CN112858521A - Method for detecting biogenic amine in aquatic product - Google Patents
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Abstract
The invention relates to the technical field of detection of biogenic amine in aquatic products, and particularly relates to a method for detecting biogenic amine in aquatic products. The method for detecting biogenic amine in aquatic products comprises the following steps: (a) mixing a sample to be detected containing biogenic amine, a formic acid acetonitrile solution and water, and then carrying out vortex treatment; then carrying out homogenization treatment and ultrasonic treatment; carrying out solid-liquid separation on the sample subjected to ultrasonic treatment; mixing the liquid with a sodium carbonate solution and a benzoyl chloride solution for incubation, mixing with an internal standard solution, and centrifuging; (b) and (b) mixing the centrifuged supernatant in the step (a) with water, and detecting by using an ultra-high liquid chromatography-tandem mass spectrometry method. The method can be used for efficiently and accurately detecting various biogenic amines at the same time.
Description
Technical Field
The invention relates to the technical field of detection of biogenic amine in aquatic products, and particularly relates to a method for detecting biogenic amine in aquatic products.
Background
In recent years, biogenic amine poisoning in aquatic products sometimes occurs, especially in coastal areas. The generation of biogenic amine not only reduces the quality of aquatic products, but also influences the health and life safety of human beings. The variety of biogenic amines is various, and the key problem of the aquatic product industry is how to accurately measure and effectively control the content of various biogenic amines simultaneously. At present, an effective method for simultaneously and accurately detecting various biogenic amines is not available, so that a method for simultaneously and accurately detecting various biogenic amines is urgently needed to be developed to provide a theoretical basis for safety control of aquatic products.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting biogenic amine in aquatic products, which is simple, efficient and high in accuracy.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the method for detecting biogenic amine in aquatic products comprises the following steps:
(a) mixing a sample to be detected containing biogenic amine, a formic acid acetonitrile solution and water, and then carrying out vortex treatment; then carrying out homogenization treatment and ultrasonic treatment; carrying out solid-liquid separation on the sample subjected to ultrasonic treatment; mixing the liquid with a sodium carbonate solution and a benzoyl chloride solution for incubation, mixing with an internal standard solution, and centrifuging;
(b) and (b) mixing the centrifuged supernatant in the step (a) with water, and detecting by using an ultra-high liquid chromatography-tandem mass spectrometry method.
Preferably, the biogenic amine comprises at least one of histamine, tyramine, dopamine, tryptamine, 5-hydroxytryptamine, putrescine, spermine, spermidine, octopamine, benzylamine, phenethylamine, cadaverine, beta-phenethylamine and agmatine.
Preferably, the phase A of the ultra-high liquid chromatography is formic acid-ammonium formate solution, the molar concentration of the ammonium formate is 1-2 mM, and the volume concentration of the formic acid is 0.08-0.15%; and the phase B of the ultra-high liquid chromatogram is acetonitrile.
Preferably, the temperature of a column incubator of the ultra-high liquid chromatography is 38-42 ℃, the temperature of a sample plate is 5-7 ℃, and the volume of sample injection is 1.8-2.2 mu L.
Preferably, the mass spectrometry conditions are electrospray ionization positive ion mode, multiple reaction monitoring scan mode, and the ion source parameters include: the ion spray voltage is 4900-5100V, the pressure of the gas curtain gas is 33-36 psi, the temperature is 395-405 ℃, the pressure of the ion source gas 1 is 58-62 psi, and the pressure of the ion source gas 2 is 58-62 psi.
Preferably, the time of the vortex treatment is 28-35 s.
Preferably, the frequency of the homogenization treatment is 35-45 Hz, and the time is 230-250 s.
Preferably, the ultrasonication treatment is carried out in an ice-water bath;
preferably, the ultrasonic treatment time is 4.5-5.5 min;
preferably, the number of the homogenization treatment and the ultrasonic treatment is 2 to 3.
