A method for enriching and detecting aromatic amine metabolites in human urine

Abstract

This invention belongs to the field of chemical detection technology, specifically relating to a method for enriching and detecting aromatic amine metabolites in human urine. The method for enriching aromatic amine metabolites in human urine provided by this invention includes the following steps: mixing human urine with an acid solution and then hydrolyzing to obtain a urine sample; adjusting the urine sample to alkaline conditions; then dispersing the urine sample in a diatomaceous earth solid-phase supported liquid-liquid extraction column and eluting with n-hexane. By employing solid-phase supported liquid-liquid extraction technology on urine samples under alkaline conditions, the alkaline conditions are conducive to the extraction of aromatic amine metabolites by n-hexane, resulting in high extraction efficiency. This achieves a highly efficient, rapid, and high-throughput sample pretreatment process, is simple to operate, has higher throughput, and is more suitable for large-scale sample testing.

Description

Technical Field

[0001] This invention relates to the field of chemical detection technology, specifically to a method for enriching and detecting aromatic amine metabolites in human urine. Background Technology

[0002] Some aromatic amines are carcinogenic and mutagenic, and can cause poisoning through inhalation, ingestion, or skin absorption. The FDA's list of hazardous and potentially hazardous substances (HPHCs) in cigarette products and smoke includes six aromatic amines: 4-aminobiphenyl, 1-aminonaphthalene, 2-aminonaphthalene, o-anisidine, 2,6-dimethylaniline, and o-toluidine. Four aromatic amines—3-aminobiphenyl, 4-aminobiphenyl, 1-aminonaphthalene, and 2-aminonaphthalene—have been included in the Hoffmann list of hazardous components in cigarette smoke. Aromatic amines are used as biomarkers of cigarette smoke exposure; reports indicate that the levels of aromatic amines in the urine of smokers are significantly higher than in non-smokers. The metabolism of aromatic amines in the human body is carried out in the liver by cellular P450s (CYP1A2), with the final metabolite being an aromatic amine-DNA adduct.

[0003] Because urine has a highly complex matrix and aromatic amine metabolites are present in extremely low concentrations and are highly polar, HPLC-MS / MS is commonly used to detect their content. Its advantage lies in eliminating the need for derivatization reactions and simplifying sample pretreatment. However, to reduce matrix effects and contamination of the ion source in HPLC-MS / MS, sample purification is usually required. Existing sample purification methods mainly include liquid-liquid extraction, molecularly imprinted solid-phase extraction, and magnetic solid-phase extraction based on magnetic COFs materials. Liquid-liquid extraction uses large amounts of organic solvents and has a cumbersome pretreatment process; molecularly imprinted solid-phase extraction offers high selectivity, but solid-phase extraction columns are expensive.

[0004] Since magnetic solid-phase extraction using magnetic COFs materials has not yet been commercialized, it is difficult to meet the needs of large-scale sample testing. For example, Chinese patent application CN108276584A, published on July 13, 2018, discloses a method for detecting aromatic amine compounds in human urine. After pretreatment of human urine, sulfonic acid-functionalized COFs materials are added for solid-phase extraction to separate the solid phase. The solid phase is then eluted with an eluent to obtain an eluent. The sulfonic acid-functionalized COFs materials include a covalent organic framework formed by the Schiff base condensation reaction of 2,4,6-tricarboxypyrogallol and 2,5-diaminobenzenesulfonic acid. Utilizing the characteristics of COFs materials, trace aromatic amine compounds can be selectively and efficiently enriched in complex matrices, which can greatly simplify the sample pretreatment process and improve the detection efficiency and accuracy of aromatic amine compounds.

[0005] Therefore, there is an urgent need for a pretreatment method that can take into account the characteristics of simple operation, high adsorption capacity, and high throughput. Summary of the Invention

[0006] The purpose of this invention is to provide a method for enriching aromatic amine metabolites in human urine, which solves the problem that existing methods for enriching aromatic amine metabolites cannot simultaneously achieve simplicity, high adsorption capacity, and high throughput.

[0007] The second objective of this invention is to provide a method for detecting aromatic amine metabolites in human urine, thereby solving the problem of low detection efficiency in existing methods for detecting aromatic amine compounds.

