Detection method of alprazolam related substances

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of alprazolam related substances, the detection method adopts high performance liquid chromatography, and the chromatographic conditions are as follows: a mobile phase A and a mobile phase B are adopted as mixed mobile phases for gradient elution; wherein the mobile phase A is a methanol-ammonium acetate solution or an acetonitrile-ammonium acetate solution, and the mobile phase B is a mixed solution of methanol and / or acetonitrile and an ammonium acetate solution, the detection method provided by the invention has the advantages that the detected impurities are more, the retention ability of main components and each impurity in the mobile phase system is stronger, the peak response is higher, each compound can be effectively separated, and the detection accuracy is high. And related substances in alprazolam can be well monitored.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and in particular relates to a method for detecting alprazolam-related substances. Background Art

[0002] Alprazolam is a benzodiazepine hypnotic sedative and antianxiety drug. It is a third-generation central nervous system depressant drug in recent years. It was developed and marketed by Upjohn (now a subsidiary of Pfizer) in 1981 to treat depression, insomnia and other diseases. Its molecular target is GABA-Areceptor. It has similar pharmacological effects to diazepam, but its adverse reactions such as fatigue, dizziness, dry mouth and nausea are milder than diazepam. Its chemical name is: 1-methyl-6-phenyl-8-chloro-4H-〔1,2,4-triazole) and [4,3-α][1,4]〕-benzodiazepine (Compound shown in Formula I) has the following structural formula:

[0003]

[0004] Patent US4000151 discloses a synthetic route of alprazolam, as shown below:

[0005]

[0006] Among them, intermediates I-1, I-2 and I-3, as key intermediates in the synthesis route, have a high risk of residues in alprazolam APIs and are listed in the European Pharmacopoeia impurities. In addition to the above three impurities, by-products of each step may also remain in the product alprazolam, thereby affecting the purity of alprazolam. Even after complicated post-processing and purification processes, it is difficult to completely remove the impurities.

[0007] There are few literature reports on the detection method of alprazolam-related substances. Among them, the detection method of alprazolam-related substances included in the European Pharmacopoeia (EP) cannot simultaneously separate and detect multiple known major related substances of alprazolam, and the impurities have a short retention time and weak response, which cannot guarantee the quality and safety of alprazolam.

[0008] The study of related substances is an extremely important project in drug quality research. Its content not only directly reflects the purity of the drug, but also has important safety significance. In order to ensure the safety and effectiveness of alprazolam preparations, it is necessary to study, detect and control the related substances of the raw materials. According to the synthetic process of this product, the related substances are mainly intermediates, reaction by-products and degradation products in the production process.

[0009] The present invention develops a new detection method for alprazolam-related substances based on the prior art, which can realize the localization and quantitative research of impurities in alprazolam and lay a foundation for the quality evaluation of alprazolam preparations. Summary of the invention

[0010] The object of the present invention is to provide a method for detecting alprazolam-related substances, wherein the method can simultaneously detect multiple alprazolam-related substances, and the retention capacity of the main component and each impurity in the mobile phase system is relatively strong, the peak response is relatively high, and each related substance can be effectively separated, thereby being able to better monitor the content of related substances in alprazolam.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] A method for detecting alprazolam-related substances, the detection method adopts high performance liquid chromatography, the liquid chromatography determination conditions are: the chromatographic column is a C18 column, methanol-ammonium acetate solution or acetonitrile-ammonium acetate solution is used as mobile phase A, and a mixture of methanol and / or acetonitrile and ammonium acetate solution is used as mobile phase B for gradient elution, the pH value of the ammonium acetate solution is 3.2-5.7, preferably the pH value is 4.6-4.8, and more preferably the pH value is 4.7, and the related substances include one or more of the following substances:

[0013]

[0014] Furthermore, the C18 chromatographic column described in the present invention is Welch AQ C18 chromatographic column, column specifications are 4.6×250mm, 5μm.

[0015] Furthermore, the mobile phase gradient elution procedure in the present invention is as follows:

[0016] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0 50-100 0-50 20 50-100 0-50 40 0-20 80-100 50 0-20 80-100 50.1 50-100 0-50 60 50-100 0-50

[0017] Preferably, the mobile phase gradient elution procedure is as follows:

[0018] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0 65 35 20 65 35 40 10 90 50 10 90 50.1 65 35 60 65 35

[0019] Further, the mobile phase A is methanol-ammonium acetate solution, wherein the volume ratio of ammonium acetate solution to methanol is 60-90:10-40, preferably 78-82:18-22, more preferably 80:20;

[0020] Further preferably, the mobile phase B is a mixed solution of ammonium acetate solution and methanol and acetonitrile, wherein the volume ratio of ammonium acetate solution to methanol and acetonitrile is 1-10:40-60:40-50, preferably 4-6:54-56:40, and more preferably 5:55:40.

