Method for detecting impurities in potassium sodium dehydroandroan drographolide succinate and application of method

Abstract

The invention provides a detection method of impurities in potassium sodium dehydroandroan drographolide succinate and application thereof, and the detection method comprises the following steps: preparing a test solution by using a sample to be detected, then carrying out liquid chromatography-mass spectrometry detection, and carrying out qualitative and / or quantitative analysis on the impurities. The detection method provided by the invention can qualitatively and quantitatively detect various small molecular impurities in potassium sodium dehydroandroan drographolide succinate at the same time, has the advantages of high impurity separation degree, good specificity, high precision, good repeatability and high sensitivity, is accurate and reliable, and can be widely applied to quality detection of potassium sodium dehydroandroan drographolide succinate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a detection method for impurities in yanhuning and an application thereof. Background Art

[0002] Yanhuning is a plant Andrographis paniculata extract - andrographolide is refined by esterification, dehydration and salt formation to form dehydrated andrographolide succinic acid half ester potassium sodium salt. Its chemical name is: 14-dehydroxy-11,12-didehydroandrographolide-3,19-disuccinic acid half ester potassium sodium salt, and its molecular formula is: C 28 H 34 KNaO 10 ·H2O, molecular weight is 610.68. Indomethacin is an antiviral drug that can inhibit early capillary permeability increase, inflammatory exudation and edema, can specifically excite pituitary-adrenal cortex function, promote ACTH release, and increase ACTH biosynthesis in the anterior pituitary; it has the effect of inactivating adenovirus, influenza virus, respiratory virus and other viruses in vitro. Indomethacin is clinically used for viral pneumonia and viral upper respiratory tract infection. It has a definite effect and is widely used, but adverse reactions are reported from time to time, mainly manifested as rash, shortness of breath, and even shock. The adverse reactions of indomethacin may be related to the impurities therein (including polymer impurities and small molecule impurities). Therefore, effective detection of impurities can help control the quality of indomethacin, but there are currently few studies on this.

[0003] Although the prior art discloses the use of liquid chromatography-mass spectrometry to detect the polymer impurities of yanhunning in the sample, wherein the molecular weight of the polymer impurities is as high as thousands, the detection of small molecule impurities is not involved at all, and comprehensive yanhunning quality control cannot be achieved. The molecular weight of the small molecule impurities of yanhunning is about 300-400. Compared with the polymer impurities, due to the large difference in molecular weight, they show different chromatographic behaviors. Therefore, in-depth research is needed in the selection of liquid chromatography columns, and the mobile phase system and chromatographic gradient conditions are also different; in addition, there are also differences in the selection of mass spectrometry parameters between small molecule impurities and polymer impurities, especially the need to ensure the integrity of the quasi-molecular ion peak of the primary fragments and the characteristic of the secondary fragments.

[0004] Therefore, there is an urgent need to develop a method that can simultaneously and accurately detect multiple small molecule impurities in indomethacin, so as to further improve the quality of indomethacin and ensure the safety of medication. Summary of the invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for detecting impurities in potassium dehydroandroan drographolide succinate and its application. The detection method provided by the present invention can qualitatively and / or quantitatively detect multiple small molecule impurities in potassium dehydroandroan drographolide succinate, and has the advantages of high impurity resolution, good specificity, high precision, good repeatability and high sensitivity. The method is accurate and reliable and can be widely applied to the quality detection of potassium dehydroandroan drographolide succinate.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted:

[0007] On the one hand, the present invention provides a method for detecting impurities in potassium dehydroandroan drographolide succinate, and the detection method includes the following steps:

[0008] Prepare a test solution from the sample to be tested, and then perform liquid chromatography-mass spectrometry (LC-MS) detection to qualitatively and / or quantitatively analyze the impurities.

[0009] Preferably, the preparation method of the test solution includes: mixing the sample to be tested and a solvent; wherein, the solvent includes an inorganic solvent and / or an organic solvent that can dissolve potassium dehydroandroan drographolide succinate and / or the impurities in potassium dehydroandroan drographolide succinate; more preferably water.

[0010] The detection method provided by the present invention combines liquid chromatography and mass spectrometry. Liquid chromatography realizes good separation of each impurity for qualitative analysis, and mass spectrometry qualitatively and quantitatively analyzes each impurity. The detection method is accurate and reliable, has the advantages of good specificity, high precision, good repeatability and high sensitivity, can simultaneously detect multiple small molecule impurities, and is widely applied to the quality detection of potassium dehydroandroan drographolide succinate.

[0011] The impurities include any one or a combination of at least two of impurities A-D, and the structures of impurities A-D are shown as follows:

[0012]

[0013] Preferably, the impurities include impurity A, impurity B, impurity C and impurity D, and this method can realize the simultaneous detection of four small molecule impurities.

