A method for detecting impurity content in tolvaptan sodium phosphate freeze-dried powder injection

Abstract

The present invention provides a method for detecting the impurity content in a tolvaptan sodium phosphate lyophilized powder injection, wherein the tolvaptan sodium phosphate lyophilized powder injection is analyzed by a high performance liquid chromatography analysis method to obtain a chromatogram, and based on the chromatogram, the content of impurities in the tolvaptan sodium phosphate lyophilized powder injection is determined, wherein the chromatographic column used in the high performance liquid chromatography analysis method is a silica gel filled chromatographic column, the mobile phase A is a 0.01-0.03 mol / L potassium dihydrogen phosphate solution, the pH value of the mobile phase A is 6.0-8.0, and the mobile phase B is acetonitrile. The detection method of the present invention is a method for detecting the impurity content in the tolvaptan sodium phosphate lyophilized powder injection, which can be used to support the research and development of pharmaceutical preparations, has strong method specificity, good repeatability, is reliable, and can save detection costs.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and in particular relates to a method for detecting impurity content in tolvaptan sodium phosphate freeze-dried powder injection. Background Art

[0002] Tolvaptan sodium phosphate is an antidiuretic drug that is currently used to treat edema caused by heart failure, cirrhosis, syndrome of inappropriate antidiuretic hormone secretion, kidney disease, etc.

[0003] Currently, there is no prior art that discloses a method for detecting the impurity content in the lyophilized powder injection of tolvaptan sodium phosphate for intravenous injection.

[0004] Therefore, providing a method for detecting the impurity content in tolvaptan sodium phosphate lyophilized powder injection is a technical problem that needs to be solved in the art. Summary of the invention

[0005] The present invention aims to provide a method for detecting the impurity content in tolvaptan sodium phosphate freeze-dried powder injection.

[0006] On the one hand, the present invention provides a method for detecting the impurity content in a tolvaptan sodium phosphate lyophilized powder injection, wherein the tolvaptan sodium phosphate lyophilized powder injection is analyzed by a high performance liquid chromatography analysis method to obtain a chromatogram, and based on the chromatogram, the content of impurities in the tolvaptan sodium phosphate lyophilized powder injection is determined, wherein the chromatographic column used in the high performance liquid chromatography analysis method is a silica gel filled chromatographic column, the mobile phase A is a 0.01-0.03 mol / L potassium dihydrogen phosphate solution, and the content of impurities in the tolvaptan sodium phosphate lyophilized powder injection is determined by a high performance liquid chromatography analysis method. The pH value is 6.0~8.0, the mobile phase B is acetonitrile, and the elution gradient is: the first stage: the ratio of mobile phase A and B is (80:20)-(50:50), elution 6min~10min; the second stage: the ratio of mobile phase A and B is (50:50)-(50:50), elution 10min~15min; the third stage: the ratio of mobile phase A and B is (50:50)-(20:80), elution 3min~5min; the fourth stage: mobile phase A, B The ratio is (20:80)-(20:80), and the elution time is 10min~15min; the fifth stage: the ratio of mobile phase A and B is (20:80)-(80:20), and the elution time is 4min-8min; the impurities are impurity 1: 5-dehydrotolvaptan, impurity 2: tolvaptan, and impurity 3: N-[4-[(5R)-7-chloro-2,3,4,5-tetrahydro-1-benzazepine-1-formyl]-3-methylphenyl]-2-methylbenzamide.

[0007] It can be understood that the applicant, through a lot of creative work, screened out a suitable mobile phase A, pH value of mobile phase A, mobile phase B and elution gradient, which can effectively achieve the separation of tolvaptan sodium phosphate peak and impurity peak under specific conditions.

[0008] In some embodiments, the pH of the mobile phase A is adjusted using triethylamine.

[0009] In some embodiments, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel chromatographic column Agilent5 HC-C18, 4.6 mm×250 mm, 5 μm.

[0010] In some embodiments, the tolvaptan sodium phosphate lyophilized powder injection is provided in the form of a test solution, wherein the test solution is an aqueous solution of tolvaptan sodium phosphate lyophilized powder injection, and based on each milliliter of the test solution, the concentration of tolvaptan sodium phosphate is 1.0 mg / mL.

[0011] In some embodiments, the detection wavelength used by the high performance liquid chromatography is 250-260 nm.

[0012] In some embodiments, the column temperature used in the HPLC is 25-35°C.