Preferably, the solid-liquid separation comprises standing precipitation and centrifugation;
preferably, the standing precipitation is carried out at-38 to-42 ℃;
preferably, the centrifugation in the solid-liquid separation is carried out at 3.5-4.4 ℃, the rotation speed of the centrifugation in the solid-liquid separation is 11500-12500 rpm, and the centrifugation time in the solid-liquid separation is 13-16 min.
Preferably, the incubation time is 25-35 min;
preferably, the centrifugation is carried out at 3.5-4.4 ℃;
preferably, the rotating speed of the centrifugation is 11500-12500 rpm, and the time of the centrifugation is 13-16 min.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting biogenic amine in aquatic products is simple and easy to implement, high in efficiency and high in accuracy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an extracted ion chromatogram of a standard solution according to the present invention;
FIG. 2 is an extracted ion chromatogram of a sample according to the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
According to one aspect of the invention, the invention relates to a method for detecting biogenic amines in aquatic products, comprising the following steps:
(a) mixing a sample to be detected containing biogenic amine, a formic acid acetonitrile solution and water, and then carrying out vortex treatment; then carrying out homogenization treatment and ultrasonic treatment; carrying out solid-liquid separation on the sample subjected to ultrasonic treatment; mixing the liquid with a sodium carbonate solution and a benzoyl chloride (L-BzCl) solution for incubation, mixing with an internal standard solution, and centrifuging;
(b) and (b) mixing the centrifuged supernatant in the step (a) with water, and detecting by using an ultra-high liquid chromatography-tandem mass spectrometry method.
Preferably, the internal standard solution is H-std-a-Bz-DF. The preparation method of the H-std-a-Bz-DF comprises the following steps:
1. preparing an internal standard mixed standard (H-std) with corresponding concentration;
2. sucking 10 μ L of 80% ACN added with 70 μ L of 0.1% FA to obtain diluted mixed standard (H-Std-a);
3.160μL H-Std-a+80μL 100mM Na2CO3+ 80. mu.L of 2% benzoyl chloride-D5 solution (deuterium isotope substitution), and incubating at room temperature for 30min to obtain derivative mixed standard H-Std-a-Bz; 320 mu L of the derived mixed standard H-Std-a-Bz is added with 1680 mu L of 65% acetonitrile (0.1% formic acid) to obtain the internal standard H-Std-a-Bz-DF.
Preferably, the biogenic amine comprises at least one of histamine, tyramine, dopamine, tryptamine, 5-hydroxytryptamine, putrescine, spermine, spermidine, octopamine, benzylamine, phenethylamine, cadaverine, beta-phenethylamine and agmatine.
The detection method can be used for efficiently and accurately detecting 14 kinds of biogenic amines including histamine, tyramine, dopamine, tryptamine, 5-hydroxytryptamine, putrescine, spermine, spermidine, octopamine, benzylamine, phenethylamine, cadaverine, beta-phenethylamine and agmatine in aquatic products.
Preferably, the phase A of the ultra-high liquid chromatography is formic acid-ammonium formate solution, the molar concentration of the ammonium formate is 1-2 mM, and the volume concentration of the formic acid is 0.08-0.15%; and the phase B of the ultra-high liquid chromatogram is acetonitrile.
The present invention employs gradient elution, with mobile phase gradients as shown in table 1. In the embodiments 1 to 3 of the invention, gradient elution is carried out by adopting the mobile phase gradient in the table 1.
TABLE 1 gradient of mobile phase
In one embodiment, the ammonium formate is present in a molar concentration of 1 to 2mM, and optionally 1mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, or 2 mM.
In one embodiment, the formic acid is present at a concentration of 0.08% to 0.15% by volume, optionally 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15%.
Preferably, the temperature of a column incubator of the ultra-high liquid chromatography is 38-42 ℃, the temperature of a sample plate is 5-7 ℃, and the volume of sample injection is 1.8-2.2 mu L.
In one embodiment, the temperature of the column oven of the ultra-high performance liquid chromatography is 38-42 ℃, and 38 ℃, 39 ℃, 40 ℃, 41 ℃ or 42 ℃ can be selected.
In one embodiment, the temperature of the sample plate is 5-7 ℃, and may be selected from 5 ℃, 6 ℃ or 7 ℃.
