An environmental sample collection and detection method

Abstract

The application discloses an environmental sample collection and detection method, aiming at the problems of loose process and poor data reliability in the prior art, and constructs a full-process standardized operation system: a sampling plan is formulated according to a task, point positions such as door handles and door gaps are defined, and sterilized swabs and blank quality controls are matched; during sampling, one-way smearing is forced, and the swab is rotated to ensure uniform extraction; the sample is transported by temperature control in a low-temperature transport box to maintain chemical stability; a fixed-volume methanol aqueous solution ice bath ultrasonic combined with vortex extraction is used in the laboratory to improve efficiency and reduce errors; an internal standard is added before sample injection to correct instrument and matrix fluctuations; liquid chromatography-tandem mass spectrometry adopts positive and negative ion segmentation mode and multiple ion monitoring, and the target object is confirmed through double threshold values of retention time and ion abundance ratio. The method significantly improves detection accuracy, repeatability and legal adoptability.

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: using ordinary cotton swab to dip ethanol or aqueous solution for random wiping, 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 number of solvent dipping, wiping direction and intensity 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 purpose, the technical scheme adopted by the present application is as follows: an environmental sample collection and detection method, comprising:

[0006] 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 clearly specifies the sampling points as door handle, door gap or floor in front of the door;

[0007] After using the prepared sampling swab to dip the methanol solution on the target surface, wipe at least three times in one direction and rotate, then break off the cotton swab head and seal it in a sealed bag and mark the information;

[0008] Place the marked sealed bag in a special clean transport box and configure a cooling ice bag for low-temperature transportation, ensure that the transportation temperature is ≤4℃ and the room temperature is placed for not more than 24 hours;

[0009] After the sample arrived by transportation was thawed in the laboratory, the cotton swab head was taken out and placed in a plastic centrifuge tube, and methanol solution was added for ice bath ultrasonic. After ultrasonic was completed, vortex for 3 to 5 seconds to mix evenly;

[0010] After 450 μL of the extraction solution was filtered, 50 μL of the internal standard solution was added to make up to 500 μL. 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 minutes to 3.0 minutes in positive ion mode and 3.0 minutes to 5.0 minutes in negative ion mode.

[0011] The target substance was confirmed according to the retention time deviation ≤2.5% and the ion abundance ratio allowed deviation threshold. The mass of the target substance on the swab head was calculated by the internal standard method.

[0012] Preferably, the sampling plan includes: at least two sampling personnel are assigned to work in cooperation, the sampling time is set to be in the early morning or during a period of low human flow, the sampling area is divided according to floor groups, three samples of door handle, door gap and floor in front of the door are collected from each household, and a blank quality control sample is prepared synchronously, the sampling frequency is set to be single or periodic according to the risk level, and all point information and numbering rules are pre-recorded in the sampling record table and correspond to the subsequent sealed bag labeling content.

[0013] Preferably, the preparation of independently packaged sampling swabs, methanol solution and blank quality control samples includes: sampling swabs made of defatted medical cotton with epoxyethane sterilization are selected, HPLC grade methanol is sub-packed in brown glass bottles, sterile clean sealed bags, disposable butyronitrile gloves, N95 masks and oily marker pens are provided, and the solvent blank detection is completed by the laboratory before sampling, and it is confirmed 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 dipped with the swab from the same batch.

[0014] Preferably, after the sampling swab is applied, the sealed labeling includes: the sampling personnel wear gloves, hold the swab tip, dip the methanol solution only once and soak the cotton head, apply the swab on the target surface in a “Z” shape path in one direction for at least three times, rotate the swab to ensure 360° contact, immediately break the cotton swab head after completion to leave the wood rod 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 to ensure consistency with the sampling record table, and the hands or other parts of the body are prohibited to touch the sampling position or the cotton head throughout the process.

[0015] Preferably, the low-temperature transportation includes: the labeled sealed bag is placed vertically in the article transfer box with the cotton head facing down, and a pre-frozen to -20℃ 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 an automatic alarm is triggered if the box temperature >8℃ or the humidity >80%RH.

