HPLC Detection Method of L-Samidorfen Malate API and Impurities

Abstract

The present invention belongs to the technical field of pharmaceutical detection, and is specifically related to an HPLC detection method for L-malic acid samidorfen bulk drug and impurities. The HPLC detection method for L-malic acid samidorfen bulk drug and impurities of the present invention, the conditions of HPLC detection thereof include: chromatographic column with octadecylsilane bonded silica gel as filler, detection wavelength 210nm, column temperature 28-32°C, 0.1wt% sodium octane sulfonate as mobile phase A, acetonitrile as mobile phase B, gradient elution. The HPLC detection method for L-malic acid samidorfen bulk drug and impurities provided by the present invention, simple and fast, strong specificity, high sensitivity, good separation, many types of impurities detected, fills the gap in drug detection, and the quality of bulk drugs is controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and in particular relates to a high performance liquid chromatography detection method for L-malic acid samidophen raw material medicine and impurities. Background Art

[0002] Samidorphan L-malate, chemical name 17-(cyclopropylmethyl)-3-carbamoyl-4,14-dihydroxymorphine-6-one L-malate, has the chemical structural formula: .

[0003] L-Samidorfen malate and olanzapine form a compound preparation that can reduce side effects such as weight gain and metabolic abnormalities caused by olanzapine. In the preparation process of L-Samidorfen malate API, there are many impurities, including process impurities, degradation impurities, etc., which need to be strictly controlled. Therefore, it is necessary to detect the related substances of L-Samidorfen malate API.

[0004] High performance liquid chromatography (HPLC) is a commonly used method for detecting malic acid content, and it is also the operating method specified in the Chinese Pharmacopoeia. This method mainly uses chiral phase separation liquid chromatography to separate malic acid in the sample under specific conditions, and then independently detects malic acid under specific detection conditions. However, there is currently no detection method for L-malic acid samidophen-related substances included in the pharmacopoeia at home and abroad. Although high performance liquid chromatography has the characteristics of high detection sensitivity, good separation effect, simple operation, and accurate data, it can complete the separation and detection of samples in a very short time. However, when detecting different substances, it is necessary to pay attention to the deviation of retention time to ensure that it is within an acceptable range. In addition, the familiarity with the method and equipment will also affect the accuracy of the test results.

[0005] Therefore, developing a method for rapid, simple and accurate detection of more related substances in L-malate samidorfen API is of great significance for the quality control of L-malate samidorfen API. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a high performance liquid chromatography detection method for L-malic acid samidorfen raw material and impurities, which is simple, fast, highly specific, highly sensitive, has good separation, and can detect a variety of impurities, thus filling the gap in drug detection and controlling the quality of the raw material.

[0007] The high performance liquid chromatography detection method of L-malate samidorfen raw material and impurities of the present invention, wherein the high performance liquid chromatography detection conditions include: the chromatographic column uses octadecylsilane bonded silica gel as filler, the detection wavelength is 210nm, the column temperature is 28-32°C, 0.08wt%-0.12wt% sodium octane sulfonate is used as mobile phase A, acetonitrile is used as mobile phase B, gradient elution, and the gradient elution conditions are:

[0008] The first gradient elution time is 0 min, mobile phase A accounts for 75%-85%, and mobile phase B accounts for 15%-25%;

[0009] The second gradient elution time is 18-22 minutes, with mobile phase A accounting for 69%-79% and mobile phase B accounting for 21%-31%;

[0010] The third gradient elution time is 28min-32min, mobile phase A accounts for 57%-67%, and mobile phase B accounts for 33%-43%;

[0011] The fourth gradient elution time is 48min-50min, the mobile phase A accounts for 57%-67%, and the mobile phase B accounts for 33%-43%;

[0012] The fifth gradient elution time is 51min-53min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%;

[0013] The sixth gradient elution time is 58min-62min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%.

