Method for simultaneously detecting dichloroacetic acid and trichloroacetic acid in urine

Abstract

The invention provides a method for simultaneously detecting dichloroacetic acid and trichloroacetic acid in urine, and belongs to the technical field of analysis detection and occupational health / environmental health. According to the method disclosed by the invention, a synergistic pretreatment strategy of acidic sample loading, WAX / reversed-phase mixed mode solid-phase extraction, water / low-proportion organic phase step-by-step leaching and ammonia-water-containing organic phase elution is adopted; a technical route of protein precipitation, acidification pH regulation, purification and enrichment by adopting WAX / reversed-phase mixed mode solid-phase extraction on a 96-well plate and stable isotope internal standard isotope dilution LC-MS / MS quantification is adopted, the method can effectively reduce the matrix effect and realize high-throughput analysis in a high-salinity urine matrix, the quantitation limits of DCEA and TCAA are not higher than 0.30 g / L and 0.20 g / L respectively, and the method has the advantages that the method is simple and convenient to operate, and the method is suitable for large-scale popularization and application. The method is suitable for biological monitoring and risk assessment of occupational and environmental low-level exposure crowds.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing and occupational health / environmental health technology, and particularly relates to a method for the simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine. Background Technology

[0002] Chlorinated solvents such as trichloroethylene (TCE) are widely used in industrial processes such as metal degreasing and cleaning, and chemical synthesis, posing potential exposure risks to both occupational and environmental populations. TCE can be metabolized in vivo to form haloacetic acid metabolites, of which dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) are excreted in urine. Therefore, urinary DCAA / TCAA is often used as a biomarker for assessing in vivo exposure. Existing methods for detecting urinary TCAA or related metabolites typically require derivatization (e.g., methylation) and organic solvent extraction, resulting in cumbersome pretreatment steps and unsuitability for large batches of samples. Some liquid chromatography-tandem mass spectrometry methods employ liquid-liquid extraction or simple dilution, which are easily affected by high-salt urinary matrices and endogenous acidic interfering substances, leading to significant matrix effects, unstable recovery, or high limits of quantitation.

[0003] Currently, published literature reports the use of LC-MS / MS with solid-phase extraction combined with stable isotope internal standards to determine disinfection byproduct metabolites such as trichloroacetic acid (TCAA) in urine, reflecting the basic technical approach of isotope dilution + solid-phase purification. Existing studies have used "solid-phase extraction + isotope dilution HPLC-MS / MS" to determine TCAA in urine, but this usually only targets TCAA and often employs single reversed-phase SPE purification. Other literature reports the determination of DCAA and TCAA after liquid-liquid extraction with organic solvents such as ether, but this suffers from problems such as large solvent consumption, easy emulsification, and difficulty in high-throughput automation for 96-well plates. However, there are still shortcomings in the simultaneous detection of DCAA and TCAA in urine, especially in high-salt urine matrices, to achieve low limits of quantitation, low matrix effects, and high-throughput sample pretreatment. There is an urgent need for a method for the simultaneous detection of DCAA and TCAA that does not require derivatization, allows for high-throughput pretreatment steps on a 96-well plate platform, and can stably achieve high recovery and low limits of quantitation. Summary of the Invention

