Method and system for trace analysis of antiviral drugs in sewage based on LC-MS / MS

Abstract

The invention relates to the field of drug analysis, and discloses a trace analysis method and system for antiviral drugs in sewage based on LC-MS / MS. The method comprises the following steps: filtering a sewage sample, analyzing the drug properties of the antiviral drugs, carrying out solid-phase extraction on the filtered sewage sample, preparing an LC-MS / MS module of the extracted sample, determining a mobile phase of a chromatographic column, and determining the mobile phase of the chromatographic column. Balancing the chromatographic column, constructing chromatographic conditions for balancing the chromatographic column, determining a tuning solution and a mass axis calibration compound of a mass spectrometer, calibrating the mass spectrometer, and constructing mass spectrum conditions of the calibrated mass spectrometer; the method comprises the following steps: preparing a standard solution containing an antiviral drug, analyzing the standard solution, constructing a standard curve of the standard solution, and calculating a response coefficient of an LC-MS / MS module; and analyzing the extracted sample to obtain a sample mass spectrum and a sample chromatogram, and calculating the content of the antiviral drug. According to the method, the sensitivity and accuracy of trace analysis of the antiviral drugs in the sewage can be improved.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method and system for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS. Background Art

[0002] Trace analysis of antiviral drugs in sewage refers to the process of using specific analytical techniques and methods to detect and quantify extremely low concentrations of antiviral drugs in sewage. Through analysis, the concentration of antiviral drugs in sewage can be understood, which helps to identify and track the pathways and sources of antiviral drugs entering the water body, and provide a basis for pollution control.

[0003] At present, the trace analysis of antiviral drugs in sewage is mainly based on chromatography, the main principle of which is that when the mixture passes through the stationary phase with the mobile phase, different components will move at different speeds and thus be separated. This method requires multiple detectors or multiple runs to detect, resulting in complex detection methods and a small dynamic range, which makes the detection cost higher and the efficiency lower. Summary of the invention

[0004] The present invention provides a method and system for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS, the main purpose of which is to improve the sensitivity and accuracy of the analysis of trace amounts of antiviral drugs in sewage.

[0005] To achieve the above object, the present invention provides a method for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS, comprising:

[0006] Obtaining a sewage sample and an antiviral drug, filtering the sewage sample to obtain a filtered sewage sample, analyzing the drug properties of the antiviral drug, and performing solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample;

[0007] configuring an LC-MS / MS module for the extracted sample, identifying a chromatographic column and a mass spectrometer of the LC-MS / MS module, determining a mobile phase of the chromatographic column based on the extracted sample, balancing the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, constructing chromatographic conditions of the balanced chromatographic column based on the extracted sample, determining a tuning solution and a mass axis calibration compound for the mass spectrometer, calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and constructing mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample;

[0008] A standard solution containing the antiviral drug is prepared, and based on the chromatographic conditions and the mass spectrometry conditions, the standard solution is analyzed using the LC-MS / MS module to obtain a standard mass spectrum and a standard chromatogram, and a standard curve of the standard solution is constructed based on the standard mass spectrum and the standard chromatogram, and a response coefficient of the LC-MS / MS module is calculated based on the standard curve;

[0009] Based on the chromatographic conditions and the mass spectrometry conditions, the extracted sample is analyzed using the LC-MS / MS module to obtain a sample mass spectrum and a sample chromatogram, and the drug content of the antiviral drug is calculated based on the sample mass spectrum, the sample chromatogram and the response coefficient.

[0010] In order to solve the above problems, the present invention also provides a sewage antiviral drug trace analysis system based on LC-MS / MS, the system comprising:

[0011] A sample extraction module is used to obtain a sewage sample and an antiviral drug, filter the sewage sample to obtain a filtered sewage sample, analyze the drug properties of the antiviral drug, and perform solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample;

[0012] a detection condition construction module, used for configuring the LC-MS / MS module of the extracted sample, identifying the chromatographic column and the mass spectrometer of the LC-MS / MS module, determining the mobile phase of the chromatographic column based on the extracted sample, balancing the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, constructing the chromatographic conditions of the balanced chromatographic column based on the extracted sample, determining the tuning solution and the mass axis calibration compound of the mass spectrometer, calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample;

[0013] a response coefficient calculation module, used for preparing a standard solution containing the antiviral drug, analyzing the standard solution using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a standard mass spectrum and a standard chromatogram, constructing a standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram, and calculating the response coefficient of the LC-MS / MS module based on the standard curve;

[0014] The drug trace analysis module is used to analyze the extracted sample using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a sample mass spectrum and a sample chromatogram, and calculate the drug content of the antiviral drug based on the sample mass spectrum, the sample chromatogram and the response coefficient.

