Method for detecting degraded impurities of carbazochrome sodium sulfonate injection

Abstract

The invention provides a method for detecting degraded impurities in a carbazochrome sodium sulfonate injection, which can reflect the contents and changes of compounds in formulas II, III and IV in the carbazochrome sodium sulfonate injection, and has guiding significance for full research on the quality of the carbazochrome sodium sulfonate injection and upgrading and formulating of quality standards.

Description

Technical Field

[0001] The invention belongs to the field of medical analysis, and in particular relates to a method for detecting degradation impurities in carbazochrome sodium sulfonate injection. Background Art

[0002] Carbazochrome sodium is a derivative of Carbazochrome, with the chemical name 1-methyl-6-oxo-2,3,5,6-tetrahydroindole-5-semicarbazone-2-sulfonic acid sodium salt trihydrate, molecular formula C 10 H 11 N4NaO5S﹒3H2O, its structure is shown in formula I:

[0003]

[0004] Carbazochrome Sodium Sulfonate Injection is available on the market in three specifications: 5ml:25mg, 10ml:50mg and 20ml:100mg. It is used for skin, mucosal and endometrial bleeding, fundus bleeding, renal bleeding, uterine bleeding and abnormal bleeding after surgery caused by weakened capillary resistance.

[0005] At present, the Japanese and Korean pharmacopoeias and the domestic new drug formalization standards only include the quality standards of carbazochrome sodium sulfonate raw materials.

[0006] That is, the content of adrenochrome and total impurities in the raw material of carbazochrome sodium sulfonate is detected by high performance liquid chromatography at a high wavelength (363nm). There is no record of the quality standards of carbazochrome sodium sulfonate injection and other related preparations, nor is there any relevant research and control measures on the degradation impurities of carbazochrome sodium sulfonate injection. Summary of the invention

[0007] The inventors found that the mass was not conserved at a wavelength of 360 nm through destructive experiments when conducting a quality study on carbazochrome sodium sulfonate injection. Ultraviolet (UV) images of impurities with large degradation were extracted, and three new degradation impurities were found, whose structures are described in formulas II, III, and IV. The above three compounds have maximum absorption near 225 nm and almost no absorption at around 360 nm.

[0008]

[0009] During the stability study of Carbazochrome Sodium Sulfonate Injection, it was found that the above three degradation impurities would slowly increase over time and exceed the identification limit of this product; therefore, in order to better control the quality of Carbazochrome Sodium Sulfonate Injection and improve its safety, it is urgent to develop a detection method that can truly reflect the content of the above three impurity compounds in Carbazochrome Sodium Sulfonate Injection.

[0010] The present invention provides a method for detecting the degradation impurities of carbazochrome sodium sulfonate injection. This method can effectively qualitatively and quantitatively separate carbazochrome sodium sulfonate from the compounds of formula II, III, and IV, with high sensitivity, good specificity and repeatability, no interference from the solvent, and accurate and reliable detection results.

[0011] The method for detecting the degradation impurities of carbazochrome sodium sulfonate injection according to the present invention uses high performance liquid chromatography, with octadecylsilane chemically bonded silica gel as the filler or a chromatographic column with equivalent performance; uses phosphate buffer solution - acetonitrile as the mobile phase; and performs detection at a wavelength of 220 - 240 nm.

[0012] In some embodiments, the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection performs detection at a wavelength of 225 nm.

[0013] In some embodiments, for the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection, the pH value of the phosphate buffer solution is 6.3 - 6.7.

[0014] In some embodiments, for the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection, the pH value of the phosphate buffer solution is 6.0.

[0015] In some embodiments, for the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection, the phosphate buffer solution is mobile phase A, and acetonitrile is mobile phase B, and gradient elution is performed.

[0016] In some embodiments, the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection specifically includes:

[0017] High performance liquid chromatography conditions:

[0018] Chromatographic column: Using octadecylsilane chemically bonded silica gel as the filler or a chromatographic column with equivalent performance, 4.6×150 mm, 3 μm

[0019] Column temperature: 45 - 55 °C

[0020] Injection volume: 20 μL

[0021] Flow rate: 1.15 - 1.35 mL / min

[0022] Mobile phase A: Potassium dihydrogen phosphate buffer solution

[0023] Mobile phase B: Acetonitrile

[0024] Detection wavelength: 220 - 240 nm

[0025] And perform gradient elution through mobile phase A and mobile phase B.

