Method for measuring content of formaldehyde in hydrochlorothiazide bulk drug
By combining high-performance liquid chromatography with 2,4-dinitrophenylhydrazine and phosphoric acid solution, the problem of formaldehyde content determination in hydrochlorothiazide raw material was solved, enabling rapid, accurate, and low-cost drug quality evaluation and ensuring drug safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-24
- Publication Date
- 2026-04-03
AI Technical Summary
There is a lack of effective methods in the current technology to determine the formaldehyde content in hydrochlorothiazide raw materials, which poses a safety hazard. Furthermore, there is a lack of standards in my country, making it impossible to evaluate the quality of the drug.
High performance liquid chromatography (HPLC) was used to detect the formaldehyde content in hydrochlorothiazide raw material by preparing reference solution, test solution and blank solution, and using 2,4-dinitrophenylhydrazine and phosphoric acid solution as derivatization reagents. The peak area was calculated by external standard method for quantification.
It enables rapid, accurate, and low-cost determination of formaldehyde content in hydrochlorothiazide raw materials, improving the reliability of drug quality evaluation, avoiding safety hazards, and has a high adoption rate.
Smart Images
Figure CN121784166A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the field of drug impurity detection and analysis, and more specifically to a method for determining the formaldehyde content in hydrochlorothiazide raw material. Background Technology
[0002] Hydrochlorothiazide is a moderately potent diuretic and antihypertensive drug that inhibits the reabsorption of Na+ and Cl- in the proximal distal convoluted tubule, thereby increasing renal excretion of sodium chloride and producing a diuretic effect. Discovered in the early 1940s by American chemists John McLeod and Robert Pratt, and developed by Merck, it was primarily used to treat edema caused by heart failure, cirrhosis, and kidney disease. In 1958, Beyer reported its natriuretic properties, and Merck subsequently marketed it as the first oral diuretic. In 1980, the prescribing practice for hydrochlorothiazide in treating hypertension shifted, and it was recommended as a first-line antihypertensive drug by the Joint Committee on Prevention, Detection, Evaluation, and Treatment of Hypertension (JNC).
[0003] Drug safety is a significant concern for the public. Current synthetic processes for hydrochlorothiazide use sulfonamides and formaldehyde as raw materials, synthesizing hydrochlorothiazide through a cyclization reaction. The synthetic process for hydrochlorothiazide is as follows:
[0004] Formaldehyde is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. The permissible deviation (PDE) of formaldehyde is 10 mg / day. Based on the maximum daily dose of hydrochlorothiazide (50 mg), the limit is equivalent to 20%, which is a relatively high safety limit. Referring to ICH Q3A, a limit of 0.10% is used to investigate the formaldehyde content in hydrochlorothiazide active pharmaceutical ingredient (API). Formaldehyde is a starting material in the synthesis of hydrochlorothiazide; therefore, there is a possibility that formaldehyde may be present in hydrochlorothiazide API.
[0005] However, my country's legal standards for hydrochlorothiazide do not include a method for testing formaldehyde in hydrochlorothiazide, resulting in a lack of standards. There are almost no literature reports on the determination of formaldehyde in hydrochlorothiazide using liquid chromatography, making it impossible to evaluate the quality of the drug and posing a safety hazard. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for determining the formaldehyde content in hydrochlorothiazide raw materials. This method is characterized by its simple process, low cost, high precision, and high accuracy, and can rapidly determine the formaldehyde content in hydrochlorothiazide raw materials.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for determining the formaldehyde content in hydrochlorothiazide raw material, the key of which includes the following steps: Preparation of reference solution: Accurately weigh formaldehyde solution, dilute with acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare reference solution; Preparation of test solution: Accurately weigh hydrochlorothiazide raw material, add acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare test solution; Preparation of blank solution: Accurately measure the acetonitrile solution of 2,4-dinitrophenylhydrazine, the phosphoric acid solution, and acetonitrile to prepare a blank solution; High-performance liquid chromatography (HPLC) was used for analysis: blank solution, test solution, and reference solution were accurately measured and injected into the HPLC instrument, and the chromatograms were recorded. The chromatograms of the blank solution and the reference solution were compared to identify the formaldehyde derivative peak. If the chromatogram of the test solution showed a peak with the same retention time as the formaldehyde derivative peak in the chromatogram of the reference solution, the formaldehyde content in the hydrochlorothiazide raw material was calculated by peak area using the external standard method.
