Detection method of nitrate and nitrite
Through high performance liquid chromatography combined with specific chromatographic columns and mobile phase conditions, the problems of high detection cost and difficulty in separation of nitrate and nitrite in isosorbate nitrate injection are solved, achieving efficient and economical detection results and reducing the Drug safety risks.
Patent Information
- Application Number
- CN202310891326.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-20
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is expensive when detecting nitrate and nitrite in isosorbate nitrate injection and is not suitable for industrial production applications. It is difficult to separate nitrate and nitrite, which limits the application of UV-HPLC method.
High performance liquid chromatography was used to bond silica gel columns by quaternary ammonium silane, the column temperature was set at 25-35°C, and the phosphate buffer solution-acetonitrile at pH 4.5 was used as the mobile phase, with a flow rate of 0.3-1.5 ml/min, and the wavelength was detected. It is 220-230 nm, combined with specific chromatographic columns and mobile phase conditions, effective separation and detection of nitrate and nitrite are achieved.
It improves the detection effectiveness and economicality of nitrate and nitrite in isosorbate nitrate injection, reduces the risk of drug safety, and has high practical value.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of pharmaceutical biotechnology, in particular to a method for detecting nitrate and nitrite in an isosorbide dinitrate raw material and an injection thereof. Background Art
[0002] Isosorbide dinitrate, chemically known as 1,4∶3,6-dianhydro-D-sorbitol dinitrate, is a nitric acid vasodilator used to treat angina pectoris, congestive heart failure and esophageal spasm. Isosorbide dinitrate preparations are mainly injection solutions, which will degrade to produce nitrate and nitrite impurities during production, storage and transportation. Nitrates are bioconverted into nitrites in the body. Nitrates are highly toxic and can react with human hemoglobin. Taking a large amount can cause hypoxia in the body's tissues. Ingestion of 0.2-0.5g in adults can cause poisoning, and 3g can be fatal. At the same time, nitrites react with amines in food in the body to transform into nitrosamines, carcinogens and teratogens, which seriously endanger human health. In 2017, the list of carcinogens published by the World Health Organization's International Agency for Research on Cancer has listed nitrates / nitrites as Class 2A carcinogens, and their content in medicines and foods must be strictly controlled.
[0003] The existing technology uses ion chromatography to detect nitrate and nitrite in isosorbide dinitrate injection, but the detection cost is high and it is not suitable for production application. At present, the generally accepted industrial production inspection is high performance liquid chromatography. In view of the weak ultraviolet absorption and similar polarity of nitrate and nitrite, it is difficult to separate them, which has become a bottleneck restricting the application of UV-HPLC. Therefore, it is of great significance to establish a UV-HPLC detection method for nitrate and nitrite in isosorbide dinitrate and its injection to improve the effectiveness and economy of the detection, which is of great significance to effectively control the quality of related drugs / foods. Summary of the invention
[0004] The purpose of the present invention is to provide a method for detecting nitrate and nitrite, so as to solve the problems existing in the current method for detecting nitrate and nitrite in isosorbide dinitrate injection.
[0005] The present invention provides a method for detecting nitrate and nitrite, comprising the following steps: (1) Preparation of test solution: Take 5-10 ml of isosorbide dinitrate injection (equivalent to 5-10 mg of isosorbide dinitrate) or 20-40 mg of isosorbide dinitrate, place in a 20 ml volumetric flask, dilute to the mark with deionized water, shake well, treat with an ion chromatography pretreatment column IC-Ag column, filter, discard 3 ml of the filtrate, and take the filtrate as the test solution; (2) Preparation of reference solution: Dissolve nitrate and nitrite in deionized water to prepare the reference solution. Preparation of single-label nitrite solution: Take an appropriate amount of sodium nitrite, weigh it accurately, and add deionized water to dissolve and dilute it to obtain a single-label nitrite solution; Preparation of single-standard nitrate solution: Take an appropriate amount of potassium nitrate, weigh it accurately, add deionized water to dissolve and dilute it, and use it as the single-standard nitrate solution.
[0006] (3) Chromatographic detection: Take the test sample solution and the reference sample solution, inject them separately, and record the chromatograms after chromatographic detection.
[0007] A method for detecting nitrate and nitrite as described above, wherein preferably, the chromatographic detection in step (3) uses a quaternary ammonium silane bonded silica gel column, the column temperature is set at 25-35°C, pH 4.5 phosphate buffer-acetonitrile is used as the mobile phase, the flow rate is 0.3-1.5 ml / min, the detection wavelength is 220-230 nm, and 20-40 μl of the test solution and the reference solution are injected respectively.
