CN111999401A - Method for detecting amine hazardous substances in food - Google Patents
Method for detecting amine hazardous substances in food Download PDFInfo
- Publication number
- CN111999401A CN111999401A CN202010708799.5A CN202010708799A CN111999401A CN 111999401 A CN111999401 A CN 111999401A CN 202010708799 A CN202010708799 A CN 202010708799A CN 111999401 A CN111999401 A CN 111999401A
- Authority
- CN
- China
- Prior art keywords
- amine
- amino
- hazards
- ammonium acetate
- acetic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for detecting amine hazardous substances in food. The detection method comprises the following steps: (1) adding a dispersing adsorbent into a sample, uniformly mixing, adding an acrylamide isotope internal standard and a heterocyclic amine compound isotope internal standard, carrying out ultrasonic extraction by using acetonitrile aqueous solution, carrying out centrifugal separation, taking supernatant nitrogen, blowing to 0.5-1mL, and carrying out constant volume by using an ammonium acetate solution containing acetic acid to obtain a purified solution; (2) adding ferroferric oxide nano particles into the purified solution, performing vortex oscillation decoloring, centrifuging, and taking the supernatant as a solution to be detected; (3) and detecting amine hazards in the liquid to be detected by using an ultra-performance liquid chromatography tandem mass spectrometry method. The method disclosed by the invention is excellent in purification effect, high in sensitivity and strong in specificity, and is suitable for simultaneous determination of various amine hazards in food.
Description
Technical Field
The invention relates to the technical field of agricultural product quality safety detection, in particular to a method for detecting amine hazards in food.
Background
The food components are complex, in the thermal processing process, reducing sugar and amino acid or protein in the food undergo Maillard reaction, caramelization reaction and other reactions, the reactions generate a series of flavor substances and also generate micromolecular ketone, aldehyde and heterocyclic compounds, wherein amine compounds such as acrylamide, heterocyclic amine and the like have carcinogenic and mutagenic effects, and the health of human bodies is harmed by eating the food containing excessive amine hazardous substances. Therefore, the research of high-throughput detection of amine hazardous substances in food is imperative.
At present, methods for detecting amine hazards in food mainly comprise gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry/mass spectrometry and the like. However, most amine hazards such as acrylamide and heterocyclic amine belong to polar and low-volatility compounds, and are easily adsorbed on a chromatographic column and a sample injector in gas chromatography and gas chromatography-mass spectrometry, so that peak shape deterioration problems such as peak broadening and tailing occur, and a target object is not easily detected at low concentration, so that a sample is often required to be subjected to a complicated derivatization step for measurement, and the sample pretreatment process is complicated; liquid chromatography is the most common analytical method, but the selectivity is poor, and the sensitivity is not required. When the liquid chromatography-mass spectrometry/mass spectrometry detection is adopted, derivatization is not needed, the operation is simple, the time is saved, the sensitivity is high, the determination of the amine compound in a target range can be achieved, and the application is more at present.
The food matrix is extremely complex and the amine based hazards are present at low levels in the food (typically ppb levels), and therefore the sample needs to be processed by appropriate extraction, enrichment and purification methods before being analyzed by the instrument. At present, the most common pretreatment methods for amine-type pests are liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Among them, LLE organic solvent consumes much and is liable to pollute the environment, and although SPE has many advantages of high recovery rate, low organic solvent consumption, etc., its adsorption effect mostly depends on the used adsorbent material. The QuEChERS technology has the characteristics of rapidness, simplicity, low price, high efficiency, durability and safety, and mainly comprises the steps of extracting a homogenized sample through acetonitrile (or acidified acetonitrile), combining a specific adsorbent with interferences such as fatty acid, organic acid, carbohydrate and the like in a matrix, and obtaining a better purification effect by using a centrifugal treatment means on the basis. However, in the actual detection process, the QuEChERS technology is found to be still incapable of effectively removing acidic substances such as caffeoylquinic acid and chlorogenic acid and impurities such as negatively charged melanoidin in the food matrix.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting amine hazards in food. The method disclosed by the invention is excellent in purification effect, high in sensitivity and strong in specificity, and is suitable for simultaneous determination of various amine hazards in food.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for detecting amine-type pests in food comprises the following steps:
(1) adding a dispersing adsorbent into a sample, uniformly mixing, adding an acrylamide isotope internal standard and a heterocyclic amine compound isotope internal standard, carrying out ultrasonic extraction by using acetonitrile aqueous solution, carrying out centrifugal separation, taking supernatant nitrogen, blowing to 0.5-1mL, and carrying out constant volume by using an ammonium acetate solution containing acetic acid to obtain a purified solution;
(2) adding ferroferric oxide nano particles into the purified solution, performing vortex oscillation decoloring, centrifuging, and taking the supernatant as a solution to be detected;
(3) detecting amine hazards in the liquid to be detected by an ultra performance liquid chromatography tandem mass spectrometry method,
wherein the acrylamide and the heterocyclic amine compound are included, the heterocyclic amine compound includes 2-amino-1, 6-dimethylimidazopyridine, 2-amino-3-methyl-3H-imidazo [4,5-F ] quinoxaline, 2-amino-3-methyl-3H-imidazo [4,5-F ] quinoline, 2-amino-3, 4-dimethyl-3H-imidazoquinoline, 2-amino-3, 8-dimethylindole [4,5-F ] quinoxaline, 2-amino-3, 7, 8-trimethyl-3H-imidazo [4,5-F ] quinoxaline, 2-amino-3, 4, 8-trimethyl-3H-imidazo [4,5-F ] quinoxaline, 9H-pyrido [3,4-B ] indole, 1-methyl-9H-pyrido [3,4-B ] indole, 3-amino-1-methyl-5H-pyrido [4,3-B ] indole, 2-amino-1-methyl-6-phenylimidazo [4,5-B ] pyridine, 3-amino-1, 4-dimethyl-5H-pyrido [4,3-B ] indoleacetic acid, 2-amino-9H-pyrido [2,3-B ] indole and 2-amino-3-methyl-9H-pyrido [2,3-B ] indole.
The invention adopts the modified QuEChERS purification, ferroferric oxide nano particle decolorization and UHPLC-MS/MS detection method, and can simultaneously and rapidly detect 15 amine hazards in food, wherein the molecular structural formula of the 15 amine hazards is shown in figure 1. The invention adopts a pretreatment method combining QuEChERS purification with ferroferric oxide nano particle decolorization, can effectively remove impurities such as pigments, acidic compounds and the like in the extracting solution, and overcomes the interference of a sample matrix, thereby ensuring that the detection method has high sensitivity, accuracy, reliability, good selectivity and specificity.
The acrylamide isotope internal standard in the invention can be selected from acrylamide-d3The isotope internal standard of the heterocyclic amine compound can be Norharman-d7。
Further, in the step (1), the mass ratio of the food sample to the dispersion adsorbent is less than or equal to 1:0.25, and more preferably 1:0.25, and the dispersion adsorbent is a PSA adsorption filler. According to the invention, PSA is used as the QuEChERS adsorbent, so that the obtained extract is lighter in color, and the matrix inhibition is obviously weakened.
Further, in the step (1), the volume percentage of acetonitrile in the acetonitrile aqueous solution is 75-90%, and more preferably 90%. The method adopts the acetonitrile-water solution to carry out ultrasonic extraction, the extraction recovery rate of the target object is higher, especially when the volume percentage of acetonitrile in the acetonitrile-water solution reaches more than 75%, the protein precipitation effect in a sample matrix is better, the acetonitrile-water extracting solution is yellow brown and slightly viscous, the matrix inhibition is serious, and the extraction recovery rate reaches more than 84%.
Further, in the step (1), the concentration of ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of acetic acid in the ammonium acetate solution containing acetic acid is 0.04%.
Further, in the step (2), the dosage ratio of the purification solution to the ferroferric oxide nanoparticles is 1.5mL to 30-50 mg, and more preferably 1.5mL to 30mg, within the range, a good decolorization effect can be obtained, and the substrate inhibition is slightly reduced.
Further, in the step (2), the vortex oscillation time is 3-5min, the centrifugation rotation speed is 10000-12000r/min, and the centrifugation time is 5-8 min.
Further, in the step (3), the chromatographic conditions are as follows:
a chromatographic column: a C18 chromatography column;
mobile phase: phase A: the ammonium acetate solution containing acetic acid, wherein the concentration of the ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of the acetic acid is 0.04%; phase B: acetonitrile;
gradient elution procedure: 0-1.0 min, 95% A; 1.0-2.0 min, 95-87% A; 2.0-5.0 min, 87-77% A; 5.0-6.0 min, 77% A; 6.0-8.5 min, 77% A-40% A; 8.5-9.0 min, 40-10% A; 9.0-9.5 min, 10% A; 9.5-10.0 min, 10% A-95% A; 10.0-12.0 min, 95% A.
Further, in the step (3), the chromatographic conditions are as follows:
a chromatographic column: phenomenex Kinetex C18 column;
flow rate: 0.3 mL/min;
the sample injection amount is 5 mu L;
column temperature: at 30 ℃.