[0021] Furthermore, the pH value of the ammonium acetate solution is 4.7, and the pH regulator of the ammonium acetate solution is selected from a mixture of one or more of glacial acetic acid and phosphoric acid, preferably glacial acetic acid.

[0022] Furthermore, the detection wavelength in the chromatographic conditions is: 252-256 nm; preferably the detection wavelength is 254 nm,

[0023] Further preferably, the flow rate in the chromatographic conditions is 0.9-1.1 ml / min; preferably the flow rate is 1.0 ml / min;

[0024] Further preferably, the column temperature in the chromatographic conditions is 33-37°C; preferably the column temperature is 35°C;

[0025] Further preferably, the injection volume in the chromatographic conditions is 10-100 μl; preferably the injection volume is 20 μl;

[0026] More preferably, the concentration of the test solution in the chromatographic conditions is 0.05-1.0 mg / ml; preferably, the concentration of the test solution is 1.0 mg / ml.

[0027] The present invention provides a detection step, comprising:

[0028] (1) Solution preparation: prepare blank solution, system suitability solution, reference solution, and test solution separately;

[0029] (2) Determination method: After the system is stable, inject blank solution, system suitability solution, reference solution, and test solution respectively, and record the chromatogram;

[0030] Among them, the chromatographic conditions are: C18 chromatographic column is Welch AQ C18 chromatographic column, column specifications are 4.6×250mm, 5μm; detection wavelength is 254nm, flow rate is 1.0ml / min, column temperature is 35℃, injection volume is 20μl, and the concentration of the test solution is 1.0mg / ml;

[0031] The mobile phase A is a methanol-ammonium acetate solution, wherein the volume ratio of the ammonium acetate solution to methanol is 80:20; the mobile phase B is a mixed solution of ammonium acetate solution, methanol and acetonitrile, wherein the volume ratio of the ammonium acetate solution to methanol and acetonitrile is 5:55:40; the pH value of the ammonium acetate solution is 4.7, and the mobile phase gradient elution procedure is as follows:

[0032] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0 50-100 0-50 20 50-100 0-50 40 0-20 80-100 50 0-20 80-100 50.1 50-100 0-50 60 50-100 0-50 ;

[0033] Preferably, the mobile phase gradient elution procedure is as follows:

[0034] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0 65 35 20 65 35 40 10 90 50 10 90 50.1 65 35 60 65 35 .

[0035] The present invention also provides a compound represented by impurity Q:

[0036]

[0037] Furthermore, the present invention also provides a method for preparing impurity Q, comprising: preparing impurity Q compound by ring-closing reaction of impurity H compound with lithium hexamethyldisilazide in a solvent.

[0038] The reaction formula is as follows:

[0039]

[0040] The preparation method of impurity Q comprises: the reaction solvent is one or more of tetrahydrofuran, cyclohexane, and n-hexane, preferably tetrahydrofuran; and the reaction temperature is -10-30°C, preferably 0-10°C.

[0041] Furthermore, the detection method is suitable for detecting related substances in alprazolam raw materials or preparations.

[0042] Beneficial Effects

[0043] The technical solution of the present invention has the following advantages:

[0044] (1) The mobile phase system adopted in the present invention can effectively improve the retention capacity of the main component and each impurity, and each component has a high response and a good peak shape, which solves the problem that alprazolam and its impurities have weak retention capacity, low response and poor peak shape in the general buffer salt mobile phase system, and can effectively detect the related substances in alprazolam, including the 6 related substances in the European Pharmacopoeia and the new compound impurity Q, so as to more accurately ensure the quality control of alprazolam raw materials and pharmaceutical preparations, and ultimately determine the safety and effectiveness of the products. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the blank solution spectrum of the present invention.

[0046] Figure 2 This is a solution spectrum of the system applicability of the present invention.

[0047] Figure 3 It is the mass spectrum of impurity Q compound of the present invention.

[0048] Figure 4 It is the hydrogen NMR spectrum of the impurity Q compound of the present invention.

[0049] Figure 5 It is the NMR carbon spectrum of the impurity Q compound of the present invention. DETAILED DESCRIPTION

[0050] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technical schemes implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0051] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0052] Example 1: Specificity test

[0053] (1) Instruments: high performance liquid chromatograph, electronic analytical balance, pH meter, etc.

