A method for detecting digoxin-related substances

Abstract

The invention discloses a method for detecting digoxin related substances. The method comprises the following steps: using a high performance liquid chromatograph to detect a test solution, adopting a C18 chromatographic column, using acetonitrile as a mobile phase A, using water as a mobile phase B, and performing gradient elution: from 0 to 23 minutes, the mobile phase A is 25%; from 23 to 45 minutes, the mobile phase A is increased from 25% to 40%; from 45 to 55 minutes, the mobile phase A is increased from 40% to 80%; from 55 to 55.5 minutes, the mobile phase A is reduced from 80% to 25%; and then balancing for 60 minutes with the mobile phase A being 25%. The detection method of the invention is used to separate and determine the related substances in digoxin raw materials and preparations, especially digoxin injections. The method has many detectable impurities, good separation between peaks, high sensitivity, good repeatability and recovery rate, and can control 10 key known impurities and 6 unknown impurities, thereby facilitating the quality control of digoxin preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection methods, and specifically relates to a method for detecting digoxin-related substances. Background Art

[0002] Digoxin, English name Digoxin, chemical name is 3β-[[O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(l→4)-O-2,6-dideoxy-β-D-ribo-hexopyranosyl-(l→4)-2,6-dideoxy-β-D-ribo-hexopyranosyl]oxy]-12β,14β-dihydroxy-5β-cardiosteroid-20(22)ene lactone, molecular formula is C 41 H 64 O 14 , molecular weight is 780.95, CAS number is 20830-75-5, and the structural formula is as follows:

[0003]

[0004] Digoxin injection is a cardiac glycoside that can be used for supraventricular arrhythmias, acute and chronic heart failure; it can control the ventricular rate and supraventricular tachycardia in patients with atrial fibrillation and atrial flutter accompanied by rapid ventricular rate.

[0005] The related substances in digoxin injection may come from two aspects, process impurities and degradation impurities. The structures of some process impurities and degradation impurities have been determined as shown in Table 0:

[0006] Table 0: Summary of Digoxin Impurity Profile Analysis

[0007]

[0008]

[0009] At present, in the inspection method for digoxin-related substances, foreign pharmacopoeias such as the European Pharmacopoeia and the United States Pharmacopoeia all use high performance liquid chromatography for the detection of digoxin raw material related substances, with octadecylsilane bonded silica gel as the filler; acetonitrile: water = 10:90 as mobile phase A; acetonitrile: water = 90:10 as mobile phase B, the detection wavelength is 220nm, and gradient elution is performed according to Table 1.

[0010] Table 1 Test methods for related substances of digoxin raw materials

[0011] Time (minutes) A％ B％ 0 78 22 5 78 22 15 30 30

[0012] It can be seen that in the detection of digoxin-related substances by the European Pharmacopoeia and the United States Pharmacopoeia, the HPLC method is consistent in the first 15 minutes, and all impurities peak within 15 minutes. However, there are still some shortcomings, such as the inability to detect all impurities, poor separation, limited separation and detection capabilities for impurities, and the quality control of products needs to be further improved. Summary of the invention

[0013] Aiming at the problems of poor separation degree of related substances in digoxin injection and less detected impurities in the prior art, the present invention provides a detection method for digoxin related substances, which separates and determines digoxin and its related substances, can detect 16 impurities at most, the separation degrees between peaks are all above 1.2, 14 peaks have a separation degree above 2.0, the sensitivity is high, the detection limit of impurities is lower than 0.04 μg / ml, the quantitative limit is lower than 0.14 μg / ml, the repeatability is good, the recovery rate of each impurity is between 93.7% and 102.1%, the RSD is 2.3% (≤10%, n=9), 10 key known impurities and 6 unknown impurities can be controlled, and the quality control of digoxin injection is beneficial.