In one embodiment, the volume of the sample injection is 1.8-2.2. mu.L, and optionally 1.8. mu.L, 1.9. mu.L, 2. mu.L, 2.1. mu.L or 2.2. mu.L.
Preferably, the mass spectrometry conditions are electrospray ionization positive ion mode, multiple reaction monitoring scan mode, and the ion source parameters include: the ion spray voltage is 4900-5100V, the pressure of the gas curtain gas is 33-36 psi, the temperature is 395-405 ℃, the pressure of the ion source gas 1 is 58-62 psi, and the pressure of the ion source gas 2 is 58-62 psi.
Preferably, the ion source parameters include: the ion spray voltage was 5000V, the gas curtain pressure was 35psi, the temperature was 400 deg.C, the ion source gas 1 pressure was 60psi, and the ion source gas 2 pressure was 60 psi.
Preferably, the time of the vortex treatment is 28-35 s.
In one embodiment, the time of the vortex treatment is 28-35 s, and 28s, 29s, 30s, 31s, 32s, 33s, 34s or 35s can be selected.
Preferably, the frequency of the homogenization treatment is 35-45 Hz, and the time is 230-250 s.
In one embodiment, the frequency of the homogenization treatment is 35-45 Hz, and 35Hz, 36Hz, 37Hz, 38Hz, 39Hz, 40Hz, 41Hz, 42Hz, 43Hz, 44Hz or 45Hz, Hz can be selected.
In one embodiment, the time for the homogenization treatment is 230 to 250s, and optionally 231s, 235s, 237s, 240s, 242s, 245s, 247s, or 250 s.
Preferably, the sonication is carried out in an ice-water bath.
Preferably, the time of the ultrasonic treatment is 4.5-5.5 min.
In one embodiment, the ultrasonic treatment time is 4.5-5.5 min, and optionally 4.5min, 4.6min, 4.7min, 4.8min, 4.9min, 5min, 5.1min, 5.2min, 5.3min, 5.4 min or 5.5 min.
Preferably, the number of the homogenization treatment and the ultrasonic treatment is 2 to 3.
In the present invention, it is preferable to repeat the homogenization treatment and the sonication treatment 3 times.
Preferably, the solid-liquid separation comprises standing precipitation and centrifugation.
Preferably, the standing precipitation is carried out at-38 to-42 ℃.
In one embodiment, the temperature of the standing precipitate is-38 to-42 ℃, and can also be selected from-38 ℃, -39 ℃, -40 ℃, -41 ℃ or-42 ℃.
Preferably, the centrifugation in the solid-liquid separation is carried out at 3.5-4.4 ℃, the rotation speed of the centrifugation in the solid-liquid separation is 11500-12500 rpm, and the centrifugation time in the solid-liquid separation is 13-16 min.
In one embodiment, the temperature of centrifugation in the solid-liquid separation is 3.5 to 4.4 ℃, and 3.5 ℃, 3.6 ℃, 3.7 ℃, 3.8 ℃, 3.9 ℃, 4 ℃, 4.1 ℃, 4.2 ℃, 4.3 ℃ or 4.4 ℃ can be selected.
In one embodiment, the rotation speed of the centrifugation in the solid-liquid separation is 11500-12500 rpm, and 11500rpm, 11600rpm, 11700rpm, 11800rpm, 11900rpm, 12000rpm, 12100rpm, 12200rpm, 12300rpm, 12400rpm or 12500rpm can be selected.
In one embodiment, the time of centrifugation in the solid-liquid separation is 13-16 min, and can be selected from 13min, 14min, 15min or 16 min.
Preferably, the incubation time is 25-35 min.
In one embodiment, the incubation time is 25-35 min, and can be 25min, 30min or 35 min.
Preferably, the centrifugation is carried out at 3.5 to 4.4 ℃.
Preferably, the rotating speed of the centrifugation is 11500-12500 rpm, and the time of the centrifugation is 13-16 min.
In the invention, the internal standard solution is added and then centrifugation is carried out.