[0054] (2) Solution preparation:

[0055] Diluent: Methanol

[0056] Test solution: Weigh an appropriate amount of alprazolam, dissolve it in methanol and dilute it to make a solution containing approximately 1 mg of alprazolam per 1 ml.

[0057] Stock solution of reference substance: Take appropriate amount of alprazolam, impurity B, C, D, F, H, Q and J reference substances respectively, weigh accurately, place in different volumetric bottles, dissolve and dilute with methanol to make a solution containing about 200 μg per 1 ml.

[0058]

[0059]

[0060] Reference solution: Accurately measure appropriate amounts of alprazolam reference stock solution and each impurity reference stock solution and dilute with methanol to make solutions containing approximately 5 μg of each substance per 1 ml.

[0061] Impurity localization solutions: Accurately measure an appropriate amount of each impurity reference stock solution, place it in different volumetric flasks, and dilute it with methanol to make a solution containing 5 μg of impurity per 1 ml.

[0062] System suitability solution: Take an appropriate amount of alprazolam test sample, weigh it accurately, add an appropriate amount of each impurity reference substance stock solution, dissolve it in methanol and dilute it to make a solution containing about 5 μg of impurities and about 1 mg of alprazolam per 1 ml.

[0063] Accurately measure 20 μl of each of the diluent, reference solution, reference positioning solution, and system suitability solution, inject them into the liquid chromatograph, and record the chromatogram.

[0064] Acceptable criteria: when injecting diluent, there should be no interfering peaks; when continuously injecting 6 needles of reference solution, the RSD of the peak area of ​​6 needles should be ≤10%; when injecting system suitability solution, the separation between each peak should meet the requirements; after the system meets the requirements, inject the test solution, system suitability solution, separation solution and each positioning solution.

[0065] Table 1 Specificity test results

[0066]

[0067]

[0068] The diluent does not interfere with the determination of related substances. In the system suitability solution, the separation between alprazolam and adjacent peaks and known impurity peaks meets the requirements; the theoretical plate number is not less than 2000 based on the alprazolam peak, and the peak shape of each impurity is good, indicating that this chromatographic condition is suitable for the determination of related substances.

[0069] Example 2: Destructive test

[0070] Take alprazolam and conduct acid, alkali, oxidation, high temperature and light tests respectively. The contents are as follows:

[0071] a. The test solution is not destroyed

[0072] Undamaged test solution: Weigh 10 mg of alprazolam into a 10 ml volumetric flask, dissolve it in methanol and make up to volume.

[0073] b. High temperature damage

[0074] High temperature destruction of the test solution: Take 10 mg of the product and place it in a 105℃ oven for 12 hours, dissolve it in methanol and make up to volume.

[0075] c. Acid-base damage

[0076] Acid-base blank: Take 1 ml of 0.1 mol / L hydrochloric acid in a 10 ml volumetric flask, add an appropriate amount of methanol, add 0.1 mol / L sodium hydroxide solution to adjust to neutrality, and dilute to the scale with methanol.

[0077] Acid destruction of test solution: Take 10 mg of the product and place it in a 10 ml volumetric flask, add 1 ml of 0.1 mol / L hydrochloric acid and let it stand for 0.5 h, add appropriate amount of methanol, add 0.1 mol / L sodium hydroxide solution to adjust to neutrality, and dilute to the scale with methanol.

[0078] Alkali destruction test solution: Take 10 mg of the product and place it in a 10 ml volumetric flask, add 1 ml of 0.1 mol / L sodium hydroxide solution and let it stand for 24 hours, add appropriate amount of methanol, add 0.1 mol / L hydrochloric acid to adjust to neutrality, and dilute to the scale with methanol.

[0079] d. Oxidative damage

[0080] Oxidative destruction blank: Take 1 ml of 0.3% hydrogen peroxide solution in a 10 ml volumetric flask and dilute to the mark with methanol.

[0081] Oxidative damage: Take 10 mg of this product and place it in a 10 ml volumetric flask, add 1 ml of 0.3% hydrogen peroxide solution, shake well, leave it at room temperature for 24 hours, and dilute it to the scale with methanol.

[0082] e. Light damage

[0083] Light irradiation damage to the test solution: Take 10 mg of the product and place it in a strong light irradiation test box (illuminance 5000±500lx, near-ultraviolet energy 90μW / cm 2 ) for 10 days, dissolved in methanol and fixed to volume

[0084] Reference solution and system suitability solution: Preparation method is the same as that of "Specificity test in Example 1".