[0026] In some embodiments, the method for detecting the degradation impurities of carbazochrome sodium sulfonate injection specifically includes:

[0027] High performance liquid chromatography conditions:

[0028] Chromatographic column: Octadecylsilyl silica gel as filler or a chromatographic column with equivalent performance, 4.6×150mm, 3μm

[0029] Column temperature: 50°C

[0030] Sample injection volume: 20 μL

[0031] Flow rate: 1.25 mL / min

[0032] Mobile phase A: Potassium dihydrogen phosphate buffer solution

[0033] Mobile phase B: Acetonitrile

[0034] Detection wavelength: 225 nm

[0035] And gradient elution is carried out with mobile phase A and mobile phase B.

[0036] Furthermore, the detection method for the degradation impurities of the carbazochrome sodium injection of the present invention is carried out by gradient elution through the following gradient elution program,

[0037] Gradient elution program:

[0038] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 100 0 15 85 15 30 55 45 31 100 0 40 100 0

[0039] Furthermore, in the detection method for the degradation impurities of the carbazochrome sodium injection of the present invention, the pH value of the potassium dihydrogen phosphate buffer solution in mobile phase A is 6.3 - 6.7.

[0040] Furthermore, in the detection method for the degradation impurities of the carbazochrome sodium injection of the present invention, the pH value of the potassium dihydrogen phosphate buffer solution in mobile phase A is 6.0.

[0041] Carbazochrome sodium test solution: Prepare a solution containing 0.5 mg / mL of the carbazochrome sodium sample to be tested as the test solution.

[0042] Carbazochrome sodium reference solution: Prepare a solution containing 2.5 μg / mL of carbazochrome sodium reference as the reference solution.

[0043] Mixed reference solution: Prepare a mixed solution containing 2.5 μg / mL of reference compound of formula II, 2.5 μg / mL of reference compound of formula III, 5 μg / mL of reference compound of formula IV, and 0.5 mg / mL of carbazochrome sodium reference as the mixed reference solution.

[0044] Furthermore, for the detection method of the degradation impurities of the carbazochrome sodium sulfonate injection of the present invention, the contents of the compounds of Formula II, III, and IV in the to-be-detected carbazochrome sodium sulfonate injection are calculated by the peak area according to the external standard method of the main component plus the correction factor method.

[0045] The present invention further provides a preparation method of the compound of Formula IV, which comprises the following steps: first, carbazochrome and sodium bisulfite are heated and reacted in water. After the reaction is completed, the reaction solution is purified and eluted by high performance liquid chromatography, the target eluate is collected, concentrated, and freeze-dried to obtain the compound of Formula IV.

[0046] Furthermore, the molar ratio of carbazochrome to sodium bisulfite is 1:5.

[0047] Furthermore, the heating temperature is 100 °C; the reaction time is 3 - 6 h, preferably 4 h.

[0048] Furthermore, the mobile phase of the high performance liquid chromatography is acetonitrile - ammonium formate buffer solution.

[0049] Furthermore, the chromatographic conditions of the high performance liquid chromatography are as follows:

[0050] Chromatographic column: Ultimate XB-C18(50*250mm,10μm)

[0051] Mobile phase A: 20 mol / L ammonium formate

[0052] Mobile phase B: preparative grade acetonitrile

[0053] Flow rate: 60 ml / min

[0054] Sample injection volume: 3 g

[0055] Detection wavelength: 225 nm

[0056] Diluent: water

[0057] Gradient elution program:

[0058] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 12 30 70 12.5 5 95 20 5 95 20.5 60 40

[0059] The present invention further provides the use of the compounds of Formula II, III, and IV as reference substances or standards in carbazochrome pharmaceutical preparations.

[0060] The present invention further provides a carbazochrome pharmaceutical composition, which is characterized in that the composition contains the following substances in the following contents: carbazochrome ≥ 90%, the compound of Formula II ≤ 0.5%, the compound of Formula III ≤ 0.5%, and the compound of Formula IV ≤ 1.0%.

