Method for detecting residual cleaning of surfaces of an azithromycin production plant

Abstract

The application belongs to the technical field of pharmaceutical analysis, and discloses a detection method for cleaning residues on the surface of azithromycin production equipment. A 25cm 2 region on the surface of the azithromycin production equipment is wiped with a cotton swab; after wiping, the cotton swab is soaked in a diluent to obtain a test sample solution; a high-performance liquid chromatograph is used to detect the blank solution, the control sample solution and the test sample solution, and a chromatogram is recorded; and the residual amount of azithromycin is calculated according to an external standard method. The detection method has stable recovery rate, high sensitivity, strong specificity and wide linear range.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting cleaning residues on the surface of azithromycin production equipment. Background Technology

[0002] Azithromycin, a second-generation macrolide antibiotic, is widely used in clinical anti-infective therapy. During drug manufacturing, residual active ingredients on equipment surfaces are a key risk factor leading to cross-contamination and affecting drug quality and safety. Currently, domestic pharmaceutical companies generally have the following technical deficiencies in detecting cleaning residues associated with azithromycin: 1. Low sampling recovery rate: Traditional wiping sampling methods lack standardized operation, resulting in large fluctuations in recovery rate and poor reliability of test results; 2. Insufficient detection sensitivity: Existing methods generally have limits of quantitation higher than 5 μg / mL, which cannot meet the requirements for low residue limits. 3. Poor specificity: Blank solvents and sampling media can easily interfere with the detection of target analytes, leading to false positive results. Summary of the Invention

[0003] The purpose of this invention is to provide a method for detecting cleaning residues on the surface of azithromycin production equipment, thereby solving the aforementioned problems in the prior art. The detection method of this invention is standardized in operation, provides reliable results, and achieves accurate quantification of azithromycin residues on the equipment surface, meeting the requirements of Good Manufacturing Practices (GMP) for pharmaceuticals.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting cleaning residues on the surface of azithromycin production equipment, comprising the following steps: Select any 25cm section on the surface of the azithromycin production equipment. 2 Wipe the area with a cotton swab; after wiping, soak the cotton swab in the diluent to obtain the test solution; Prepare blank solution and control solution separately; The blank solution, reference solution, and test solution were detected by high performance liquid chromatography, and the chromatograms were recorded. The residual amount of azithromycin was calculated according to the external standard method, thus completing the detection of cleaning residues on the surface of the azithromycin production equipment.

[0005] Preferably, the cotton swab is soaked in a 95% ethanol solution before wiping.

[0006] Preferably, the wiping is performed by first wiping horizontally and then wiping vertically.

[0007] Preferably, the diluent is a mixture of ammonium dihydrogen phosphate buffer, methanol, and acetonitrile at pH 10.0 with a volume ratio of 7:7:6.

[0008] Preferably, the conditions for soaking the cotton swab in the diluent are: soaking time of 20-30 minutes, and the soaking process also includes ultrasound, with ultrasound for 30 seconds every 2 minutes.

[0009] Preferably, the chromatographic conditions of the high performance liquid chromatograph are as follows: using an Xbridge C18 column, mobile phase A is a disodium hydrogen phosphate solution with pH=8.9, mobile phase B is acetonitrile-methanol with a volume ratio of 3:1, gradient elution, detection wavelength is 210 nm, column temperature is 60 ℃, flow rate is 1 mL / min, and injection volume is 50 μL.

[0010] Preferably, the gradient elution program is as follows: at 0 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%; at 25 min, the volume fraction of mobile phase A is 45% and the volume fraction of mobile phase B is 55%; at 30 min, the volume fraction of mobile phase A is 40% and the volume fraction of mobile phase B is 60%; at 80 min, the volume fraction of mobile phase A is 25% and the volume fraction of mobile phase B is 75%; at 81 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%, and this is maintained until 93 min.

