Method for rapid purification and detection of antiviral drugs in disinfectants or antiseptics

Abstract

The application provides a rapid purification and detection method of an antiviral drug in a disinfectant or an antiseptic, comprising the following steps: adding an extraction agent into a sample to be tested, obtaining supernatant containing a to-be-tested substance after vortex oscillation, ultrasonic extraction, freezing and centrifugation; then adding mixed internal standard working solution into the supernatant containing the to-be-tested substance and uniformly mixing, filtering after passing through a one-step rapid purification column to obtain a to-be-tested liquid; detecting the to-be-tested liquid and a to-be-tested substance standard liquid by using a liquid chromatography-mass spectrometry internal standard method, and drawing a standard curve according to a detection result; finally, comparing the detection result of the to-be-tested liquid with the standard curve, determining the type of the to-be-tested substance in the to-be-tested liquid and calculating the content of the to-be-tested substance. The pretreatment of the application has high extraction efficiency and good purification effect, the determination method is rapid, accurate, stable, has strong anti-interference ability and wide sample matrix applicability, and is suitable for rapid screening and accurate quantitative analysis of the antiviral drug residues in disinfectants and antiseptics with large sample quantity.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents. Background Technology

[0002] Disinfectants and antibacterial agents, due to their convenient use, have become deeply integrated into people's lives and work. However, they are not ordinary daily chemical products; their effectiveness and safety are directly related to public health. Therefore, the regulation of these two types of disinfectants is particularly important. The standard WS / T 686—2020, "Detection Methods and Evaluation Requirements for Antiviral Drugs in Disinfectants or Antibacterial Agents," stipulates that antiviral drugs must not be added to disinfectants or antibacterial agents. The content or total amount of ganciclovir, acyclovir, penciclovir, and ribavirin in disinfectants or antibacterial agents should not exceed 100 mg / kg. The standard also specifies the detection methods for antiviral drugs in disinfectants or antibacterial agents: the four components need to be determined separately, including the detection methods for ganciclovir, acyclovir, and penciclovir, and the detection method for ribavirin. The detection methods for ganciclovir, acyclovir, and penciclovir are divided into three methods: high performance liquid chromatography (HPLC), micellar electrokinetic capillary chromatography (MCGC), and ultra-high performance liquid chromatography-tandem mass spectrometry (LC / MS). The detection methods for ribavirin include: 1. electroosmotic flow reverse capillary electrophoresis; 2. LC-MS confirmatory method: ribavirin in the sample is extracted ultrasonically with a methanol-water mixture, purified using a C18 solid-phase extraction column, separated using a C18 chromatographic column, and confirmed by LC-MS.

[0003] In recent years, domestic companies have added various traditional Chinese medicines, plant extracts, surfactants, and oils to their disinfectant and antibacterial agent products to achieve superior performance, resulting in complex matrices and making component content determination difficult. Current national standards require various extraction and purification methods, followed by chromatographic quantification and mass spectrometry qualitative confirmation using different instruments, to accurately evaluate and determine the content of antiviral drugs such as ganciclovir, acyclovir, penciclovir, and ribavirin added to disinfectants or antibacterial agents. Because ribavirin has weak retention on C18 columns, its determination is particularly susceptible to matrix interference, requiring special solid-phase extraction purification pretreatment for separate ribavirin determination. Furthermore, quantification, qualitative analysis, and chromatographic separation must be performed separately, making it impossible to simultaneously determine all four antiviral drugs, resulting in a highly complex process. Moreover, among the reported methods for detecting antiviral drugs, LC-MS is the most widely used. Reports on detection methods mainly focus on the illegal addition of drugs to biological fluids such as plasma and urine, as well as veterinary drugs, including drug residues in biological samples, food, traditional Chinese medicine preparations, or animal feed. However, there are no literature reports on methods for detecting the illegal addition of antiviral drugs to disinfectants or antibacterial agents.

[0004] Furthermore, the strong matrix inhibition effect of co-extractants in complex matrix samples can contaminate liquid chromatography and mass spectrometry systems, severely affecting instrument performance and consequently the sensitivity and accuracy of trace determination results. Therefore, necessary purification measures are required before mass spectrometry analysis. Existing pretreatment methods mainly rely on solid-phase extraction columns. However, solid-phase extraction columns generally require activation, rinsing, elution, and concentration procedures, which are cumbersome, affect detection efficiency, have high purification costs, and require highly skilled operators. Although some impurities in the extractant can be removed, reducing the influence of the matrix on the ionization efficiency of the target component, the purification process is complex and time-consuming, costly, and requires highly skilled operators. Moreover, due to the different chemical properties and polarities of various antiviral drugs, some components are easily lost, thus affecting the accuracy of quantitative results. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents. After extraction with an extractant, the drug is rapidly purified using a one-step rapid purification column. Then, liquid chromatography-tandem mass spectrometry with internal standard method can be used to rapidly detect the antiviral drugs ribavirin, ganciclovir, acyclovir, and penciclovir in the disinfectant or antibacterial agent. This method has a simple pretreatment process and high sensitivity, precision, accuracy, and specificity.

[0006] The technical problem solved by this invention is achieved by the following technical solution.

[0007] This invention proposes a rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents, comprising the following steps:

[0008] S1. Sample extraction: Add the extractant to the sample to be tested, and after vortexing, ultrasonic extraction, freezing, and centrifugation, obtain the supernatant containing the analyte;

[0009] S2. Sample purification: Add mixed internal standard working solution to the supernatant containing the analyte and mix well. Then, filter through a one-step rapid purification column to obtain the analyte solution.