[0004] Existing solid-phase extraction (SPE) plates mostly employ a single reversed-phase or single-ion exchange mechanism, which may lead to insufficient target analyte retention, co-elution interference, and batch-to-batch recovery fluctuations due to salt precipitation / plate blockage in complex high-salt matrices such as urine. This is especially true for small-molecule, highly polar haloacetic acids like DCAA and TCAA, where the single reversed-phase mechanism is more prone to penetration and co-elution interference in high-salt urine. Therefore, a pretreatment scheme suitable for high-throughput processing of 96-well plates and capable of maintaining stable purification effects in high-salt urine matrices is needed. To this end, this invention proposes a method for the simultaneous detection of dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in urine. This method requires no derivatization, is simple to operate, has high sensitivity, and is suitable for the detection of dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in large batches of urine samples, meeting the application needs of occupational and environmental low-level exposure biomonitoring.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for the simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine, comprising the following steps: (1) Take a urine sample and add a stable isotope internal standard mixed solution, wherein the stable isotope internal standard is dichloroacetic acid-D2 (DCAA-D2) and trichloroacetic acid-D2. 13 C2 (TCAA- 13 C2); (2) Add protein precipitant to the system obtained in step (1) to precipitate, centrifuge and take the supernatant, and adjust the pH of the supernatant to 1.8-3.0; (3) The supernatant obtained in step (2) is loaded into a 96-well plate solid-phase extraction device for purification and enrichment. Each well of the 96-well plate solid-phase extraction device is filled with weak anion exchange (WAX) packing material or a mixed-mode composite packing material composed of WAX and reversed-phase packing material. The solid-phase extraction process includes activation, equilibration, sample loading, rinsing, drying and elution in sequence. The rinsing is a stepwise rinsing, which includes at least water rinsing and low-proportion organic phase aqueous solution rinsing. The low-proportion organic phase aqueous solution rinsing is performed after water rinsing. The elution uses an organic solvent containing ammonia. (4) After concentrating the eluent obtained in step (3), add a redissolving solution to redissolve it; (5) The complex solution obtained in step (4) was used to detect DCAA, TCAA and their corresponding internal standards in the negative ion electrospray ionization mode by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and multiple reaction monitoring (MRM). The content of DCAA and TCAA in the urine sample was calculated based on the isotope dilution quantification principle.

[0006] Furthermore, in step (1), the final concentration of each stable isotope internal standard in the urine sample system is independently 5-20 μg / L.

[0007] Further, in step (2), the pH adjustment is adjusted to 2.0-2.5; the protein precipitant is methanol or acetonitrile, and the amount added is 1.0-2.0 times the volume of the urine sample; the centrifugation conditions are centrifugation at 10000-20000g for 3-8min, or centrifugation at 12000-16000rpm for 3-8min.

[0008] Further, in step (2), the pH adjustment is performed using a 1%-5% volume fraction of formic acid, hydrochloric acid, phosphoric acid, or sulfuric acid solution.

[0009] Further, in step (3), the low-proportion organic phase aqueous solution is an aqueous solution containing 1%-8% by volume of methanol or acetonitrile.

[0010] Further, in step (3), the organic solvent containing ammonia used for elution is a methanol, acetonitrile, or methanol / acetonitrile mixed solution containing 1%-5% ammonia by volume. The elution is carried out using a methanol solution containing 3% ammonia by volume, or a methanol / acetonitrile mixed solution with a volume ratio of 1:1 containing 3% ammonia by volume. The elution is performed twice.

[0011] Further, in step (4), the complex solution is a mixed solution of water and acetonitrile containing 0.01% formic acid, wherein the volume ratio of water to acetonitrile is (80-90):(10-20).

[0012] Further, in step (3), the filling method of the mixed-mode composite packing is as follows: a) A mixed-mode packing material in which weak anion exchange functional groups and reverse hydrophobic groups are introduced simultaneously on the same carrier; or, b) A mixed packing material in which weak anion exchange packing material and reverse packing material are mixed and packed in the same extraction well at a mass ratio of (1:9)-(9:1); or, c) A layered tandem packing bed in which the upstream is a weak anion exchange packing material layer and the downstream is a reverse packing material layer; wherein the reverse packing material is C18, C8, polymer reverse packing material or polar-reinforced reverse packing material.

[0013] Further, in step (3), the amount of packing material per well of the 96-well plate solid phase extraction device is 10-60 mg, and the particle size of the packing material is 5-80 μm; each extraction well is provided with a sieve plate or filter membrane with a pore size of 5-30 μm above and below the packing bed to limit the packing material.

[0014] Furthermore, in step (3), the anion exchange functional group of the WAX ​​packing is a tertiary amine group or its salt form.

