Method for detecting metronidazole in gynecological medical instrument

Abstract

The invention discloses a method for detecting metronidazole in a gynecological medical instrument, and relates to the technical field of medical instrument detection, HPLC-DAD is adopted for preliminary screening, HPLC-MS is adopted for verification, a positive test sample obtained after verification is subjected to quantitative determination through HPLC-DAD, the detection limit of the detection method is low, and the detection limit of metronidazole is 0.05 microgram / mL; the metronidazole detection method is simple in operation, good in repeatability, good in stability and high in recovery rate, can be used as a supplementary detection method for metronidazole in gynecological medical instruments, and makes up the detection blank in the field.

Description

Technical Field

[0001] The present application relates to the technical field of medical device detection, and in particular to a method for detecting metronidazole in gynecological medical devices. Background Art

[0002] According to Article 103 of the Regulations on the Supervision and Administration of Medical Devices, medical devices refer to instruments, equipment, appliances, in vitro diagnostic reagents and calibrators, materials and other similar or related items used directly or indirectly on the human body, including the required computer software; their effectiveness is mainly obtained through physical means, not through pharmacological, immunological or metabolic means.

[0003] According to the "Medical Device Classification Catalogue", gel-type gynecological medical devices such as vaginal antibacterial gel and carbomer gynecological gel belong to the Class II management category in the medical device classification catalogue; they are usually composed of carbomer, gelatin, oligosaccharides, etc., and the ingredients contained have no pharmacological effects and are for single use; by forming a protective film on the vaginal wall, the vaginal wall is physically isolated from external bacteria, thereby preventing the colonization of pathogenic microorganisms.

[0004] These widely used gynecological medical devices, in addition to gels, also include washes, specifically carbomer gynecological washes, chitosan vaginal lavage solutions, medical dressing vaginal repair solutions, carbomer gynecological gels, and medical gynecological dressing gels. Neither the registration details nor the instructions for use for these gynecological medical devices indicate the presence of chemical drugs. However, during testing, the inventors discovered that metronidazole was present in multiple batches of these gynecological medical devices. The addition of the chemical metronidazole imparts temporary, rapid, and effective properties to these gynecological medical devices. However, this illegal addition, without rigorous scientific testing and risk assessment, poses a significant threat to public health and poses serious social hazards.

[0005] Furthermore, as previously mentioned, the ingredients in these gynecological medical devices registered as medical devices should not have pharmacological effects and should physically isolate the vaginal wall from external bacteria by forming a protective membrane. The addition of metronidazole to these gynecological medical devices does not comply with the provisions and requirements of the Regulations on the Supervision and Administration of Medical Devices and the Medical Device Classification Catalogue.

[0006] If metronidazole is added to this type of gynecological medical device and its efficacy is mainly achieved through pharmacological means, it should be approved in accordance with the management regulations for "drugs" rather than the management regulations for "medical devices".

[0007] According to Article 76 of the Regulations on the Supervision and Administration of Medical Devices, if medical devices that may contain harmful substances or have their designs, raw materials, and production processes changed without authorization and pose safety hazards cannot be inspected according to the inspection items and methods specified in the national standards and industry standards for medical devices, medical device inspection institutions may use supplementary inspection items and methods for inspection; the inspection conclusions obtained using the supplementary inspection items and methods may be used as the basis for the department responsible for drug supervision and administration to determine the quality of medical devices.

[0008] At present, the State Food and Drug Administration has only approved two supplementary inspection methods for medical devices, namely the "Supplementary Inspection Methods for the Identification and Content Determination of 17 Chemical Drugs in Patching Medical Devices" and the "Supplementary Inspection Methods for the Identification and Quantification of Fluorescent Substances in Polyvinyl Chloride Infusion and Blood Transfusion Devices". There is no supplementary inspection method for metronidazole in such gynecological medical devices. Therefore, it is very urgent to establish a supplementary inspection method for metronidazole in gynecological medical devices, aiming to protect the health and safety of the people, provide technical support for medical device supervision, and effectively promote corporate self-discipline and the healthy development of the medical device industry. Summary of the Invention

[0009] In view of this, the present application provides a method for detecting metronidazole in gynecological medical devices, which uses HPLC-DAD for primary screening and further verifies the positive test samples by HPLC-MS; this method fills the gap in the detection of metronidazole in gynecological medical devices. The specific scheme is as follows:

[0010] The specific steps for the detection of metronidazole in gynecological medical devices are as follows:

[0011] S1. Preparation of Metronidazole Reference Solution: Prepare a metronidazole reference solution using 20% ​​methanol solution;