[0008] Furthermore, the preparation process of the reference solution is as follows: accurately weigh an appropriate amount of formaldehyde solution, place it in a 100ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, accurately measure a certain amount of formaldehyde acetonitrile solution into a 20ml volumetric flask, accurately add phosphoric acid solution and acetonitrile solution of 2,4-dinitrophenylhydrazine, add acetonitrile to dilute to the mark, shake well, and the reference solution is obtained; The volume ratio of the formaldehyde acetonitrile solution, the phosphoric acid solution, and the acetonitrile solution of 2,4-dinitrophenylhydrazine is 1:4~8:8~12.
[0009] Furthermore, the preparation process of the test solution is as follows: accurately weigh 0.2g of hydrochlorothiazide raw material, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve and dilute to the mark, shake well, and the test solution is obtained.
[0010] Furthermore, the blank solution is prepared as follows: accurately measure acetonitrile, place it in a 20ml volumetric flask, accurately add phosphoric acid solution and acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute with acetonitrile to 20ml, shake well, and the blank solution is obtained. The volume ratio of the formaldehyde acetonitrile solution, the phosphoric acid solution, and the acetonitrile solution of 2,4-dinitrophenylhydrazine is 1:4~8:8~12.
[0011] Furthermore, the preparation process of the acetonitrile solution of 2,4-dinitrophenylhydrazine is as follows: Accurately measure 0.5 ml of 2,4-dinitrophenylhydrazine and place it in a 100 ml volumetric flask. Dilute with acetonitrile to 100 ml and shake well to obtain an acetonitrile solution of 2,4-dinitrophenylhydrazine.
[0012] Furthermore, the preparation process of the phosphoric acid solution is as follows: accurately measure 4.5 ml of pure phosphoric acid solution, place it in a 50 ml volumetric flask, dilute with water to 50 ml, and shake well to obtain the phosphoric acid solution.
[0013] Furthermore, the chromatographic conditions for the high performance liquid chromatography are as follows: octadecylsilane-bonded silica gel is used as the packing material; 0.6% 50:50 phosphoric acid solution-acetonitrile is used as the mobile phase; the flow rate is 1.0 ml per minute; the detection wavelength is 336 nm; and the injection volume is 20 μl.
[0014] The significant effects of this invention are: 1. The method provided by this invention uses liquid chromatography for separation and detection. The equipment is widely available and has low operating costs, which can effectively expand the applicable occasions and scope of this method.
[0015] 2. In the detection method provided by the present invention, the processing of the reference standard and the test sample is simple, and the amount of reference standard and test sample used is small, which can be conveniently and quickly prepared into reference standard solution, test sample solution and blank solution.
[0016] 3. Under the chromatographic conditions provided by this invention, the separation between impurities and main components is good, the system has good stability and repeatability, high accuracy, fast peak elution time, short analysis time, and saves detection costs.
[0017] 4. The method provided by this invention can quickly, effectively and easily determine the formaldehyde impurity content in hydrochlorothiazide. It can be used to evaluate or assess drug components, avoiding potential safety hazards, and can also provide reliable data monitoring for the quality and safety of the active pharmaceutical ingredient.
