Preparation method of meloxicam degradation impurity
Through light, heat or oxidative degradation methods combined with column chromatography separation, the problem of preparation of meloxicam degraded impurities is solved, and high-purity impurity preparation is achieved, which meets the needs of drug quality control and ensures the safety and stability of drugs.
Patent Information
- Application Number
- CN202411098110.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-12
- Publication Date
- 2024-12-17
AI Technical Summary
Meloxicam will produce degraded impurities in stability experiments, resulting in the impurity content exceeding the limit specified by ICH, affecting the safety and quality controllability of drugs. The existing technology lacks effective preparation methods.
Meloxicam is dissolved in organic solvent by light degradation, high temperature degradation or oxidation degradation methods, and then separated and purified by column chromatography to prepare high-purity degradation impurities. The specific steps include light degradation, column chromatography separation and drying treatment, and optimize the conditions. Includes light intensity, time, temperature and solvent selection.
It has achieved efficient preparation of meloxicam degraded impurities, with a purity of no less than 98.0%, providing a basis for drug quality research and quality control, and simply and accurately measuring the purity of impurities.
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Figure CN119143692A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a method for preparing meloxicam degradation impurities. Background Art
[0002] Meloxicam is a cyclooxygenase-2 (COX-2) inhibitor and a nonsteroidal anti-inflammatory drug (NSAID). Its mechanism of action is to inhibit the synthesis of inflammatory prostaglandins by inhibiting cyclooxygenase (COX). Compared with traditional NSAIDs, meloxicam has similar anti-inflammatory and analgesic effects and significantly improved gastrointestinal tolerance. The COX-2 / COX-1 value of meloxicam is much lower than that of other commonly used NSAIDs in clinical practice, so meloxicam is more widely used in clinical practice.
[0003] In order to ensure the safety of drug use, each impurity in the raw material / preparation must be evaluated for safety, that is, the impurity limit to ensure safety must be established. The Chinese Pharmacopoeia limits the total amount of meloxicam impurities: if there are impurity peaks in the chromatogram of the test solution, the area of a single impurity peak shall not be greater than 0.5 times (0.5%) the area of the main peak of the control solution, and the sum of the areas of each impurity peak shall not be greater than the main peak area of the control solution (1.0%). The ICH guidelines require: the limit of impurities in drugs is 0.1% (lower for toxic drugs), all unknown impurities above this level should be identified, and more importantly, all impurities above 0.15% should be studied for their toxicity.
[0004]
[0005] At present, there are few studies on the degradation impurities of meloxicam. Studies have found that during the stability test of meloxicam, it will degrade to produce impurity compounds as shown in Formula 1, and the impurity content gradually increases with the extension of the stability test time, and it is easy to exceed the definition limit (0.15%) specified in the ICH guidelines, which has an important impact on the safety, efficacy and quality controllability of the drug. Therefore, it is necessary to provide a method for preparing the degradation impurities of meloxicam in the art. Summary of the invention
[0006] The present invention aims at the problems existing in the prior art and provides a preparation method of meloxicam by degrading impurities.
[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0008] A method for preparing meloxicam degraded impurities comprises the following steps:
[0009] (1) dissolving meloxicam in an organic solvent and degrading it to obtain a degradation product;
[0010] (2) Separating and purifying the degradation products by column chromatography to obtain:
[0011] Wherein, the degradation in step (1) is light degradation, high temperature degradation or oxidative degradation,
[0012] The structural formula of the meloxicam degradation impurity is shown in Formula 1:
[0013]
[0014] Preferably, the light intensity of the light degradation is 4000-8000Lx, the light degradation time is 5-11 days, and the total illumination of the light source is not less than 1.2×10 6 Lux·hr, near-ultraviolet lamp energy is not less than 200W·hr / m 2 .
[0015] More preferably, the light intensity of the light degradation is 5500-6500Lx, the light degradation time is 8-10 days, and the total illumination of the light source is 1.3×10 6 Lux·hr, near-ultraviolet lamp energy is 250W·hr / m 2 .
[0016] Further preferably, the light intensity of the light degradation is 6000Lx, and the time of the light degradation is 9 days.
[0017] Preferably, the organic solvent in step (1) is selected from one or more of dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetone, isopropanol and hexafluoroisopropanol.
[0018] More preferably, the organic solvent is dimethyl sulfoxide, isopropanol or hexafluoroisopropanol.
[0019] Preferably, the concentration of meloxicam after being dissolved in an organic solvent is 0.1-10 mg / ml.
[0020] More preferably, the concentration of meloxicam after being dissolved in an organic solvent is 5-10 mg / ml.
