Evaluation of simultaneous detection methods for urinary biomarkers of exposure to multiple thermal process contaminants and applications

Abstract

The application discloses a kind of synchronous detection method and application of evaluating multiple thermal processing pollutant exposure urine biomarker, belong to analytical detection technical field, and thermal processing pollutant includes acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD, method includes: taking urine sample, adding each thermal processing pollutant corresponding urine biomarker internal standard, then adding formic acid and ammonium formate, vortex centrifugation is taken supernatant after activation balance solid phase extraction column, then elution liquid is obtained by elution and elution, and elution liquid is obtained after treatment to obtain sample solution to be measured;Then the ultra-high performance liquid chromatography tandem mass spectrometry technique is used to detect each urine biomarker in sample solution to be measured.The method is short in analysis time, high in sensitivity, high in detection accuracy, can realize the synchronous quantitative analysis of AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA 8 kinds of urine biomarkers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical detection technology, and particularly relates to a synchronous detection method for evaluating urine biomarkers of exposure to various thermal processing pollutants and application thereof. BACKGROUND

[0002] In the process of food thermal processing, non-enzymatic browning such as Maillard reaction and caramelization reaction occurs to form color and flavor substances specific to food, which endows food with color and flavor. However, thermal processing pollutants such as acrylamide, furfural, furfuryl alcohol, 5-hydroxymethyl furfural (5-HMF) and 3-chloro-1,2-propanediol (3-MCPD) are generated in the process, which have attracted widespread attention. Therefore, it is of great significance to evaluate the safety risk of pollutants in thermal processed food for food safety.

[0003] For the risk assessment of furfural, furfuryl alcohol and 5-HMF, the existing technology mainly adopts the external exposure evaluation method, that is, the intake level of dietary furfural, furfuryl alcohol and 5-HMF is investigated by determining the content of furfural, furfuryl alcohol and 5-HMF in various foods. However, this evaluation method cannot accurately reflect the true exposure level of furfural, furfuryl alcohol and 5-HMF into the human body. In order to accurately evaluate the exposure risk of dietary furfural, furfuryl alcohol and 5-HMF to the human body, the internal exposure evaluation method should be adopted, that is, the risk assessment is carried out by determining the in vivo metabolic biomarkers of furfural, furfuryl alcohol and 5-HMF. Compared with the external exposure of dietary furfural, furfuryl alcohol and 5-HMF, the internal exposure level based on biomarkers can more accurately evaluate the daily intake of furfural, furfuryl alcohol and 5-HMF and provide more reliable basis for epidemiological research.

[0004] For the risk assessment of acrylamide and 3-MCPD, the Chinese patent document with publication number CN105158398A and the Chinese patent document with publication number CN110208406A respectively disclose a detection method for evaluating N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA), N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (iso-GAMA) and N-acetyl-S-(2-carbamoylethyl)-L-cysteine-sulfoxide (AAMA-sul) as thiol uric acid adducts of short-term exposure of acrylamide and 2,3-dihydroxypropylmercapturic acid (DHPMA) as thiol uric acid adducts of short-term exposure of 3-MCPD, which respectively realize the internal exposure analysis of acrylamide and 3-MCPD.

[0005] At present, there is no quantitative analysis method for furoyl urine biomarkers (N-2-furoylglycine (N2FG)), furfuryl alcohol urine biomarkers (N-2-furoylglycine (N2FG)) and 5-HMF urine biomarkers (5-hydroxymethylfuroic acid (HMFA), 5-hydroxymethyl-2-furoylglycine (HMFG)) in China. At the same time, the currently disclosed methods for human biomonitoring and internal exposure level detection and analysis of acrylamide and 3-MCPD have the following disadvantages: the detectable metabolite species in the pretreated sample are less or the target detection substance is single; or the detection method, instrument, etc. are not sufficient; the detection is respectively for a single kind of urine biomarker of acrylamide and 3-MCPD; the processing time is long, the solvent amount is large, the processing cost and analysis time are large, and the method cannot be used for the detection and analysis of a large number of samples in epidemiology; at the same time, it is not conducive to green environmental protection, and the synchronous analysis of internal exposure of multiple heat processing pollutants cannot be realized. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a synchronous detection method for urine biomarkers for evaluating exposure to multiple thermal processing pollutants, including acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD, which has short analysis time, high sensitivity, high detection accuracy and high detection efficiency, and can realize synchronous quantitative analysis of AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA, eight kinds of urine biomarkers.