[0018] 5. The detection method provided by the present invention was verified by experiments. The results showed that the method of the present invention has strong specificity, good precision, and high accuracy, and can rapidly determine the amount of formaldehyde in hydrochlorothiazide raw material. Attached Figure Description
[0019] Figure 1 This is a flowchart of the method of the present invention; Figure 2 The spectrum obtained in Example 1 of the present invention; Figure 3 The spectrum obtained in Example 2 of the present invention; Figure 4 The spectrum obtained in Example 3 of the present invention; Figure 5 This is the standard curve for formaldehyde. Detailed Implementation
[0020] The specific embodiments and working principles of the present invention will be further described in detail below with reference to the accompanying drawings.
[0021] First, the instruments and chromatographic columns used in the embodiments of the present invention are as follows: instrument: Instrument 1: Shimadzu LC-20A, serial number: JQ1320; Instrument 2: Shimadzu LC-20A, serial number: JQ1323; Chromatographic column: Column 1: Welch Ultimate XB-C18 (4.6 mm × 150 mm, 5 μm), SN: USSE037561; Column 2: Welch Ultimate XB-C18 (4.6 mm × 150 mm, 5 μm), SN: USSE037676; Electronic balance: METTLER TOLEDO ML204T, part number: JQ1405; Reagents:
[0022] Example 1: like Figure 1 As shown, a method for determining the formaldehyde content in hydrochlorothiazide raw material includes the following specific steps: Preparation of reference solution: Accurately weigh formaldehyde solution, dilute with acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare reference solution; Preparation of test solution: Accurately weigh hydrochlorothiazide raw material, add acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare test solution; Preparation of blank solution: Accurately measure the acetonitrile solution of 2,4-dinitrophenylhydrazine, the phosphoric acid solution, and acetonitrile to prepare a blank solution; High-performance liquid chromatography (HPLC) was used for analysis: blank solution, test solution, and reference solution were accurately measured and injected into the HPLC instrument, and the chromatograms were recorded. The chromatograms of the blank solution and the reference solution were compared to identify the formaldehyde derivative peak. If the chromatogram of the test solution showed a peak with the same retention time as the formaldehyde derivative peak in the chromatogram of the reference solution, the formaldehyde content in the hydrochlorothiazide raw material was calculated by peak area using the external standard method.
[0023] In this example, the preparation process of the reference solution is as follows: accurately weigh an appropriate amount of formaldehyde solution equivalent to 20 mg of formaldehyde, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the 100 ml mark, shake well, accurately measure 1 ml of formaldehyde acetonitrile solution into a 20 ml volumetric flask, accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute to the 20 ml mark with acetonitrile, shake well, and the reference solution is obtained.
[0024] In this example, the preparation process of the test solution is as follows: accurately weigh 0.2g of hydrochlorothiazide raw material, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve and dilute to the 20ml mark, shake well, and the test solution is obtained.
[0025] In this example, the blank solution is prepared as follows: accurately measure 1 ml of acetonitrile and place it in a 20 ml volumetric flask. Accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the 20 ml mark and shake well to obtain the blank solution.
[0026] In this example, the preparation process of the acetonitrile solution of 2,4-dinitrophenylhydrazine is as follows: Accurately measure 0.5 ml of 2,4-dinitrophenylhydrazine and place it in a 100 ml volumetric flask. Dilute with acetonitrile to the 100 ml mark and shake well to obtain an acetonitrile solution of 2,4-dinitrophenylhydrazine.
[0027] In this example, the preparation process of the phosphoric acid solution is as follows: accurately measure 4.5 ml of pure phosphoric acid solution, place it in a 50 ml volumetric flask, dilute with water to the 50 ml mark, and shake well to obtain the phosphoric acid solution.
[0028] In this example, the chromatographic conditions for the high performance liquid chromatography (HPLC) are as follows: octadecylsilane-bonded silica gel as the stationary phase (Welch Ultimate XB-C18, 4.6 mm × 150 mm, 5 μm); 0.6% 50:50 phosphoric acid solution-acetonitrile as the mobile phase; flow rate of 1.0 mL per minute; detection wavelength of 336 nm; and injection volume of 20 μL.