[0085] Take 20 μl of system suitability solution, reference solution, undamaged solution and each of the above-mentioned damaged test solutions and inject them into the liquid chromatograph.

[0086] Calculation formula:

[0087]

[0088] C 对 : Concentration of known impurities in reference solution, μg / ml;

[0089] C 样 : Alprazolam concentration in the test solution, μg / ml;

[0090] A 样 : Peak area of ​​known impurities in the test solution;

[0091] A 对 : Peak area of ​​known impurities in reference solution;

[0092]

[0093] C 对 : Alprazolam concentration in the reference solution, μg / ml;

[0094] C 样 : Alprazolam concentration in the test solution, μg / ml;

[0095] A 样 : The peak area of ​​unknown impurities in the test solution;

[0096] A 对 : Peak area of ​​alprazolam in reference solution;

[0097] Total impurities (%) = known impurities % + unknown impurities %

[0098]

[0099] A 未破 : The peak area of ​​alprazolam in the test solution was not destroyed;

[0100] A 破 : Peak area of ​​alprazolam in the destruction test solution;

[0101] m 未破 : The sample weight of the test solution without destroying it;

[0102] A 破 : The sample amount of the destruction test solution;

[0103] Acceptable standard: The degradation products produced by this product after being destroyed by acid, alkali, oxidation, high temperature and light can be well detected, and the separation degree between each degradation product and the main component peak should meet the requirements; the purity of the main peak should meet the requirements, and the percentage of total material should be between 90% and 110%.

[0104] Table 2 Summary of destructive test results

[0105]

[0106] Impurity B ND ND ND ND ND ND Impurity C ND ND ND ND ND ND Impurity D ND ND ND ND ND ND Impurity F ND ND ND ND ND ND Impurity Q ND ND ND ND ND ND Impurity H ND ND ND ND ND ND Impurity J 0.064 0.065 0.072 0.065 0.062 0.065 Maximum single impurity (%) 0.027 0.123 0.030 0.031 0.029 0.028 Total impurities (%) 0.091 0.231 0.102 0.096 0.091 0.093

[0107] Table 3 Destruction test material balance

[0108]

[0109] Conclusion: 1. It is unstable under acid destruction, but relatively stable under alkali destruction, oxidation destruction, light and high temperature.

[0110] 2. No new impurities were found in this product after alkali destruction, oxidation destruction, light destruction and high temperature destruction. Under acidic conditions, the seven-membered ring in the alprazolam structure will open to generate open-ring products. Above pH 6.0, the open-ring products will automatically close to alprazolam. Under 0.1 mol / L hydrochloric acid for 0.5 h, impurities increase slightly. In the test solution, the degradation products produced under each destruction condition can be well separated from alprazolam, and the peak purity factor of the main peak meets the requirements. After destruction, the materials are basically balanced.

[0111] 3. Under each destruction condition, each impurity can be well separated from the main component, and the material before and after destruction is basically balanced; the main peak purity of each destroyed sample and undestroyed sample was analyzed, and the results all met the requirements.

[0112] Example 3: Sensitivity

[0113] The preparation method of each reference substance stock solution is the same as that of "Example 1: Specificity Test".

[0114] Quantitation limit solution: Accurately measure an appropriate amount of reference stock solution and dilute it step by step with methanol. If the signal-to-noise ratio S / N is not less than 10, the amount of sample added is the quantitation limit.

[0115] Detection limit solution: accurately measure an appropriate amount of the quantitative limit solution and dilute it step by step with methanol. When the signal-to-noise ratio S / N is not less than 3, the amount of sample injected is the detection limit. Repeat the injection of the quantitative limit solution for 6 times, and the RSD of the peak area is required to be ≤10%.

[0116] Table 4 Quantitation limit determination results

[0117]

[0118]

[0119]

[0120] Table 5 Detection limit determination results

[0121]

[0122] The test results show that the quantitative limit concentration and detection limit concentration of each impurity and alprazolam are low, and can be well detected and accurately quantified; the quantitative limit solution was repeatedly injected 6 times, and the RSD of the peak area was less than 10%, indicating that the method has a high sensitivity.

[0123] Example 4: Linearity and Range

[0124] Take 5 concentration points within the range of the quantitative limit and the 150% limit, perform linear regression on the peak area y against the concentration x, calculate the linear equation and correlation coefficient, and the correlation coefficient r should be greater than 0.99.