[0021] During the dissolution process of meloxicam, dissolution can be assisted by strong shaking, vortexing or ultrasound.
[0022] Preferably, the temperature of the high temperature degradation is 40-150° C., and the time of the high temperature degradation is 5-12 hours.
[0023] More preferably, the temperature of the high temperature degradation is 130-140° C., and the time of the high temperature degradation is 9-11 hours.
[0024] Further preferably, the high temperature degradation temperature is 135° C., and the high temperature degradation time is 10 h.
[0025] The oxidation degradation process includes mixing with hydrogen peroxide with a volume concentration of 3-10%, the oxidation degradation temperature is 30-50° C., and the oxidation degradation time is 3-8 hours.
[0026] Preferably, the oxidative degradation process includes mixing with hydrogen peroxide with a volume concentration of 6-10%, the oxidative degradation temperature is 40-50° C., and the oxidative degradation time is 4-6 hours.
[0027] More preferably, the oxidative degradation process includes mixing with hydrogen peroxide having a volume concentration of 10%, the oxidative degradation temperature is 50° C., and the oxidative degradation time is 5 hours.
[0028] By separating impurities using the following chromatographic parameters, the interference of other degradation components on the target substance is minimized, and the obtained impurities are purer.
[0029] Preferably, the conditions for the column chromatography separation in step (2) include: mobile phase A is a mixture of phosphate buffer and methanol in a volume ratio of 70:30-95:5, mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 6:4-8:2, and the elution mode of the column chromatography separation is gradient elution.
[0030] More preferably, mobile phase A is a mixture of phosphate buffer and methanol in a volume ratio of 90:10, and mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 7:3.
[0031] Preferably, the phosphate buffer is potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, ammonium dihydrogen phosphate solution or diammonium hydrogen phosphate solution, and the pH of the phosphate buffer is 4.5-6.5.
[0032] More preferably, the phosphate buffer is diammonium hydrogen phosphate solution, and the pH of the phosphate buffer is 5.5.
[0033] Preferably, the elution procedure of the gradient elution is:
[0034] 0-10min, A:B is 100-80:0-20, V / V;
[0035] 15-25min, A:B is 80-60:20-40, V / V;
[0036] 25.1-35min, A:B is 100-80:0-20, V / V.
[0037] More preferably, the elution procedure of the gradient elution is:
[0038] 0-10min, A:B is 95:5, V / V;
[0039] 15-25min, A:B is 70:30, V / V;
[0040] 25.1-35min, A:B is 95:5, V / V.
[0041] Preferably, the column chromatography separation conditions in step (2) also include: the chromatographic column is Shim-pack GIS C18 or USHA C18, the detection wavelength is 250-270nm, the flow rate is 1-20ml / min, and the column temperature is 25-45°C.
[0042] More preferably, the chromatographic column is Shim-pack GIS C18, the detection wavelength is 260 nm, the flow rate is 10 ml / min, and the column temperature is 30°C.
[0043] Preferably, after the column chromatography separation in step (2) is completed, the method further comprises: collecting liquid components with a relative retention time of 1.05-1.10.
[0044] The impurity outflow component liquid received is further purified and dried in the following manner. This purification and drying method is easier to implement in a pharmaceutical research and development laboratory, has a higher purification and drying efficiency, and can better protect the quality of the separated and purified impurities.
[0045] Preferably, the purification comprises dissolving the liquid component in a non-polar solvent, extracting, evaporating the non-polar solvent, and drying to obtain the product.
[0046] Preferably, the non-polar solvent is ethyl acetate, toluene, n-hexane, dichloromethane, chloroform or n-heptane.
[0047] More preferably, the non-polar solvent is ethyl acetate, toluene or n-heptane.
[0048] Preferably, the method of volatilizing the non-polar solvent is nitrogen purging or rotary evaporation, the gas flow rate of the nitrogen purging is 1-20lpm, the temperature of the nitrogen purging is 25-60°C; the rotation speed of the rotary evaporation is 20-180rpm, the temperature of the rotary evaporation is 35-60°C, and the vacuum degree of the rotary evaporation is 250-500mbar.
[0049] More preferably, the gas flow rate of the nitrogen purge is 8-12 lpm, and the temperature of the nitrogen purge is 35-45°C.
[0050] More preferably, the rotation speed of the rotary evaporation is 40-180 rpm, the temperature of the rotary evaporation is 40-50° C., and the vacuum degree of the rotary evaporation is 300-450 mbar.
[0051] Preferably, the drying is reduced-pressure drying, the drying temperature is 45-80° C., the drying pressure is less than 2.67 kPa, and the drying time is 5-24 h.
[0052] Preferably, the drying requires the use of a desiccant, which is phosphorus pentoxide, anhydrous calcium chloride or silica gel.