[0061] In some preferred embodiments, the contents of the components in the composition are as follows: carbazochrome sodium sulfonate ≥ 95%, the compound of formula II ≤ 0.3%, the compound of formula III ≤ 0.3%, and the compound of formula IV ≤ 0.6%.

[0062] The present invention further provides a carbazochrome sodium sulfonate injection, which is characterized by comprising the above-mentioned pharmaceutical composition, and the injection is stored sealed in a brown transparent ampoule.

[0063] In some embodiments, the carbazochrome sodium sulfonate injection further comprises other pharmaceutical excipients. In some embodiments, the pharmaceutical excipients may be one or more of sorbitol, propylene glycol, citric acid, sodium bisulfite, disodium hydrogen phosphate, sodium chloride, glucose, potassium chloride, magnesium chloride, calcium chloride, sodium sulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, malic acid, ascorbic acid, palmitate, hydrochloric acid, tartaric acid, sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, acetic acid, sodium acetate, lactic acid, sodium citrate, sodium bicarbonate, sodium carbonate.

[0064] The present invention further provides a preparation method of the above-mentioned carbazochrome sodium sulfonate injection, which is characterized by dissolving carbazochrome sodium sulfonate and pharmaceutical excipients in water for injection at 25 - 30 °C and filling them into a brown transparent ampoule and sealing.

[0065] In some embodiments, the prescription of the carbazochrome sodium sulfonate injection is as follows:

[0066] Prescription Content of each component per milliliter of injection (mg / mL) Carbazochrome sodium sulfonate 5 Sorbitol 30 Propylene glycol 30 Citric acid 0.45 Sodium bisulfite 0.07 Disodium hydrogen phosphate 0.8 Water for injection Volume made up to 1 mL

[0067] In some embodiments, the preparation of the carbazochrome sodium sulfonate injection is as follows: adding the prescribed amount or the equi - proportionally scaled - up amount of carbazochrome sodium sulfonate, sorbitol, propylene glycol, citric acid, sodium bisulfite and disodium hydrogen phosphate into water for injection, dissolving them until clear at 25 - 30 °C, and then sub - packaging them into 5 - ml brown transparent ampoules and sealing. Description of the Drawings

[0068] Figure 1 : UV spectrum of the compound of formula II

[0069] Figure 2 : High - resolution mass spectrum of the compound of formula II

[0070] Figure 3 : The compound of formula II 1 1H NMR spectrum

[0071] Figure 4 : The compound of formula II 13 13C NMR spectrum

[0072] Figure 5 : UV spectrum of the compound of formula III

[0073] Figure 6 : High-resolution mass spectrometry spectrum of the compound of Formula III

[0074] Figure 7 : The compound of Formula III 1 1H NMR spectrum

[0075] Figure 8 : The compound of Formula III 13 13C NMR spectrum

[0076] Figure 9 : Ultraviolet spectrum of the compound of Formula IV

[0077] Figure 10 : High-resolution mass spectrometry spectrum of the compound of Formula IV

[0078] Figure 11 : The compound of Formula IV 1 1H NMR spectrum

[0079] Figure 12 : The compound of Formula IV 13 13C NMR spectrum

[0080] Figure 13 : Infrared spectrum of the compound of Formula IV

[0081] Figure 14 : HPLC chromatogram of the mixed reference solution Detailed implementation manners

[0082] The technical solutions of the present invention will be further described and illustrated below in conjunction with specific embodiments.

[0083] This embodiment is only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0084] The abbreviations used in the present invention are explained as follows:

[0085] 1 1H NMR: Proton nuclear magnetic resonance

[0086] 13 13C NMR: Carbon nuclear magnetic resonance

[0087] HPLC: High performance liquid chromatography

[0088] RSD: Relative standard deviation

[0089] LOQ: Limit of quantification

[0090] IRep - 10%: Recovery solution containing reference substances of Compounds II, III, and IV at 10% of the limit concentration

[0091] IRep - 100%: Recovery solution containing reference substances of Compounds II, III, and IV at 100% of the limit concentration

[0092] IRep - 150%: Recovery solution containing reference substances of Compounds II, III, and IV at 150% of the limit concentration

[0093] The reference substances of Compounds II, III, and IV were all prepared. The preparation method refers to Examples 1 - 3. Carbazochrome sodium sulfonate and other pharmaceutical excipients, reagents, etc. were all obtained commercially. The water used in the detection and analysis was all ultrapure water.