[0008] To solve the above-mentioned technical problems, the technical solution of the method for enriching aromatic amine metabolites in human urine of the present invention is as follows: A method for enriching aromatic amine metabolites in human urine includes the following steps: mixing human urine with acid and then hydrolyzing to obtain a urine sample; adjusting the urine sample to alkaline; then dispersing the urine sample in a diatomaceous earth solid-phase supported liquid-liquid extraction column and eluting with n-hexane.

[0009] This invention improves upon existing technology and provides a method for enriching aromatic amine metabolites in human urine. By employing solid-phase supported liquid-liquid extraction technology on urine samples under alkaline conditions, the extractant is distributed on the surface of diatomaceous earth in the form of a thin liquid film. Liquid-liquid extraction occurs immediately upon contact between the extractant and n-hexane on the diatomaceous earth surface. The alkaline conditions are conducive to the extraction of aromatic amine metabolites by n-hexane, resulting in high extraction efficiency.

[0010] The enrichment method provided by this invention achieves an efficient, rapid, and high-throughput sample pretreatment process. It is simple to operate, has higher throughput, and is more suitable for large-scale sample testing. Furthermore, the SLE solid-supported liquid-liquid extraction column used is a commercially available solid-phase extraction column, which is cheaper than molecularly imprinted solid-phase extraction columns, resulting in lower experimental costs.

[0011] To further improve the enrichment effect, preferably, the diatomaceous earth solid-phase supported liquid-liquid extraction column is ISOLUTE. ® SLE+.

[0012] To further ensure complete extraction of the target pollutants from human urine, preferably, 10-50 mL of n-hexane is added for every 4-5 mL of human urine.

[0013] To further improve the extraction effect of n-hexane, preferably, the urine sample is adjusted to be alkaline by adding an alkaline solution, namely sodium hydroxide solution, to adjust the pH of the urine sample to 10-11.

[0014] To further improve hydrolysis efficiency, preferably, the hydrolysis temperature is 60~100°C. o C, the hydrolysis time is 0.5~2h.

[0015] To further improve the degree of hydrolysis of human urine, preferably, the acid solution is concentrated hydrochloric acid with a mass concentration of 36-38%; 0.5-2 mL of concentrated hydrochloric acid is added for every 4-5 mL of human urine.

[0016] To further improve the enrichment effect of the target analyte, preferably, the aromatic amine metabolites include 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, and 4-aminobiphenyl.

[0017] The technical solution of the method for detecting aromatic amine metabolites in human urine of the present invention is as follows: A method for detecting aromatic amine metabolites in human urine, comprising enriching the aromatic amine metabolites in human urine using the aforementioned enrichment method, followed by HPLC-MS / MS analysis.

[0018] The present invention provides a method for detecting aromatic amine metabolites in human urine. This method utilizes an enrichment method for aromatic amine metabolites in human urine, achieving a highly efficient, rapid, and high-throughput sample pretreatment process. It is simple to operate, has higher throughput, and is more suitable for large-scale sample testing. Using the detection method provided by this invention, the detection limit for aromatic amine compounds is 0.003~0.015 ng / mL, and the quantitation limit is 0.009~0.049 ng / mL.

[0019] To further improve the separation capability of aromatic amine compounds, the preferred chromatographic conditions for HPLC-MS / MS analysis are: Column: Waters Symmetry Shield TM RP18; Column temperature: 30~40 o C; Injection volume: 10~15 μL; Pre-equilibration time: 10~15 min; Mobile phase A: 0.1 vol % formic acid-water solution, Mobile phase B: 0.1 vol % formic acid-acetonitrile solution; Gradient elution conditions: 0~3 min, 100% A; 3.1~17 min, 78% A; 17.1~22 min, 100% B; 22.1~33 min, 100% A.