[0015] Furthermore, in step (3), the solid-phase extraction uses vacuum negative pressure or centrifugation to pass the liquid through the extraction plate, and the flow rate of each step is 0.2-2.0 mL / min.

[0016] Further, in step (3), the activation includes activation with 100-300 μL of methanol; the equilibration includes equilibration with 100-300 μL of water.

[0017] Further, in step (3), the solid-phase extraction uses vacuum negative pressure or centrifugation to pass the liquid through the extraction plate at a flow rate of 0.2-2.0 mL / min; the activation includes activation with methanol, and the equilibration includes equilibration with water.

[0018] Further, in step (4), after reconstitution, centrifuge at 12000-16000 rpm for 2-5 min, and take the supernatant as the injection solution with an injection volume of 5-20 μL.

[0019] Further, in step (5), the chromatographic column used for liquid chromatography-tandem mass spectrometry detection is a C18 reversed-phase column, mobile phase A is an aqueous solution containing 0.01% formic acid, mobile phase B is an acetonitrile solution containing 0.01% formic acid, and a gradient elution program is used.

[0020] Furthermore, the method has a limit of quantitation (LOQ, S / N=10) of no more than 0.30 μg / L for dichloroacetic acid and a limit of quantitation (LOQ, S / N=10) of no more than 0.20 μg / L for trichloroacetic acid; and a matrix effect (ME) of 96.7%-101.3% in high-salt urine matrix, with a relative standard deviation (RSD) ≤2.4%.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects: ① No derivatization is required, reducing the use of derivatization reagents and lowering operational risks; ② Sample pretreatment can be completed in high throughput on a 96-well plate platform, which is compatible with vacuum manifolds or centrifugal liquid flow, facilitating large-scale monitoring; ③ By using the WAX / reverse-phase mixing mode retention and stepwise rinsing strategy, the matrix effect can be significantly reduced and the recovery stability can be improved in high-salt urine matrix; and by comparing with the closest existing technology (pure reverse-phase SPE), it is demonstrated that it has a non-obvious comprehensive technical effect in terms of flowability (pore blockage rate / flow time) and matrix effect stability. ④ By combining stable isotope internal standard correction, simultaneous and accurate quantification of DCAA and TCAA can be achieved, and a low limit of quantification can be obtained (DCAA≤0.30 µg / L, TCAA≤0.20 µg / L). ⑤ The method has a wide parameter window and good repeatability, making it suitable for biomonitoring and risk assessment of populations with low levels of occupational and environmental exposure. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic flowchart of the method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to the present invention; Figure 2 MRM chromatograms of DCAA, TCAA and their stable isotope internal standards; Figure 3 Distribution of urinary DCAA and TCAA concentrations in 42 individuals with low-level occupational exposure to trichloroethylene. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine according to embodiments of the present invention, sample precipitation can be performed by using methanol or acetonitrile for protein precipitation followed by centrifugation to collect the supernatant, or by centrifugation precipitation or filtration to remove particulate matter; without affecting the permeability of solid-phase extraction, 0-1.0 mol / L inorganic salt can be added to adjust the ionic strength. Precipitation and pH adjustment can be performed in centrifuge tubes or 96-well plates to accommodate batch operations of solid-phase extraction.

[0029] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine in this embodiment of the invention, the acid used to adjust the sample pH can be formic acid, hydrochloric acid, phosphoric acid or sulfuric acid, preferably 1%-5% (volume fraction) formic acid or hydrochloric acid, to control the sample pH at 1.8-3.0.

[0030] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine according to embodiments of the present invention, when the solid-phase extraction packing is WAX (weak anion exchange) / anion exchange / composite packing, the elution solvent can be methanol, acetonitrile, or a mixture of both, and 1%-5% ammonia or other organic base is added to promote the elution of the target analyte; when using pure reversed-phase packing, an organic solvent containing 0.1%-2% acid can be used for elution. The elution volume, number of elution cycles, and acidity / alkalinity can be routinely optimized while ensuring that the recovery rate and matrix effect meet the requirements.