[0014] Preferably, the mobile phase in the liquid chromatography includes mobile phase A and mobile phase B. Mobile phase A includes an aqueous formic acid solution, and mobile phase B is acetonitrile and / or methanol.

[0015] Preferably, the volume concentration of the aqueous formic acid solution is 0.03-0.07%, such as 0.03%, 0.04%, 0.05%, 0.06% or 0.07%, etc., but not limited to the above-listed values. Other unlisted values within the above numerical range are equally applicable, and preferably 0.04-0.06%.

[0016] Preferably, the purity of the acetonitrile and / or methanol is mass spectrometry purity, with a purity of more than 99%, more preferably 99.9%.

[0017] Preferably, the mobile phase B is acetonitrile.

[0018] Preferably, the liquid chromatography uses gradient elution, and the gradient elution program is as follows:

[0019] At 0 - 10 min, the volume ratio of mobile phase A is 65 - 75%, and the volume ratio of mobile phase B is 25 - 35%;

[0020] At 10 - 40 min, the volume ratio of mobile phase A uniformly changes to 45 - 55%, and the volume ratio of mobile phase B uniformly changes to 45 - 55%;

[0021] At 40 - 50 min, the volume ratio of mobile phase A uniformly changes to 18 - 28%, and the volume ratio of mobile phase B uniformly changes to 72 - 82%;

[0022] At 50 - 51 min, the volume ratio of mobile phase A uniformly changes to 65 - 75%, and the volume ratio of mobile phase B uniformly changes to 25 - 35%;

[0023] After 51 min, the volume ratio of mobile phase A is 65 - 75%, and the volume ratio of mobile phase B is 25 - 35%.

[0024] Preferably, at 10 - 40 min, the change rate of mobile phase A is 0.33 - 1.0% / min, preferably 0.50 - 0.83% / min.

[0025] Preferably, the chromatographic column used in the liquid chromatography is an octadecylsilane-bonded chromatographic column, and the preferred chromatographic column is ACE Excel 3C18-AR.

[0026] Preferably, the particle size of the chromatographic column used in the liquid chromatography is 1.5 - 5 μm; preferably, the column length of the chromatographic column is 100 - 300 mm; preferably, the inner diameter of the chromatographic column is 2 - 4.6 mm.

[0027] Preferably, the column temperature of the liquid chromatography is 30 - 40 °C, such as 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C, etc., but not limited to the above-listed values, and other unlisted values within the above numerical range are equally applicable, preferably 30 - 35 °C.

[0028] Preferably, the injection flow rate of the liquid chromatography is 0.4 - 0.8 mL / min, such as 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min or 0.8 mL / min, etc., but not limited to the values listed above. Other unlisted values within the above numerical range are equally applicable, and more preferably it is 0.5 - 0.7 mL / min.

[0029] Preferably, the ultraviolet detection wavelength of the liquid chromatography is 246 - 256 nm, such as 246 nm, 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, 254 nm, 255 nm or 256 nm, etc., but not limited to the values listed above. Other unlisted values within the above numerical range are equally applicable.

[0030] Preferably, the mass analyzer of the mass spectrometry is a triple quadrupole mass spectrometer.

[0031] Preferably, the detector of the mass spectrometry is an ESI mass spectrometer.

[0032] Preferably, the ion source of the mass spectrometry is set to the ESI negative ion mode.

[0033] Preferably, the sheath gas pressure of the mass spectrometry is 25 - 35 arb, such as 25 arb, 26 arb, 27 arb, 28 arb, 29 arb, 30 arb, 31 arb, 32 arb, 33 arb, 34 arb or 35 arb, etc., but not limited to the values listed above. Other unlisted values within the above numerical range are equally applicable.

[0034] Preferably, the auxiliary gas pressure of the mass spectrometry is 10 - 14 arb, such as 10 arb, 11 arb, 12 arb, 13 arb or 14 arb, etc., but not limited to the values listed above. Other unlisted values within the above numerical range are equally applicable.

[0035] The above characteristic detection parameters can effectively improve the separation degree, specificity, precision, repeatability and sensitivity of the detection, and effectively realize the simultaneous detection of four impurities.

[0036] The method for qualitative analysis is: determining the types of impurities according to the relative retention time of each impurity relative to potassium dehydroandroan drographolide succinate in the liquid chromatography;

[0037] Preferably, the relative retention time of impurity A is 0.23 ± 5% - 10%, and more preferably it is 0.23 ± 2% - 5%.

[0038] Preferably, the relative retention time of impurity B is 0.40 ± 5% - 10%, and more preferably it is 0.40 ± 2% - 5%.