In one embodiment, the centrifugation temperature is 3.5-4.4 deg.C, and optionally 3.5 deg.C, 3.6 deg.C, 3.7 deg.C, 3.8 deg.C, 3.9 deg.C, 4 deg.C, 4.1 deg.C, 4.2 deg.C, 4.3 deg.C or 4.4 deg.C.
In one embodiment, the rotation speed of the centrifugation is 11500-12500 rpm, and 11500rpm, 11600rpm, 11700rpm, 11800rpm, 11900rpm, 12000rpm, 12100rpm, 12200rpm, 12300rpm, 12400rpm or 12500rpm can be selected.
In one embodiment, the centrifugation time is 13-16 min, and can be selected from 13min, 14min, 15min or 16 min.
In a preferred embodiment, the method for detecting biogenic amines in aquatic products comprises the following steps:
(I) sample pretreatment
(a) Weighing a proper amount of sample to be detected in a 2.0mL EP tube, and adding a steel ball;
(b) adding 80 μ L of extractive solution (acetonitrile containing 0.1% formic acid, precooling at-20 deg.C) and 20 μ L of ethanol2O, uniformly mixing by swirling for 28-35 s;
(c) homogenizing at 35-45 Hz for 230-250 s, performing ice-water bath ultrasonic treatment for 4.5-5.5 min, and repeating the steps of homogenizing and ultrasonic treatment for 3 times;
(d) precipitating the sample after ultrasonic treatment at-38 to-42 ℃ overnight;
(e) centrifuging the sample at 3.5-4.4 ℃ and 11500-12500 rpm for 13-16 min, taking 80 mu L of supernatant (Spl), and adding 40 mu L of 100mM/L Na2CO3And 40 mu L of 2% L-BzCl, and incubating for 25-35 min at room temperature; taking Spl 160 mu L after derivation, adding 10 mu L H-std-a-Bz-DF internal standard;
(f) centrifuging the sample at 3.5-4.4 ℃ and 11500-12500 rpm for 13-16 min, taking 40 mu L of the sample, and adding 20 mu L H2O, detecting on a computer;
(II) detection on computer
1. Mobile phase conditions:
the present invention uses SCIEX ExionLC ultra high performance liquid chromatograph, and uses a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm) liquid chromatographic column to perform chromatographic separation on the target compound. Liquid chromatography phase A was 0.1% formic acid &1mM aqueous ammonium formate and phase B was acetonitrile. The temperature of the column incubator is 38-42 ℃, the sample tray is set to be 5-7 ℃, and the sample introduction volume is 1.8-2.2 mu L;
AB Sciex QTrap 6500 Mass Spectrometry instrument parameter settings:
the mass spectrum conditions are an electrospray ionization negative ion mode and a multi-reaction monitoring scanning mode, and the ion source parameters comprise: the ion spray voltage is 4900-5100V, the pressure of the gas curtain gas is 33-36 psi, the temperature is 395-405 ℃, the pressure of the ion source gas 1 is 58-62 psi, and the pressure of the ion source gas 2 is 58-62 psi.
All target compound data analysis qualitative and quantitative work was done by analysis 1.63 and Multiquant 3.03 software.
The invention will be further explained with reference to specific examples.