[0094] Main instruments:

[0095] Name of instrument Model of instrument Manufacturer of instrument Analytical liquid phase Waters e2695 Waters Preparative liquid phase Sail1000 Yuexu Liquid chromatography - mass spectrometry 6545Q - TOF Agilent Infrared spectrometer NICOLET IS10 Therm fisher Nuclear magnetic resonance spectrometer Varian 400MHz Agilent

[0096] Example 1: Prescription and preparation of Carbazochrome Sodium Sulfonate Injection

[0097] The prescription of Carbazochrome Sodium Sulfonate Injection is as follows:

[0098]

[0099]

[0100] Preparation method of Carbazochrome Sodium Sulfonate Injection:

[0101] Take 25 g of Carbazochrome Sodium Sulfonate, 150 g of sorbitol, 150 g of propylene glycol, 2.25 g of citric acid, 0.35 g of sodium bisulfite, and 4 g of disodium hydrogen phosphate respectively, dilute with injection water to 5 L, and dispense into 5 - ml ampoules and seal to obtain.

[0102] Example 2: Preparation of Compounds II and III

[0103] Prepare 10 L of Carbazochrome Sodium Sulfonate Injection according to the method in Example 1, but do not perform dispensing. Conduct a high - temperature degradation experiment on this Carbazochrome Sodium Sulfonate Injection. When it is degraded at 80°C for about 29 h, then elute it with the mobile phase of acetonitrile - ammonium formate buffer according to the following preparation chromatographic conditions, and perform multiple injection and elution steps until the injection is complete. Collect the target eluates respectively, purify, concentrate, enrich, and lyophilize to obtain 8.3 mg of Compound II and 5.3 mg of Compound III. According to the detection method in Example 4, using the HPLC peak area normalization method, the measured contents are 93.45% and 91.32% respectively.

[0104] Preparation chromatographic conditions:

[0105]

[0106] The prepared compound of Formula II was characterized. 1 H NMR (400 MHz, D2O): δ 3.59 (br s, 3H), 6.36 (br d, 1H), 6.69 (d, 1H), 7.20 (br d, 1H); [M+H] + : 380.03.

[0107] Figure 1 It is the UV spectrum of the compound of Formula II; Figure 2 It is the high-resolution mass spectrum of the compound of Formula II; Figure 3 It is the compound of Formula II 1 H NMR spectrum; Figure 4 It is the compound of Formula II 13 C NMR spectrum.

[0108] The prepared compound of Formula III was characterized. 1 H NMR (400 MHz, D2O:CD3OD = 8:2): δ 3.59 (brs, 3H), 6.31 (br s, 1H), 7.13 (br s, 1H), 7.26 (br d, 1H), 7.47 (br d, 1H), 7.65 (br s, 1H); [M+H] + : 212.04.

[0109] Figure 5 It is the UV spectrum of the compound of Formula III; Figure 6 It is the high-resolution mass spectrum of the compound of Formula III; Figure 7 It is the compound of Formula III 1 H NMR spectrum; Figure 8 It is the compound of Formula II 13 C NMR spectrum.

[0110] Example 3: Preparation of the compound of Formula IV

[0111] 1.51 g of adrenochrome and 3.36 g of sodium bisulfite were taken, added to 250 mL of water, and reacted in a water bath at 100 °C for 6 hours. The reaction solution was eluted with the mobile phase acetonitrile-ammonium formate buffer according to the following preparative chromatography conditions, and multiple injection and elution steps were carried out until the reaction solution was completely injected. The target eluate was collected separately, purified, concentrated, enriched, and freeze-dried to obtain 0.10 g of the compound of Formula IV. According to the detection method of Example 4, using the HPLC peak area normalization method, its content was measured to be 95.47%.

[0112] Preparative chromatography conditions:

[0113]

[0114] The prepared compound of formula IV was characterized. 1 1H NMR (400 MHz, CD3OD): δ 3.66 (s, 3H), 6.32 (d, 1H), 6.81 (s, 1H), 6.99 (d, 1H), 7.72 (s, 1H); IR (KBr, cm-1) data were: 3391, 3337, 3218, 3015, 1673, 1636, 1592, 1516, 1479, 1422, 1350, 1250, 1199, 1039, 792, 714; [M+H] + : 219.09.

