chromatographic column specification was Waters XBridge BEH C18 5 μm 4.6 mm × 250 mm, and the column temperature was 25°C. The injection volume was 20 μL, a PDA detector was used, the starting wavelength was 190 nm, and the ending wavelength was 800 nm. The mobile phase was a binary high-pressure gradient, phase A was H2O, phase B was acetonitrile, and the mobile phase gradient was the same as Table 1 in Example 1.

[0069] Under the three mobile phase conditions, the liquid-phase purity of the samples collected at the front, middle, and rear segments of the main component chromatographic peak in the supercritical fluid chromatogram exceeded 99.5% (Table 4).

[0070]

[0071] Figure 4 HPLC purity analysis chart of the samples at the front, middle, and rear segments of the main component chromatographic peak under the isocratic mobile phase condition with 25% ethanol as the modifier. It can be seen that the liquid-phase purity of digitoxin in the effluent during the flat-top period was the highest, reaching 99.83%.

[0072] To ensure the accuracy of the digitoxin purity determination result, the pure digitoxin fraction collected in the flat-top region of the chromatographic peak in Example 2 was dried using the nitrogen blowing method to ensure the signal-to-noise ratio of the subsequent qNMR detection. Taking the isocratic elution condition with 25% ethanol as the modifier as an example, the fraction collected in the flat-top section contained approximately 1 mL of residual ethanol, and the sample was completely dried using the nitrogen blowing method under a 30°C water bath heating. The total time for collection and drying was approximately 25 minutes.

[0073] Comparative Example 2

[0074] This comparative example provides an HPLC-based digitoxin purification method, which includes the following steps:

[0075] 60 mg of digitoxin crude product was weighed and dissolved in an equal-proportion mixed solution of 1 mL of methanol and dichloromethane to prepare a mother liquor with a concentration of 60 mg / mL.

[0076] In this comparative example, an LC-20AD liquid chromatograph from Shimadzu Corporation was selected. The chromatographic column had a specification of Waters XBridge BEH C18 5 μm 4.6 mm × 250 mm, and the column temperature was 25°C. The injection volume was 20 μL. A PDA detector was used, with the starting wavelength at 190 nm and the ending wavelength at 800 nm. The analysis wavelength was 220 nm. The mobile phase was a binary high-pressure gradient, with phase A being H2O and phase B being acetonitrile. The mobile phase gradient is shown in Table 5.

[0077]

[0078] Figure 5 It is the chromatogram and fraction collection interval for the separation and purification of digitoxin by liquid chromatography. The effluent under the flat-top period of the chromatographic peak was collected and dried under nitrogen assistance to obtain high-purity digitoxin.

[0079] Using a high-performance liquid chromatograph to Figure 6 perform liquid-phase purity analysis on the samples at the front, middle, and rear sections of the main component chromatographic peak in . In this example, an LC-20AD liquid chromatograph from Shimadzu Corporation was selected. The chromatographic column had a specification of Waters XBridge BEH C18 5 μm 4.6 mm × 250 mm, and the column temperature was 25°C. The injection volume was 20 μL. A PDA detector was used, with the starting wavelength at 190 nm and the ending wavelength at 800 nm. The mobile phase was a binary high-pressure gradient, with phase A being H2O and phase B being acetonitrile. The mobile phase gradient was the same as Table 1 in Example 1.

[0080] Figure 6 It is the HPLC purity analysis chart of the middle-section sample of the main component chromatographic peak collected by liquid chromatography purification. It can be seen that the liquid-phase purity of digitoxin in the effluent under the flat-top period was the highest, at 98.43%. The purification effect was lower than that of the supercritical fluid chromatography system.

[0081] To ensure the accuracy of the digitoxin purity determination result, the pure digitoxin fraction collected in the flat-top region of the chromatographic peak in Comparative Example 2 was dried using the nitrogen blowing method to ensure the signal-to-noise ratio for subsequent qNMR detection. The fraction collected in the flat-top section contained approximately 1 mL of acetonitrile aqueous solution with 65% residue. The sample was completely dried using the nitrogen blowing method under a 30°C water bath heating. The total time for collection and drying was approximately 90 minutes.

[0082] Example 3

[0083] This example provides a digitoxin purity determination method named SFC-ISC-qNMR. Based on the high-purity digitoxin prepared in Example 1, a quantity traceability chain was established, enabling rapid and accurate purity determination of digitoxin in samples. The specific operation steps are as follows:

[0084] According to the basic principles for selecting NMR internal standards, ethyl paraben national standard substance (EP, GBW06120, 99.97%, U = 0.05%) was selected as the internal standard for determining the purity of digitoxin. The pure digitoxin fraction collected from the flat-top region of the chromatographic peak in Example 1 was dried by nitrogen blowing (about 0.92 mg), and then transferred and dissolved in 500 μL of deuterated methanol (Methanol-d4) containing about 0.2 mg of the internal standard to obtain a calibration solution.

