Ultraviolet spectrophotometric detection method for metronidazole cleaning verification
By using ethanol solvent and ultraviolet spectrophotometry to detect metronidazole residues, the problems of poor water solubility of metronidazole and the time-consuming and expensive HPLC method were solved, achieving rapid and accurate cleaning verification and ensuring the safety of drug production.
Patent Information
- Application Number
- CN202511720167.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-02-24
AI Technical Summary
In existing technologies, the poor water solubility of metronidazole leads to low detection results, and the HPLC method is time-consuming, expensive, and difficult to meet the needs of rapid screening, and cannot effectively verify the cleaning effect of the equipment.
Ethanol was used as a solvent, and metronidazole residues were detected at a wavelength of 320 nm using ultraviolet spectrophotometry. The residue amount was calculated by wiping the sample and establishing a standard curve.
It enables rapid and accurate detection of metronidazole residues on equipment surfaces, ensuring the authenticity and reliability of cleaning validation results, reducing testing costs and improving efficiency.
Smart Images
Figure CN121558657A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical production quality control, and in particular to an ultraviolet spectrophotometric detection method for cleaning validation of metronidazole. Background Technology
[0002] Metronidazole is a commonly used antibacterial drug. After metronidazole production, the production equipment must be thoroughly cleaned to prevent cross-contamination of subsequent batches. Therefore, it is necessary to verify the amount of metronidazole residue on the equipment surfaces after cleaning.
[0003] Currently, high-performance liquid chromatography (HPLC) is commonly used for the determination of metronidazole content. Although HPLC offers good separation and high accuracy, it has the following drawbacks: it is time-consuming, requires expensive instruments and columns, is complex to operate, requires specialized technicians, has high operating costs, and is difficult to meet the needs of rapid screening of large numbers of samples in cleanliness validation.
[0004] In addition, metronidazole has poor solubility in water. If water is used as a solvent to prepare the sampling solution for cleaning verification, the residual metronidazole on the equipment surface cannot be completely dissolved, which will lead to significantly lower test results and fail to truly reflect the cleaning effect, thus posing a risk to product quality. Summary of the Invention
[0005] This invention provides a UV spectrophotometric detection method for the cleaning verification of metronidazole, which overcomes the aforementioned shortcomings of existing detection methods. This method utilizes ethanol as a solvent and combines UV spectrophotometry for detection at a specific wavelength, solving the problems of poor water solubility of metronidazole and the time-consuming and expensive nature of HPLC methods.
[0006] To address the aforementioned technical problems, this invention provides a UV spectrophotometric detection method for metronidazole cleaning verification, comprising the following steps: (1) Sampling and solution preparation: Using ethanol as a solvent, samples were taken from the surface of the device to be verified and prepared into a test solution; (2) Determination: Using ethanol as a blank control, the absorbance of the test solution was measured at a wavelength of 320 nm using a UV spectrophotometer; (3) Calculation: Based on the absorbance value measured in step (2), the amount of metronidazole residue on the surface of the equipment is calculated.
[0007] In a preferred embodiment of the present invention, in step (3), the amount of metronidazole residue is calculated by using a pre-established standard curve of metronidazole in ethanol.
[0008] In a preferred embodiment of the present invention, the standard curve is established by the following method: at least five metronidazole standard solutions with concentrations ranging from 0.75 μg / ml to 22.5 μg / ml are prepared with ethanol, the absorbance is measured at a wavelength of 320 nm, and the standard curve is obtained by linear regression of concentration and absorbance values.
[0009] In a preferred embodiment of the present invention, in step (1), the sampling method is the wiping method.
[0010] In a preferred embodiment of the present invention, the wiping method includes the following steps: (a) Use ethanol-moistened swabs for sampling; (b) Within the pre-defined sampling area, gently bend the cotton swab tip and wipe the surface smoothly and slowly, moving from one side to the other while moving forward to cover the entire sampling area. (c) Flip the cotton swab so that the other side is perpendicular to the previous direction for wiping.
[0011] In a preferred embodiment of the present invention, the concentration of metronidazole in the test solution prepared in step (1) is between 0.75 μg / ml and 22.5 μg / ml.