[0015] The embodiment of the present invention performs solid phase extraction on the filtered sewage sample according to the properties of the drug to obtain an extracted sample, which can significantly increase the concentration of trace antiviral drugs in the sewage sample to a level that can be accurately measured by a detection instrument; optionally, the embodiment of the present invention determines the mobile phase of the chromatographic column based on the extracted sample to improve the separation effect of the target compound and other components, reduce peak overlap, and improve resolution; the embodiment of the present invention constructs the chromatographic conditions of the balanced chromatographic column based on the extracted sample to reduce peak tailing, front extension or splitting, and obtain a more symmetrical and sharp peak shape, which helps to improve qualitative and quantitative The accuracy of the analysis; the embodiment of the present invention can provide more consistent and reliable data by constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample, thereby improving the reproducibility of the experiment; the embodiment of the present invention can help optimize the detection conditions and improve the detection sensitivity by calculating the response coefficient of the LC-MS / MS module based on the standard curve, thereby reducing the detection limit, so that trace analytes can also be accurately detected. Finally, the embodiment of the present invention can achieve high-precision quantitative analysis of antiviral drugs by calculating the drug content of the antiviral drugs based on the sample mass spectrum, the sample chromatogram and the response coefficient. Therefore, the present invention can improve the sensitivity and accuracy of trace analysis of antiviral drugs in sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of a process for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS according to an embodiment of the present invention;

[0017] Figure 2 A functional module diagram of a sewage antiviral drug trace analysis system based on LC-MS / MS provided in one embodiment of the present invention;

[0018] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0019] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0020] The embodiment of the present application provides a method for trace analysis of antiviral drugs in sewage based on LC-MS / MS. The execution subject of the trace analysis method of antiviral drugs in sewage based on LC-MS / MS includes but is not limited to at least one of the electronic devices such as a server, a terminal, etc. that can be configured to execute the method provided in the embodiment of the present application. In other words, the trace analysis method of antiviral drugs in sewage based on LC-MS / MS can be executed by software or hardware installed on a terminal device or a server device, and the software can be a blockchain platform. The server includes but is not limited to: a single server, a server cluster, a cloud server or a cloud server cluster, etc. The server can be an independent server, or it can be a cloud server that provides basic cloud computing services such as cloud services, cloud databases, cloud computing, cloud functions, cloud storage, network services, cloud communications, middleware services, domain name services, security services, content delivery networks (Content Delivery Network, CDN), and big data and artificial intelligence platforms.

[0021] Reference Figure 1 FIG. 1 is a flow chart of a method for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS according to an embodiment of the present invention. In this embodiment, the method for analyzing trace amounts of antiviral drugs in sewage based on LC-MS / MS includes:

[0022] S1. Obtain a sewage sample and an antiviral drug, filter the sewage sample to obtain a filtered sewage sample, analyze the drug properties of the antiviral drug, and perform solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample.

[0023] The embodiments of the present invention can monitor the distribution and concentration of these drugs in the environment and evaluate the degree of pollution to the water environment by obtaining sewage samples and antiviral drugs. The sewage samples refer to water samples collected from sewage treatment plants, industrial discharge outlets, domestic drainage systems or other water environments. The antiviral drugs refer to drugs specifically used to treat or prevent viral infections.

[0024] The embodiment of the present invention filters the sewage sample to obtain a filtered sewage sample, which can remove suspended solids, particulate matter and some plankton in the sewage, thereby reducing the interference of these substances on subsequent analysis steps. The filtered sewage sample refers to a sewage sample that has been filtered.

[0025] Optionally, as an embodiment of the present invention, the sewage sample is filtered to obtain a filtered sewage sample by filtering using a filter membrane with a pore size of 0.45 microns or 0.22 microns.

[0026] The embodiment of the present invention can improve the sensitivity and specificity of the detection method by analyzing the drug properties of the antiviral drugs, ensuring that trace amounts of drugs can be accurately detected even in a complex sewage matrix. The drug properties refer to a series of characteristics of an object in terms of chemistry, physics, biology, etc.

[0027] Optionally, the analysis of the drug properties of the antiviral drugs can be performed by nuclear magnetic resonance spectroscopy.

[0028] The embodiment of the present invention performs solid phase extraction on the filtered sewage sample according to the properties of the drug to obtain an extracted sample, which can significantly increase the concentration of trace antiviral drugs in the sewage sample to a level that can be accurately measured by a detection instrument. The extracted sample refers to a sewage sample that has been processed by a solid phase extraction process.

[0029] As an embodiment of the present invention, the solid phase extraction of the filtered sewage sample is performed according to the properties of the drug to obtain an extracted sample, including:

[0030] Determining the solid phase extraction column for filtering the sewage sample according to the properties of the drug;

[0031] Activating the solid phase extraction column with a preset organic solvent to obtain an activated extraction column;

[0032] Determining an addition flow rate of the filtered sewage sample, and based on the addition flow rate, loading the filtered sewage sample into the activated extraction column to obtain an initial sample extraction column;

[0033] Washing the initial sample extraction column with a preset washing liquid to obtain a washed sample extraction column;

[0034] Drying the washed sample cloth extraction column to obtain a sample extraction column;

[0035] An eluent is prepared for the sample extraction column, and the sample extraction column is eluted with the eluent to obtain an extracted sample.

[0036] Wherein, the solid phase extraction column refers to a columnar device used to separate, enrich and purify target compounds in samples. The preset organic solvent refers to an organic solvent used for activation, such as methanol, acetonitrile, etc. The activated extraction column refers to an extraction column after the air and impurities in the solid phase extraction column are removed by using an organic solvent. The addition flow rate refers to the speed at which the filtered sewage sample is loaded into the activated solid phase extraction column. The initial sample extraction column refers to an extraction column containing antiviral drugs after extraction. The preset washing liquid refers to a liquid used to clean the solid phase extraction column, the purpose of which is to remove interfering substances non-specifically adsorbed on the column, such as water, saline, a specific pH buffer solution, etc. The washed sample cloth extraction column refers to an extraction column that has been treated with a washing step during the solid phase extraction process. The sample extraction column refers to an extraction column after drying and removing the washing liquid. The eluent refers to a solution used to extract target compounds from solid phase materials, such as a mixture of methanol or acetonitrile and water.