[0010] S3. Sample detection: The test solution was detected by liquid chromatography-mass spectrometry with internal standard method;

[0011] S4. Standard curve plotting: Dissolve the analyte standard and the mixed internal standard working solution in the extractant to prepare a series of analyte standard solutions of different concentrations. Test the analyte standard solutions of different concentrations under the same conditions as in step S3, and plot the standard curve based on the test results.

[0012] S5. Qualitative and quantitative analysis: The detection results of the test solution are compared with the standard curve to determine the type of analyte in the test solution and to calculate the content of the analyte in the test sample.

[0013] The beneficial effects of the rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to embodiments of the present invention are:

[0014] 1. This invention uses a mixed solution of methanol, acetonitrile, and 1% formic acid water as the extractant to extract the sample to be tested. After freezing and centrifugation, bound residues are effectively released, the target drug is extracted, and some impurities are precipitated. Freezing and centrifugation facilitates the precipitation of some lipid components. Further one-step purification using a SHIMSEN QVet-NM rapid purification column removes lipids and surfactants, thereby effectively reducing the matrix inhibition effect of the LC-MS co-effluent. This extraction method has higher extraction efficiency, shorter pretreatment time, better purification effect, and higher recovery rate.

[0015] 2. This invention utilizes an internal standard method combined with a one-step small column purification process, which is stable and rapid, requiring no activation elution, and improving the overall efficiency of sample pretreatment by more than 10 times. No rinsing is needed after sample loading; the resulting purified solution is colorless, transparent, and clear, with no impurities interfering near the target peak in the obtained chromatogram, enabling accurate quantification. This also facilitates the long-term use of the chromatographic column and LC-MS instrument. After column chromatography, conventional steps such as nitrogen blowing, concentration, and solvent conversion are unnecessary; samples can be directly injected for analysis, simplifying pretreatment operations and reducing the need for excessive sophisticated equipment and specialized technicians, thus reducing labor costs by 90%. Furthermore, the pretreatment method of this invention reduces the amount of organic solvent used, lowering experimental costs while reducing waste generation, protecting the environment, and ensuring the health and safety of practitioners.

[0016] 3. This invention establishes a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of residues of prohibited antiviral drugs such as ribavirin, ganciclovir, acyclovir, and penciclovir in disinfectants or antibacterial agents. This method overcomes the limitation of current standard methods requiring separate determinations, utilizes an instrument-friendly mobile phase to achieve higher analytical sensitivity, and has a minimum limit of quantitation of 1 μg / kg. This significantly improves work efficiency, effectively shortens detection time, is environmentally friendly, and saves reagent consumption and labor costs. This method is rapid, accurate, stable, highly resistant to interference, and has broad sample matrix applicability, making it suitable for rapid screening and precise quantitative analysis of antiviral drug residues in large sample volumes of disinfectants and antibacterial agents. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A flowchart for the rapid purification and detection of antiviral drugs in disinfectants or antibacterial agents;

[0019] Figure 2 The multiple reaction monitoring (MRG) spectra of the target antiviral drugs ribavirin, acyclovir, ganciclovir, and penciclovir, and the internal standard compound, as described in Example 1 of this invention.

[0020] Figure 3 The image shows the MRM chromatogram of purified ribavirin (with internal standard) after purification of the blank matrix sample (with internal standard) of the herbal antibacterial cream of Example 1 of the present invention (with internal standard added).

[0021] Figure 4 The image shows the MRM chromatogram of acyclovir (with internal standard) extracted from the blank matrix sample (with internal standard) of the herbal antibacterial cream of Example 1 of the present invention after purification. The ion currents (quantitative, qualitative and internal standard quantitative ions) are as follows:

[0022] Figure 5 The image shows the MRM chromatogram of ganciclovir (with internal standard) extracted from the blank matrix sample (with internal standard) of the herbal antibacterial cream in Example 1 of this invention after purification. (Quantitative, qualitative and internal standard quantitative ions)

[0023] Figure 6 The MRM chromatogram of purified penciclovir (with internal standard) after purification of the blank matrix sample (with internal standard) of the herbal antibacterial cream in Example 1 is shown.

[0024] Figure 7 MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg ribavirin (internal standard) to the blank matrix sample of herbal antibacterial cream;

[0025] Figure 8 MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg acyclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream;

[0026] Figure 9MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg ganciclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream;

[0027] Figure 10 MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg penciclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream;

[0028] Figure 11 MRM chromatogram of the extraction ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with added 1.2 mg / kg ribavirin (with internal standard) and not passed through the purification column;

[0029] Figure 12 MRM chromatogram of the extract ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with 1.2 mg / kg acyclovir (with internal standard) added and not passed through the purification column;

[0030] Figure 13 MRM chromatogram of the extract ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with added 1.2 mg / kg ganciclovir (with internal standard) and not passed through the purification column;

[0031] Figure 14 MRM chromatogram of the extracted ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with 1.2 mg / kg penciclovir (with internal standard) added and not passed through the purification column. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] The following is a detailed description of the rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to embodiments of the present invention.

[0034] See Figure 1 As shown in the embodiment of the present invention, a rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents includes the following steps:

[0035] S1. Sample extraction: Add the extractant to the sample to be tested, and after vortexing, ultrasonic extraction, freezing, and centrifugation, obtain the supernatant containing the analyte.