[0008] A method for detecting nitrate and nitrite as described above, wherein preferably, the concentrations of nitrite and nitrate in the reference solution, the nitrite single-label solution and the nitrate single-label solution are preferably that the nitrite concentration is 0.3-1 μg / ml, and the nitrate concentration is 1-2 μg / ml.
[0009] In the above-mentioned method for detecting nitrate and nitrite, preferably, the volume ratio of the phosphate buffer to acetonitrile is 70:25-35.
[0010] In the above-mentioned method for detecting nitrate and nitrite, preferably, the concentration of the phosphate buffer is 0.05-0.15 mol / L.
[0011] In the above-mentioned method for detecting nitrate and nitrite, preferably, the pH adjuster of the pH 4.5 phosphate buffer is phosphoric acid or potassium hydroxide.
[0012] In the above-mentioned method for detecting nitrate and nitrite, preferably, the phosphate is a mixture of one or more of potassium phosphate, potassium dihydrogen phosphate and potassium hydrogen phosphate.
[0013] Compared with the prior art, the invention has the following beneficial effects: the invention adopts high performance liquid chromatography to quantitatively analyze nitrate and nitrite nitrate in isosorbide dinitrate and isosorbide dinitrate injection, which is beneficial to controlling the quality of isosorbide dinitrate and isosorbide dinitrate injection, reducing the risk of drug safety, and having high practical value. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1is a chromatogram of the reference substances of nitrate and nitrite in Example 1 of the present invention; Figure 2 is a mixed chromatogram of isosorbide dinitrate, nitrate and nitrite in Example 1 of the present invention; Figure 3 is the isosorbide dinitrate chromatogram in Example 1 of the present invention; Figure 4 is the reference substance chromatogram of nitrate and nitrite in Example 2 of the present invention; Figure 5 is a mixed chromatogram of isosorbide dinitrate, nitrate and nitrite in Example 2 of the present invention; Figure 6 It is the chromatogram of isosorbide nitrate injection in Example 2 of the present invention. Implementation
[0015] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, but should not be construed as limiting the present invention.
[0016] The present invention provides a method for detecting nitrate and nitrite, comprising the following steps: (1) Preparation of test solution: Take 5-10 ml of isosorbide dinitrate injection (equivalent to 5-10 mg of isosorbide dinitrate) or 20-40 mg of isosorbide dinitrate, place in a 20 ml volumetric flask, dilute to the mark with deionized water, shake well, treat with an ion chromatography pretreatment column IC-Ag column, filter, discard 3 ml of the filtrate, and take the filtrate as the test solution; (2) Preparation of reference solution: Dissolve nitrate and nitrite in deionized water to prepare the reference solution. Preparation of single-label nitrite solution: Take an appropriate amount of sodium nitrite, weigh it accurately, and add deionized water to dissolve and dilute it to obtain a single-label nitrite solution.
[0017] Preparation of single-standard nitrate solution: Take an appropriate amount of potassium nitrate, weigh it accurately, add deionized water to dissolve and dilute it, and use it as the single-standard nitrate solution.
[0018] (3) Chromatographic detection: Measure the test solution and the reference solution, inject them separately, and record the chromatogram. In the embodiments provided in this application, the chromatographic detection uses a quaternary ammonium silane bonded silica gel column, the column temperature is set at 25-35°C, pH 4.5 phosphate buffer-acetonitrile is used as the mobile phase, the flow rate is 0.3-1.5 ml / min, the detection wavelength is 220-230 nm, and 20-40 μl of the test solution and the reference solution are injected separately.
[0019] In a feasible embodiment, the nitrite concentration is 0.3-1 μg / ml, the nitrate concentration is 1-2 μg / ml; the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 70:25-35; the concentration of the phosphate buffer is 0.05-0.15 mol / L; the pH adjuster of the pH 4.5 phosphate buffer is phosphoric acid or potassium hydroxide; the phosphate is a mixture of one or more of potassium phosphate, potassium dihydrogen phosphate, and potassium hydrogen phosphate.
[0020] Several embodiments are listed below to further illustrate the content of the present invention. Those skilled in the art will know that more chromatographic conditions can be designed according to the following embodiments, all of which belong to the protection scope of the present invention. Example
[0021] (1) Sample preparation: Reference solution: Take about 122 mg of potassium nitrate and about 37.5 mg of sodium nitrite (dried at 105°C for 3 hours), accurately weigh, place in the same 100 ml volumetric flask, dissolve and dilute to the scale with deionized water, shake well, accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the scale with deionized water, shake well, as the stock solution; accurately measure 2 ml of the stock solution, place in a 20 ml volumetric flask, dilute to the scale with deionized water, shake well, and obtain.