By adopting the optimized chromatographic conditions, the invention can avoid the co-outflow of alkaloids (the chemical properties of which are similar to those of amine compounds) such as trigonelline, caffeine and the like and amine to-be-detected substances, and reduce the inhibition effect generated by competitive ionization of the impurities and the to-be-detected substances to the maximum extent.
Further, in the step (3), the mass spectrum conditions are as follows:
electrospray positive ion (ESI)+) Ionizing; spray voltage: 5.5 kV; temperature of the auxiliary gas: 400 ℃; the flow rate of the sheath gas: nitrogen gas, 30L/min; auxiliary air flow rate: nitrogen gas, 50L/min; collision gas: ar; the detection mode is as follows: the multi-reaction monitoring scanning mode adopts the optimized mass spectrum conditions, so that the detection method disclosed by the invention has better selectivity and higher sensitivity on the target compound.
The method adopts a modified QuEChERS purification-ferroferric oxide nanoparticle decolorization-UHPLC-MS/MS method to simultaneously detect various amine hazards in food, and compared with the prior detection technology, the method has the following prominent substantive advantages and remarkable progress:
(1) compared with the existing detection technology for detecting only one type of amine hazard, the analysis method has obvious high-throughput advantage and can realize the purpose of one-time sample injection detection of the two types of substances.
(2) Aiming at the complexity of the food matrix, the invention adopts a double purification means of modified QuEChERS dispersion purification and ferroferric oxide nano particle decolorization, can realize the high-efficiency impurity removal of the food matrix, and particularly comprises the following steps: 1) fe in ferroferric oxide2+、Fe3+The valence electron of (2) is easily coordinated with an atom containing a lone pair electron such as N, O, S to form specific adsorption, and the removal of the dye can be realized by the advantages of large specific surface area, many adsorption sites and the like; 2) ferroferric oxide and water generate coordination to be subjected to surface hydroxylation, so that the ferroferric oxide is positively charged, and can adsorb negatively charged substances and carboxyl-containing acidic compounds through electrostatic force, hydrogen bonds or covalent bonds.
(3) The method has high sensitivity, accuracy, reliability, good selectivity and specificity, and can overcome the interference of sample matrix. The method has the detection limit of 0.10-0.15 mu g/kg for various amine hazards in foods such as coffee and the like.
(4) The invention adopts isotope internal standard correction, and the matrix effect is obviously improved, thereby more accurately quantifying the target.
(5) The method is simple, convenient and quick, has strong operability, is suitable for detecting mass samples, provides important technical support for quality control and hazard detection of hot processed foods such as coffee and the like, and has good economic and social benefits.
Drawings
FIG. 1 is a molecular structural formula of the amine based hazard of the present invention.
FIG. 2 is an extracted ion chromatogram of 15 target amine compounds after the chromatographic mass spectrometry conditions are optimized.
FIG. 3 is a graph showing the purification and decolorization effects of an extract at different stages of extraction and purification, a-an extract obtained after extraction with 90% by volume aqueous acetonitrile; b-adsorbing the purified extracting solution by adopting PSA (pressure swing adsorption) filler; c, adopting an extracting solution treated by ferroferric oxide nano particles.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1: determination of Mass Spectrometry conditions
Selecting a positive ion mode according to the structural characteristics of acrylamide and heterocyclic amine compounds; respectively carrying out full scanning on acrylamide and isotope internal standard thereof, heterocyclic amine compounds and single standard solution of isotope internal standard thereof by adopting a peristaltic pump direct sample injection mode to obtain molecular ion peaks [ M + H ] of the compounds]+(ii) a Then, carrying out secondary mass spectrum full scanning on the molecular ions, and finding that the cracking rules of various amine compounds are similar; 2-3 main characteristic fragment ions are selected as qualitative and quantitative ions for each compound, the strength of the characteristic fragment ions is maximized by optimizing collision voltage, de-clustering voltage and the like, and the optimized mass spectrum conditions are shown in table 1.
TABLE 1 abbreviations for amine hazards and their isotopic internal standards and optimized mass spectrometry parameters
Example 2: determination of chromatographic conditions
This experiment compared the separation effect of a Phenomenex Kinetex C18 column (2.6 μm, 2.1 mm. times.100 mm) and an Acquity UPLC BEH C18 column (1.7 μm, 100 mm. times.2.1 mm). The result shows that the retention time of the micromolecule acrylamide with large polarity on a BEH C18 chromatographic column is shorter than that of a Kinetex C18 chromatographic column, and the micromolecule acrylamide is more easily interfered by polar impurities flowing out from the early stage, so the detection method in the invention preferably uses a Phenomenex Kinetex C18 chromatographic column.