[0014] In order to achieve the above object, the present invention provides the following technical solutions:

[0015] A method for detecting digoxin-related substances comprises the following steps:

[0016] The test solution was detected by high performance liquid chromatography, using a C18 chromatographic column, acetonitrile as mobile phase A, water as mobile phase B, and gradient elution at a flow rate of 0.8-1.2 ml / min. The test solution was an aqueous solution containing digoxin;

[0017] Among them, the gradient elution conditions are: 0-23min, mobile phase A is 25%, mobile phase B is 75%; 23-45min, mobile phase A increases from 25% to 40%, mobile phase B decreases from 75% to 60%; 45-55min, mobile phase A increases from 40% to 80%, mobile phase B decreases from 60% to 20%; 55-55.5min, mobile phase A decreases from 80% to 25%, mobile phase B increases from 20% to 75%; then continue to balance with mobile phase A at 25% and mobile phase B at 75% for 60min.

[0018] Further, the related substances include: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, unknown impurity 1, unknown impurity 2, unknown impurity 3, unknown impurity 4, unknown impurity 5 and unknown impurity 6;

[0019] The impurity A is digitoxin, the impurity B is gemtocine, the impurity C is isohydroxydigitoxin, the impurity D is digoxin monosaccharide, the impurity E is dihydroxydigitoxin, the impurity F is isohydroxydigitoxin glycoside, the impurity G is digoxin glycoside, the impurity H is lanatin C, the impurity J is β-acetyldigoxin, and the impurity K is digoxigenin tetraketide;

[0020] The structural formulas and names of the known impurities are as follows:

[0021]

[0022]

[0023] Preferably, the C18 chromatographic column has a length of 2-30 cm.

[0024] Furthermore, the particle size of the C18 chromatographic column filler is 1.7-5 μm.

[0025] Furthermore, the C18 chromatographic column is a chromatographic column selected from the following brand series: Agilent ZORBAX, Agilent InfinityLab Poroshell 120, Agilent Zorbax, Agilent Pursuit, Agilent XRs, Agilent XRs Ultra, Agilent Polaris, waters Symmetry, waters Sunfire, waters Xterra, waters XBridge, waters XSelect, waters CORTECS, Shimadzu Inertsil, Shimadzu wandasil, Shimadzu WondaCract or Shimadzu Shim-pack.

[0026] Furthermore, the high performance liquid chromatograph uses a diode array or ultraviolet absorption as a detector, the column temperature is 25 to 35° C., and the detection wavelength is 200 to 240 nm.

[0027] Furthermore, the concentration of digoxin in the test solution is 0.1-0.75 mg / ml.

[0028] Again, the injection volume was 10-50 μl, the flow rate during gradient elution was 1.0 ml / min, and the concentration of digoxin in the test solution was 0.25 mg / ml.

[0029] Preferably, the test solution is digoxin injection or digoxin aqueous solution containing an organic solvent, the organic solvent is selected from ethanol, methanol, DMSO or acetonitrile, and the added amount of the organic solvent is 20-50 v / v%.

[0030] The detection method of digoxin-related substances of the present invention can be used for detecting or separating impurities in digoxin raw materials or digoxin preparations.

[0031] The inventors screened a suitable mobile phase and optimized the ratio of each component, using acetonitrile as mobile phase A and water as mobile phase B for gradient elution. At 0-23 min, the mobile phase ratio was 25:75, and this setting can make impurities C, impurity D, unknown impurity 1, impurity E, unknown impurity 2, impurity F, and unknown impurity 3 all well separated. At 23-45 min, the mobile phase ratio changed from 25:75 to 40:60, and this setting can make impurities G, impurity H, digoxin, unknown impurity 4, impurity K, unknown impurity 5, unknown impurity 6, and impurity F all well separated. At 45-55 min, the mobile phase ratio changed from 40:60 to 80:20, and this setting can make impurities B and impurity A all well separated. At 55-55.5 min, the mobile phase ratio changed from 80:20 to 25:75. At 55.5-60 min, the mobile phase ratio was 25:75, and this setting can make the mobile phase ratio return to the initial ratio balance for 4.5 min. This method can separate and determine digoxin and its related substances, and can detect 16 impurities. The separation degrees between peaks are all above 1.2, and there are 14 peaks with a separation degree above 2.0. It has high sensitivity, the detection limit of impurities is lower than 0.04μg / ml, the quantification limit is lower than 0.14μg / ml, and the repeatability is good. The recovery rate of each impurity is between 93.7% and 102.1%, and the RSD is 2.3% (≤10%, n=9). It can control 10 key known impurities and 6 unknown impurities, which is beneficial to the quality control of digoxin preparations, especially injections.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] The detection method of the invention has a high detection rate for digoxin related substances, can detect and control 10 key known impurities and 6 unknown impurities, has high sensitivity, a detection limit of impurities lower than 0.04 μg / ml, a quantitative limit lower than 0.14 μg / ml, good repeatability, a recovery rate of each impurity between 93.7% and 102.1%, and an RSD of 2.3% (≤10%, n=9), thereby ensuring the quality of digoxin raw materials and digoxin preparations, especially the final product of digoxin injection.