[0053] The present invention also provides the use of the meloxicam degradation impurities prepared by the above preparation method as a meloxicam impurity reference substance.
[0054] The present invention also provides a method for detecting the purity of the meloxicam degradation impurities prepared by the above preparation method, comprising mixing the meloxicam degradation impurities purified by the above preparation method with a diluent, and performing liquid chromatography detection to obtain the product.
[0055] Preferably, the conditions of the liquid chromatography include: the chromatographic column is Welch Ultimate XB-C18 (150mm×4.6mm, 5μm) or YMC-Triart-C18 (150mm×4.6mm, 3μm); the mobile phase A is a potassium dihydrogen phosphate solution or an ammonium acetate solution with a concentration of 20-40mmol / L, and the mobile phase B is acetonitrile; the detection wavelength is 250-270nm; the flow rate is 0.8-1.2ml / min; and the column temperature is 25-45°C.
[0056] Preferably, mobile phase A is a potassium dihydrogen phosphate solution or an ammonium acetate solution with a concentration of 30 mmol / L, the detection wavelength is 260 nm, the flow rate is 1 ml / min, and the column temperature is 30°C.
[0057] Preferably, the diluent is a mixture of ethanol and isopropanol in a volume ratio of 2:8-8:2, and the mass concentration of the meloxicam degradation impurities in the diluent is 0.5-5 mg / ml.
[0058] Preferably, the elution mode of the liquid chromatography is gradient elution, and the elution procedure of the gradient elution is:
[0059] 0-15min, A:B is 90-70:10-30, V / V;
[0060] 15-25min, A:B is 70-90:30-10, V / V;
[0061] 25-35.1min, A:B is 90-70:10-30, V / V;
[0062] 35.1-40min, A:B is 90-70:10-30, V / V.
[0063] Compared with the prior art, the present invention has the following beneficial effects:
[0064] (1) The present invention provides a method for preparing meloxicam degradation impurities. The degradation products are refined by preparing liquid phase separation conditions and purification methods, and meloxicam degradation impurities with a purity of not less than 98.0% can be efficiently obtained. The meloxicam degradation impurities can be used as reference substances in the quality standard research and quality control of raw material and preparation production, and provide a basis for the quality research, standard research, stability research and mechanism research of adverse drug reactions of meloxicam.
[0065] (2) The present invention also provides a method for detecting the purity of meloxicam degradation impurities. The detection method is simple to operate, has accurate results, and can quickly and accurately determine the purity of meloxicam degradation impurities. BRIEF DESCRIPTION OF THE DRAWINGS
[0066] Figure 1 This is the preparative liquid chromatogram of the target impurity under the light destruction conditions of Example 1.
[0067] Figure 2 This is a chromatogram of the purity determination of impurities obtained by purification under light degradation conditions in Example 1.
[0068] Figure 3 This is the preparative liquid chromatogram of the target impurity under high temperature destruction conditions in Example 2.
[0069] Figure 4 This is a chromatogram of the purity determination of impurities obtained by purification under high temperature degradation conditions in Example 2.
[0070] Figure 5 This is the preparative liquid chromatogram of the target impurity under the oxidative destruction conditions of Example 3.
[0071] Figure 6 This is a chromatogram showing the purity of impurities obtained by purification under oxidative degradation conditions in Example 3.
[0072] Figure 7 This is the chromatogram of purity determination of the impurity mixture obtained by purification under different degradation conditions.
[0073] Figure 8 For the degradation of meloxicam impurities 1 H-NMR spectrum.
[0074] Fig. 9 For the degradation of meloxicam impurities 13 C-NMR spectrum.
[0075] Fig.10 LC-MS mass spectrum of meloxicam degradation impurities.
[0076] Fig.11 This is the UV absorption diagram of meloxicam degradation impurities.
[0077] Fig.12 This is the preparative liquid chromatogram of the target impurity under light destruction conditions in Comparative Example 1.
[0078] Fig.13 This is the preparative liquid chromatogram of the target impurity under high temperature destruction conditions in Comparative Example 2.
[0079] Fig.14 This is the preparative liquid chromatogram of the target impurity under oxidative destruction conditions in Comparative Example 3.
[0080] Fig.15 This is the preparative liquid chromatogram of the target impurity in Comparative Example 4. DETAILED DESCRIPTION
[0081] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which meloxicam is purchased from Shandong Xinhua Pharmaceutical Co., Ltd.; dimethyl sulfoxide is an AR grade reagent purchased from Sinopharm Holdings Co., Ltd.; n-hexane is an HPLC grade reagent purchased from Merck; methanol is an HPLC grade reagent purchased from Merck; isopropanol is an HPLC grade reagent purchased from Merck; hexafluoroisopropanol is an HPLC grade reagent purchased from Merck; acetonitrile is an HPLC grade reagent purchased from Merck; ethyl acetate is an HPLC reagent purchased from Merck; toluene is an AR grade reagent purchased from Chongqing Chuandong Chemical Co., Ltd.