In the ESI + mode, the invention also researches the influence of different acetic acid concentrations (0.02%, 0.04%, 0.06%, 0.1%, 0.2% acetic acid, volume percentage) in the ammonium acetate solution containing acetic acid on the separation effect of the substance to be detected, and as a result, the amine compounds have better response and separation conditions when the volume percentage of the acetic acid in the ammonium acetate solution is 0.04%. Under these conditions, the ammonium acetate solutions were further examined for the improvement in peak shape at different ammonium acetate concentrations (0.25mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L, 5mmol/L, 10mmol/L ammonium acetate). The results show that the addition of ammonium acetate at a certain concentration, especially when the ammonium acetate concentration in the ammonium acetate solution is 1mmol/L ammonium acetate, can effectively improve the peak shape. Therefore, 1mmol/L ammonium acetate is finally preferred in the invention, and the mobile phase system is determined as ammonium acetate solution containing acetic acid (phase A) -acetonitrile (phase B), and the concentration of ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of acetic acid is 0.04%.
In the gradient elution procedure, a large proportion of phase A (95%) is used as an initial gradient to delay the retention time of acrylamide with the maximum polarity, and the phase A can be effectively separated from trigonelline to eliminate ionization competition of the two phases which flow out together; then slowly increasing phase B to avoid high content of caffeine and target substance co-outflow, and obtaining good separation of two isomers of 7,8-DiMeIQx and 4,8-DiMeIQx, then increasing phase B ratio to rapidly elute less polar compounds. The optimized gradient elution procedure is as follows: 0-1.0 min, 95% A; 1.0-2.0 min, 95-87% A; 2.0-5.0 min, 87-77% A; 5.0-6.0 min, 77% A; 6.0-8.5 min, 77% A-40% A; 8.5-9.0 min, 40-10% A; 9.0-9.5 min, 10% A; 9.5-10.0 min, 10% A-95% A; 10.0-12.0 min, 95% A.
After the chromatographic mass spectrometry conditions are optimized, the extracted ion chromatogram of the target amine compound is shown in figure 2.
Example 3: optimization of purification conditions
In this experiment, the sample was dissolved in water, and the liquid-liquid extraction effect of acetonitrile (NaCl salting out) was examined. As a result, the acetonitrile can effectively precipitate the proteins in the sample matrix, but the target substances are not easily distributed in the acetonitrile layer, and the extraction recovery rate is low. Then, the extraction effect of acetonitrile aqueous solutions (the sample is firstly dissolved by water and then added with acetonitrile, so that the acetonitrile volume percentage is respectively 90%, 80%, 75% and 50%) with different proportions is observed, and the result shows that the precipitation effect of the acetonitrile aqueous solution with the volume percentage of more than 75% is better, the acetonitrile-water extracting solution is yellowish brown and slightly viscous, the matrix inhibition is serious, and the extraction recovery rate is about 84% or more. Finally, acetonitrile water solution with the acetonitrile volume percentage of 90 percent is selected as an extraction solvent for further optimization.
PSA adsorption filler is selected as a QuEChERS adsorbent in the experiment, and the influence of different PSA adsorption filler addition amounts on the extracting solution is examined. The result shows that the extracting solution without adding PSA adsorbing filler is dark brown, the matrix inhibition is obvious, and the matrix effect is between 0.13 and 0.44; after the PSA adsorption filler is added, the color of the extracting solution is lightened and is in a tea yellow color, the matrix inhibition is obviously weakened, and especially when the mass ratio of the sample to the PSA adsorption filler is less than or equal to 1:0.25, the color removal and the matrix effect of the final extracting solution are not obviously different (the matrix effect value is between 0.61 and 0.88). Thus, finally, the present invention prefers PSA sorptive fillers as the dispersing adsorbent, with the mass ratio of sample to PSA sorptive filler being 1: 0.25.
Example 4: further decolorization of the purified liquor
As shown in figure 3, after QuEChERS extraction and purification are carried out by adopting PSA adsorption packing, the extracting solution is still in a tea yellow color after being concentrated to a constant volume. Therefore, the invention continuously considers the decolorization effect of the ferroferric oxide nanoparticles (the dosage ratio of the purification solution to the ferroferric oxide nanoparticles is 1.5mL:20mg, 1.5mL:30mg, 1.5mL:40mg and 1.5mL:50 mg). The results show that when the dosage ratio of the purifying solution to the ferroferric oxide nano particles is 1.5mL to 30mg, better decolorizing effect is obtained (figure 3), and simultaneously matrix inhibition is slightly further reduced (matrix effect value is between 0.76 and 1.11). Therefore, the ferroferric oxide nano particles are selected for further adsorption and decoloration, and the dosage ratio of the purifying solution to the ferroferric oxide nano particles is preferably 1.5mL to 30 mg.