[0029] Example 2: The difference between this embodiment and Embodiment 1 is that: In this example, the preparation process of the reference solution is as follows: accurately weigh an appropriate amount of formaldehyde solution equivalent to 20 mg of formaldehyde, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the 100 ml mark, shake well, accurately measure 1 ml of formaldehyde acetonitrile solution into a 20 ml volumetric flask, accurately add 4 ml of phosphoric acid solution and 8 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute to the 20 ml mark with acetonitrile, shake well, and the reference solution is obtained.
[0030] In this example, the preparation process of the test solution is as follows: accurately weigh 0.2g of hydrochlorothiazide raw material, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve and dilute to the 20ml mark, shake well, and the test solution is obtained.
[0031] In this example, the blank solution is prepared as follows: accurately measure 1 ml of acetonitrile and place it in a 20 ml volumetric flask. Accurately add 8 ml of phosphoric acid solution and 12 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the 20 ml mark and shake well to obtain the blank solution.
[0032] Example 3: The difference between this embodiment and Embodiment 1 is that: In this example, the preparation process of the reference solution is as follows: accurately weigh an appropriate amount of formaldehyde solution equivalent to 20 mg of formaldehyde, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the 100 ml mark, shake well, accurately measure 1 ml of formaldehyde acetonitrile solution into a 20 ml volumetric flask, accurately add 8 ml of phosphoric acid solution and 12 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute to the 20 ml mark with acetonitrile, shake well, and the reference solution is obtained.
[0033] In this example, the preparation process of the test solution is as follows: accurately weigh 0.2g of hydrochlorothiazide raw material, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve and dilute to the 20ml mark, shake well, and the test solution is obtained.
[0034] In this example, the blank solution is prepared as follows: accurately measure 1 ml of acetonitrile and place it in a 20 ml volumetric flask. Accurately add 5 ml of phosphoric acid solution and 10 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the 20 ml mark and shake well to obtain the blank solution.
[0035] The spectra obtained from the three embodiments are shown below. Figures 2 to 4 The formaldehyde content was calculated, and the results are shown in Table 1: Table 1. Results of formaldehyde determination in samples
[0036] The experimental results showed that the formaldehyde content in all three batches of samples met the requirements.
[0037] Method verification: 1. Exclusivity (1) Blank solution: Accurately measure 1 ml of acetonitrile and place it in a 20 ml volumetric flask. Accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the 20 ml mark and shake well to obtain the blank solution.
[0038] (2) Reference stock solution: Take an appropriate amount of formaldehyde solution (equivalent to 20 mg of formaldehyde), accurately weigh it, put it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the 100 ml mark, shake well, and use it as the reference stock solution.
[0039] (3) Reference solution: Accurately measure 1 ml of the reference stock solution into a 20 ml volumetric flask, accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute with acetonitrile to the 20 ml mark, shake well, and use as the reference solution.
[0040] (4) Test solution: Accurately weigh 0.2g of this product, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve, dilute with acetonitrile to the 20ml mark, shake well, and use as the test solution.
[0041] (5) Spiked solution for test sample: Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1 ml of reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample and dilute to the 20 ml mark with acetonitrile. Shake well to obtain the spiked solution for test sample.
[0042] Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms and examine the suitability of the formaldehyde derivative peak system. The results are shown in Table 2. Table 2 Results of the applicability study of the formaldehyde derivative peak system
[0043] The results showed that the formaldehyde derivative peaks were identified by comparing the chromatograms of the blank solution and the reference solution; neither the blank solution nor the test solution interfered with the determination of the formaldehyde derivative peaks. The tailing factor and theoretical plate number of the formaldehyde derivative peaks both met the requirements; the method for determining the formaldehyde content in hydrochlorothiazide API showed good specificity.
