Environmental sample collection and detection method

Abstract

The invention discloses an environmental sample collection and detection method, aiming at the problems of loose process and poor data reliability in the prior art, a whole-process standardized operation system is constructed: a sampling plan is formulated according to a task, point locations such as a door handle and a door slot are determined, and a matched sterilization swab and blank quality control are performed; during sampling, the swab is forcibly smeared in one direction and rotated to ensure uniform extraction; carrying out temperature-controlled transportation on the sample through a low-temperature transfer box to maintain chemical stability; fixed-volume methanol aqueous solution ice-bath ultrasound is combined with vortex extraction in a laboratory, so that the efficiency is improved and errors are reduced; an internal calibration volume is added before sample introduction, and instrument and matrix fluctuation is corrected; the liquid chromatography-tandem mass spectrometry adopts a positive and negative ion segmented mode and multi-ion monitoring, and a target object is confirmed through double thresholds of retention time and ion abundance ratio. According to the method, the detection accuracy, repeatability and legal accessibility are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of sample detection, and particularly relates to an environmental sample collection and detection method. BACKGROUND

[0002] In the current environmental sample collection and detection, the mainstream technology adopts traditional cotton swab sampling combined with laboratory gas chromatography-mass spectrometry (GC-MS) or enzyme-linked immunoassay (ELISA) analysis. The typical process is as follows: on-site random wiping with ordinary cotton swab dipped in ethanol or aqueous solution, sample transportation at room temperature, and then solvent soaking, centrifugation, nitrogen blowing concentration and other pretreatments in the laboratory, and then instrument detection.

[0003] However, this method has a prominent technical problem: lack of standardization control in the whole process, resulting in high false positive rate of detection results and poor quantitative repeatability, which cannot meet the strict requirements of judicial expertise on data accuracy and traceability. The specific performance is as follows: there is no unified standard for sampling operation, the solvent dipping frequency, wiping direction and force are random, and the extraction efficiency of target substances in the sample fluctuates greatly; there is no temperature guarantee in the transportation process, and the target substances are easily affected by environmental factors to cause degradation or transformation; no internal standard correction is introduced in the pretreatment link, and the response value drifts due to different matrix interference; the instrument analysis mode is single, which cannot consider both polar and non-polar compounds, and some target substances are missed or misjudged due to low ionization efficiency or co-flow interference. SUMMARY

[0004] The purpose of the present application is to provide an environmental sample collection and detection method, which solves the problem of unreliable data caused by loose process and lack of control in the prior art, and makes the detection results have high accuracy, repeatability and legal adoptability.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: an environmental sample collection and detection method, comprising: According to the detection task, a sampling plan is formulated, and independent packaging sampling swabs, methanol solution and blank quality control samples are prepared, wherein the sampling plan specifies the sampling points as door handles, door gaps or floor in front of the door; After the prepared sampling swab is used to dip the methanol solution on the target surface, it is wiped at least three times and rotated, then the cotton swab head is sealed in a sealed bag and labeled; The labeled sealed bag is placed in a special clean transport box and configured with a cooling ice bag for low-temperature transportation, ensuring that the transportation temperature is ≤4℃ and the room temperature is placed for not more than 24 hours; After the sample arrives at the laboratory, the cotton swab head is taken out and placed in a plastic centrifuge tube, methanol aqueous solution is added for ice bath ultrasonic, and after ultrasonic is completed, it is vortexed for 3 to 5 seconds for mixing; 450 μL of the extract was filtered and 50 μL of an internal standard solution was added to make up to 500 μL, and a liquid chromatography-tandem mass spectrometry system equipped with a C18 reversed-phase chromatographic column was used to perform multiple ion monitoring under a gradient elution program of 0.1 min to 3.0 min in positive ion mode and 3.0 min to 5.0 min in negative ion mode; The target substance was confirmed according to a retention time deviation of ≤2.5% and an ion abundance ratio allowed deviation threshold, and the mass of the target substance on the swab head was calculated by an internal standard method.

[0006] Preferably, the sampling plan includes: assigning at least two sampling personnel to work in cooperation, setting the sampling time to be early morning or a period of low human flow, grouping the sampling area according to floor, collecting samples from door handles, door gaps, and floor in front of the door of each household, and synchronously preparing blank quality control samples, setting the sampling frequency to be single or periodic re-sampling according to the risk level, and pre-entering all point information and numbering rules into the sampling record table and corresponding to the subsequent sealed bag labeling content.

[0007] Preferably, the preparation of independently packaged sampling swabs, methanol solution, and blank quality control samples includes: selecting sampling swabs made of defatted medical cotton with an epoxyethane sterilized wooden handle, dispensing HPLC grade methanol in brown glass bottles, providing sterile clean sealed bags, disposable butyronitrile gloves, N95 masks, and oily marker pens, and completing solvent blank detection by the laboratory before sampling to confirm that the benzene content is <0.1 ppb and the phthalate content is <0.5 ppb before it can be used, and the blank quality control sample is prepared by directly sealing the swab dipped in methanol.

[0008] Preferably, after applying the sampling swab, the sealing and labeling includes: the sampling personnel wearing gloves hold the swab tip and dip it in the methanol solution only once and soak the cotton head, apply it evenly on the target surface at least three times in a one-way "Z" shape path, while rotating the swab to ensure 360° contact, immediately break the cotton tip after completion to leave the wooden handle outside the bag, clearly label the sample number, sampling point, and date on the surface of the sealed bag with an oily pen, the labeling content needs to be checked in real time with the sampling record table to ensure consistency, and the hands or other parts of the body are prohibited from touching the sampling position or the cotton head throughout the process.

[0009] Preferably, the low-temperature transportation includes: placing the labeled sealed bag vertically in the item transfer box with the cotton head facing down, placing a pre-frozen to -20℃ cooling ice bag between each layer of samples, and configuring a wireless temperature and humidity recorder in the box for real-time monitoring, and automatically alarming if the box temperature >8℃ or the humidity >80%RH.

[0010] Preferably, the process of ultrasonic and vortex extraction includes: breaking the wooden stick outside the sealed bag with tweezers in a biological safety cabinet, cutting the bag corner to pour the cotton head into a 10 mL polypropylene centrifuge tube, adding 1.00 mL of 20% methanol aqueous solution to completely cover the cotton head, tightly covering the tube cap, and placing it in an ice water bath ultrasonic cleaner at 40 kHz for 15 minutes with a program setting of 1 minute pause for every 5 minutes of ultrasonic, immediately vortexing at 3000 rpm for 30 seconds to fully desorb the adsorbate after ultrasonic, and then centrifuging at 4000 rpm for 5 minutes to take the supernatant for subsequent filtration operation.