[0034] The present invention can ensure the separation degree between peaks when detecting more impurities. The separation degrees between peaks of 16 detectable impurities are all above 1.2, and 14 peaks have a separation degree above 2.0. The detection limit of impurities is lower than 0.04 μg / ml, and the quantitative limit is lower than 0.14 μg / ml. In specific detection, the limits of key impurities can be formulated according to the detection results of each batch, which greatly improves the economic applicability in the actual production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The HPLC spectrum of the impurity mixed solution in Example 1 of the present invention.

[0036] Figure 2 This is the system applicability spectrum in Example 1 of the present invention.

[0037] Figure 3 This is the spectrum of the test solution when the column temperature of the chromatographic column is 25°C in Example 1 of the present invention.

[0038] Figure 4 This is the spectrum of the test solution when the column temperature of the chromatographic column is 30°C in Example 1 of the present invention.

[0039] Figure 5 This is the spectrum of the test solution when the column temperature of the chromatographic column is 35°C in Example 1 of the present invention.

[0040] Figure 6 The HPLC spectrum of the impurity mixed solution in Comparative Example 1 of the present invention.

[0041] Figure 7 The HPLC spectrum of impurities H and digoxin in Comparative Example 1 of the present invention is shown.

[0042] Figure 8 The HPLC spectrum of the impurity mixed solution with extended elution time in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the embodiments.

[0044] The experimental methods in the following examples without specifying specific conditions were selected according to conventional methods and conditions, or according to the product instructions; in the chromatograms of the examples and comparative examples, the unit of peak time is min.

[0045] The sources of impurity reference substances and mixed standard substances are shown in Table 2:

[0046] Table 2

[0047]

[0048] Example 1

[0049] This example is an example of system adaptability and detection of related substances in digoxin injection, and the experimental conditions are as follows:

[0050] Instrument: Waters e2695 / 2489 high performance liquid chromatograph, with diode array or UV absorption as detector;

[0051] Chromatographic column: Agilent ZORBAX SB C18 (4.6 mm × 250 mm, filler particle size 3.5 μm);

[0052] Mobile phase A: acetonitrile;

[0053] Mobile phase B: water;

[0054] Flow rate: 1.0 ml / min;

[0055] The column temperature was 30°C;

[0056] The detection wavelength is 220nm;

[0057] The injection volume was 20 μl;

[0058] Gradient elution was performed according to the elution program shown in Table 3.

[0059] Table 3 Gradient elution program

[0060] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 25 75 23 25 75 45 40 60 55 80 20 55.5 25 75 60 25 75

[0061] Blank solvent: Take 2500ml of anhydrous ethanol, add 2500ml of water, mix well.

[0062] Impurity mixed solution: Take 5 mg of digoxin mixed reference substance and place it in a 20 ml volumetric flask, dissolve it with the above blank solvent and dilute to the scale, shake well.

[0063] Impurity H reference substance stock solution: Take 5.680 mg of impurity H reference substance, accurately weigh it, put it in a 20 ml volumetric flask, dissolve it with the above blank solvent and dilute it to the scale, shake well.