[0020] To further improve detection accuracy, the preferred mass spectrometry conditions for HPLC-MS / MS analysis are as follows: ion source: electrospray ionization source; scanning mode: positive ion scan; detection mode: multiple reaction monitoring (MRM); electrospray voltage: 5000~5500 V; auxiliary gas 1 pressure: 70~80 psi; auxiliary gas 2 pressure: 70~80 psi; ion source temperature: 500~550 °C.o C. Attached Figure Description

[0021] Figure 1 This is an MRM diagram of typical aromatic amine metabolites in human urine, as shown in Example 1. Detailed Implementation

[0022] The technical concept of the method for enriching aromatic amine metabolites in human urine according to the present invention is as follows: Because human urine has a complex matrix and the content of the target analyte, aromatic amine metabolites, is extremely low, sample enrichment is necessary. Existing enrichment methods include liquid-liquid extraction, which is complex and uses a large amount of solvent; molecularly imprinted solid-phase extraction columns have low adsorption capacity and high cost; and novel solid-phase extraction materials based on organic frameworks have poor enrichment effects and low throughput. This invention uses solid-phase supported liquid-liquid extraction technology to improve extraction efficiency by controlling the alkaline conditions and eluent during liquid-liquid extraction, achieving rapid, effective, high-throughput, and low-cost enrichment.

[0023] The method for enriching aromatic amine metabolites in human urine provided by the present invention includes the following steps: Thaw human urine stored at -80℃ to room temperature. Take 4-5 mL of human urine and mix it with 0.5-2 mL of concentrated hydrochloric acid (36-38% by mass). Incubate at 60-100℃. o Hydrolyze at C for 0.5-2 hours to obtain a urine sample. Add sodium hydroxide solution to the urine sample to adjust the pH of the urine sample to 10-11. Then disperse the urine sample in a diatomaceous earth solid-phase supported liquid-liquid extraction column and elute with n-hexane.

[0024] In a specific implementation, the urine sample is dispersed in the diatomaceous earth solid-phase supported liquid-liquid extraction column by loading the urine sample into the diatomaceous earth solid-phase supported liquid-liquid extraction column and allowing it to stand for 5-10 minutes.

[0025] In a specific implementation, before HPLC-MS / MS analysis, the eluent obtained by elution with n-hexane is dried using a nitrogen concentrator, and then redissolved with 100~500 µL of 5~10% ammonia-methanol solution to obtain the test solution.

[0026] In a specific implementation, after adjusting the pH of the urine sample to 10-11, 50-100µL of a mixed internal standard solution (d7-1-aminonaphthalene, d7-2-aminonaphthalene, d9-3-aminobiphenyl, d9-4-aminobiphenyl) is added.

[0027] In a specific embodiment, the concentration of the sodium hydroxide solution is 10~15 mol / L.

[0028] In a specific implementation, human urine was collected in accordance with the Technical Specifications for On-site Survey Management of Large Population Cohorts (T / CPMA 001-2019).

[0029] The embodiments of the present invention will be further described below with reference to specific examples. Unless otherwise specified, the chemical reagents involved in the following examples are all commercially available conventional products.

[0030] I. Specific Embodiments of the Method for Enriching Aromatic Amine Metabolites in Human Urine of the Present Invention Example 1 The method for enriching aromatic amine metabolites in human urine in this embodiment is as follows: Human urine samples were collected in accordance with the Technical Specifications for the Management of Field Surveys of Large Populations (T / CPMA 001-2019), collecting 24-hour urine samples from non-smokers, regular cigarette smokers, and slim cigarette smokers. Collected human urine samples were stored in an ultra-low temperature freezer at -80°C.

[0031] Human urine samples were thawed to room temperature, and five urine samples each were collected from non-smokers, regular cigarette smokers, and slim cigarette smokers. Pretreatment of these 15 urine samples was conducted simultaneously. 4 mL of each sample was placed in a 15 mL centrifuge tube, and 0.8 mL of concentrated hydrochloric acid (36–38%) was added. After thorough mixing, the tube was placed in a constant temperature water bath at 80°C for 1 h to hydrolyze the samples. The urine samples were then cooled to room temperature, and 950 µL of 12 mol / L NaOH solution was added to adjust the pH to 10–11. Then, 50 µL of a mixed internal standard solution (d7-1-NA, d7-2-NA, d9-3-ABP, d9-4-ABP) was added to obtain a mixed solution. This mixed solution was then loaded onto an ISOLUTE solid-phase supported liquid-liquid extraction column. ® In SLE+, after standing for 10 min, elute with 25 mL of n-hexane to obtain the eluent. Dry the eluent using a nitrogen concentrator, and then redissolve it with 100 µL of 5% ammonia-methanol solution to obtain the test solution.