[0044] 2. Limit of detection and limit of quantitation (1) Limit of quantitation The limit of quantitation (LOQ) was determined using the signal-to-noise ratio (SNR) method, with the LOQ set at a SNR of 10:1. Accurately measure 0.57 ml of formaldehyde stock solution into a 50 ml volumetric flask, dilute to the mark with acetonitrile, and mix well. Accurately measure 1 ml of this solution into a 20 ml volumetric flask, dilute to the mark with acetonitrile, and mix well. This solution serves as the LOQ solution for formaldehyde. The resulting concentration was 0.1169 g / ml, which is 1.14% of the limit.
[0045] The results showed that the formaldehyde quantitation limit solution had good precision.
[0046] (2) Detection limit The detection limit was determined using the signal-to-noise ratio method, with the corresponding concentration at a signal-to-noise ratio of 3:1. 3 ml of the formaldehyde quantitation limit solution was accurately measured and placed in a 10 ml volumetric flask. The solution was diluted to the mark with acetonitrile and shaken well. This was used as the formaldehyde detection limit solution, and the resulting concentration was 0.0351 g / ml, which is 0.34% of the limit.
[0047] 3. Linearity and Range Take an appropriate amount of formaldehyde solution (equivalent to 20 mg of formaldehyde), accurately weigh it, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark of 100 ml, shake well, and use it as the reference stock solution. Accurately measure 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.5 ml, and 2.0 ml of the reference stock solution and place them in 20 ml volumetric flasks respectively. Add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the 20 ml mark and shake well. These are used as 20%, 50%, 80%, 100%, 120%, 150%, and 200% linear solutions, respectively.
[0048] Accurately measure 20 μl each of the above linear solution and limit of quantitation solution, and inject them into the liquid chromatograph. The results are shown in Table 3. The standard curve for formaldehyde is shown in [Table 3]. Figure 5 .
[0049] Table 3. Results of formaldehyde linearity determination
[0050] The results showed that formaldehyde exhibited good linearity within the concentration range of 0.1169 μg / ml to 15.3808 μg / ml. The regression equation was y = 346169x - 1485.6, and the correlation coefficient was R0. 2 =0.9999.
[0051] 4. Precision (1) Instrument precision Accurately measure 20 μl of the reference solution and inject it repeatedly 6 times. Calculate the RSD (%) of the peak area of the formaldehyde derivative. The results are shown in Tables 4 and 5. Table 4. Results of Instrument Precision Measurement (JQ1320)
[0052] Table 5. Results of Instrument Precision Measurement (JQ1323)
[0053] The results show that the instrument precision of this method is good.
[0054] (2) Repeatability Take an appropriate amount of formaldehyde solution (equivalent to 20 mg of formaldehyde), accurately weigh it, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the reference stock solution.
[0055] Accurately measure 1 ml of the reference stock solution into a 20 ml volumetric flask, accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute with acetonitrile to the 20 ml mark, and shake well to obtain the reference solution.
[0056] Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the product and dilute to the 20 ml mark with acetonitrile. Shake well to obtain the test solution.
[0057] Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1 ml of the reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample, and dilute to the 20 ml mark with acetonitrile. Shake well to obtain the spiking solution for the test sample (prepare 6 parallel portions).
[0058] Accurately measure 20 μl each of the reference solution, the test solution, and the spiked test solution, and calculate the formaldehyde content RSD (%). The results are shown in Table 6. Table 6. Repeatability test results
[0059] The results show that the method has good repeatability.
[0060] (3) Intermediate precision Prepare the reference solution, the test solution, and the spiked solution of the test solution separately, using the same method as under the repeatability section.
[0061] The RSD (%) of formaldehyde content in spiked solutions of test samples was investigated using different instruments, by different personnel, and on different dates. The results are shown in Table 7. Table 7 Results of intermediate precision determination
[0062] The results show that the intermediate precision of this method is good.
[0063] 4. Accuracy Take an appropriate amount of formaldehyde solution (equivalent to 20 mg of formaldehyde), accurately weigh it, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the reference stock solution.
[0064] Accurately measure 1 ml of the reference stock solution into a 20 ml volumetric flask, accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute with acetonitrile to the 20 ml mark, and shake well to obtain the reference solution.