[0022] Preparation of single-label nitrite solution: Take an appropriate amount of sodium nitrite, weigh it accurately, and add deionized water to dissolve and dilute it to make a solution containing about 0.5ug / ml of nitrite.
[0023] Preparation of single-label nitrate solution: Take an appropriate amount of potassium nitrate, weigh it accurately, and dissolve and dilute it with deionized water to make a solution containing about 1.5ug / ml of nitrate.
[0024] Mixed solution: Take about 20 mg of isosorbide dinitrate, accurately weigh it, put it in a 20 ml volumetric flask, accurately add 2 ml of the above reference substance stock solution, dilute to the scale with deionized water, shake well, filter through an ion chromatography pretreatment column IC-Ag column (1.0 cc or equivalent column), take the filtrate, shake well, and obtain.
[0025] Test solution: Take about 20 mg of isosorbide dinitrate, accurately weigh it, put it in a 20 ml volumetric flask, dissolve it with deionized water and dilute it to the scale, shake it well, filter it through an ion chromatography pretreatment column IC-Ag column (1.0 cc or equivalent column), and take the filtrate to obtain the test solution.
[0026] (2) Chromatographic detection: Take the above sample solution, inject 30 μl of each solution, and record the chromatograms respectively. The chromatographic column used is ChromCore SAX (4.6 mm × 250 mm, 5 μm). The chromatographic conditions are pH 4.5 phosphate buffer (take an appropriate amount of potassium dihydrogen phosphate, add water to dissolve and dilute to make a 0.1 mol / L solution, adjust the pH to 4.5 with phosphoric acid, and filter to obtain.)-acetonitrile (70:30) as the mobile phase; detection wavelength: 228 nm; flow rate: 1.0 ml / min; column temperature: 30 °C; injection volume: 30 μl.
[0027] (3) System suitability: In the chromatogram of the mixed solution, the main peak, nitrate and nitrite appear in sequence, and the separation between the peaks should be greater than 1.5; the theoretical plate number should be no less than 10,000 based on the nitrate peak and nitrite peak.
[0028] Reference Figures 1 to 3 As shown in Table 1, the method for detecting nitrate and nitrite provided in this embodiment adopts high performance liquid chromatography, with specific chromatographic conditions such as mobile phase, chromatographic column, column temperature, etc., to achieve effective separation and detection of nitrate and nitrite in isosorbide dinitrate, and the separation between the nitrate peak, the nitrite peak and the main peak is greater than 1.5, and the number of theoretical plates is not less than 10000 based on the nitrate peak and the nitrite peak.
[0029] Table 1 Statistics of chromatographic separation parameters of nitrate and nitrite name Retention time (min) Relative retention time Theoretical plate number Separation Isosorbide dinitrate 3.203 1 13626 19.858 Nitrates 5.893 1.84 21205 2.416 Nitrite 6.303 1.97 19972 / The analytical method of the present invention has high detection sensitivity. Take appropriate amounts of nitrite single-label solution and nitrate single-label solution, dilute them step by step with deionized water to prepare a series of solutions, inject 30 μl of them for detection, record the chromatogram, and define the detection limit and quantitative limit according to the signal-to-noise ratio S / N≈3 and S / N≈10. As a result, the quantitative limit of nitrate was 0.010 μg / ml and the detection limit was 0.003 μg / ml, and the quantitative limit of nitrite was 0.025 μg / ml and the detection limit was 0.008 μg / ml.
[0030] The analytical method of the present invention has a wide detection concentration range and strong applicability. Take appropriate amounts of sodium nitrite and potassium nitrate, accurately weigh them, dilute them with deionized water to prepare a series of linear concentration solutions, inject 30 μl of them for detection, record the chromatogram, and perform linear regression on the peak area of the sample solution concentration. The results show that the nitrate is in the range of 0.1502-3.004 μg / ml, and the nitrite is in the range of 0.0506-1.011 μg / ml, and the detection concentration and the chromatographic response show a good linear relationship (r>0.9998).
[0031] The analytical method of the present invention has high accuracy. Take 6 portions of isosorbide dinitrate raw materials of the same batch with known nitrate and nitrite content, each portion is about 20 mg, put them in a 20 ml volumetric bottle, accurately add 2 ml of reference substance stock solution, dissolve and dilute to the scale with mobile phase, shake well, and use it as the sample solution. Take 30 μl of the reference solution and the sample solution, respectively, detect them, and calculate the recovery rates of nitrate and nitrite by the external standard method. As a result, the average recovery rates of nitrate and nitrite were 99.59% and 99.98%, respectively, both greater than 95%. Example
[0032] (1) Sample preparation: Reference solution: Take about 122 mg of potassium nitrate and about 37.5 mg of sodium nitrite (dried at 105°C for 3 hours), accurately weigh, place in the same 100 ml volumetric flask, dissolve and dilute to the scale with deionized water, shake well, accurately measure 1 ml, place in a 25 ml volumetric flask, dilute to the scale with deionized water, shake well, as the stock solution; accurately measure 2 ml of the stock solution, place in a 20 ml volumetric flask, dilute to the scale with deionized water, shake well, and obtain.