[0031] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine in this embodiment of the invention, the reversed-phase column can be selected from C18, C8 or polar-enhanced reversed-phase packing; the organic phase can be selected from acetonitrile or methanol; the additive can be selected from formic acid or acetic acid and adjusted within the range of 0.005%-0.05%.

[0032] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine according to embodiments of the present invention, different manufacturers' triple quadrupole platforms can all achieve this, as long as ESI is met. - It can be collected with MRM and isotope dilution quantification can be performed based on internal standards.

[0033] In the simultaneous detection method of dichloroacetic acid and trichloroacetic acid in urine in the embodiments of the present invention, the WAX ​​packing is a weak anion exchange packing with tertiary amine groups on its surface; the anion exchange packing can be a strong anion exchange (SAX) packing with quaternary ammonium salt groups on its surface; the composite packing is a mixed-mode packing containing anion exchange groups and reverse hydrophobic groups (e.g., C18 / C8 or polymer reverse groups), or is obtained by mixing anion exchange packing and reverse packing in a mass ratio of (1:9)-(9:1).

[0034] In the simultaneous detection method of dichloroacetic acid and trichloroacetic acid in urine in this embodiment of the invention, a sieve plate or filter membrane is respectively set above and below each extraction well of a 96-well plate; 10-60 mg of any of the above-mentioned fillers are added to each well and gently vibrated and compacted to form a filler bed, and then covered with an upper sieve plate or filter membrane to limit the filler; the sieve plate pore size is preferably 5-30 μm to balance liquid permeability and material leakage prevention.

[0035] In the simultaneous detection method for dichloroacetic acid and trichloroacetic acid in urine according to embodiments of the present invention, under the acidic conditions, the retention / purification behavior of the target analyte and the packing system in the sample can be improved, and the interference of high-salt matrix can be reduced; rinsing is performed using water or a low proportion of organic phase to remove salt and polar interference; elution can be performed using methanol, acetonitrile, or a mixture thereof containing 1%-5% ammonia water, so as to achieve efficient elution of the target analyte and reduce matrix effect without increasing the derivatization step.

[0036] All raw materials used in the embodiments of this invention were purchased commercially.

[0037] Figure 1 This is a schematic flowchart of the method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to the present invention.

[0038] The technical solution of the present invention will be further illustrated by the following embodiments.

[0039] Example 1 A method for the simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine includes the following steps: (1) Reagents and standard / internal standard solutions: The concentration of DCAA and TCAA standard stock solutions is 1000 mg / L; stable isotope internal standards (DCAA-D2 and TCAA-) 13 C2) The stock solution concentration is 100 mg / L. Dilute the standard stock solution to prepare a mixed standard working solution (1.0 mg / L each), and dilute the internal standard stock solution to prepare a mixed internal standard working solution (1.0 mg / L each). Store at low temperature and protected from light for later use.

[0040] (2) Sample pretreatment: Take 400.0 µL of urine sample into a 2 mL polypropylene centrifuge tube or a 96-well plate. Add the mixed internal standard working solution to achieve a target concentration of 10.0 µg / L for both internal standards. Then add 500.0 µL of methanol or acetonitrile and vortex for 1 min to precipitate the protein. Centrifuge at 12000 rpm for 5 min and collect the supernatant. Add 3% (v / v) formic acid to the supernatant to adjust the pH to 3.0 and mix well. The treated sample was loaded into a 96-well solid-phase extraction plate (20 mg packing per well) containing a hybrid composite packing material consisting of WAX and C18 reversed-phase packing at a mass ratio of 1:1. The sample was activated and equilibrated sequentially with 200 µL of methanol and 200 µL of water before loading. It was then rinsed with 200 µL of water, followed by 200 µL of an aqueous solution containing 1% methanol, and dried under vacuum or by centrifugation for 2 min. The eluent was collected and eluted with a methanol solution containing 3% ammonia (2 × 100 µL). The eluent was concentrated to near dryness by purging under a nitrogen stream or by vacuum centrifugation at 40 °C. The residue was reconstituted with 200 µL of a reconstitution solution, which was a mixture of water (containing 0.01% formic acid) and acetonitrile (containing 0.01% formic acid) in a ratio of 85:15 (v / v). After reconstitution, mix thoroughly and centrifuge at 15000 rpm for 3 min. Transfer the supernatant to a sample vial for testing; the injection volume is 10.0 µL.