[0082] Example 1
[0083] A method for preparing meloxicam degraded impurities, comprising the following steps:
[0084] (1) Meloxicam 100 mg was dissolved in 20 ml of dimethyl sulfoxide and solubilized by 300 W ultrasound for 10 min to obtain a 5 mg / ml meloxicam solution. The meloxicam solution was photodegraded at a light intensity of 6000 Lx for 9 days and a total light source illumination of 1.3 × 10 6 Lux·hr, near-ultraviolet lamp energy is 250W·hr / m 2 , to obtain degradation products;
[0085] (2) The degradation products were separated by column chromatography using a preparative liquid chromatograph of model LC-16P. The chromatographic parameters were as follows. The preparative chromatogram was as follows: Figure 1 As shown, a small beaker was used to collect the degradation impurity peak eluting component after the main peak of meloxicam, and the relative retention time of the impurity peak was 1.07.
[0086] The parameters of the preparative liquid chromatograph were set as follows:
[0087] Chromatographic column: Shim-pack GIS C18;
[0088] Chromatographic column stationary phase: octadecylsilane bonded silicon;
[0089] Chromatographic column inner diameter: 50mm;
[0090] Column length: 250mm;
[0091] Stationary phase filler particle size: 5μm;
[0092] Pore diameter: 10nm;
[0093] Detection wavelength: 260nm;
[0094] Flow rate: 10ml / min;
[0095] Column temperature: 30°C;
[0096] Sample volume: 2.5 ml;
[0097] Mobile phase A: a mixed solution of pH 5.5 diammonium hydrogen phosphate solution and methanol in a volume ratio of 90:10;
[0098] Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 7:3;
[0099] The elution program is shown in Table 1.
[0100] Table 1 Liquid phase elution procedure for photodegradation sample preparation
[0101] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 10 90 10 15 60 40 25 60 40 25.1 90 10 35 90 10
[0102] (3) The impurity outflow component liquid collected in the preparative liquid phase was transferred to a 150 ml clean separatory funnel, and then 10 ml of the extracting solution ethyl acetate was added for extraction. The mixture was shaken vigorously for more than half a minute to allow the impurity outflow component liquid and the extracting solution to be fully mixed. The mixture was then allowed to stand to separate into layers, and the upper organic phase solution was collected. 10 ml of the extracting solution was added to the lower aqueous phase solution, and the above extraction operation was repeated twice. The upper organic phase solutions extracted three times were combined.
[0103] (4) The organic phase solution extracted three times was transferred to another 150 ml clean separatory funnel, and then the same volume of ultrapure water was added for washing. The solution was shaken vigorously for more than half a minute, and then allowed to stand to separate. The upper organic phase solution was collected; the lower aqueous phase solution was discarded.
[0104] (5) The extracted organic phase solution is purged with nitrogen, the nitrogen purging gas flow rate is about 10 lpm, and the purging temperature is 40°C. The solid material obtained after the solvent is evaporated is placed in a vacuum dryer for drying to remove residual water or organic solvent; the vacuum drying temperature is 70°C, the pressure is 2.67 kPa, the desiccant is phosphorus pentoxide, the drying time is 24 hours, and the impurity yield is 5.6%.
[0105] (6) Take 5 mg of the purified and dried impurity sample (prepared in multiple liquid phases) and mix it with 10 ml of diluent (the diluent is a mixed solution of ethanol and isopropanol in a volume ratio of 8:2). The purity of the impurity is determined using the following chromatographic parameters. The determination spectrum is shown in Figure 2 As shown, the purity of the target impurity is 99.7% calculated based on the chromatogram area normalization method.
[0106] Chromatographic column: Welch Ultimate XB-C18 (4.6×150mm×5μm);
[0107] Mobile phase A: 30 mmol / L potassium dihydrogen phosphate solution at pH 5.5;
[0108] Mobile phase B: acetonitrile;
[0109] Detection wavelength: 260nm;
[0110] Flow rate: 1.0ml / min;
[0111] Column temperature: 30°C;
[0112] Injection volume: 10 μL;
[0113] The elution program is shown in Table 2.