[0033] Mixed solution: Accurately measure 10 ml of isosorbide dinitrate injection and place it in a 20 ml volumetric flask. Accurately add 2 ml of the above reference substance stock solution, dilute to the scale with deionized water, shake well, filter through an ion chromatography pretreatment column IC-Ag column (1.0 cc or equivalent column), and obtain the filtrate.
[0034] Test solution: Accurately measure 10 ml of isosorbide dinitrate injection, place it in a 20 ml volumetric flask, dilute to the scale with deionized water, shake well, filter through an ion chromatography pretreatment column IC-Ag column (1.0 cc or equivalent column), and obtain the filtrate.
[0035] (2) Chromatographic detection: Take 30 μl of each of the above sample solutions and inject them respectively, and record the chromatograms respectively. The chromatographic column used is Ulimate XB-SAX (4.6 mm × 250 mm, 5 μm); the chromatographic conditions are pH 4.5 phosphate buffer (take an appropriate amount of potassium hydrogen phosphate, add water to dissolve and dilute to make a 0.05 mol / L solution, adjust the pH to 4.5 with phosphoric acid solution, and filter to obtain.)-acetonitrile (70:30) as the mobile phase; detection wavelength: 228 nm; flow rate: 1.0 ml / min; column temperature: 30 °C; injection volume: 30 μl.
[0036] (3) System suitability: In the chromatogram of the mixed solution, the main peak, nitrate and nitrite appear in sequence, and the separation between the peaks should be greater than 1.5; the theoretical plate number should be no less than 10,000 based on the nitrate peak and nitrite peak.
[0037] Reference Figures 4 to 6The method for detecting nitrate and nitrite provided in this embodiment adopts the chromatographic conditions of Example 1 to achieve effective separation and detection of nitrate and nitrite in isosorbide dinitrate injection. The separation between the nitrate peak, the nitrite peak and the main peak is greater than 1.5, and the theoretical plate number is not less than 10,000 based on the nitrate peak and the nitrite peak. Example
[0038] (1) Sample preparation: Reference solution: Take about 73 mg of potassium nitrate and about 22.5 mg of sodium nitrite (dried at 105°C for 3 hours), accurately weigh, place in the same 100 ml volumetric flask, dissolve and dilute to the scale with deionized water, shake well, accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the scale with deionized water, shake well, as the stock solution; accurately measure 2 ml of the stock solution, place in a 20 ml volumetric flask, dilute to the scale with water, shake well, and obtain.
[0039] Test solution: Accurately measure 5 ml of isosorbide dinitrate injection, place it in a 20 ml volumetric flask, dilute to the scale with deionized water, shake well, filter through an ion chromatography pretreatment column IC-Ag column (1.0 cc or equivalent column), and obtain the filtrate.
[0040] (2) Chromatographic detection: Take the above sample solution, inject 40 μl respectively, and record the chromatogram respectively. The chromatographic column is Elite Supersil SAX (4.6 mm × 250 mm, 5 μm); the chromatographic conditions are pH 4.5 phosphate buffer (take an appropriate amount of potassium phosphate, add water to dissolve and dilute to make a 0.05 mol / L solution, adjust the pH to 4.5 with phosphoric acid solution, filter, and obtain.)-acetonitrile (70:25) as the mobile phase; detection wavelength: 220 nm; flow rate: 1.5 ml / min; column temperature: 25 °C; injection volume: 20 μl.
[0041] (3) System suitability: In the chromatogram of the reference solution, nitrate and nitrite appear in sequence, and the separation between the two peaks should be greater than 1.5; the theoretical plate number should be no less than 2000 based on the nitrate peak and nitrite peak.
[0042] Results: After replacing chromatographic columns of different brands and adjusting chromatographic conditions (column temperature, flow rate, wavelength, salt concentration, etc.), the system applicability still met the detection requirements, the main peak and the impurity peaks were completely separated, the chromatographic retention time of each peak did not change much, and had no effect on the measurement results. Example
[0043] (1) Sample preparation: Reference solution: Take about 144 mg of potassium nitrate and about 75 mg of sodium nitrite (dried at 105℃ for 3 hours), weigh accurately, place in the same 100 ml volumetric flask, dissolve and dilute to the scale with water, shake well, accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the scale with water, shake well, as the stock solution; accurately measure 2 ml of the stock solution, place in a 20 ml volumetric flask, dilute to the scale with water, shake well, and obtain.