[0013] In some embodiments, based on the chromatogram, the impurity content in the tolvaptan sodium phosphate is determined by calculating the peak area according to the external standard method, and the calculation formula is as follows:

[0014] ,

[0015] A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance,

[0016] N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

[0017] In the second aspect of the present invention, a method for detecting the impurity content in tolvaptan sodium phosphate lyophilized powder injection is more specifically proposed, comprising:

[0018] (1) Chromatographic conditions

[0019] Chromatographic column: Octadecylsilane bonded silica gel chromatographic column,

[0020] Detector: DAD,

[0021] Detection wavelength: 250~260nm,

[0022] Column temperature: 25~35℃,

[0023] Mobile phase A: 0.01-0.03 mol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 6.0-8.0,

[0024] Mobile phase B: acetonitrile,

[0025] Flow rate: 0.9~1.1mL / min,

[0026] Injection volume: 20 μL,

[0027] Running time: 33~53min;

[0028] The elution gradient is:

[0029] The first stage: the ratio of mobile phase A and B is (80:20)-(50:50), and the elution time is 6-10 minutes; the second stage: the ratio of mobile phase A and B is (50:50)-(50:50), and the elution time is 10-15 minutes; the third stage: the ratio of mobile phase A and B is (50:50)-(20:80), and the elution time is 3-5 minutes; the fourth stage: the ratio of mobile phase A and B is (20:80)-(20:80), and the elution time is 10-15 minutes; the fifth stage: the ratio of mobile phase A and B is (20:80)-(80:20), and the elution time is 4-8 minutes;

[0030] (2) Preparation of blank solution

[0031] Blank solution: 50% acetonitrile;

[0032] (3) Preparation of test solution

[0033] Take 25 mg of Tolvaptan Sodium Phosphate Lyophilized Powder Injection, accurately weigh it, put it into a 25 mL volumetric flask, dilute it to the mark with blank solution, shake well, and obtain;

[0034] (4) Preparation of reference solution

[0035] Impurity 1 stock solution: Take 3 mg of impurity 1 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well;

[0036] Impurity 2 stock solution: Take 3 mg of impurity 2 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the mark with blank solution, and shake well;

[0037] Impurity 3 stock solution: Take 3 mg of impurity 3 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the mark with blank solution, and shake well;

[0038] Tolvaptan sodium phosphate stock solution: Take 3 mg of tolvaptan sodium phosphate reference substance, accurately weigh it, put it into a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well;

[0039] Reference solution: Take 1 mL of impurity 1 stock solution, 1 mL of impurity 2 stock solution, 1 mL of impurity 3 stock solution, and 1 mL of tolvaptan sodium phosphate stock solution, respectively, place them in a 100 mL volumetric flask, dilute to the mark with blank solution, and shake well;

[0040] (5) Sample injection

[0041] After the instrument is stable, inject samples in sequence: 1 empty needle, 1 to 3 blank solution needles, 6 reference solution needles, and 1 test solution needle. Before the end of the sequence and every 12 hours, inject 1 needle of the reference solution for back calibration and record the chromatogram.

[0042] (6) Calculation of impurity content

[0043] The impurity content is calculated by peak area according to the external standard method, and the calculation formula is as follows:

[0044] ,

[0045] A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance,

[0046] N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

[0047] In some embodiments, a method for detecting the impurity content in tolvaptan sodium phosphate lyophilized powder injection is more specifically proposed, comprising:

[0048] (1) Chromatographic conditions

[0049] Chromatographic column: Agilent5 HC-C18, 4.6mm×250mm, 5μm,

[0050] Detector: DAD,

[0051] Detection wavelength: 254nm,

[0052] Column temperature: 30°C,

[0053] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 7.0,

[0054] Mobile phase B: acetonitrile,

[0055] Flow rate: 0.9~1.1mL / min,

[0056] Injection volume: 20 μL,

[0057] Running time: 50min;

[0058] The elution gradient is:

[0059] ;

[0060] (2) Preparation of blank solution

[0061] Blank solution: 50% acetonitrile;

[0062] (3) Preparation of test solution

[0063] Take 25 mg of Tolvaptan Sodium Phosphate Lyophilized Powder Injection, accurately weigh it, put it into a 25 mL volumetric flask, dilute it to the mark with blank solution, shake well, and obtain;

[0064] (4) Preparation of reference solution

[0065] Impurity 1 stock solution: Take 3 mg of impurity 1 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well;

[0066] Impurity 2 stock solution: Take 3 mg of impurity 2 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well;

[0067] Impurity 3 stock solution: Take 3 mg of impurity 3 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to the mark with blank solution, and shake well;

[0068] Tolvaptan sodium phosphate stock solution: Take 3 mg of tolvaptan sodium phosphate reference substance, accurately weigh it, put it into a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well;

[0069] Reference solution: Take 1 mL of impurity 1 stock solution, 1 mL of impurity 2 stock solution, 1 mL of impurity 3 stock solution, and 1 mL of tolvaptan sodium phosphate stock solution respectively, place them in a 100 mL volumetric flask, add blank solution to the scale, and shake well;

[0070] (5) Sample injection

[0071] After the instrument is stable, inject samples in sequence: 1 empty needle, 1 to 3 blank solution needles, 6 reference solution needles, and 1 test solution needle. Before the end of the sequence and every 12 hours, inject 1 needle of the reference solution for back calibration and record the chromatogram.