[0114] Table 2 Elution program
[0115] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 15 70 30 25 70 30 35.1 90 10 45 90 10
[0116] Example 2
[0117] A method for preparing meloxicam degraded impurities, comprising the following steps:
[0118] (1) Dissolve 200 mg of meloxicam in 20 ml of hexafluoroisopropanol solution and shake vigorously to dissolve to obtain a 10 mg / ml meloxicam solution. The meloxicam solution is subjected to high temperature forced destruction at a temperature of 135° C. for a destruction time of 10 h to obtain a degradation product.
[0119] (2) The degradation products were separated by column chromatography using a preparative liquid chromatograph of model LC-16P. The chromatogram is shown in FIG. Figure 3 As shown, a small beaker was used to collect the degradation impurity peak eluting component after the main peak of meloxicam, and the relative retention time of the impurity peak was 1.08.
[0120] The parameters of the preparative liquid chromatography were set as follows:
[0121] Chromatographic column: Shim-pack GIS C18;
[0122] Chromatographic column stationary phase: octadecylsilane bonded silicon;
[0123] Chromatographic column inner diameter: 50mm;
[0124] Column length: 250mm;
[0125] Stationary phase filler particle size: 2μm;
[0126] Pore diameter: 5nm;
[0127] Detection wavelength: 260nm;
[0128] Flow rate: 10ml / min;
[0129] Column temperature: 30°C;
[0130] Sample volume: 4.0 ml;
[0131] Mobile phase A: a mixed solution of potassium dihydrogen phosphate solution with a pH of 4.5 and methanol in a volume ratio of 70:30;
[0132] Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 6:4;
[0133] The elution program is shown in Table 3.
[0134] Table 3 Elution program
[0135] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 15 60 40 25 60 40 25.1 95 5 45 95 5
[0136] (3) The impurity outflow component liquid collected in the preparative liquid phase was transferred to a 250 ml clean separatory funnel, and then 20 ml of toluene extract was added for extraction. The mixture was shaken vigorously for more than half a minute to allow the impurity outflow component liquid and the extract to be fully mixed. The mixture was then allowed to stand for stratification, and the upper organic phase solution was collected. 20 ml of the extract was added to the lower aqueous phase solution, and the above extraction operation was repeated twice. The upper organic phase solutions extracted three times were combined.
[0137] (4) The organic phase solution after three extractions was transferred to another 250 ml clean separatory funnel, and then the same volume of ultrapure water was added for washing. The solution was shaken vigorously for more than half a minute, and then allowed to stand to separate. The upper organic phase solution was collected, and the lower aqueous phase solution was discarded.
[0138] (5) The extracted organic phase solution is subjected to rotary evaporation at a rotary evaporation speed of 180 rpm, a rotary evaporation temperature of 50° C., and a rotary evaporation vacuum of 450 mbar. The solid material obtained after the solvent is evaporated is placed in a vacuum dryer for drying to remove residual water or organic solvent; the vacuum drying temperature is 80° C., the pressure is 2.67 kPa, the desiccant is anhydrous calcium chloride, the drying time is 24 h, and the impurity yield is 4.7%.
[0139] (6) Take 5 mg of the purified and dried impurity sample (prepared by multiple liquid phases) and mix it with 5 ml of diluent (the diluent is a mixed solution of ethanol and isopropanol in a volume ratio of 1:1). The purity of the impurity is determined using the following chromatographic parameters. The determination spectrum is shown in Figure 4 As shown, the purity of the target impurity is 99.4% calculated based on the chromatogram area normalization method.
[0140] Chromatographic column: Welch Ultimate XB-C18 (4.6×150mm×5μm);
[0141] Mobile phase A: 30 mmol / L potassium dihydrogen phosphate solution at pH 5.5;
[0142] Mobile phase B: acetonitrile;
[0143] Detection wavelength: 260nm;
[0144] Flow rate: 1.0ml / min;
[0145] Column temperature: 30°C;
[0146] Injection volume: 10 μL;
[0147] The elution program is shown in Table 4.
[0148] Table 4 Elution program
[0149] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 15 70 30 25 70 30 35.1 90 10 45 90 10
[0150] Example 3
[0151] A method for preparing meloxicam degraded impurities, comprising the following steps:
[0152] (1) Weigh 10 mg of meloxicam into a 25 ml volumetric flask, add 15 ml of isopropyl alcohol, shake vigorously to dissolve, then dilute to scale with 10% hydrogen peroxide, shake well, seal, and place in a 50°C thermostat for 5 hours to obtain a degradation product;
[0153] (2) The degradation products were separated by column chromatography using a preparative liquid chromatograph of model LC-16P. The chromatogram is shown in FIG. Figure 5 As shown, a small beaker was used to collect the degradation impurity peak eluting component after the main peak of meloxicam, and the relative retention time of the impurity peak was 1.08.