[0007] The specific technical solutions adopted are as follows:

[0008] A synchronous detection method for urine biomarkers for evaluating exposure to multiple thermal processing pollutants, wherein the thermal processing pollutants include acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD, and the corresponding urine biomarkers include AAMA (N-acetyl-S-(2-carbamoyl ethyl)-L-cysteine), GAMA (N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine), iso-GAMA (N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine), AAMA-sul (N-acetyl-S-(2-carbamoyl ethyl)-L-cysteine sulfoxide), N2FG (N-(2-furoyl) glycine), HMFA (5-hydroxymethyl-2-furoic acid), HMFG (5-hydroxymethyl-2-furoyl glycine) and DHPMA (2,3-dihydroxypropyl mercapturic acid); the method comprises the following steps:

[0009] (1) Sample pretreatment: take a urine sample, add urine biomarker internal standards corresponding to each thermal processing pollutant, then add formic acid and ammonium formate, vortex and centrifuge, take the supernatant, pass through an activated and balanced Isolute ENV+ solid-phase extraction column, then elute and wash to obtain an eluate, and then perform nitrogen blowing, redissolution and membrane filtration to obtain a sample solution to be tested;

[0010] (2) Use ultra-high performance liquid chromatography tandem mass spectrometry to detect the sample solution to be tested, obtain the peak areas of each urine biomarker and urine biomarker internal standard in the sample solution to be tested, and substitute them into the corresponding standard curve to obtain the contents of each urine biomarker in the urine sample;

[0011] Ultra-high performance liquid chromatography conditions: use formic acid aqueous solution as mobile phase A and formic acid acetonitrile solution as mobile phase B, and the elution program is as follows: 0-4.5 min, 2% mobile phase B; 4.5-7.0 min, 2%-100% mobile phase B; 7.0-9.0 min, 100% mobile phase B; 9.0-9.1 min, 100%-2% mobile phase B; 9.1-15.0 min, 2% mobile phase B;

[0012] Mass spectrometry conditions: ionization by electrospray, negative ion scanning in multiple reaction monitoring mode.

[0013] The urine sample is purified and enriched by the Isolute ENV+ solid phase extraction column, and AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA all have good purification and enrichment effect, which is time-saving, convenient, and greatly improves the accuracy of quantitative analysis. Meanwhile, combined with ultra-high performance liquid chromatography tandem mass spectrometry technology, the urine biomarkers of acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD are quantified, the analysis time is greatly shortened, and the synchronous detection and quantitative analysis of AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA are realized.

[0014] The urine sample includes human urine sample, high-level mammal urine sample, etc.

[0015] Preferably, the urine biomarker internal standard is D3-AAMA, D3-GAMA, D3-iso-GAMA, D3-AAMA-sul, D3-N2FG, 13 C6-HMFA, 13 C2, 15 N-HMFG and D5-DHPMA.

[0016] Specifically, the volume ratio of the urine sample, formic acid and ammonium formate is 1:0.0025-0.01:0.5-2; the ammonium formate is preferably 50mmol / L in concentration and pH 2.5.

[0017] Preferably, the Isolute ENV+ solid phase extraction column is activated with methanol and equilibrated with water and formic acid solution.

[0018] Specifically, 0.05-0.2% formic acid solution is used for elution; formic acid acetonitrile solution is used for elution, and the volume fraction of formic acid in the formic acid acetonitrile solution is 0-4%.

[0019] Preferably, the ultra-high performance liquid chromatography conditions further include:

[0020] Chromatographic column ACQUTITY HSS T3, 2.1x150mm i.d., 1.8μm; the column pressure that the chromatographic column can bear is much higher than that of the ordinary liquid chromatographic column, so that the detection efficiency is greatly improved;

[0021] Column temperature 30-40℃;

[0022] Flow rate 0.1-0.2mL·min-1 ;

[0023] Injection volume 1-10 μL.

[0024] The mass spectrometry conditions further include:

[0025] Spray voltage -4500 V;

[0026] Ion source heating temperature 500℃;

[0027] Gas curtain gas is 40 Psi;

[0028] Atomization gas is 50 Psi;

[0029] Auxiliary heating gas is 50 psi.