[0039] Preferably, the relative retention time of the impurity C is 0.84 ± 5% to 10%, more preferably 0.84 ± 2% to 5%.

[0040] Preferably, the relative retention time of the impurity D is 0.86 ± 5% to 10%, more preferably 0.86 ± 2% to 5%.

[0041] Preferably, the relative retention time of the impurity A is 0.23.

[0042] Preferably, the relative retention time of the impurity B is 0.40.

[0043] Preferably, the relative retention time of the impurity C is 0.84.

[0044] Preferably, the relative retention time of the impurity D is 0.86.

[0045] Preferably, the relative retention time deviation is ≤ 5% to 10%.

[0046] Preferably, the relative retention time deviation is ≤ 6%.

[0047] Preferably, the relative retention time deviation is ≤ 5%.

[0048] Preferably, the relative retention time deviation is ≤ 3%.

[0049] Preferably, the relative retention time deviation is ≤ 2%.

[0050] Preferably, the relative retention time deviation is ≤ 1%.

[0051] Preferably, the relative retention time deviation is 0.

[0052] On the other hand, the present invention also provides the application of the detection method as described above in the quality control of potassium dehydroandroan drographolide succinate.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention provides a method for detecting impurities in potassium dehydroandroan drographolide succinate, which realizes the simultaneous qualitative and / or quantitative detection of multiple impurities in potassium dehydroandroan drographolide succinate by using the method of liquid chromatography and mass spectrometry in combination; wherein, liquid chromatography realizes good separation of each impurity, and mass spectrometry qualitatively analyzes each impurity. The detection method is accurate and reliable, and has the advantages of good specificity, high precision, good repeatability and high sensitivity. It can simultaneously detect multiple small molecule impurities and is widely used in the quality detection of potassium dehydroandroan drographolide succinate. Description of the Drawings

[0055] Figure 1 It is the liquid chromatogram using the Thermo Hypersil GOLD PFP chromatographic column in Example 2;

[0056] Figure 2 It is the liquid chromatogram obtained by using the ACE Excel 3C18-AR chromatographic column in Example 2;

[0057] Figure 3 It is the liquid chromatogram obtained by using the Ultimate LP C8 chromatographic column in Example 2;

[0058] Figure 4 It is the liquid chromatogram obtained by using the mobile phase ratio of Group 4 in Example 4;

[0059] Figure 5 It is the mass spectrometry qualitative chromatogram of impurity A in the specificity test of Example 8;

[0060] Figure 6 It is the mass spectrometry qualitative chromatogram of impurity B in the specificity test of Example 8;

[0061] Figure 7 It is the mass spectrometry qualitative chromatogram of impurity C in the specificity test of Example 8;

[0062] Figure 8 It is the mass spectrometry qualitative chromatogram of impurity D in the specificity test of Example 8;

[0063] Figure 9 It is the second-order mass spectrometry chromatogram of impurity A in the specificity test of Example 8;

[0064] Figure 10 It is the second-order mass spectrometry chromatogram of impurity B in the specificity test of Example 8;

[0065] Figure 11 It is the second-order mass spectrometry chromatogram of impurity C in the specificity test of Example 8;

[0066] Figure 12 It is the second-order mass spectrometry chromatogram of impurity D in the specificity test of Example 8. Detailed implementation manners

[0067] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0068] Example 1

[0069] A method for detecting impurities in potassium sodium dehydroandroan drographolide succinate, comprising the following steps:

[0070] (1) Preparation of solutions

[0071] Solvent: water;

[0072] Test solution: Take 10 mg of Potassium Sodium Dehydroandrographolide Succinate, weigh accurately, place it in a 20 mL volumetric flask, dissolve and dilute with the solvent to prepare a solution containing 0.5 mg of Potassium Sodium Dehydroandrographolide Succinate (calculated as C 28 H 34 KNaO 10 ·H2O) per 1 mL as the test solution;

[0073] Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain the control solution;

[0074] System suitability solution: Weigh accurately appropriate amounts of reference substances of impurities A, B, C, and D respectively, dissolve and dilute with the solvent to prepare a solution containing 500 μg per 1 mL as the stock solutions of impurities A, B, C, and D; Take 10 mg of Potassium Sodium Dehydroandrographolide Succinate, weigh accurately, place it in a 20 mL volumetric flask, accurately measure 200 μL of each of the stock solutions of impurities A, B, C, and D, place them in the same 20 mL volumetric flask, dissolve and dilute to the mark with the solvent, and shake well to obtain the system suitability solution.