[0036] Further, in a preferred embodiment of the present invention, the extractant is a mixed solution of methanol, acetonitrile, and formic acid solution, wherein the formic acid solution has a mass percentage of 1%, and the volume ratio of methanol, acetonitrile, and formic acid solution in the extractant is 45:45:10. The present invention uses an optimized extractant to extract samples, and combined with subsequent steps such as heating, ultrasonication, freezing, and centrifugation, can effectively release bound residues into the extractant and precipitate some impurities, thereby improving sample extraction efficiency and the purification efficiency of the rapid purification column.

[0037] Commonly used solvents for extracting antiviral drugs include methanol, acetonitrile, and water. Disinfectants or antibacterial agents contain a large amount of surfactants, oils, natural plant ingredients, and other macromolecular substances. Extraction using these solutions alone results in turbid extracts, making sample purification difficult and leading to low recovery rates. Methanol and water have good solubility for the target analyte. Acetonitrile has the effect of precipitating proteins and carries away few weakly polar components, resulting in high mass spectrometry response and minimal matrix interference. However, acetonitrile has poor solubility for the target analyte, so its proportion should not be too high; otherwise, the sample will quickly agglomerate, hindering the extractant from penetrating and acting. Adding a certain proportion of water can achieve a dispersing effect; therefore, this invention selects methanol, acetonitrile, and water as the main extraction solvents. Furthermore, nucleoside analogues contain multiple amino and hydroxyl groups, which readily react with one or more H+ molecules. + These compounds combine to form positively charged single or multiple ions. Adding a certain proportion of acid to the organic solvent during extraction can significantly improve the recovery rate. This invention investigated the extraction effects of different ratios of extractants, and through specific analysis and repeated experiments, it was found that when a mixed solution of methanol, acetonitrile, and 1% formic acid solution was used as the extractant, the recovery rate of each compound was high, and the supernatant after extraction and freezing was relatively clear. The acidic extractant provided by this invention (methanol:acetonitrile:1% formic acid solution = 45:45:10) can achieve even better recovery rates and is beneficial for the implementation of subsequent purification methods.

[0038] Further, in a preferred embodiment of the present invention, the mass-to-volume ratio of the test sample to the extractant is 1:18-22 (g / mL). When the sample amount of disinfectant or antibacterial agent is 0.5 g and the volume is adjusted to 10 mL, the limit of detection (LOD) for ribavirin is 4 μg / kg, and the LOD for acyclovir, ganciclovir, and penciclovir is 0.6 μg / kg; the limit of quantitation (LOQ) for ribavirin is 10 μg / kg, and the LOD for acyclovir, ganciclovir, and penciclovir is 2 μg / kg. The average recoveries of the four antiviral drugs are 75.8%–117.6%, with relative standard deviations not exceeding 10.9%.

[0039] Further, in a preferred embodiment of the present invention, the steps of ultrasonic extraction, freezing, and centrifugation are as follows: the sample of the test reagent with the added extractant is vortexed and mixed, then ultrasonicated at 35–45°C for 10–20 min, then frozen at -20°C for at least 1 h, and immediately centrifuged at 7500–8500 rpm for 4–6 min. Preferably, the sample is ultrasonicated at 40°C for 15 min, then frozen at -20°C for 1 h, and immediately centrifuged at 8000 rpm for 5 min. Freezing and centrifugation facilitates the precipitation of some lipid components, and subsequent one-step purification using a SHIMSENQVet-NM rapid purification column can remove lipids and surfactants, effectively reducing the matrix inhibition effect of the LC-MS co-effluent.

[0040] Furthermore, in a preferred embodiment of the present invention, the test substance includes ribavirin, acyclovir, ganciclovir, and penciclovir.

[0041] S2. Sample purification: Take an appropriate amount of the supernatant containing the analyte, add the mixed internal standard working solution to the supernatant and mix well. Then, filter it through a one-step rapid purification column to obtain the analyte solution.

[0042] In analytical testing, sample preparation is a crucial step in achieving satisfactory recovery rates for drug components with different physicochemical properties. Disinfectants or antibacterial agents often contain various traditional Chinese medicines, plant extracts, or surfactants, resulting in exceptionally complex matrices. These components can adversely affect the performance of LC-MS instruments; therefore, it is essential to minimize or remove them as much as possible during pretreatment. The rapid purification method of this invention uses multiple matrices to adsorb impurities and employs a one-step rapid extraction purification process, thereby efficiently removing impurities. The entire process involves only simple steps such as sample weighing, pipetting, vortexing, sonication, freezing, centrifugation, and filtration, eliminating the need for conventional solid-phase extraction steps such as activation, rinsing, elution, nitrogen blowing, concentration, and solvent conversion. Pretreatment operations are simpler and require less sophisticated equipment and specialized technical personnel training. The freezing and column purification pretreatment processes require minimal time from the laboratory and hardly extend the experimental procedure, potentially saving at least half the manpower and time compared to traditional SPE techniques.

[0043] The single-pass purification column of this invention uses less packing material and less organic solvent, overcoming the problems of excessive solvent use, numerous solvent conversion and extraction operations, and cumbersome pretreatment in existing solid-phase extraction methods. This reduces experimental costs and waste generation, making it environmentally friendly and ensuring the health and safety of personnel. The purified solution obtained after direct passage through the one-step rapid purification column is colorless, transparent, and clear, with no impurities interfering with the target peak in the obtained chromatogram, enabling accurate quantification. It also facilitates the long-term use of the chromatographic column and LC-MS instrument.

