Method for controlling quality of nitrite in ornidazole injection

Abstract

The invention discloses a method for detecting nitrite in ornidazole injection by ion chromatography. The method comprises the following steps: setting chromatographic conditions: using an anion exchange chromatographic column; a KOH solution is used as an eluent, and the concentration of the KOH solution is 6.0 mmol / L; performing gradient elution; the column temperature is 25-35 DEG C; an electrical conductivity detector (ECD); the flow velocity is 0.8 ml to 1.2 ml per minute; and injecting 20 microliters of the blank solution, 20 microliters of the reference solution and 20 microliters of the test solution into an ion chromatograph, recording chromatograms, and calculating according to an external standard method. The method for determining the nitrite in the ornidazole injection by using the ion chromatography has the advantages of strong specificity, fast analysis speed and high detection sensitivity, and is a brand new method for controlling the quality of the nitrite in the ornidazole injection.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection, and in particular to a quality detection method for nitrite in ornidazole injection. Background Art

[0002] The chemical name of ornidazole is 1-(3-chloro-2-hydroxypropyl)-2-methyl-5-nitroimidazole. It is a third-generation nitroimidazole derivative with strong antibacterial ability, high antiprotozoal activity, long half-life and good tissue permeability. Ornidazole injection is mainly used clinically for various diseases caused by anaerobic bacterial infections and is included in Part II of the 2020 edition of the "Chinese Pharmacopoeia".

[0003] Hemoglobin is a protein responsible for carrying oxygen in the body, and nitrite (NO 2- Once it enters the blood, it is easy to combine with hemoglobin in the human body, causing normal hemoglobin to deform and lose its oxygen transport function, further leading to hypoxia in human tissues, causing dizziness, sore limbs, shock and death. Animal tests or in vitro measurements have shown that this product has carcinogenic and mutagenic effects, and can cause tumors in various tissues and organs of different experimental animals. Therefore, controlling the content of nitrite in ornidazole injection is of great significance to the clinical safety of drugs.

[0004] At present, the methods used for quantitative determination of nitrite in food and medicine at home and abroad mainly include ion chromatography (IC), spectrophotometry (SP), fluorescence spectroscopy (FS), colorimetry, etc. Ion chromatography is the preferred method for conventional anion detection. This method has good stability, high sensitivity, fast analysis speed, good selectivity and can detect multiple anions at the same time. Summary of the invention

[0005] The object of the present invention is to provide a quality detection method for nitrite in ornidazole injection, so that the nitrite in ornidazole injection has good specificity and accuracy, and is convenient for rapid, accurate and sensitive mass content analysis of nitrite.

[0006] The technical solution adopted by the present invention is: ion chromatography is used to determine nitrite in ornidazole injection, comprising the following steps: (1) Blank solvent: purified water; (2) Preparation of test solution: Accurately measure 1.0 ml of ornidazole injection and place it in a 10 ml plastic volumetric flask. Add blank solvent to dilute to the mark and shake well. (3) Preparation of reference solution: Take an appropriate amount of sodium nitrite reference solution (equivalent to 33.3 mg of nitrite), accurately weigh it, place it in a 100 ml plastic volumetric flask, add blank solvent to dissolve and dilute to the scale, shake well, accurately measure 0.5 ml, place it in a 10 ml plastic volumetric flask, add blank solvent to dilute to the scale, shake well, and obtain; (4) Injection: Take 20 μl of the control solution, 20 μl of the test solution, and 20 μl of the blank solvent and inject them into the ion chromatograph, and record the chromatogram. According to the external standard method and calculated by peak area, the nitrite content of ornidazole injection shall not exceed 0.1%; The chromatographic conditions of step (4) are as follows: the chromatographic column is an anion exchange chromatographic column; KOH solution is used as the eluent; the column temperature is 25°C-35°C; an electroconductivity detector (ECD); and the flow rate is 0.8ml-1.2ml per minute.

[0007] Preferably, in step (4), the quantitative limit of nitrite is 1.64 μg / ml (0.01%); the detection limit is 0.82 μg / ml (0.005%).