[0014] The conditions for high performance liquid chromatography detection also include: using a PDA or UV detector; injection volume of 5-20 μL; flow rate of 0.9-1.1 mL / min. The elution time of the different gradients mentioned above, that is, the point value, such as setting the mobile phase A to account for 75%-85% and the mobile phase B to account for 15%-25% at the first gradient elution time of 0 min, and eluting all the time, the second gradient elution time of 18 min-22 min means that from the first gradient elution to the 18th min, the mobile phase A is set to account for 69%-79% and the mobile phase B to account for 21%-31%, or it means that from the first gradient elution to the 22nd min, it is also possible, that is, at any time point within 18 min-22 min, the mobile phase ratio of the next gradient can be changed, which will not change the peak shape, but will only change the overall peak appearance time, and the same is true for the elution time of other gradients.

[0015] The high performance liquid chromatography detection method of L-malic acid samidophen raw material and impurities comprises the following steps:

[0016] 1) Preparation of reference solution:

[0017] Take L-malic acid samidophen reference substance, dissolve it in solvent and dilute it quantitatively to prepare reference substance solution;

[0018] 2) Preparation of test solution:

[0019] Take L-malic acid samidophen test sample, dissolve it in solvent and dilute it quantitatively to prepare the test sample solution;

[0020] 3) System suitability solution preparation:

[0021] Take L-malic acid samidophen reference substance, impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J reference substances, dissolve and dilute with solvent to prepare system suitability solutions;

[0022] 4) Detection:

[0023] The solvent, reference solution, test solution and system suitability solution were respectively taken and injected into the liquid chromatograph, the chromatogram was recorded, and L-samidorfen malate and various impurities were separated.

[0024] In the chromatogram of the test solution, the chromatographic peaks that are consistent with the retention time of each impurity are calculated by the peak area according to the external standard method.

[0025] The impurity A is 17-(cyclopropylmethyl)-3-trifluoromethanesulfonyl-4,14-dihydroxymorphinan-6-one, and its chemical structure is .

[0026] Impurity B is 17-(cyclopropylmethyl)-3-cyano-4,14-dihydroxymorphinan-6-one, with the chemical formula .

[0027] Impurity C is 17-(cyclopropylmethyl)-4,14-dihydroxymorphinan-6-one, with the chemical formula .

[0028] Impurity D is 17-(cyclopropylmethyl)-3-carboxy-4,14-dihydroxymorphinan-6-one, with the chemical formula .

[0029] Impurity G is 17-(cyclopropylmethyl)-3-carbamoyl-4,14-hydroxymorphinan, with the chemical formula .

[0030] Impurity J is 17-oxo-(cyclopropylmethyl)-3-carbamoyl-4,14-dihydroxymorphinan-6-one, with the chemical formula .

[0031] The solvent is water.

[0032] The concentration of L-samidorfen malate in the reference solution is 0.8 μg / mL-1.2 μg / mL.

[0033] The concentration of L-samidorfen malate in the test solution is 0.8 μg / mL-1.2 mg / mL.

[0034] The concentration of L-samidorfen malate in the system suitability solution is 0.8 μg / mL-1.2 mg / mL, and the concentrations of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J are each 0.8 μg / mL-1.2 μg / mL.

[0035] Specifically, the high performance liquid chromatography detection method of L-malate samidophen raw material and impurities comprises the following steps:

[0036] 1) Preparation of reference solution:

[0037] Take L-malate samidophen reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 1µg per 1mL as reference substance solution;

[0038] 2) Preparation of test solution:

[0039] Take L-malate samidophen test sample, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 1 mg per 1 mL as the test sample solution;

[0040] 3) System suitability solution preparation:

[0041] Take L-malate samidorfen reference substance, impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J respectively, weigh accurately, dissolve in solvent and dilute to make a mixed solution containing 1 mg of L-malate samidorfen and 1 µg of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J per 1 mL as the system suitability solution;

[0042] 4) Detection:

[0043] Accurately measure the solvent, system suitability solution, reference solution and test solution respectively, inject them into a liquid chromatograph, record the chromatogram, and separate L-samidophen malate and its related substances;

[0044] The chromatographic conditions were as follows: chromatographic column: InertSustain AQ-C18, specification 4.6 mm × 250 mm, 5 µm, filler octadecylsilane bonded silica gel;

[0045] Column temperature: 28-32°C;