[0016] 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 then 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 after ultrasonic to fully desorb the adsorbate, and then centrifuging at 4000 rpm for 5 minutes to take the supernatant for subsequent filtration operation.

[0017] 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.

[0018] Preferably, the verification of the ion abundance ratio allowed deviation threshold includes: calculating the relative abundance Ksam of the qualitative sub-ion in the sample, (A2 / A1) x 100%, where A2 is the peak area of the qualitative sub-ion and A1 is the peak area of the quantitative sub-ion;

[0019] calculating the relative abundance Kstd of the qualitative sub-ion in the standard sample, (Astd2 / Astd1) x 100%, where Astd2 is the peak area of the qualitative sub-ion in the standard sample and Astd1 is the peak area of the quantitative sub-ion in the standard sample;

[0020] 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.

[0021] 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;

[0022] 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 to the constant volume, and then re-injection is performed, and the result is multiplied by the dilution factor;

[0023] The final report includes target mass, expanded uncertainty, risk level and blockchain storage number.

[0024] Preferably, the internal standard method calculates the mass formula as: m=(ρ1×f×V) / V1, wherein m is the target mass on the wiping head, ρ1 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.

[0025] The technical effects and advantages of the present application: the environmental sample collection and detection method provided by the present application has the following advantages compared with the prior art:

[0026] 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 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 segmented 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

[0027] Figure 1 The flow chart of the environmental sample collection and detection method of the present application. DETAILED DESCRIPTION

[0028] 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.

[0029] 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

[0030] 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.

[0031] Preparation of independently packaged sampling swabs, methanol solution and blank quality control samples includes: selecting sampling swabs made of degreased 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.

[0032] 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 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.

[0033] 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 with the cotton head downward, and 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 >8°C or the humidity >80%RH, an automatic alarm is generated.

[0034] 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;

[0035] The ultrasonic and vortex extraction process includes: in the biological safety cabinet, the wooden rod is broken from the outside of the sealed bag with tweezers, the bag corner is cut open, 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 placed in a 40kHz ultrasonic cleaner for ice water bath ultrasonic 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 centrifuged at 4000rpm for 5 minutes to take the supernatant for subsequent filtration operation.

[0036] 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 decreasing 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 5mSec, and the setting of the target compound monitoring parameters includes: the first column of each substance is the quantitative ion pair.

[0037] 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.

[0038] The confirmation of the ion abundance ratio allowable deviation threshold includes:

[0039] The relative abundance of the qualitative sub-ion in the sample is calculated as Ksam=(A2 / A1) x 100%, wherein A2 is the peak area of the qualitative sub-ion, and A1 is the peak area of the quantitative sub-ion.

[0040] The relative abundance of the qualitative sub-ion in the standard sample is calculated as Kstd=(Astd2 / Astd1) x 100%, wherein Astd2 is the peak area of the qualitative sub-ion in the standard sample, and Astd1 is the peak area of the quantitative sub-ion in the standard sample.

[0041] The deviation of Ksam and Kstd is compared, and when Kstd>50%, the maximum deviation is allowed to be 20%, when 20%<Kstd≤50%, the maximum deviation is allowed to be 25%, when 10%<Kstd≤20%, the maximum deviation is allowed to be 30%, and when Kstd≤10%, the maximum deviation is allowed to be 50%, and at the same time, the absolute value of the relative deviation of the target substance is required to be less than 2.5% to determine that the corresponding target substance exists.

[0042] 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 the value;

[0043] The standard curve needs to be fitted by the weighted least squares method and the correlation coefficient is greater than or equal to 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 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 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.

[0044] Specifically, the mass calculation formula of the internal standard method is: m=(ρ1 x f x V) / V1, wherein m is the mass of the target substance on the wiping head, ρ1 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.

[0045] 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 a fixed volume of 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.