[0125] Table 6 Impurity B and impurity C linearity test results

[0126]

[0127] Table 7 Results of linearity study of alprazolam and impurity D

[0128]

[0129] Table 8 Impurity F and impurity Q linearity test results

[0130]

[0131] Table 9 Impurity H and impurity J linearity test results

[0132]

[0133]

[0134] Impurity B has a good linear relationship in the concentration range of 0.10μg / ml to 7.85μg / ml (n=5); the correlation coefficient r is 0.99988; Impurity C has a good linear relationship in the concentration range of 0.11μg / ml to 8.07μg / ml (n=5); the correlation coefficient r is 0.99995; Alprazolam has a good linear relationship in the concentration range of 0.10μg / ml to 7.83μg / ml (n=5); the correlation coefficient r is 0.99990; Impurity D has a good linear relationship in the concentration range of 0.04μg / ml to 7.61μg / ml (n=5); the correlation coefficient r is 0.999988 ; Impurity F has a good linear relationship in the concentration range of 0.04μg / ml to 7.74μg / ml (n=5); the correlation coefficient r is 0.999989; Impurity Q has a good linear relationship in the concentration range of 0.03μg / ml to 7.91μg / ml (n=5); the correlation coefficient r is 0.99997; Impurity H has a good linear relationship in the concentration range of 0.04μg / ml to 8.34μg / ml (n=5); the correlation coefficient r is 0.99995; Impurity J has a good linear relationship in the concentration range of 0.04μg / ml to 7.83μg / ml (n=5); the correlation coefficient r is 0.99998.

[0135] Example 5: Correction Factor

[0136] Different people used different instruments and different chromatographic columns to perform linear determinations of alprazolam and each impurity. The slope of the alprazolam linear equation was divided by the slope of the impurity linear equation to calculate the correction factor of each impurity relative to alprazolam.

[0137] Table 10 Results of determination of each impurity correction factor

[0138]

[0139] It can be seen from the above table that the correction factors of impurities measured by the three different liquid phases are basically the same. The correction factors of impurities B, C, D, F, Q, H, and J are 0.98, 0.95, 0.65, 1.05, 1.01, 1.19, and 1.54, respectively.

[0140] Example 6: Accuracy

[0141] The accuracy is achieved by measuring the recovery rate between the actual concentration and the theoretical concentration of each component. It is required that the recovery rate of each component in the solution with 80% limit concentration, 100% limit concentration and 120% limit concentration recovery rate should be between 80% and 120%.

[0142] Table 11 Impurity B average recovery test results

[0143]

[0144]

[0145] Table 12 Impurity C average recovery test results

[0146]

[0147] Table 13 Impurity D average recovery test results

[0148]

[0149] Table 14 Impurity F average recovery test results

[0150]

[0151]

[0152] Table 15 Impurity Q average recovery test results

[0153]

[0154] Table 16 Impurity H average recovery test results

[0155]

[0156] Table 17 Impurity J average recovery test results

[0157]

[0158]

[0159] The recovery rates of each impurity at each concentration level were all within 80%-120% and the RSDs were all less than 10% (n=9), indicating that the chromatographic conditions used to determine the content of related substances in this product had a high recovery rate and good accuracy.

[0160] Example 7: Precision

[0161] Repeatability was achieved by measuring the mean and RSD of the test results of 6 replicates of the test solution.

[0162] The intermediate precision was determined by different testers using different instruments at different times to test 12 samples of the test product, and the mean value and RSD of the detected values ​​were calculated.

[0163] Acceptable criteria; related substance detection values: LOQ to 0.1%, RSD not exceeding 40.0%; >0.1% to 0.2%, RSD not exceeding 30.0%; >0.2% to 0.5%, RSD not exceeding 15.0%; >0.5% to 5%, RSD not exceeding 7.5%; ≥5%, RSD not exceeding 4.0%.

[0164] Table 18 Repeatability and intermediate precision test results

[0165]

[0166]

[0167] In the repeatability test, the RSDs of known impurities, maximum unknown impurities and total impurities in 6 samples all met the requirements, and the contents of known impurities, maximum unknown impurities and total impurities were basically consistent; in the intermediate precision test, the RSDs of known impurities, maximum unknown impurities and total impurities in 12 samples all met the requirements, and the contents of known impurities, maximum unknown impurities and total impurities were basically consistent, indicating that the method has good precision.