Example 1
The method for detecting biogenic amine in aquatic products comprises the following steps:
(I) sample pretreatment
(a) Weighing a proper amount of sample to be detected in a 2.0mL EP tube, and adding a steel ball;
(b) adding 80 μ L of extractive solution (acetonitrile containing 0.1% formic acid, precooling at-20 deg.C) and 20 μ L of ethanol2O, uniformly mixing by swirling for 30 s;
(c) homogenizing at 40Hz for 240s, performing ultrasonic treatment in ice water bath for 5min, and repeating the steps of homogenizing and ultrasonic treatment for 3 times;
(d) samples were precipitated overnight at-40 ℃;
(e) the sample was centrifuged at 12000rpm for 15min at 4 ℃ to give 80. mu.L of supernatant (Spl), and 40. mu.L of 100mM/L Na was added2CO3And 40. mu.L of 2% L-BzCl, incubated at room temperature for 30 min; taking Spl 160 mu L after derivation, adding 10 mu L H-std-a-Bz-DF internal standard;
(f) the sample was centrifuged at 12000rpm for 15min at 4 ℃ to obtain 40. mu.L of the mixture, and 20. mu. L H was added2O, detecting on a computer;
(II) detection on computer
1. Mobile phase conditions:
the present invention uses SCIEX ExionLC ultra high performance liquid chromatograph, and uses a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm) liquid chromatographic column to perform chromatographic separation on the target compound. Liquid chromatography phase A was 0.1% formic acid &1mM aqueous ammonium formate and phase B was acetonitrile. The temperature of the column incubator is 40 ℃, the sample tray is set to be 6 ℃, and the sample injection volume is 2 mu L;
AB Sciex QTrap 6500 Mass Spectrometry instrument parameter settings:
the mass spectrum conditions are an electrospray ionization positive ion mode and a multi-reaction monitoring scanning mode, and the ion source parameters comprise: the ion spray voltage was 5000V, the gas curtain pressure was 35psi, the temperature was 400 deg.C, the ion source gas 1 pressure was 60psi, and the ion source gas 2 pressure was 60 psi.
Example 2
The method for detecting biogenic amine in aquatic products comprises the following steps:
(I) sample pretreatment
(a) Weighing a proper amount of sample to be detected in a 2.0mL EP tube, and adding a steel ball;
(b) adding 80 μ L of extractive solution (acetonitrile containing 0.1% formic acid, precooling at-20 deg.C) and 20 μ L of ethanol2O, uniformly mixing by swirling for 28 s;
(c) homogenizing at 35Hz for 250s, performing ultrasonic treatment in ice water bath for 4.5min, and repeating the homogenizing ultrasonic step for 3 times;
(d) samples were precipitated overnight at-42 ℃;
(e) the sample was centrifuged at 11500rpm for 16min at 3.5 ℃ to obtain 80. mu.L of supernatant (Spl), 40. mu.L of 100mM/L Na was added2CO3And 40. mu.L of 2% L-BzCl, incubated at room temperature for 25 min; taking Spl 160 mu L after derivation, adding 10 mu L H-std-a-Bz-DF internal standard;
(f) the sample was centrifuged at 12500rpm for 13min at 3.5 deg.C, 40. mu.L was taken and 20. mu. L H was added2O, detecting on a computer;
(II) detection on computer
1. Mobile phase conditions:
the present invention uses SCIEX ExionLC ultra high performance liquid chromatograph, and uses a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm) liquid chromatographic column to perform chromatographic separation on the target compound. Liquid chromatography phase A was 0.1% formic acid &1mM aqueous ammonium formate and phase B was acetonitrile. The temperature of the column incubator is 42 ℃, the sample tray is set to be 7 ℃, and the sample introduction volume is 2.2 mu L;
AB Sciex QTrap 6500 Mass Spectrometry instrument parameter settings:
the mass spectrum conditions are an electrospray ionization positive ion mode and a multi-reaction monitoring scanning mode, and the ion source parameters comprise: the ion spray voltage was 4900V, the gas curtain pressure was 36psi, the temperature was 395 deg.C, the pressure of the ion source gas 1 was 58psi, and the pressure of the ion source gas 2 was 58 psi.