[0012] S2. Preparation of test solution: Take an appropriate amount of gel test sample or wash test sample and add anhydrous ethanol, mix thoroughly in a water bath, centrifuge to obtain the supernatant, dilute with 20% methanol solution, filter, and obtain the filtrate; the test solution is obtained;

[0013] S3. Perform HPLC-DAD detection on the reference solution and the test solution, and compare the results. If the retention time and the UV spectrum from 190 nm to 400 nm are consistent, it is determined to be a positive test sample in the initial screening;

[0014] S4. Take the test solution and the reference solution, perform HPLC-MS detection and analysis, compare the primary mass spectrum and secondary mass spectrum information of the test solution and the reference solution, and if the two are consistent, it is confirmed that the test sample contains metronidazole, that is, it is confirmed as a positive test sample;

[0015] The conditions for HPLC detection in step S3 are:

[0016] Column: ThermoAcclaim120 C 18 The column was eluted with water as mobile phase A and methanol as mobile phase B for gradient elution; the column temperature was 30°C.

[0017] Preferably, the conditions for HPLC-MS detection in step S4 include:

[0018] Chromatographic conditions: The chromatographic column was Poroshell 120SB-C18, the mobile phase was 0.1% formic acid solution-methanol, the volume ratio of the two was 60:40, the flow rate was 0.2 mL / min, and the injection volume was 1 μL.

[0019] Further preferably, the conditions for HPLC-MS detection in step S4 further include:

[0020] Mass spectrometry conditions: ESI + Scanning: capillary voltage: 3.5 kV, capillary outlet voltage: 120 V, drying gas temperature: 325°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psig, collision energy: 15 V; scanning range: 50-500 m / z.

[0021] The method for detecting metronidazole in gynecological medical devices described in the present application further includes quantitative detection of the metronidazole in the confirmed positive test sample after the positive test sample is confirmed. The quantitative detection method includes: preparing metronidazole reference solution of different concentrations with 20% methanol solution, performing HPLC-DAD detection, recording the peak area in the absorption spectrum, performing linear regression with the reference concentration as the abscissa and the peak area as the ordinate to obtain a linear regression equation; performing HPLC-DAD detection on the test sample solution, substituting the peak area of ​​the absorption spectrum of the corresponding wavelength obtained into the linear regression equation to obtain the concentration of metronidazole in the test sample solution.

[0022] Beneficial effects:

[0023] The detection method described in this application fills the gap in the detection of metronidazole in gynecological medical devices.

[0024] The detection method described in this application has low limits of detection and quantification, with a detection limit of 0.05 μg / mL and a quantification limit of 0.21 μg / mL for metronidazole; this method is sufficient to detect whether such gynecological medical devices sold on the market contain metronidazole.

[0025] The linear regression equation for quantitative detection of metronidazole obtained by the detection method described in the present application has a good linear relationship, r = 0.9999;

[0026] The detection method described in this application has good reproducibility, with RSDs of 1.36% and 1.76% for the wash solution and gel, respectively;

[0027] The detection method described in this application has good stability, with good stability within 24 hours;

[0028] The detection method described in this application has a high recovery rate, with average recovery rates of 98.17% and 98.85% for the wash solution and gel respectively;

[0029] The chromatographic conditions described in this application are robust and applicable to different chromatographic columns;

[0030] In summary, the detection method described in this application can be used as a supplementary detection method for metronidazole in gynecological medical devices, filling the detection gap in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chromatogram of the metronidazole reference substance;

[0032] Figure 2 This is the solution chromatogram of the initially screened positive test product (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620);

[0033] Figure 3 This is the chromatogram of the solution of the initially screened positive test product (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Device Research Institute, batch number 20210301);

[0034] Figure 4 This is the solution chromatogram of the negative test product in the initial screening (chitosan gynecological antibacterial spray, Jiangxi Lvyuantang Pharmaceutical Co., Ltd., batch number 20220101);

[0035] Figure 5 This is the chromatogram of the solution of the negative test product in the initial screening (medical antibacterial gel dressing, Zhengzhou Heya Medical Instrument Co., Ltd., batch number 210701);

[0036] Figure 6 This is the spectrum of metronidazole reference substance;

[0037] Figure 7 This is the solution spectrum of the initial screening positive test product (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620);

[0038] Figure 8 This is the solution spectrum of the initially screened positive test product (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Device Research Institute, batch number 20210301);

[0039] Figure 9 This is the spectrum of metronidazole at the detection limit;

[0040] Figure 10 This is the spectrum of metronidazole at the limit of quantification;

[0041] Figure 11 is the standard curve of metronidazole;