[0072] (6) Calculation of impurity content

[0073] The impurity content is calculated by peak area according to the external standard method, and the calculation formula is as follows:

[0074] ,

[0075] A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance,

[0076] N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

[0077] The method described in the present invention for detecting the content of impurities in tolvaptan sodium phosphate lyophilized powder injection has the following advantages:

[0078] The detection method of the present invention is a method for detecting the impurity content in tolvaptan sodium phosphate lyophilized powder injection, which can be used to support the research and development of pharmaceutical preparations;

[0079] The method described in the present invention has strong specificity and good repeatability, and the detection method is reliable and saves detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is the chromatogram under chromatographic condition 1 in Example 1;

[0081] Figure 2 is the chromatogram under chromatographic condition 2 in Example 1;

[0082] Figure 3 It is the chromatogram under chromatographic condition 4 in Example 1;

[0083] Figure 4 It is the chromatogram under chromatographic condition 5 in Example 1;

[0084] Figure 5 is the selective solution chromatogram of Example 3;

[0085] Figure 6 It is the standard curve chromatogram of impurity 1 of Example 4;

[0086] Figure 7 It is the standard curve chromatogram of impurity 2 of Example 4;

[0087] Figure 8 It is the standard curve chromatogram of impurity 3 of Example 4;

[0088] Fig. 9 It is the tolvaptan sodium phosphate standard curve chromatogram of Example 4;

[0089] Reference numerals:

[0090] 1: Tolvaptan sodium phosphate peak; 2: Impurity 1 peak; 3: Impurity 2 peak; 4: Impurity 3 peak. DETAILED DESCRIPTION

[0091] For ease of understanding, the application will be described more fully below, and preferred embodiments of the application are provided. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In the embodiment, if specific techniques or conditions are not indicated, the techniques or conditions described in the document in this area or according to the product specification are carried out. Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained through the market.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the technical field of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0093] Example 1 Screening process of chromatographic conditions

[0094] 1. Screening of mobile phase

[0095] Chromatographic conditions 1:

[0096] Chromatographic column: Octadecylsilane bonded silica gel chromatographic column ZORBAX ValueLab 5 LC C18-HD (4.6mm×250mm, 5μm),

[0097] Detector: DAD,

[0098] Detection wavelength: 254nm,

[0099] Column temperature: 30°C,

[0100] Mobile phase: acetonitrile-water (55:45),

[0101] Flow rate: 1mL / min,

[0102] Injection volume: 10 μL,

[0103] Elution procedure: isocratic elution,

[0104] Running time: 35min;

[0105] The lyophilized powder injection of tolvaptan sodium phosphate was analyzed using chromatographic condition 1, and the chromatogram was obtained as follows: Figure 1 As shown, the peak shape of tolvaptan sodium phosphate has poor symmetry and severe tailing, which does not meet the requirements.

[0106] Chromatographic conditions 2:

[0107] Chromatographic column: Octadecylsilane bonded silica gel column ZORBAX ValueLab 5 LC C18-HD (4.6mm×250mm, 5μm),

[0108] Detector: DAD,

[0109] Detection wavelength: 254nm,

[0110] Column temperature: 30°C,

[0111] Mobile phase: 0.1% trifluoroacetic acid aqueous solution-acetonitrile (55:45),

[0112] Flow rate: 1mL / min,

[0113] Injection volume: 10 μL,

[0114] Elution procedure: isocratic elution,

[0115] Running time: 40 minutes;

[0116] The lyophilized powder injection of tolvaptan sodium phosphate was analyzed using chromatographic condition 2, and the chromatogram was obtained as follows: Figure 2 As shown, the peak shape of tolvaptan sodium phosphate is good, but the retention time is too short and it cannot be effectively separated from the adjacent impurity peaks, which does not meet the requirements.

[0117] Chromatographic conditions 3:

[0118] Chromatographic column: Octadecylsilane bonded silica gel column ZORBAX ValueLab 5 LC C18-HD (4.6mm×250mm, 5μm),

[0119] Detector: DAD,

[0120] Detection wavelength: 254nm,

[0121] Column temperature: 30°C,

[0122] Mobile phase A: 0.1% trifluoroacetic acid aqueous solution at pH 7.0 (measure 1 mL of trifluoroacetic acid, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine).

[0123] Mobile phase B: acetonitrile,

[0124] Flow rate: 1mL / min,

[0125] Injection volume: 10 μL,

[0126] Elution gradient:

[0127] ;

[0128] Tolvaptan sodium phosphate lyophilized powder injection was analyzed using chromatographic condition 3. It was found that after changing the isocratic elution to gradient elution, the peak shape of tolvaptan sodium phosphate was good and the separation degree from adjacent impurity peaks met the requirements. However, the 0.1% trifluoroacetic acid aqueous solution lacked buffering capacity, and the process of adjusting the pH value to 7.0 was difficult to control and unstable.

[0129] Chromatographic conditions 4:

[0130] Chromatographic column: Octadecylsilane bonded silica gel column ZORBAX ValueLab 5 LC C18-HD (4.6mm×250mm, 5μm),

[0131] Detector: DAD,

[0132] Detection wavelength: 254nm,

[0133] Column temperature: 30°C,

[0134] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution at pH 7.0 (weigh about 2.7 g of potassium dihydrogen phosphate, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine).