[0154] The parameters of the preparative liquid chromatography were set as follows:
[0155] Chromatographic column: Shim-pack GIS C18;
[0156] Chromatographic column stationary phase: octadecylsilane bonded silicon;
[0157] Chromatographic column inner diameter: 50mm;
[0158] Column length: 250mm;
[0159] Stationary phase filler particle size: 2μm;
[0160] Pore diameter: 5nm;
[0161] Detection wavelength: 260nm;
[0162] Flow rate: 10ml / min;
[0163] Column temperature: 30°C;
[0164] Sample volume: 2.0 ml;
[0165] Mobile phase A: a mixed solution of potassium dihydrogen phosphate solution with a pH of 4.5 and methanol in a volume ratio of 70:30;
[0166] Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 6:4;
[0167] The elution program is shown in Table 5.
[0168] Table 5 Elution program
[0169] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 15 60 40 25 60 40 25.1 95 5 45 95 5
[0170] (3) The impurity outflow component liquid collected in the preparative liquid phase was transferred to a 250 ml clean separatory funnel, and then extracted with 20 ml of n-heptane extract. The mixture was shaken vigorously for more than half a minute to allow the impurity outflow component liquid and the extract to be fully mixed. The mixture was then allowed to stand to separate into layers, and the upper organic phase solution was collected. 20 ml of the extract was added to the lower aqueous phase solution, and the above extraction operation was repeated twice. The upper organic phase solutions extracted three times were combined.
[0171] (4) The organic phase solution after three extractions was transferred to another 250 ml clean separatory funnel, and then the same volume of ultrapure water was added for washing. The solution was shaken vigorously for more than half a minute, and then allowed to stand to separate. The upper organic phase solution was collected, and the lower aqueous phase solution was discarded.
[0172] (5) The extracted organic phase solution is subjected to rotary evaporation at a rotary evaporation speed of 50 rpm, a rotary evaporation temperature of 45° C., and a rotary evaporation vacuum of 400 mbar. The solid material obtained after the solvent is evaporated is placed in a vacuum dryer for drying to remove residual water or organic solvent; the vacuum drying temperature is 60° C., the pressure is 2.67 kPa, the desiccant is silica gel, the drying time is 24 h, and the impurity yield is 1.8%.
[0173] (6) Take 2.5 mg of the purified and dried impurity sample (prepared by multiple liquid phases) and mix it with 1 ml of diluent (the diluent is a mixed solution of ethanol and isopropanol in a volume ratio of 2:8). The purity of the impurity is determined using the following chromatographic parameters. The determination spectrum is shown in Figure 6 As shown, the purity of the target impurity is 98.6% calculated based on the chromatogram area normalization method.
[0174] Chromatographic column: YMC-Triart-C18 (100 mm × 4.6 mm, 3 μm) chromatographic column;
[0175] Mobile phase A: 30 mmol / L ammonium acetate buffer, adjusted to pH 5.5 with ammonia or glacial acetic acid;
[0176] Mobile phase B: ethanol;
[0177] Detection wavelength: 260nm;
[0178] Flow rate: 1.0ml / min;
[0179] Column temperature: 35°C;
[0180] Injection volume: 20 μL;
[0181] The elution program is shown in Table 6.
[0182] Table 6 Elution program
[0183] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 85 15 15 75 25 25 75 25 35.1 85 15 40 85 15
[0184] Example 4
[0185] A structural identification of a meloxicam degradation impurity, the steps are as follows:
[0186] (1) The degradation impurities purified in Examples 1-3 were mixed, 10 mg of the mixture was taken and placed in a 5 ml volumetric flask, ethanol-isopropanol mixed solution (5:5, V / V) was added to dissolve and dilute to scale, and shaken to obtain an impurity sample solution with a concentration of 2 mg / ml. The impurity sample solution was taken for purity determination under the following chromatographic conditions. The chromatogram is shown in Figure 7 As shown. From the measurement results, it can be seen that after the degradation impurities purified by the three different preparation methods of Examples 1-3 are mixed, they are the same peak in the high performance liquid chromatography, indicating that the three substances are the same substance, and the purity of the sample is 98.9%, which meets the requirements for compound structure identification. Then the mixture is tested by superconducting pulse Fourier transform nuclear magnetic resonance (NMR), triple quadrupole liquid phase mass spectrometry (LC-MS) and ultraviolet visible spectrophotometry (UV).
[0187] Chromatographic column: YMC-Triart-C18 (100 mm × 4.6 mm, 3 μm) chromatographic column;
[0188] Mobile phase A: 30 mmol / L ammonium acetate buffer, adjusted to pH 5.5 with ammonia or glacial acetic acid;
[0189] Mobile phase B: ethanol;
[0190] Detection wavelength: 260nm;
[0191] Flow rate: 1.0ml / min;
[0192] Column temperature: 35°C;
[0193] Injection volume: 20 μL;
[0194] The elution program is shown in Table 7.