[0042] Figure 12 This is the chromatogram of the Waters column (top: reference substance; middle: Carbomer gynecological wash; bottom: medical gynecological dressing gel);

[0043] Figure 13 This is the chromatogram of the Phenomenex column (top: reference substance; middle: Carbomer gynecological wash; bottom: medical gynecological dressing gel);

[0044] Figure 14 This is the chromatogram of the Thermo column (top: reference substance; middle: Carbomer gynecological wash; bottom: medical gynecological dressing gel);

[0045] Figure 15 This is the ion chromatogram of the metronidazole reference substance;

[0046] Figure 16 This is the ion chromatogram of the positive test product (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620);

[0047] Figure 17 This is the ion chromatogram of the positive test product (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Device Research Institute, batch number 20210301);

[0048] Figure 18 This is the primary mass spectrum of the metronidazole reference substance;

[0049] Figure 19 This is the primary mass spectrum of the positive test product (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620);

[0050] Figure 20 This is the primary mass spectrum of the positive test product (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Device Research Institute, batch number 20210301);

[0051] Figure 21 This is the MS / MS spectrum of the metronidazole reference substance;

[0052] Figure 22 This is the secondary mass spectrum of the positive test product (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620);

[0053] Figure 23This is the secondary mass spectrum of the positive test sample (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Equipment Research Institute, batch number 20210301). DETAILED DESCRIPTION

[0054] The following will be combined with the drawings of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described in this application are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0055] Example 1

[0056] 1. Instrument, reagent and test sample information

[0057] U3000 high performance liquid chromatograph with diode array detector (Thermo Fisher Scientific Inc.); Sartorius BP211D electronic balance (Sartorius, Germany).

[0058] Metronidazole reference substance (Source: China Food and Drug Inspection Institute, Batch No. 100191-201808, content 100.0% as C6H9N3O3); ethanol and methanol were chromatographically pure; and water was ultrapure. Information on gynecological medical device test products is shown in Table 1 below. Test products numbered 1-14 are lotions, and test products numbered 15-34 are gels.

[0059] Table 1 Gynecological medical device test sample information

[0060]

[0061]

[0062]

[0063]

[0064] 2 Chromatographic conditions

[0065] Column: ThermoAcclaim120 C 18 Use a 250 × 4.6 mm, 5 μm column with water as mobile phase A and methanol as mobile phase B, using gradient elution as specified in the table below; detection wavelength at 320 nm; column temperature at 30°C; injection volume at 10 μL. The theoretical plate number, calculated based on the metronidazole peak, should be no less than 2000.

[0066] Table 2 Gradient elution program

[0067]

[0068] 3. Solution Preparation

[0069] 3.1 Preparation of reference solution: Take an appropriate amount of metronidazole reference substance, accurately weigh it, and add 20% methanol solution to make a solution containing approximately 50 μg of metronidazole per 1 mL.

[0070] 3.2 Preparation of Test Solution: Take 1 mL of the sample for lotion preparations (0.5 g for gel preparations) as listed in Table 1 and place it in a 50 mL stoppered colorimetric tube. Add 8 mL of anhydrous ethanol (for gel preparations, place in an 80°C water bath). Shake to thoroughly mix the sample and extraction solvent. Make up to 10 mL with anhydrous ethanol, shake well, and centrifuge at 15,000 rpm for 5 minutes. Take 5 mL of the supernatant and place it in a 10 mL volumetric flask. Dilute to the mark with 20% methanol solution, shake well, filter, and collect the filtrate to obtain the test solution. Dilutions may be performed by appropriate multiples if necessary.

[0071] 4HPLC-DAD detection

[0072] Take 10 μL of metronidazole reference solution and test solution respectively and measure them according to the chromatographic conditions under item 2. The results are as follows: Figures 1 to 5 , some test solution showed chromatographic peaks with the same retention time as the metronidazole reference substance, and, Figures 6 to 8 , the UV-visible absorption spectrum of the corresponding chromatographic peak is the same as that of the metronidazole reference substance; if the absorption spectrum of the chromatographic peak with the same retention time in the test solution is the same as that of the reference substance, it is judged to be a positive test sample in the initial screening.

[0073] 5. Detection limit and quantification limit

[0074] The reference solution was diluted to different concentrations, and the detection limit of metronidazole was 0.05 μg / mL, with a signal-to-noise ratio of about 3, and the quantification limit was 0.21 μg / mL, with a signal-to-noise ratio of about 10. Figures 9 and 10 .