[0075] (2) Liquid chromatography conditions:

[0076] Mobile phase A: 0.05% formic acid aqueous solution;

[0077] Mobile phase B: Acetonitrile;

[0078] Flow rate: 0.6 mL / min;

[0079] Chromatographic column: ACE Excel 3C18-AR (4.6×100 mm, 3 μm);

[0080] Detection wavelength: 251 nm;

[0081] Column temperature: 35°C;

[0082] Gradient elution program:

[0083] Time (min) Mobile Phase B (vt%) Mobile Phase A (vt%) 0 70 30 10 70 30 40 50 50 50 23 77 51 70 30 60 70 30

[0084] (3) Mass spectrometry conditions

[0085] Ion source: ESI-;

[0086] Spray voltage: 3000 V (-);

[0087] Sheath gas pressure: 30 arb;

[0088] Auxiliary gas pressure: 12 arb;

[0089] Auxiliary gas temperature: 320°C;

[0090] Ion transfer tube temperature: 450°C;

[0091] Scanning mode: FulMS-ddMS2;

[0092] Ful MS: The scan range is m / z 100 - 1500, and the resolution is 70000;

[0093] MS 2 : The resolution is 35000;

[0094] Step NCE: 30;

[0095] TopN: 5.

[0096] (4) Using the above liquid chromatography and mass spectrometry conditions, the solution obtained in step (1) was detected by a liquid chromatography - mass spectrometry instrument;

[0097] (5) Sample test results and conclusions

[0098] Qualitative analysis of impurities: Qualify each impurity peak according to the following criteria: The relative retention time of impurity A is 0.23; the relative retention time of impurity B is 0.40; the relative retention time of impurity C is 0.84; the relative retention time of impurity D is 0.86. Among them, the relative retention time of the characteristic peak (main peak) corresponding to potassium sodium dehydroandroan drographolide succinate is 1.00. And the resolution R between each peak is controlled above 2.2 - 32, all significantly higher than 1.5, showing complete separation.

[0099] Quantitative analysis of impurities: Calculated by the self - control method based on the peak area, in the test solution, the content of impurity B is 0.08%, and impurities A, C, and D are not detected; the system suitability solution is a solution with impurity reference substances added to potassium sodium dehydroandroan drographolide succinate, and impurities A, B, C, and D can be accurately detected, and the contents are 0.94%, 1.04%, 0.95%, and 0.94% respectively.

[0100]

[0101] Example 2 Screening of chromatographic columns

[0102] In this example, the chromatographic column models in liquid chromatography were screened, specifically including:

[0103] (1) Solution preparation

[0104] System suitability solution: Weigh appropriate amounts of reference substances of Impurity A, B, C, and D respectively, accurately weigh them, dissolve them with a solvent and dilute to make a solution containing 500 μg per 1 mL as the stock solutions of Impurity A, B, C, and D; Weigh 10 mg of Potassium Sodium Dehydroandrographolide Succinate accurately, place it in a 20 mL volumetric flask, accurately pipette 200 μL of the stock solutions of Impurity A, B, C, and D respectively, place them in the same 20 mL volumetric flask, dissolve with a solvent and dilute to the mark, and shake well to obtain;

[0105] Based on Example 1, changing the type of chromatographic column, the detection results are shown in the following table:

[0106]

[0107] The detection results of the impurity mixed solution are shown in the following table (Resolution: The resolution between the peak to be measured and the adjacent chromatographic peak with the earlier retention time, the same hereinafter):

[0108]

[0109] The results of the retention time (six repetitions) of the impurity mixed solution in Group 2 are shown in the following table:

[0110]

[0111] Conclusion: Using chromatographic column 2, the resolution between Impurity A and Impurity B is 14.2, and the minimum value of the theoretical plate number of each impurity is 13002; Using chromatographic column 1, the resolution between Impurity C and Impurity D is only 1.3, which does not meet the resolution standard (above 1.5), and the minimum value of the theoretical plate number of each impurity is 9791; Using chromatographic column 3, Impurity A and B overlap. It can be concluded that the resolution and theoretical plate number between impurities obtained using chromatographic column 2 are better than those obtained using chromatographic column 1 and chromatographic column 3; Continuously detecting the impurity mixed solution using chromatographic column 2, the maximum RSD of the retention time of each impurity and Potassium Sodium Dehydroandrographolide Succinate is 0.09%, which is lower than 1.0%, indicating that the method has good precision.