[0046] 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:

[0047] Step one: sampling scheme design and material preparation

[0048] 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.

[0049] Step 1.1: Develop a sampling plan and divide the area

[0050] The plan needs to clarify the division of labor of sampling personnel (at least 2 people in a group, 1 person sampling, 1 person recording), sampling time (it is recommended to be in the early morning or during the off-peak period to reduce interference), sampling points (such as the door handle, door gap, and door tile in front of the door of 502 rooms in 3 buildings in a certain community), sampling frequency (single sampling for initial investigation, and re-sampling every week for key areas), and sample quantity (at least 3 swab samples per household + 1 blank control). Area division needs to be combined with building structure, such as grouping by floor in high-rise residential buildings, and selecting 3-5 households per floor to avoid sample confusion. This plan will serve as a direct basis for the calculation of consumable quantity in "Step 1.2" and on-site operation in "Step 1.3".

[0051] Systematic sampling plan ensures the spatial representativeness and temporal comparability of samples. Stratified sampling (by floor, house type) can improve the detection rate in hot areas.

[0052] Step 1.2: Prepare standardized sampling consumables and quality control

[0053] 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 bag (10 cm x 15 cm, with self-sealing strip); 4. Blank quality control swab (unused swab of the same batch); 5. Disposable butyronitrile gloves, N95 mask, oil-based marker; 6. Cooling ice bag (-20°C pre-frozen), special transport box (lined with aluminum foil to prevent contamination).

[0054] 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".

[0055] Ethylene oxide sterilization avoids microbial degradation of target objects; wooden swab prevents plastic components (such as phthalates) from interfering with mass spectrometry; solvent blank test intercepts batch contamination, avoiding the waste of the entire batch of samples.

[0056] Step 1.3: On-site sampling operation specification

[0057] 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 the swab at the cotton head end (wooden rod left 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 pre-treatment in "Step Three".

[0058] 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 in this method makes the recovery rate of methamphetamine stable at 92.4±3.1% (n=30).

[0059] Step 1.4: Sample identification, temporary storage, and transportation handover

[0060] Immediately after sampling, check the label information (number, site, time) 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 the transportation time 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 the form. 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".

[0061] 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.

[0062] Step Two: Sample Transportation and Laboratory Reception

[0063] 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 samples are maintained stable during transportation, providing "original state" samples for subsequent high-precision analysis.

[0064] Step 2.1: Real-time monitoring of temperature and humidity during transportation

[0065] During the 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".

[0066] Step 2.2: Laboratory Reception and Preliminary Sorting

[0067] The laboratory reception personnel check the "Sample Handover Form" and temperature and humidity records. If the temperature exceeds the standard or the label is unclear, 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.

[0068] Step 2.3: Sample Thawing and Appearance Inspection

[0069] 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 legible? Qualified samples are transferred to a biological safety 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.

[0070] The biological safety cabinet provides a sterile environment to prevent laboratory air pollutants (such as phthalates in dust) from interfering.

[0071] Step 2.4: Sample pre-registration and blind sample coding

[0072] In the LIMS system, a "blind sample code" (such as BLXX-XX-001) is generated for each sample, hiding the original information (address, homeowner), and only retaining the parameters required for detection (sampling point type, receipt date). Print the blind sample label and attach it to 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.

[0073] Step Three: Sample pre-treatment and extraction

[0074] This step is the core of the detection sensitivity, through the standardized extraction process to efficiently release and enrich trace target substances from complex matrix (cotton fiber, dust, sebum), while removing interferents, providing "clean" on-machine liquid for instrument analysis.

[0075] Step 3.1: Cotton head transfer and anti-pollution operation

[0076] In the biological safety 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.

[0077] Polypropylene tubes have an adsorption rate of <2% for target substances (vs. 5-8% for glass tubes) and are resistant to organic solvents.

[0078] Step 3.2: Solvent extraction and ultrasonic enhancement

[0079] 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".