[0064] System suitability solution: Accurately measure 1.0 ml of impurity H stock solution and test solution, place them in the same 10 ml volumetric flask, dilute to volume with the above blank solvent, and shake well.

[0065] Digoxin injection: Take this product directly as the test solution;

[0066] Digoxin reference substance stock solution: Accurately weigh 6.404 mg of digoxin reference substance, place it in a 25 ml volumetric flask, dissolve and dilute to the scale with solvent, and shake well.

[0067] Impurity A reference substance stock solution: Accurately weigh 6.517 mg of impurity A reference substance, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake well.

[0068] Impurity B reference substance stock solution: Accurately weigh 6.299 mg of impurity B reference substance, place it in a 250 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake well.

[0069] Impurity C reference substance stock solution: Accurately weigh about 6.543 mg of impurity C reference substance, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake well.

[0070] Impurity D reference substance stock solution: Accurately weigh 6.482 mg of impurity D reference substance, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake well.

[0071] Impurity F reference substance stock solution: Accurately weigh 6.280 mg of impurity F reference substance, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake well.

[0072] Impurity J reference substance stock solution: Accurately weigh 6.604 mg of impurity J reference substance, place it in a 25 ml volumetric flask, dissolve it with solvent and dilute it to the scale, and shake well.

[0073] Linear stock solution: Accurately measure 2.5 ml of digoxin reference stock solution and impurity A reference stock solution, 5.0 ml of impurity C and impurity D reference stock solutions, 25.0 ml of impurity B reference stock solution, and 12.5 ml of impurity F reference stock solution, and place them in the same 100 ml volumetric flask. Dilute to the scale with solvent and shake well.

[0074] Take 20 μl of blank solvent, system suitability solution, each impurity stock solution and impurity mixed solution respectively, inject them into high performance liquid chromatograph, record the chromatogram, and the HPLC spectrum of impurity mixed solution is shown in Figure 1 , chromatographic peak attribution see Table 4, system suitability spectrum see Figure 2 The chromatographic peaks are listed in Table 5.

[0075] Table 4 Chromatographic peaks attribution

[0076]

[0077]

[0078] Table 5 System suitability results

[0079] Chromatographic peak Retention time / min Impurity Attribution Separation 1 25.952 Impurity H N / A 2 28.198 Main Peak 3.2

[0080] The results in Table 4-5 show that the detection method meets the system suitability requirements. This method can detect 10 known impurities and 6 unknown impurities in the impurity mixed solution. The separation degree of each of the 16 detectable impurities is greater than 1.2, which meets the requirements. There are 14 peaks with a separation degree above 2.0.

[0081] The test solution was tested under different column temperatures (25°C, 30°C, 35°C) and the chromatograms were recorded. The spectrum of the test solution at 25°C column temperature is shown in Figure 3 , column temperature 30℃, the spectrum of the test solution is shown in Figure 4 , column temperature 35℃, the spectrum of the test solution is shown in Figure 5 It can be seen that when the test solution is tested at different column temperatures, all 9 impurities in the test sample, including 7 known impurities and 2 unknown impurities, can be detected. The peak order is impurity C, impurity D, impurity E, impurity F, unknown impurity 3, unknown impurity, impurity K, impurity B, and impurity A.

[0082] Example 2

[0083] This example studies the detection limit and quantification limit of digoxin and the obtained impurity reference substances impurity A, impurity B, impurity C, impurity D, and impurity F under the condition of solution concentration of 0.25 mg / ml:

[0084] The experimental conditions, liquid chromatography method and solution preparation are as shown in Example 1. The solution is prepared according to methods well known to those skilled in the art.

[0085] For known impurities: the reference solutions of impurities A, B, C, D and F with known concentrations in Example 1 and the digoxin reference solution are diluted to 0.12 μg / ml, and the measured signals are compared with the baseline signal (baseline noise). The concentration corresponding to the signal-to-noise ratio s / n≥10 of the response on the instrument among the known impurities is the quantitative limit of the method; the concentration corresponding to the signal-to-noise ratio s / n≥3 of the lowest response on the instrument among the known impurities is the detection limit of the method.