[0008] Preferably, in step (4), the nitrite has a good linear relationship in the range of 1.64-32.73 μg / ml.

[0009] Preferably, the anion analysis chromatographic column in step (4) is a Thermo Dionex IonPac AS9-HC, RFIC, 4×250 mm ion chromatography analysis column.

[0010] Preferably, the column temperature in step (4) is 30°C.

[0011] Preferably, the flow rate of the eluent in step (4) is 1.0 ml / min.

[0012] Preferably, the elution gradient of the eluent in step (4) is:

[0013] Beneficial effects of the present invention: The method for detecting nitrite in ornidazole injection provided by the present invention provides a rapid, highly specific and highly accurate detection method, which is suitable for detecting nitrite in ornidazole injection. Implementation

[0014] The present invention is further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0015] Experimental Example 1 Detection of nitrite content in ornidazole injection by the method of the present invention 1. Test materials and reagents (1) Test materials: Ornidazole injection (our company, specification 3 ml: 0.5 g, batch number 210901, 210621, 210623, 210625; specification 6 ml: 1.0 g, 210901, 210701, 210703, 210705; reference preparation, specification 3 ml: 0.5 g, batch number 2635; specification 6 ml: 1.0 g, batch number 2700).

[0016] (2) Reagents: Sodium nitrite [Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity 99.6%, batch number BCCC3243]; water was ultrapure water.

[0017] 2. Instruments and equipment Ion chromatograph (ICS2100, equipped with conductivity detector, AERS suppressor, CR-TC capture column), RP column and Na column were purchased from Thermo Fisher, USA; analytical balance (XS205DU) was purchased from Mettler 3 Experimental methods (3) Establishment of chromatographic conditions Chromatographic column: Thermo Dionex IonPac AS9-HC, RFIC, ion chromatography analysis column (4×250mm); equipped with Thermo Dionex IonPac AG18, HC RFIC, (HFTR) type guard column (4mm×50mm) The detection conditions were as follows: column temperature, 30°C; suppressor current, 149 mA; flow rate, 1.0 ml / min, injection volume, 20 μl. Potassium hydroxide eluent, eluent conditions are shown in Table 1.

[0018] Table 1 Gradient elution conditions

[0019] (4) Determination of linearity, limit of quantification and limit of detection According to the best detection conditions determined by the above experiments, nitrite was prepared into a series of concentration working solutions. The concentrations of nitrite were 1.64, 8.18, 13.09, 16.36, 24.55, and 32.73 μg of nitrite per 1 ml of linear series standard solutions. Detection was performed on the machine. A standard curve was drawn with nitrite ion concentration (μg / ml) as the horizontal axis (X) and peak area (μS) as the vertical axis (Y) to determine the best linear range and correlation coefficient. The detection limit (LOD) was calculated with a signal-to-noise ratio of 3 times and a signal-to-noise ratio of 10 times. Calculate the limit of quantitation (LOQ).

[0020] (5) Precision experiment Accurately measure 1.0 ml of ornidazole injection and place it in a 10 ml plastic volumetric flask to prepare a solution containing 100% nitrite. Prepare 6 copies in parallel as repeatability test solutions and calculate the nitrite content.

[0021] (6) Accuracy experiment Accurately measure 1.0 ml of ornidazole injection for a total of 3 portions, and place them in 10 ml plastic volumetric bottles respectively to prepare solutions containing 50%, 100%, and 150% nitrite. Prepare 3 portions for each concentration as 50% to 150% recovery test solutions.

[0022] (7) Durability test Accurately measure 1.0 ml of ornidazole injection and place it in a 10 ml plastic volumetric flask to prepare a solution containing 100% nitrite. The differences in nitrite content determination under different column temperatures and flow rates were investigated.

[0023] 4. Data Analysis The data were analyzed using the Chromeleon Client Program software from Thermo Fisher Scientific, USA.

[0024] 5. Test results and analysis (1) Detection limit, quantification limit and linear range The ion chromatogram of nitrite shows that the retention times of nitrite and nitrate are 14.637min, good separation.