[0037] Optionally, the solid phase extraction column for filtering the sewage sample according to the properties of the drug can be confirmed by microfluidic solid phase extraction.

[0038] S2. Configure the LC-MS / MS module of the extracted sample, identify the chromatographic column and mass spectrometer of the LC-MS / MS module, determine the mobile phase of the chromatographic column based on the extracted sample, balance the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, construct the chromatographic conditions of the balanced chromatographic column based on the extracted sample, determine the tuning solution and mass axis calibration compound of the mass spectrometer, calibrate the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and construct the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample.

[0039] The embodiment of the present invention can obtain accurate molecular weight information by configuring the LC-MS / MS module of the extracted sample, which is helpful for the identification of the compound. Wherein, the LC-MS / MS module refers to a liquid chromatography-tandem mass spectrometry analysis system, which is an integrated analysis platform composed of a liquid chromatography (LC) unit and a tandem mass spectrometry (MS / MS) detection unit.

[0040] The embodiment of the present invention can optimize the separation effect, reduce the interference of co-eluting substances, and improve the resolution by identifying the chromatographic column and mass spectrometer of the LC-MS / MS module. The chromatographic column refers to the column used in the liquid chromatography part. The mass spectrometer refers to an instrument used to detect and quantify compounds in a sample.

[0041] Optionally, as an embodiment of the present invention, the chromatographic column and mass spectrometer of the LC-MS / MS module may be identified by integrated identification technology.

[0042] The embodiment of the present invention can improve the separation effect of the target compound and other components, reduce peak overlap, and improve resolution by determining the mobile phase of the chromatographic column based on the extracted sample. The mobile phase refers to a liquid mixture flowing in the chromatographic column, which is used to carry the sample through the chromatographic column and is a key component for separating and detecting different components in the sample.

[0043] As an embodiment of the present invention, determining the mobile phase of the chromatographic column based on the extracted sample includes:

[0044] Determining the type of the chromatographic column according to the antiviral drugs corresponding to the extracted sample;

[0045] Determining a mixed solvent for the chromatographic column based on the type of the chromatographic column and the corresponding drug properties of the antiviral drug;

[0046] Determining additives for the mixed solvent, wherein the additives include: acid-base regulators, ion pair reagents, and pH buffers;

[0047] Determining the initial mobile phase of the chromatographic column according to the mixed solvent and the additive;

[0048] Performing a chromatographic running test on the initial mobile phase to obtain a chromatographic test result;

[0049] Based on the chromatographic test results, calculating the separation coefficient of the antiviral drug;

[0050] When the separation coefficient meets a preset separation coefficient threshold, the initial mobile phase is used as the mobile phase of the chromatographic column.

[0051] Among them, the chromatographic column type refers to the specific type of chromatographic column, which determines the stationary phase and separation mechanism of the chromatographic column. The mixed solvent refers to a mixture of two or more solvents used for the mobile phase. The additive refers to a chemical substance added to the mobile phase to improve the chromatographic separation effect, increase the detection sensitivity of the analyte or stabilize the properties of the mobile phase. The acid-base regulator refers to a chemical substance used to adjust the pH value (pH value) of the mobile phase, such as formic acid, acetic acid, ammonia water, triethylamine, etc. The ion pair reagent refers to a chemical substance used to form a neutral complex with ions in the sample, such as sodium pentane sulfonate, sodium heptane sulfonate, sodium bisulfate, hydrochloric acid, etc. The pH buffer refers to a chemical substance used to maintain the pH value of the mobile phase stable, such as phosphate buffer, acetate buffer, citrate buffer, etc. The initial mobile phase refers to the mobile phase obtained by preliminary mixing of the mixed solvent and the additive. The chromatographic test results refer to the data and related information obtained by the chromatograph, which reflect the separation and analysis of each component in the sample. The separation coefficient refers to the relative degree of separation of two components during the chromatographic separation process. The preset separation coefficient threshold refers to a minimum value of a separation coefficient set in advance in order to achieve a specific separation effect.

[0052] Optionally, the mixed solvent of the chromatographic column based on the type of the chromatographic column and the corresponding drug properties of the antiviral drug can be determined by molecular dynamics simulation.

[0053] Optionally, the calculating the separation coefficient of the antiviral drug based on the chromatographic test result comprises:

[0054] Extracting a test chromatogram of the chromatographic test result, and identifying a first peak and a secondary peak of the test chromatogram;

[0055] Determine the first peak retention time and the second peak retention time of the first peak and the second peak respectively;

[0056] Analyzing the first peak baseline peak width and the second peak baseline peak width of the first peak and the second peak respectively;

[0057] According to the first peak retention time, the second peak retention time, the first peak baseline peak width and the second peak baseline peak width, the drug resolution of the antiviral drug is calculated using the following formula:

[0058]

[0059] Where F represents the drug resolution, t c represents the secondary peak retention time, t s represents the first peak retention time, K s Indicates the first peak baseline width, K c Indicates the secondary peak baseline width;

[0060] The separation coefficient of the antiviral drug is determined according to the drug resolution.