[0041] (3) The reconstituted solution was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in negative ion electrospray ionization mode using multiple reaction monitoring (MRM) to detect DCAA, TCAA and their corresponding internal standards. The content of DCAA and TCAA in the urine sample was calculated based on the isotope dilution quantification principle. The LC-MS / MS detection conditions were as follows: Chromatographic column: Agilent Eclipse Plus C18 RRHD (150 mm × 3.0 mm, 1.8 μm) or equivalent reversed-phase column; column temperature 40 ℃; flow rate 0.30 mL / min. Mobile phase A: water + 0.01% formic acid; mobile phase B: acetonitrile + 0.01% formic acid.

[0042] Gradient program: 0-0.5 min, B is 15%; 0.5-3.0 min, B rises to 95%; 3.0-4.0 min, B remains at 95%; 4.01 min, B drops to 15% and balances to 6.5 min; single run time is 6.5 min.

[0043] Mass spectrometry conditions: Triple quadrupole mass spectrometry, negative ion electrospray ionization (ESI-), MRM mode acquisition. Example ion source parameters: CUR 50 psi; CAD 8 psi; GS1 50 psi; GS2 50 psi; IS -4500 V; TEM 350 ℃; MRM residence time 80 ms. MRM parameters are shown in Table 1. Under the above conditions, DCAA, TCAA, and their corresponding internal standards were well separated. Typical MRM chromatograms of samples are shown below. Figure 1 As shown. By Figure 2 It can be seen that the retention times of DCAA and DCAA-D2 are approximately 2.82 min, while those of TCAA and TCAA-D2 are... 13 The retention time of C2 was approximately 3.29 min, with a symmetrical peak shape and no interference, indicating that the method has good separation and detection specificity.

[0044] Table 1. MRM parameters for target and internal standard

[0045] Example 2 Same as Example 1, except that in step (2), the treated sample is loaded into a solid phase extraction 96-well plate filled with WAX (weak anion exchange) packing material (20 mg packing material per well), and the rest of the steps are the same as in Example 1.

[0046] Example 3 Same as Example 1, except that in step (2), the processed sample is loaded into a solid phase extraction 96-well plate (20 mg of packing per well) filled with a mixed-mode composite packing composed of WAX and C18 packing, wherein WAX and C18 reverse-phase packing are layered in a mass ratio of 1:1 (upper layer WAX, lower layer C18).

[0047] The remaining steps are consistent with those in Example 1.

[0048] Layered packing is more advantageous for selectively retaining the target analyte first using ion exchange mechanism and then capturing some hydrophobic interfering substances using reflection mechanism, thereby reducing co-elution; mixed packing is easier to control in terms of flowability and packing consistency. Both packing methods in Examples 1 and 3 can achieve stable recovery of DCAA / TCAA, but a more suitable packing method can be selected according to the sample salinity, throughput and automated platform conditions.