[0032] Example 2 The method for enriching aromatic amine metabolites in human urine in this embodiment is as follows: Human urine samples were collected in accordance with the Technical Specifications for the Management of Field Surveys of Large Populations (T / CPMA 001-2019), collecting 24-hour urine samples from non-smokers, regular cigarette smokers, and heated cigarette smokers. Collected human urine samples were stored in an ultra-low temperature freezer at -80°C.

[0033] Human urine samples were thawed to room temperature, and five urine samples each were collected from non-smokers, regular cigarette smokers, and heated cigarette smokers. Pretreatment of these 15 urine samples was conducted simultaneously. 4 mL of each sample was placed in a 15 mL centrifuge tube, and 0.8 mL of concentrated hydrochloric acid (36–38%) was added. After thorough mixing, the tube was placed in an 80°C water bath for hydrolysis for 1 h. The resulting urine samples were cooled to room temperature, and a certain amount of 12 mol / L NaOH solution was added to adjust the pH to 10–11. Then, 50 µL of a mixed internal standard solution (d7-1-NA, d7-2-NA, d9-3-ABP, d9-4-ABP) was added to obtain a mixed solution. This mixed solution was then loaded onto an ISOLUTE solid-phase supported liquid-liquid extraction column. ® On SLE+, let stand for 5 min, then elute with 50 mL of n-hexane to obtain the eluent. Dry the eluent using a nitrogen concentrator, and then redissolve it with 200 µL of 5% ammonia-methanol solution to obtain the test solution.

[0034] II. Specific Embodiments of the Method for Detecting Aromatic Amine Metabolites in Human Urine of the Present Invention Example 3 The method for detecting aromatic amine metabolites in human urine in this embodiment is as follows: The test solution obtained after enrichment using the enrichment method for aromatic amine metabolites in human urine as described in Examples 1-2 was analyzed by HPLC-MS / MS.

[0035] The chromatographic conditions for HPLC-MS / MS analysis were as follows: Column: Waters Symmetry Shield TM RP18 (150 mm × 2.1 mm id, 3.5 μm); Column temperature: 30 °C o C; Injection volume: 10 μL; Pre-equilibration time: 10 min; Mobile phase A: 0.1% (v / v) formic acid-water solution, Mobile phase B: 0.1% (v / v) formic acid-acetonitrile solution; Gradient elution conditions: 0~3 min, 100% A; 3.1~17 min, 78% A; 17.1~22 min, 100% B; 22.1~33 min, 100% A. The gradient elution conditions are shown in Table 1.

[0036] Table 1. Gradient elution conditions for chromatography The mass spectrometry conditions for HPLC-MS / MS analysis were as follows: ion source: electrospray ionization (ESI); scan mode: positive ion scan; detection mode: multiple reaction monitoring (MRM); electrospray voltage: 5000 V; ion source temperature: 500 °C.o C; Auxiliary gas Gas1 pressure: 70 psi; Auxiliary gas Gas2 pressure: 70 psi; The quantitative ion pairs, qualitative ion pairs, residence time, collision energy (CE), and declustering voltage (DP) of each compound are shown in Table 2.

[0037] Table 2. Quantitative ion pairs, qualitative ion pairs, residence time, collision energy (CE), and declustering voltage (DP) for each compound. a Quantitative ions III. Experimental Examples (1) Tests for recovery rate, limit of detection, and limit of quantitation A mixed standard stock solution containing four aromatic amine compounds (concentrations of 0.5 µg / mL for 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, and 4-aminobiphenyl, respectively) was prepared using 5% ammonia / methanol. A mixed internal standard stock solution was also prepared. Different volumes of the mixed standard stock solution were then added to form a series of mixed standard working solutions. These mixed standard working solutions contained internal standards d7-1-aminonaphthalene, d7-2-aminonaphthalene, d9-3-aminobiphenyl, and d9-4-aminobiphenyl at concentrations of 10 ng / mL, 10 ng / mL, 5 ng / mL, and 5 ng / mL, respectively. The series of mixed standard working solutions were analyzed by HPLC-MS / MS, and linear regression was performed on the peak area ratio of aromatic amine compounds to internal standards against the concentration ratio to obtain standard working curves for each target compound. Among them, d7-1-aminonaphthalene is the internal standard for 1-aminonaphthalene; d7-2-aminonaphthalene is the internal standard for 2-aminonaphthalene; d9-3-aminobiphenyl is the internal standard for 3-aminobiphenyl; and d9-4-aminobiphenyl is the internal standard for 4-aminobiphenyl.

