CN117214114B - Method for detecting concentration of trimethylamine - Google Patents
Method for detecting concentration of trimethylamine Download PDFInfo
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- CN117214114B CN117214114B CN202311164681.0A CN202311164681A CN117214114B CN 117214114 B CN117214114 B CN 117214114B CN 202311164681 A CN202311164681 A CN 202311164681A CN 117214114 B CN117214114 B CN 117214114B
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- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 title claims abstract description 426
- 238000000034 method Methods 0.000 title claims abstract description 33
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims abstract description 92
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 claims abstract description 90
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 84
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- PFEFOYRSMXVNEL-UHFFFAOYSA-N 2,4,6-tritert-butylphenol Chemical compound CC(C)(C)C1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 PFEFOYRSMXVNEL-UHFFFAOYSA-N 0.000 claims abstract description 65
- 238000002835 absorbance Methods 0.000 claims abstract description 58
- 239000000243 solution Substances 0.000 claims abstract description 50
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 42
- 239000012085 test solution Substances 0.000 claims abstract description 41
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims abstract description 38
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- 239000000523 sample Substances 0.000 description 20
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 18
- 239000006286 aqueous extract Substances 0.000 description 15
- 235000013372 meat Nutrition 0.000 description 12
- -1 2,4, 6-tri-tert-butylphenol trimethylamine Chemical compound 0.000 description 11
- 239000004312 hexamethylene tetramine Substances 0.000 description 9
- 235000010299 hexamethylene tetramine Nutrition 0.000 description 9
- 230000006872 improvement Effects 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000003988 headspace gas chromatography Methods 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 7
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- 239000008098 formaldehyde solution Substances 0.000 description 3
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 3
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- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
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- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 description 1
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Abstract
The application relates to the technical field of trimethylamine detection, in particular to a method for detecting trimethylamine concentration. The detection method comprises the following steps: a standard curve of trimethylamine concentration versus absorbance was prepared. Trimethylamine concentration in test aquatic products: firstly, extracting trimethylamine in an aquatic product to obtain an extracting solution; obtaining a plurality of extracting solutions, and adding formaldehyde to remove the interference effect of ammonia; then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate are added, thus eliminating the interference of methylamine and dimethylamine; and finally, adding 2,4, 6-tri-tert-butylphenol and trimethylamine for complexing to obtain the test solution. And (3) taking the 2,4, 6-tri-tert-butylphenol solution as a blank test control group, testing the relative absorbance of the test solution, finding out the trimethylamine concentration corresponding to the measured relative absorbance on a standard curve, and calculating the trimethylamine concentration contained in the water product according to the found trimethylamine concentration, wherein the obtained result is accurate.
Description
Technical Field
The application relates to the technical field of trimethylamine detection, in particular to a method for detecting trimethylamine concentration.
Background
Trimethylamine oxide (TMAO) is widely found in fish muscle. TMAO has effects of stabilizing protein structure, regulating osmotic pressure, resisting ion instability, and resisting water pressure. TMAO itself is odorless and has a special fresh sweet taste. However, after fish death, proteins start to decompose under the action of enzymes, bacteria, and high temperature environment, and most of trimethylamine oxide is reduced to Trimethylamine (TMA) having fishy smell. The longer the fish death time, the higher the concentration of trimethylamine and the greater the fishy smell.
A small portion of trimethylamine oxide in fish bodies can also be reduced and decomposed to dimethylamine or methylamine. Ammonia is also a substance that gives a putrefactive odor when the freshness of fish meat or the like is lowered. In the early post-mortem stage of fish, ammonia is produced during the conversion of adenosine to inosinic acid, and more ammonia is derived from deamination of amino acids in fish meat. And urea existing in fish muscle can generate ammonia under the action of bacterial urease, so that frozen fish and the like can generate obvious ammonia odor.
TMA is a biochemical indicator of freshness of fish. The higher the TMA content, the less fresh the fish. The fresh index of the common freshwater fish is TMA content not higher than 6mg/100g, and the fresh index of the sea fish is TMA content below 39mg/100 g.
One of the methods for detecting the trimethylamine content in fish is a spectrophotometry method, and a plurality of chemical reagents and the like are adopted to combine with the trimethylamine for reaction, but the method has interference of ammonia, methylamine and dimethylamine at present, so that the detection result is inaccurate.