[0195] Table 7 Elution program
[0196] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 85 15 15 70 30 25 70 30 35.1 85 15 40 85 15
[0197] (2) In the superconducting pulse Fourier transform nuclear magnetic resonance (NMR) measurement, the instrument model used was BrukerAVANCE AV400, and the solvent for dissolving the sample was deuterated chloroform (CDCl3). 1 H-NMR spectrum (such as Figure 8 shown) and 13 C-NMR spectrum (such as Fig. 9 shown).
[0198] (3) In the triple quadrupole liquid phase mass spectrometry (LC-MS) determination, the instrument model used was Agilent 1260TSQQuantum Ultra, the ionization source was ESI, the positive ion scanning mode, the capillary voltage was 4000v, the drying gas flow rate was 12L / min, the drying gas temperature was 400°C, and the nebulizer pressure was 70psi. Fig.10 Mass spectrum shown.
[0199] (4) In the UV-visible spectrophotometry (UV) determination, the instrument model used was PerkinElmer Lambda950 UV-visible spectrophotometer, the solvent for dissolving the sample was anhydrous ethanol, the scanning wavelength range was 200-400 nm, the wavelength scanning rate was 100 nm / min, and the following was obtained: Fig.11 UV absorption diagram shown.
[0200] (5) Through the above 1 H-NMR spectrum, 13 C-NMR spectrum, LC-MS and UV spectra were analyzed in combination with the structure and properties of meloxicam. The structure of the degradation impurity is shown in Formula 1.
[0201]
[0202] Comparative Example 1
[0203] The same as Example 1, the only difference is that the illumination conditions in step (1) are different, specifically, the illumination intensity is 3500Lx, the illumination time is 5 days, and the total illumination of the light source is 4.2×10 5 Lux·hr, near-ultraviolet lamp energy is 250W·hr / m 2 .
[0204] The yield of the degradation impurity of meloxicam was 0.003%. The preparative liquid chromatography of the degraded sample was shown in Fig.12 ,Depend on Fig.12 It can be seen that only a trace amount of target impurities were degraded at the relative retention time of 1.08, and the impurity preparation yield was too low to meet the experimental requirements.
[0205] Comparative Example 2
[0206] The same as Example 2, the only difference is that the high temperature conditions in step (1) are different, specifically, the destruction is carried out at a temperature of 35° C. for 5 hours.
[0207] The yield of the degradation impurity of meloxicam was 0.002%. The preparative liquid chromatography spectrum of the degraded sample was shown in Fig.13 ,Depend on Fig.13 It can be seen that only a trace amount of target impurities were degraded at the relative retention time of 1.08, and the impurity preparation yield was too low to meet the experimental requirements.
[0208] Comparative Example 3
[0209] The method is the same as Example 3, except that the oxidation conditions in step (1) are different, specifically, the sample is diluted with 1% by volume hydrogen peroxide, shaken well, sealed, and placed at 25° C. for 2 h.
[0210] The yield of the degradation impurity of meloxicam was 0.003%. The preparative liquid chromatography spectrum of the degraded sample was shown in Fig.14 ,Depend on Fig.14 It can be seen that only a trace amount of target impurities were degraded at the relative retention time of 1.08, and the impurity preparation yield was too low to meet the experimental requirements.
[0211] Comparative Example 4
[0212] The same as Example 1, the only difference is that the chromatographic conditions in step (2) are different, specifically:
[0213] Mobile phase A: a mixture of pH 5.5 diammonium hydrogen phosphate solution and methanol in a volume ratio of 50:50;
[0214] Mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 4:6;
[0215] The elution program is shown in Table 8.
[0216] Table 8 Elution program
[0217]
[0218]
[0219] The preparative liquid chromatogram of the degraded sample is shown in Fig.15 ,Depend on Fig.15 It can be seen that due to the adjustment of the component ratio of mobile phase A and mobile phase B, as well as the change of the elution gradient conditions, the peak of the target impurity after degradation appeared too early, and it could not be effectively separated from other degraded impurities, making it impossible to access the target impurity elution component from the preparative chromatography.
[0220] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.
Claims
1. A method for preparing meloxicam by degrading impurities, characterized in that: The steps include: (1) dissolving meloxicam in an organic solvent and degrading it to obtain a degradation product; (2) Separating and purifying the degradation products by column chromatography to obtain: Wherein, the degradation in step (1) is light degradation, high temperature degradation or oxidative degradation, The light intensity of the light degradation is 4000-8000Lx, the light degradation time is 5-11 days, and the total illumination of the light source is not less than 1.2×10 6 Lux·hr.