[0011] Preferably, the multiple ion monitoring includes: setting the ultra-high performance liquid chromatography flow rate to 0.3 mL / min, the column temperature to 35°C, the mobile phase A to 30 mM ammonium formate and 0.1% formic acid aqueous solution, the mobile phase B to HPLC grade methanol, the gradient elution program to 20% B phase at 0.1 minutes, increasing to 80% at 1.5 minutes, increasing to 95% at 2.25 minutes, switching to negative ion mode at 3.0 minutes and maintaining 95% B phase to 3.1 minutes, then decreasing B phase to 20% and stopping at 5.0 minutes; the mass spectrometry is set to ESI ion source voltage 5500V in positive ion mode and -4500V in negative ion mode, the acquisition type is multiple ion monitoring mode and the residence time is 5 mSec, and the setting of the target compound monitoring parameters includes: the first column of each substance is the quantitative ion pair.

[0012] Preferably, the verification of the ion abundance ratio allowed deviation threshold includes: calculating the relative abundance of the qualitative daughter ion in the sample Ksam=(A2 / A1) x 100%, wherein A2 is the peak area of the qualitative daughter ion and A1 is the peak area of the quantitative daughter ion; calculating the relative abundance of the qualitative daughter ion in the standard sample Kstd=(Astd2 / Astd1) x 100%, wherein Astd2 is the peak area of the qualitative daughter ion in the standard sample and Astd1 is the peak area of the quantitative daughter ion in the standard sample; comparing the deviation of Ksam and Kstd, when Kstd>50%, the maximum allowed deviation is 20%, when 20%<Kstd≤50%, the maximum allowed deviation is 25%, when 10%<Kstd≤20%, the maximum allowed deviation is 30%, when Kstd≤10%, the maximum allowed deviation is 50%, and at the same time, the absolute value of the relative deviation of the retention time of the target substance is required to be less than 2.5% to determine that the corresponding analyte exists.

[0013] Preferably, the internal standard method requires deducting the response value of the blank sample before calculating the mass, that is, if the target substance concentration is detected in the blank sample, the concentration results of all samples are deducted by this value; The standard curve is established by using the weighted least square method fitting and the correlation coefficient is greater than or equal to 0.995, the quality control points include 5 ng / mL and 50 ng / mL concentration points, and the measured value must be within the range of the nominal value ± 15%; if the response value of the sample exceeds 80% of the highest point 100 ng / mL of the standard curve, dilution is triggered, 100 muL of the original extract is diluted to 1000 muL, then the internal standard is added, and the result is multiplied by the dilution factor; The final report includes target mass, expanded uncertainty, risk level and blockchain storage number.

[0014] Preferably, the internal standard method calculates the mass formula as: m=(p1xfxV) / V1, wherein m is the target mass on the wiping head, p1 is the target concentration in the sample obtained from the standard curve, f is the dilution factor, V is the sample extraction volume 1 mL, and V1 is the constant volume 0.5 mL.

[0015] The technical effects and advantages of the present application are as follows: The present application first formulates a structured sampling plan according to the detection task, clearly defines the sampling points and uses independent sterilized sampling swabs and blank quality control samples, and controls the variables from the source; secondly, the one-way smearing and rotating swab operation specification is strictly implemented during the sampling process to ensure uniform and stable extraction of the target; thirdly, the special transport box with cooling ice bags is configured to realize the whole process of low-temperature transportation, effectively maintaining the chemical stability of the sample; in the laboratory pretreatment stage, the fixed volume of methanol aqueous solution is combined with ice bath ultrasonic and vortex mixing to improve the extraction efficiency and reduce the operation difference; the internal standard solution is added for constant volume before injection, and the internal standard response ratio is used to correct the instrument fluctuation and matrix effect; finally, the positive and negative ion segment monitoring mode is adopted in the liquid chromatography-tandem mass spectrometry system, combined with multiple ion reaction monitoring, to realize the synchronous high selectivity identification of different physicochemical properties of the target, and the qualitative confirmation is carried out through the double threshold of retention time and ion abundance ratio, to ensure that the result determination is rigorous and reliable. The data unreliability problem caused by loose process and control loss in the prior art is solved, so that the detection result has high accuracy, repeatability and legal adoptability. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The flowchart of the environmental sample collection and detection method of the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. The specific embodiments described herein are only used to explain the present application, and are not used to limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present application.

[0018] The present application provides an environmental sample collection and detection method as shown in Figure 1 The present application provides an environmental sample collection and detection method as shown in According to the detection task, a sampling plan is formulated, and independently packaged sampling swabs, methanol solution and blank quality control samples are prepared. The sampling plan specifies that the sampling points are door handles, door gaps or floor in front of the door. The sampling plan includes: at least two sampling personnel are assigned to work in cooperation, the sampling time is set as early morning or low-traffic period, the sampling area is divided by floor, three samples of door handle, door gap and floor in front of the door are collected for each household, and blank quality control samples are prepared synchronously. The sampling frequency is set as single or periodic re-sampling according to the risk level. All point information and numbering rules are pre-recorded in the sampling record table and correspond to the subsequent sealed bag labeling content.

[0019] Preparation of independently packaged sampling swabs, methanol solution and blank quality control samples includes: selecting sampling swabs made of defatted medical cotton with epoxyethane sterilization, dispensing HPLC grade methanol in brown glass bottles, providing sterile clean sealed bags, disposable nitrile gloves, N95 masks and oily marker pens, and completing solvent blank detection in the laboratory before sampling to confirm that the benzene content is <0.1 ppb and the phthalate content is <0.5 ppb before it can be used, and the blank quality control sample is prepared by directly sealing the methanol after dipping with the swab from the same batch.

[0020] After using the prepared sampling swab to dip the methanol solution on the target surface, it is rubbed at least three times in one direction and rotated, then the cotton tip is broken and sealed in the sealed bag and the information is labeled; after rubbing the sampling swab, the sealing and labeling include: the sampler wears gloves and holds the swab tip to dip the methanol solution only once and soak the cotton head, rubs the target surface at least three times in one direction with "Z" shape path, rotates the swab to ensure 360° contact, breaks the cotton tip immediately after completion to leave the wood rod outside the bag, and clearly labels the sample number, sampling point and date on the surface of the sealed bag with an oily pen. The labeling content needs to be checked in real time with the sampling record table to ensure consistency. Hands or other parts of the body are prohibited from touching the sampling position or the cotton head throughout the process.

[0021] The sealed bag after labeling is placed in a special clean article transfer box and a cooling ice bag is configured for low-temperature transportation, to ensure that the transportation temperature is ≤4°C and the room temperature is placed for not more than 24 hours; the low-temperature transportation includes: the sealed bag after labeling is placed vertically in the article transfer box and the cotton head is downward, a pre-frozen to -20°C cooling ice bag is placed between each layer of samples, a wireless temperature and humidity recorder is configured in the box for real-time monitoring, and if the box temperature is >8°C or the humidity is >80%RH, an automatic alarm is generated.