[0025] The standard curves of nitrate and nitrite were drawn to determine the linear range of nitrite. As shown in Table 2, the limit of quantification of nitrite was 1.64 μg / ml and the limit of detection was 0.82 μg / ml. Nitrite showed a good linear relationship in the range of 1.64~32.73 μg / ml, and its correlation coefficient R 2 is 0.9978.

[0026] Table 2 Linear equation, linear coefficient, quantification limit and detection limit of nitrite

[0027] From the precision experiment, it can be seen from Table 3 that the precision of the present invention is better.

[0028] Table 3 Precision experiment of nitrite

[0029] Accuracy experiment: From Table 4, it can be seen that the accuracy of the present invention is better.

[0030] Table 4 Accuracy and RSD of the present invention

[0031]

[0032] Durability test: From Table 5, it can be seen that the durability of the present invention is better.

[0033] Table 5, Durability test results

[0034] Test Example 2: Practical application of the detection method of the present invention Six batches of ornidazole injection prepared by our company and two batches of reference preparations were selected to detect the nitrite content therein. The detection conditions were referred to Experimental Example 1, and the actual application effect of the method of the present invention in detecting the nitrite content in ornidazole injection was evaluated.

[0035] As shown in 6, both the ornidazole injection prepared by our company and the reference preparation contain a certain amount of nitrite, both lower than 0.1%; the nitrite content in the ornidazole injection product prepared by our company is much lower than the nitrite content in the reference preparation.

[0036] Table 6 Nitrite content in ornidazole injection prepared by our company and reference preparation

Claims

1. A method for detecting the quality of nitrite in ornidazole injection, characterized in that: The following steps are involved: Blank solvent: purified water; Preparation of test solution: Accurately measure 1.0 ml of ornidazole injection and place it in a 10 ml plastic volumetric flask. Add blank solvent to dilute to scale and shake well; Preparation of reference solution: Take an appropriate amount of sodium nitrite reference (equivalent to 33.3 mg of nitrite), accurately weigh, place in a 100 ml plastic volumetric flask, add blank solvent to dissolve and dilute to scale, shake well, accurately measure 0.5 ml, place in a 10 ml plastic volumetric flask, add blank solvent to dilute to scale, shake well, and obtain; The chromatographic columns were: anion exchange column (Thermo Dionex IonPac AS9-HC, RFIC, 4 × 250 mm); Chromatographic conditions: KOH solution as eluent, KOH solution concentration of 6.0 mmol / L; gradient elution; column temperature of 25℃-35℃; conductivity detector (ECD); flow rate of 0.8ml-1.2ml per minute; Under the above chromatographic conditions, accurately pipette 20 μl of control solution, 20 μl of test solution and 20 μl of blank solvent into the ion chromatograph, record the chromatogram and calculate the peak area using the external standard method. The nitrite content of ornidazole injection shall not exceed 0.1%.

2. The quality inspection method according to claim 1, characterized in that Chromatographic conditions: KOH solution as eluent, KOH solution concentration of 6.0 mmol / L; gradient elution; column temperature of 25°C; electroconductivity detector (ECD); flow rate of 0.8 ml per minute.

3. The quality inspection method according to claim 1, characterized in that Chromatographic conditions: KOH solution as eluent, KOH solution concentration of 6.0 mmol / L; gradient elution; column temperature of 30°C; electroconductivity detector (ECD); flow rate of 1.0 ml per minute.

4. The quality inspection method according to claim 1, characterized in that Chromatographic conditions: KOH solution as eluent, KOH solution concentration of 6.0 mmol / L; gradient elution; column temperature of 35°C; electroconductivity detector (ECD); flow rate of 1.2 ml per minute.

5. The detection method according to claim 1, characterized in that: The elution gradient of the potassium hydroxide eluent in steps (2) and (3) is: ① 6mmol / L, 0-20min; ② 70mmol / L, 25-30min; ③ 6mmol / L, 35-40min.

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