[0085] A Bruker ASCENDTM 800 spectrometer was used, with a working frequency of 800.13 MHz (1H). The pulse width was set to 30°. The number of scans was set to 32 times. The relaxation delay was set to 32 s, and the acquisition time was set to 2 s. The nuclear magnetic resonance signals at chemical shifts of 5.92 ppm and 6.84 ppm were selected as the characteristic hydrogen quantitative peaks of digitoxin and internal standard EP, respectively.

[0086] Figure 7 This is the integration interval used in the quantitative NMR experiment for the calibration solution in Example 3. The quantitative nuclear magnetic resonance spectroscopy proton NMR technique was used to measure the integration areas corresponding to the characteristic hydrogen quantitative peaks of digitoxin and the internal standard in the calibration solution, which were and . The 13C satellite signals of digitoxin and the internal standard were excluded from the integration interval. 13

[0087] About 1 mg of crude digitoxin and 0.2 mg of the internal standard were weighed using a high-precision balance and after mixing them, they were prepared into a test solution with 500 μL of methanol.

[0088] The liquid chromatography method was used to completely separate digitoxin and the internal standard from other impurities in the test solution and the calibration solution. The liquid chromatography parameters were the same as those in Table 1 of Example 1. The liquid chromatography peak areas of digitoxin and the internal standard in the test solution and the calibration solution were successively , , , . The accurate purity of digitoxin in the unknown concentration crude sample was calculated by Formula 1.

[0089]

[0090] Among them, is the accurate purity of digitoxin in the tested crude sample, is the liquid chromatography peak area of digitoxin in the tested crude sample, is the liquid chromatography peak area of the internal standard mixed with the tested crude sample, is the liquid chromatography peak area of the pure digitoxin in the calibration solution, is the liquid chromatography peak area of the internal standard mixed with pure digitoxin in the calibration solution, is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of digitoxin, is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of the internal standard, is the molar mass of digitoxin, is the molar mass of the internal standard, is the accurately weighed mass of digitoxin, is the accurately weighed mass of the internal standard, is the integral area corresponding to the nuclear magnetic resonance signal of digitoxin, is the integral area corresponding to the nuclear magnetic resonance signal of the internal standard, The purity of the internal standard.

[0091] After calculation, the purity of the crude digitoxin sample obtained by the SFC-ISC-qNMR method is 94.17% ± 0.15%. This purity value is consistent with the results obtained by the two direct quantitative NMR methods in Comparative Example 3, and the measurement error is smaller, indicating that the result of this method for determining the value is accurate and reliable.

[0092] Comparative Example 3

[0093] In this comparative example, the direct quantitative NMR method was used to determine the purity of the crude digitoxin in Example 3. Ethyl paraben national standard substance (EP, GBW06120, 99.97%, U = 0.05%) was selected as the internal standard for the purity determination of digitoxin.

[0094] The Bruker ASCENDTM 800 spectrometer was used, with a working frequency of 800.13 MHz (1H). The pulse width was set to 30°. The number of scans was set to 32 times. The relaxation delay was set to 32 s, and the acquisition time was set to 2 s. The nuclear magnetic resonance signals at chemical shifts of 5.92 ppm and 6.84 ppm were selected as the quantitative peaks of digitoxin and the internal standard EP, respectively.

[0095] The Bruker ASCENDTM 800 spectrometer was used, with a working frequency of 800.13 MHz (1H). The pulse width was set to 30°. The number of scans was set to 32 times. The relaxation delay was set to 32 s, and the acquisition time was set to 2 s. The nuclear magnetic resonance signals at chemical shifts of 5.92 ppm and 6.84 ppm were selected as the characteristic hydrogen quantitative peaks of digitoxin and the internal standard EP, respectively.

[0096] Figure 8The first integration method used in the quantitative NMR experiment on the calibration solution for Comparative Example 3. The characteristic hydrogen quantitative peak corresponding integral areas of digitoxin and the internal standard in the calibration solution were measured using quantitative nuclear magnetic resonance spectroscopy proton NMR technology, which were respectively and . The 13 C satellite signals of digitoxin and the internal standard, as well as the impurity peak signals adjacent to the digitoxin qNMR quantitative peak, were excluded from the integration interval. The purity value of the digitoxin crude product was calculated using Formula 2.

[0097] Among them, the said Formula 2 is:

[0098]

[0099] Among them, the descriptions of each parameter can refer to Formula 1.

[0100] After calculation, the purity of the tested digitoxin crude product obtained by the first integration method of the direct quantitative NMR method was 94.31% ± 0.28%. This purity value was consistent with the SFC-ISC-qNMR calibration result in Example 3, but had a larger measurement error, indicating that this calibration method might be interfered by factors such as impurities, affecting the accuracy and stability of the purity measurement result.