Disclosure of Invention
In view of the situation that the existing spectrophotometry for detecting the trimethylamine content in fish has interference of ammonia, methylamine and dimethylamine, so that the detection result is inaccurate, the application provides a trimethylamine concentration detection method and adopts the following technical scheme.
A method for detecting trimethylamine concentration, the method comprising:
making a standard curve of trimethylamine concentration corresponding to absorbance: preparing a plurality of groups of trimethylamine standard solutions with different concentrations, and respectively adding 2,4, 6-tri-tert-butylphenol; 2,4, 6-tri-tert-butylphenol solution is used as a blank standard control group, and the absorbance of a plurality of groups of trimethylamine standard solutions with different concentrations relative to the blank standard control group is tested to obtain a plurality of relative absorbance values; and preparing the standard curve according to the relation that a plurality of concentration values of a plurality of groups of trimethylamine standard solutions are in one-to-one correspondence with a plurality of relative absorbance values.
Trimethylamine concentration in test aquatic products: firstly, extracting trimethylamine in an aquatic product to obtain an extracting solution; obtaining a plurality of extracting solutions, adding formaldehyde to fully react ammonia in the extracting solutions, then adding maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate to fully react methylamine and dimethylamine in the extracting solutions, and finally adding 2,4, 6-tri-tert-butylphenol to react with trimethylamine in the extracting solutions to obtain test solutions; and (3) taking the 2,4, 6-tri-tert-butylphenol solution as a blank test control group, testing the relative absorbance of the test solution, finding out the trimethylamine concentration corresponding to the measured relative absorbance on the standard curve, and calculating the trimethylamine concentration contained in the water product according to the found trimethylamine concentration.
By adopting the technical scheme, the standard curve of the absorbance corresponding to the trimethylamine concentration prepared in advance can be used as a reference standard of the test liquid, and the trimethylamine concentration of the test liquid can be conveniently obtained on the standard curve according to the measured relative absorbance of the test liquid. Because the aquatic products contain not only trimethylamine, but also ammonia, methylamine and dimethylamine, the interference of the ammonia, the methylamine and the dimethylamine on the test needs to be eliminated during the test. According to the scheme, formaldehyde is added into the sample liquid, the formaldehyde can react with ammonia to generate hexamethylenetetramine, the formaldehyde cannot react with trimethylamine, and the interference of ammonia is eliminated. Then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate are added into the sample liquid, the 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate can dissociate H on N of methylamine and transfer the H to anhydride oxygen, so that N of methylamine can be combined on carbonyl carbon of maleic anhydride, and epoxy bond of maleic anhydride is opened to generate amidated product; the 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate can dissociate and transfer H on N of dimethylamine to anhydride oxygen, so that N of dimethylamine is combined with carbonyl carbon of maleic anhydride, an epoxy bond of maleic anhydride is opened, an amidation product is generated, and interference of methylamine and dimethylamine is eliminated. Since trimethylamine has no H on N, 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, maleic anhydride cannot react with trimethylamine. And finally, adding 2,4, 6-tri-tert-butylphenol into the sample liquid, wherein the 2,4, 6-tri-tert-butylphenol can be complexed with trimethylamine to generate yellow 2,4, 6-tri-tert-butylphenol trimethylamine, and detecting absorbance according to the yellow 2,4, 6-tri-tert-butylphenol trimethylamine, so as to calculate the concentration of the trimethylamine in the sample. Wherein formaldehyde, maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, and the generated hexamethylenetetramine and the generated amidated product do not react with 2,4, 6-tri-tert-butylphenol and trimethylamine, so the scheme of adding the reagent can eliminate the interference of ammonia, methylamine and dimethylamine and accurately obtain the concentration of trimethylamine. Wherein, the standard curve can be drawn on rectangular coordinates. The aquatic product can be animal aquatic product such as sea fish, freshwater fish, etc.
As an improvement of the method for detecting the concentration of trimethylamine, when a standard curve of the concentration of trimethylamine corresponding to absorbance is manufactured, preparing a plurality of groups of trimethylamine standard solutions in the concentration range of 0.002-0.2 mg/mL, respectively adding 2,4, 6-tri-tert-butylphenol with the same mass and excessive quantity, and calculating the concentration of the added 2,4, 6-tri-tert-butylphenol in the trimethylamine standard solution according to an unreacted state to be 1-3 mg/mL; the concentration of 2,4, 6-tri-tert-butylphenol in the blank standard control group is the same as the concentration of 2,4, 6-tri-tert-butylphenol in the trimethylamine standard solution calculated in an unreacted state.