Example 3
The method for detecting biogenic amine in aquatic products comprises the following steps:
(I) sample pretreatment
(a) Weighing a proper amount of sample to be detected in a 2.0mL EP tube, and adding a steel ball;
(b) adding 80 μ L of extractive solution (acetonitrile containing 0.1% formic acid, precooling at-20 deg.C) and 20 μ L of ethanol2O, uniformly mixing by vortex for 35 s;
(c) homogenizing at 35Hz for 250s, performing ultrasonic treatment in ice water bath for 4.5min, and repeating the homogenizing ultrasonic step for 3 times;
(d) samples were precipitated overnight at-38 ℃;
(e) the sample was centrifuged at 12500rpm for 13min at 4.4 deg.C, 80. mu.L of the supernatant (Spl) was added 40. mu.L of 100mM/L Na2CO3And 40. mu.L of 2% L-BzCl, incubated at room temperature for 35 min; taking Spl 160 mu L after derivation, adding 10 mu L H-std-a-Bz-DF internal standard;
(f) the sample was centrifuged at 11500rpm for 16min at 4.4 ℃ to obtain 40. mu.L of a mixture, and 20. mu. L H was added2O, detecting on a computer;
(II) detection on computer
1. Mobile phase conditions:
the present invention uses SCIEX ExionLC ultra high performance liquid chromatograph, and uses a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm) liquid chromatographic column to perform chromatographic separation on the target compound. Liquid chromatography phase A was 0.1% formic acid &1mM aqueous ammonium formate and phase B was acetonitrile. The temperature of the column incubator is 38 ℃, the sample tray is set to be 5 ℃, and the sample injection volume is 1.8 mu L;
AB Sciex QTrap 6500 Mass Spectrometry instrument parameter settings:
the mass spectrum conditions are an electrospray ionization positive ion mode and a multi-reaction monitoring scanning mode, and the ion source parameters comprise: the ion spray voltage was 5100V, the pressure of the gas curtain gas was 33psi, the temperature was 405 ℃, the pressure of the ion source gas 1 was 62psi, and the pressure of the ion source gas 2 was 62 psi.
Examples of the experiments
First, the information of 14 kinds of biogenic amines in the present invention is shown in table 2.
TABLE 2 information on 14 biogenic amines of the invention
Second, chromatographic separation
The standard solution Extraction Ion Chromatograms (EICs) are shown in FIG. 1, and the sample extraction ion chromatograms are shown in FIG. 2. It can be seen that: 1) the research adopts an analysis method, and the chromatographic separation of each target compound is well realized; 2) the retention time and the chromatographic peak shape of the target compound in the biological sample and the standard solution have no obvious difference.
Third, detection limit and quantification limit of the method
The detection limit and the quantitative limit of the target compound are shown in a table 3, the lowest detection limit (LLOD) of the target compound is between 0.136 and 69.755nmol/L, and the lowest quantitative limit (LLOQ) of the target compound is between 0.273 and 139.509 nmol/L; the correlation coefficient (R2) of all target compounds is larger than 0.9961, which shows that the peak area of the chromatographic peak and the concentration of the compounds have good quantitative relation and can meet the analysis requirement of the target compounds.
Detection and quantitation limits for the detection of 314 biogenic amines in Table
Fourthly, precision and accuracy of method
The precision of the method was assessed by the standard relative deviation (RSD) of QC sample replicate injections. The accuracy was assessed by the Recovery (Recovery) of the QC sample, and the percentage value of the measured concentration to the spiked concentration was the spiked Recovery. The recovery (recovery) and the standard relative deviation (RSD) of the QC samples are shown in Table 4, and the number of times of repeated injection of the QC samples is 6. As shown in table 4, the average recovery of all target compounds was 110.5 ± 25.0%.
TABLE 414 accuracy and precision of the biogenic amine detection methods
| Compound Name | Concentration(nmol/L) | Recovery | RSD |
| Histamine | 1000.00 | 101.2％ | 6.7％ |
| Tyramine | 1000.00 | 187.4％ | 18.7％ |
| Dopamine | 1000.00 | 108.6％ | 13.1％ |
| Tryptamine | 1000.00 | 92.6％ | 6.8％ |
| Serotonin | 1000.00 | 148.4％ | 37.0％ |
| Putrescine | 1000.00 | 103.7％ | 9.9％ |
| Spermine | 1000.00 | 97.0％ | 15.0％ |
| Spermidine | 1000.00 | 100.7％ | 12.3％ |
| Octopamine | 1000.00 | 110.9％ | 16.9％ |
| Benzylamine | 1000.00 | 93.6％ | 7.4％ |
| 1-Phenylethanamine | 1000.00 | 102.2％ | 6.1％ |
| Cadaverine | 1000.00 | 99.2％ | 9.3％ |
| 2-Phenethylamine | 1000.00 | 99.8％ | 6.1％ |
| Agmatine | 1000.00 | 101.9％ | 6.2％ |
As can be seen from the data in Table 4, the method of the present invention can accurately and reliably detect the content of the target metabolite in the sample within the concentration range shown above.