[0075] 6 Linear relationship

[0076] Accurately weigh an appropriate amount of metronidazole reference substance and prepare a series of linear reference substance solutions with metronidazole mass concentrations of 2.572, 5.144, 10.288, 25.72, 51.44, 128.6, and 257.2 μg / mL using a 20% methanol solution. The samples were injected and analyzed according to the chromatographic conditions under item 2. The peak area of ​​the absorption spectrum was recorded. A linear regression was performed with the reference substance mass concentration (μg / mL) as the abscissa and the peak area as the ordinate. The regression equation for metronidazole was: y = 0.3674x – 0.0201, r = 0.9999. The results showed that metronidazole had a good linear relationship in the range of 2.572 to 257.2 μg / mL. Figure 11 The spectra of detection limit and quantification limit of metronidazole are as follows: Figure 9 、 Figure 10 The regression equation of metronidazole is used for the quantitative detection of positive test samples.

[0077] 7 Precision test

[0078] Take the positive test solution (such as: Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620; medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Equipment Research Institute, batch number 20210301) and inject 10 μL under the chromatographic conditions under item 2. Inject 6 times continuously and record the peak area. See Table 3. The measurement results show that the instrument precision is good, with RSDs of 0.19% and 0.17%, respectively.

[0079] Table 3 Precision test

[0080]

[0081] 8 Repeatability test

[0082] Six samples of the initially screened positive test samples (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620; medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Equipment Research Institute, batch number 20210301) were prepared in parallel according to the method under "3.2 Preparation of Test Sample Solution". The metronidazole content was determined according to the proposed determination method. The method had good reproducibility. The results are shown in Table 4.

[0083] Table 4 Repeatability test

[0084]

[0085] 9 Recovery test

[0086] Nine aliquots (0.5 mL) of a prescreened positive test sample (Carbomer Gynecological Wash, Jilin Guoda Bioengineering Co., Ltd., Batch No. 2020110620) with a known content of metronidazole (0.2794 mg / mL) were precisely measured and added with appropriate amounts of the metronidazole reference substance. The solution was prepared according to the method described in "3.2 Preparation of Test Solution" and assayed and calculated according to the proposed method. The results are shown in Table 5. The results showed that the average recovery of metronidazole was 98.17% with an RSD of 1.77%, indicating good sample recovery for this method.

[0087] Table 5 Carbomer gynecological lotion recovery test

[0088]

[0089] Nine portions of a prescreened positive test sample (medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical Equipment Research Institute, batch number 20210301) with a known content (3.1325 mg / g of metronidazole) were accurately weighed. An appropriate amount of a metronidazole reference substance was added to each portion. The sample was prepared according to the method described under "Test Sample Solution Preparation." The solution was determined and calculated according to the proposed assay method. The results are shown in Table 5. The results showed that the average recovery of metronidazole was 98.85% with an RSD of 1.41%, indicating good sample recovery.

[0090] Table 6 Medical gynecological dressing gel recovery test

[0091]

[0092]

[0093] 10. Stability test

[0094] The positive test samples in the initial screening (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620; medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical and Medical Equipment Research Institute, batch number 20210301) were taken and the test sample solutions were prepared according to the "3.2 Preparation of Test Sample Solution". The samples were injected at 0 h, 4 h, 8 h, 12 h, and 24 h after preparation. The results are shown in Table 7, indicating that the test samples had good stability within 24 hours.

[0095] Table 7 Stability test

[0096]

[0097] 11. Durability

[0098] To investigate the reproducibility of this method on different brands of chromatographic columns, three different models of chromatographic columns, Waters Symmetry C 18 (250×4.6mm, 5μm), Phenomenex Luna C 18 (250×4.6mm, 5μm), ThermoAcclaim120 C 18 (250×4.6mm, 5μm), metronidazole reference solution and positive test solution (Carbomer gynecological wash, Jilin Guoda Bioengineering Co., Ltd., batch number 2020110620; medical gynecological dressing gel, Changchun Kexin Biochemical Pharmaceutical Equipment Research Institute, batch number 20210301) were injected according to the chromatographic conditions, and the chromatograms were recorded. Results The chromatographic peak of the positive test solution had the same retention time as the chromatographic peak of the metronidazole reference solution, and it could achieve good chromatographic separation with other components, meeting the experimental requirements and showing good robustness of the method. Figures 12 to 14 .

[0099] Example 2

[0100] HPLC-MS confirmation

[0101] 1. Instruments and reagents

[0102] Instruments: Agilent 6530B Accurate-Mass Q-TOF, Sartorius BP211D electronic analytical balance (Sartorius, Germany).

[0103] Reagents: ethanol, methanol, and formic acid were all of chromatographic grade; water was ultrapure water.