[0065] Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the product and dilute to the 20 ml mark with acetonitrile. Shake well to obtain the test solution.
[0066] Using the standard addition method, accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 0.8 ml of the reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the solution, dilute with acetonitrile to the 20 ml mark, and shake well to obtain the 80% recovery solution (prepare 3 parallel portions). Using the standard addition method, accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1.0 ml of the reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample, dilute to the 20 ml mark with acetonitrile, and shake well to obtain a 100% recovery solution (prepare 3 parallel portions). Using the standard addition method, accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1.2 ml of the reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample, dilute to the 20 ml mark with acetonitrile, and shake well to obtain a 120% recovery solution (prepare 3 parallel portions). Accurately measure 0.57 ml of the reference stock solution and place it in a 50 ml volumetric flask. Dilute with acetonitrile to the 20 ml mark and shake well to obtain the quantitation limit reference stock solution. Using the standard addition method, accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1.0 ml of the stock solution of the limit of quantitation reference, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample, dilute to the 20 ml mark with acetonitrile, and shake well. This solution is used as the limit of quantitation recovery solution (prepare 3 parallel portions).
[0067] Accurately measure 20 μl each of the reference solution, test solution, limit of quantitation recovery solution, 80% recovery solution, 100% recovery solution, and 120% recovery solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the formaldehyde recovery rate RSD (%).
[0068] The results are shown in Table 8: Table 8. Accuracy Measurement Results
[0069] The results show that the method has good accuracy. 1. Durability Take an appropriate amount of formaldehyde solution (equivalent to 20 mg of formaldehyde), accurately weigh it, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and use it as the reference stock solution.
[0070] Accurately measure 1 ml of the reference stock solution into a 20 ml volumetric flask, accurately add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, dilute with acetonitrile to the 20 ml mark, and shake well to obtain the reference solution.
[0071] Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Add 6 ml of phosphoric acid solution and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the product and dilute to the 20 ml mark with acetonitrile. Shake well to obtain the test solution.
[0072] Accurately weigh 0.2 g of this product and place it in a 20 ml volumetric flask. Accurately add 1 ml of the reference stock solution, 6 ml of phosphoric acid solution, and 9 ml of acetonitrile solution of 2,4-dinitrophenylhydrazine. Add an appropriate amount of acetonitrile to dissolve the sample, dilute to the 20 ml mark with acetonitrile, and shake well to prepare the spiking solution for the test sample.
[0073] The formaldehyde content RSD (%) in the spiked solution of the test sample was determined by changing the flow rate (1.0±0.1 ml / min), column temperature (30±2℃), and detection wavelength (336±2℃). The results are shown in Table 9. Table 9. Durability Test Results
[0074] The results show that the proposed method has good durability.
[0075] 6. Solution stability Prepare the spiked solution for the test sample using the same method as described under the specificity section.
[0076] Accurately measure 20 μl of the spiked solution of the test sample and inject it into the liquid chromatograph at 1-hour intervals over 24 hours. Record the chromatograms and calculate the RSD (%) of the peak area of the formaldehyde derivative. The results are shown in Table 10: Table 10 Results of solution stability test (spiked solution of test sample)
[0077] The results showed that the spiked solution of the test sample had good stability within 24 hours.
[0078] Table 11 summarizes the validation of the formaldehyde content determination method: Table 11 Summary of Method Validation for Formaldehyde Content Determination
[0079] In summary, the method for determining formaldehyde content in hydrochlorothiazide raw material proposed in this invention employs high-performance liquid chromatography (HPLC). Blank solution, test solution, and reference solution are precisely measured and injected into the HPLC instrument, and the chromatograms are recorded. The formaldehyde content is calculated based on peak area using the external standard method. This method can rapidly determine the amount of formaldehyde in hydrochlorothiazide raw material, which can be used for evaluating or assessing drug components, avoiding potential safety hazards, and providing reliable data monitoring for the quality and safety of the raw material.