[0086] The test results are shown in Table 6-8:

[0087] Table 6 Detection limit signal-to-noise ratio is as follows:

[0088]

[0089] Table 7 The signal-to-noise ratio of the quantitative limit is as follows:

[0090]

[0091] Table 8 Quantitative Limit Peak Area is as follows:

[0092]

[0093] It can be seen that the detection limit of impurity C is 0.0371μg / ml, and the quantification limit is 0.1237μg / ml; the detection limit of impurity D is 0.0366μg / ml, and the quantification limit is 0.1220μg / ml; the detection limit of impurity F is 0.0369μg / ml, and the quantification limit is 0.1228μg / ml; the detection limit of digoxin is 0.0380μg / ml, and the quantification limit is 0.1265μg / ml; the detection limit of impurity B is 0.0378μg / ml, and the quantification limit is 0.1260μg / ml; the detection limit of impurity A is 0.0391μg / ml, and the quantification limit is 0.1303μg / ml. It can be seen that the detection limits of the above impurities are all below 0.04μg / ml, and the quantification limits are all below 0.14μg / ml.

[0094] Example 3

[0095] This example studies the linearity of digoxin and impurity A, impurity B, impurity C, impurity D, and impurity F for which impurity reference substances are available:

[0096] The experimental conditions, HPLC method and solution preparation were the same as in Example 1. The solution was prepared according to methods well known to those skilled in the art.

[0097] For known impurities A, impurity B, impurity C, impurity D and impurity F, 6 concentration points were taken for study within the range of LOQ (LOQ limit of quantification) concentration to not less than 150% of the index concentration. The linear concentrations included 6 series of concentrations: limit of quantification (LOQ) concentration, 50% impurity limit concentration, 100% impurity limit concentration, 120% impurity limit concentration, 150% impurity limit concentration, and 200% impurity limit concentration.

[0098] Inject 2 injections of each linear concentration solution and record the mean peak area; use the peak area as the ordinate (Y-axis) and the concentration as the abscissa (X-axis) to perform linear regression to calculate the correlation coefficient and the Y-axis intercept. The linear relationship is plotted as a function of the measured response signal (peak area) versus the analyte concentration. Perform linear regression using the least squares method and report at least the correlation coefficient R2 to confirm a good linear relationship, which should be between 0.990 and 1.000.

[0099] Table 9

[0100]

[0101] Using this analytical method, the linear relationship of each substance was determined and the results are shown in Tables 10 to 15.

[0102] Table 10 Linearity determination results of digoxin

[0103]

[0104]

[0105] Table 11 Linear determination results of impurity A

[0106]

[0107] Table 12 Linear determination results of impurity B

[0108]

[0109] Table 13 Linear determination results of impurity C

[0110]

[0111] Table 14 Linear determination results of impurity D

[0112]

[0113]

[0114] Table 15 Linear determination results of impurity F

[0115]

[0116] It can be seen that within the range of digoxin concentration from 0.13 μg / ml to 2.53 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=21011.7543x-517.9766, the linear correlation coefficient r is 0.9998, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is -0.3%, and the residual sum of squares is 929539;

[0117] The concentration of impurity A is in the range of 0.13 μg / ml to 2.61 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=20889.1027x-89.8899, the linear correlation coefficient r is 1.0000, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is 2.0%, and the residual sum of squares is 61570;

[0118] The concentration of impurity B is in the range of 0.13 μg / ml to 2.52 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=19062.5827x-59.5090, the linear correlation coefficient r is 0.9999, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is 1.2%, and the residual sum of squares is 246892;

[0119] The concentration of impurity C is in the range of 0.12 μg / ml to 4.95 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=42554.0431x-748.4617, the linear correlation coefficient r is 1.0000, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is -0.2%, and the residual sum of squares is 862875;