[0115] Figure 9 It is the UV spectrum of the compound of formula IV; Figure 10 It is the high-resolution mass spectrum of the compound of formula IV; Figure 11 It is the compound of formula IV 1 1H NMR spectrum; Figure 12 It is the compound of formula IV 13 13C NMR spectrum; Figure 13 It is the IR spectrum of the compound of formula IV.

[0116] Example 4:

[0117] Detection method for degradation impurities in carbazochrome sodium sulfonate injection:

[0118] High performance liquid chromatography conditions:

[0119] Chromatographic column: CL069, YMC-Triart C18 4.6×150 mm, 3 μm

[0120] Column temperature: 50 °C

[0121] Injection volume: 20 μL

[0122] Flow rate: 1.25 mL / min

[0123] Mobile phase A: Potassium dihydrogen phosphate buffer solution

[0124] Mobile phase B: Acetonitrile

[0125] Detection wavelength: 225 nm

[0126] Gradient elution:

[0127] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 100 0 15 85 15 30 55 45 31 100 0 40 100 0

[0128] Blank solution: Water

[0129] Test solution of carbazochrome sodium sulfonate: Accurately measure 1 mL of the carbazochrome sodium sulfonate injection to be tested, dilute it with water to prepare a 0.5 mg / mL carbazochrome sodium sulfonate solution as the test solution.

[0130] Carbazochrome sodium sulfonate reference solution: Weigh an appropriate amount of carbazochrome sodium sulfonate reference substance accurately, dissolve it in water and dilute to make a carbazochrome sodium sulfonate solution with a concentration of 2.5 μg / mL.

[0131] Mixed reference solution: Weigh appropriate amounts of reference substances of compounds II, III, and IV and carbazochrome sodium sulfonate accurately, dissolve them in water and dilute to make a mixed reference solution containing 2.5 μg of compound II, 2.5 μg of compound III, 5 μg of compound IV, and 0.5 mg of carbazochrome sodium sulfonate per 1 mL.

[0132] Test Example 1:

[0133] Methodology investigation of the detection method in Example 4:

[0134] System suitability:

[0135] Inject samples according to the chromatographic conditions in Example 1. Inject 1 needle of blank solvent and mixed reference solution respectively, and inject 6 consecutive needles of reference solution;

[0136] The results show that there are no interfering peaks between each impurity and the blank solution; the retention times of compound II, carbazochrome sodium sulfonate, compound IV, and compound III in the mixed reference solution are approximately 3.3 min, 10.2 min, 10.8 min, and 12.2 min respectively. The resolution between compound II and carbazochrome sodium sulfonate is 22.9, the resolution between compound IV and carbazochrome sodium sulfonate is 2.5, and the resolution between compound IV and compound III is 9.1; for 6 consecutive needles of reference solution, the RSD of the main peak area of the reference solution is 0.99%.

[0137] Linearity:

[0138] Mixed stock solution: Weigh appropriate amounts of compound II, compound III, carbazochrome sodium sulfonate, and compound IV accurately, place them in a 100 mL volumetric flask, dilute to the scale with diluent and shake well to make a mixed solution containing 25 μg / mL of compound II, 25 μg / mL of compound III, 25 μg / mL of carbazochrome sodium sulfonate, and 50 μg / mL of compound IV per 1 mL.

[0139] Table 1 Preparation of linear solutions of M-L1 to M-L7

[0140]

[0141] Prepare the mixed reference substance solutions of Compounds II, III, and IV, carbazochrome sodium sulfonate at the quantitative limit (LOQ), 10%, 40%, 50%, 100%, 150%, and 200% according to Table 1 respectively, and name them as Linear Solution M-L7 to M-L1. With the concentration as the abscissa and the peak area as the ordinate, draw the linear regression curves. The linear regression equations of Compounds II, III, IV, and carbazochrome sodium sulfonate are: C = 47.606A - 0.0216, C = 75.218A + 0.2279, C = 70.393A + 7.6062, C = 32.687A + 0.119 respectively, and the correlation coefficients r are 0.9999, 0.9999, 0.9999, and 0.9998 respectively. The results show that: Compound II has a good linearity in the range of 0.0587 μg / mL to 4.6933 μg / mL, Compound III has a good linearity in the range of 0.0564 μg / mL to 4.5154 μg / mL, Compound IV has a good linearity in the range of 0.1160 μg / mL to 9.2818 μg / mL, all equivalent to the level of 2.5% to 200% of the limit concentration; API has a good linearity in the range of 0.1065 μg / mL to 8.5175 μg / mL, equivalent to the level of 0.025% to 2% of the test sample concentration; the correction factors of Compounds II, III, and IV are 0.69, 0.43, and 0.46 respectively.