Fifth, the detection result of 14 kinds of biogenic amine in the fish sample
In total, 3 samples of tuna and black carp species were purchased from the market, and 14 kinds of biogenic amines were tested according to the method of example 1, and the results are shown in Table 5. R1, R2, R3 are three replicates of each sample, respectively; "-" indicates that the detection value is 0; dopamine and Octopamine were not detected in each sample and the results are not shown in the table.
TABLE 5 detection results of 14 biogenic amines in fish samples
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for detecting biogenic amine in aquatic products is characterized by comprising the following steps:
(a) mixing a sample to be detected containing biogenic amine, a formic acid acetonitrile solution and water, and then carrying out vortex treatment; then carrying out homogenization treatment and ultrasonic treatment; carrying out solid-liquid separation on the sample subjected to ultrasonic treatment; mixing the liquid with a sodium carbonate solution and a benzoyl chloride solution for incubation, mixing with an internal standard solution, and centrifuging;
(b) and (b) mixing the centrifuged supernatant in the step (a) with water, and detecting by using an ultra-high liquid chromatography-tandem mass spectrometry method.
2. The method of claim 1, wherein the biogenic amine comprises at least one of histamine, tyramine, dopamine, tryptamine, 5-hydroxytryptamine, putrescine, spermine, spermidine, octopamine, benzylamine, phenethylamine, cadaverine, β -phenethylamine, and agmatine.
3. The method for detecting biogenic amines in aquatic products according to claim 1, wherein the phase A of the ultra-high liquid chromatography is formic acid-ammonium formate solution, the molar concentration of the ammonium formate is 1-2 mM, and the volume concentration of the formic acid is 0.08-0.15%; and the phase B of the ultra-high liquid chromatogram is acetonitrile.
4. The method for detecting biogenic amine in aquatic products according to claim 1, wherein the temperature of a column incubator of the ultra-high liquid chromatography is 38-42 ℃, the temperature of a sample plate is 5-7 ℃, and the volume of sample injection is 1.8-2.2 μ L.
5. The method for detecting biogenic amines in aquatic products according to claim 1, wherein the mass spectrometry conditions are electrospray ionization positive ion mode and multi-reaction monitoring scanning mode, and the ion source parameters comprise: the ion spray voltage is 4900-5100V, the pressure of the gas curtain gas is 33-36 psi, the temperature is 395-405 ℃, the pressure of the ion source gas 1 is 58-62 psi, and the pressure of the ion source gas 2 is 58-62 psi.
6. The method for detecting biogenic amines in aquatic products according to any one of claims 1 to 5, wherein the time of the vortex treatment is 28 to 35 s.
7. The method for detecting biogenic amines in aquatic products according to any one of claims 1 to 5, wherein the frequency of the homogenization treatment is 35 to 45Hz, and the time is 230 to 250 s.
8. The method for detecting biogenic amines in aquatic products according to any one of claims 1 to 5, wherein the ultrasonic treatment is carried out in an ice-water bath;
preferably, the ultrasonic treatment time is 4.5-5.5 min;
preferably, the number of the homogenization treatment and the ultrasonic treatment is 2 to 3.
9. The method for detecting biogenic amines in aquatic products according to any one of claims 1 to 5, wherein the solid-liquid separation comprises standing precipitation and centrifugation;
preferably, the standing precipitation is carried out at-38 to-42 ℃;
preferably, the centrifugation in the solid-liquid separation is carried out at 3.5-4.4 ℃, the rotation speed of the centrifugation in the solid-liquid separation is 11500-12500 rpm, and the centrifugation time in the solid-liquid separation is 13-16 min.
10. The method for detecting biogenic amines in aquatic products according to any one of claims 1 to 5, wherein the incubation time is 25 to 35 min;
preferably, the centrifugation is carried out at 3.5-4.4 ℃;
preferably, the rotating speed of the centrifugation is 11500-12500 rpm, and the time of the centrifugation is 13-16 min.
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