High performance liquid chromatography quantitative analysis method of methoxyamine hydrochloride

Abstract

The invention provides a high performance liquid chromatography quantitative analysis method of methoxyamine hydrochloride, which comprises the following steps: preparation of solutions: preparation of reference solutions with a series of concentration gradients: weighing methoxyamine hydrochloride standard substances according to different concentrations respectively, adding a sodium hydroxide solution for dissolving, then adding an organic solvent, and fixing the volume; preparing a test solution, namely weighing a test sample, neutralizing the test sample to a specific pH value by using a sodium hydroxide solution, then adding an organic solvent, and fixing the volume; high performance liquid chromatography is adopted for quantitative detection, and quantitative analysis is carried out according to a linear regression curve; wherein the mobile phase is deionized water, and the mobile phase B is chromatographic methanol. Compared with traditional acid-base titration method quantification and potentiometric titration method quantification, the method provided by the invention has the advantages that the result accuracy is not interfered by the main impurities ammonium chloride and N, O-dimethylhydroxylamine hydrochloride in the methoxylamine hydrochloride finished product, the sensitivity is higher, and the accuracy is higher. The method has important practical significance in the quality control aspect of industrial production of methoxyamine hydrochloride.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride. Background Art

[0002] Methoxyamine hydrochloride is an important chemical raw material used in the production of antibiotics such as cefuroxime acid (ester) and fenoxystrobin fungicides such as trifloxystrobin, fenoxystrobin, and fenoxystrobin; methoxyamine hydrochloride is also a drug commonly used in surgical operations to maintain or restore arterial pressure, especially for the reduction of blood pressure caused by spinal anesthesia.

[0003] At present, the methods used by domestic factories to determine methoxyamine hydrochloride usually adopt sodium hydroxide titration, potentiometric titration, gas phase method and chemical derivatization method, etc. These quantitative methods are often interfered by the main impurities ammonium chloride and N,O-dimethylhydroxylamine hydrochloride in methoxyamine hydrochloride, and the quantitative results are prone to deviation.

[0004] Tian Tieniu and others from Hebei University of Science and Technology investigated that when methoxyamine hydrochloride does not contain free acid and the ammonium chloride content is greater than 3%, the sodium hydroxide titration method has a large deviation in the measurement results and is not suitable for factory quality inspection. The potentiometric method of secondary titration is relatively complex to operate and has high equipment requirements, making it unsuitable for factory use. Both titration methods titrate the hydrochloric acid in methoxyamine hydrochloride, and the hydrochloric acid in the impurity N,O-dimethylhydroxylamine hydrochloride in industrial methoxyamine hydrochloride will also be titrated, and the interference of the impurity N,O-dimethylhydroxylamine hydrochloride cannot be ruled out.

[0005] The chemical industry standard HG / T4712-2014 for methoxyamine hydrochloride uses gas chromatography to determine the content of methoxyamine hydrochloride and N,O-dimethylhydroxylamine hydrochloride. This method has three problems. First, the area normalization method is used for quantification, and the difference in the response ratio of the two substances is not considered, so the measured content value has deviations; second, the sample solvent is water, and the expansion volume of water is large, so the repeatability of the peak area is poor; long-term use of water as a solvent will damage general non-water-resistant chromatographic columns, and water-resistant chromatographic columns must be used, which is not universal.

[0006] Ying Liming and others from Hangzhou Normal University investigated and used the liquid phase derivatization method to determine the content of methoxyamine hydrochloride. The derivatization method has a derivatization reaction time of up to 2 hours, which is time-consuming and relatively complicated to operate. The spot plate method is not accurate in determining the reaction endpoint. This derivatization method will also derive the impurity N,O-dimethylhydroxylamine hydrochloride in industrial methoxyamine hydrochloride, and the interference of the impurity N,O-dimethylhydroxylamine hydrochloride cannot be eliminated.

[0007] Therefore, the present invention designs a high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride, in order to achieve accurate quantification of methoxyamine hydrochloride and eliminate interference from impurities, and the sensitivity of the high performance liquid chromatography method is higher than that of the titration method. Summary of the invention

[0008] In order to overcome the defects in the prior art, the present invention provides a high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride, the accuracy of the method result is not interfered by the main impurities ammonium chloride and N, O-dimethylhydroxylamine hydrochloride in the methoxyamine hydrochloride product.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention is to provide a high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride, comprising the following steps:

[0011] Step 1, preparation of solution:

[0012] Preparation of a series of reference solutions with a concentration gradient: weigh methoxyamine hydrochloride standards according to different concentrations, add sodium hydroxide solution to dissolve, then add organic solvent to make up to volume;

[0013] Preparation of test solution: Weigh the test sample, neutralize it to a specific pH with sodium hydroxide solution, then add organic solvent to make up the volume;

[0014] Step 2: Use high performance liquid chromatography for quantitative detection and perform quantitative analysis based on the linear regression curve; wherein the mobile phase is deionized water and mobile phase B is chromatographic methanol.