[0046] Detection wavelength: 210nm;

[0047] Flow rate: 0.9-1.1 mL / min;

[0048] Injection volume: 5-20 μL;

[0049] Blank solvent: water;

[0050] Detector: PDA or UV detector;

[0051] Mobile phase A: 0.08wt%-0.12wt% sodium octane sulfonate;

[0052] Mobile phase B: acetonitrile;

[0053] Gradient elution conditions:

[0054] The first gradient elution time is 0 min, mobile phase A accounts for 75%-85%, and mobile phase B accounts for 15%-25%;

[0055] The second gradient elution time is 18-22 minutes, with mobile phase A accounting for 69%-79% and mobile phase B accounting for 21%-31%;

[0056] The third gradient elution time is 28min-32min, mobile phase A accounts for 57%-67%, and mobile phase B accounts for 33%-43%;

[0057] The fourth gradient elution time is 48min-50min, the mobile phase A accounts for 57%-67%, and the mobile phase B accounts for 33%-43%;

[0058] The fifth gradient elution time is 51min-53min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%;

[0059] The sixth gradient elution time is 58min-62min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%.

[0060] In the chromatogram of the test solution, if there is a chromatographic peak in the obtained test solution that is consistent with the retention time of the related substance, the mass percentage of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J shall not exceed 0.1% by calculating the peak area using the external standard method, and the mass percentage of other individual impurities shall not exceed 0.10%, and the total amount of impurities shall not exceed 0.5%.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The high performance liquid chromatography detection method for L-malate samidorfen raw material and impurities of the present invention coordinates the preparation of the test solution with the chromatographic conditions, so that the method has high sensitivity, good separation and strong specificity, and can accurately qualitatively and quantitatively detect various impurities in the L-malate samidorfen raw material, thereby objectively, accurately and comprehensively evaluating the quality of the L-malate samidorfen raw material, which has important practical significance for the control of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 A high performance liquid chromatogram of a blank solvent in a specificity experiment in the methodological validation of the present invention;

[0064] Figure 2 A high performance liquid chromatogram of a specific mixed solution in a specific experiment in the methodology validation of the present invention;

[0065] Figure 3 A high performance liquid chromatogram of a reference solution for a system precision experiment in the methodology validation of the present invention;

[0066] Figure 4 is the HPLC chromatogram of the test solution of Example 1;

[0067] Figure 5 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 2;

[0068] Figure 6 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 3;

[0069] Figure 7 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 4;

[0070] Figure 8 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 5;

[0071] Fig. 9 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 6;

[0072] Fig.10 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 7;

[0073] Fig.11 The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 8;

[0074] Fig.12The HPLC chromatogram of the reference solution of L-samidorfen malate and six impurities in Example 9;

[0075] Fig.13 The HPLC chromatogram of the reference solution of L-samidophen malate and six impurities in Comparative Example 1;

[0076] Fig.14 The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in Comparative Example 2 is shown. DETAILED DESCRIPTION

[0077] The present invention will be further described below in conjunction with specific embodiments.

[0078] The raw materials used in the examples, unless otherwise specified, are all commercially available conventional raw materials; the process methods used in the examples, unless otherwise specified, are all conventional methods in the art.

[0079] Example 1

[0080] The high performance liquid chromatography detection method of L-malate samidophen raw material and impurities comprises the following steps:

[0081] 1) Preparation of reference solution:

[0082] Take L-malate samidophen reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 1µg per 1mL as the reference substance solution;

[0083] 2) Preparation of test solution:

[0084] Take L-malate samidophen test sample, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 1 mg per 1 mL as the test solution;

[0085] 3) System suitability solution preparation:

[0086] Take L-malic acid samidorfen reference substance, impurity A, impurity B, impurity C, impurity D, impurity G, impurity J, and reference substance respectively, weigh accurately, dissolve in solvent and dilute to make a mixed solution containing 1 mg of L-malic acid samidorfen, 1 µg of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J per 1 mL as the system suitability solution;

[0087] 4) Detection:

[0088] The solvent, system suitability solution, reference solution and test solution were accurately measured respectively, injected into the liquid chromatograph, the chromatogram was recorded, and L-samidorfen malate and its related substances were separated. The chromatographic conditions were as follows: chromatographic column: InertSustain AQ-C18, specification 4.6mm×250mm, 5µm, filler octadecylsilane bonded silica gel;

[0089] Column temperature: 30°C;

[0090] Detection wavelength: 210nm;

[0091] Flow rate: 1.0 mL / min;

[0092] Injection volume: 10 μL;

[0093] Detector: PDA;

[0094] Blank solvent: water;

[0095] Mobile phase A: 0.1wt% sodium octane sulfonate solution, pH 4.5;

[0096] Mobile phase B: acetonitrile;

[0097] The gradient elution conditions are shown in Table 1.

[0098] Table 1 Gradient elution conditions

[0099]

[0100] The chromatogram of the test solution is as follows Figure 4 As shown, the chromatographic peak in the obtained test solution that is consistent with the retention time of the related substance is determined, and the impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J are calculated by peak area using the external standard method, and the impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J shall not exceed 0.1%, and other individual impurities shall not exceed 0.10%, and the total amount of impurities shall not exceed 0.5%.

[0101] Example 2

[0102] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column temperature is replaced with 28° C. After the measurement, the chromatogram is recorded.

[0103] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Figure 5 As shown by Figure 5 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 2. Figure 5 It can be seen from Table 2 that the separation degree of each substance meets the requirements, so as to more accurately evaluate the product quality.

[0104] Table 2 Peak table of L-samidophen malate and six impurities in the reference solution

[0105]

[0106] Example 3

[0107] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column temperature is replaced with 32° C. After the measurement, the chromatogram is recorded.

[0108] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Figure 6 As shown by Figure 6 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 3. Figure 6 It can be seen from Table 3 that the separation degree of each substance meets the requirements, thus more accurately evaluating the product quality.

[0109] Table 3 Peak table of L-samidorfen malate and six impurities in the reference solution

[0110]

[0111] Example 4

[0112] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the flow rate is replaced with 0.9 mL / min. After the measurement, the chromatogram is recorded.

[0113] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Figure 7 As shown by Figure 7 It can be seen that impurity J, L-samidophen malate, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 4. Figure 7 It can be seen from Table 4 that the separation degree of each substance meets the requirements, thus more accurately evaluating the product quality.

[0114] Table 4 Peak table of L-samidorfen malate and six impurities in the reference solution

[0115]

[0116] Example 5

[0117] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the flow rate is replaced with 1.1 mL / min. After the measurement, the chromatogram is recorded.

[0118] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Figure 8 As shown by Figure 8 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 5. Figure 8 It can be seen from Table 5 that the separation degree of each substance meets the requirements, so as to more accurately evaluate the product quality.

[0119] Table 5 Peak table of L-samidorfen malate and six impurities in the reference solution

[0120]

[0121] Example 6

[0122] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the gradient elution conditions are replaced with the gradient in Table 6. After the measurement, the chromatogram is recorded.

[0123] The gradient elution conditions are shown in Table 6.

[0124] Table 6 Elution conditions

[0125]

[0126] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Fig. 9 As shown by Fig. 9 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 7. Fig. 9 It can be seen from Table 7 that the separation degree of each substance meets the requirements, so as to more accurately evaluate the product quality.

[0127] Table 7 Peak table of reference solution of L-malic acid samidophen and six impurities in Example 6

[0128]

[0129] Example 7

[0130] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the gradient elution condition is replaced with the gradient in the table below. After the measurement, the chromatogram is recorded.

[0131] The gradient elution conditions are shown in Table 8.

[0132] Table 8 Elution conditions

[0133]

[0134] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Fig.10 As shown by Fig.10 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 9. Fig.10 It can be seen from Table 9 that the separation degree of each substance meets the requirements, so as to more accurately evaluate the product quality.

[0135] Table 9 Peak table of reference solution of L-malic acid samidophen and six impurities in Example 7

[0136]

[0137] Example 8

[0138] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the salt concentration in mobile phase A is adjusted to 0.08 wt % sodium octane sulfonate. After the measurement, the chromatogram is recorded.