[0022] After the sample transported arrives is thawed in the laboratory, the cotton swab head is taken out and placed in a plastic centrifuge tube, methanol solution is added for ice bath ultrasonic, after ultrasonic is completed, vortex is 3 to 5 seconds for mixing; 450μL of the extraction liquid is filtered, 50μL of the internal standard solution is added, and the volume is made up to 500μL, a liquid chromatography-tandem mass spectrometry system equipped with a C18 reversed-phase chromatographic column is used to perform multiple ion monitoring under a gradient elution program of 0.1 minutes to 3.0 minutes in positive ion mode and 3.0 minutes to 5.0 minutes in negative ion mode; The ultrasonic and vortex extraction process includes: in the biological safety cabinet, the wooden rod is broken from the outside of the sealed bag, the bag corner is cut, the cotton head is poured into a 10mL polypropylene centrifuge tube, 1.00mL of 20% methanol solution is added to completely cover the cotton head, the tube cap is tightly covered, and then the ice water bath ultrasonic is placed in a 40kHz ultrasonic cleaner for 15 minutes, and the program is set to stop for 1 minute every 5 minutes of ultrasonic; after ultrasonic is completed, vortex oscillation is immediately performed at 3000rpm for 30 seconds to fully desorb the adsorbed material, and then centrifugation is performed at 4000rpm for 5 minutes to take the supernatant for subsequent filtration operation.

[0023] Further, the multiple ion monitoring includes: setting the ultra-high performance liquid chromatography flow rate to 0.3mL / min, the column temperature to 35°C, the mobile phase A to 30mM ammonium formate and 0.1% formic acid aqueous solution, the mobile phase B to HPLC grade methanol, and the gradient elution program to 20% B phase at 0.1 minutes, rising to 80% at 1.5 minutes, rising to 95% at 2.25 minutes, switching to negative ion mode at 3.0 minutes and maintaining 95% B phase to 3.1 minutes, and then B phase is reduced to 20% and stopped at 5.0 minutes; the mass spectrometry is set to ESI ion source voltage 5500V in positive ion mode and -4500V in negative ion mode, the acquisition type is multiple ion monitoring mode and the residence time is 5mSec, and the setting of the target compound monitoring parameters includes: the first column of each substance is the quantitative ion pair.

[0024] According to the retention time deviation ≤2.5% and the ion abundance ratio allowable deviation threshold, the target substance is verified, and the mass of the target substance on the swab head is calculated by the internal standard method.

[0025] Among them, the verification of the ion abundance ratio allowable deviation threshold includes: The relative abundance of the qualitative daughter ions in the sample is calculated as Ksam = (A2 / A1) x 100%, wherein A2 is the peak area of the qualitative daughter ions, and A1 is the peak area of the quantitative daughter ions. The relative abundance of the qualitative daughter ions in the standard sample is calculated as Kstd = (Astd2 / Astd1) x 100%, wherein Astd2 is the peak area of the qualitative daughter ions in the standard sample, and Astd1 is the peak area of the quantitative daughter ions in the standard sample. The deviation of Ksam and Kstd is compared, and when Kstd > 50%, the maximum deviation allowed is 20%, when 20% < Kstd ≤ 50%, the maximum deviation allowed is 25%, when 10% < Kstd ≤ 20%, the maximum deviation allowed is 30%, and when Kstd ≤ 10%, the maximum deviation allowed is 50%, and at the same time, the absolute value of the relative deviation of the target substance retention time is required to be less than 2.5% to determine that the corresponding target substance exists.

[0026] In the internal standard method, the response value of the blank sample needs to be deducted before mass calculation, that is, if the target substance concentration is detected in the blank sample, the concentration results of all samples are reduced by this value. The standard curve needs to be fitted by weighted least squares method and the correlation coefficient is ≥0.995, and the quality control points include 5 ng / mL and 50 ng / mL concentration points, and the measured value must be within the range of ± 15% of the nominal value; if the response value of the sample exceeds 80% of the highest point 100 ng / mL of the standard curve, dilution is triggered, 100 μL of the original extract is diluted to 1000 μL, and then the internal standard is added to the constant volume and re-injected, and the result is multiplied by the dilution factor; the final report includes the mass of the target substance, the expanded uncertainty, the risk level and the blockchain storage number.

[0027] Specifically, the mass calculation formula of the internal standard method is: m = (p1 x f x V) / V1, wherein m is the mass of the target substance on the wiping head, p1 is the concentration of the target substance in the sample obtained from the standard curve, f is the dilution factor, V is the sample extraction volume 1 mL, and V1 is the constant volume 0.5 mL.

[0028] This method formulates a sampling plan according to the task, clearly defines the points such as door handles and door gaps, and matches sterilization swabs and blank quality control; during sampling, one-way smearing is forced and the swab is rotated to ensure uniform extraction; the sample is transported in a low-temperature transport box to maintain chemical stability; the laboratory uses fixed-volume methanol water solution ice bath ultrasonic combined with vortex extraction to improve efficiency and reduce errors; the internal standard is added before injection to constant volume to correct the instrument and matrix fluctuations; liquid chromatography-tandem mass spectrometry uses positive and negative ion segmentation mode and multiple ion monitoring to confirm the target substance through double threshold values of retention time and ion abundance ratio.

[0029] The method has made a number of technical innovations on the basis of traditional environmental sampling, including: standardized processing of sampling swabs, solvent blank quality control, ultrasonic-vortex collaborative extraction, positive and negative ion segment MRM scanning, internal standard method quantitative correction, ion abundance deviation threshold control, etc., which significantly improves the detection sensitivity (LOD reaches 0.05 ng / mL), specificity (false positive rate <1%) and throughput (single sample detection period ≤5 min). The following will be described in detail in combination with specific examples: Step one: sampling scheme design and material preparation This step can minimize human error, environmental interference and cross contamination risk by systematic design of sampling strategy and standardized preparation of materials, laying a foundation for subsequent high-precision detection.

[0030] Step 1.1: Develop a sampling plan and divide the area The plan should specify the division of labor among sampling personnel (at least two people per group, one person sampling and one person recording), sampling time (recommended in the early morning or during off-peak hours to reduce interference), sampling points (e.g. door handles, door gaps, and door tiles in front of 502 rooms in three buildings in a certain community), sampling frequency (single sampling for initial screening, and weekly re-sampling for key areas), and sample quantity (at least 3 swab samples per household + 1 blank control). Area division should be based on building structure, such as grouping by floor in high-rise residential buildings, with 3-5 households selected per floor to avoid sample confusion. This plan will serve as a direct basis for the calculation of consumable quantities in "Step 1.2" and the on-site operation in "Step 1.3".

[0031] The system sampling plan ensures the spatial representativeness and temporal comparability of the samples. Stratified sampling (by floor and house type) can improve the detection rate in hotspots.

[0032] Step 1.2: Prepare standardized sampling consumables and quality control materials According to the plan in Step 1.1, the following consumables are prepared: 1. Independent sterilized sampling swabs (degreasing cotton head + wooden rod, ethylene oxide sterilization, single head easy break design); 2. HPLC grade methanol (or unopened ethanol) packaged in 5 mL brown glass bottles; 3. Sterile sealed bags (10 cm x 15 cm with self-sealing strips); 4. Blank quality control swabs (unused swabs from the same batch); 5. Disposable butyronitrile gloves, N95 masks, and oil-based markers; 6. Cooling ice packs (-20°C pre-frozen), and dedicated transport boxes (lined with aluminum foil to prevent contamination).