[0012] The beneficial effects of this invention are as follows: This invention provides an ultraviolet spectrophotometric detection method for metronidazole cleaning verification. Using ultraviolet spectrophotometry with ethanol as the solvent and detection at a wavelength of 320 nm, it completely solves the industry problem of incomplete dissolution of residues and serious deviations in detection results caused by the poor water solubility of metronidazole, ensuring the authenticity and reliability of the cleaning verification results. The analysis time is shortened to several minutes, significantly improving detection efficiency and reducing detection costs. This method has been systematically verified and possesses excellent performance such as high sensitivity, good precision, and stable recovery rate. It can accurately and effectively determine trace amounts of metronidazole residues on the surface of equipment, providing strong technical support for the safe production of pharmaceuticals. Attached Figure Description
[0013] Figure 1 This is a schematic diagram of the horizontal direction of cotton swab sampling; Figure 2 This is a schematic diagram of a cotton swab being used for sampling and wiping in the vertical direction. Detailed Implementation
[0014] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.
[0015] Example 1 Step (1): Sampling and solution preparation Define the sampling area: Define a sampling area of 100 cm² on the surface of the stainless steel equipment to be verified.
[0016] Moisten the cotton swab: Take a laboratory-grade cotton swab, thoroughly wet its tip with anhydrous ethanol, and then squeeze it through the mouth of the solvent bottle to remove excess solvent.
[0017] Perform wiping: a. Hold the cotton swab and apply pressure within the designated area to slightly bend the tip of the swab to create appropriate friction.
[0018] b. Wipe smoothly and slowly in a "bow" shaped pattern (i.e., from left to right while moving from top to bottom), ensuring the entire area is covered (e.g., ...). Figure 1 (As shown).
[0019] c. Flip the cotton swab over, and repeat the wiping once with the other side in a path perpendicular to the direction of the first wiping (i.e., from top to bottom while moving from left to right). Figure 2 (As shown).
[0020] Sample collection: Cut off the cotton swab tip with clean scissors and put it into a 50 ml clean volumetric flask.
[0021] Dissolution and dilution: Add approximately 30 ml of anhydrous ethanol to the volumetric flask and sonicate for 10 minutes to ensure complete dissolution of metronidazole. After cooling to room temperature, dilute to the mark with anhydrous ethanol and mix well to obtain the test solution.
[0022] Step (2): Measurement Start and preheat the UV spectrophotometer.
[0023] Take a pair of quartz cuvettes, fill one of them with anhydrous ethanol as a blank solution, place it in the optical path, and zero it at a wavelength of 320 nm.
[0024] Pour another cuvette into the prepared test solution, place it in the optical path, and read and record its absorbance value at a wavelength of 320 nm, denoted as A. sample .
[0025] Step (3): Calculation Calculate the solution concentration: Measure the A sample Substituting into the pre-established standard curve equation C = (A sample +0.0008) / 0.0445 (the establishment of this equation is shown in Verification Example 2), calculate the concentration C of metronidazole in the test solution. sample (Unit: μg / mL).
[0026] Calculate surface residue: Calculate the amount of residue per unit area based on the sampling parameters.
[0027] Surface residue (μg / cm²) = (C sample × Constant volume V) / Sampling area S In this example, V = 50ml, S = 100cm².
[0028] Result determination: The calculated surface residue is compared with the preset acceptable standard to determine whether the cleaning is qualified.
[0029] Verification Example 1: Confirmation of Specificity and Maximum Absorption Wavelength Accurately weigh 0.15 g of metronidazole sample and place it in a 100 ml volumetric flask. Add ethanol to dissolve and dilute to the mark, then shake well to obtain the test sample stock solution. Accurately measure 1 ml of the test sample stock solution and place it in another 100 ml volumetric flask. Add ethanol to dilute to the mark, then shake well to obtain the test sample solution.
[0030] The spectrum was scanned in the wavelength range of 200 nm to 600 nm. Ethanol was used as a blank solution. After zeroing, the sample solution was loaded and the spectrum was scanned.
[0031] The results showed that the sample spectrum had maximum absorption at 320 nm and a single peak shape, indicating that the ethanol solvent did not interfere at this wavelength and that the method had good specificity.