Example 4: detection of amine-type hazardous substances in instant coffee and coffee powder
(1) Preparation of Standard solutions
Respectively weighing a proper amount of each amine hazardous substance standard, dissolving with methanol, and fixing the volume to prepare a single-standard stock solution; diluting with acetonitrile step by step to prepare mixed standard solution with mass concentration of 1.0mg/L and acylamide-d with mass concentration of 5.0mg/L3And Norharman-d7Mixing the internal standard working solution. When the preparation is used, the preparation is diluted into mixed standard working solution with required concentration by using ammonium acetate solution containing acetic acid in initial mobile phase, namely acetonitrile 95:5(v: v), and each working solution contains 10 mu g/L of acylamide-d3And 10. mu.g/L Norharman-d7Wherein the concentration of ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of acetic acid in the ammonium acetate solution containing acetic acid is 0.04%.
(2) Sample extraction and purification
Weighing 0.2g (accurate to 0.001g) of the mixed sample into a 50mL plastic centrifuge tube, adding 50mg of PSA adsorption filler, mixing by vortex, adding 20 μ L of acylamide-d with concentration of 5.0mg/L3And Norharman-d7Mixing internal standard working solution and 10mL acetonitrile aqueous solution with the volume percentage of 90%, performing vortex oscillation for 1min, performing ultrasonic extraction for 20min, centrifuging for 5min at 4000 r/min, and sucking supernatant toBlowing the solution to about 0.7mL (not less than 0.7mL) in a nitrogen blowing tube at 40 ℃ in a water bath, using an ammonium acetate solution containing acetic acid to perform constant volume to 2.0mL, wherein the concentration of ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, the volume percentage of acetic acid is 0.04%, uniformly mixing by vortex, transferring 1.5mL into a 2mL plastic centrifuge tube filled with 30mg of ferroferric oxide nanoparticles, performing vortex oscillation for 1min, centrifuging at 12000r/min for 3min, and taking the supernatant as a liquid to be detected.
(3) UHPLC-MS/MS assay
Respectively injecting the matrix standard solution and the solution to be detected under the following UHPLC-MS/MS conditions, making a matrix standard curve, and calculating the content of corresponding amine hazards in the solution to be detected according to the matrix standard curve and an internal standard method.
A chromatographic column: phenomenex Kinetex C18 column (2.6 μm, 2.1 mm. times.100 mm); mobile phase: phase A: the ammonium acetate solution containing acetic acid, wherein the concentration of the ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of the acetic acid is 0.04%; phase B: acetonitrile; gradient elution procedure: 0-1.0 min, 95% A; 1.0-2.0 min, 95-87% A; 2.0-5.0 min, 87-77% A; 5.0-6.0 min, 77% A; 6.0-8.5 min, 77% A-40% A; 8.5-9.0 min, 40-10% A; 9.0-9.5 min, 10% A; 9.5-10.0 min, 10% A-95% A; 10.0-12.0 min, 95% A. Flow rate: 0.3 mL/min; sample size 5 μ L, column temperature: at 30 ℃.
Mass spectrum conditions: electrospray positive ion (ESI)+) Ionizing; spraying voltage: 5.5 kV; temperature of the auxiliary gas: 400 ℃; the flow rate of the sheath gas: nitrogen gas, 30L/min; auxiliary air flow rate: nitrogen gas, 50L/min; collision gas: ar; the detection mode is as follows: multiple reaction monitoring scan mode (MRM); the mass spectrometry acquisition parameters are shown in table 1.
(4) Selectivity of the process
In the experiment, 10 coffee powder and instant coffee samples are selected, pretreatment and detection are carried out according to the sample pretreatment method and the instrument conditions, and whether interference of other components in the samples on the determination of an object to be detected exists or not is examined by combining a labeling experiment. The result shows that the coexisting substance in the sample solution has no interference to the qualitative and quantitative determination of the substance to be detected because of the high selectivity of the triple quadrupole mass spectrometry.
(5) Detection limit and matrix Effect of the method
The instrument detection limit (LOD, S/N is more than or equal to 3) and the quantification limit (LOQ, S/N is more than or equal to 10) are calculated by the signal-to-noise ratio (S/N). As can be seen from Table 2, the linear relationship of the amine-type noxious substances in the corresponding concentration ranges is good, and the correlation coefficients are all larger than 0.996; the detection limit is 0.02-0.15 mug/kg.
Table 2 detection limits and matrix effects of amine hazards
The ME of DMIP, PhIP, AaC and MeAaC is between 0.95 and 1.09, and basically has no matrix effect, so that the experiment adopts an external standard method for quantification; ME of IQ, MeIQx, Harman and Trp-P-2 is between 0.81 and 0.87, the matrix inhibition effect is weak and can be basically ignored, but the internal standard method is adopted for quantification in the experiment, so that the accuracy of the quantification result is further corrected; ME of other 7 amines is between 0.74 and 0.80, which indicates that stronger matrix inhibition exists, and the invention adopts an internal standard method for quantification.