[0033] Key requirement: All solvents must be accompanied by a Certificate of Analysis (COA) and a "solvent blank test" (GC-MS screening for common interferents) must be completed by the laboratory before sampling. The results must meet the "Standard for Solvent Blank Limits of Environmental Samples" (benzene <0.1 ppb, phthalate <0.5 ppb), otherwise the batch is prohibited from leaving the warehouse. This step provides a pollution-free tool for the on-site operation of "Step 1.3", and the quality control results will directly affect the blank test judgment of "Step Four".

[0034] Ethylene oxide sterilization avoids microbial degradation of target objects; wooden swab prevents plastic components (such as phthalates) from interfering with mass spectrometry; solvent blank testing intercepts batch contamination, avoiding the waste of the entire batch of samples.

[0035] Step 1.3: On-site sampling operation specification execution The sampling personnel wear gloves and masks and operate according to the points designated in "Step 1.1": 1. Prepare a blank quality control - take a new swab and dip it in methanol, then directly into a sealed bag (without touching any surface); 2. Formal sampling - hold the swab tip, dip it in methanol (only once, soak the cotton head), and draw a "Z" shape on the target surface (such as a doorknob) with one-way back and forth for ≥3 times (about 10 cm 2 area), while rotating the swab to ensure 360° contact; 3. Immediately after sampling, break off the cotton head end of the swab (the wooden rod remains outside the bag), seal the bag, and mark "sample number - sampling point - date" with an oily pen; 4. Repeat the above operation to complete sampling at 3 points in the same household. Throughout the process, hands must not touch the sampling surface or the cotton head, and if accidental contact occurs, gloves must be replaced and recorded. The samples generated in this step will be directly used for transportation in "Step Two" and pretreatment in "Step Three".

[0036] One-way smearing avoids cross-contamination; rotating the swab improves extraction efficiency; breaking the design prevents secondary contamination. Experimental comparison: The traditional "repeated dipping of solvent" method leads to sample dilution (recovery rate reduced by 15-20%), while the "single dipping + sufficient smearing" method of this method makes the recovery rate of methamphetamine stable at 92.4±3.1% (n=30).

[0037] Step 1.4: Sample identification, temporary storage, and transportation handover Check the label information (number, site, time) immediately after sampling to ensure consistency with the sampling record table. Place the sealed bag vertically into the transport box (cotton head facing down to prevent liquid leakage), and place an ice bag (-20°C) on each layer. The temperature monitor in the box should display ≤4°C. The transportation time limit is ≤24 hours to reach the laboratory at room temperature (25°C). If it exceeds 24 hours or crosses provincial boundaries, use dry ice (-78°C) for frozen transportation. Fill out the "Sample Handover Form" and note the transportation conditions, time, and temperature control records. Both the sampling party and the laboratory should sign it. This step ensures that the samples received in "Step Three" are not degraded or contaminated, and the temperature control data will be used as a basis for determining the effectiveness of the results in "Step Five".

[0038] Low temperature inhibits microbial growth and chemical degradation (e.g., cocaine degradation rate reaches 35% at 25°C for 48 hours); vertical placement prevents solvent soaking of labels, which can cause information loss.

[0039] Step Two: Sample Transportation and Laboratory Reception This step is the key link between the field and the laboratory. Through strict temperature control, shock prevention, and cross-contamination prevention measures, the physical and chemical properties of the sample are maintained stable during transportation, providing "original state" samples for subsequent high-precision analysis.

[0040] Step 2.1: Real-time monitoring of temperature and humidity during transportation When packing in "Step 1.4", place a wireless temperature and humidity recorder (accuracy ±0.5°C, sampling interval 5 minutes) in the center of the transport box. The transportation vehicle should be equipped with GPS and temperature control alarm system. If the temperature in the box is >8°C or the humidity is >80%RH, an automatic alarm will be sent to the laboratory. The driver needs to check the ice bag status every 2 hours and supplement if necessary. Upon arrival at the laboratory, the temperature and humidity curve is exported to confirm that the entire process meets the requirements of "Step 1.4" (≤4°C). This monitoring data will be the core basis for the reception judgment in "Step 2.2".

[0041] Step 2.2: Laboratory Reception and Preliminary Sorting The laboratory reception personnel check the "Sample Handover Form" and temperature and humidity records. If the temperature exceeds the standard or the label is blurred, the sample is rejected. Qualified samples are sorted according to the floor grouping principle in "Step 1.1": 1. Place them in -20°C refrigerator according to the number prefix (e.g. A building, B building); 2. Take out the blank quality control samples and store them separately; 3. Record the sample information in the LIMS system (Laboratory Information Management System) to generate a unique sample ID; 4. Notify the pre-processing group to prepare consumables. This step ensures that the pre-processing in "Step Three" is traceable and free of cross-contamination.

[0042] Step 2.3: Sample Thawing and Appearance Inspection The pre-treatment group takes the sample from the refrigerator and thaws it for 2 hours in a 4°C environment (cold room). After thawing, check: 1. Is the sealed bag damaged? (If so, mark "contamination" and discard); 2. Is the cotton head intact? (If broken, record); 3. Is there solvent leakage? (Weigh the bag, if the weight is >0.1 g than the initial weight, it is considered contaminated); 4. Is the label clear and readable. Qualified samples are transferred to the biosafety cabinet (Class II) for operation, and unqualified samples are filled in the "Abnormal Sample Report" for archiving. This check result directly affects the feasibility of "Step 2.4" pre-treatment.

[0043] The biosafety cabinet provides a sterile environment to prevent laboratory air pollutants (such as phthalates in dust) from interfering.

[0044] Step 2.4: Sample pre-registration and blind sample coding In the LIMS system, generate a "blind sample code" (such as BLXX-XX-001) for each sample, hide the original information (address, homeowner), and only keep the parameters required for detection (sampling point type, reception date). Print the blind sample label and paste it on the centrifuge tube, and store it together with the original sealed bag in the 4°C treatment area. This code will run through "Step Three" to "Step Six", ensuring that the detection personnel operate "double-blind", avoiding subjective bias.

[0045] Step Three: Sample pre-treatment and extraction This step is the core of the detection sensitivity. Through the standardized extraction process, trace amounts of target substances are efficiently released and enriched from complex matrices (cotton fibers, dust, sebum), while interfering substances are removed, providing "clean" on-machine liquid for instrument analysis.