[0135] Mobile phase B: acetonitrile,

[0136] Flow rate: 1mL / min,

[0137] Injection volume: 10 μL,

[0138] Elution gradient:

[0139] ;

[0140] Tolvaptan sodium phosphate lyophilized powder injection was analyzed using chromatographic condition 4, and the resulting chromatogram is shown in FIG. Figure 3 As shown, the peak shape of tolvaptan sodium phosphate is good, the separation degree from adjacent impurity peaks meets the requirements, and the experimental operation is simple and convenient.

[0141] Chromatographic conditions 5:

[0142] Chromatographic column: Octadecylsilane bonded silica gel column ZORBAX ValueLab 5 LC C18-HD (4.6mm×250mm, 5μm),

[0143] Detector: DAD,

[0144] Detection wavelength: 254nm,

[0145] Column temperature: 30°C,

[0146] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution at pH 7.0 (weigh about 2.7 g of potassium dihydrogen phosphate, dilute to 1000 mL with water, and adjust the pH to 7.0 with triethylamine)

[0147] Mobile phase B: methanol,

[0148] Flow rate: 1mL / min,

[0149] Injection volume: 10 μL,

[0150] Elution gradient:

[0151] ;

[0152] Tolvaptan sodium phosphate lyophilized powder injection was analyzed using chromatographic condition 5, and the resulting chromatogram is shown in FIG. Figure 4 As shown, mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution at pH 7.0, and mobile phase B is methanol. The separation effect between the main peak and impurities is poor, and the elution ability is poor, which does not meet the requirements.

[0153] 2. Selection of chromatographic column

[0154] Octadecylsilane bonded silica gel column ZORBAX ValueLab 5 LC C18-HD, 4.6mm×250mm, 5μm, octadecylsilane bonded silica gel column Agilent5 HC-C18, 4.6mm×250mm, 5μm, and octyl bonded silica gel column Welch Ultimate XB-C8, 4.6mm×250mm, 5μm were used to analyze the tolvaptan sodium phosphate lyophilized powder injection by liquid chromatography. The results showed that the main peak and the adjacent impurity peaks and the specific impurity peaks could be effectively separated, and the separation effect was equivalent. Since the octyl bonded silica gel column is an unconventional column and the Agilent5 HC-C18, 4.6mm×250mm, 5μm has more stable performance, the octadecylsilane bonded silica gel column Agilent5 HC-C18, 4.6mm×250mm, 5μm is preferred as the chromatographic column.

[0155] 3. pH screening of mobile phase A

[0156] Since tolvaptan sodium phosphate is alkaline, in order to better inhibit the dissociation of sample components and increase the retention of components on the stationary phase, and the pH tolerance range of conventional chromatographic columns is 2-8, the pH of the mobile phase A used in this method is 6.0-8.0.

[0157] 4. Selection of flow rate

[0158] According to the packing and specifications of the preliminary planned chromatographic column, flow rates of 0.9mL / min, 1.0mL / min and 1.1mL / min were tested respectively. The main peak and adjacent impurity peaks or specific impurity peaks were all effectively separated experimentally at the three flow rates, and the separation effects were comparable.

[0159] Example 2 System suitability test

[0160] Chromatographic column: Agilent5 HC-C18, 4.6mm×250mm, 5μm,

[0161] Detector: DAD,

[0162] Detection wavelength: 254nm,

[0163] Column temperature: 30°C,

[0164] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 7.0,

[0165] Mobile phase B: acetonitrile,

[0166] Flow rate: 1mL / min,

[0167] Injection volume: 20 μL,

[0168] Running time: 50min;

[0169] The elution gradient is:

[0170] ;

[0171] (2) Preparation of blank solution

[0172] Blank solution: 50% acetonitrile;

[0173] (3) Preparation of test solution

[0174] Take 25 mg of Tolvaptan Sodium Phosphate Lyophilized Powder Injection, accurately weigh it, put it into a 25 mL volumetric flask, dilute it to the mark with blank solution, shake well, and obtain;

[0175] (4) Preparation of reference solution

[0176] Impurity 1 stock solution: Take 3 mg of impurity 1 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to scale with blank solution, and shake well; (concentration: containing impurity 1 about 150 μg / mL)

[0177] Impurity 2 stock solution: Take 3 mg of impurity 2 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to scale with blank solution, and shake well; (concentration: contains impurity 2 about 150 μg / mL)

[0178] Impurity 3 stock solution: Take 3 mg of impurity 3 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to scale with blank solution, and shake well; (concentration: contains impurity 3 about 150 μg / mL)

[0179] Tolvaptan sodium phosphate stock solution: Take 3 mg of tolvaptan sodium phosphate reference substance, accurately weigh it, place it in a 20 mL volumetric flask, dilute it to the mark with blank solution, and shake well; (concentration: about 150 μg / mL of tolvaptan sodium phosphate)