[0061] Among them, the test chromatogram refers to a chart recorded and generated by a chromatographic instrument in a test experiment, which shows the movement of each component in the sample in the chromatographic column over time. The first peak refers to the first peak that appears on the chromatogram, that is, the peak with the shortest retention time. The secondary peak refers to the second peak that follows the first peak on the chromatogram. The retention time of the first peak refers to the time from the injection of the sample into the chromatographic system to the time when the peak top of the first peak reaches the detector. The retention time of the secondary peak refers to the time from the injection of the sample into the chromatographic system to the time when the peak top of the second peak reaches the detector. The first peak baseline peak width refers to the distance between the points where the baselines on both sides of the first peak intersect with the peak in the chromatogram (that is, the starting point and end point of the peak). The secondary peak baseline peak width refers to the distance between the points where the baselines on both sides of the secondary peak intersect with the peak in the chromatogram (that is, the starting point and end point of the peak).

[0062] In the embodiment of the present invention, the chromatographic column is balanced according to the mobile phase to obtain a balanced chromatographic column, which can ensure that the stationary phase and the mobile phase reach a dynamic balance, thereby improving the separation efficiency of the components. The balanced chromatographic column refers to a process in which the chromatographic column reaches a stable state through specific operating steps.

[0063] Optionally, as an embodiment of the present invention, the chromatographic column is balanced according to the mobile phase, and the balanced chromatographic column can be obtained by balancing a multiphase mobile phase.

[0064] In the embodiment of the present invention, by constructing the chromatographic conditions of the equilibrium chromatographic column based on the extracted sample, the tailing, fronting or splitting of the peak can be reduced, and a more symmetrical and sharp peak shape can be obtained, which helps to improve the accuracy of qualitative and quantitative analysis. The chromatographic conditions refer to a series of parameters and conditions set to effectively separate and detect components in the sample.

[0065] As an embodiment of the present invention, the chromatographic conditions of the equilibrium chromatographic column are constructed based on the extracted sample, including:

[0066] Determining the mobile phase flow rate of the mobile phase corresponding to the balanced chromatographic column according to the extracted sample and the balanced chromatographic column;

[0067] Analyzing the column temperature of the equilibrium chromatographic column according to the drug properties of the extracted sample;

[0068] Analyzing the tolerance of the balanced chromatographic column, and determining the sample dilution factor of the extracted sample based on the tolerance;

[0069] Determining a detector of the equilibrium chromatographic column, and determining detection parameters of the detector according to the properties of the drug;

[0070] constructing a gradient elution program for the equilibrium chromatographic column, and determining the elution time of the equilibrium chromatographic column;

[0071] The chromatographic conditions of the equilibrium chromatographic column are determined based on the mobile phase flow rate, the chromatographic column temperature, the sample dilution factor, the detection parameters, the gradient elution program and the elution time.

[0072] Among them, the mobile phase flow rate refers to the speed at which the mobile phase passes through the chromatographic column during the chromatographic analysis. The chromatographic column temperature refers to the temperature inside the chromatographic column during the chromatographic analysis. The tolerance refers to the range of operating conditions that the chromatographic column can withstand in chromatographic analysis without damaging its performance or structure. The sample dilution multiple refers to the multiple to which the concentration of the original sample solution is reduced to a certain degree. The detector refers to an instrument used to detect the components after separation by the chromatographic column. The detection parameters refer to a set of set values ​​used to configure and optimize the performance of the detector. The gradient elution program refers to an elution method in which the composition of the mobile phase gradually changes over time. The elution time refers to the time from the injection of the sample into the chromatographic system to the time when a specific component reaches the detector and is recorded during the chromatographic analysis.

[0073] Optionally, the mobile phase flow rate of the mobile phase corresponding to the balanced chromatographic column is determined based on the extracted sample and the balanced chromatographic column by a high-precision pump and a flow sensor.

[0074] Optionally, the sample dilution factor of the extracted sample determined based on the tolerance may be determined by a dynamic dilution control method.

[0075] The embodiment of the present invention can optimize the parameters of the mass spectrometer by determining the tuning solution and mass axis calibration compound of the mass spectrometer, obtain clearer ion peaks, improve resolution, reduce the interference of co-eluting substances, and thus improve the sensitivity when detecting low-concentration samples. The tuning solution refers to a solution specifically used to adjust and optimize the performance of the mass spectrometer. The mass axis calibration compound refers to a standard compound used to calibrate the mass axis of the mass spectrometer.

[0076] Optionally, as an embodiment of the present invention, the determination of the tuning solution and mass axis calibration compound of the mass spectrometer may be determined by high-precision measurement technology.

[0077] The embodiment of the present invention calibrates the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, which can help maintain the long-term stability of the mass spectrometer and improve the repeatability of experimental results, thereby improving the accuracy and reliability of quantitative analysis. The calibrated mass spectrometer refers to a mass spectrometer that has been systematically calibrated by using a specific tuning solution and a mass axis calibration compound.

[0078] As an embodiment of the present invention, the step of calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer includes:

[0079] Analyzing mass spectrometry parameters of the mass spectrometer, wherein the mass spectrometry parameters include: source parameters, collision chamber parameters, and detector parameters;

[0080] determining a fragmentation coefficient of the mass spectrometer according to the collision cell parameters;

[0081] Using the mass spectrometer to detect the ionic strength and ionic stability of the tuning solution;

[0082] Determining the ionization efficiency of the mass spectrometer based on the ion intensity and the ion stability;

[0083] Detecting the mass-to-charge ratio of the mass axis calibration compound using the mass spectrometer, and calculating the detection sensitivity of the mass spectrometer based on the detected mass-to-charge ratio;

[0084] Analyzing the detection performance of the mass spectrometer based on the ionization efficiency, the fragmentation coefficient, and the detection sensitivity;

[0085] When the detection performance does not meet the preset detection performance standard, optimizing the mass spectrometry parameters to obtain optimized mass spectrometry parameters, and returning to the step of determining the fragmentation coefficient of the mass spectrometer according to the collision chamber parameters to obtain optimized detection performance;

[0086] When the optimized detection performance meets the preset detection performance standard, the mass spectrometer is calibrated according to the optimized mass spectrometry parameters to obtain a calibrated mass spectrometer.