[0046] Step 3.1: Cotton head transfer and anti-pollution operation In the biosafety cabinet, put on new gloves and use tweezers to pick up the sealed bag of "Step 2.4" and break the wooden rod from the outside (keep the cotton head in the bag). Cut the bag corner and pour the cotton head into a 10 mL polypropylene centrifuge tube (pre-marked blind sample code), avoiding the gloves touching the cotton head or the tube wall at all times. If accidentally touched, change gloves and record. This operation ensures that the extraction liquid in "Step 3.2" is not contaminated by external sources.

[0047] Polypropylene tubes have an adsorption rate of <2% for target substances (vs. 5-8% for glass tubes) and are resistant to organic solvents.

[0048] Step 3.2: Solvent extraction and ultrasonic enhancement Add 1.00 mL of 20% methanol in water (LCMS grade methanol + ultrapure water) to the centrifuge tube, ensuring the liquid completely covers the cotton tip. Tighten the tube cap and place in an ultrasonic cleaner (power 200 W, frequency 40 kHz) with ice water bath for 15 minutes (program: 5 min ultrasonic, 1 min standing, repeat 3 times). After ultrasonication, vortex for 30 seconds (3000 rpm) to fully desorb the adsorbate. This extract will be used for filtration in "Step 3.3" and internal standard addition in "Step 3.4".

[0049] 20% methanol in water balances the polarity between polar and non-polar (lipid-soluble) components; ultrasonic cavitation disrupts the cotton fiber-target binding; Table 1: Ice bath prevents thermal degradation, comparative data: Step 3.3: Centrifugal filtration and supernatant transfer Centrifuge the extract at 4000 rpm for 5 minutes, and transfer 450 μL of the supernatant to a 1.5 mL injection vial. Filter the supernatant through a 0.22 μm water filter (nylon material), discard the first 100 μL of the filtrate (to rinse the filter), and collect the subsequent filtrate into the injection vial. This filtrate is the "pure extract", which will be used for internal standard addition in "Step 3.4".

[0050] Centrifugation removes cotton fiber debris; 0.22 μm filtration traps >0.22 μm particles (such as dust, bacteria), preventing column blockage.

[0051] Step 3.4: Internal standard addition and constant volume for instrument analysis Add 50 μL of internal standard solution (mixed internal standards containing 13 deuterated compounds such as METH-D8, MOR-D3, etc., at a concentration of 100 ng / mL) to the injection vial, and dilute to 500 μL with 20% methanol in water, then mix for 10 seconds. Cap with an aluminum cap and store at -20°C in the dark until analysis (within 48 hours). The internal standard concentration must be consistent with the internal standard of the standard curve in "Step Five" to ensure accurate quantification.

[0052] Internal standard method corrects matrix effects and instrument fluctuations. For example, sebum in the sample may inhibit ionization, but the internal standard and target undergo the same process, and their response ratios are stable.

[0053] Step Four: Instrument analysis condition setting and calibration This step optimizes UHPLC-MS / MS parameters to achieve simultaneous detection of 13 samples within 5 minutes, and uses positive and negative ion segment scanning and MRM mode to improve selectivity and sensitivity.

[0054] Step 4.1: Chromatographic column and mobile phase configuration A C18 reversed-phase chromatographic column (50 mm x 2.1 mm, 1.7 μm) was installed with a column temperature of 35 °C. Mobile phase A: 30 mM ammonium formate + 0.1% formic acid in water; mobile phase B: HPLC grade methanol. Elution was performed according to the following gradient (flow rate 0.3 mL / min): Table 2: Gradient elution program: This gradient separates polar (e.g. BE) and non-polar (e.g. THC-COOH) compounds in the injection from "Step 3.4".

[0055] Step 4.2: Mass spectrometry parameter optimization and MRM settings ESI source was set to positive ion voltage 5500 V, negative ion -4500 V; gas curtain gas 35 psi, nebulizer gas 55 psi, auxiliary gas 50 psi. MRM channels were set (e.g. METH: 150.1-91.0, DP 50, CE 27) with a dwell time of 5 ms. Key optimization: 5 CE values were tested for each compound (±5 V), and the one with the highest response was selected. This parameter was used for the standard curve in "Step 4.3" and sample detection in "Step 4.4".

[0056] MRM enhances selectivity (excludes isomer interference); optimization of CE value maximizes fragment ion yield.

[0057] Step 4.3: Mass axis calibration and sensitivity verification After starting up each day, the mass axis was calibrated with a calibration solution (containing caffeine, reserpine, etc.) to ensure a deviation of <±0.1 Da. Then, a 1 ng / mL mixed standard was injected to verify that the S / N of each compound was >10 (LOD standard). If not, the ion source was cleaned or the capillary was replaced. The calibration data were archived as a prerequisite for the effectiveness of the results in "Step 4.4".

[0058] Mass axis deviation caused MRM channel failure (e.g. target m / z 150.1 shifted to 150.3, response went to zero). A certain uncalibration caused all samples to be false negatives, and after calibration the detection rate was restored.

[0059] Step 4.4: Blank sample and system suitability test A blank sample prepared in "Step 3.4" (solvent + internal standard) was injected to confirm that there were no target peaks (response < LOD). A system suitability solution containing 10 ng / mL of each target was then injected to verify: 1. retention time RSD < 1%; 2. peak area RSD < 5%; 3. resolution > 1.5. After passing, actual samples could be detected.

[0060] Blank sample monitoring laboratory background contamination; system suitability ensures stable instrument state.

[0061] Step five: Standard curve establishment and sample detection Step 5.1: Standard preparation and internal standard addition This example requires that all standards must be the same batch, high purity (≥98%), solid powder with NMR and HPLC purity certificates (such as Cerilliant or Sigma-Aldrich certified products), and the stock solution (1.0 mg / mL) must be prepared with methanol (LCMS grade), divided into brown glass bottles, stored at -80°C in the dark, and valid for 6 months. Before use, it needs to be warmed to room temperature and vortexed for 30 seconds.

[0062] The standard curve concentration points are designed as 11 points: 0.1, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 20.0, 50.0, 75.0, 100.0 ng / mL, covering the expected concentration range of environmental samples (0.5-50 ng / swab). Take 450 μL of standard solution for each concentration point, add 50 μL of mixed internal standard working solution (containing 13 deuterated internal standards, each 100 ng / mL, solvent is methanol:water=1:1), and finally constant volume is 500 μL, so that the final concentration of internal standard is constant 10 ng / mL (not original 20 ng / mL, after optimization, the concentration of internal standard is reduced to avoid ion suppression). Vortex for 15 seconds, stand for 5 minutes.

[0063] Step 5.2: Standard curve injection and data acquisition The injection sequence strictly follows the "from low to high" principle: blank-0.1-0.5-1.0-…-100.0 ng / mL-blank (needle washing). Each concentration point is injected 3 times, and the last 2 are averaged (the first one is discarded to eliminate possible residues). The injection volume is 2 μL, and the "needle washing program" is used: before injection, wash the outer wall of the injection needle with 90% methanol water for 3 times, and after injection, wash the inner wall with 100% methanol for 5 times.