[0180] Reference solution: Take 1 mL of impurity 1 stock solution, 1 mL of impurity 2 stock solution, 1 mL of impurity 3 stock solution, and 1 mL of tolvaptan sodium phosphate stock solution, respectively, place in a 100 mL volumetric flask, add blank solution to the scale, and shake well; (Concentration: impurity 1 about 1.5 μg / mL, impurity 2 about 1.5 μg / mL, impurity 3 about 1.5 μg / mL, tolvaptan sodium phosphate about 1.5 μg / mL)

[0181] (5) Sample injection

[0182] After the instrument is stable, inject samples in sequence, 1 empty needle, 1 to 3 blank solution needles, 6 reference solution needles, and 1 test solution needle. Before the end of the sequence and every 12 hours, inject 1 needle of the reference solution for back calibration, record the chromatogram, and calculate the peak area RSD of impurity 1 peak, impurity 2 peak, impurity 3 peak, and tolvaptan sodium phosphate peak, respectively. The results are shown in Table 1;

[0183] Table 1 System suitability test results

[0184] ;

[0185] From the results in Table 1, it can be seen that the peak area RSDs of impurity 1 peak, impurity 2 peak, impurity 3 peak and tolvaptan sodium phosphate peak are all ≤2.0%, and the system suitability meets the requirements.

[0186] Example 3 Specificity Test

[0187] (1) Chromatographic conditions

[0188] Same as Example 2;

[0189] (2) Solution preparation

[0190] Blank solution: 50% acetonitrile;

[0191] Impurity 1 stock solution: the same as in Example 2;

[0192] Impurity 2 stock solution: same as Example 2;

[0193] Impurity 3 stock solution: the same as in Example 2;

[0194] Tolvaptan sodium phosphate stock solution: the same as in Example 2;

[0195] Reference solution: same as in Example 2;

[0196] Impurity 1 positioning solution: Accurately measure 0.2 mL of impurity 1 stock solution, place it in a 20 mL volumetric flask, dilute to the scale with blank solution, and shake well; (concentration: containing impurity 1 about 1.5 μg / mL)

[0197] Impurity 2 location solution: Accurately measure 0.2 mL of impurity 2 stock solution, place in a 20 mL volumetric flask, dilute to scale with blank solution, and shake well; (concentration: contains impurity 2 about 1.5 μg / mL)

[0198] Impurity 3 positioning solution: Accurately measure 0.2 mL of impurity 3 stock solution, place it in a 20 mL volumetric flask, dilute to the scale with blank solution, and shake well; (concentration: contains impurity 3 about 1.5 μg / mL)

[0199] Tolvaptan sodium phosphate positioning solution: accurately measure 0.2 mL of tolvaptan sodium phosphate stock solution, place it in a 20 mL volumetric flask, dilute to the scale with blank solution, and shake well; (concentration: about 1.5 μg / mL of tolvaptan sodium phosphate)

[0200] Selective solution: Take about 20 mg of the test sample, weigh accurately, place in a 20 mL volumetric flask, add 0.2 mL of impurity 1 stock solution, 0.2 mL of impurity 2 stock solution, 0.2 mL of impurity 3 stock solution, dilute to the scale with blank solution, and shake well; (Concentration: contains about 1000 μg / mL of tolvaptan sodium phosphate, about 1.5 μg / mL of impurity 1, about 1.5 μg / mL of impurity 2, about 1.5 μg / mL of impurity 3)

[0201] (3) Accurately measure 20 μL each of the blank solution, impurity 1 localization solution, impurity 2 localization solution, impurity 3 localization solution, and tolvaptan sodium phosphate localization solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 2;

[0202] Table 2 Specificity determination results

[0203] ;

[0204] As shown in Table 2, the blank solution had no interference with the detection. In the selective solution chromatogram, the peak shape of tolvaptan sodium phosphate was good, and the separation degree from the adjacent impurity peaks met the requirements.

[0205] Example 4 Linearity Test

[0206] (1) Chromatographic conditions

[0207] Same as Example 2;

[0208] (2) Solution preparation

[0209] Blank solution: 50% acetonitrile;

[0210] Impurity 1 stock solution: the same as in Example 2;

[0211] Impurity 2 stock solution: the same as in Example 2;

[0212] Impurity 3 stock solution: the same as in Example 2;

[0213] Tolvaptan sodium phosphate stock solution: the same as in Example 2;

[0214] Reference solution: same as in Example 2;

[0215] Linear stock solution: measure 2 mL of impurity 1 stock solution, 2 mL of impurity 2 stock solution, 2 mL of impurity 3 stock solution, and 2 mL of tolvaptan sodium phosphate stock solution respectively, place them in a 20 mL volumetric flask, dilute to the scale with blank solution, and shake well; (concentration: impurity 1 about 15 μg / mL, impurity 2 about 15 μg / mL, impurity 3 about 15 μg / mL, tolvaptan sodium phosphate about 15 μg / mL)

[0216] Prepare linear solutions of impurity 1, impurity 2, impurity 3 and tolvaptan sodium phosphate according to the requirements of Table 3 below:

[0217] Table 3 Linear solution preparation

[0218] ;

[0219] (3) Accurately measure 20 μL of each of the above linear solutions, inject into the liquid chromatograph, record the chromatogram, and calculate the linear relationship between the peak area and concentration of tolvaptan sodium phosphate and each impurity. The results are shown in Table 4;

[0220] Table 4 Linearity determination results

[0221] ;

[0222] Note: Actual concentration = linear stock solution sample volume × reference substance content / dilution multiple.