[0044] Furthermore, considering that the extraction rate of antiviral drugs from disinfectants or antibacterial agents is very high, with extraction recovery rates generally within 75-120%, and that most isotope internal standards are expensive, we chose to add ten equal parts of the extractant (1 mL) to the isotope internal standard after extraction and before the purification column. Compared to adding the isotope internal standard before sample extraction, this greatly reduces the amount of stable isotope used (saving 90%), thus saving detection costs and effectively compensating for the influence of matrix effects, ensuring the accuracy of detection results.

[0045] Further, in a preferred embodiment of the present invention, the mixed internal standard working solution is a methanol solution of isotope internal standards, wherein the isotope internal standards include ribavirin-13C5, acyclovir-D4, ganciclovir-D5, and penciclovir-D4, wherein the concentrations of ribavirin-13C5, acyclovir-D4, ganciclovir-D5, and penciclovir-D4 in the mixed internal standard working solution are all 200 ng / mL.

[0046] Further, in a preferred embodiment of the present invention, the one-step rapid purification column is a SHIMS EN QVet-NM (3 mL, 0.5 g) rapid purification column. The supernatant containing the analyte, after being mixed with internal standard working solution, is passed through the one-step rapid purification column under pressure or atmospheric pressure, and then filtered through a 0.22 μm hydrophilic PTFE membrane to obtain the analyte solution. After passing through the one-step rapid purification column, impurities such as lipids, proteins, and surfactants in the sample are adsorbed and retained, while the target analyte passes through the packing material along with the sample solution. The filtrate can be directly used for analysis.

[0047] The present invention employs a low-temperature cryogenic purification technique (freezing at -20°C for at least 1 hour) after sample extraction to reduce the solubility of oils in the extractant, promoting their coagulation and precipitation. This method also has minimal impact on antiviral drugs. Although some oils precipitate after cryogenic treatment, many coexisting components remain in the sample extractant. These substances not only interfere with LC-MS / MS analysis but also affect the active sites of the chromatographic column stationary phase, reducing its resolution and even contaminating the mass spectrometer ion source, requiring further purification. The QVet-NM one-step rapid purification column can better remove oils and surfactants from disinfectant or antibacterial agent samples, effectively reducing matrix effects. Therefore, the present invention uses low-temperature cryogenic purification combined with the QVet-NM purification column for rapid and effective purification of disinfectant or antibacterial agent samples, improving the sensitivity of the method and extending the lifespan of the chromatographic column and mass spectrometer. The through-feed purification strategy eliminates the need for conventional solid-phase extraction steps such as activation, rinsing, elution, nitrogen blowing, concentration, and solvent conversion. The sample solution is directly loaded through the purification column, resulting in a colorless, transparent, and clear purified solution.

[0048] S3. Sample detection: The test solution was detected by liquid chromatography-mass spectrometry with internal standard method.

[0049] Further, in a preferred embodiment of the present invention, the liquid chromatography-mass spectrometry (LC-MS) internal standard method uses an amide-bonded stationary phase column to separate the analyte. The amide-bonded stationary phase column is an ACQUITY UPLC BEH Amide (1.7 μm, 2.1 × 100 mm), with an ACQUITY UPLC BEH Amide (1.7 μm, 2.1 × 5 mm) guard column at the front end. Gradient elution is performed using 0.05% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phase. The flow rate is 0.50 mL / min, the injection volume is 2 μL, the column temperature is 40 °C, and the gradient elution program is: 0–1 min, 90% B; 1–3 min, 90%–70% B; 3–3.9 min, 70%–30% B; 4–5 min, 90% B. This invention employs an amide-bonded stationary phase column, ACQUITY UPLCBEH Amide (1.7 μm, 2.1 × 100 mm), to separate the analyte solution, achieving ideal resolution and peak shape. The liquid chromatography uses a gradient elution with 0.05% formic acid aqueous solution and acetonitrile as the mobile phase, eliminating the need for buffer salts or commonly used chromatographic modifiers. This not only reduces reagent preparation steps for operators but also improves the environmental friendliness of the instrument system. With an injection volume of 2 μL, solvent effects are negligible, and the peak shapes of all analyte components in the spiked sample are acceptable.

[0050] Furthermore, in a preferred embodiment of the present invention, in liquid chromatography, the running time (including washing and equilibration) for simultaneous determination of the analyte does not exceed 5 minutes, and the retention time of the analyte is dispersed between 1 and 4 minutes.

[0051] Furthermore, in a preferred embodiment of the present invention, the mass spectrometry conditions for detection using the liquid chromatography-mass spectrometry coupled with internal standard method are as follows: the ion source is an electrospray ion source, using positive ion mode and multiple reaction monitoring; nebulizer gas flow rate: 3 L / min; heating gas flow rate: 25 L / min; interface temperature: 300 °C; DL temperature: 250 °C; heating block temperature: 400 °C; drying gas flow rate: 3 L / min.

[0052] S4. Standard Curve Plotting: Dissolve the analyte standard and the mixed internal standard working solution in the extraction solvent to prepare a series of analyte standard solutions of various concentrations. Detect the analyte standard solutions of each concentration under the same conditions as in step S3, and plot the standard curve based on the detection results. Specifically, take ribavirin, ganciclovir, acyclovir, and penciclovir standards and internal standards, dilute them with the above extraction solvent to prepare a series of analyte standard solutions, and detect them by LC-MS. Plot the ratio of the corresponding compound concentration to the internal standard concentration on the x-axis and the ratio of the peak area of ​​the quantitative ion to the peak area of ​​the internal standard on the y-axis to obtain the standard curve.