[0064] Key pollution prevention measures: 1. Injection needle residue monitoring: After the highest concentration (100 ng / mL) is injected, inject blank solvent, if the target response is >10% of LOD, perform "deep cleaning" (wash with 50% isopropyl alcohol:water for 10 times).

[0065] 2. Column equilibration: Before each batch of samples, equilibrate the column with the initial mobile phase (20%B) for 15 minutes, and monitor the baseline noise <1 mV.

[0066] 3. System pressure monitoring: Record the column pressure in real time, if the fluctuation is >±50 psi, pause the injection, check the filter or replace the guard column.

[0067] The average of the last two injections was taken because the first injection is often affected by residual samples from the previous injection or the initial state of the injection needle. Data shows that the RSD for the first injection was 8.7%, while the RSD for the next two injections decreased to 3.2%.

[0068] Traditional methanol flushing only removes 70% of lipid-soluble substances (such as THC-COOH), while using isopropanol:water results in a removal rate >95%. In one instance of insufficient flushing, 0.3 ng / mL of cocaine was detected in a subsequent blank sample. After 100 consecutive injections, the column pressure increased from 2100 psi to 2350 psi (an increase of 12%), but remained within the safe range (<3000 psi), demonstrating the robustness of the method.

[0069] Table 3: Data Record Table Step 5.3: Linear Regression and Quality Control Point Validation Linear regression was performed using weighted least squares (WLS), with a weight factor of 1 / X. 2 (X represents concentration) to compensate for the large variability in response at low concentrations. Regression equation: Y = aX + b, where Y is the peak area of ​​the target analyte / the peak area of ​​the internal standard, and X is the concentration. Mandatory requirement: Correlation coefficient R ≥ 0.995 (non-R 2 (because R is more sensitive to low concentrations). The 95% confidence interval of intercept b must include 0 (to prove there is no systematic bias). The absolute value of the residual (measured Y - predicted Y) at each concentration point is <15%.

[0070] The quality control points (QC-Mid: 5 ng / mL, QC-High: 50 ng / mL) must meet the following requirements: The measured concentration was within ±15% of the nominal value; The response value RSD is less than 10% (n=3).

[0071] If it fails, proceed with a "three-level investigation": 1. Re-enter the quality control point to confirm whether it is a random error; 2. Re-prepare the standard curve and check the preparation process; 3. Check the instrument status (ion source, pump, chromatographic column).

[0072] The response variation of low concentration points (0.1-1 ng / mL) was large (RSD 10-20%) and the variation of high concentration points was small (RSD 3-5%) when using WLS instead of OLS. WLS gave more weight to high concentration points, making the curve more accurate in the key high concentration area. Comparative data: the deviation of OLS method at 0.1 ng / mL point was as high as -25%, while the deviation of WLS method was only -8%.

[0073] The meaning of the intercept confidence interval containing 0: if the intercept is significantly not 0, it means that there is a systematic error (such as background pollution or internal standard mismatch). The intercept b = 0.05 (95% CI: 0.03-0.07) of a batch, and the internal standard bottle was found to be contaminated with methanol, and after replacement, b = 0.002 (CI: -0.01-0.01).

[0074] The average R of 100 curve fitting of 13 compounds was 0.9982 by WLS method and 0.9931 by OLS method (p < 0.001).

[0075] Table 4: statistical output representation (METH): Step 5.4: actual sample detection and dilution retest Before sample injection, a "pre-scan" was performed: a rough estimate of the concentration was made using a fast gradient (0-2 min, 20-95% B). If the response value exceeds 80% of the highest point of the standard curve (100 ng / mL), "intelligent dilution" is automatically triggered: 1. Take 100 μL of the original extract and dilute it to 1000 μL (10 times) with 20% methanol water; 2. Add internal standard again (add 50 μL of 100 ng / mL internal standard solution, and dilute to 500 μL); 3. Re-inject, and multiply the result by the dilution factor.

[0076] All sample data must be bound to the "blind sample code" in the LIMS system to generate an unalterable electronic record, including: injection time, operator, instrument serial number, original data file path. If the sample response is abnormal (such as peak distortion, retention time drift > 0.1 min), the system automatically marks "to be reviewed" and is reviewed by a senior analyst.

[0077] The meaning of pre-scan: to avoid detector saturation (>1 x 10 6 cps linear distortion) or column overload caused by direct injection of high concentration samples.

[0078] The accuracy of intelligent dilution: traditional "visual dilution" is prone to error, and this scheme quantitatively estimates the dilution factor through pre-scan, with an error of <5%.

[0079] Table 5: Smart dilution decision table Step six: Data qualification and quantitative calculation Step 6.1: Retention time alignment and deviation calculation In addition to the calculation of relative deviation (≤2.5%), a "peak purity check" is added: 1. Extract the "extracted ion chromatogram" (XIC) of the target peak using mass spectrometry software (e.g. Analyst®) to check if it is a single peak (no shoulder or bifurcation); 2. Calculate the "peak symmetry factor" (As): (As = (Rt+10%*Rt) - (Rt-10%*Rt) where Rt is the retention time, Rt+10%*Rt is the front half width, Rt-10%*Rt is the back half width, and 0.8 < As < 1.5 is required.

[0080] 3. If As < 0.8 or > 1.5, or the XIC shows multiple peaks, mark "co-elution interference" and further confirm using "library matching of secondary mass spectra" (e.g. NIST or self-built library).

[0081] Necessity of peak purity check: Environmental sample matrix is complex, and co-elution substances (e.g. cleaning agents, cosmetic ingredients) may have similar RT but different mass spectra to the target. A sample had METH RT deviation of only 1.2%, but XIC showed double peaks, which was later confirmed to be surfactant interference.

[0082] Significance of peak symmetry factor: As < 0.8 indicates column head collapse or sample overload, and As > 1.5 indicates column efficiency decline. Data: Quantitative error of abnormal samples with As is 3 times higher than that of normal samples (25% vs 8%).

[0083] Peak purity check was performed on 500 samples, and 12 (2.4%) were marked due to co-elution, of which 8 were excluded as false positives by library matching.

[0084] Table 6: Peak purity check examples Step 6.2: Ion abundance ratio verification and threshold determination For each target, at least two ion pairs are monitored (one for quantification and one or two for qualification). Calculate the relative abundance of the qualitative ion Ksam and compare it with the standard Kstd on the same day. Introduce the concept of "statistical tolerance": allow deviation not only according to the fixed value in Table 4, but also consider measurement uncertainty.

[0085] Calculation: Allowable deviation = Table 4 threshold + k x u(Ksam); Where u(Ksam) is the standard uncertainty of Ksam (calculated by 6 replicates), k is the coverage factor (k = 2, confidence level 95%).

[0086] For example, METH qualitative ion m / z 119.2, Kstd = 65%, u(Ksam) = 3.5%, then allowable deviation = 20% + 2 x 3.5% = 27%. If Ksam = 58% (deviation 10.8%), within the tolerance, the determination is passed.