[0223] The standard curves of impurity 1, impurity 2, impurity 3 and tolvaptan sodium phosphate are shown in Figure 6-9 shown.

[0224] From the results in Table 4, it can be seen that within the linear concentration range of 10%~150%, the correlation coefficient of the linear equation is r≥0.999, and the linear relationship is good.

[0225] Example 5 Quantitation Limit and Detection Limit Test

[0226] (1) Chromatographic conditions

[0227] Same as Example 2;

[0228] (2) Solution preparation

[0229] Blank solution: 50% acetonitrile;

[0230] LOQ solution (limit of quantitation solution): prepared in the same manner as the LOQ solution in the linearity test of Example 4;

[0231] LOD solution (detection limit solution): Measure 5 mL of LOQ solution, place it in a 10 mL volumetric flask, dilute to the mark with blank solution, and shake well; (concentration: contains impurity 1 about 0.075 μg / mL, impurity 2 about 0.075 μg / mL, impurity 3 about 0.075 μg / mL, tolvaptan sodium phosphate about 0.075 μg / mL)

[0232] (3) LOQ solution was injected 6 times and LOD solution was injected 3 times, the chromatogram was recorded, and the signal-to-noise ratio RSD was calculated. The results are shown in Table 5;

[0233] Table 5 Results of quantification limit and detection limit of impurities 1-3

[0234] ;

[0235] Table 6 Results of limit of quantification and limit of detection of tolvaptan sodium phosphate

[0236] ;

[0237] It can be seen from the results in Tables 5 and 6 that the RSDs of the peak areas of impurity 1 peak, impurity 2 peak, impurity 3 peak and tolvaptan sodium phosphate peak in the chromatograms of the six quantitative limit solutions are all ≤10.0%, and the signal-to-noise ratios S / N of the peak heights of impurity 1 peak, impurity 2 peak, impurity 3 peak and tolvaptan sodium phosphate peak in the quantitative limit solutions are all ≥10, which meets the requirements.

[0238] Example 6 Accuracy Test

[0239] (1) Chromatographic conditions

[0240] Same as Example 2;

[0241] (2) Solution preparation

[0242] Blank solution: 50% acetonitrile;

[0243] Impurity 1 stock solution: the same as in Example 2;

[0244] Impurity 2 stock solution: the same as in Example 2;

[0245] Impurity 3 stock solution: the same as in Example 2;

[0246] Tolvaptan sodium phosphate stock solution: the same as in Example 2;

[0247] Reference solution: same as in Example 2;

[0248] Accuracy stock solution: Take 5 mL of impurity 1 stock solution, 5 mL of impurity 2 stock solution, and 5 mL of impurity 3 stock solution, place them in a 50 mL volumetric flask, dilute to the scale with blank solution, and shake well; (Concentration: impurity 1 about 15 μg / mL, impurity 2 about 15 μg / mL, impurity 3 about 15 μg / mL)

[0249] Blank test solution: Take about 20 mg of the test sample, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the mark with the blank solution, and shake well; (Concentration: Contains about 1000 μg / mL of tolvaptan sodium phosphate)

[0250] Accuracy (LOQ) solution: Take about 100 mg of the test sample, accurately weigh it, place it in a 100 mL volumetric flask, add 1 mL of the accuracy stock solution, dilute to the scale with the blank solution, shake well, and prepare three parallel copies; (concentration: containing about 1000 μg / mL of tolvaptan sodium phosphate, about 0.15 μg / mL of impurity 1, about 0.15 μg / mL of impurity 2, and about 0.15 μg / mL of impurity 3)

[0251] Accuracy (100%) solution: Take about 20 mg of the test sample, accurately weigh it, place it in a 20 mL volumetric flask, add 2 mL of the accuracy stock solution, dilute to the scale with the blank solution, shake well, and prepare three parallel copies; (Concentration: containing about 1000 μg / mL of tolvaptan sodium phosphate, about 1.5 μg / mL of impurity 1, about 1.5 μg / mL of impurity 2, and about 1.5 μg / mL of impurity 3)

[0252] Accuracy (150%) solution: Take about 20 mg of the test sample, accurately weigh it, place it in a 20 mL volumetric flask, add 3 mL of the accuracy stock solution, dilute to the scale with the blank solution, shake well, and prepare three copies in parallel; (concentration: containing about 1000 μg / mL of tolvaptan sodium phosphate, about 2.25 μg / mL of impurity 1, about 2.25 μg / mL of impurity 2, and about 2.25 μg / mL of impurity 3)