[0139] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Fig.11 As shown by Fig.11 It can be seen that impurity J, L-malic acid samidophen, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 10. Fig.11 It can be seen from Table 10 that the separation degree of each substance meets the requirements, thereby more accurately evaluating the product quality.

[0140] Table 10 Peak table of reference solution of L-malic acid samidophen and six impurities in Example 8

[0141]

[0142] Example 9

[0143] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the salt concentration in mobile phase A is adjusted to 0.12 wt % sodium octane sulfonate. After the measurement, the chromatogram is recorded.

[0144] The HPLC chromatogram of the reference solution of L-malic acid samidophen and six impurities in this example is as follows Fig.12 As shown by Fig.11 It can be seen that impurity J, L-samidophen malate, impurity B, impurity C, impurity D, impurity G, and impurity A are peaked in sequence, and the data table of the separation degree is shown in Table 11. Fig.12 It can be seen from Table 11 that the separation degree of each substance meets the requirements, thereby more accurately evaluating the product quality.

[0145] Table 11 Peak table of reference solution of L-malic acid samidophen and six impurities in Example 7

[0146]

[0147] Comparative Example 1

[0148] The detection method and chromatographic conditions used in this comparative example are the same as those in Example 1, except that the 0.1 wt % sodium octane sulfonate solution in the mobile phase is replaced by a 0.01 mol / L potassium dihydrogen phosphate solution.

[0149] Take the test solution in Example 1 and inject it into the liquid chromatograph with water as the blank solvent. The other chromatographic conditions are the same as those in Example 1. The obtained HPLC chromatogram is as follows: Fig.13 As shown, the main peak appeared within 5 minutes without any retention, which indicated that this comparative example was not suitable for the determination of L-malic acid samidophen and the six impurities in the present invention.

[0150] Comparative Example 2

[0151] The detection method and chromatographic conditions used in this comparative example are the same as those in Example 1, and the mobile phase A is adjusted to a pH value of 6.5 using a 0.1 mol / L potassium hydroxide solution.

[0152] Take the test solution in Example 1 and inject it into the liquid chromatograph with water as the blank solvent. The HPLC chromatogram is as follows: Fig.14 As shown by Fig.14 It can be seen that the main peak has a poor shape, indicating that this comparative example is not suitable for the determination of L-malic acid samidophen and the six impurities in the present invention.

[0153] Table 12 Peak table of test solution

[0154]

[0155] Methodological verification of the present invention:

[0156] 1. Specificity experiment

[0157] Stock solutions of each impurity: Take impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J respectively, place them in different 100mL volumetric flasks, dissolve and dilute to the scale with solvent respectively, shake well, and obtain.

[0158] Specific mixed solution: Accurately weigh 10 mg of L-malic acid samidophen reference substance, place it in a 10 mL volumetric flask, add an appropriate amount of solvent to dissolve it, add 0.1 mL of each of the above impurity stock solutions, dilute to the scale with solvent, shake well, and obtain.

[0159] Precisely measure the blank solvent and the specific mixed solution and inject them according to the chromatographic conditions of Example 1, record the chromatogram, and the results are shown in Table 11. Figure 1 , Figure 2 shown.

[0160] Table 11 Test results of specific mixed solution

[0161]

[0162] Conclusion: The blank solvent does not interfere with the detection of L-malic acid samidophen and its impurities. The separation degree between the main peak and the adjacent impurity peaks is 3.328, which is greater than 1.5, and the separation effect is good.

[0163] 2. Experiments on methods in different experimental environments

[0164] Reference solution: Take 10 mg each of L-malic acid samidophen, impurity A, impurity B, impurity C, impurity D, impurity G and impurity J, place them in the same 100 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, add solvent to dilute to the scale, mix well, and obtain.