[0080] 20% methanol in water balances the polarity between polar and non-polar (lipid-soluble) components; ultrasonic cavitation disrupts the cotton fiber-target binding;

[0081] Table 1: Ice bath prevents thermal degradation, comparative data:

[0082]

[0083] Step 3.3: Centrifugal filtration and supernatant transfer

[0084] 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".

[0085] Centrifugation removes cotton fiber debris; 0.22 μm filtration traps >0.22 μm particles (such as dust, bacteria), preventing column blockage.

[0086] Step 3.4: Internal standard addition and constant volume for instrument analysis

[0087] 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 constant volume to 500 μL with 20% methanol in water, vortex for 10 seconds to mix. Cap with an aluminum cap, 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.

[0088] 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.

[0089] Step Four: Instrument analysis condition setting and calibration

[0090] 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.

[0091] Step 4.1: Chromatographic column and mobile phase configuration

[0092] A C18 reversed-phase chromatographic column (50 mm x 2.1 mm, 1.7 pm) 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 with the following gradient (flow rate 0.3 mL / min):

[0093] Table 2: Gradient elution program:

[0094]

[0095] This gradient separates polar (e.g. BE) and non-polar (e.g. THC-COOH) compounds in the injection from Step 3.4.

[0096] Step 4.2: Mass spectrometry parameter optimization and MRM setup

[0097] 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.

[0098] MRM enhances selectivity (excludes isomer interference); optimized CE value maximizes fragment ion yield.

[0099] Step 4.3: Mass axis calibration and sensitivity verification

[0100] After daily startup, 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. Calibration data were archived as a prerequisite for the validity of the results in Step 4.4.

[0101] Mass axis deviation caused MRM channel failure (e.g. target m / z 150.1 shifted to 150.3, response went to zero). Un-calibration once caused all samples to be false negatives, and the detection rate returned to normal after calibration.

[0102] Step 4.4: Blank sample and system suitability test

[0103] Injection of blank sample prepared in "Step 3.4" (solvent + internal standard) to confirm no target peaks (response < LOD). Injection of system suitability solution (containing 10 ng / mL each target) to verify: 1. retention time RSD < 1%; 2. peak area RSD < 5%; 3. resolution > 1.5. Actual samples can be injected after.

[0104] Blank sample to monitor laboratory background contamination; system suitability to ensure instrument stability.

[0105] Step Five: Standard Curve Establishment and Sample Detection

[0106] Step 5.1: Standard Gradient Preparation and Internal Standard Addition

[0107] 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 the expiration date is 6 months. Before use, it needs to be warmed to room temperature and vortexed for 30 seconds.

[0108] 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). 450 μL of standard solution was taken for each concentration point, 50 μL of mixed internal standard working solution (containing 13 deuterated internal standards, each 100 ng / mL, solvent methanol: water = 1:1) was added, and the final constant volume was 500 μL, so that the final concentration of internal standard was 10 ng / mL (not original 20 ng / mL, after optimization, the internal standard concentration was reduced to avoid ion suppression). Vortex for 15 seconds, and stand for 5 minutes.

[0109] Step 5.2: Standard Curve Injection and Data Collection

[0110] 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 needles are averaged (the first needle is discarded to eliminate possible residues). The injection volume is 2 μL, and the "needle washing program" is used: before injection, the injection needle is washed with 90% methanol water for 3 times, and after injection, the inner wall is washed with 100% methanol for 5 times.

[0111] Key Anti-pollution Measures:

[0112] 1. Needle residue monitoring: Inject blank solvent after the highest concentration (100 ng / mL). If the target analyte response is >10% of the LOD, perform "deep cleaning" (rinse 10 times with 50% isopropanol:water).

[0113] 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 to be <1mV.

[0114] 3. System pressure monitoring: Record column pressure in real time. If the fluctuation is > ±50 psi, pause the injection and check the filter or replace the guard column.

[0115] 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%.

[0116] 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.