By adopting the technical scheme, the added 2,4, 6-tri-tert-butylphenol is excessive, so that trimethylamine can be completely reacted. And after eliminating the interference absorbance of the blank standard control group, the absorbance of the obtained trimethylamine standard solution linearly changes along with the trimethylamine concentration to obtain a linear standard curve. And (3) referring to a standard curve, the error of the trimethylamine concentration of the test solution obtained by the calibration is small.
As an improvement of the method for detecting the concentration of trimethylamine, a plurality of groups of trimethylamine standard solutions in the concentration range of 0.002-0.02 mg/mL are respectively configured as 0.002mg/mL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL, 0.010mg/mL, 0.012mg/mL, 0.014mg/mL, 0.016mg/mL, 0.018mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.10mg/mL, 0.11mg/mL, 0.12mg/mL, 0.13mg/mL, 0.14mg/mL, 0.15mg/mL, 0.16mg/mL, 0.17mg/mL, 0.18mg/mL, 0.19mg/mL and 0.20mg/mL trimethylamine.
By adopting the technical scheme, the trimethylamine standard solution has smaller concentration progressive difference in a smaller concentration area, so that the test error in the small concentration area is small; the trimethylamine standard solution has larger concentration progressive difference in a larger concentration area, and can obtain a highly linear standard curve by controlling the number of test groups.
As an improvement of the method for detecting the concentration of trimethylamine, when the concentration of trimethylamine in the aquatic product is tested, the method for extracting trimethylamine in the aquatic product comprises the following steps: mashing aquatic product with mass of M, and adding volume of V 1 Stirring, centrifuging to obtain volume V 1 Supernatant, adding V to the supernatant 2 To precipitate protein in the clear liquid, centrifuging, and collecting the solution with volume of V 3 As the extract, the extract contains trimethylamine.
By adopting the technical scheme, the trimethylamine is easy to dissolve in water, and the trimethylamine in the water product can be extracted by using the water. The clear extracting solution can be obtained through centrifugation, trichloroacetic acid solution addition and the like, and the detection interference on absorbance is small.
As an improvement of the method for detecting the concentration of trimethylamine, the mass of formaldehyde added is 0.001M [ V ] 3 /(V 1 +V 2 )]10 to 70 times of the total weight of the steel sheet.
By adopting the technical proposal, the ammonia content in the aquatic product is generally not more than 100mg/100g, namely in the improved proposal, the ammonia content in the aquatic product is 0.001M at maximum, and the ammonia content in the extracting solution is 0.001M [ V ] at maximum 3 /(V 1 +V 2 )]. Formaldehyde (30 g/mol) and ammonia (17 g/mol) react to generate hexamethylenetetramine, the reaction ratio of formaldehyde to ammonia is 3:2, and the reaction mass ratio of formaldehyde to ammonia is 90:34, so that 10-70 times of formaldehyde can be used for completely reacting ammonia.
As an improvement of the method for detecting the concentration of trimethylamine, maleic anhydride is added with the mass of 0.001M [ V ] 3 /(V 1 +V 2 )]10 to 70 times of the total weight of the steel sheet.
By adopting the technical scheme, the methylamine content in the aquatic product is generally not more than 100mg/100g, and the dimethylamine content in the aquatic product is generally not more than 100mg/100g, namely in the improved scheme, the methylamine content in the aquatic product is maximally 0.001M, the dimethylamine content in the aquatic product is maximally 0.001M, and the methylamine content in the extracting solution is maximally 0.001M [ V ] 3 /(V 1 +V 2 )]The maximum dimethylamine content in the extract is 0.001M [ V ] 3 /(V 1 +V 2 )]. The reaction ratio of methylamine (molar mass 31 g/mol) to maleic anhydride (molar mass 98 g/mol) was 1:1. The reaction ratio of dimethylamine (molar mass 45 g/mol) to maleic anhydride (molar mass 98 g/mol) was 1:1. The reaction mass ratio of maleic anhydride to methylamine was 98:31, and the reaction mass ratio of maleic anhydride to dimethylamine was 98:45. The maleic anhydride may be used in an amount of 10 to 70 times the mass of the reaction mixture to react methylamine and dimethylamine.