[0112] Determination of the chromatographic mobile phase in Example 3

[0113] In this example, different mobile phases in the gradient elution of high-performance liquid chromatography were compared, specifically including:

[0114] (1) On the basis of the detection conditions in Example 1, change the type of mobile phase according to the conditions described below respectively, and keep other conditions unchanged:

[0115] Group 1: The same mobile phase as in Example 1;

[0116] Group 2: The difference from the mobile phase in Example 1 is only that mobile phase B is replaced with methanol;

[0117] (2) Experimental procedures and conclusions

[0118] The corresponding detection results of the mobile phases in Group 1 and Group 2 above are shown in the following table:

[0119]

[0120] The corresponding detection results of the impurity mixed solution using the mobile phase in Group 2 above are shown in the following table:

[0121] Name Retention Time Relative Retention Time Theoretical Plate Number Tailing Factor Resolution Impurity A 15.221 0.43 7002 1.2 / Impurity B 19.045 0.49 8554 1.4 4.5 Impurity D 29.014 0.77 10872 1.4 30.5 Impurity C 30.034 0.77 11309 1.4 1.1(＜1.5) Potassium Sodium Dehydroandrographolide Succinate 35.024 1.00 9588 1.5 15.2

[0122] Conclusion: When using 0.05% formic acid aqueous solution - methanol as the mobile phase system to detect the impurity mixed solution, the resolution between impurity D and impurity C is 1.1, which is less than 1.5 and does not meet the resolution standard (above 1.5), indicating poor resolution. The peak resolution is better when using 0.05% formic acid aqueous solution - acetonitrile as the mobile phase system.

[0123] Example 4 Determination of the proportion of the mobile phase in liquid chromatography

[0124] In this example, the proportions of the mobile phase in the gradient elution of high-performance liquid chromatography are compared, specifically including:

[0125] (1) On the basis of the detection conditions in Example 1, the proportions of the mobile phase are changed according to the conditions described below, and other conditions remain unchanged:

[0126] Group 1: The difference from the mobile phase elution gradient in Example 1 is only that at the 40th minute, the volume ratio of mobile phase A is 55% and the volume ratio of mobile phase B is 45%, that is, within 10 - 40 minutes, the change rate of mobile phase A is 0.50% / min;

[0127] Group 2: The difference from the mobile phase elution gradient in Example 1 is only that at the 40th minute, the volume ratio of mobile phase A is 45% and the volume ratio of mobile phase B is 55%, that is, within the 10 - 40th minutes, the change rate of mobile phase A is 0.83% / min;

[0128] Group 3: The same as the mobile phase elution gradient in Example 1;

[0129] Group 4: The difference from the mobile phase elution gradient in Example 1 is only that at the 0 - 10th minutes and the 51 - 60th minutes, the volume ratio of mobile phase A is 65% and the volume ratio of mobile phase B is 35%, and at the 40th minute, the volume ratio of mobile phase A is 55% and the volume ratio of mobile phase B is 45%;

[0130] Group 5: The difference from the mobile phase elution gradient in Example 1 is only that from 0 to 10 minutes and from 51 to 60 minutes, the volume ratio of mobile phase A is 75%, and the volume ratio of mobile phase B is 25%; at 40 minutes, the volume ratio of mobile phase A is 45%, and the volume ratio of mobile phase B is 55%.

[0131] (2) Experimental procedures and conclusions

[0132] The detection results under the gradient elution programs of different groups are shown in the following table:

[0133]

[0134]

[0135] The detection results of the impurity mixed solution under the gradient elution programs of different groups are shown in the following table:

[0136]

[0137] Conclusion: When the change rate of mobile phase A is between 0.50% and 0.83% from 10 to 40 minutes, the resolution between each impurity peak and the adjacent peak is greater than 1.5, the resolution is good, and the optimal change rate is 0.67%.

[0138] Determination of the chromatographic column temperature range in Example 5

[0139] In this example, the chromatographic column temperature in high performance liquid chromatography was compared, specifically including:

[0140] (1) On the basis of Example 1, the column temperature of the chromatographic column was changed to 30 °C, 35 °C and 40 °C respectively, and other conditions remained unchanged;

[0141] (2) Experimental procedures and conclusions

[0142] The detection effects at different column temperatures are shown in the following table:

[0143]

[0144] The results of detecting the impurity mixed solution at different column temperatures are shown in the following table:

[0145]

[0146]

[0147] Conclusion: When the chromatographic column temperature is 40 °C, the resolution between impurity D and impurity C is less than 1.5, and the resolution is poor. When the column temperature of the chromatographic column is between 30 and 35 °C, the resolution between each impurity is good.

[0148] Determination of the flow rate of the chromatographic conditions in Example 6

[0149] In this embodiment, the injection flow rate in high performance liquid chromatography detection was compared, specifically including:

[0150] (1) On the basis of Example 1, the injection flow rates were changed to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, and 0.8 mL / min respectively, and other conditions remained unchanged;

[0151] (2) Experimental procedures and conclusions

[0152] The detection results at different injection flow rates are shown in the following table:

[0153]

[0154] The detection results of the impurity mixed solution at different injection flow rates are shown in the following table:

[0155]

[0156]

[0157] Conclusion: When the flow rate is 0.4 mL / min and 0.8 mL / min, the resolution between impurity D and impurity C is lower than 1.5, and the resolution is poor. When the flow rate is between 0.5 - 0.7 mL / min, the peak shape, resolution, and sensitivity of the chromatographic method are better, and the optimal flow rate is 0.6 mL / min.