[0011] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Stable recovery rate: The average recovery rate of swab sampling can reach 88.62%, RSD≤0.58%, and the test results have good repeatability; (2) High sensitivity: The limit of quantitation is as low as 3.29 μg / mL and the limit of detection is as low as 0.82 μg / mL, which can meet the detection requirements of low residue limit; (3) High specificity: Neither blank solvent nor wiping medium interferes with the detection of azithromycin, effectively avoiding false positive results; (4) Wide linear range: It has good linearity in the concentration range of 3.08~385.42μg / mL, with a correlation coefficient r≥0.9999, which can cover the detection needs of different residue levels. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0013] Figure 1 The chromatogram is of the reference solution; Figure 2The chromatogram is for the elution of the blank solution; Figure 3 The chromatogram of the blank solution after wiping; Figure 4 This is the chromatogram of the blank diluted solution. Detailed Implementation

[0014] This invention provides a method for detecting cleaning residues on the surface of azithromycin production equipment, comprising the following steps: Select any 25cm section on the surface of the azithromycin production equipment. 2 Wipe the area with a cotton swab; after wiping, soak the cotton swab in the diluent to obtain the test solution; Prepare blank solution and control solution separately; The blank solution, reference solution, and test solution were detected by high performance liquid chromatography, and the chromatograms were recorded. The residual amount of azithromycin was calculated according to the external standard method, thus completing the detection of cleaning residues on the surface of the azithromycin production equipment.

[0015] In this invention, the cotton swab is further soaked in a 95% ethanol solution before wiping.

[0016] In this invention, the wiping is performed by first wiping horizontally and then wiping vertically.

[0017] In this invention, the diluent is a mixture of ammonium dihydrogen phosphate buffer, methanol, and acetonitrile at pH 10.0 with a volume ratio of 7:7:6.

[0018] In this invention, the blank solution includes a washing blank solution, a wiping blank solution, and a blank diluent; the washing blank solution is a mixture of 95% ethanol solution and water in a volume ratio of 50:50; the wiping blank solution is a blank cotton swab immersed in a mixture of 95% ethanol solution and water in a volume ratio of 50:50; the blank diluent is a mixture of ammonium dihydrogen phosphate buffer, methanol, and acetonitrile in a volume ratio of 7:7:6 at pH=10.0.

[0019] In this invention, the reference solution is a mixture of azithromycin and diluent; the concentration of azithromycin in the reference solution is 200 μg / mL.

[0020] In this invention, the conditions for soaking the cotton swab in the diluent are as follows: the soaking time is 20-30 minutes, and the soaking process also includes ultrasound, which is performed for 30 seconds every 2 minutes.

[0021] In this invention, the chromatographic conditions of the high performance liquid chromatograph are as follows: an Xbridge C18 column is used, mobile phase A is a disodium hydrogen phosphate solution with pH=8.9, mobile phase B is acetonitrile-methanol with a volume ratio of 3:1, gradient elution is used, the detection wavelength is 210 nm, the column temperature is 60 °C, the flow rate is 1 mL / min, and the injection volume is 50 μL.

[0022] In this invention, the gradient elution procedure is as follows: at 0 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%; at 25 min, the volume fraction of mobile phase A is 45% and the volume fraction of mobile phase B is 55%; at 30 min, the volume fraction of mobile phase A is 40% and the volume fraction of mobile phase B is 60%; at 80 min, the volume fraction of mobile phase A is 25% and the volume fraction of mobile phase B is 75%; at 81 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%, and this is maintained until 93 min.

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] (1) The instruments and reagents used in the embodiments of the present invention include: High-performance liquid chromatograph: Agilent 1260 Infinity II, equipped with a UV detector; Electronic balance: Mettler XS205DU, accuracy 0.01mg; Column: Xbridge C18, 4.6 mm × 250 mm, 5 μm; Standard laboratory equipment such as ultrasonic cleaners, volumetric flasks, and pipettes.