[0253] (3) Accurately measure 20 μL of each of the above accuracy solutions, inject into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of the peak area of ​​impurity 1, impurity 2 and impurity 3. The results are shown in Tables 7, 8 and 9;

[0254] Table 7 Accuracy determination results of impurity 1

[0255] ;

[0256] Table 8 Accuracy determination results of impurity 2

[0257] ;

[0258] Table 9 Accuracy determination results of impurity 3

[0259] ;

[0260] From the results in Tables 7, 8 and 9, it can be seen that when the concentrations of impurities 1, impurity 2 and impurity 3 were in the range of 10% to 150%, the average recoveries of the accuracy (LOQ) solutions were all between 70% and 130.0%, and the recovery RSD was ≤5.0%; the average recoveries of the accuracy (100%) solutions were all between 90.0% and 130.0%, and the recovery RSD was ≤5.0%; the average recoveries of the accuracy (LOQ) solutions were all between 70% and 130.0%, and the recovery RSD was ≤5.0%; the accuracy of this method was good.

[0261] Example 6 Repeatability Test

[0262] (1) Chromatographic conditions

[0263] Same as Example 2;

[0264] (2) Solution preparation

[0265] Repeatability test solution: Prepare 6 portions in the same manner as the accuracy (100%) solution in Example 5;

[0266] (3) Accurately measure 20 μL of each test solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of tolvaptan sodium phosphate. The results are shown in Tables 10, 11, and 12;

[0267] Table 10 Repeatability test results of impurity 1

[0268] ;

[0269] Table 11 Repeatability determination results of impurity 2

[0270] ;

[0271] Table 12 Repeatability test results of impurity 3

[0272] ;

[0273] From the results in Tables 10, 11 and 12, it can be seen that the RSD (%) of impurity 1, impurity 2 and impurity 3 (n=6) is ≤2.0%, and the repeatability of the method is good.

[0274] The above embodiments are only used to illustrate the technical solutions of the present invention and should not be used to limit the scope of the present invention. That is, any simple modifications made according to the claims of the present invention and the contents of the present invention description should fall within the scope covered by the patent of the present invention.

Claims

1. A method for detecting impurity content in tolvaptan sodium phosphate freeze-dried powder injection, characterized in that: The tolvaptan sodium phosphate lyophilized powder injection is analyzed by a high performance liquid chromatography analysis method to obtain a chromatogram, and based on the chromatogram, the content of impurities in the tolvaptan sodium phosphate lyophilized powder injection is determined, wherein the chromatographic column used in the high performance liquid chromatography analysis method is an octadecylsilane bonded silica gel chromatographic column or an octyl bonded silica gel column, the mobile phase A is a 0.01-0.03 mol / L potassium dihydrogen phosphate solution, the pH value of the mobile phase A is 6.0-8.0, the mobile phase B is acetonitrile, and the elution gradient is: The first stage: the ratio of mobile phase A and B is (80:20)-(50:50), and the elution time is 6-10 minutes; the second stage: the ratio of mobile phase A and B is (50:50)-(50:50), and the elution time is 10-15 minutes; the third stage: the ratio of mobile phase A and B is (50:50)-(20:80), and the elution time is 3-5 minutes; the fourth stage: the ratio of mobile phase A and B is (20:80)-(20:80), and the elution time is 10-15 minutes; the fifth stage: the ratio of mobile phase A and B is (20:80)-(80:20), and the elution time is 2 minutes; The impurities are impurity 1: 5-dehydrotolvaptan, impurity 2: tolvaptan, and impurity 3: N-[4-[(5R)-7-chloro-2,3,4,5-tetrahydro-1-benzazepine-1-formyl]-3-methylphenyl]-2-methylbenzamide.

2. The detection method according to claim 1, characterized in that: The pH value of the mobile phase A was adjusted using triethylamine.

3. The detection method according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel chromatographic column Agilent5 HC-C18, 4.6 mm×250 mm, 5 μm.

4. The detection method according to claim 1, characterized in that: The tolvaptan sodium phosphate lyophilized powder injection is provided in the form of a test solution, wherein the test solution is an aqueous solution of tolvaptan sodium phosphate lyophilized powder injection, and based on each milliliter of the test solution, the concentration of tolvaptan sodium phosphate is 1.0 mg / mL.

5. The detection method according to claim 1, characterized in that: The detection wavelength adopted by the high performance liquid chromatography is 250-260 nm.

6. The detection method according to claim 1, characterized in that: The column temperature used in the high performance liquid chromatography is 25-35°C.