(5) Recovery and precision of the process
Method recovery, accuracy and precision were investigated by an additive recovery test (n ═ 6) of 1 instant coffee sample. That is, 3 concentration levels of the mixed standard solutions were added to each sample, sample treatment and measurement were performed under the present experimental conditions, 6 tests were performed in parallel, the second addition level was selected and continuously tested for 5 days, and the recovery rate (calculated by external standard method, and the rest by internal standard method, for DMIP, MeAaC, AaC, and PhIP), the in-day precision (n ═ 6), and the in-day precision (n ═ 5) were calculated, and the results are shown in table 3. It can be seen that at 3 addition levels, the daily average recovery rate is 81.6% to 100%, and the daily precision (n ═ 6) is between 4.3% to 9.0%; the average recovery rate in the daytime is 81.0-101%, and the precision in the daytime (n is 5) is 5.0-7.8%. The method has the advantages of high recovery rate, high accuracy and high precision.
TABLE 3 recovery and precision of the process
(6) Detection of actual coffee powder and instant coffee samples
The detection method established in the invention is used for detecting and analyzing 12 parts of coffee powder and instant coffee samples. The detection result shows that AA, Harman and Norharman are detected in the sample, and the content is respectively between 5.0 and 35 mu g/kg, 550 mu g/kg and 460 mu g/kg, wherein the content is between 120 and 150.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A method for detecting amine-type hazards in food is characterized by comprising the following steps:
(1) adding a dispersing adsorbent into a sample, uniformly mixing, adding an acrylamide isotope internal standard and a heterocyclic amine compound isotope internal standard, carrying out ultrasonic extraction by using acetonitrile aqueous solution, carrying out centrifugal separation, taking supernatant nitrogen, blowing to 0.5-1mL, and carrying out constant volume by using an ammonium acetate solution containing acetic acid to obtain a purified solution;
(2) adding ferroferric oxide nano particles into the purified solution, performing vortex oscillation decoloring, centrifuging, and taking the supernatant as a solution to be detected;
(3) detecting amine hazards in the liquid to be detected by an ultra performance liquid chromatography tandem mass spectrometry method,
wherein the amine based hazard comprises acrylamide and a heterocyclic amine based compound comprising 2-amino-1, 6-dimethylimidazopyridine, 2-amino-3-methyl-3H-imidazo [4,5-F ] quinoxaline, 2-amino-3-methyl-3H-imidazo [4,5-F ] quinoline, 2-amino-3, 4-dimethyl-3H-imidazoquinoline, 2-amino-3, 8-dimethylindol [4,5-F ] quinoxaline, 2-amino-3, 7, 8-trimethyl-3H-imidazo [4,5-F ] quinoxaline, 2-amino-3, 4, 8-trimethyl-3H-imidazo [4,5-F ] quinoxaline, 5-F ] quinoxaline, 9H-pyrido [3,4-B ] indole, 1-methyl-9H-pyrido [3,4-B ] indole, 3-amino-1-methyl-5H-pyrido [4,3-B ] indole, 2-amino-1-methyl-6-phenylimidazo [4,5-B ] pyridine, 3-amino-1, 4-dimethyl-5H-pyrido [4,3-B ] indoleacetic acid, 2-amino-9H-pyrido [2,3-B ] indole and 2-amino-3-methyl-9H-pyrido [2,3-B ] indole.
2. The method for detecting amine-type hazards in food according to claim 1, wherein in the step (1), the mass ratio of the food sample to the dispersed adsorbent is less than or equal to 1:0.25, preferably 1:0.25, and the dispersed adsorbent is PSA adsorption packing.
3. The method for detecting amine-type hazards in food according to claim 1, wherein in said step (1), the volume percentage of acetonitrile in the acetonitrile water solution is 75% to 90%.
4. The method for detecting amine-type hazards in food according to claim 3 wherein in said step (1), the volume percentage of acetonitrile in the acetonitrile water solution is 90%.
5. The method for detecting amine-type hazardous substances in food according to claim 1, wherein in the step (1), the concentration of ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of acetic acid in the ammonium acetate solution containing acetic acid is 0.04%.
6. The method for detecting amine hazards in food according to claim 1, wherein in the step (2), the dosage ratio of the purification solution to the ferroferric oxide nanoparticles is 1.5mL: 30-50 mg.