[0087] Among them, the mass spectrometer parameters refer to multiple parameters that can be adjusted and set during the operation of the mass spectrometer. The source parameters refer to the parameters of the ion source part of the mass spectrometer, such as cone voltage, capillary voltage, nebulizer pressure, etc. The collision chamber parameters refer to the parameters used to control and optimize the behavior and reaction of ions in the collision chamber, such as collision voltage, collision chamber pressure, collision chamber temperature, etc. The detector parameters refer to the parameters used to detect and record ion signals in the mass spectrometer, such as detector threshold, detector dynamic range, peak detection algorithm, etc. The fragmentation coefficient refers to the efficiency of a molecule to produce fragment ions under the action of collision-induced dissociation or other dissociation techniques in mass spectrometry. The ionic strength refers to a parameter that measures the total concentration of ions in a solution. The ionic stability refers to the ability of an ion to maintain its original structure and properties without change. The ionization efficiency refers to the proportion or efficiency of molecules in a sample being converted into ions. The detection mass-to-charge ratio refers to the ratio of the mass of an ion to its charge. The detection sensitivity refers to the minimum difference between the concentration of an analyte and a blank sample (without analyte) that the system can reliably identify and distinguish at a given confidence level (e.g., 95%). The detection performance refers to the ability of a mass spectrometer to identify, quantify and confirm a target under specific conditions. The preset detection performance standard refers to a series of performance index requirements set in advance to ensure the effectiveness and reliability of a detection method or system in a specific application. The optimized mass spectrometer parameters refer to the mass spectrometer parameters that reach the preset detection performance standards after a series of experiments and adjustments.

[0088] Optionally, the fragmentation coefficient of the mass spectrometer determined according to the collision chamber parameters can be determined by a collision induced dissociation method.

[0089] Optionally, the detection sensitivity of the mass spectrometer calculated based on the detected mass-to-charge ratio can be calculated by a method for determining the limit of detection and the limit of quantitation.

[0090] The embodiment of the present invention can provide more consistent and reliable data by constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample, thereby improving the reproducibility of the experiment. The mass spectrometry conditions refer to various parameters and conditions that need to be set and optimized in order to obtain the best detection effect and analysis data when performing mass spectrometry analysis.

[0091] As an embodiment of the present invention, the step of constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample includes:

[0092] configuring samples of different concentrations of the extracted sample, and constructing quantitative analysis conditions of the calibrated mass spectrometer based on the samples of different concentrations;

[0093] Determining a parent ion full scan method of the calibrated mass spectrometer;

[0094] Determining the parent ion and the daughter ion of the parent ion full scan method according to the extracted sample;

[0095] determining a collision energy and a fragmentor voltage of the calibrated mass spectrometer based on the parent ions and the daughter ions;

[0096] The mass spectrometry conditions of the calibrated mass spectrometer are constructed based on the quantitative analysis conditions, the collision energy and the fragmentation voltage.

[0097] Wherein, the different concentration samples refer to a series of extracted samples with different concentration gradients prepared in the experiment. The quantitative analysis conditions refer to a series of experimental and operational requirements that need to be met when performing quantitative analysis. The parent ion full scan method refers to a mass spectrometry analysis technology, which is mainly used to detect and identify compounds in samples on a mass spectrometer. The parent ion refers to the original ion that is ionized in the ion source of the mass spectrometer without further fragmentation or reaction in mass spectrometry. The daughter ion refers to the charged particle generated in the mass spectrometer after the parent ion undergoes a specific fragmentation process (such as collision induced dissociation CID, electron capture dissociation ECD, electron transfer dissociation ETD, etc.) in mass spectrometry. The collision energy refers to the energy applied when the parent ion collides with the atoms or molecules of the collision gas (usually an inert gas, such as argon) during the collision induced dissociation process. The rupture voltage refers to the maximum voltage value that a dielectric (insulating material) can withstand under certain conditions.

[0098] Optionally, the collision energy and the fragmentation voltage of the calibrated mass spectrometer based on the parent ions and the daughter ions may be determined by collision induced dissociation technology.

[0099] S3. Prepare a standard solution containing the antiviral drug, analyze the standard solution using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a standard mass spectrum and a standard chromatogram, construct a standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram, and calculate the response coefficient of the LC-MS / MS module based on the standard curve.

[0100] The embodiment of the present invention can be used to construct a standard curve by configuring a standard solution containing the antiviral drug, and the curve can be used to determine the drug concentration in an unknown sample, which is a key step in quantitative analysis. The standard solution refers to a solution of one or more antiviral drugs of known concentration.

[0101] The embodiment of the present invention analyzes the standard solution using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a standard mass spectrum and a standard chromatogram that can provide the retention time of the drug at different concentrations, which is used to determine the position of the drug peak in the sample, and provide the characteristic ions of the drug and its corresponding mass spectrometry peak for qualitative confirmation and quantitative analysis. The standard mass spectrum refers to a mass spectrum obtained by detecting the analyte in the standard solution by a mass spectrometer under specific LC-MS / MS analysis conditions. The standard chromatogram refers to a chromatogram obtained under specific chromatographic conditions using a standard sample of known concentration in chromatographic analysis.