[0053] S5. Qualitative and quantitative analysis: The detection results of the test solution are compared with the standard curve to determine the type of analyte in the test solution and to calculate the content of the analyte in the test sample.

[0054] This invention employs liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) to simultaneously determine the antiviral drugs ribavirin, acyclovir, ganciclovir, and penciclovir in disinfectants or antibacterial agents. Under optimized chromatographic and mass spectrometric conditions, a series of mixed standard solutions of the antiviral drugs ribavirin, acyclovir, ganciclovir, and penciclovir, along with the sample solution, are sequentially injected for analysis. Linear regression analysis is performed with the area ratio of each component in the standard solution to its corresponding internal standard as the ordinate and the mass concentration of each component to its corresponding internal standard as the abscissa. The peak area of ​​the sample solution is then substituted into the linear regression equation standard curve to obtain the content of the target analyte in the sample.

[0055] The qualitative determination method of this invention conforms to the EU EC / 657 / 2002 requirement for four identification points (IPs) in mass spectrometry qualitative analysis. Selecting one parent ion (1 point) and two daughter ions (1.5 points each) achieves the four-point requirement. The presence of antiviral drugs in the sample is determined by the liquid chromatography retention time and the relative abundance of each ion pair. Quantitative analysis is performed using the internal standard method. The concentration C of the antiviral drug in the test solution is obtained from the aforementioned standard curve, and then the residual amount of antiviral drug in the disinfectant or antibacterial agent to be detected is calculated. The calculation formula is as follows:

[0056]

[0057] V—Volume at constant volume (mL);

[0058] C—The mass concentration (μg / L) of the analyte in the test solution obtained from the standard curve;

[0059] m — Sample weight (g);

[0060] f—dilution factor;

[0061] X — The residual amount of the analyte in the test sample (μg / kg).

[0062] This invention establishes a rapid purification and pretreatment method for the detection of prohibited antiviral drugs ribavirin, ganciclovir, acyclovir, and penciclovir residues in disinfectants or antibacterial agents, followed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). This rapid purification and detection method offers higher extraction efficiency and purification effect, overcoming the limitation of current standard methods requiring separate determination. The use of an instrument-friendly mobile phase during operation results in higher analytical sensitivity, with a minimum limit of quantitation as low as 2 μg / kg, significantly improving work efficiency, effectively shortening detection time, being environmentally friendly, and saving reagent consumption and labor costs. This invention fills the technological gap for the simultaneous rapid detection and analysis of antiviral drugs in disinfectants or antibacterial agents, possessing strong practical application significance. It allows for the rapid detection of common antiviral drugs in disinfectants or antibacterial agents in a single step, with high sensitivity, strong specificity, accuracy, and a simple pretreatment process. The optimized LC-MS analysis can complete the separation and determination of antiviral drugs in disinfectants or antibacterial agents within 5 minutes, simultaneously improving both quantitative and qualitative accuracy and precision. It can be used for the detection of various disinfectant and antibacterial agent samples, providing manufacturers and testing institutions with a simpler, faster, greener, and more efficient detection method. This invention's detection method has significant reference and promotion value for subsequent standard revisions and related scientific research.

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1

[0065] This embodiment provides a rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents, which includes the following steps:

[0066] (1) Instruments and test materials

[0067] (1.1) LCMS-8060NX liquid chromatography-mass spectrometry system (Shimadzu Corporation, Japan); Allegra X-15R high-speed centrifuge (Beckman Corporation, USA); G-560E vortex mixer (Scientific Industries, USA); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water system (Millipore Corporation, USA).

[0068] (1.2) Relevant information on standard substances and isotopic internal standards is shown in Table 1. The standard substances ribavirin, acyclovir, ganciclovir, and penciclovir all have a purity greater than 98% (CATO, USA); the internal standards ribavirin-13C5 (100.1 μg / mL), acyclovir-D4 (100.0 μg / mL), ganciclovir-D5 (100.2 μg / mL), and penciclovir-D4 (99.9 μg / mL) all have a purity greater than 95% (Tianjin Alta Company) and are soluble in methanol solution.

[0069] Table 1. Information on Standard Materials and Isotope Internal Standards

[0070] Table 1. Relevant information on ribavirin, acyclovir, ganciclovir, and penciclovir reference materials and their isotopic internal standards.

[0071] Chinese name English name CAS serial number Molecular formula molecular weight Ribavirin Ribavirin 36791-04-5 <![CDATA[C8H 12 N4O5]]> 244.20 Acyclovir Acyclovir 59277-89-3 <![CDATA[C8H 11 N5O3]]> 225.20 Gancilovy Vinegar Ganciclovir 82410-32-0 <![CDATA[C9H 13 N5O4]]> 255.23 Penciclovir Penciclovir 39809-25-1 <![CDATA[C 10 H 15 N5O3]]> 253.26 Ribavirin-13C5 Ribavirin-13C5 1646818-35-0 <![CDATA[ 13 C5C8H 12 N4O5]]> 249.17 Acyclovir-D4 Acyclovir-D4 1185179-33-2 <![CDATA[C8H7D4N5O3]]> 229.23 Gancilove-D5 Ganciclovir-D5 1189966-73-1 <![CDATA[C9H8D5N5O4]]> 260.26 Penciclovir-D4 Penciclovir-D4 1020719-72-5 <![CDATA[C 10 H 11 D4N5O3]]> 257.28

[0072] (1.3) Methanol and acetonitrile (HPLC grade, Merk, USA); formic acid (HPLC grade, CNW, USA); SHIMSENQVet-NM rapid purification column (3 mL, 0.5 g, Shimadzu Shanghai); hydrophilic PTFE filter membrane (0.22 μm, Shimadzu Shanghai); ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 × 100 mm, Waters, USA).