[0158] Determination of sheath gas pressure in mass spectrometry detection in Example 7

[0159] In this embodiment, the corresponding detection sensitivities at different sheath gas pressures in mass spectrometry detection were compared, specifically including:

[0160] (1) On the basis of Example 1, column 2 was used, and the sheath gas pressures were changed to 10 arb, 25 arb, 30 arb, and 35 arb respectively, and other conditions remained unchanged.

[0161] (2) Experimental procedures and conclusions

[0162] The detection sensitivity results at different sheath gas pressures are as follows:

[0163]

[0164] Conclusion: When the sheath gas pressure is between 25 - 35 arb, the mass spectrometry response sensitivity is good.

[0165] Specificity test in Example 8

[0166] (1) Chromatographic conditions

[0167] Mobile phase A: 0.05% formic acid aqueous solution;

[0168] Mobile phase B: acetonitrile;

[0169] Flow rate: 0.6 mL / min;

[0170] Chromatographic column: ACE Excel 3C18-AR (4.6×100 mm, id);

[0171] Detection wavelength: 251 nm;

[0172] Column temperature: 35 °C;

[0173] Gradient elution program:

[0174] Time (min) Mobile Phase B (vt%) Mobile Phase A (vt%) 0 70 30 10 70 30 40 50 50 50 23 77 51 70 30 60 70 30

[0175] (2) Mass spectrometry conditions

[0176] Ion source: ESI-;

[0177] Spray voltage: 3000 V(-);

[0178] Sheath gas pressure: 30 arb;

[0179] Auxiliary gas pressure: 12 arb;

[0180] Auxiliary gas temperature: 320 °C;

[0181] Ion transfer tube temperature: 450 °C;

[0182] Scan mode: FulMS-ddMS2;

[0183] Ful MS: The scan range is m / z 100 - 1500, and the resolution is 70000;

[0184] MS 2 : The resolution is 35000;

[0185] Step NCE: 30;

[0186] TopN: 5.

[0187] (3) Test method

[0188] Blank solvent: water.

[0189] System suitability solution: Weigh appropriate amounts of reference substances of impurity A, B, C, and D respectively, accurately weigh them, dissolve and dilute with solvent to prepare a solution containing 500 μg per 1 mL as the stock solutions of impurity A, B, C, and D; weigh 10 mg of potassium sodium dehydroandroan drographolide succinate accurately, place it in a 20 mL volumetric flask, accurately measure 200 μL of the stock solutions of impurity A, B, C, and D respectively, place them in the same 20 mL volumetric flask, dissolve with solvent and dilute to the mark, shake well to obtain the solution.

[0190] The test results are shown in the following table:

[0191] Item Situation Blank Solvent Does not interfere with the detection of each impurity System Suitability Solution The resolution of each impurity peak is good

[0192] Conclusion: The blank solvent does not interfere with the impurity detection in potassium sodium dehydroandroan drographolide succinate, and the resolution between each impurity peak is good, indicating that the detection method of the present invention has good specificity. The mass spectrometry qualitative spectra corresponding to impurity A, B, C, and D are shown in Figure 5 、 Figure 6 、 Figure 7 and Figure 8 , and the secondary mass spectrometry spectra are shown in Figure 9 、 Figure 10 、 Figure 11 and Figure 12 . In the negative ion mode of the first ionization mass spectrometry, Figure 5 the peak at m / z 395.1972 is the formic acid adduct of impurity A (molecular weight 350.46). Secondary mass spectrometry analysis of this molecular ion gives main fragments such as m / z 287.1946 and 331.1833; Figure 6 the peak at m / z 563.1989 in Figure 7 is the dehydrogenation peak of impurity B (molecular weight 564.58). Secondary mass spectrometry analysis of this molecular ion gives main fragments of m / z 319.1836 and 99.0051, and the fragment at m / z 99.0051 is the succinic anhydride fragment ion; Figure 8 the peak at m / z 431.1962 in

[0193] Example 9 Sensitivity test

[0194] Test method:

[0195] Blank solvent: water;

[0196] Sensitivity test solution: Take the system suitability solution described in Example 1 and dilute it for determination when the signal-to-noise ratio (S / N) = 10:1 and the signal-to-noise ratio (S / N) = 3:1. That is, the quantitative limit and detection limit of the method of the present invention;

[0197] The chromatographic conditions and mass spectrometry conditions refer to Example 1.