[0049] Comparative Example 1 Pure inverting SPE: Same as Example 1, except that each well was packed with 20 mg of C18 reversed-phase packing material; the conventional reversed-phase SPE process (methanol activation-water equilibration-sample loading-water rinsing-high proportion organic solvent elution) was followed, specifically: the sample was loaded after activation and equilibration with 200 µL of methanol and 200 µL of water respectively; it was rinsed with 200 µL of water, and dried under vacuum or by centrifugation for 2 min; then eluted twice with acetonitrile solution containing 0.1% formic acid, 100 µL each time, and the eluent was collected. The eluent was then purged under a nitrogen stream at 40 °C or concentrated to near dryness by vacuum centrifugation, and the residue was reconstituted with 200 µL of a reconstitution solution; the reconstitution solution was a mixture of water (containing 0.01% formic acid) and acetonitrile (containing 0.01% formic acid) in a ratio of 85:15 (v / v). After reconstitution, mix thoroughly and centrifuge at 15000 rpm for 3 min. Transfer the supernatant to a sample vial for analysis. The remaining chromatographic and mass spectrometric conditions are the same as in Example 1.

[0050] The recovery rate, matrix effect (ME), precision (RSD), and batch processing throughput (risk of salt precipitation / plate blockage) of Examples 1-2 and Comparative Example 1 were verified as follows: (1) Calibration curve and sensitivity: A mixed calibration standard of DCAA and TCAA was prepared using a reconstituted solution / initial mobile phase at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 µg / L; an equal amount of internal standard was added at each point to make the internal standard concentration 10.0 µg / L. A linear regression was performed on the analyte / internal standard peak area ratio against the concentration, and the correlation coefficient r>0.999. Examples of detection limits (S / N=3) are DCAA 0.10 µg / L and TCAA 0.07 µg / L; examples of quantitation limits (S / N=10) are DCAA 0.30 µg / L and TCAA 0.20 µg / L. The calibration curve and sensitivity results are shown in Table 2.

[0051] Table 2 Calibration Curves and Sensitivity

[0052] (2) Matrix effect (ME): Six blank urine samples from different sources were pretreated in step (2) of Example 1 to obtain extracts. DCAA and TCAA (1.0, 10.0, 40.0 µg / L) were added to the extracts respectively, and corresponding isotopic internal standards were added. Using the peak area ratio of the same concentration level in the pure solvent as a control, ME (%) was calculated as (peak area ratio of matrix spiked peaks / peak area ratio of pure solvent) × 100. The matrix effect evaluation results are shown in Table 3.

[0053] Table 3 Matrix effect assessment results

[0054] (3) Accuracy and precision: Blank urine samples were spiked at low / medium / high levels (1.0, 10.0, 40.0 µg / L) and tested according to Example 1, with 6 parallel samples per level. The recovery rate ranged from 96.7% to 102.0%; the intra-day and inter-day precision RSD was ≤2.9%. The spiked recovery and precision results are shown in Table 4.

[0055] Table 4 Spike Recovery and Precision Results

[0056] As can be seen, this invention, through a synergistic design of acidic loading + WAX / reverse-phase mixed mode retention + water / low-proportion organic phase stepwise rinsing + ammonia-containing organic phase elution (after acidifying the sample to pH 1.8-3.0, it is loaded onto a 96-well SPE plate packed with a 1:1 mixture of WAX and C18, and sequentially washed with water and rinsed with 1% methanol aqueous solution, and eluted twice with 3% ammonia methanol solution), controls ME at approximately 96.7%-101.3% and RSD≤2.4% in a high-salt urine matrix (Table 3), and obtains spiked recovery rates of approximately 96.7%-102.0% and good intra-day / inter-day precision (Table 4). Thus, it achieves low LOQ and high throughput without the need for derivatization, which is a comprehensive technical effect that is difficult for those skilled in the art to expect based on a single reverse-phase SPE or conventional purification process.

[0057] The comparison results of Example 1 and Comparative Example 1 are shown in Table 5.