[0038] The recovery rate of the detection method was investigated. Following Example 1, urine samples from non-smokers were used to determine the recovery rate at low, medium, and high spiked levels. The lowest concentration of the mixed standard working solution was used for 10 parallel determinations, and the standard deviation was calculated. Three times the standard deviation was defined as the limit of detection, and ten times the standard deviation was defined as the limit of quantitation. The intra-day and inter-day precision of aromatic amine compounds was also investigated. The results are shown in Table 3.

[0039] Table 3. Linear regression equation, recovery rate, limit of detection, limit of quantitation, and precision test results. (2) Actual sample test results The results of the test for aromatic amine metabolites in urine samples from non-smokers and smokers of different types of cigarettes in Example 1 are shown in Table 4. The MRM diagram of aromatic amine metabolites in typical human urine (regular cigarette smoker 1) from Example 1 is shown below. Figure 1 As shown, where, Figure 1 a is the MRM diagram of 1-aminonaphthalene (1-NA) and 2-aminonaphthalene (2-NA). Figure 1 b is the MRM diagram of 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP). Figure 1 c represents the MRM diagrams for d7-1-NA and d7-2-NA. Figure 1 d represents the MRM diagrams for d9-3-ABP and d9-4-ABP.

[0040] Table 4. Content of aromatic amine metabolites (pg / mL) in urine samples from non-smokers and smokers of different types of cigarettes. b Not detected The results of the test for aromatic amine metabolites in urine samples from non-smokers, regular cigarette smokers, and heated cigarette smokers in Example 2 are shown in Table 5.

[0041] Table 5. Content of aromatic amine metabolites (pg / mL) in urine samples from non-smokers, regular cigarette smokers, and heated cigarette smokers. Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention 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 substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enriching aromatic amine metabolites in human urine, characterized in that, Includes the following steps: Human urine and acid were mixed and hydrolyzed to obtain a urine sample. The urine sample was then adjusted to be alkaline and dispersed in a diatomaceous earth solid-phase supported liquid-liquid extraction column, and eluted with n-hexane.

2. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, The diatomaceous earth solid-phase supported liquid-liquid extraction column is ISOLUTE. ® SLE+.

3. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, Add 10-50 mL of n-hexane for every 4-5 mL of human urine.

4. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, Adjusting the pH of a urine sample to alkaline is achieved by adding an alkaline solution, specifically sodium hydroxide solution, to the urine sample to a pH of 10-11.

5. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, The hydrolysis temperature is 60~100℃. o C, the hydrolysis time is 0.5~2h.

6. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, The acid solution is concentrated hydrochloric acid with a mass concentration of 36-38%; 0.5-2 mL of concentrated hydrochloric acid is added for every 4-5 mL of human urine.

7. The method for enriching aromatic amine metabolites in human urine as described in claim 1, characterized in that, The aromatic amine metabolites include 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, and 4-aminobiphenyl.

8. A method for detecting aromatic amine metabolites in human urine, characterized in that, After enrichment using the method described in any one of claims 1-7 for the enrichment of aromatic amine metabolites in human urine, HPLC-MS / MS analysis was performed.

9. The method for detecting aromatic amine metabolites in human urine as described in claim 8, characterized in that, The chromatographic conditions for HPLC-MS / MS analysis were as follows: Column: Waters Symmetry Shield TM RP18; Column temperature: 30~40 o C; Injection volume: 10~15 μL; Pre-equilibration time: 10~15 min; Mobile phase A: 0.1 vol % formic acid-water solution, Mobile phase B: 0.1 vol % formic acid-acetonitrile solution; Gradient elution conditions: 0~3 min, 100% A; 3.1~17 min, 78% A; 17.1~22min, 100%B; 22.1~33min, 100%A.

10. The method for detecting aromatic amine metabolites in human urine as described in claim 8, characterized in that, The mass spectrometry conditions for HPLC-MS / MS analysis were as follows: ion source: electrospray ionization source; scanning mode: positive ion scan; detection mode: multiple reaction monitoring mode; electrospray voltage: 5000~5500 V; auxiliary gas pressure: 70~80 psi. Assist gas pressure: 70~80 psi; Ion source temperature: 500~550℃ o C.

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