[0032] Verification Example 2: Establishment of Standard Line and Linearity Verification Accurately measure 5 ml of the test sample stock solution prepared in Example 1, place it in a 100 ml volumetric flask, add ethanol to make up to the mark, shake well, and prepare a linear stock solution with a concentration of 50 μg / mL.
[0033] Accurately measure appropriate amounts of the above linear stock solutions and dilute them with ethanol to prepare a series of standard solutions with concentrations of 0.75 μg / mL, 7.5 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL and 22.5 μg / mL, respectively.
[0034] Specifically: 5% (0.75 μg / ml) solution: Accurately measure 1 ml of the linear stock solution into a 100 ml volumetric flask, add ethanol to the mark and shake well.
[0035] 50% (7.5 μg / ml) solution: Accurately measure 1 ml of the linear stock solution into a 10 ml volumetric flask, add ethanol to the mark and shake well.
[0036] 80% (12 μg / ml) solution: Accurately measure 1.6 ml of the linear stock solution into a 10 ml volumetric flask, add ethanol to the mark and shake well.
[0037] 100% (15 μg / ml) solution: Accurately measure 2 ml of the linear stock solution into a 10 ml volumetric flask, add ethanol to the mark and shake well.
[0038] 120% (18 μg / ml) solution: Accurately measure 2.4 ml of the linear stock solution into a 10 ml volumetric flask, add ethanol to the mark and shake well.
[0039] 150% (22.5 μg / ml) solution: Accurately measure 3 ml of the linear stock solution into a 10 ml volumetric flask, add ethanol to the mark and shake well.
[0040] Using ethanol as a blank, the absorbance values of the above standard solutions at a wavelength of 320 nm were measured by ultraviolet spectrophotometry. Each concentration was measured twice and the average value was taken. The results are shown in Table 1 below.
[0041] Table 1 The results clearly showed that metronidazole had maximum absorption at 320 nm in ethanol solvent without interference, demonstrating the good specificity of the method. Linear regression was performed with concentration (C, μg / mL) on the x-axis and absorbance (A) on the y-axis, yielding the standard curve equation: A = 0.0445C - 0.0008, with a linear correlation coefficient r = 0.9999. The established standard curve exhibits a wide linear range and excellent correlation coefficient, providing a reliable basis for accurate quantification.
[0042] Verification Example 3: Limits of Detection and Limits of Quantification Place the blank solvent ethanol into the absorption cell, adjust the detection wavelength to 320nm, zero the cell, and scan the fluctuation value of the absorbance of the blank solution over a period of time as the baseline of the blank solution. Determine the noise of the blank solution based on the fluctuation of the baseline.
[0043] The test solution from Example 1 was serially diluted, and its absorbance was measured. Based on the signal-to-noise ratio (S / N) of the measurement results, the limit of quantitation was determined according to a signal-to-noise ratio of 10:1 and the limit of detection was determined according to a signal-to-noise ratio of 3:1. The results are shown in Table 2 below.
[0044] Table 2 The results showed that when the signal-to-noise ratio (S / N) was approximately 3:1, the corresponding concentration was 0.335 μg / mL, which was determined to be the limit of detection (LOD); when the S / N was approximately 10:1, the corresponding concentration was 1.000 μg / mL, which was determined to be the limit of quantitation (LOQ). This indicates that the method has extremely high sensitivity.
[0045] Verification Example 4: Repeatability Verification Accurately weigh 0.15 g of metronidazole test sample and place it in a 250 ml stoppered conical flask. Add 100 ml of ethanol to dissolve it and shake well to obtain the test sample stock solution. Then, accurately measure 1 ml of the treated test sample stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with ethanol and shake well to obtain the test solution. Using ethanol as a blank, measure the absorbance at a wavelength of 320 nm using ultraviolet spectrophotometry. The results are shown in Table 3 below.
[0046] Table 3 The results showed that the relative standard deviation (RSD) of the absorbance of the six samples was 0.08% (n=6). This indicates that the method has excellent repeatability, ensuring the stability and reliability of the detection results.