[0044] Test solution: Take about 40 mg of isosorbide dinitrate, weigh accurately, place in a 20 ml volumetric flask, dissolve and dilute to the scale with mobile phase, shake well, and obtain.
[0045] (2) Chromatographic detection: Take the above sample solutions, inject 20 μl of each solution, and record the chromatograms respectively. The chromatographic column used is ChromCore SAX (4.6 mm × 250 mm, 5 μm). The chromatographic conditions are pH 4.5 phosphate buffer (take appropriate amounts of potassium hydrogen phosphate and potassium phosphate, add water to dissolve and dilute to make a solution containing 0.1 mol / L potassium, adjust the pH to 4.5 with phosphoric acid, and filter to obtain.) -acetonitrile (70:35) as the mobile phase; detection wavelength: 230 nm; flow rate: 0.5 ml / min; column temperature: 35 °C; injection volume: 40 ml.
[0046] (3) System suitability: In the chromatogram of the reference solution, nitrate and nitrite appear in sequence, and the separation between the two peaks should be greater than 1.5; the theoretical plate number should be no less than 2000 based on the nitrate peak and nitrite peak.
[0047] Results: After replacing chromatographic columns of different brands and adjusting chromatographic conditions (column temperature, flow rate, wavelength, salt concentration, etc.), the system applicability still met the detection requirements, the main peak and the impurity peaks were completely separated, the chromatographic retention time of each peak did not change much, and had no effect on the measurement results.
[0048] The detection methods of nitrate and nitrite provided in Examples 3 and 4, by replacing chromatographic columns of different brands and adjusting chromatographic conditions (column temperature, flow rate, wavelength, salt concentration, etc.), the system applicability meets the detection requirements, the main peak and each impurity peak are completely separated, the chromatographic retention time of each peak does not change much, and has no effect on the measurement results. It shows that the analytical method of the present invention has good durability and can meet different detection requirements.
[0049] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for detecting nitrate and nitrite, characterized in that: The following steps are involved: (1) Preparation of test solution: Take 5-10 ml of isosorbide dinitrate injection or 20-40 mg of isosorbide dinitrate, place in a 20 ml volumetric flask, dilute to the mark with deionized water and shake well, treat with an ion chromatography pretreatment column IC-Ag column, filter, discard 3 ml of the filtrate, and take the filtrate as the test solution; (2) Preparation of reference solution: Dissolve nitrate and nitrite in deionized water to prepare the reference solution. Preparation of single-label nitrite solution: Take an appropriate amount of sodium nitrite, weigh it accurately, and add deionized water to dissolve and dilute it to obtain a single-label nitrite solution; Preparation of single-standard nitrate solution: Take an appropriate amount of potassium nitrate, weigh it accurately, and add deionized water to dissolve and dilute it to obtain a single-standard nitrate solution. (3) Chromatographic detection: Take the test sample solution and the reference sample solution, inject them separately, and record the chromatograms after chromatographic detection.
2. The method for detecting nitrate and nitrite according to claim 1, characterized in that: The chromatographic detection in step (3) uses a quaternary ammonium silane bonded silica gel column, the column temperature is set at 25-35°C, pH 4.5 phosphate buffer-acetonitrile is used as the mobile phase, the flow rate is 0.3-1.5 ml / min, the detection wavelength is 220-230 nm, and 20-40 μl of the test solution and the reference solution are injected respectively.
3. The method for detecting nitrate and nitrite according to claim 1, characterized in that: In the reference solution, the nitrite single-label solution and the nitrate single-label solution, the nitrite concentration is 0.3-1 μg / ml, and the nitrate concentration is 1-2 μg / ml.
4. The method for detecting nitrate and nitrite according to claim 2, characterized in that: The volume ratio of the phosphate buffer to acetonitrile is 70:25-35.
5. The method for detecting nitrate and nitrite according to claim 2, characterized in that: The concentration of the phosphate buffer is 0.05-0.15 mol / L.
6. The method for detecting nitrate and nitrite according to claim 2, characterized in that: The pH regulator of the pH 4.5 phosphate buffer is phosphoric acid or potassium hydroxide.
7. The method for detecting nitrate and nitrite according to claim 2, characterized in that: The phosphate is a mixture of one or more of potassium phosphate, potassium dihydrogen phosphate and potassium hydrogen phosphate.