[0015] Furthermore, when preparing the reference solution, the molar ratio of the added methoxyamine hydrochloride standard to sodium hydroxide is 1 to 2:1, preferably 1:1.

[0016] Furthermore, the organic solvent is a chromatographic methanol solution, and its added volume ratio is 5-20%, preferably 10%.

[0017] Furthermore, the specific pH value is 4.0 to 6.5, preferably 5.0 to 6.0.

[0018] Furthermore, the chromatographic column used for quantitative detection and analysis is a chromatographic column with octadecylsilane bonded silica gel as a filler, 250 mm×4.6 mm, 5 μm.

[0019] Furthermore, the column temperature is 20-30°C, preferably 25°C.

[0020] Furthermore, the detection wavelength is 210 nm.

[0021] Furthermore, in step 2, the gradient elution procedure is: 0-10 min, 5% B phase; 10-20 min, 5%-100% B phase; the flow rate of the mobile phase is 0.5-1.5 ml / min, preferably 1.0 ml / min.

[0022] Furthermore, the injection volume is 4-10 μl, preferably 5.0 μl.

[0023] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0024] Compared with conventional quantitative acid-base titration and potentiometric titration, the method provided by the present invention has a higher sensitivity than conventional quantitative acid-base titration and potentiometric titration, and the method is not affected by the main impurities ammonium chloride and N,O-dimethylhydroxylamine hydrochloride in the finished product of methoxyamine hydrochloride, and has important practical significance in quality control of industrial production of methoxyamine hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the HPLC chromatogram of the test sample methoxyamine hydrochloride;

[0026] Figure 2 It is the HPLC chromatogram of methoxyamine hydrochloride and N,O-dimethylhydroxylamine hydrochloride;

[0027] Figure 3 is the HPLC chromatogram of ammonium chloride;

[0028] Figure 4 This is the standard curve of methoxyamine hydrochloride. DETAILED DESCRIPTION

[0029] The present invention is described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

[0030] The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0031] Example 1

[0032] This embodiment provides a high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride, which comprises the following steps:

[0033] Step 1: Solution preparation: including the preparation of reference solution and test solution

[0034] With reference to the preparation of sample solution in the chemical industry standard HG / T4712-2014, a comparison of neutralization temperature and neutralization pH conditions was carried out; the optimized neutralization temperature condition was room temperature, which did not affect the test results compared with an ice bath and was simpler to operate than an ice bath; the neutralization method was to add an equimolar amount of sodium hydroxide solution, at which time the pH value was 5.0-6.0.

[0035] Preparation of reference solution: Accurately weigh 40.0mg, 80.0mg, 120.0mg, 160.0mg, and 200.0mg (accurate to 0.0001g) of methoxyamine hydrochloride standard into a 50ml brown volumetric flask, add an equal molar amount of 0.08mol / L sodium hydroxide solution to dissolve, shake vigorously for 2 minutes, add 5ml of methanol, and dilute with pure water to prepare a series of methoxyamine hydrochloride standard solutions.

[0036] Preparation of test solution: Weigh 100 mg (accurate to 0.0001 g) of the sample solution into a 50 mL brown volumetric flask, add 0.08 mol / L sodium hydroxide solution dropwise while shaking, monitor with pH paper, when the pH value reaches 5.0-6.0, add 5 ml of methanol and make up to volume with pure water.

[0037] Step 2, using high performance liquid chromatography for quantitative detection and analysis; the chromatographic conditions are: the chromatographic column is a chromatographic column (250mm×4.6mm, 5μm) using octadecylsilane bonded silica as a filler; the mobile phase A is deionized water, the mobile phase B is chromatographic methanol, the elution program is: 0-10min, 5% B phase; 10-20min, 5%-100% B phase; the mobile phase flow rate is 1.0ml / min, the detection wavelength is 210nm; the column temperature is 25°C; the injection volume is 5.0μl.