2. The preparation method according to claim 1, characterized in that: The organic solvent in step (1) is selected from one or more of dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetone, isopropanol and hexafluoroisopropanol.
3. The preparation method according to claim 2, characterized in that: The concentration of meloxicam after being dissolved in an organic solvent is 0.1-10 mg / ml.
4. The preparation method according to claim 1, characterized in that: The temperature of the high temperature degradation is 40-150° C., and the time of the high temperature degradation is 5-12 hours.
5. The preparation method according to claim 1, characterized in that: The oxidation degradation process includes mixing with hydrogen peroxide with a volume concentration of 3-10%, the oxidation degradation temperature is 30-50° C., and the oxidation degradation time is 3-8 hours.
6. The preparation method according to claim 1, characterized in that: The conditions for the column chromatography separation in step (2) include: mobile phase A is a mixture of phosphate buffer and methanol in a volume ratio of 70:30-95:5, mobile phase B is a mixture of methanol and acetonitrile in a volume ratio of 6:4-8:2, and the elution mode of the column chromatography separation is gradient elution.
7. The preparation method according to claim 6, characterized in that: The phosphate buffer is potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, ammonium dihydrogen phosphate solution or diammonium hydrogen phosphate solution, and the pH of the phosphate buffer is 4.5-6.
5.
8. The preparation method according to claim 6, characterized in that: The elution procedure of the gradient elution is: 0-10min, A:B is 100-80:0-20, V / V; 15-25min, A:B is 80-60:20-40, V / V; 25.1-35min, A:B is 100-80:0-20, V / V.
9. The preparation method according to claim 6, characterized in that: The conditions for column chromatography separation in step (2) also include: the chromatographic column is Shim-pack GIS C18 or USHA C18, the detection wavelength is 250-270nm, the flow rate is 1-20ml / min, and the column temperature is 25-45°C.
10. The preparation method according to claim 1, characterized in that: After the column chromatography separation in step (2) is completed, the method further comprises: collecting liquid components with a relative retention time of 1.05-1.
10.
11. The preparation method according to claim 1, characterized in that: The purification comprises dissolving the liquid component in a non-polar solvent, extracting, evaporating the non-polar solvent, and drying to obtain the product.
12. The preparation method according to claim 11, characterized in that: The non-polar solvent is ethyl acetate, toluene, n-hexane, dichloromethane, chloroform or n-heptane.
13. The preparation method according to claim 11, characterized in that: The method of volatilizing the non-polar solvent is nitrogen purging or rotary evaporation, the gas flow rate of the nitrogen purging is 1-20lpm, the temperature of the nitrogen purging is 25-60°C; the rotation speed of the rotary evaporation is 20-180rpm, the temperature of the rotary evaporation is 35-60°C, and the vacuum degree of the rotary evaporation is 250-500mbar.
14. The preparation method according to claim 13, characterized in that: The drying temperature is 45-80° C., the drying pressure is less than 2.67 kPa, the drying time is 5-24 hours, and a desiccant is required for the drying, and the desiccant is phosphorus pentoxide, anhydrous calcium chloride or silica gel.
15. Use of the meloxicam degradation impurity prepared by the preparation method according to any one of claims 1 to 14 as a meloxicam impurity reference substance.
16. A purity detection method for meloxicam degradation impurities, characterized in that: The method comprises mixing the meloxicam degradation impurities prepared by the preparation method according to any one of claims 1 to 14 with a diluent, and performing liquid chromatography detection to obtain: The conditions of the liquid chromatography include: the chromatographic column is Welch Ultimate XB-C18 or YMC-Triart-C18, the mobile phase A is a potassium dihydrogen phosphate solution or an ammonium acetate solution with a concentration of 20-40 mmol / L, the mobile phase B is acetonitrile, the detection wavelength is 250-270 nm, the flow rate is 0.8-1.2 ml / min, and the column temperature is 25-45°C.
17. The detection method according to claim 16, characterized in that: The diluent is a mixture of ethanol and isopropanol in a volume ratio of 2:8-8:2, and the mass concentration of the meloxicam degradation impurities in the diluent is 0.5-5 mg / ml.
18. The detection method according to claim 16, characterized in that: The elution mode of the liquid chromatography is gradient elution, and the elution procedure of the gradient elution is: 0-15min, A:B is 90-70:10-30, V / V; 15-25min, A:B is 70-90:30-10, V / V; 25-35.1min, A:B is 90-70:10-30, V / V; 35.1-40min, A:B is 90-70:10-30, V / V.