[0073] (2) Sample pretreatment

[0074] (2.1) Sample extraction: Weigh 0.5 g (accurate to 1 mg) of homogenized sample into a centrifuge tube, add extraction solution (methanol:acetonitrile:1% formic acid water = 45:45:10) to the 10 mL mark, vortex to mix for 1 min, sonicate at 40 °C for 15 min, freeze at -20 °C for 1 h, and immediately centrifuge at 8000 rpm for 5 min to obtain the supernatant containing the analyte. The sample used in this example is a herbal antibacterial cream, purchased from Fujian Zhenghe Liangong Pharmaceutical Co., Ltd., batch number 20211201.

[0075] (2.2) Sample purification: Take 1 mL of supernatant and add 100 μL of mixed internal standard working solution. Mix well and load the entire solution directly into the SHIMSEN QVet-NM one-step rapid purification column. Pass through the column under pressure or at normal pressure and filter through a 0.22 μm hydrophilic PTFE membrane. The filtrate is ready for injection.

[0076] (3) Preparation of standard solutions

[0077] (3.1) Mixed standard stock solution: Weigh 1 mg of each of the four antiviral drug standards (accurate to 0.01 mg), place them in a 10 mL brown volumetric flask, dissolve them in methanol, and dilute to the mark. Shake well to prepare a mixed standard stock solution with a concentration of 100 μg / mL. Store in a -20℃ freezer for later use.

[0078] (3.2) Mixed standard working solution: Accurately transfer 50 μL of the above mixed standard stock solution into a 10 mL brown volumetric flask, dilute with the extraction solvent, and bring to the mark. Shake well to prepare a mixed standard working solution with a concentration of 0.5 μg / mL. Prepare mixed internal standard working solutions with concentrations of 0.5 μg / mL using the same method and store them in a -20℃ refrigerator for later use.

[0079] (4) Instrumental analysis conditions

[0080] (4.1) Liquid chromatography conditions:

[0081] Chromatographic column: ACQUITY UPLC BEH Amide (1.7μm, 2.1×100mm) amide-bonded stationary phase column with ACQUITY UPLC BEH Amide (1.7μm, 2.1×5mm) guard column at the front end. Gradient elution was performed using 0.05% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases at a flow rate of 0.5mL / min, an injection volume of 2μL, and a column temperature of 40℃. The elution program is shown in Table 2.

[0082] Table 2 Gradient elution program

[0083] Time (min) Phase A (%) Phase B (%) 0.0 10.0 90.0 1.0 10.0 90.0 3.0 30.0 70.0 3.9 30.0 30.0 4.0 10.0 90.0 5.0 10.0 90.0

[0084] (4.2) Mass spectrometry conditions:

[0085] The ion source was an electrospray ionization source, using positive ion mode and multiple reaction monitoring; nebulizer gas flow rate: 3 L / min; heating gas flow rate: 25 L / min; interface temperature: 300℃; DL temperature: 250℃; heating block temperature: 400℃; drying gas flow rate: 5 L / min. The qualitative and quantitative ion assays of the compounds are shown in Table 3.

[0086] Table 3. Mass spectrometry parameters of antiviral compounds and internal standards

[0087]

[0088] * indicates quantitative ions

[0089] like Figure 2 The image shows the multiple reaction monitoring (MRM) spectra of the target antiviral drugs ribavirin, acyclovir, ganciclovir, and penciclovir, as well as the internal standard compound, from Example 1. Figure 2It can be seen that the retention and dispersion times of ribavirin, acyclovir, ganciclovir, and penciclovir are 1.62 min, 2.13 min, 2.80 min, and 3.05 min, respectively. The analytical run time for each sample, which is being analyzed simultaneously with the analytes, does not exceed 5 min.

[0090] like Figure 3 The image shows the MRM chromatogram of the herbal antibacterial cream blank matrix sample (with internal standard added) after purification of ribavirin (with internal standard added) in Example 1. Figure 4 The image shows the MRM chromatogram of purified acyclovir (with internal standard) after purification of the blank matrix sample (with internal standard) of the herbal antibacterial cream in Example 1. Figure 5 The image shows the MRM chromatogram of purified ganciclovir (with internal standard) after purification of the blank matrix sample (with internal standard) of the herbal antibacterial cream in Example 1, which includes quantitative, qualitative and internal standard quantitative ions. Figure 6 The image shows the MRM chromatogram of the purified penciclovir (with internal standard) after purification of the blank matrix sample (with internal standard) of the herbal antibacterial cream in Example 1. (Quantitative, qualitative and internal standard quantitative ions)

[0091] The residue levels of ribavirin, acyclovir, ganciclovir, and penciclovir in the herbal antibacterial cream sample of this embodiment were found to be <4 μg / kg, <0.6 μg / kg, <0.6 μg / kg, and <0.6 μg / kg, respectively. This herbal antibacterial cream sample was used as a blank matrix sample for subsequent method validation experiments.