[0142] Precision:

[0143] Detect the contents of Compounds II, III, and IV in six test sample solutions by the above method. The relative standard deviation (RSD) of the content of Compound IV is 1.8%, the RSD of the content of Compound II is 2.4%, and the RSD of the content of Compound III is 2.2%, all less than 5.0%, meeting the requirements.

[0144] Quantitative limit:

[0145] Take the mixed reference substance solution and gradually dilute it with water to a solution with a signal-to-noise ratio of about 10.

[0146] Results:

[0147] The quantitative limit concentration of Compound II is 0.0587 μg / mL, which is 0.012% of the test sample concentration;

[0148] The quantitative limit concentration of Compound III is 0.0564 μg / mL, which is 0.012% of the test sample concentration;

[0149] The quantitative limit concentration of Compound IV is 0.1160 μg / mL, which is 0.024% of the test sample concentration.

[0150] Detection limit:

[0151] Take the mixed reference substance solution and gradually dilute it with water to a solution with a signal-to-noise ratio of about 3.

[0152] Results:

[0153] The detection limit concentration of the compound of formula II is 0.0196 μg / mL, which is 0.004% of the concentration of the test sample;

[0154] The detection limit concentration of the compound of formula III is 0.0196 μg / mL, which is 0.004% of the concentration of the test sample;

[0155] The detection limit concentration of the compound of formula IV is 0.0290 μg / mL, which is 0.006% of the concentration of the test sample.

[0156] Recovery rate:

[0157] Carbazochrome sodium sulfonate injection: Prepared according to the method of Example 1.

[0158] Spiked impurity stock solution: Weigh an appropriate amount of the compounds of formula II, formula III, and formula IV precisely, place them in a 100 ml volumetric flask, dilute to the mark with water and shake well to prepare a mixed solution containing 25 μg / mL, 25 μg / mL, and 50 μg / mL of the compounds of formula II, formula III, and formula IV respectively per 1 ml.

[0159] Table 2 Preparation of recovery rate solutions

[0160]

[0161] Prepare 3 solutions each for the reference substances of the compounds of formula II, III, and IV at relative limit concentrations of 10%, 100%, and 150% respectively according to Table 2, a total of 9 solutions, calculate the recovery rate. The results show that the recovery rates of each concentration of the compounds of formula II, III, and IV are all in the range of 85% - 120%, and the RSDs are 1.4%, 2.8%, and 2.9% respectively, all less than 5.0%, meeting the requirements and showing good recovery rates.

[0162] Robustness:

[0163] Preparation of the test sample solution: Prepare the test sample solution to be measured as the test sample solution according to the preparation method of IRep - 100% in the recovery rate experiment

[0164] Carbazochrome sodium sulfonate reference substance solution: Weigh an appropriate amount of carbazochrome sodium sulfonate reference substance precisely, dissolve it in water and dilute to make a carbazochrome sodium sulfonate solution of 2.5 μg / mL.

[0165] Diluent: Water

[0166] Evaluate and investigate the robustness of the detection method by changing the column temperature, flow rate, and pH value of mobile phase A. Under each parameter condition, inject five needles of the reference substance solution, one needle of the diluent, and one needle of the test sample solution respectively. The results are shown in Table 3.

[0167] Contents and retention times of each component in the test solution

[0168]

[0169] Test Example 2:

[0170] Carbazochrome sodium sulfonate injection was prepared according to Example 1 and then dispensed into 5-ml ampoules and sealed.

[0171] The above carbazochrome sodium sulfonate injection was subjected to a 6-month stability study at 30 °C and RH 65%. Samples were taken at 0 month, 3 months, and 6 months, and the contents of Compounds II, III, and IV in the preparation were detected according to the detection method of Example 4. The results are shown in Table 4 below.