[0087] The meaning of statistical tolerance: a fixed threshold (such as 20%) is too strict at low repeatability, which may misjudge true positives. The introduction of uncertainty is more scientific.

[0088] Multiple ion pairs for confirmation: for key samples, require 2 qualitative ion pairs to pass (such as m / z 211.1 and 165.2), reduce the risk of misjudgment.

[0089] For 1000 times of abundance ratio test of 13 kinds of compounds, after introducing statistical tolerance, the confirmation pass rate is increased from 89.3% to 98.7%, and there is no false positive increase.

[0090] Table 7: Ion abundance confirmation table (METH example): Step 6.3: Absolute mass calculation and unit conversion Calculate the mass of the target on the swab m (ng): m = (p1 x f x V) / V1; Where p1 is the sample concentration (ng / mL), f is the dilution factor, V is the extraction volume (1.00 mL), and V1 is the constant volume (0.500 mL).

[0091] Introduce "measurement uncertainty evaluation": 1. Type A uncertainty: calculate the standard deviation s(p1) by 6 replicates; 2. Type B uncertainty: from instrument calibration (±2%), pipette accuracy (±1%), volume measurement (±0.5%); 3. Combined uncertainty: u(m) = m x √[(s(p1) / p1) 2 + (2%) 2 + (1%) 2 + (0.5%) 2 ]; 4. Expanded uncertainty: U(m) = k x u(m) (k = 2).

[0092] The result is reported as: m ± U(m) ng, and "risk classification" is made according to the mass: Low risk: <5 ng / swab; Medium risk: 5-20 ng / swab; High risk: >20 ng / swab.

[0093] Data support: 6 replicates on the same swab, METH mass = 16.4 ng, s = 0.8 ng, u(m) = 16.4 x sqrt[(0.8 / 16.4) 2 + 0.0004 + 0.0001 + 0.000025] = 0.92 ng, U(m) = 1.84 ng. Report: 16.4 ± 1.8 ng (high risk).

[0094] Table 8: Uncertainty evaluation table: Step 6.4: Blank deduction and result reporting Blank sample response must be deducted: if the blank detects the target (such as 0.1 ng / mL METH), all sample concentrations are reduced by this value. Report generation uses an "automated template" that includes: Sample information (blind code, sampling point, date); Test results (compound, mass ± uncertainty, risk level); Method parameters (LOD, LOQ, recovery rate); Quality control data (blank, QC point, curve R value); Audit electronic signature (two people).

[0095] Introduce "blockchain notarization": upload the report hash value to the judicial alliance chain to ensure that the data cannot be tampered with. Scanning the QR code can verify the authenticity of the report.

[0096] Table 9: Core fields of automated report: Step seven: quality control and method validation Step 7.1: Spiked recovery experiment Spiked on 6 typical substrate surfaces: stainless steel (door handle), wood (door frame), ceramic tile (floor), plastic (switch), glass (mirror), fabric (sofa). Each substrate is spiked at 3 levels (low: 1 ng, medium: 10 ng, high: 50 ng), with 6 replicates for each level. Calculate the recovery rate and RSD.

[0097] Conduct "long-term stability test" at the same time: spiked samples are detected after 0, 24, 48, and 72 hours of storage at 4°C to evaluate the degradation rate.

[0098] Influence of matrix difference: fabric has lower recovery due to porous structure (75-85%), stainless steel is the highest (95-105%). Matrix type should be noted in the report.

[0099] Stability data guide the transportation time limit: morphine degrades 15% in 72 hours, so the transportation is limited to ≤24 hours. Data: 48 hours vs 24 hours, morphine recovery rate 92.1% vs 98.5% (p<0.05).

[0100] Table 10: Data support table (METH recovery, n=6): Step 7.2: Precision test (intra-day / inter-day) In addition to intra-day (n=6), inter-day (n=3 days) precision, increase: 1. Multi-laboratory comparison: 3 laboratories simultaneously detect the same batch of samples (10 ng / mL), calculate the inter-laboratory RSD (requirement <15%); 2. Robustness test: slight change in parameters (such as ultrasonic time ±2 min, methanol concentration ±2%), evaluate the change in results (requirement deviation <10%).

[0101] Significance of multi-laboratory comparison: to prove the transferability of the method and meet the standardization requirements (such as GA / T standard).

[0102] Value of robustness test: to ensure that slight fluctuations in daily operation do not affect the results.

[0103] Table 11: Robustness test table: Step 7.3: Determination of limit of detection (LOD) and limit of quantification (LOQ) LOD and LOQ must be determined under "matrix matching" conditions: low concentration standards are prepared with blank matrix (such as no standard door handle extract) to avoid solvent effect underestimating difficulty.

[0104] Calculation method: LOD=3.3×σ / S (σ is the standard deviation of the response of low concentration samples, S is the slope of the curve); LOQ=10×σ / S.

[0105] Optimize "signal-to-noise ratio acquisition parameters" at the same time: adjust the mass spectrometry dwell time (5-10 ms), collision energy (CE ±2 V), so that the LOD is reduced by 20-30%.

[0106] Necessity of matrix matching: LOD in solvent may be 50% lower than in matrix, leading to false positives.

[0107] Residence time optimization: prolonging residence time improves sensitivity but reduces the number of compounds that can be monitored. This method monitors 13 compounds in 5 min, with 5 ms residence time as the optimal balance point.

[0108] Table 12: Data support table: Step 7.4: Method comparison and continuous improvement Compare 200 samples with methods such as GC-MS, ELISA, etc. Calculate sensitivity, specificity, and compliance rate. Introduce "AI-assisted parameter optimization": use machine learning algorithms (such as random forest) to analyze historical data and recommend optimal ultrasonic time, methanol concentration, etc.

[0109] Establish "annual review mechanism": every year, the technical committee reviews the method, and updates the target list and MRM parameters according to new samples.

[0110] Efficiency of AI optimization: traditional "trial and error" method takes 2 weeks to optimize one parameter, while AI analysis of historical data gives the optimal solution in 3 days.

[0111] Prospective of annual review: in 2024, a new fentanyl MRM channel (m / z 337.2-105.1) will be added, and in XXX, the detection rate will be improved by 12%.

[0112] Table 13: Method comparison data table: This embodiment improves the efficiency of sample detection from the weighted regression of standard curve and intelligent dilution, to the statistical tolerance and uncertainty evaluation of qualitative confirmation, to the multi-laboratory comparison and AI optimization of method validation.

[0113] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features, as long as they are within the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the scope of the present application should be included in the protection scope of the present application.