[0092] Experimental Example 1

[0093] This experimental example studies the effect of the purification method on the detection results by comparing the extraction ion chromatograms of the herbal antibacterial cream blank matrix spiked sample before and after purification. The specific steps are as follows:

[0094] Blank matrix of herbal antibacterial cream was taken separately and divided into experimental group and control group. After the blank matrix of herbal antibacterial cream in experimental group and control group was spiked, it was tested according to the steps (2) to (4) of Example 1. Among them, the blank matrix of herbal antibacterial cream in control group was not purified by SHIMSEN QVet-NM one-step rapid purification column after being spiked.

[0095] like Figure 7 The image shows the MRM chromatogram of the extracted ion currents (quantitative, qualitative, and internal standard quantitative ions) after purification of the blank matrix sample of herbal antibacterial cream with 1.2 mg / kg ribavirin (with internal standard added); Figure 8MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg acyclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream; Figure 9 MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg ganciclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream; Figure 10 MRM chromatogram of the purified extract ion flow (quantitative, qualitative and internal standard quantitative ions) after adding 1.2 mg / kg penciclovir (with internal standard) to the blank matrix sample of herbal antibacterial cream; Figure 11 MRM chromatogram of the extraction ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with added 1.2 mg / kg ribavirin (with internal standard) and not passed through the purification column; Figure 12 MRM chromatogram of the extract ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with 1.2 mg / kg acyclovir (with internal standard) added and not passed through the purification column; Figure 13 MRM chromatogram of the extract ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with added 1.2 mg / kg ganciclovir (with internal standard) and not passed through the purification column; Figure 14 MRM chromatogram of the extracted ion current (quantitative, qualitative and internal standard quantitative ions) of the blank matrix sample of herbal antibacterial cream with 1.2 mg / kg penciclovir (with internal standard) added and not passed through the purification column.

[0096] from Figures 7-14 It can be seen that all antiviral drug components in the unpurified sample were inhibited by the matrix, especially ribavirin, which was significantly inhibited. The matrix effect could reach over 90%, even leading to false negative results. Figure 11 This severely affects the sensitivity of the method and the accuracy of low-concentration determination results. Purification columns are extremely helpful for analyzing antiviral drug components in disinfectant or antibacterial agent samples with complex matrices, significantly improving detection sensitivity and ensuring data reliability.

[0097] Experimental Example 2

[0098] Antiviral drugs such as ribavirin, ganciclovir, acyclovir, and penciclovir are highly polar. Ribavirin, in particular, exhibits poor retention in the most commonly used reversed-phase C18 column. When using this column, the mobile phase must contain 95%–100% (v / v) aqueous phase, and the peak shape is poor with a low response. Hydrophilic interaction chromatography is an effective supplement to reversed-phase chromatography, providing strong retention of polar compounds. The mobile phase typically uses a high proportion of acetonitrile aqueous solution, and the composition of the mobile phase significantly affects the retention and selectivity of polar compounds.

[0099] In this experiment, two different hydrophilic interaction columns of the same specification, ACQUITY UPLC BEH, were used to analyze the test solutions: the amide-bonded ACQUITY UPLC BEH Amide (1.7 μm, 2.1 × 100 mm) and the non-bonded silica-based ACQUITY UPLC BEH HILIC (1.7 μm, 2.1 × 100 mm). The results showed that the Amide column with the amide-bonded stationary phase exhibited better retention, peak shape, sensitivity, and resolution for the four antiviral drug components. In contrast, the HILIC column with the non-bonded silica-based stationary phase showed poorer peak shape and response, especially severe tailing of the ribavirin peak. By changing the composition and ratio of the mobile phase, it was found that there was no need to add buffer salts, which are commonly used chromatographic modifiers in liquid chromatography. Using 0.05% formic acid aqueous solution and acetonitrile as the mobile phase for gradient elution, all four target peaks achieved good response and separation. At the same time, the running time of the analysis (including washing and equilibration) did not exceed 5 minutes, the retention times of all analytes were dispersed in the range of 1 to 4 minutes, and the detection limit of the detection method was lower than that of existing literature methods.

[0100] Experimental Example 3

[0101] This experimental example studies the sensitivity of the detection method of the present invention. The specific steps are as follows:

[0102] Take a blank sample, add the minimum amount of the mixed standard working solution, and process it according to the sample pretreatment steps in Example 1 before injection and testing. Calculate the limit of detection (LOD) of each analyte using a signal-to-noise ratio of 3, and the limit of quantitation (LOQ) of each analyte using a signal-to-noise ratio of 10. The linearity, LOD, and LOQ of antiviral drugs are shown in Table 4.

[0103] Table 4. Linearity, Limit of Detection, and Limit of Quantification of Antiviral Drugs

[0104]

[0105] As shown in Table 4, the four antiviral drugs exhibited good linearity within their respective ranges (R² ≥ 0.999), with method detection limits ranging from 0.6 to 4 μg / kg and quantitation limits ranging from 2 to 10 μg / kg.

[0106] Subsequently, the blank sample was spiked and the experiment was carried out according to steps (2) to (4) of Example 1. The MRM spectra of the blank sample and the blank matrix spiked solution were compared. Figures 7-14 The results showed that no interfering peaks were found near the retention times of the target drugs in typical disinfectant or antibacterial agent matrices, indicating that the established experimental conditions could distinguish between analytes and interfering matrix components, and the method had good selectivity.