[0038] Accurately weigh 180.0 mg of the sample to be tested, measure according to the above chromatographic conditions, inject the sample 3 times, record the chromatogram, record the peak area, and calculate the average content of the sample to be 98.7%.

[0039] Example 2

[0040] This example conducts a methodological investigation on the method provided in Example 1, and the specific steps and results are as follows:

[0041] 1. Specificity test

[0042] Take the methoxyamine hydrochloride test solution, the mixed solution of methoxyamine hydrochloride and N,O-dimethylhydroxylamine hydrochloride, and the ammonium chloride solution respectively, and measure according to the chromatographic conditions in Example 1.

[0043] Results Figure 1-Figure 3The retention time of methoxyamine hydrochloride after neutralization is 3.625min, the retention time of N,O-dimethylhydroxylamine hydrochloride after neutralization is 6.816min, and the retention time of ammonium chloride is 2.385min. The results show that the main impurities N,O-dimethylhydroxylamine hydrochloride and ammonium chloride in the finished product of industrial methoxyamine hydrochloride will not interfere with the determination of methoxyamine hydrochloride.

[0044] 2. Investigation of linear relationship

[0045] Take the reference solution of the series of concentrations described in Example 1, measure according to the above chromatographic conditions, and record the peak area; perform linear regression with the reference solution concentration (C) as the abscissa and the peak area (A) as the ordinate, and see Figure 4 , the regression equation of methoxyamine hydrochloride is y=338.61047x–6.30615, R 2 =0.99996, linear range: 0.82mg / ml~3.99mg / ml.

[0046] 3. Precision test

[0047] A reference solution of 2.41 mg / ml of methoxyamine hydrochloride was selected, and the sample was injected 6 times, and the peak area was recorded and the relative standard deviation RSD was calculated to determine the intra-day precision; three different concentrations of reference solutions of low (0.82 mg / ml), medium (2.41 mg / ml), and high (3.99 mg / ml) were selected, and each concentration was injected twice a day for 3 days, and the peak area was recorded and the relative standard deviation RSD was calculated to determine the inter-day precision. The results showed that the intra-day precision of methoxyamine hydrochloride was less than 0.20%, and the inter-day precision was less than 2%, which met the requirements.

[0048] 4. Accuracy test

[0049] Take 9 portions of the test sample, each portion is about 50 mg, divided into 3 groups, add three high, medium and low concentration reference solution of 0.87 mg / ml, 1.68 mg / ml and 2.65 mg / ml respectively, and measure according to the chromatographic conditions in Example 1, inject each sample 3 times, record the chromatogram, and calculate the average recovery rate and RSD of methoxyamine hydrochloride. The results show (Table 1) that the average recovery rate of methoxyamine hydrochloride is 99.1% (RSD1.84%), which meets the requirements.

[0050] Table 1 Sample recovery of methoxyamine hydrochloride

[0051]

[0052] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A high performance liquid chromatography quantitative analysis method for methoxyamine hydrochloride, characterized in that: The steps include: Step 1, preparation of solution: Preparation of a series of reference solutions with a concentration gradient: weigh methoxyamine hydrochloride standards according to different concentrations, add sodium hydroxide solution to dissolve, then add organic solvent to make up to volume; Preparation of test solution: Weigh the test sample, neutralize it to a specific pH with sodium hydroxide solution, then add organic solvent to make up the volume; Step 2: Use high performance liquid chromatography for quantitative detection and perform quantitative analysis based on the linear regression curve; wherein the mobile phase is deionized water and mobile phase B is chromatographic methanol.

2. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: When preparing the reference solution, the molar ratio of the added methoxyamine hydrochloride standard to sodium hydroxide is 1-2:1, preferably 1:

1.

3. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The organic solvent is a chromatographic methanol solution, and its added volume ratio is 5-20%, preferably 10%.

4. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The specific pH value is 4.0 to 6.5, preferably 5.0 to 6.

0.

5. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The chromatographic column used for quantitative detection and analysis is a chromatographic column with octadecylsilane bonded silica gel as a filler, 250mm×4.6mm, 5μm.

6. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The column temperature is 20-30°C, preferably 25°C.

7. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The detection wavelength is 210 nm.

8. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: In step 2, the gradient elution program is: 0-10 min, 5% B phase; 10-20 min, 5%-100% B phase; The flow rate of the mobile phase is 0.5 to 1.5 ml / min, preferably 1.0 ml / min.

9. The high performance liquid chromatography quantitative analysis method according to claim 1, characterized in that: The injection volume is 4-10 μl, preferably 5.0 μl.

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