[0168] Example 8: Solution Stability

[0169] Place the system suitability solution, reference solution and test solution in an injector controlled at 15°C, accurately measure 20 μl at different time points and inject into the liquid chromatograph, record the chromatogram, and count the changes in the peak area of ​​each impurity.

[0170] Table 19 Peak area determination results of the reference solution 15°C storage stability test

[0171]

[0172] Table 20 System suitability solution 15℃ storage stability test separation results

[0173]

[0174] Table 21 Determination results of related substances in the stability of the test solution at 15°C

[0175]

[0176] At 15°C, the reference solution is stable for 59 hours, the system suitability solution is stable for 69 hours, and the test solution is stable for 69 hours.

[0177] Example 9: Durability

[0178] Purpose of the durability test: To determine whether the determination of sample-related substances is affected by slight changes in chromatographic conditions.

[0179] The determination conditions are: slight changes in wavelength, column temperature, flow rate, mobile phase ratio, pH value of the salt solution and the use of different chromatographic columns.

[0180] Table 22 Parameters of chromatographic condition changes

[0181]

[0182] Investigate the separation between the main peak, known impurity peak and adjacent impurity peak in the system suitability solution under various conditions, the retention time, tailing factor and theoretical plate number of the main peak and known impurity peak; calculate the impurity determination results in the sampled test solution, use the external standard method to calculate the known impurities, use the main component external standard method to calculate the unknown impurities, the sum of the total impurities, and calculate the ratio of the content of the relevant substances under various changing conditions to the initial conditions.

[0183] Example 10 Effect of wavelength variation on the determination results of related substances

[0184] Accurately measure 20 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph at wavelengths of 252 nm, 254 nm, and 256 nm, and record the chromatograms.

[0185] Table 23 Effect of the change of detection wavelength of related substances on the measurement results

[0186]

[0187]

[0188] From the data in the table above, we can see that the content of related substances in this product is determined using three different wavelengths of 252nm, 254nm and 256nm. The retention time and theoretical plate number of each component peak do not change significantly with the change of wavelength. The separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in wavelength basically does not affect the determination results of related substances in this product.

[0189] Example 11 Effect of pH Change of Ammonium Acetate Solution in Mobile Phase on Determination Results of Related Substances

[0190] Accurately measure 20 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the condition of mobile phase acetate pH 4.6, 4.7, and 4.8, and record the chromatograms.

[0191] Table 24 Effect of pH changes of buffer solutions on the determination results

[0192]

[0193]

[0194] From the data in the above table, we can see that when using three different pH acetate solutions to prepare mobile phases to determine the related substances of this product, the retention time of each component peak and the number of theoretical plates change slightly, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in the pH value of the acetate solution in the mobile phase does not basically affect the determination results of the related substances of this product.

[0195] Example 12 Effect of a small change in the mobile phase ratio on the determination results of related substances

[0196] Accurately measure 20 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the condition of changing the mobile phase ratio, and record the chromatogram.

[0197] Table 25 Effect of changes in mobile phase A of related substances on the determination results

[0198]

[0199]

[0200] Table 26 Effect of changes in mobile phase B of related substances on the determination results

[0201]

[0202]

[0203] From the data in the above table, we can see that when different proportions of mobile phase are used to determine the content of related substances in this product, the retention time and theoretical plate number of each component peak basically do not change with the change of mobile phase ratio, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in the mobile phase ratio basically does not affect the determination results of the content of related substances in this product.

[0204] Example 13 Effect of flow rate change on the determination results of related substances

[0205] Accurately measure 2 μl of system suitability solution, test solution, and reference solution, and inject them into the liquid chromatograph at flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively, and record the chromatograms.

[0206] Table 27 Effect of the flow rate change of related substances on the measurement results

[0207]

[0208]

[0209] From the data in the table above, we can see that when three different flow rates of 0.9ml / min, 1.0ml / min and 1.1ml / min were used to determine the content of related substances in this product, the retention time of each component peak slightly advanced with the increase of flow rate, the number of theoretical plates slightly changed, the separation of each chromatographic peak met the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions did not change significantly. This shows that a slight change in flow rate basically does not affect the determination results of related substances in this product.

[0210] Example 14 Effect of column temperature change on related substance determination results

[0211] Accurately measure 20 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph at column temperatures of 33°C, 35°C, and 37°C, and record the chromatograms.

[0212] Table 28 Effect of column temperature changes on determination results of related substances

[0213]

[0214]

[0215] Conclusion: From the data in the above table, we can see that when the content of related substances of this product is determined at three different column temperatures of 33℃, 35℃ and 37℃, the retention time and theoretical plate number of each component peak do not change significantly, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that a slight change in column temperature basically does not affect the determination results of related substances of this product.