[0117] Table 3: Data Record Table

[0118]

[0119] Step 5.3: Linear Regression and Quality Control Point Validation

[0120] 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:

[0121] Correlation coefficient R ≥ 0.995 (non-R 2 (because R is more sensitive to low concentrations).

[0122] The 95% confidence interval of intercept b must include 0 (to prove there is no systematic bias).

[0123] The absolute value of the residual (measured Y - predicted Y) at each concentration point is <15%.

[0124] The quality control points (QC-Mid: 5 ng / mL, QC-High: 50 ng / mL) must meet the following requirements:

[0125] Measured concentration within ±15% of nominal value;

[0126] Response value RSD <10% (n=3).

[0127] If failed, perform "three-level troubleshooting":

[0128] 1. Re-enter quality control point, confirm whether it is accidental error;

[0129] 2. Reconfigure standard curve, check the preparation process;

[0130] 3. Check the instrument state (ion source, pump, chromatographic column).

[0131] When using WLS instead of ordinary least squares, the response variation of low concentration points (0.1-1 ng / mL) of environmental samples is large (RSD 10-20%), and the variation of high concentration points is small (RSD 3-5%). WLS gives more weight to high concentration points, making the curve more accurate in the key high concentration area. Comparison data: OLS method deviates by-25% at 0.1 ng / mL, and WLS method only by-8%.

[0132] 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 after tracing, it was found that the internal standard bottle was contaminated by methanol, and after replacement, b=0.002 (CI:-0.01-0.01).

[0133] For 13 kinds of compounds, 100 times of curve fitting, the average R of WLS method is 0.9982, and the R of OLS method is 0.9931 (p<0.001).

[0134] Table 4: statistical output representation example (METH):

[0135]

[0136] Step 5.4: actual sample detection and dilution retest

[0137] Before the sample is injected, a "pre-scan" is performed: use a fast gradient (0-2 min, 20-95% B) to roughly estimate the concentration. If the response value exceeds 80% of the highest point of the standard curve (100 ng / mL), "intelligent dilution" is automatically triggered:

[0138] 1. Take 100 μL of the original extract, dilute to 1000 μL (10 times) with 20% methanol water;

[0139] 2. Add internal standard again (add 50 μL of 100 ng / mL internal standard solution, and dilute to 500 μL);

[0140] 3. Re-inject, result multiplied by dilution factor.

[0141] All sample data must be bound with "Blind Sample Code" in LIMS system, to generate an unalterable electronic record, containing: injection time, operator, instrument serial number, raw data file path. If sample response is abnormal (e.g. peak shape distortion, retention time drift >0.1 min), system will automatically mark "To be reviewed", and rechecked by senior analysts.

[0142] Purpose of pre-scan: avoid detector saturation (>1x10 6 cps linear distortion) or column overload caused by direct injection of high concentration sample.

[0143] Accuracy of intelligent dilution: traditional "visual dilution" is error-prone, this solution estimates dilution factor quantitatively through pre-scan, with error <5%.

[0144] Table 5: Intelligent dilution decision table:

[0145]

[0146] Step six: qualitative confirmation and quantitative calculation of data

[0147] Step 6.1: retention time comparison and deviation calculation

[0148] In addition to calculating the relative deviation (≤2.5%), "peak purity check" is added:

[0149] 1. Use mass spectrometry software (e.g. Analyst®) to extract the "extracted ion chromatogram" (XIC) of the target peak, check if it is a single peak (no shoulder peak or bifurcation);

[0150] 2. Calculate the "peak symmetry factor" (As): The front half width is 10% of the peak height, The back half width is 90% of the peak height), and the requirement is 0.8<As<1.5;

[0151] 3. If As<0.8 or >1.5, or XIC shows multiple peaks, mark "co-elution interference", and further confirmed by "secondary mass spectrum library matching" (e.g. NIST or self-built library).

[0152] Retention time correction: due to column aging, RT drift may occur, daily use "lock mass" (LockMass, e.g. m / z 609.3 of ristocetin) to correct the system time axis, to ensure the stability of RT.