As an improvement of the method for detecting the concentration of trimethylamine, the mass of the added 2,4, 6-tri-tert-butylphenol is 0.001M [ V ] 3 /(V 1 +V 2 )]5 to 15 times of the total weight of the steel sheet.
By adopting the technical scheme, the content of the trimethylamine in the aquatic product is generally not more than 100mg/100g, namely in the improved scheme, the content of the trimethylamine in the aquatic product is 0.001M at the maximum, and the content of the trimethylamine in the extracting solution is 0.001M [ V ] at the maximum 3 /(V 1 +V 2 )]. Trimethylamine (59 g/mol in molar mass) is complexed with 2,4, 6-tri-tert-butylphenol (262 g/mol in molar mass) in a 1:1 ratio to form yellow trimethylamine of 2,4, 6-tri-tert-butylphenol. The complexing mass ratio of the 2,4, 6-tri-tert-butylphenol and the trimethylamine is 262:59, so that the trimethylamine can be fully reacted by using the 2,4, 6-tri-tert-butylphenol with the mass of 5-15 times.
As an improvement of the method for detecting the concentration of the trimethylamine, when the concentration of the trimethylamine in the aquatic product is tested, adding the formaldehyde to completely react the ammonia in the extracting solution to obtain a primary reaction solution, and adding toluene into the primary reaction solution to extract to obtain an extracting solution; and then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate are added into the extract liquid to completely react methylamine and dimethylamine in the extract liquid, thus obtaining a secondary reaction liquid.
By adopting the technical scheme, formaldehyde reacts with ammonia to generate hexamethylenetetramine, the hexamethylenetetramine is insoluble in toluene, and the interference of ammonia is eliminated after toluene extraction is used.
As the concentration of the trimethylamineAn improvement of the detection method is that 2,4, 6-tri-tert-butylphenol and trimethylamine are added into the secondary reaction liquid to react, so as to obtain the test liquid; the concentration of the 2,4, 6-tri-tert-butylphenol in the blank test control group is the same as the concentration of the 2,4, 6-tri-tert-butylphenol in the test solution calculated according to the unreacted state; testing the relative absorbance of the test solution, and finding out that the trimethylamine concentration corresponding to the relative absorbance is n on the standard curve, wherein the volume of the test solution is V, and the trimethylamine concentration X=NV [ (V) in the aquatic product 1 +V 2 )/V 3 ]/M。
By adopting the technical scheme, the obtained relative absorbance of the test solution basically counteracts the interference of the 2,4, 6-tri-tert-butylphenol, and the concentration value of the trimethyl amine can be accurately corresponding to the standard curve. NV is the mass of trimethylamine in the test solution, NV [ (V) 1 +V 2 )/V 3 ]The concentration of the trimethylamine in the aquatic product is obtained according to the formula.
As an improvement of the method for detecting the concentration of the trimethylamine, the relative absorbance of the trimethylamine standard solution and the test solution is tested, and the relative absorbance of the test solution is the absorbance of the test solution at 452 nm.
By adopting the technical scheme, the absorption peak of the 2,4, 6-tri-tert-butylphenol trimethylamine is at 452nm, the interference of other substances on the absorption peak is small, and the absorbance change is larger than that at other wavelengths along with the change of the concentration of the 2,4, 6-tri-tert-butylphenol trimethylamine, so that the method has the most indication significance. The change of the absorbance at 452nm is tested to obtain the concentration of the trimethylamine of the 2,4, 6-tri-tert-butylphenol, so that the concentration of the trimethylamine in the water product can be calculated, and the freshness of the water product can be judged. The trimethylamine concentration in the test liquid is measured by using a headspace-gas chromatography, and the measurement process parameters are referred to HJ 1042-2019, and experiments show that the trimethylamine concentration difference measured by using the headspace-gas chromatography and an absorbance method is small and is within 1%, so that the detection result obtained by the trimethylamine concentration detection method in the scheme is accurate.
In summary, the method for detecting the concentration of trimethylamine of the present application has the following advantages:
the standard curve of the absorbance corresponding to the trimethylamine concentration prepared in advance can be used as a reference standard of the test liquid, and the trimethylamine concentration of the test liquid can be conveniently obtained on the standard curve according to the measured relative absorbance of the test liquid.