[0030] Preferably, a series of standard solutions containing each urine biomarker internal standard at a concentration of 1-50000 ng / mL are prepared for detection, the standard solution is detected under the conditions of the sample solution to be detected, the ratio of the concentration of the standard solution to the concentration of the urine biomarker internal standard is taken as the abscissa (X), and the ratio of the peak area of the standard to the peak area of the urine biomarker internal standard is taken as the ordinate (Y) to obtain a standard curve.

[0031] The application also provides application of the urine biomarker synchronous detection method in the field of food safety risk analysis.

[0032] Compared with the prior art, the application has the beneficial effects that:

[0033] (1) The method of the application realizes synchronous detection of eight urine biomarkers, AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA, compared with the food matrix detection method of acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD, the internal exposure analysis is realized by directly detecting urine metabolites, which can more accurately evaluate the daily intake of the above heat processing pollutants and provide more reliable basis for epidemiological research.

[0034] (2) The pretreatment step of the method of the application is simple, the process analysis time of ultra-high performance liquid chromatography tandem mass spectrometry is short, and the efficiency is high; and the detection limit of the method of the application can reach the level of 0.2-1.2 ng / mL, and has the advantages of high sensitivity and high detection precision. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a total ion chromatogram of eight urine biomarker standards;

[0036] Figure 2 is an extracted ion chromatogram of eight urine biomarker standards;

[0037] Figure 3 Total ion chromatogram of 8 urinary biomarkers in human urine sample. DETAILED DESCRIPTION

[0038] The application will be further illustrated by the following examples and figures. It should be understood that these examples are only used to illustrate the application, and are not used to limit the scope of the application.

[0039] Specifically, by chemical synthesis and structural identification, the standards of AAMA, GAMA, iso-GAMA, AAMA-sul, D3-AAMA, D3-GAMA, D3-iso-GAMA and D3-AAMA-sul were obtained. HMFA, HMFG, N2FG, 13 C6-HMFA, 13 C2, 15 N-HMFG and D3-N2FG were purchased from Toronto Research Chemicals (Toronto, Canada). DHPMA and D5-DHPMA were synthesized by Tianjin Bonar Eagle Technology Co., Ltd. on commission.

[0040] Example 1

[0041] The simultaneous detection method of urinary biomarkers for evaluating various thermal processing contaminant exposures was evaluated using human urine as the urine sample, and the following steps were performed in turn:

[0042] (1) Sample pretreatment:

[0043] Take 1 mL of human urine sample, add 10 μL of mixed urinary biomarker internal standard (D3-AAMA, D3-GAMA, D3-iso-GAMA, D3-AAMA-sul, 13 C6-HMFA, 13 C2, 15N-HMFG, D3-N2FG and D5-DHPMA, urine biomarker internal standard concentration was 10 pg / mL, with diluent 0.1% formic acid aqueous solution as solvent, 10 pL of formic acid and 2 mL of ammonium formate (50 mmol / L, pH 2.5) were added in turn, vortexed for 30 s and centrifuged at 10000 r / min for 5 min. Then the supernatant was transferred to an Isolute ENV+ solid phase extraction column (100 mg, 3 cc, Biotage, Sweden) which was previously activated with 3 mL of methanol, equilibrated with 1.5 mL of water and 1.5 mL of 0.1% formic acid aqueous solution, eluted with 2 mL of 0.1% formic acid aqueous solution after the flow was dry, impurities were removed and the eluent was discarded, and finally eluted with 2 mL of 1% formic acid acetonitrile solution (the volume concentration of formic acid was 1%), and the eluent was collected. The eluent was blown dry with nitrogen at 40°C, and the initial mobile phase solution (mobile phase A 0.1% formic acid aqueous solution, mobile phase B 0.1% formic acid acetonitrile solution, 98% mobile phase A, 2% mobile phase B) was used to make up to 1 mL, vortexed for 1 min, filtered through a 0.22 pm microporous filter to obtain the sample solution for analysis.