[0216] Example 15 Effect of different batches of chromatographic columns of the same model on relevant measurement results

[0217] Accurately measure 20 μl of system suitability solution, test solution, and reference solution, respectively, and inject them into the liquid chromatograph under the same model but different batches of chromatographic columns to record the chromatograms.

[0218] Table 29 Effect of replacing chromatographic column on the determination results of related substances

[0219]

[0220]

[0221] From the data in the table above, we can see that when using the same model but different batches of chromatographic columns to determine the related substances of this product, the retention time and theoretical plate number of each component peak slightly change, the separation of each chromatographic peak meets the requirements, and the ratio of each known impurity, the maximum unknown impurity and the total impurity content to the initial conditions does not change significantly. This shows that using the same model but different batches of chromatographic columns basically does not affect the determination results of the related substances of this product.

[0222] Example 16 (Preparation of Impurity Q Compound):

[0223] Under nitrogen protection, 2.00g of compound impurity H and 50ml of tetrahydrofuran were added to the reaction bottle, cooled in an ice-water bath, and 30ml of lithium hexamethyldisilazide (1mol / L in THF) was added by syringe at 0-10°C. The reaction process was monitored by HPLC. After the reaction was complete, 30ml of 10% ammonium chloride solution was added to quench the reaction. After stirring for 10 minutes, the upper organic phase was collected and dried by adding 5.0g of anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3.14g of yellow solid. It was separated and purified by silica gel column chromatography to obtain 0.16g of the target product (eluent was dichloromethane: methanol = 15:1-10:1, volume ratio), and then the preparative column was separated to obtain high-purity impurity Q, HPLC purity: 99.479%. MS (m / z): 636.1676 [M+H].

[0224] 1 HNMR(d6-DMSO)δppm:7.836(d,1H),7.746(d,1H),7.661(m,1H),7.655(m, 1H),7.642(m,2H),7.517(m,2H),7.493(m,1H),7.415(dd,1H),7.333(m,1H ),7.323(m,2H),7.286(m,2H),7.219(d,1H),3.960(dd,1H),3.859(dd,1H ),3.636(m,1H),3.517(dd,1H),3.612(m,1H),3.317(d,1H),2.089(s,3H).

[0225] Embodiment 17:

[0226] (1) Solution preparation: prepare blank solution, system suitability solution, reference solution, and test solution separately;

[0227] (2) Determination method: After the system is stable, inject blank solution, system suitability solution, reference solution, and test solution respectively, and record the chromatogram;

[0228] (3) The instrument and chromatographic conditions used in the method of the present invention are as follows:

[0229] Instruments: High performance liquid chromatograph, electronic balance, pH meter, etc.

[0230] Column: Welch AQ C18 (4.6mm×250mm, 5μm)

[0231] Mobile phase A: ammonium acetate buffer (take 0.77g of ammonium acetate, add 1000ml of water and shake to dissolve, adjust the pH value to 4.7 with glacial acetic acid) - methanol (volume ratio 80:20)

[0232] Mobile phase B: ammonium acetate buffer (take 0.77g of ammonium acetate, add 1000ml of water and shake to dissolve, adjust the pH value to 4.7 with glacial acetic acid) - methanol - acetonitrile (volume ratio 5:55:40)

[0233] Elution method: gradient elution

[0234] Flow rate: 1.0ml / min

[0235] Detection wavelength: 254nm

[0236] Column temperature: 35°C

[0237] Injection volume: 20 μl

[0238] Perform gradient elution according to the following table:

[0239] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 20 65 35 40 10 90 50 10 90 50.1 65 35 60 65 35

[0240] Blank solution: methanol;

[0241] Stock solution of reference substance: Take appropriate amount of alprazolam, impurity B, C, D, F, H, Q and J reference substances respectively, weigh accurately, place in different volumetric bottles, dissolve and dilute with methanol to make a solution containing about 200 μg per 1 ml.

[0242] Reference solution: Accurately measure appropriate amounts of alprazolam reference stock solution and each impurity reference stock solution and dilute with methanol to make solutions containing approximately 5 μg of each substance per 1 ml.

[0243] System suitability solution: Take an appropriate amount of alprazolam test sample, weigh it accurately, add an appropriate amount of each impurity reference substance stock solution, dissolve it in methanol and dilute it to make a solution containing about 5 μg of impurities and about 1 mg of alprazolam per 1 ml.