[0104] 2. Detection conditions

[0105] Chromatographic conditions: the chromatographic column was Poroshell 120SB-C18 (50×2.1 mm, 2.7 μm), the mobile phase was 0.1% formic acid solution-methanol (60:40), the flow rate was 0.2 mL / min, and the injection volume was 1 μL.

[0106] Mass spectrometry conditions: using mass spectrometry detector, ESI + Scanning: capillary voltage: 3.5 kV, capillary outlet voltage: 120 V, drying gas temperature: 325°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psig, collision energy: 15 V; scanning range: 50-500 m / z.

[0107] 3. Solution Preparation

[0108] A solution was prepared according to the method described in item 3 of Example 1 and diluted with 20% methanol solution to make the concentration of metronidazole about 2 μg / mL.

[0109] 4 Confirmation experiment

[0110] Take the metronidazole reference solution and the test solution prepared in Item 3 of this Example and inject them according to the detection conditions under Item 2 of this Example. The results of the initial screening showed that the positive test sample showed an ion peak with the same retention time as the metronidazole reference sample, and the primary mass spectrum and the secondary mass spectrum were consistent with the metronidazole reference sample, confirming that it was a positive test sample. The negative test sample did not have the same mass spectrum peak, as shown in FIG. Figures 15 to 23 and Table 8.

[0111] Table 8 Data of Metronidazole Reference Substance and Positive Test Substances for Gynecological Medical Devices

[0112]

[0113]

[0114] Measurement results

[0115] According to the detection method described in this application, 34 batches of gynecological medical devices were tested respectively, and 18 batches of initially screened positive test samples were detected. The initially screened positive test samples were confirmed by mass spectrometry. The results showed that the primary mass spectrometry parent ion peaks and the main fragment ion peaks of the secondary mass spectrometry of the 18 batches of initially screened positive test samples were consistent with those of the metronidazole reference substance, and they were confirmed as positive test samples, as shown in Tables 9 and 10.

[0116] Table 9 Determination results of metronidazole in gynecological medical devices

[0117]

[0118]

[0119] Table 10 Mass spectrometry confirmation results of metronidazole in gynecological medical devices

[0120]

[0121]

Claims

1. The specific steps for the detection of metronidazole in gynecological medical devices are as follows: S1. Preparation of Metronidazole Reference Solution: Prepare a metronidazole reference solution using 20% ​​methanol solution; S2. Preparation of test solution: Take an appropriate amount of gel test sample or wash test sample and add anhydrous ethanol, mix thoroughly in a water bath, centrifuge to obtain the supernatant, dilute with 20% methanol solution, filter, and obtain the filtrate; the test solution is obtained; S3. Perform HPLC-DAD detection on the reference solution and the test solution, and compare the results. If the retention time and the UV spectrum from 190 nm to 400 nm are consistent, it is determined to be a positive test sample in the initial screening; S4. Take the test solution and the reference solution, perform HPLC-MS detection and analysis, compare the primary mass spectrum and secondary mass spectrum information of the test solution and the reference solution, and if the two are consistent, it is confirmed that the test sample contains metronidazole, that is, it is confirmed as a positive test sample; The conditions for HPLC detection in step S3 are: Column: ThermoAcclaim120 C 18 The column was used for gradient elution with water as mobile phase A and methanol as mobile phase B; the detection wavelength was 320 nm; and the column temperature was 30°C.

2. The method for detecting metronidazole in a gynecological medical device according to claim 1, wherein: The conditions for HPLC-MS detection in step S4 include: Chromatographic conditions: The chromatographic column was Poroshell 120SB-C18, the mobile phase was 0.1% formic acid solution-methanol, the volume ratio of the two was 60:40, the flow rate was 0.2 mL / min, and the injection volume was 1 μL.

3. The method for detecting metronidazole in a gynecological medical device according to claim 2, wherein: The conditions for HPLC-MS detection in step S4 also include: Mass spectrometry conditions: ESI+ scan, capillary voltage: 3.5 kV, capillary outlet voltage: 120 V, drying gas temperature: 325°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psig, collision energy 15 V; scanning range 50-500 m / z.

4. The method for detecting metronidazole in a gynecological medical device according to claim 1, wherein: The method also includes quantitative detection of metronidazole in a confirmed positive test sample, and the quantitative detection method includes: preparing metronidazole reference solution of different concentrations with 20% methanol solution, performing HPLC-DAD detection, recording the peak area in the absorption spectrum, performing linear regression with the reference concentration as the horizontal coordinate and the peak area as the vertical coordinate to obtain a linear regression equation; performing HPLC-DAD detection on the test sample solution, and substituting the peak area of ​​the absorption spectrum of the corresponding wavelength obtained into the linear regression equation to obtain the concentration of metronidazole in the test sample solution.

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