[0153] ​Necessity of peak purity check: environmental sample matrix is complex, co-eluting substances (e.g. cleaning agents, cosmetic ingredients) might have similar RT but different MS to target. One sample METH RT deviation is only 1.2%, but XIC shows doublet, later confirmed as surfactant interference.

[0154] Significance of peak symmetry factor: As<0.8 suggests column collapse or sample overload, As>1.5 suggests column efficiency drop. Data: quantification error of abnormal samples is 3 times higher than normal samples (25% vs 8%).

[0155] Peak purity check was performed on 500 samples, 12 (2.4%) were flagged due to co-elution, 8 of which were excluded as false positive by library matching.

[0156] Table 6: peak purity check examples:

[0157]

[0158] Step 6.2: ion abundance ratio verification and threshold determination

[0159] For each target, at least 2 ion pairs were monitored (1 quantitative, 1-2 qualitative). The relative abundance of the qualitative ion Ksamwas calculated and compared with the standard Kstdof the day. The concept of “statistical tolerance” was introduced: the allowed deviation was not only the fixed value in Table 4, but also the measurement uncertainty.

[0160] Calculation formula: allowed deviation = Table 4 threshold + k x u(Ksam);

[0161] where u(Ksam) is the standard uncertainty of Ksam(calculated by 6 replicate determinations), k is the coverage factor (k=2, confidence level 95%).

[0162] For example, for METH qualitative ion m / z 119.2, Kstd=65%, u(Ksam)=3.5%, then allowed deviation = 20% + 2 x 3.5% = 27%. If Ksam=58% (deviation 10.8%), within tolerance, pass.

[0163] Significance of statistical tolerance: the fixed threshold (e.g. 20%) is too strict when the repeatability is low, which might misjudge true positives. The introduction of uncertainty is more scientific.

[0164] Multiple ion pair confirmation: for critical samples, both 2 qualitative ion pairs were required to pass (e.g. m / z 211.1 and 165.2), to reduce the risk of misjudgment.

[0165] For 1000 abundance ratio tests of 13 compounds, after the introduction of statistical tolerance, the confirmation pass rate increased from 89.3% to 98.7%, without false positive increase.

[0166] Table 7: Ion abundance confirmation table (METH example):

[0167]

[0168] Step 6.3: Absolute mass calculation and unit conversion

[0169] 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 final volume (0.500 mL).

[0170] Introduce the “measurement uncertainty evaluation”:

[0171] 1. Class A uncertainty: Calculate the standard deviation s(p1) by 6 replicate determinations;

[0172] 2. Class B uncertainty: From instrument calibration (±2%), pipette accuracy (±1%), volume measurement (±0.5%);

[0173] 3. Combined uncertainty: u(m) = m x V [(s(p1) / p1) 2 + 2% 2 + 1% 2 + 0.5% 2 ];

[0174] 4. Expanded uncertainty: U(m) = k x u(m) (k = 2).

[0175] Report the results as: m ± U(m) ng, and perform “risk classification” according to the mass:

[0176] Low risk: <5 ng / swab;

[0177] Medium risk: 5-20 ng / swab;

[0178] High risk: >20 ng / swab.

[0179] Data support: 6 replicate determinations on the same swab, METH mass = 16.4 ng, s = 0.8 ng, u(m) = 16.4 x V [(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).

[0180] Table 8: Uncertainty evaluation table:

[0181]

[0182] Step 6.4: Blank subtraction and result reporting

[0183] Blank sample response must be subtracted: if blank detects target (e.g. 0.1 ng / mL METH), then all sample concentrations minus this value. Report generation uses "automated template" containing:

[0184] Sample information (blind code, sampling point, date);

[0185] Detection results (compound, mass ± uncertainty, risk level);

[0186] Method parameters (LOD, LOQ, recovery);

[0187] Quality control data (blank, QC points, curve R value);

[0188] Audit electronic signature (two people).