[0058] Table 5 Comparison results of Example 1 and Comparative Example 1

[0059] As shown in Table 5, Example 1 of the present invention exhibits a near 100% median eluent (ME) and good precision in a high-salt urine matrix. In contrast, Comparative Example 1, relying solely on the reverse phase mechanism, is more prone to target analyte penetration and co-elution interference when faced with various endogenous acidic interfering substances and salt-carrying components in high-salt urine. This manifests as an ME deviating from 100%, decreased repeatability at low concentrations, and increased risk of poor flow / plate clogging. The above comparison demonstrates that the combination of "acidic loading + WAX / reverse phase mixing mode retention + water / low-proportion organic phase stepwise elution + ammonia-containing organic phase elution" described in this invention has a comprehensive technical effect of synergistic purification and resistance to matrix effects, which cannot be expected simply by replacing packing material or optimizing conventional parameters. To further enhance comparability with the closest existing technology (pure reversed-phase SPE), the "instability of liquid flow / plate clogging risk" and system stability can be quantified as follows: (1) Liquid flow time: Record the average liquid flow time (unit s / well, it is recommended that n≥3 96-well plates for each method) for each step of sample loading, water rinsing, low-proportion organic phase rinsing and elution, and calculate the RSD within the same plate; (2) Clogging rate: Count those liquid flow times exceeding twice the median of the same plate or those without effluent within the specified time as clogging, clogging rate = number of clogging wells / 96 × 100%; (3) Recovery and precision: Compare the intra-plate / inter-plate recovery and RSD under the same urine source and the same spiking level (e.g. 10.0 µg / L); (4) Chromatographic system stability: Record the column pressure (initial and after 300 consecutive injections) and the column pressure abnormality rate (e.g., the column pressure increases by 20%–30% from the baseline or the guard column needs to be replaced / the ion source needs to be cleaned) as abnormal.

[0060] To verify the selective retention and purification stability of Examples 1, 2, and Comparative Example 1 in a high-salt urine matrix, blank urine samples from the same batch were selected and spiked at three levels: DCAA and TCAA concentrations of 1.0 µg / L, 10.0 µg / L, and 40.0 µg / L, respectively. Stable isotope internal standards of the same concentration as in Example 1 were also added. After protein precipitation and pH adjustment (1.8–3.0) as per step (2), solid-phase extraction was performed according to the above-described route, with the remaining chromatographic and mass spectrometric conditions the same as in Example 1.

[0061] Quantitative comparison of flowability and chromatographic system stability: Under the same urine source and spiked level (10.0 µg / L), at least three 96-well plates of Example 1 and Comparative Example 1 of the present invention were batch-processed according to the above method. The flow time, pore blockage rate and column pressure change of each step were recorded. The statistical results are summarized in Table 6 to more intuitively reflect the batch processing stability and anti-system contamination ability of the present invention in high-salt urine matrix.

[0062] Table 6 Comparison of results for indicators such as liquid flow time, pore blockage rate, and column pressure changes at each step

[0063] In practical applications, using the pretreatment process of this invention, samples can be purified and eluted in parallel on a 96-well plate, allowing a single person to preprocess approximately 96-192 urine samples per day; the LC-MS / MS analysis time is approximately 6.5 minutes, which can meet the requirements for large-scale continuous detection. This method was applied to urine samples from subjects with low-level occupational trichloroethylene exposure (n=42). TCAA was detected in 33 / 42 samples, with concentrations ranging from 0.2 to 6.7 µg / L (median 1.97 µg / L); DCAA was detected in 19 / 42 samples, with concentrations ranging from 0.3 to 4.8 µg / L (median 1.79 µg / L). The distribution of urinary DCAA and TCAA concentrations in 42 individuals with low-level occupational trichloroethylene exposure is shown in the figure. Figure 3 .

[0064] The present invention relates to a method for the simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine. The main technical features are a combination of acidic loading, a 1:1 mixture of WAX and C18 filler, stepwise rinsing with 1% methanol aqueous solution after water washing, and two elutions with 3% ammonia and methanol. Compared with existing technologies, the present invention has at least the following advantages: (1) no derivatization is required, simplifying the process and making it suitable for batch operation; (2) 96-well plate solid-phase extraction improves purification efficiency and facilitates high-throughput processing; (3) isotope dilution quantification improves accuracy and reduces matrix effects; (4) low quantitation limit, suitable for monitoring low-level occupational / environmental exposures; (5) short single analysis cycle and high throughput, suitable for routine monitoring in public health and occupational health laboratories. The 96-well plate uses WAX (weak anion exchange) / anion exchange / composite filler, which further enhances the selective retention of haloacetic acid targets, resistance to matrix interference, and batch-to-batch recovery stability, while reducing the risk of plate clogging.