[0101] Figure 9 The second integration method used in the quantitative NMR experiment on the calibration solution for Comparative Example 3. The characteristic hydrogen quantitative peak corresponding integral areas of digitoxin and the internal standard in the calibration solution were measured using quantitative nuclear magnetic resonance spectroscopy proton NMR technology, which were respectively and . The characteristic hydrogen nuclear magnetic quantitative signals of digitoxin and the internal standard, including all of their 13 C satellite signals and related impurity peak signals, were all included in the integration interval, and the impurity peak signals adjacent to the digitoxin qNMR quantitative peak were excluded from the integration interval.

[0102] Figure 10 is the liquid chromatogram of the tested digitoxin crude product. The liquid phase purity of digitoxin in the tested crude product was measured to be xxx% using the liquid chromatography conditions described in Table 1 of Example 1. The purity result obtained by the second integration method was corrected using this liquid phase purity value, that is, the purity value of the digitoxin crude product was calculated using Formula 3.

[0103] Among them, the said Formula 3 is:

[0104]

[0105] Among them, is the liquid phase purity of digitoxin in the tested crude product, and the descriptions of the remaining parameters can refer to Formula 1.

[0106] After calculation, the purity of the crude digitoxin sample obtained by the second integration method using direct quantitative nuclear magnetic method is 94.61% ± 0.58%. This purity value is consistent with the SFC-ISC-qNMR determination result in Example 3, but has a larger measurement error, indicating that this determination method may also be interfered by factors such as impurities, affecting the accuracy and stability of the purity measurement result.

[0107] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A method for determining the purity of digitoxin, comprising the following steps: using the pure digitoxin obtained by purification through the following method as a control, and using chromatography and nuclear magnetic resonance spectroscopy to determine the purity of the crude digitoxin; The method for purifying digitoxin comprises the following steps: Dissolve digitoxin in an organic solvent, and perform separation and purification on a chromatographic column by supercritical fluid chromatography to obtain pure digitoxin; The mobile phase for the separation and purification is a mixture of supercritical carbon dioxide phase A and modifier phase B; The stationary phase for the separation and purification is normal-phase silica gel; The modifier is one or both of methanol and ethanol; The mobile phase elution conditions are: 0 - 25 min, isocratic elution with 23% B, or 0 - 25 min, isocratic elution with 25% B, or 0 - 25 min, gradient elution with 24 - 26% B; The back pressure of the supercritical fluid chromatograph is 8 - 12 MPa; The flow rate of the mobile phase in the chromatographic column of the supercritical fluid chromatograph is 3 - 10 mL / min, and the detection wavelength is 220 nm; The injection volume is 10 - 200 μL; The temperature of the chromatographic column is maintained at 32 - 35 °C; The method for determining the purity of digitoxin comprises the following steps: collect and dry the pure digitoxin fraction and replace it into a deuterated reagent containing an internal standard to obtain a calibration solution, precisely measure the crude digitoxin and the internal standard and mix them to prepare a test solution, use quantitative nuclear magnetic resonance spectroscopy proton nuclear magnetic resonance method to measure the characteristic hydrogen nuclear integral areas of digitoxin and the internal standard in the calibration solution, use liquid chromatography to measure the liquid phase peak areas of digitoxin and the internal standard in the calibration solution and the test solution, and calculate the accurate purity of digitoxin in the crude product with unknown concentration through a formula.

2. The method according to claim 1, characterized in that, The internal standard is the national standard substance of ethyl paraben; The mass of the crude digitoxin and the internal standard is 0.1 - 10 mg, and the volumes of the calibration solution and the test solution are 0.5 - 1 mL; The deuterated reagent is selected from at least one of deuterated methanol, deuterated DMSO, and deuterated dichloromethane.

3. The method according to claim 1, characterized in that, The formula is: Wherein, is the accurate purity of digitoxin in the crude product to be measured; is the liquid chromatography peak area of digitoxin in the crude product to be measured; is the liquid chromatography peak area of the internal standard mixed with the crude product to be measured; is the liquid chromatography peak area of pure digitoxin in the calibration solution; is the liquid chromatography peak area of the internal standard mixed with pure digitoxin in the calibration solution; is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of digitoxin; is the number of hydrogen atoms corresponding to the nuclear magnetic resonance signal of the internal standard; is the molar mass of digitoxin; is the molar mass of the internal standard; is the accurately weighed mass of digitoxin; is the accurately weighed mass of the internal standard; is the integral area corresponding to the nuclear magnetic resonance signal of digitoxin; is the integral area corresponding to the nuclear magnetic resonance signal of the internal standard; is the purity of the internal standard.

4. Application of the method for determining the purity of digitoxin according to any one of claims 1 - 3 in the quality control of digoxin.

Images