[0047] Verification Example 5: Wipe Recovery Rate (1) Sampling and solution preparation: Accurately weigh 0.15 g of metronidazole test sample and place it in a 250 ml stoppered conical flask. Add 100 ml of ethanol and shake well to obtain the test sample stock solution. Then, accurately measure 1 ml of the test sample stock solution and place it in a 100 ml volumetric flask. Dilute to the mark with ethanol and shake well to obtain the test sample solution, preparing a solution containing approximately 15 μg per ml. Accurately measure 1 ml of this sample solution and place it in a 25 cm... 2 On a stainless steel plate, apply the coating evenly with a coating rod, let it dry, and prepare two copies using the same method. Then, using the same procedure on an enamel plate, prepare three copies.
[0048] Take a confirmed cotton swab, wet its tip with anhydrous ethanol, and squeeze it at the bottle opening to remove excess solvent.
[0049] Mark the sampling area on the stainless steel plate. Press the cotton swab tip onto the sampling surface, slightly bending the tip to create friction on the device. Wipe smoothly and slowly from left to right, while moving from top to bottom to ensure complete coverage of the area. Figure 1 As shown.
[0050] Flip the swab over and wipe the area once more using the other side in a path perpendicular to the previous direction (i.e., wiping from top to bottom while moving from left to right). Then, using a second moistened swab, wipe the same area a second time in the same manner.
[0051] Cut off the cotton swab tips after two swabs and place them in a clean 25ml volumetric flask. Dilute with ethanol to the mark, shake well, and obtain the test solution.
[0052] Samples were taken from the surface of the enamel-coated equipment using the same method. Three sampling points were taken from both the stainless steel and enamel-coated surfaces.
[0053] (2) Measurement: Turn on the ultraviolet spectrophotometer and preheat for 30 minutes.
[0054] Take a quartz cuvette, fill it with anhydrous ethanol as a blank solution, place it in the sample cell, and zero it at a wavelength of 320 nm.
[0055] Replace the blank solution with the test solution prepared in step 1, measure its absorbance at a wavelength of 320 nm, and record the reading.
[0056] (3) Calculation: The concentration of metronidazole in the test solution was calculated using the linear equation established in Verification Example 2, and the wiping recovery rate of metronidazole was calculated. The results are shown in Table 4 below.
[0057] Table 4 The results showed that the average recovery rate was 69.3% on stainless steel surfaces and 73.3% on enamel surfaces, with RSDs less than 5.0% for both. These stable and compliant recovery rates demonstrate that the method of this invention can accurately and effectively recover residues from equipment surfaces in practical applications, ensuring the authenticity and reliability of the cleaning validation results.
[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.
Claims
1. A UV spectrophotometric detection method for metronidazole cleaning verification, characterized in that, Includes the following steps: (1) Sampling and solution preparation: Using ethanol as a solvent, samples were taken from the surface of the device to be verified and prepared into a test solution; (2) Determination: Using ethanol as a blank control, the absorbance of the test solution was measured at a wavelength of 320 nm using a UV spectrophotometer; (3) Calculation: Based on the absorbance value measured in step (2), the amount of metronidazole residue on the surface of the equipment is calculated.
2. The method according to claim 1, characterized in that, In step (3), the residual amount of metronidazole is calculated using a pre-established standard curve of metronidazole in ethanol.
3. The method according to claim 2, characterized in that, The standard curve was established by preparing at least five metronidazole standard solutions with concentrations ranging from 0.75 μg / ml to 22.5 μg / ml using ethanol, measuring the absorbance at a wavelength of 320 nm, and obtaining the standard curve by performing linear regression between concentration and absorbance values.
4. The method according to claim 1, characterized in that, In step (1), the sampling method is the wiping method.
5. The method according to claim 4, characterized in that, The wiping method includes the following steps: (a) Use ethanol-moistened swabs for sampling; (b) Within the pre-defined sampling area, gently bend the cotton swab tip and wipe the surface smoothly and slowly, moving from one side to the other while moving forward to cover the entire sampling area. (c) Flip the cotton swab so that the other side is perpendicular to the previous direction for wiping.
6. The method according to claim 1, characterized in that, The concentration of metronidazole in the test solution prepared in step (1) ranges from 0.75 μg / ml to 22.5 μg / ml.