[0198] The test results are shown in the following table:

[0199] Name Quantitation Limit (ng / mL) Detection Limit (ng / mL) Impurity A 15 4 Impurity B 9 3 Impurity C 17 5 Impurity D 25 7

[0200] Conclusion: The quantitative limits of impurities A, B, C, and D are 15, 9, 17, and 25 ng / mL respectively, and the detection limits are 4, 3, 5, and 7 ng / mL respectively. The sensitivity of this method is higher than that of high performance liquid chromatography.

[0201] Example 10 Repeatability test

[0202] Blank solvent: water;

[0203] Repeatability test solution: Take the system suitability solution described in Example 1 as the repeatability test solution and prepare six portions in parallel; denoted as solution - 1, solution - 2, solution - 3, solution - 4, solution - 5, solution - 6;

[0204] The chromatographic conditions and mass spectrometry conditions refer to Example 1.

[0205] The test results are shown in the following table:

[0206]

[0207]

[0208] Conclusion: The maximum RSD of the impurity content in the six repeatability solutions is 1.7%, all less than 3%, indicating that the method of the present invention has good repeatability.

[0209] Example 11 Accuracy test

[0210] Blank solvent: water;

[0211] Background solution: Take the test solution described in Example 1 as the background solution;

[0212] Accuracy stock solution: Stock solutions of impurities A, B, C, and D: Weigh 5 mg of each of the reference substances of impurities A, B, C, and D respectively, accurately weigh, place in a 10 mL volumetric flask, dissolve with solvent and dilute to the mark, shake well, then obtain; accurately measure 5 mL of each of the above impurity stock solutions respectively, place in a 50 mL volumetric flask, dilute to the mark with solvent, and shake well as the accuracy stock solution;

[0213] 20% Recovery Solution: Accurately weigh 10 mg of this product, place it in a 20-mL volumetric flask, accurately add 0.4 mL of the accuracy stock solution, dissolve and dilute to the mark with the solvent, and shake well. Prepare 3 parallel portions.

[0214] 100% Recovery Solution: Accurately weigh 10 mg of this product, place it in a 20-mL volumetric flask, accurately add 2 mL of the accuracy stock solution, dissolve and dilute to the mark with the solvent, and shake well. Prepare 3 parallel portions.

[0215] 150% Recovery Solution: Accurately weigh 10 mg of this product, place it in a 20-mL volumetric flask, accurately add 3 mL of the accuracy stock solution, dissolve and dilute to the mark with the solvent, and shake well. Prepare 3 parallel portions.

[0216] The chromatographic conditions and mass spectrometry conditions refer to Example 1.

[0217] The test results are shown in the following table:

[0218]

[0219]

[0220]

[0221] Conclusion: The recovery rate of impurity A is between 97.3% and 103.9%, the recovery rate of impurity B is between 94.9% and 102.0%, the recovery rate of impurity C is between 96.8% and 102.9%, and the recovery rate of impurity D is between 96.1% and 103.1%. All are between 92% and 105%. The maximum RSD value of the recovery rate of each impurity in 9 samples is 3.0%, all less than 10.0%, meeting the requirements, and the method accuracy is good.

[0222] Example 12 Stability Test

[0223] Blank Solvent: Water;

[0224] Stability Test Solution: Take the test solution described in Example 1 as the stability test solution;

[0225] The chromatographic conditions and mass spectrometry conditions refer to Example 1.

[0226] The test results are shown in the following table:

[0227]

[0228] Conclusion: The results show that impurities A, C, and D in the test solution are not detected, and the change value of the peak area of impurity B is less than 5%, and the solution is stable.

[0229] Comparative Example 1

[0230] The following Test was carried out using the HPLC chromatographic conditions of Comparative Example 1 and Comparative Example 2 and the conditions of Example 1 respectively:

[0231] Comparative Example 1

[0232] The HPLC chromatographic conditions are shown in the following table:

[0233]

[0234]

[0235] Example 1: For the HPLC chromatographic conditions, refer to the above part.

[0236] Sensitivity results:

[0237]

[0238] Conclusion: In the HPLC method of Comparative Example 1, the lowest detection limit of impurities is 22.7 ng / mL. In the present invention, the lowest detection limit of impurities is 3 ng / mL. Compared with the HPLC method of Comparative Example 1, the sensitivity of this method is significantly improved.