[0244] Test solution: Weigh an appropriate amount of alprazolam, dissolve it in methanol and dilute it to make a solution containing approximately 1 mg of alprazolam per 1 ml;

[0245] The solution was injected into a liquid chromatograph for determination. The results were as follows:

[0246]

[0247] The above is a description of a specific embodiment of the present invention. It should be understood that the present invention is not limited to the above embodiment, and the above embodiment and description are only for illustrating the principle of the present invention.

[0248] Without departing from the concept of the present invention, those skilled in the art may also make various non-substantial changes and improvements to the present invention, all of which fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting alprazolam-related substances, the detection method adopts high performance liquid chromatography, the liquid chromatography determination conditions are: the chromatographic column is a C18 column, methanol-ammonium acetate solution or acetonitrile-ammonium acetate solution is used as mobile phase A, and a mixture of methanol and / or acetonitrile and ammonium acetate solution is used as mobile phase B for gradient elution, the pH value of the ammonium acetate solution is 3.2 to 5.7, preferably the pH value is 4.6 to 4.8, and the related substances include one or more of the following substances:

2. The detection method according to claim 1, characterized in that The C18 chromatographic column is Welch AQC18 chromatographic column, the column specifications are 4.6×250mm, 5μm.

3. The detection method according to claim 1, characterized in that: The mobile phase gradient elution procedure is as follows: ； Preferably, the mobile phase gradient elution procedure is as follows:

4. The detection method according to claim 1, characterized in that: The mobile phase A is methanol-ammonium acetate solution, wherein the volume ratio of ammonium acetate solution to methanol is 60-90:10-40, preferably 78-82:18-22, more preferably 80:20; Further preferably, the mobile phase B is a mixed solution of ammonium acetate solution and methanol and acetonitrile, wherein the volume ratio of ammonium acetate solution to methanol and acetonitrile is 1-10:40-60:40-50, preferably 4-6:54-56:40, and more preferably 5:55:

40.

5. The detection method according to claim 1, characterized in that: The pH value of the ammonium acetate solution is 4.7, and the pH regulator of the ammonium acetate solution is selected from a mixture of one or more of glacial acetic acid and phosphoric acid, preferably glacial acetic acid.

6. The detection method according to claim 1, characterized in that: The detection wavelength in the chromatographic conditions is: 252-256 nm; the preferred detection wavelength is 254 nm; Further preferably, the flow rate in the chromatographic conditions is 0.9-1.1 ml / min; preferably the flow rate is 1.0 ml / min; Further preferably, the column temperature in the chromatographic conditions is 33-37°C; preferably the column temperature is 35°C; Further preferably, the injection volume in the chromatographic conditions is 10-100 μl; preferably the injection volume is 20 μl; More preferably, the concentration of the test solution in the chromatographic conditions is 0.05-1.0 mg / ml; preferably, the concentration of the test solution is 1.0 mg / ml.

7. The detection method according to claim 1, characterized in that: The detection steps include: (1) Solution preparation: prepare blank solution, system suitability solution, reference solution, and test solution separately; (2) Determination method: After the system is stable, inject blank solution, system suitability solution, reference solution, and test solution respectively, and record the chromatogram; Among them, the chromatographic conditions are: C18 chromatographic column is Welch AQ C18 chromatographic column, column specifications are 4.6×250mm, 5μm; detection wavelength is 254nm, flow rate is 1.0ml / min, column temperature is 35℃, injection volume is 20μl, and the concentration of the test solution is 1.0mg / ml; The mobile phase A is a methanol-ammonium acetate solution, wherein the volume ratio of the ammonium acetate solution to methanol is 80:20; the mobile phase B is a mixed solution of ammonium acetate solution, methanol and acetonitrile, wherein the volume ratio of the ammonium acetate solution to methanol and acetonitrile is 5:55:40; the pH value of the ammonium acetate solution is 4.7, and the mobile phase gradient elution procedure is as follows: ； Preferably, the mobile phase gradient elution procedure is as follows: 。 8. A compound represented by an impurity Q:

9. The method for preparing impurity Q according to claim 8, characterized in that: include: The impurity compound H is reacted with lithium hexamethyldisilazide in a solvent to prepare the impurity compound Q: The reaction solvent is one or more of tetrahydrofuran, cyclohexane, and n-hexane, preferably tetrahydrofuran; the reaction temperature is -10-30°C, preferably 0-10°C.

10. The detection method according to any one of claims 1 to 7 is suitable for detecting related substances in alprazolam raw materials or preparations.

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