[0044] (2), UPLC-MS / MS detection (2.1) chromatographic conditions:

[0045] The chromatographic column type and size was ACQUTITY HSS T3 (2.1 x 150 mm i.d., 1.8 pm);

[0046] Flow rate: 0.2 mL / min;

[0047] Column temperature: 40°C;

[0048] Injection volume was 5 pL;

[0049] 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution, the gradient elution program was shown in Table 1;

[0050] Table 1 Gradient elution program of mobile phase

[0051]

[0052] (2.2) Mass spectrometry conditions:

[0053] Instrument: triple quadrupole mass spectrometer; mass spectrometry quantification method: multiple reaction monitoring (MRM); ion source: electrospray (ESI) negative ion scanning mode; ion source temperature: 500°C; spray voltage: -4500V; curtain gas: 40 psi; atomization gas: 50 psi; auxiliary heating gas: 50 psi, the mass spectrometry parameters of 8 urine biomarkers were shown in Table 2.

[0054] Table 2 Mass spectrometry parameters of 8 urinary biomarkers

[0055]

[0056] Note: a quantitative ion channel, b qualitative ion channel

[0057] When mass spectrometry is used, AAMA, GAMA, iso-GAMA, AAMA-sul, HMFA, HMFG, N2FG and DHPMA are adducts to be analyzed, D3-AAMA, D3-GAMA, D3-iso-GAMA, D3-AAMA-sul, 13 C6-HMFA, 13 C2, 15 N-HMFG, D3-N2FG and D5-DHPMA are corresponding urinary biomarker internal standards (internal standard method quantification for adducts), and Table 2 shows a total of 32 mass spectrometry detection channels, i.e., 2 channels for each compound, the first being a quantitative ion channel, and the second being a qualitative ion channel (auxiliary quantification and confirmation). For example, for AAMA, 233.1 is the molecular ion peak of AAMA, and 103.9 and 161.9 are both characteristic fragment ion peaks of AAMA. 233.1 > 103.9 is selected as the quantitative ion channel, and 233.1 > 161.9 is selected as the qualitative ion channel, and so on. The de-clustering voltage and the collision energy are the main parameters that determine the quantitative and qualitative ion response values.

[0058] (2.3) Detection results

[0059] The detection results are quantitatively analyzed using the standard curve method. A series of standard solutions containing 100 ng of urinary biomarker internal standards at concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL, 10000 ng / mL and 50000 ng / mL are prepared, and analysis is performed using the above conditions. The total ion chromatograms and extracted ion chromatograms of the 8 urinary biomarker standards are shown in Figure 1 and 2 The ratio of the standard concentration to the urinary biomarker internal standard concentration is taken as the abscissa (X), and the ratio of the standard peak area to the urinary biomarker internal standard peak area is taken as the ordinate (Y), to obtain the corresponding linear regression equation.

[0060] The above ultra-high performance liquid chromatography tandem mass spectrometry conditions are used to detect each urine biomarker in the sample solution to be tested, and the total ion chromatogram of the eight urine biomarkers in the human urine sample is shown in Figure 3 The peak area of each urine biomarker and the urine biomarker internal standard in the sample solution to be tested is substituted into the corresponding standard curve to obtain the content of each urine biomarker in the urine sample.

[0061] (2.4) Method analysis

[0062] The LOD of the method of the present application is 0.2 ng / mL (AAMA), 0.3 ng / mL (GAMA), 0.7 ng / mL (iso-GAMA), 0.4 ng / mL (AAMA-sul), 1.2 ng / mL (HMFA), 0.9 ng / mL (HMFG), 0.9 ng / mL (N2FG) and 0.2 ng / mL (DHPMA), and a high precision has been achieved in the chromatography mass spectrometry method. The median in 5 non-smoking male populations is 83.7 ng / mL (AAMA, 39.8-173.2 ng / mL), 8.1 ng / mL (GAMA, 5.9-20.6 ng / mL), 8.6 ng / mL (iso-GAMA 5.3-9.8 ng / mL), 21.9 ng / mL (AAMA-sul, 14.7-51.1 ng / mL), 2927.2 ng / mL (HMFA, 1914.0-3386.1 ng / mL), 218.0 ng / mL (HMFG, 100.4-675.8 ng / mL), 1014.3 ng / mL (N2FG, 492.2-1383.2 ng / mL), 652.3 ng / mL (DHPMA, 357.2-1105.9 ng / mL).

[0063] The above embodiments are used to explain the technical solutions of the present application in detail, and it should be understood that the above embodiments are only specific embodiments of the present application and are not used to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection range of the present application.