[0165] Preparation of undegraded test sample solution: Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0166] Preparation of acid degradation test solution: Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, leave it at room temperature for 4 hours, then add 1 mL of 1 mol / L sodium hydroxide solution to neutralize it, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0167] Preparation of alkaline degradation test solution: Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add 1 mL of 1 mol / L sodium hydroxide solution, leave it at room temperature for 4 hours, then add 1 mL of 1 mol / L hydrochloric acid solution to neutralize it, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0168] Preparation of oxidative degradation test solution: Accurately weigh 10 mg of the test sample, place it in a 10 mL volumetric flask, add 1 mL of 3% hydrogen peroxide solution, leave it at room temperature for 2 hours, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0169] Preparation of high temperature degradation test solution: Take an appropriate amount of the test sample, place it in a 60℃ oven, leave it for 30 days, take it out, cool it to room temperature, accurately weigh 10 mg, place it in a 10mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0170] Preparation of high-humidity degradation test sample solution: Take an appropriate amount of the test sample and place it at a relative humidity of 92.5% for 30 days. Take it out, accurately weigh 10 mg, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0171] Preparation of light degradation test sample solution: Take an appropriate amount of the test sample and place it under light conditions (1.2×10 6 Lux•hr), place for 30 days, take out, accurately weigh 10 mg, put into a 10 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and the product is obtained.

[0172] Acid and alkali degradation blank solvent: Take 1mL of 1mol / L hydrochloric acid solution and 1mL of 1mol / L sodium hydroxide solution, place in a 10mL volumetric flask, add solvent to dilute to the scale, shake well, and use as the acid and alkali degradation blank solvent;

[0173] Oxidative degradation blank solvent: Take 1 mL of 3% hydrogen peroxide solution, place it in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and use it as the oxidative degradation blank solvent.

[0174] Accurately measure each of the above solutions and inject them according to the above chromatographic conditions. Record the chromatogram and calculate the content of each impurity by the external standard method. The results are shown in Table 12.

[0175] Table 12 Forced degradation test results

[0176]

[0177] Conclusion: The solvent and blank solvents had no interference on the sample determination. Under each degradation condition, the purity similarity of the main peak was greater than the purity threshold, and the separation between the main peak and the adjacent peaks was greater than 1.5, indicating that the method had strong specificity.

[0178] 3. System precision experiment

[0179] Reference solution: Take 10 mg each of L-malic acid samidophen, impurity A, impurity B, impurity C, impurity D, impurity G and impurity J, place them in the same 100 mL volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, add solvent to dilute to the scale, mix well, and obtain.

[0180] Accurately measure the reference solution and inject it according to the above chromatographic conditions, record the chromatogram, and calculate the relative standard deviation (RSD) of the peak area and retention time of each peak after 6 consecutive injections of the reference solution. The results are shown in Table 13. Figure 3 .

[0181] Table 13 System precision test results

[0182]

[0183]

[0184] Conclusion: The reference solution was injected 6 times continuously, the RSD of the reference peak area was less than 10.0%, and the RSD of the retention time was less than 1.0%, indicating that the system precision was good.

[0185] 4. Detection limit / quantification limit

[0186] Use water as blank solvent, adjust the instrument sensitivity, inject samples three times continuously, record the noise level of the instrument within the peak time range of the analyte, and calculate the average noise.

[0187] Detection limit: Accurately prepare the reference solution of the test object, gradually dilute it to a certain concentration and inject it, and measure it three times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 3 is the detection limit concentration, and its ratio to the theoretical sample concentration is the detection limit.

[0188] Limit of quantitation: Accurately prepare the reference solution of the substance to be measured, gradually dilute it to a certain concentration and inject it, and measure it 6 times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 10 is the limit of quantitation concentration, and its ratio to the theoretical sample concentration is the limit of quantitation.

[0189] Quantitation limit solution: Accurately measure 0.1 mL of impurity J and L-samidophen malate stock solutions, 50 µL of impurities B, G, and D, 0.5 mL of impurity C stock solution, and 0.25 mL of impurity A stock solution in the specificity experiment in (I), place them in the same 100 mL volumetric flask, add solvent to dilute to the scale, mix well, and use as the quantitation limit solution.

[0190] Detection limit solution: Accurately measure 5 mL of the quantitative limit solution, place it in a 10 mL volumetric flask, dilute to the scale with solvent, shake well, and use it as the detection limit solution.