[0107] Test Example 4

[0108] This experimental example uses a blank matrix spike test in typical dosage forms of three disinfectant or antibacterial agent matrices (liquid, gel, and cream) to determine the accuracy and precision of the detection method of the present invention. The disinfectant liquid was purchased from Hubei Aibang Biotechnology Co., Ltd., batch number 20210928; the hand sanitizer gel was purchased from Dezhou Anweishi Disinfection Products Co., Ltd., batch number 20210902; and the herbal antibacterial cream was purchased from Fujian Zhenghe Liangong Pharmaceutical Co., Ltd., batch number 20211201. The specific steps are as follows: spike the above three blank matrix samples and conduct the experiment according to steps (2) to (4) of Example 1. Table 5 shows the recovery rate and precision of antiviral drugs in liquid dosage forms; Table 6 shows the recovery rate and precision of antiviral drugs in gel dosage forms; and Table 7 shows the recovery rate and precision of antiviral drugs in cream dosage forms.

[0109] Table 5 Recovery rate and precision of antiviral drugs in liquid dosage forms

[0110]

[0111] Table 6 Recovery rate and precision of antiviral drugs in gel formulations

[0112]

[0113] Table 7 Recovery rate and precision of antiviral drugs in ointment formulations

[0114]

[0115]

[0116] As shown in Tables 5-7, the recoveries of all target analytes ranged from 75.8% to 117.6%, with RSDs all less than 10.9%. The accuracy and precision of the detection method of this invention are satisfactory.

[0117] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for rapid decontamination and detection of antiviral drugs in disinfectants or antiseptics, characterized in that, The method comprises the following steps: S1, sample extraction: adding an extraction agent to a sample to be tested, vortexing, ultrasonic extraction, freezing, centrifugation, and obtaining a supernatant containing a substance to be tested; The substance to be tested is ribavirin, acyclovir, ganciclovir, and penciclovir; The extraction agent is a mixed solution of methanol, acetonitrile, and formic acid water; S2, sample purification: adding a mixed internal standard working solution to the supernatant containing the substance to be tested, mixing, passing through a one-step rapid purification column, and filtering to obtain a test solution; The mixed internal standard working solution is an isotopic internal standard methanol solution, and the isotopic internal standard includes ribavirin-13C5, acyclovir-D4, ganciclovir-D5, and penciclovir-D4; The one-step rapid purification column is a SHIMSEN QVet-NM3mL, 0.5g rapid purification column, S3, sample detection: detecting the test solution by liquid chromatography-mass spectrometry internal standard method; The liquid chromatography-mass spectrometry internal standard method separates the test solution by using an amide-bonded stationary phase column, the amide-bonded stationary phase column is ACQUITY UPLC BEH Amide 1.7μm, 2.1×100mm, and an ACQUITY UPLC BEH Amide 1.7μm, 2.1×5mm guard column is matched at the front end of the column, 0.05% formic acid aqueous solution A and acetonitrile B are used as the mobile phase for gradient elution, and the gradient elution program is: 0-1min, 90% B; 1-3min, 90%-70% B; 3-3.9min, 70%-30% B; 4-5min, 90% B; S4, standard curve drawing: dissolving substance to be tested standard and mixed internal standard working solution in an extraction agent to prepare a series of substance to be tested standard solutions with different concentrations, detecting each concentration of the substance to be tested standard solution under the same conditions as step S3, and drawing a standard curve according to the detection results; S5, qualitative and quantitative: comparing the detection results of the test solution with the standard curve to determine the type of substance to be tested in the test solution and calculate the content of the substance to be tested in the test sample.

2. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S1, the mass percentage of formic acid water is 1%, and in the extraction agent, the volume ratio of methanol, acetonitrile, and formic acid water is 45:45:

10.

3. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S1, the mass-volume ratio of the test sample to the extraction agent is 1:18-22g / mL.

4. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S1, the ultrasonic extraction, freezing, and centrifugation steps are as follows: after vortexing and mixing the test sample with the extraction agent, ultrasonic extraction is performed at 35-45℃ for 10-20min, then freezing at -20℃ for at least 1h, and immediately centrifuging at 7500-8500rpm for 4-6min.

5. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S2, the concentration of ribavirin-13C5, acyclovir-D4, ganciclovir-D5, and penciclovir-D4 in the mixed internal standard working solution is 200ng / mL.

6. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, The supernatant containing the analyte is added to the mixed internal standard working solution, and then is filtered through a 0.22 μm hydrophilic PTFE filter membrane after being passed through the one-step rapid purification column under pressure or at normal pressure to obtain the sample solution.

7. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S3, the flow rate is 0.50 mL / min, the injection volume is 2 μL, and the column temperature is 40℃.

8. The method of rapid purification and detection of antiviral drugs in disinfectants or antiseptics according to claim 1, characterized in that, In step S3, the running time of the simultaneous determination of the analytes in the liquid chromatography is not more than 5 min, and the retention time of the analytes is dispersed in 1-4 min.

9. The rapid purification and detection method for antiviral drugs in disinfectants or antibacterial agents according to claim 1, characterized in that, In step S3, the mass spectrometry conditions of the liquid chromatography-mass spectrometry internal standard method are as follows: the ion source is an electrospray ion source, a positive ion mode is adopted, and multiple reaction monitoring is adopted; the flow rate of atomization gas is 3 L / min; the flow rate of heating gas is 25 L / min; the interface temperature is 300℃; the DL temperature is 250℃; the heating block temperature is 400℃; and the flow rate of dry gas is 3 L / min.

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