Claims

1. A method for the simultaneous detection of urinary biomarkers of exposure to multiple thermal processing contaminants, comprising, The heat processing contaminants include acrylamide, furfural, furfuryl alcohol, 5-HMF and 3-MCPD, and the corresponding urine biomarkers include N-acetyl-S-(2-carbamoyl ethyl)-L-cysteine AAMA, N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine GAMA, N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine iso-GAMA, N-acetyl-S-(2-carbamoyl ethyl)-L-cysteine sulfoxide AAMA-sul, N-(2-furoyl) glycine N2FG, 5-hydroxymethyl-2-furancarboxylic acid HMFA, 5-hydroxymethyl-2-furoyl glycine HMFG and 2,3-dihydroxypropyl mercapturic acid DHPMA; The method comprises the following steps: (1) Sample pretreatment: take the urine sample, add the urine biomarker internal standard corresponding to each heat processing contaminant, then add formic acid and ammonium formate, vortex and centrifuge, take the supernatant, pass through the activated and balanced Isolute ENV+ solid phase extraction column, then perform elution and washing to obtain the eluate, and then perform nitrogen blowing, redissolution and membrane filtration to obtain the sample solution to be tested; (2) The sample solution to be tested is detected by using the ultra-high performance liquid chromatography tandem mass spectrometry technology, the peak areas of each urine biomarker and the urine biomarker internal standard in the sample solution to be tested are obtained, and the peak areas are substituted into the corresponding standard curve to obtain the content of each urine biomarker in the urine sample; The ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is ACQUTITY HSS T3, 2.1*150mm i.d., 1.8μm; formic acid aqueous solution is used as the mobile phase A, formic acid acetonitrile solution is used as the mobile phase B, and the elution program is as follows: 0-4.5 min, 2% mobile phase B; 4.5-7.0 min, 2%-100% mobile phase B; 7.0-9.0 min, 100% mobile phase B; 9.0-9.1 min, 100%-2% mobile phase B; 9.1-15.0 min, 2% mobile phase B; The mass spectrometry conditions are as follows: ionization is performed by using the electrospray mode, and negative ion scanning is performed in the multiple reaction monitoring mode.

2. The method for simultaneous detection of urine biomarkers according to claim 1, characterized in that, Urine biomarker internal standards are D3-AAMA, D3-GAMA, D3-iso-GAMA, D3-AAMA-sul, D3-N2FG, 13 C6-HMFA, 13 C2, 15 N-HMFG and D5-DHPMA.

3. The method for simultaneous detection of urine biomarkers according to claim 1, characterized in that, The volume ratio of the urine sample, formic acid and ammonium formate is 1:0.0025-0.01:0.5-2.

4. The method of claim 1, wherein the method is characterized by, The Isolute ENV+ solid phase extraction column is activated by using methanol, and balanced by using water and formic acid aqueous solution.

5. The method of claim 1, wherein the method is performed simultaneously. 0.05-0.2% formic acid aqueous solution is used for elution, and formic acid acetonitrile solution is used for elution, and the volume fraction of formic acid in the formic acid acetonitrile solution is 0-4%.

6. The method of claim 1, wherein the method is performed simultaneously. The ultra-high performance liquid chromatography conditions further include: The column temperature is 30-40℃; Flow rate 0.1-0.2 mL•min -1 ; The injection amount is 1-10 μL.

7. The method of claim 1, wherein the method is performed simultaneously. The mass spectrometry conditions further include: The spray voltage is-4500 V; The ion source heating temperature is 500℃; The curtain gas is 40 Psi; The atomization gas is 50 Psi; The auxiliary heating gas is 50 psi.

8. The method for simultaneous detection of urine biomarkers according to claim 1, characterized in that, A series of standard solutions containing each urine biomarker internal standard at a concentration of 1-50000 ng / mL is prepared. The standard solutions are detected under the same conditions as the sample solution to be detected. The ratio of the standard solution concentration to the urine biomarker internal standard concentration is used as the abscissa, and the ratio of the standard peak area to the urine biomarker internal standard peak area is used as the ordinate to obtain a standard curve.

9. Use of the urine biomarker simultaneous detection method according to any one of claims 1-8 in the field of food safety risk analysis.

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