[0189] Introduce "blockchain notarization": upload report hash value to judicial alliance chain to ensure data cannot be tampered with. Scan the QR code to verify the authenticity of the report.

[0190] Table 9: Core fields of automated report:

[0191]

[0192] Step seven: quality control and method validation

[0193] Step 7.1: Spiked recovery experiment

[0194] Spiked on 6 typical substrates: 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 recovery and RSD.

[0195] 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 degradation rate.

[0196] Effect of substrate difference: fabric has lower recovery rate due to porous structure (75-85%), while stainless steel has the highest recovery rate (95-105%). The type of substrate needs to be noted in the report.

[0197] Stability data guide transportation time limit: morphine degrades by 15% in 72 hours, so the transportation time is limited to ≤24 hours. Data: 48 hours vs 24 hours, morphine recovery rate 92.1% vs 98.5% (p<0.05).

[0198] Table 10: Data support table (METH recovery rate, n=6):

[0199]

[0200] Step 7.2: Precision test (intra-day / inter-day)

[0201] In addition to intra-day (n=6), inter-day (n=3 days) precision, increase:

[0202] 1. Multi-lab comparison: 3 labs test the same batch of samples (10 ng / mL) simultaneously, calculate inter-lab RSD (require <15%);

[0203] 2. Robustness test: slight change parameters (e.g. sonication time ±2 min, methanol concentration ±2%), evaluate result change (require bias <10%).

[0204] Significance of multi-lab comparison: prove the method transferability, meet the standardization requirements (e.g. GA / T standard).

[0205] Value of robustness test: ensure that slight fluctuations in daily operation do not affect the results.

[0206] Table 11: Robustness test table:

[0207]

[0208] Step 7.3: Determination of limit of detection (LOD) and limit of quantification (LOQ)

[0209] LOD and LOQ must be determined under "matrix matching" conditions: prepare low concentration standards with blank matrix (e.g. without adding standard door handle extract) to avoid solvent effect underestimating difficulty.

[0210] Calculation method: LOD = 3.3 x σ / S (σ is the standard deviation of the response of low concentration samples, S is the slope of the curve); LOQ = 10 x σ / S.

[0211] Optimize "signal-to-noise ratio acquisition parameters" at the same time: adjust the mass spectrometry dwell time (5-10 ms), collision energy (CE ±2V) to reduce LOD by 20-30%.

[0212] Necessity of matrix matching: LOD in solvent may be 50% lower than in matrix, leading to false positives.

[0213] Dwell time optimization: extending dwell time improves sensitivity, but reduces the number of compounds that can be monitored. This method monitors 13 compounds within 5 minutes, and 5 ms is the optimal balance point.

[0214] Table 12: Data support table:

[0215]

[0216] Step 7.4: Method comparison and continuous improvement

[0217] Compare 200 samples with 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.

[0218] Establish "annual review mechanism": every year, the technical committee reviews the method, updates the target list and MRM parameters based on new samples.

[0219] Efficiency of AI optimization: traditional "trial and error" method takes 2 weeks to optimize one parameter, while AI analyzes historical data and provides optimal solution in 3 days.

[0220] 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%.

[0221] Table 13: Method comparison data table

[0222]

[0223] 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.

[0224] Finally, it should be noted that the above description is only the preferred embodiment 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: Comprise: 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 clearly specifies the sampling points as 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, it is painted at least three times in one direction 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 a cooling ice bag is configured 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, and methanol solution is added for ice bath ultrasonic, then after ultrasonic is completed, vortex for 3 to 5 seconds for mixing; 450μL of the extracted 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, and 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 for multiple ion monitoring; 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; The ion abundance ratio allowable deviation threshold includes: The relative abundance of the qualitative sub-ion in the sample Ksam=(A2 / A1)×100% is calculated, wherein A2 is the peak area of the qualitative sub-ion, and A1 is the peak area of the quantitative sub-ion; the relative abundance of the qualitative sub-ion in the standard sample Kstd=(Astd2 / Astd1)×100% is calculated, wherein Astd2 is the peak area of the qualitative sub-ion of the standard sample, and Astd1 is the peak area of the quantitative sub-ion of the standard sample; the deviation of Ksam and Kstd is compared, when Kstd>50%, the maximum deviation is allowed to be 20%, when 20%<Kstd≤50%, the maximum deviation is allowed to be 25%, when 10%<Kstd≤20%, the maximum deviation is allowed to be 30%, when Kstd≤10%, the maximum deviation is allowed to be 50%, and at the same time, the relative deviation absolute value of the target substance retention time is required to be less than 2.5% to determine that the corresponding target substance exists.