7. The method for detecting amine hazards in food according to claim 6, wherein in the step (2), the dosage ratio of the purification solution to the ferroferric oxide nanoparticles is 1.5mL to 30 mg.
8. The method for detecting amine-type hazards in food according to claim 1, wherein in said step (3), the chromatographic conditions are as follows:
a chromatographic column: a C18 chromatography column;
mobile phase: phase A: the ammonium acetate solution containing acetic acid, wherein the concentration of the ammonium acetate in the ammonium acetate solution containing acetic acid is 1mmol/L, and the volume percentage of the acetic acid is 0.04%; phase B: acetonitrile;
gradient elution procedure: 0-1.0 min, 95% A; 1.0-2.0 min, 95-87% A; 2.0-5.0 min, 87-77% A; 5.0-6.0 min, 77% A; 6.0-8.5 min, 77% A-40% A; 8.5-9.0 min, 40-10% A; 9.0-9.5 min, 10% A; 9.5-10.0 min, 10% A-95% A; 10.0-12.0 min, 95% A.
9. The method for detecting amine-type hazards in food according to claim 8, wherein in said step (3), the chromatographic conditions are as follows:
a chromatographic column: phenomenex Kinetex C18 column;
flow rate: 0.3 mL/min;
the sample injection amount is 5 mu L;
column temperature: at 30 ℃.
10. The method for detecting amine-type hazards in food according to claim 1, wherein in said step (3), the mass spectrometric conditions are as follows:
electrospray positive ion ionization; spraying voltage: 5.5 kV; temperature of the auxiliary gas: 400 ℃; the flow rate of the sheath gas: nitrogen gas, 30L/min; auxiliary air flow rate: nitrogen gas, 50L/min; collision gas: ar; the detection mode is as follows: multiple reaction monitoring scan mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010708799.5A CN111999401A (en) | 2020-07-21 | 2020-07-21 | Method for detecting amine hazardous substances in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010708799.5A CN111999401A (en) | 2020-07-21 | 2020-07-21 | Method for detecting amine hazardous substances in food |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111999401A true CN111999401A (en) | 2020-11-27 |
Family
ID=73467313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010708799.5A Pending CN111999401A (en) | 2020-07-21 | 2020-07-21 | Method for detecting amine hazardous substances in food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111999401A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112881568A (en) * | 2021-01-15 | 2021-06-01 | 大连工业大学 | Method for simultaneously determining multiple associated harmful substances in food thermal processing |
| CN113156034A (en) * | 2021-01-17 | 2021-07-23 | 北京工业大学 | Method for rapidly detecting various coffee flavor substances |
| CN113189231A (en) * | 2021-04-27 | 2021-07-30 | 中国农业科学院农业质量标准与检测技术研究所 | Method for determining content of 14 heterocyclic amines in food |
| CN113419018A (en) * | 2021-07-28 | 2021-09-21 | 福州海关技术中心 | Pretreatment method of incense sample and method for detecting various cannabinoids in incense smoke |
| CN114646706A (en) * | 2022-03-24 | 2022-06-21 | 沈阳农业大学 | Detection method for simultaneously detecting heterocyclic amine, acrylamide and nitrosamine in meat product |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467654A (en) * | 2013-09-12 | 2013-12-25 | 厦门大学 | Magnetic heterocyclic amine molecularly imprinted polymer and preparation method thereof |
| CN111398458A (en) * | 2020-04-01 | 2020-07-10 | 山东省食品药品检验研究院 | Improved method for rapidly determining acrylamide in vegetable oil by QuEChERS-L C-MS/MS |
-
2020
- 2020-07-21 CN CN202010708799.5A patent/CN111999401A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467654A (en) * | 2013-09-12 | 2013-12-25 | 厦门大学 | Magnetic heterocyclic amine molecularly imprinted polymer and preparation method thereof |
| CN111398458A (en) * | 2020-04-01 | 2020-07-10 | 山东省食品药品检验研究院 | Improved method for rapidly determining acrylamide in vegetable oil by QuEChERS-L C-MS/MS |
Non-Patent Citations (3)
| Title |
|---|
| YANPING XIAN 等: "Modified QuEChERS purification and Fe3O4 nanoparticle decoloration for robust analysis of 14 heterocyclic aromatic amines and acrylamide in coffee products using UHPLC-MS/MS", 《FOOD CHEMISTRY》 * |
| 张峰 等: "固相萃取-超高效液相色谱-电喷雾串联质谱法同时测定烤鱼中15种杂环胺含量", 《分析化学》 * |
| 阴永泼 等: "QuEChERS前处理结合HPLC-MS法检测淀粉类食品中丙烯酰胺", 《食品科技》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112881568A (en) * | 2021-01-15 | 2021-06-01 | 大连工业大学 | Method for simultaneously determining multiple associated harmful substances in food thermal processing |
| CN112881568B (en) * | 2021-01-15 | 2023-11-17 | 大连工业大学 | Method for simultaneously measuring multiple associated harmful substances in food thermal processing |