[0102] Optionally, as an embodiment of the present invention, based on the chromatographic conditions and the mass spectrometry conditions, the standard solution is analyzed using the LC-MS / MS module, and the standard mass spectrum and standard chromatogram can be generated by a liquid chromatography detector and a mass spectrometer.

[0103] The embodiment of the present invention provides a method for associating the chromatographic peak area or peak height of the target analyte in the sample with its concentration by constructing the standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram. The standard curve refers to a curve graph that represents the linear relationship between the analyte signal (such as chromatographic peak area or peak height) detected by the instrument and the actual concentration of the analyte in a series of standard solutions of known concentrations.

[0104] Optionally, as an embodiment of the present invention, the standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram can be constructed by a linear regression analysis method.

[0105] The embodiment of the present invention can help optimize the detection conditions and improve the detection sensitivity by calculating the response coefficient of the LC-MS / MS module based on the standard curve, thereby reducing the detection limit, so that trace analytes can also be accurately detected. Wherein, the response coefficient refers to the ratio of the detector response caused by the unit concentration of the analyte in chromatography-mass spectrometry analysis.

[0106] As an embodiment of the present invention, the calculation of the response coefficient of the LC-MS / MS module based on the standard curve includes:

[0107] Extracting standard solution concentration data of the standard curve;

[0108] According to the standard curve, mapping the detector response data corresponding to the standard solution concentration data;

[0109] Determining the number of data mapping groups of the standard solution concentration data and the detector response data;

[0110] According to the standard solution concentration data, the detector response data and the number of data mapping groups, the response coefficient of the LC-MS / MS module is calculated using the following formula:

[0111]

[0112] Where X represents the response coefficient, n represents the number of data mapping groups, and U i represents the detector response data of the i-th group, Q i Represents the concentration data of the i-th group of standard solutions.

[0113] The standard solution concentration data refers to the solution data of known concentration used in constructing the standard curve. The detector response data refers to the signal data obtained from the detector when analyzing the standard solution. The number of data mapping groups refers to the number of standard solutions of different concentrations used to construct the standard curve. Each standard solution group contains a specific concentration and corresponding detector response data.

[0114] S4. Based on the chromatographic conditions and the mass spectrometry conditions, the extracted sample is analyzed using the LC-MS / MS module to obtain a sample mass spectrum and a sample chromatogram, and based on the sample mass spectrum, the sample chromatogram and the response coefficient, the drug content of the antiviral drug is calculated.

[0115] The embodiment of the present invention can achieve efficient, accurate and sensitive qualitative and quantitative analysis of drugs by analyzing the extracted sample using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a sample mass spectrum and a sample chromatogram. The sample mass spectrum refers to a spectrum generated after the compounds in the sample are ionized, separated and detected in a mass spectrometer during liquid chromatography-tandem mass spectrometry analysis. The sample chromatogram refers to a spectrum detected and recorded by a detector after the compounds in the sample are separated in a chromatographic column during liquid chromatography analysis.

[0116] The embodiment of the present invention can achieve high-precision quantitative analysis of antiviral drugs by calculating the drug content of the antiviral drugs based on the sample mass spectrum, the sample chromatogram and the response coefficient. Due to the high sensitivity and selectivity of mass spectrometry detection, even low-concentration drugs can be accurately measured. Among them, the drug content refers to the concentration of antiviral drugs in the sample after extraction.

[0117] As an embodiment of the present invention, the method of calculating the drug content of the antiviral drug based on the sample mass spectrum, the sample chromatogram and the response coefficient includes:

[0118] Analyzing the sample pretreatment efficiency of the extracted sample corresponding to the sample mass spectrum to determine the dilution factor of the extracted sample;

[0119] Determining the drug structural characteristics of the antiviral drug according to the mass spectrum of the sample;

[0120] Analyzing the drug type of the antiviral drug based on the structural characteristics of the drug;

[0121] Determining the peak area of ​​the antiviral drug based on the chromatogram and the drug type;

[0122] Calculating the intercept of the standard curve corresponding to the response coefficient;

[0123] According to the peak area, the response coefficient, the intercept, the sample pretreatment efficiency and the dilution factor, the drug content of the antiviral drug is calculated using the following formula:

[0124]

[0125] Among them, H represents the drug content, M f represents the peak area, b represents the intercept, X represents the response coefficient, E represents the sample pretreatment efficiency, and D represents the dilution factor.

[0126] Among them, the sample pretreatment efficiency refers to the efficiency of the target compound being extracted, purified and transferred from the original sample to the final detection medium during the entire sample pretreatment process. The dilution factor refers to the dilution multiple used when the sample is diluted in order to adjust the sample concentration to the detection range of the analytical equipment or for ease of operation. The drug structural characteristics refer to the characteristics such as the specific atomic arrangement, functional groups, stereochemical configuration and intermolecular interactions in the drug molecule. The drug type refers to the category to which it belongs based on its chemical structure, pharmacological action, therapeutic use or other classification standards. The peak area refers to the area enclosed by the chromatographic peak of a specific component on the chromatogram. The intercept refers to the intercept term of the standard curve.

[0127] Optionally, the drug structural characteristics of the antiviral drug can be determined based on the sample mass spectrum by a spectrum analysis method.

[0128] Optionally, the peak area of ​​the antiviral drug determined based on the chromatogram and the drug type can be calculated by a peak area integration method.