[0025] Azithromycin reference standard: batch number WS-AZ-2020-06, purity 94.79%; Acetonitrile and methanol: chromatographic grade; Disodium hydrogen phosphate, ammonium dihydrogen phosphate, ammonia, phosphoric acid, sodium hydroxide: analytical grade; Water used in the experiment: ultrapure water.

[0026] (2) The chromatographic conditions of this invention are as follows: Mobile phase A: Weigh 1.8g of anhydrous disodium hydrogen phosphate, dissolve it in 1L of water, and adjust the pH to 8.9 with 1N sodium hydroxide or 10% phosphoric acid; Mobile phase B: Acetonitrile-methanol (volume ratio 3:1); Diluent: Buffer salt solution (1.73g ammonium dihydrogen phosphate dissolved in 1L of water, pH adjusted to 10.0±0.05 with ammonia): Methanol: Acetonitrile = 7:7:6 (volume ratio); Detection wavelength: 210nm; Injection volume: 50 μL; Column temperature: 60℃; Sample chamber temperature: 4℃; Flow rate: 1.0 mL / min; Running time: 93 minutes; The gradient elution procedure is shown in Table 1 below: Table 1 Gradient elution program

[0027] (3) The solution preparation in this embodiment of the invention includes: Blank solution: Wash blank solution: 95% ethanol-water (volume ratio 50:50); Wipe the blank solution: Place a blank cotton swab in a 25mL volumetric flask containing 95% ethanol-water (50:50), add 95% ethanol-water (50:50) to submerge the cotton swab and make up to the mark, sonicate and shake well; Blank diluent: Buffer salt solution (1.73g ammonium dihydrogen phosphate dissolved in 1L of water, pH adjusted to 10.0±0.05 with ammonia): Methanol: Acetonitrile = 7:7:6 (volume ratio).

[0028] Reference solution: Accurately weigh 20 mg of azithromycin reference standard, place it in a 100 mL volumetric flask, dissolve it with diluent and bring the volume to the mark, shake well to obtain a reference solution with a concentration of approximately 200 μg / mL.

[0029] Test solution: Sample preparation: Take a cotton swab soaked in 95% ethanol and apply it to a 25cm swab. 2 Wipe the surface of the equipment, first horizontally and then vertically. After wiping, place the cotton swab in a 25mL volumetric flask containing 25mL of diluent, let it stand for 20 minutes, sonicate for 30 seconds every 2 minutes, and shake well to obtain the solution. Preparation of leaching sample: Take the leaching water sample and filter it through an organic phase needle filter to obtain the sample.

[0030] (4) Calculation method of the present invention embodiment: The azithromycin residue was calculated based on peak area using the external standard method, and the formula is as follows:

[0031] in: C rThe concentration of the reference solution is expressed in mg / mL. A x The peak area of ​​azithromycin in the test solution; A r This represents the average peak area of ​​azithromycin in the reference solution.

[0032] (5) Methodological validation

[0033] Specificity test: Inject blank solution, wash blank solution, blank diluent, and reference solution separately. Chromatograms are shown below. Figures 1-4 As shown in the figure, the results indicate that there is no interference in the peak position of azithromycin with various blank solutions, indicating that the method has good specificity.

[0034] System suitability test: Six consecutive injections of the reference solution were performed. The relative standard deviation (RSD) of the main peak area of ​​azithromycin was 0.25%, which is less than the acceptable standard of 10.0%, indicating that the system suitability is good.

[0035] Linearity and range tests: A series of linear solutions with concentrations ranging from 3.08 to 385.42 μg / mL were prepared. Linear regression was performed with concentration as the abscissa and peak area as the ordinate, yielding the regression equation: Y = 4686.8167X + 8360.7172; the correlation coefficient r = 0.9999, and the ratio of the Y-intercept to the 100% concentration response value was 0.92%, within ±20%, indicating a good linear relationship. The linear range of the method is 3.08–385.42 μg / mL (3.08–385.42 ppm), the limit of quantitation is 3.29 μg / mL, and the limit of detection is 0.82 μg / mL.