7. The detection method according to claim 1, characterized in that: Based on the chromatogram, the impurity content in the tolvaptan sodium phosphate was determined by calculating the peak area according to the external standard method, and the calculation formula is as follows: ， A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance, N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

8. The detection method according to any one of claims 1 to 7, characterized in that: include: (1) Chromatographic conditions Chromatographic column: Octadecylsilane bonded silica gel chromatographic column, Detector: DAD, Detection wavelength: 250~260nm, Column temperature: 25~35℃, Mobile phase A: 0.01-0.03 mol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 6.0-8.0, Mobile phase B: acetonitrile, Flow rate: 0.9~1.1mL / min, Injection volume: 20 μL, Running time: 33~53min; The elution gradient is: The first stage: the ratio of mobile phase A and B is (80:20)-(50:50), and the elution time is 6-10 minutes; the second stage: the ratio of mobile phase A and B is (50:50)-(50:50), and the elution time is 10-15 minutes; the third stage: the ratio of mobile phase A and B is (50:50)-(20:80), and the elution time is 3-5 minutes; the fourth stage: the ratio of mobile phase A and B is (20:80)-(20:80), and the elution time is 10-15 minutes; the fifth stage: the ratio of mobile phase A and B is (20:80)-(80:20), and the elution time is 2 minutes; (2) Preparation of blank solution Blank solution: 50% acetonitrile; (3) Preparation of test solution Take 25 mg of Tolvaptan Sodium Phosphate Lyophilized Powder Injection, accurately weigh it, put it into a 25 mL volumetric flask, dilute it to the mark with blank solution, shake well, and obtain; (4) Preparation of reference solution Impurity 1 stock solution: Take 3 mg of impurity 1 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well; Impurity 2 stock solution: Take 3 mg of impurity 2 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well; Impurity 3 stock solution: Take 3 mg of impurity 3 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to the mark with blank solution, and shake well; Tolvaptan sodium phosphate stock solution: Take 3 mg of tolvaptan sodium phosphate reference substance, accurately weigh it, put it into a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well; Reference solution: Take 1 mL of impurity 1 stock solution, 1 mL of impurity 2 stock solution, 1 mL of impurity 3 stock solution, and 1 mL of tolvaptan sodium phosphate stock solution, respectively, place them in a 100 mL volumetric flask, dilute to the mark with blank solution, and shake well; (5) Sample injection After the instrument is stable, inject samples in sequence: 1 empty needle, 1 to 3 blank solution needles, 6 reference solution needles, and 1 test solution needle. Before the end of the sequence and every 12 hours, inject 1 needle of the reference solution for back calibration and record the chromatogram. (6) Calculation of impurity content The impurity content is calculated by peak area according to the external standard method, and the calculation formula is as follows: ， A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance, N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

9. The detection method according to claim 8, characterized in that: include: (1) Chromatographic conditions Chromatographic column: Agilent5 HC-C18, 4.6mm×250mm, 5μm, Detector: DAD, Detection wavelength: 254nm, Column temperature: 30°C, Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 7.0, Mobile phase B: acetonitrile, Flow rate: 0.9~1.1mL / min, Injection volume: 20 μL, Running time: 50min; The elution gradient is: ; (2) Preparation of blank solution Blank solution: 50% acetonitrile; (3) Preparation of test solution Take 25 mg of Tolvaptan Sodium Phosphate Lyophilized Powder Injection, accurately weigh it, put it into a 25 mL volumetric flask, dilute it to the mark with blank solution, shake well, and obtain; (4) Preparation of reference solution Impurity 1 stock solution: Take 3 mg of impurity 1 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the mark with blank solution, and shake well; Impurity 2 stock solution: Take 3 mg of impurity 2 reference substance, accurately weigh it, put it in a 20 mL volumetric flask, dilute it to the mark with blank solution, and shake well; Impurity 3 stock solution: Take 3 mg of impurity 3 reference substance, accurately weigh, place in a 20 mL volumetric flask, dilute to the mark with blank solution, and shake well; Tolvaptan sodium phosphate stock solution: Take 3 mg of tolvaptan sodium phosphate reference substance, accurately weigh it, put it into a 20 mL volumetric flask, dilute it to the scale with blank solution, and shake well; Reference solution: Take 1 mL of impurity 1 stock solution, 1 mL of impurity 2 stock solution, 1 mL of impurity 3 stock solution, and 1 mL of tolvaptan sodium phosphate stock solution, respectively, place them in a 100 mL volumetric flask, dilute to the mark with blank solution, and shake well; (5) Sample injection After the instrument is stable, inject samples in sequence: 1 empty needle, 1 to 3 blank solution needles, 6 reference solution needles, and 1 test solution needle. Before the end of the sequence and every 12 hours, inject 1 needle of the reference solution for back calibration and record the chromatogram. (6) Calculation of impurity content The impurity content is calculated by peak area according to the external standard method, and the calculation formula is as follows: ， A S : Peak area of ​​impurities in the test solution, M R1 :Sampling amount of reference substance / mg, C R :Content of reference substance, N S : Dilution multiple of the test solution, A R1 : Average peak area of ​​5 consecutive injections of reference solution, M S : Sampling amount of test sample / mg, N R : Dilution multiple of reference solution.

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