[0239] Comparative Example 2

[0240] The following Test was carried out using the conditions of Comparative Example 2 and Example 1 respectively:

[0241] The detection conditions of Comparative Example 2 are as follows

[0242]

[0243]

[0244] Mass spectrometry conditions: Ion source: ESI+; Spray voltage: 3500 V (+); Sheath gas pressure: 40 arb; Auxiliary gas pressure: 10 arb; Evaporation temperature: 350 °C; Ion transfer tube temperature: 350 °C; Scanning mode: FulMS-ddMS2; Ful MS: Scan range is m / z 200 - 3000, Resolution is 70000; MS2: Resolution is 17500; Step NCE: 20, 40, 60; TopN: 3.

[0245] Example 1: For the detection conditions, refer to the above part.

[0246] Resolution results:

[0247]

[0248] Conclusion: In the detection method of Comparative Example 2, the resolution between impurity C and D is 0.9, and baseline separation cannot be achieved. In the present invention, the lowest resolution is 2.2. Compared with the detection method of Comparative Example 2, the resolution of this method is also significantly improved.

[0249] The applicant declares that the present invention illustrates the detection method and its application of impurities in potassium dehydroandrographolide succinate by the above-mentioned examples, but the present invention is not limited to the above-mentioned examples, that is, it does not mean that the present invention must rely on the above-mentioned examples to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0250] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0251] In addition, it should be noted that in the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without conflict. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for detecting impurities in yanhuning, characterized in that: The detection method comprises the following steps: The test sample is used to prepare a test solution, which is then tested by liquid chromatography-mass spectrometry to perform qualitative and / or quantitative analysis of impurities; The impurities include any one of impurities A to D or a combination of at least two of them. The structure of impurity A to D is as follows:

2. The detection method according to claim 1, characterized in that: The impurities include impurity A, impurity B, impurity C and impurity D.

3. The detection method according to claim 1 or 2, characterized in that: The mobile phase in the liquid chromatography comprises mobile phase A and mobile phase B; the mobile phase A comprises aqueous formic acid solution, and the mobile phase B is acetonitrile and / or methanol; Preferably, the volume concentration of the formic acid aqueous solution is 0.03-0.07%, preferably 0.04-0.06%; Preferably, the mobile phase B is acetonitrile.

4. The detection method according to any one of claims 1 to 3, characterized in that: The liquid chromatography adopts gradient elution, and the procedure of the gradient elution is as follows: From 0 to 10 minutes, the volume of mobile phase A accounts for 65-75%, and the volume of mobile phase B accounts for 25-35%; From 10 to 40 minutes, the volume proportion of mobile phase A changes at a constant rate to 45-55%, and the volume proportion of mobile phase B changes at a constant rate to 45-55%; At 40-50 minutes, the volume proportion of mobile phase A changes uniformly to 18-28%, and the volume proportion of mobile phase B changes uniformly to 72-82%; At 50-51 minutes, the volume proportion of mobile phase A changes uniformly to 65-75%, and the volume proportion of mobile phase B changes uniformly to 25-35%; After the 51st minute, the volume proportion of mobile phase A was 65-75%, and the volume proportion of mobile phase B was 25-35%.

5. The detection method according to any one of claims 1 to 4, characterized in that: The chromatographic column used in the liquid chromatography is an octadecylsilane bonded chromatographic column, and the preferred chromatographic column is ACE Excel 3C18-AR.

6. The detection method according to any one of claims 1 to 5, characterized in that: The column temperature of the liquid chromatography is 30-40°C, preferably 30-35°C; Preferably, the injection flow rate of the liquid chromatography is 0.4-0.8 mL / min, more preferably 0.5-0.7 mL / min.

7. The detection method according to any one of claims 1 to 6, characterized in that: The ultraviolet detection wavelength of the liquid chromatography is 246-256 nm.

8. The detection method according to any one of claims 1 to 7, characterized in that: The mass analyzer of the mass spectrometer is a triple quadrupole mass spectrometer; Preferably, the mass spectrometer is an ESI mass spectrometer; Preferably, the ion source of the mass spectrometer is set to ESI negative ion mode; Preferably, the sheath gas pressure of the mass spectrometer is 25-35arb; Preferably, the auxiliary gas pressure of the mass spectrometer is 10-14arb.

9. The detection method according to any one of claims 1 to 8, characterized in that: The qualitative method is: determining the type of impurity according to the relative retention time of each impurity relative to yanhuning in liquid chromatography; Preferably, the relative retention time of impurity A is 0.23±5% to 10%, more preferably 0.23±2% to 5%; Preferably, the relative retention time of impurity B is 0.40±5% to 10%, more preferably 0.40±2% to 5%; Preferably, the relative retention time of the impurity C is 0.84±5% to 10%, more preferably 0.84±2% to 5%; Preferably, the relative retention time of the impurity D is 0.86±5% to 10%, more preferably 0.86±2% to 5%.

10. Use of the detection method according to any one of claims 1 to 9 in the quality control of Yanhuning.

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