When the concentration of trimethylamine in a sample is tested, formaldehyde is added into the sample liquid, and the formaldehyde can react with ammonia to generate hexamethylenetetramine. Then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate are added into the sample liquid, so that maleic anhydride, methylamine and dimethylamine form amidated products, and the interference of methylamine and dimethylamine is eliminated. And finally, adding 2,4, 6-tri-tert-butylphenol into the sample liquid, wherein the 2,4, 6-tri-tert-butylphenol can be complexed with trimethylamine to generate yellow 2,4, 6-tri-tert-butylphenol trimethylamine, and detecting absorbance according to the yellow 2,4, 6-tri-tert-butylphenol trimethylamine, so as to calculate the concentration of the trimethylamine in the sample. Wherein formaldehyde, maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, and the generated hexamethylenetetramine and the generated amidated product do not react with 2,4, 6-tri-tert-butylphenol and trimethylamine, so the scheme of adding the reagent can eliminate the interference of ammonia, methylamine and dimethylamine and accurately obtain the concentration of trimethylamine.
Drawings
FIG. 1 is a graph showing the concentration of trimethylamine-relative absorbance standard, which was prepared in example 1.
Detailed Description
Some examples of the method for detecting trimethylamine concentration of the present application are specifically described below.
Example 1
The trimethylamine solutions of 0.002mg/mL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL, 0.010mg/mL, 0.012mg/mL, 0.014mg/mL, 0.016mg/mL, 0.018mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.10mg/mL, 0.11mg/mL, 0.12mg/mL, 0.13mg/mL, 0.14mg/mL, 0.15mg/mL, 0.16mg/mL, 0.17mg/mL, 0.18mg/mL, 0.19mg/mL, 0.20mg/mL and toluene were prepared, and 2,4, 6-tri-tert-butylphenol was added to each trimethylamine solution. The concentration of 2,4, 6-tri-tert-butylphenol in each group of solutions was 1.5mg/mL calculated as the unreacted state of 2,4, 6-tri-tert-butylphenol and trimethylamine.
2,4, 6-tri-tert-butylphenol (molar mass 262 g/mol) and trimethylamine (molar mass 59 g/mol) can be complexed in a 1:1 ratio to form yellow 2,4, 6-tri-tert-butylphenol trimethylamine. The complexing mass ratio of 2,4, 6-tri-tert-butylphenol and trimethylamine is 262:59, so that the concentration of 2,4, 6-tri-tert-butylphenol (1.5 mg/mL) is sufficient to completely react trimethylamine (maximum 0.2 mg/mL).
The absorption peak of the 2,4, 6-tri-tert-butylphenol trimethylamine is 452nm, and other substances have little interference on the absorption peak.
2,4, 6-tri-tert-butylphenol with the concentration of 1.5mg/mL is used as a blank control group, and the relative absorbance of each group of reaction liquid at 452nm is tested to obtain 28 relative absorbance values. As shown in table 1 below.
Table 1 trimethylamine concentration corresponding to relative absorbance
Establishing a rectangular coordinate system by taking the relative absorbance value as an ordinate and the trimethylamine concentration value as an abscissa, wherein 28 relative absorbance values and 28 trimethylamine concentration values are in one-to-one correspondence, each absorbance value and the trimethylamine concentration value corresponding to each absorbance value are taken as a coordinate point, and a trimethylamine concentration-relative absorbance standard curve is drawn, as shown in figure 1, and the curve fitting degree R 2 0.9977, which shows a highly linear correlation between the relative absorbance and trimethylamine concentration values.
Example 2
Taking 20g of mackerel meat, mashing, adding 30mL of water, stirring for 10min, centrifuging, and taking supernatant. The remaining solid residue was repeatedly added with 30mL of water twice, shaken and centrifuged to obtain a supernatant, and the three supernatants were combined and added with 90mL of water three times. 10mL of an aqueous solution of trichloroacetic acid having a concentration of 0.2g/mL was added to the combined 90mL of the supernatant, the mixture was shaken, the protein in the supernatant was completely precipitated with trichloroacetic acid, and the mixture was centrifuged to obtain about 100mL of a total supernatant of the upper layer, which was an aqueous extract containing ammonia, methylamine, dimethylamine and trimethylamine, followed by adding the reagent as follows.