[0120] The concentration of impurity D is in the range of 0.12 μg / ml to 4.88 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=25498.3581x-676.3647, the linear correlation coefficient r is 1.0000, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is 0.6%, and the residual sum of squares is 170026;

[0121] The concentration of impurity F is in the range of 0.12 μg / ml to 12.28 μg / ml (quantitative limit concentration to 200% limit concentration), the linear equation is: y=21011.7543x-517.9766, the linear correlation coefficient r is 1.0000, and the linear relationship is good; the ratio of the y-axis intercept to the 100% limit concentration response value is 0.5%, and the residual sum of squares is 3522717.

[0122] Example 4

[0123] This example studies the accuracy of digoxin and the impurity reference substances available, including impurity A, impurity B, impurity C, impurity D, and impurity F:

[0124] The linear stock solution in Example 2 was used as the recovery stock solution.

[0125] Prepare the sample recovery solution according to Table 16: add different volumes of recovery stock solution to different volumetric bottles, add digoxin injection to the scale, shake well, and use them as sample recovery solutions of different concentrations. Prepare 3 sample recovery solutions for each concentration level. The sample recovery solution concentrations include 3 series of concentrations: 50% impurity limit concentration, 100% impurity limit concentration and 150% impurity limit concentration.

[0126] Table 16

[0127]

[0128] Test sample: Take an appropriate amount of digoxin injection.

[0129] Control solution: Accurately measure 1.0 ml of the above test solution and place it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and use as the control solution.

[0130] Reference solution: accurately measure 2.0 ml of the linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and use as the reference solution.

[0131] One injection was made for each control solution, reference substance solution, and sample recovery solution, and the chromatogram and peak area were recorded. The results are shown in Table 17.

[0132] It can be seen that the recovery rate of impurity C is between 98.6% and 100.5%, and the RSD is 0.7% (≤10%, n=9); the recovery rate of impurity D is between 99.1% and 102.1%, and the RSD is 1.1% (≤10%, n=9); the recovery rate of impurity F is between 98.1% and 100.7%, and the RSD is 1.0% (≤10%, n=9); the recovery rate of impurity B is between 94.0% and 97.7%, and the RSD is 1.3% (≤10%, n=9); the recovery rate of impurity A is between 93.7% and 101.2%, and the RSD is 2.3% (≤10%, n=9); all meet the requirements.

[0133] Comparative Example 1: Chromatographic methods for determination of related substances in digoxin injection according to the European Pharmacopoeia and the United States Pharmacopoeia

[0134] Detection solution: the impurity mixed solution in Example 1;

[0135] Instrument: Waters e2695 / 2489 high performance liquid chromatograph;

[0136] Chromatographic column: Agilent ZORBAX SB C18 (4.6 mm × 250 mm, 3.5 μm)

[0137] Mobile phase A: acetonitrile: water = 10:90;

[0138] Mobile phase B: acetonitrile: water = 90:10;

[0139] The flow rate was 1.0 ml / min; the column temperature was 30 °C; the detection wavelength was 220 nm; the injection volume was 20 μl;

[0140] Gradient elution, the elution program is shown in Table 1:

[0141] Table 1

[0142] Time (minutes) A％ B％ 0 78 22 5 78 22 15 30 30

[0143] The chromatogram of the test results is shown in Figure 6 , see Table 18 for the attribution of chromatographic peaks.

[0144] Table 18 Chromatographic peak attribution

[0145]

[0146]

[0147] It can be seen that there is no separation between impurity D and unknown impurity 1, unknown impurity 1 and impurity E, impurity E and unknown impurity 3, unknown impurity 3 and impurity F, impurity K and unknown impurity 4, unknown impurity 4 and unknown impurity 5, impurity A and unknown impurity 6. This is because the impurity peaks are too close to each other and the software cannot calculate the separation. There is also unknown impurity 2 in impurity F that is not separated.