[0080] The technical solution provided by this invention has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make several improvements and modifications to this invention without departing from the principles of this invention, and these improvements and modifications also fall within the protection scope of the claims of this invention.
Claims
1. A method for determining the formaldehyde content in hydrochlorothiazide raw material, characterized in that, Includes the following steps: Preparation of reference solution: Accurately weigh formaldehyde solution, dilute with acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare reference solution; Preparation of test solution: Accurately weigh hydrochlorothiazide raw material, add acetonitrile solution and phosphoric acid solution of 2,4-dinitrophenylhydrazine to prepare test solution; Preparation of blank solution: Accurately measure the acetonitrile solution of 2,4-dinitrophenylhydrazine, the phosphoric acid solution, and acetonitrile to prepare a blank solution; High-performance liquid chromatography (HPLC) was used for analysis: blank solution, test solution, and reference solution were accurately measured and injected into the HPLC instrument, and the chromatograms were recorded. The chromatograms of the blank solution and the reference solution were compared to identify the formaldehyde derivative peak. If the chromatogram of the test solution showed a peak with the same retention time as the formaldehyde derivative peak in the chromatogram of the reference solution, the formaldehyde content in the hydrochlorothiazide raw material was calculated by peak area using the external standard method.
2. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to claim 1, characterized in that: The preparation process of the reference solution is as follows: accurately weigh an appropriate amount of formaldehyde solution, place it in a 100ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, accurately measure a certain amount of formaldehyde acetonitrile solution into a 20ml volumetric flask, accurately add phosphoric acid solution and acetonitrile solution of 2,4-dinitrophenylhydrazine, add acetonitrile to dilute to the mark, shake well, and the reference solution is obtained. The volume ratio of the formaldehyde acetonitrile solution, the phosphoric acid solution, and the acetonitrile solution of 2,4-dinitrophenylhydrazine is 1:4~8:8~12.
3. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to claim 1, characterized in that: The preparation process of the test solution is as follows: accurately weigh 0.2g of hydrochlorothiazide raw material, place it in a 20ml volumetric flask, add 6ml of phosphoric acid solution and 9ml of acetonitrile solution of 2,4-dinitrophenylhydrazine, add an appropriate amount of acetonitrile to dissolve and dilute to the mark, shake well, and the test solution is obtained.
4. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to claim 1, characterized in that: The blank solution was prepared as follows: Accurately measure acetonitrile and place it in a 20 ml volumetric flask. Accurately add phosphoric acid solution and acetonitrile solution of 2,4-dinitrophenylhydrazine. Dilute with acetonitrile to the mark and shake well to obtain the blank solution. The volume ratio of the formaldehyde acetonitrile solution, the phosphoric acid solution, and the acetonitrile solution of 2,4-dinitrophenylhydrazine is 1:4~8:8~12.
5. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to any one of claims 1-4, characterized in that: The preparation process of the acetonitrile solution of 2,4-dinitrophenylhydrazine is as follows: Accurately measure 0.5 ml of 2,4-dinitrophenylhydrazine and place it in a 100 ml volumetric flask. Dilute with acetonitrile to 100 ml and shake well to obtain an acetonitrile solution of 2,4-dinitrophenylhydrazine.
6. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to any one of claims 1-4, characterized in that: The preparation process of the phosphoric acid solution is as follows: accurately measure 4.5 ml of pure phosphoric acid solution, place it in a 50 ml volumetric flask, dilute with water to 50 ml, shake well, and the phosphoric acid solution is obtained.
7. The method for determining the formaldehyde content in hydrochlorothiazide raw material according to any one of claims 1 to 4, characterized in that: The chromatographic conditions for the high performance liquid chromatography method are as follows: octadecylsilane-bonded silica gel is used as the packing material; 0.6% 50:50 phosphoric acid solution-acetonitrile is used as the mobile phase; the flow rate is 1.0 ml per minute; the detection wavelength is 336 nm; and the injection volume is 20 μl.