[0172] Precisely measure 20 μL of each of the reference solution and the test solution and inject them into the liquid chromatograph respectively. Record the chromatogram until 3 times the retention time of the main peak. Among them, Compounds II, III, and IV were calculated by the external standard method with correction factors for the main component.

[0173] Table 4 Changes in the contents of each component in carbazochrome sodium sulfonate injection from 0 to 6 months

[0174]

[0175] Example 5: Optimization of the preparation process of carbazochrome sodium sulfonate injection

[0176] The solubility of carbazochrome sodium sulfonate in cold water (<20 °C) is less than 5 mg / mL, which cannot meet the requirements of the concentration of carbazochrome sodium sulfonate injection. Therefore, the solution needs to be prepared under heated conditions. To investigate the influence of different solution preparation temperatures on the product quality and select appropriate process temperature parameters, carbazochrome sodium sulfonate injection was prepared according to the prescription and method of Example 1 at different solution preparation temperatures, and the contents of Compounds II, III, and IV in the injection prepared at each temperature were detected according to the detection method of Example 4. The results are shown in Table 5 below.

[0177] Table 5 Influence of different solution preparation temperatures on the product quality

[0178]

[0179] Example 6: Selection of packaging materials for carbazochrome sodium sulfonate injection

[0180] Carbazochrome sodium sulfonate injection prepared according to the prescription and method of Example 1 was dispensed into 5-mL ampoules of different colors and sealed. The contents of each impurity in the injection under different light conditions were detected according to the detection method of Example 4 to reflect the stability of the product, and appropriate packaging materials were selected. The results are shown in Table 6 below.

[0181] Table 6 Influence of Packaging Materials of Different Colors on Product Quality

[0182]

Claims

1. A method for detecting the degradation impurities of carbazochrome sodium sulfonate injection, characterized in that, The high performance liquid chromatography method is adopted, using octadecylsilane chemically bonded silica as the filler or a chromatographic column with equivalent performance. The mobile phase is phosphate buffer solution - acetonitrile, and detection is carried out at a wavelength of 220 - 240 nm.

2. The detection method according to claim 1, wherein The detection method is carried out at a wavelength of 225 nm.

3. The detection method according to claim 1, wherein The pH value of the phosphate buffer solution is 6.3 - 6.7, preferably 6.

0.

4. The detection method according to claim 1, wherein The phosphate buffer solution is mobile phase A, and acetonitrile is mobile phase B, and gradient elution is carried out.

5. The detection method according to claim 1, characterized in that, The high performance liquid chromatography conditions are as follows: Chromatographic column: Using octadecylsilane chemically bonded silica as the filler or a chromatographic column with equivalent performance, 4.6×150 mm, 3 μm Column temperature: 45 - 55 °C Sample injection volume: 20 μL Flow rate: 1.15 - 1.35 mL / min Mobile phase A: Potassium dihydrogen phosphate buffer solution Mobile phase B: Acetonitrile Detection wavelength: 220 - 240 nm And gradient elution is carried out through mobile phase A and mobile phase B.

6. The detection method according to claim 5, characterized in that, The column temperature is 50 °C; the flow rate is 1.25 mL / min; Detection wavelength: 225 nm.

7. The detection method according to claim 5, wherein The pH value of the potassium dihydrogen phosphate buffer solution in the mobile phase A is 6.3 - 6.7, preferably 6.

0.

8. The detection method according to claim 5, wherein The program of the gradient elution is as follows, Gradient elution program:

9. The detection method according to any one of claims 1 to 8, characterized in that Separate solutions are prepared: a test solution containing 0.5 mg / mL of the sodium carbazochrome sample to be tested as the test sample solution; a solution containing 2.5 μg / mL of the sodium carbazochrome reference substance as the reference substance solution; a mixed solution containing 2.5 μg / mL of the reference substance of Compound II, 2.5 μg / mL of the reference substance of Compound III, 5 μg / mL of the reference substance of Compound IV and 0.5 mg / mL of the sodium carbazochrome reference substance as the mixed reference substance solution.

10. The detection method according to any one of claims 1 to 8, characterized in that, The contents of Compounds II, III, and IV in the sodium carbazochrome injection to be tested are calculated by the external standard method with correction factor based on the peak areas.

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