[0148] When the test solution containing impurity H and digoxin was tested under the same conditions as above in this comparative example, the peak time of digoxin was 4.518min, the peak time of impurity H was 4.023min, and the separation degree was 1.89, see Figure 7 .

[0149] The same chromatographic method and conditions as those in the European Pharmacopoeia and the United States Pharmacopoeia were used to test the samples with extended elution time. The elution procedure is shown in Table 19 and the results are shown in Table 19. Figure 8 , see Table 20 for the attribution of chromatographic peaks.

[0150] Table 19 Elution gradient

[0151] Time (minutes) A％ B％ 0 78 22 10.5 78 22 32 30 30 32.1 78 22 37 78 22

[0152] Table 20 Chromatographic peak attribution

[0153]

[0154]

[0155] It can be seen that under this method, the peak position of the known impurity F (5.131min) contains the unknown impurity 2, which cannot be separated and detected. In addition, the peak of impurity H should be between the impurity G and digoxin peaks. Under this condition, the impurity H peak and the digoxin peak cannot be separated.

[0156]

Claims

1. A method for detecting digoxin-related substances, comprising the following steps: The test solution was detected by high performance liquid chromatography, using a C18 chromatographic column, acetonitrile as mobile phase A, water as mobile phase B, and gradient elution at a flow rate of 0.8-1.2 ml / min. The test solution was an aqueous solution containing digoxin; The gradient elution conditions are: 0-23min, mobile phase A is 25%, mobile phase B is 75%; 23-45min, mobile phase A increases from 25% to 40%, mobile phase B decreases from 75% to 60%; 45-55min, mobile phase A increases from 40% to 80%, mobile phase B decreases from 60% to 20%; 55-55.5min, mobile phase A decreases from 80% to 25%, mobile phase B increases from 20% to 75%; then continue to balance with mobile phase A at 25% and mobile phase B at 75% for 60min; The related substances include: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J and impurity K; impurity A is digitoxin, impurity B is gemtuximab, impurity C is isohydroxydigitoxin, impurity D is digoxin monosaccharide, impurity E is dihydroxydigitoxin, impurity F is isohydroxydigitoxin glycoside, impurity G is digoxin, impurity H is lanatin C, impurity J is β-acetyldigoxin, and impurity K is digoxigenin tetraketide; The C18 chromatographic column has a length of 2-30 cm and a filler particle size of 3.5 μm.

2. The method for detecting digoxin-related substances according to claim 1, characterized in that: The C18 chromatographic column is a chromatographic column selected from the following brand series: Agilent ZORBAX, Agilent Infinity Lab Poroshell 120, Agilent Zorbax, Agilent Pursuit, Agilent XRs, Agilent XRs Ultra, Agilent Polaris, waters Symmetry, waters Sunfire, waters Xterra, waters XBridge, waters XSelect, waters CORTECS, Shimadzu Inertsil, Shimadzu wandasil, Shimadzu WondaCract or Shimadzu Shim-pack.

3. The method for detecting digoxin-related substances according to claim 1, characterized in that: The high performance liquid chromatograph uses a diode array or ultraviolet absorption as a detector, the temperature of the chromatographic column is 25-35° C., and the detection wavelength is 200-240 nm.

4. The method for detecting digoxin-related substances according to claim 1, characterized in that: During the detection, the concentration of digoxin in the test solution is 0.1-0.75 mg / ml.

5. The method for detecting digoxin-related substances according to claim 1, characterized in that: The injection volume was 10-50 μl, the flow rate during gradient elution was 1.0 ml / min, and the concentration of digoxin in the test solution was 0.25 mg / ml.

6. The method for detecting digoxin-related substances according to any one of claims 1 to 5, characterized in that: The test solution is digoxin injection or digoxin aqueous solution containing an organic solvent, the organic solvent is selected from ethanol, methanol, DMSO or acetonitrile, and the content of the organic solvent is 20-50 v / v%.

7. Use of the method for detecting digoxin-related substances as claimed in claim 1 in separating impurities from digoxin bulk drugs or digoxin preparations.

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