[0036] Accuracy test: The spiking recovery method was used. A known concentration of azithromycin solution was coated onto the surface of a stainless steel plate, dried, and then sampled and tested using the wiping sampling method. Three parallel tests were conducted, with six samples in each batch. The results showed: The average recovery rate for the first batch was 88.64%, with an RSD of 0.35%. The average recovery rate for the second batch was 88.10%, with an RSD of 0.43%. The average recovery rate for the third batch was 89.10%, with an RSD of 0.21%. The average recovery rate of the 18 samples was 88.62%, and the total RSD was 0.58%, which met the recovery rate requirement of 85-108% and the acceptable standard of RSD ≤ 10.0%, indicating that the method was accurate.

[0037] Example 1

[0038] A method for detecting cleaning residues on the surface of azithromycin production equipment, comprising the following steps: After cleaning the reaction vessel in the azithromycin production workshop of a pharmaceutical company, three 25cm samples were taken from the inner surface of the reaction vessel. 2 For the area, use cotton swabs soaked in 95% ethanol solution to wipe and collect samples, first wiping horizontally and then vertically; after wiping, soak the cotton swabs in the diluent for 20 minutes, sonicate for 30 seconds every 2 minutes, shake well, and obtain the test solution; The blank solution, reference solution, and test solution were analyzed by high performance liquid chromatography under the above chromatographic conditions. The chromatograms were recorded, and the residual amount of azithromycin was calculated according to the external standard method. The residual amounts of azithromycin at the three sampling points were 12.3 ppm, 10.5 ppm, and 11.8 ppm, respectively, all of which were lower than the residual limit of 118.6 ppm, indicating that the cleaning effect met the requirements.

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting cleaning residues on the surface of azithromycin production equipment, characterized in that, Includes the following steps: Select any 25cm section on the surface of the azithromycin production equipment. 2 Wipe the area with a cotton swab; After wiping, soak the cotton swab in the diluent to obtain the test solution; Prepare blank solution and control solution separately; The blank solution, reference solution, and test solution were detected by high performance liquid chromatography, and the chromatograms were recorded. The residual amount of azithromycin was calculated according to the external standard method, thus completing the detection of cleaning residues on the surface of the azithromycin production equipment.

2. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 1, characterized in that, The cotton swabs are also soaked in a 95% ethanol solution before wiping.

3. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 1, characterized in that, The wiping process involves wiping horizontally first and then vertically.

4. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 1, characterized in that, The diluent is a mixture of ammonium dihydrogen phosphate buffer, methanol, and acetonitrile at pH 10.0 with a volume ratio of 7:7:

6.

5. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 1, characterized in that, The conditions for soaking the cotton swabs in the diluent are as follows: the soaking time is 20-30 minutes, and the soaking process also includes ultrasound, with ultrasound for 30 seconds every 2 minutes.

6. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 1, characterized in that, The chromatographic conditions of the high performance liquid chromatograph are as follows: Xbridge C18 column, mobile phase A is disodium hydrogen phosphate solution with pH=8.9, mobile phase B is acetonitrile-methanol with a volume ratio of 3:1, gradient elution, detection wavelength is 210 nm, column temperature is 60 ℃, flow rate is 1 mL / min, and injection volume is 50 μL.

7. The method for detecting surface cleaning residues in azithromycin production equipment according to claim 6, characterized in that, The gradient elution program is as follows: at 0 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%; at 25 min, the volume fraction of mobile phase A is 45% and the volume fraction of mobile phase B is 55%; at 30 min, the volume fraction of mobile phase A is 40% and the volume fraction of mobile phase B is 60%; at 80 min, the volume fraction of mobile phase A is 25% and the volume fraction of mobile phase B is 75%; at 81 min, the volume fraction of mobile phase A is 50% and the volume fraction of mobile phase B is 50%, and this is maintained until 93 min.

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