2. The method of claim 1, wherein, The sampling plan includes: At least two sampling personnel are assigned to work together, the sampling time is set to 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 according to the risk level as single or periodic re-sampling, all point information and number rules are pre-recorded in the sampling record table and correspond to the subsequent sealed bag label content.

3. The method of claim 1, wherein The preparation of independent packaged sampling swabs, methanol solution and blank quality control samples includes: Selecting sampling swabs made of defatted medical cotton with epoxyethane sterilization, packaging HPLC grade methanol in brown glass bottles, providing sterile clean sealed bags, disposable nitrile gloves, N95 masks and oil-based markers, 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 The sample is directly sealed after the same batch of unused swabs are dipped in methanol.

4. The method of claim 1, wherein The sealed label includes: After wearing gloves, the sampler dips the swab tip in the methanol solution only once and soaks the cotton head, then uniformly applies it on the target surface in a "Z" shape path for at least three times, while rotating the swab to ensure 360° contact. After completion, immediately break the cotton tip to leave the wooden rod outside the bag, and clearly mark the sample number, sampling point and date on the surface of the sealed bag with an oily pen. The marked content needs to be checked with the sampling record table in real time to ensure consistency. Throughout the process, hands or other parts of the body are prohibited from touching the sampling position or the cotton head.

5. The method of claim 1, wherein, The low-temperature transportation includes: placing the labeled sealed bag vertically in the article transfer box with the cotton head facing down, and placing a pre-frozen to -20℃ cooling ice bag between each layer of samples. A wireless temperature and humidity recorder is configured in the box for real-time monitoring. If the box temperature is >8℃ or the humidity is >80%RH, an automatic alarm will be triggered.

6. The method of claim 1, wherein, The process of ultrasonic and vortex extraction includes: In the biological safety cabinet, the wooden rod is broken from the outside of the sealed bag with tweezers, the bag corners are cut open, and the cotton head is poured into a 10mL polypropylene centrifuge tube. 1.00mL of 20% methanol aqueous solution is added to completely cover the cotton head. After tightly covering the tube, it is placed in a 40kHz ultrasonic cleaner with ice water bath for 15 minutes, and the program is set to stop for 1 minute every 5 minutes of ultrasonic. After ultrasonic is completed, immediately vortex for 30 seconds at 3000rpm to fully desorb the adsorbed material. Then centrifuge at 4000rpm 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 is set to 0.3mL / min, the column temperature is 35℃, the mobile phase A is 30mM ammonium formate and 0.1% formic acid aqueous solution, the mobile phase B is HPLC grade methanol, the gradient elution program is 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, then B phase is reduced to 20% and stopped at 5.0 minutes; the mass spectrometry is set to ESI ion source voltage positive ion mode 5500V, negative ion mode -4500V, the acquisition type is multiple ion monitoring mode and the residence time is 5mSec, the target compound monitoring parameter setting includes: the first column of each substance is the quantitative ion pair.

8. The method of claim 1, wherein, The mass before the internal standard method calculation needs to deduct the response value of the blank sample, 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 establishment needs to use weighted least squares method fitting and the correlation coefficient is ≥0.995, the quality control points include 5ng / mL and 50ng / 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 100ng / mL of the standard curve, dilution is triggered, 100μL of the original extract is diluted to 1000μL, 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.

9. 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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