[0065] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for the simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine, characterized in that, Includes the following steps: (1) Take a urine sample and add a stable isotope internal standard mixed solution, wherein the stable isotope internal standard is dichloroacetic acid-D2 and trichloroacetic acid-D2. 13 C2; (2) Add protein precipitant to the system obtained in step (1) to precipitate, centrifuge and take the supernatant, and adjust the pH of the supernatant to 1.8-3.0; (3) The supernatant obtained in step (2) is loaded into a 96-well plate solid-phase extraction device for purification and enrichment. Each well of the 96-well plate solid-phase extraction device is filled with a weak anion exchange packing or a mixed-mode composite packing composed of a weak anion exchange packing and a reversed-phase packing. The solid-phase extraction process includes activation, equilibration, sample loading, rinsing, drying and elution in sequence. The rinsing is a stepwise rinsing, which includes at least water rinsing and low-proportion organic phase aqueous solution rinsing. The low-proportion organic phase aqueous solution rinsing is performed after water rinsing. The elution uses an organic solvent containing ammonia. (4) After concentrating the eluent obtained in step (3), add a redissolving solution to redissolve it; (5) The complex solution obtained in step (4) was subjected to liquid chromatography-tandem mass spectrometry in negative ion electrospray ionization mode to detect dichloroacetic acid and trichloroacetic acid and their corresponding internal standards in multiple reaction monitoring mode, and the content of dichloroacetic acid and trichloroacetic acid in urine sample was calculated based on the isotope dilution quantification principle.

2. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (2), the pH is adjusted to 2.0-2.5; the protein precipitant is methanol or acetonitrile, and the amount added is 1.0-2.0 times the volume of the urine sample.

3. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (3), the low-proportion organic phase aqueous solution is an aqueous solution containing 1%-8% by volume of methanol or acetonitrile.

4. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (3), the organic solvent containing ammonia used for elution is a methanol, acetonitrile, or methanol / acetonitrile mixed solution containing 1%-5% ammonia by volume.

5. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 4, characterized in that, The elution is performed using a methanol solution containing 3% ammonia by volume, or a methanol / acetonitrile mixture with a volume ratio of 1:1 containing 3% ammonia by volume, and the elution is performed twice.

6. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (4), the complex solution is a mixed solution of water and acetonitrile containing 0.01% formic acid, wherein the volume ratio of water to acetonitrile is (80-90):(10-20).

7. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (3), the filling method of the mixed-mode composite packing is as follows: a mixed-mode packing that simultaneously introduces weak anion exchange functional groups and reverse hydrophobic groups on the same carrier; or a mixed packing packing in which weak anion exchange packing and reverse packing are mixed and filled in the same extraction pore at a mass ratio of (1:9)-(9:1); or a layered series packing bed with a weak anion exchange packing layer upstream and a reverse packing layer downstream.

8. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 7, characterized in that, The reversed-phase packing is C18, C8, polymer reversed-phase, or polar-reinforced reversed-phase packing.

9. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, In step (3), the solid-phase extraction uses vacuum negative pressure or centrifugation to pass the liquid through the extraction plate at a flow rate of 0.2-2.0 mL / min; the activation includes activation with methanol, and the equilibration includes equilibration with water.

10. The method for simultaneous detection of dichloroacetic acid and trichloroacetic acid in urine according to claim 1, characterized in that, The method has a limit of quantification (LOQ) of no more than 0.30 μg / L for dichloroacetic acid and no more than 0.20 μg / L for trichloroacetic acid; and the matrix effect in high-salt urine matrix is ​​96.7%-101.3%, with a relative standard deviation of ≤2.4%.

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