| CN113156034A (en) * | 2021-01-17 | 2021-07-23 | 北京工业大学 | Method for rapidly detecting various coffee flavor substances |
| CN113156034B (en) * | 2021-01-17 | 2023-07-21 | 北京工业大学 | Method for rapidly detecting various coffee flavor substances |
| CN113189231A (en) * | 2021-04-27 | 2021-07-30 | 中国农业科学院农业质量标准与检测技术研究所 | Method for determining content of 14 heterocyclic amines in food |
| CN113419018A (en) * | 2021-07-28 | 2021-09-21 | 福州海关技术中心 | Pretreatment method of incense sample and method for detecting various cannabinoids in incense smoke |
| CN113419018B (en) * | 2021-07-28 | 2022-06-17 | 福州海关技术中心 | Pretreatment method of incense sample and method for detecting various cannabinoids in incense smoke |
| CN114646706A (en) * | 2022-03-24 | 2022-06-21 | 沈阳农业大学 | Detection method for simultaneously detecting heterocyclic amine, acrylamide and nitrosamine in meat product |
| CN114646706B (en) * | 2022-03-24 | 2023-09-29 | 沈阳农业大学 | Detection method for simultaneously detecting heterocyclic amine, acrylamide and nitrosamine in meat product |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111999401A (en) | Method for detecting amine hazardous substances in food | |
| Dong et al. | Modified QuEChERS combined with ultra high performance liquid chromatography tandem mass spectrometry to determine seven biogenic amines in Chinese traditional condiment soy sauce | |
| Alaejos et al. | Analytical methods applied to the determination of heterocyclic aromatic amines in foods | |
| Gökmen et al. | Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry | |
| Rubert et al. | Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples | |
| Tölgyesi et al. | Determination of tetracyclines in pig and other meat samples using liquid chromatography coupled with diode array and tandem mass spectrometric detectors | |
| Kokkonen et al. | Determination of ergot alkaloids from grains with UPLC‐MS/MS | |
| CN110243980B (en) | High-throughput detection method for 92 forbidden industrial dyes in food | |
| Kataoka et al. | Determination of the oxidative stress biomarker urinary 8-hydroxy-2⿲-deoxyguanosine by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography⿿ tandem mass spectrometry | |
| Guo et al. | A reliable and accurate UHPLC-MS/MS method for screening of Aspergillus, Penicillium and Alternaria mycotoxins in orange, grape and apple juices | |
| CN110501438B (en) | Detection method of sodium picosulfate in weight-reducing tea | |
| Ruan et al. | Determination of ochratoxin A and citrinin in fruits using ultrasound-assisted solvent extraction followed by dispersive liquid–liquid microextraction with HPLC with fluorescence detection | |
| Chen et al. | Determination of ellagic acid in wine by solid-phase extraction–ultra-high performance liquid chromatography–tandem mass spectrometry | |
| Chen et al. | Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken | |
| Cheng et al. | Sensitive analysis of phthalate esters in plastic bottled water via on-line capillary solid-phase microextraction liquid chromatography electrospray ionization-ion trap mass spectrometry | |
| CN110702816B (en) | Liquid chromatography-mass spectrometry detection method for detecting acidic azo colorant in dry animal aquatic product | |
| Li et al. | Determination of 4 psychoactive substances in tea using ultra high performance liquid chromatography combined with the quadrupole time-of-flight mass spectrometry | |
| Yao et al. | HILIC‐UPLC‐MS/MS combined with hierarchical clustering analysis to rapidly analyze and evaluate nucleobases and nucleosides in Ginkgo biloba leaves | |
| CN105938102B (en) | Method for rapidly determining pesticide residues in fruits and vegetables by chemical color development method | |
| CN112834680B (en) | Method for determining concentrations of tegafur, gimeracil and 5-fluorouracil in blood plasma of tumor patient | |
| CN111474279B (en) | Method and kit for detecting macrolide antibiotic compounds | |
| CN112198249B (en) | Detection method of ethanolamine compound in soil | |
| CN111307992B (en) | Pre-column derivative liquid chromatography-mass spectrometry analysis method for quantitatively detecting organic acid in PM2.5 | |
| CN111323510B (en) | Method for determining quinolone veterinary drug residues in chicken | |
| Zhang et al. | Melatonin in different food samples: Recent update on distribution, bioactivities, pretreatment and analysis techniques |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201127 |
|
| RJ01 | Rejection of invention patent application after publication |