[0129] The embodiment of the present invention performs solid phase extraction on the filtered sewage sample according to the properties of the drug to obtain an extracted sample, which can significantly increase the concentration of trace antiviral drugs in the sewage sample to a level that can be accurately measured by a detection instrument; optionally, the embodiment of the present invention determines the mobile phase of the chromatographic column based on the extracted sample to improve the separation effect of the target compound and other components, reduce peak overlap, and improve resolution; the embodiment of the present invention constructs the chromatographic conditions of the balanced chromatographic column based on the extracted sample to reduce peak tailing, front extension or splitting, and obtain a more symmetrical and sharp peak shape, which helps to improve qualitative and quantitative The accuracy of the analysis; the embodiment of the present invention can provide more consistent and reliable data by constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample, thereby improving the reproducibility of the experiment; the embodiment of the present invention can help optimize the detection conditions and improve the detection sensitivity by calculating the response coefficient of the LC-MS / MS module based on the standard curve, thereby reducing the detection limit, so that trace analytes can also be accurately detected. Finally, the embodiment of the present invention can achieve high-precision quantitative analysis of antiviral drugs by calculating the drug content of the antiviral drugs based on the sample mass spectrum, the sample chromatogram and the response coefficient. Therefore, the present invention can improve the sensitivity and accuracy of trace analysis of antiviral drugs in sewage.

[0130] like Figure 2 , is a functional module diagram of a sewage antiviral drug trace analysis system based on LC-MS / MS provided by one embodiment of the present invention.

[0131] The sewage antiviral drug trace analysis system 200 based on LC-MS / MS of the present invention can be installed in an electronic device. According to the functions to be implemented, the sewage antiviral drug trace analysis system 200 based on LC-MS / MS can include a sample extraction module 201, a detection condition construction module 202, a response coefficient calculation module 203 and a drug trace analysis module 204. The module of the present invention can also be called a unit, which refers to a series of computer program segments that can be executed by an electronic device processor and can complete fixed functions, which are stored in the memory of the electronic device.

[0132] In this embodiment, the functions of each module / unit are as follows:

[0133] The sample extraction module 201 is used to obtain a sewage sample and an antiviral drug, filter the sewage sample to obtain a filtered sewage sample, analyze the drug properties of the antiviral drug, and perform solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample;

[0134] The detection condition construction module 202 is used to configure the LC-MS / MS module of the extracted sample, identify the chromatographic column and the mass spectrometer of the LC-MS / MS module, determine the mobile phase of the chromatographic column based on the extracted sample, balance the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, construct the chromatographic conditions of the balanced chromatographic column based on the extracted sample, determine the tuning solution and mass axis calibration compound of the mass spectrometer, calibrate the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and construct the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample;

[0135] The response coefficient calculation module 203 is used to prepare a standard solution containing the antiviral drug, analyze the standard solution using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a standard mass spectrum and a standard chromatogram, construct a standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram, and calculate the response coefficient of the LC-MS / MS module based on the standard curve;

[0136] The drug trace analysis module 204 is used to analyze the extracted sample based on the chromatographic conditions and the mass spectrometry conditions using the LC-MS / MS module to obtain a sample mass spectrum and a sample chromatogram, and calculate the drug content of the antiviral drug based on the sample mass spectrum, the sample chromatogram and the response coefficient.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for trace analysis of antiviral drugs in sewage based on LC-MS / MS, characterized in that: The method comprises: Obtaining a sewage sample and an antiviral drug, filtering the sewage sample to obtain a filtered sewage sample, analyzing the drug properties of the antiviral drug, and performing solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample; configuring an LC-MS / MS module for the extracted sample, identifying a chromatographic column and a mass spectrometer of the LC-MS / MS module, determining a mobile phase of the chromatographic column based on the extracted sample, balancing the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, constructing chromatographic conditions of the balanced chromatographic column based on the extracted sample, determining a tuning solution and a mass axis calibration compound for the mass spectrometer, calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and constructing mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample; A standard solution containing the antiviral drug is prepared, and based on the chromatographic conditions and the mass spectrometry conditions, the standard solution is analyzed using the LC-MS / MS module to obtain a standard mass spectrum and a standard chromatogram, and a standard curve of the standard solution is constructed based on the standard mass spectrum and the standard chromatogram, and a response coefficient of the LC-MS / MS module is calculated based on the standard curve; Based on the chromatographic conditions and the mass spectrometry conditions, the extracted sample is analyzed using the LC-MS / MS module to obtain a sample mass spectrum and a sample chromatogram, and the drug content of the antiviral drug is calculated based on the sample mass spectrum, the sample chromatogram and the response coefficient.

2. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 1, characterized in that: According to the properties of the drug, the filtered sewage sample is subjected to solid phase extraction to obtain an extracted sample, including: Determining the solid phase extraction column for filtering the sewage sample according to the properties of the drug; Activating the solid phase extraction column with a preset organic solvent to obtain an activated extraction column; The addition flow rate of the filtered sewage sample is determined, and based on the addition flow rate, the filtered sewage sample is loaded into the activated extraction column to obtain an initial sample extraction column.

3. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 2, characterized in that: According to the properties of the drug, the filtered sewage sample is subjected to solid phase extraction to obtain an extracted sample, including: Washing the initial sample extraction column with a preset washing liquid to obtain a washed sample extraction column; Drying the washed sample cloth extraction column to obtain a sample extraction column; An eluent is prepared for the sample extraction column, and the sample extraction column is eluted with the eluent to obtain an extracted sample.

4. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 1, characterized in that: Determining the mobile phase of the chromatographic column based on the extracted sample comprises: Determining the type of the chromatographic column according to the antiviral drugs corresponding to the extracted sample; Determining a mixed solvent for the chromatographic column based on the type of the chromatographic column and the corresponding drug properties of the antiviral drug; Determine the additives of the mixed solvent, wherein the additives include: acid-base regulators, ion pair reagents and pH buffers.

5. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 4, characterized in that: Determining the mobile phase of the chromatographic column based on the extracted sample comprises: Determining the initial mobile phase of the chromatographic column according to the mixed solvent and the additive; Performing a chromatographic running test on the initial mobile phase to obtain a chromatographic test result; Based on the chromatographic test results, calculating the separation coefficient of the antiviral drug; When the separation coefficient meets a preset separation coefficient threshold, the initial mobile phase is used as the mobile phase of the chromatographic column.

6. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 5, characterized in that: The step of calculating the separation coefficient of the antiviral drug based on the chromatographic test result comprises: Extracting a test chromatogram of the chromatographic test result, and identifying a first peak and a secondary peak of the test chromatogram; Determine the first peak retention time and the second peak retention time of the first peak and the second peak respectively; Analyzing the first peak baseline peak width and the second peak baseline peak width of the first peak and the second peak respectively; Calculating the drug resolution of the antiviral drug according to the first peak retention time, the second peak retention time, the first peak baseline peak width, and the second peak baseline peak width; The separation coefficient of the antiviral drug is determined according to the drug resolution.

7. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 1, characterized in that: The step of constructing the chromatographic conditions of the equilibrium chromatographic column based on the extracted sample comprises: Determining the mobile phase flow rate of the mobile phase corresponding to the balanced chromatographic column according to the extracted sample and the balanced chromatographic column; Analyzing the column temperature of the equilibrium chromatographic column according to the drug properties of the extracted sample; The tolerance of the balanced chromatographic column is analyzed, and based on the tolerance, the sample dilution factor of the extracted sample is determined.

8. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 7, characterized in that: The step of constructing the chromatographic conditions of the equilibrium chromatographic column based on the extracted sample comprises: Determining a detector of the equilibrium chromatographic column, and determining detection parameters of the detector according to the properties of the drug; constructing a gradient elution program for the equilibrium chromatographic column, and determining the elution time of the equilibrium chromatographic column; The chromatographic conditions of the equilibrium chromatographic column are determined based on the mobile phase flow rate, the chromatographic column temperature, the sample dilution factor, the detection parameters, the gradient elution program and the elution time.

9. The method for trace analysis of antiviral drugs in sewage based on LC-MS / MS as claimed in claim 1, characterized in that: The step of calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer comprises: Analyzing mass spectrometry parameters of the mass spectrometer, wherein the mass spectrometry parameters include: source parameters, collision chamber parameters, and detector parameters; determining a fragmentation coefficient of the mass spectrometer according to the collision cell parameters; Using the mass spectrometer to detect the ionic strength and ionic stability of the tuning solution; Determining the ionization efficiency of the mass spectrometer based on the ion intensity and the ion stability; Detecting the mass-to-charge ratio of the mass axis calibration compound using the mass spectrometer, and calculating the detection sensitivity of the mass spectrometer based on the detected mass-to-charge ratio; Analyzing the detection performance of the mass spectrometer based on the ionization efficiency, the fragmentation coefficient, and the detection sensitivity; When the detection performance does not meet the preset detection performance standard, optimizing the mass spectrometry parameters to obtain optimized mass spectrometry parameters, and returning to the step of determining the fragmentation coefficient of the mass spectrometer according to the collision chamber parameters to obtain optimized detection performance; When the optimized detection performance meets the preset detection performance standard, the mass spectrometer is calibrated according to the optimized mass spectrometry parameters to obtain a calibrated mass spectrometer.

10. A sewage antiviral drug trace analysis system based on LC-MS / MS, characterized in that: Used to perform the LC-MS / MS-based trace analysis method for antiviral drugs in sewage as described in any one of claims 1 to 9, the system comprising: A sample extraction module is used to obtain a sewage sample and an antiviral drug, filter the sewage sample to obtain a filtered sewage sample, analyze the drug properties of the antiviral drug, and perform solid phase extraction on the filtered sewage sample according to the drug properties to obtain an extracted sample; a detection condition construction module, used for configuring the LC-MS / MS module of the extracted sample, identifying the chromatographic column and the mass spectrometer of the LC-MS / MS module, determining the mobile phase of the chromatographic column based on the extracted sample, balancing the chromatographic column according to the mobile phase to obtain a balanced chromatographic column, constructing the chromatographic conditions of the balanced chromatographic column based on the extracted sample, determining the tuning solution and the mass axis calibration compound of the mass spectrometer, calibrating the mass spectrometer based on the tuning solution and the mass axis calibration compound to obtain a calibrated mass spectrometer, and constructing the mass spectrometry conditions of the calibrated mass spectrometer based on the extracted sample; a response coefficient calculation module, used for preparing a standard solution containing the antiviral drug, analyzing the standard solution using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a standard mass spectrum and a standard chromatogram, constructing a standard curve of the standard solution based on the standard mass spectrum and the standard chromatogram, and calculating the response coefficient of the LC-MS / MS module based on the standard curve; The drug trace analysis module is used to analyze the extracted sample using the LC-MS / MS module based on the chromatographic conditions and the mass spectrometry conditions to obtain a sample mass spectrum and a sample chromatogram, and calculate the drug content of the antiviral drug based on the sample mass spectrum, the sample chromatogram and the response coefficient.

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