[0191] Accurately measure the solvent and inject it continuously for 3 times according to the above chromatographic conditions, inject the detection limit solution continuously for 3 times and the quantification limit solution continuously for 6 times. Record the chromatogram and calculate the signal-to-noise ratio. The results are shown in Tables 14, 15 and 16.

[0192] Table 14 Baseline noise measurement results

[0193]

[0194] Table 15 Quantitation limit determination results

[0195]

[0196] Table 16 Detection limit determination results

[0197]

[0198] Conclusion: Under this method, the sensitivity of L-malic acid samidophen and its impurities meets the requirements.

Claims

1. A high performance liquid chromatography method for detecting L-malic acid samidophen bulk drug and impurities, characterized in that: The conditions for high performance liquid chromatography detection include: the chromatographic column is filled with octadecylsilane bonded silica gel, the column temperature is 28-32°C, 0.08wt%-0.12wt% sodium octane sulfonate is used as mobile phase A, acetonitrile is used as mobile phase B, gradient elution, and the gradient elution conditions are: The first gradient elution time is 0 min, mobile phase A accounts for 75%-85%, and mobile phase B accounts for 15%-25%; The second gradient elution time is 18-22 minutes, with mobile phase A accounting for 69%-79% and mobile phase B accounting for 21%-31%; The third gradient elution time is 28min-32min, mobile phase A accounts for 57%-67%, and mobile phase B accounts for 33%-43%; The fourth gradient elution time is 48min-50min, the mobile phase A accounts for 57%-67%, and the mobile phase B accounts for 33%-43%; The fifth gradient elution time is 51min-53min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%; The sixth gradient elution time is 58min-62min, the mobile phase A accounts for 75%-85%, and the mobile phase B accounts for 15%-25%; the high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities comprises the following steps: 1) Preparation of reference solution: Take L-malic acid samidophen reference substance, dissolve it in solvent and dilute it quantitatively to prepare reference substance solution; 2) Preparation of test solution: Take L-malate samidophen test sample, dissolve it in solvent and dilute it quantitatively to prepare the test sample solution; 3) System suitability solution preparation: Take L-malic acid samidophen reference substance, impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J reference substances, dissolve and dilute with solvent to prepare system suitability solutions; 4) Detection: The solvent, the reference solution, the test solution, and the system suitability solution were respectively taken and injected into the liquid chromatograph, the chromatogram was recorded, and L-samidorfen malate and the impurities were separated; The impurity A is 17-(cyclopropylmethyl)-3-trifluoromethanesulfonyl-4,14-dihydroxymorphinan-6-one, the impurity B is 17-(cyclopropylmethyl)-3-cyano-4,14-dihydroxymorphinan-6-one, the impurity C is 17-(cyclopropylmethyl)-4,14-dihydroxymorphinan-6-one, the impurity D is 17-(cyclopropylmethyl)-3-carboxy-4,14-dihydroxymorphinan-6-one, the impurity G is 17-(cyclopropylmethyl)-3-carbamoyl-4,14-hydroxymorphinan; the impurity J is 17-oxo-(cyclopropylmethyl)-3-carbamoyl-4,14-dihydroxymorphinan-6-one.

2. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: The conditions for high performance liquid chromatography detection also include: using a PDA or ultraviolet detector; an injection volume of 5-20 μL; and a flow rate of 0.9-1.1 mL / min.

3. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: In the chromatogram of the test solution, the chromatographic peaks that are consistent with the retention time of each impurity are calculated by the peak area according to the external standard method.

4. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: The solvent is water.

5. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: The concentration of L-samidorfen malate in the reference solution is 0.8 μg / mL-1.2 μg / mL.

6. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: The concentration of L-samidorfen malate in the test solution is 0.8 mg / mL-1.2 mg / mL.

7. The high performance liquid chromatography detection method of L-malic acid samidophen bulk drug and impurities according to claim 1, characterized in that: The concentration of L-samidorfen malate in the system suitability solution is 0.8 mg / mL-1.2 mg / mL, and the concentrations of impurity A, impurity B, impurity C, impurity D, impurity G, and impurity J are each 0.8 μg / mL-1.2 μg / mL.

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