Claims

1. An environmental sample collection and detection method, comprising: The method comprises the following steps: According to the detection task, a sampling plan is formulated, and independent packaged sampling swabs, methanol solution and blank quality control samples are prepared, wherein the sampling plan specifies that the sampling points are door handles, door gaps or floor in front of the door; After the target surface is dipped in the prepared sampling swab and methanol solution, the sampling swab is rubbed at least three times in one direction and rotated, then the cotton swab head is broken and sealed in a sealed bag, and information is marked; The sealed bag after marking is placed in a special clean goods transfer box and a cooling ice bag is configured for low-temperature transportation, to ensure that the transportation temperature is ≤4℃ and the room temperature is placed for not more than 24 hours; After the sample transported to the laboratory is thawed, the cotton swab head is taken out and placed in a plastic centrifuge tube, methanol solution is added for ice bath ultrasonic, and after ultrasonic is completed, it is vortexed for 3 to 5 seconds for mixing; 450μL of the extraction solution is filtered, 50μL of the internal standard solution is added, and the volume is made up to 500μL, a liquid chromatography-tandem mass spectrometry system equipped with a C18 reversed-phase chromatographic column is used, a gradient elution program of 0.1 minutes to 3.0 minutes in positive ion mode and 3.0 minutes to 5.0 minutes in negative ion mode is performed, and multiple ion monitoring is performed; The target substance is confirmed according to the retention time deviation ≤2.5% and the ion abundance ratio allowable deviation threshold, and the mass of the target substance on the swab head is calculated by the internal standard method.

2. The method of claim 1, wherein, The formulation of the sampling plan comprises: At least two sampling personnel are assigned to work in cooperation, the sampling time is set to be in the morning or during a period of low human flow, the sampling area is divided according to floors, three samples of door handles, door gaps and floor in front of the door are collected for each household, and blank quality control samples are prepared synchronously, the sampling frequency is set according to the risk level as single or periodic re-sampling, all point information and numbering rules are pre-recorded in the sampling record table and correspond to the subsequent sealed bag marking content.

3. The method of claim 1, wherein the method further comprises: The preparation of independent packaged sampling swabs, methanol solution and blank quality control samples comprises: The sampling swab is selected from a defatted medical cotton wood handle sterilized by ethylene oxide, HPLC grade methanol is packaged in a brown glass bottle, a sterile clean sealed bag, disposable butyronitrile gloves, an N95 mask and an oily marker pen are provided, and the solvent blank detection is completed by the laboratory before sampling, and it is confirmed that the benzene content is <0.1ppb and the phthalate content is <0.5ppb before it can be used, and the blank quality control sample is prepared by directly sealing the methanol taken by the swab from the same batch.

4. The method of claim 1, wherein, The sealing and marking after rubbing the sampling swab comprises: The sampling personnel wear gloves, hold the swab tip, dip the methanol solution only once and soak the cotton head, rub the target surface at least three times in one direction in a "Z" shape path, rotate the swab to ensure 360° contact, immediately break the cotton swab head after completion, leave the wood rod outside the bag, clearly mark the sample number, sampling point and date on the surface of the sealed bag with an oily pen, the marking content needs to be checked in real time with the sampling record table to ensure consistency, and the hands or other parts of the body are prohibited from touching the sampling position or the cotton head throughout the process.

5. The method of claim 1, wherein the method further comprises: The low-temperature transportation comprises: The sealed bag after marking is placed vertically in the goods transfer box with the cotton head downward, a cooling ice bag pre-frozen to-20℃ is placed between each layer of samples, a wireless temperature and humidity recorder is configured in the box for real-time monitoring, and an automatic alarm is triggered if the box temperature is >8℃ or the humidity is >80%RH.

6. The method of claim 1, wherein, In the process of ultrasonic and vortex extraction, In a biological safety cabinet, the cotton head was poured into a 10 mL polypropylene centrifuge tube after the wooden rod was broken from the outside of the sealed bag with tweezers and the bag corners were cut. 1.00 mL of 20% methanol aqueous solution was added to completely cover the cotton head, the tube cap was tightly covered, and then placed in an ice water bath ultrasonic cleaner for 15 minutes with a program setting of 5 minutes of ultrasonic and 1 minute of pause. After ultrasonic completion, the adsorbed substance was fully desorbed by vortex oscillation at 3000 rpm for 30 seconds, and then centrifuged at 4000 rpm for 5 minutes to take the supernatant for subsequent filtration operation.

7. The method of claim 1, wherein the method further comprises: The multiple ion monitoring includes: The ultra-high performance liquid chromatography flow rate was set to 0.3 mL / min, the column temperature was 35°C, the mobile phase A was 30 mM ammonium formate and 0.1% formic acid aqueous solution, the mobile phase B was HPLC grade methanol, the gradient elution program was 20% B phase at 0.1 minutes, increased to 80% at 1.5 minutes, increased to 95% at 2.25 minutes, switched to negative ion mode at 3.0 minutes and maintained 95% B phase to 3.1 minutes, then B phase decreased to 20% and stopped at 5.0 minutes; the mass spectrometry was set to ESI ion source voltage 5500V in positive ion mode and -4500V in negative ion mode, the acquisition type was multiple ion monitoring mode and the residence time was 5 mSec, and the setting of target compound monitoring parameters included: the first column of each substance was the quantitative ion pair.

8. The method of claim 1, wherein, The verification of the ion abundance ratio allowed deviation threshold includes: The relative abundance of the qualitative daughter ion in the sample was calculated as Ksam=(A2 / A1)×100%, where A2 was the peak area of the qualitative daughter ion and A1 was the peak area of the quantitative daughter ion; The relative abundance of the qualitative daughter ion in the standard sample was calculated as Kstd=(Astd2 / Astd1)×100%, where Astd2 was the peak area of the qualitative daughter ion in the standard sample and Astd1 was the peak area of the quantitative daughter ion in the standard sample; The deviation of Ksam and Kstd was compared, and when Kstd>50%, the maximum allowed deviation was 20%, when 20%<Kstd≤50%, the maximum allowed deviation was 25%, when 10%<Kstd≤20%, the maximum allowed deviation was 30%, and when Kstd≤10%, the maximum allowed deviation was 50%, and at the same time, the relative deviation absolute value of the target retention time was required to be less than 2.5% to determine the presence of the corresponding target.

9. The method of claim 1, wherein, The blank sample response value was deducted before the mass was calculated by the internal standard method, that is, if the target concentration was detected in the blank sample, the concentration results of all samples were reduced by this value; The standard curve was established by weighted least squares fitting with a correlation coefficient of ≥0.995, and the quality control points included 5 ng / mL and 50 ng / mL concentration points, and the measured values must be within ±15% of the nominal value; if the sample response value exceeded 80% of the highest point of the standard curve 100 ng / mL, dilution was triggered, 100 μL of the original extract was diluted to 1000 μL, then the internal standard was added to the constant volume and re-injected, and the result was multiplied by the dilution factor; The final report includes the mass of the target, the expanded uncertainty, the risk level, and the blockchain storage number.

10. The method of claim 1, wherein, The internal standard method calculates the mass formula as: m=(p1xfxV) / V1, wherein m is the target mass on the wiping head, p1 is the target concentration in the sample obtained from the standard curve, f is the dilution multiple, V is the sample extraction volume 1 mL, and V1 is the constant volume 0.5 mL.

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