(1) Taking 10mL of water extract, adding 1mL of formaldehyde solution with the concentration of 0.1g/mL, stirring for 1min, adding 10mL of toluene, oscillating, separating the solution into a toluene layer and a water layer, obtaining the toluene layer, adding anhydrous sodium sulfate into the toluene layer to dry the toluene layer to obtain an extract, wherein the toluene layer contains substances such as methylamine, dimethylamine, trimethylamine and the like;
(2) 1mL of maleic anhydride solution with the concentration of 0.1g/mL is added into the extract, the solvent is toluene, and the solution also contains 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate with the mass fraction of 0.001g/mL (catalytic action);
(3) Finally, adding 2mL of 2,4, 6-tri-tert-butylphenol solution with the concentration of 0.01g/mL, taking toluene as a solvent, and carrying out oscillation reaction for 1min to obtain the test solution.
In the step (1), formaldehyde (molar mass: 30 g/mol) is reacted with ammonia (molar mass: 17 g/mol) to form hexamethylenetetramine, which cannot react with 2,4, 6-tri-tert-butylphenol, and which is insoluble in toluene, and the interference of ammonia is eliminated after extraction with toluene. Wherein formaldehyde does not react with trimethylamine. The concentration of ammonia in fish meat is below 100mg/100g, the mass of ammonia in 20g fish meat is below 20mg, 10mL of water extract is 1/10 of 100mL of total clear liquid, so that the mass of ammonia in 10mL of water extract is below 2mg, the reaction ratio of formaldehyde to ammonia is 3:2, the reaction mass ratio of formaldehyde to ammonia is 90:34, and the added amount of formaldehyde (100 mg) is enough to completely react ammonia (2 mg).
In step (2), the catalyst of 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate: (1) reacting methylamine (31 g/mol in terms of molar mass) with maleic anhydride (98 g/mol in terms of molar mass), and binding N of methylamine to carbonyl carbon of maleic anhydride to produce acetamido product; (2) dimethylamine (molar mass 45 g/mol) was reacted with maleic anhydride and N of dimethylamine was bound to the carbonyl carbon of maleic anhydride to produce the acetaminated product. The concentration of methylamine and dimethylamine in fish flesh is below 100mg/100g, the mass of methylamine and dimethylamine in fish flesh is below 20mg, 10mL of water extract is 1/10 of 100mL of total clear liquid, so that 10mL of water extract contains methylamine and dimethylamine, the mass of methylamine and dimethylamine is below 2mg, the reaction ratio of methylamine to dimethylamine to maleic anhydride is 1:1, the reaction mass ratio of maleic anhydride to methylamine is 98:31, the reaction mass ratio of maleic anhydride to dimethylamine is 98:45, and the added maleic anhydride (100 mg) is enough to completely react methylamine (2 mg) and dimethylamine (2 mg).
Formaldehyde, maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate added in the step (1) and the step (2) are not reacted with 2,4, 6-tri-tert-butylphenol and trimethylamine in the hexamethylenetetramine formed in the step (1) and the two acetamides formed in the step (2). So that the step (1) and the step (2) can eliminate the interference of ammonia, methylamine and dimethylamine.
In the step (3), 2,4, 6-tri-tert-butylphenol (molar mass 262 g/mol) and trimethylamine (molar mass 59 g/mol) can be complexed in a ratio of 1:1 to form yellow 2,4, 6-tri-tert-butylphenol trimethylamine. The concentration of trimethylamine in fish meat is generally 100mg/100g or less, the mass of trimethylamine contained in 20g of fish meat is 20mg or less, 10mL of aqueous extract is 1/10 of 100mL of total clear liquid, so that the mass of trimethylamine contained in 10mL of aqueous extract is 2mg or less, the complexing mass ratio of 2,4, 6-tri-tert-butylphenol and trimethylamine is 262:59, and the above-mentioned added amount of 2,4, 6-tri-tert-butylphenol (20 mg) is sufficient to react trimethylamine (2 mg) completely.
Since the volume of the test solution was changed to 13mL after adding 2mL of the 2,4, 6-tri-tert-butylphenol solution having a concentration of 0.01g/mL, and the concentration of 20mg of 2,4, 6-tri-tert-butylphenol in the test solution was 1.5mg/mL, the absorbance of the test solution at 452nm wavelength was measured using a spectrophotometer as a blank control group with the concentration of 1.5mg/mL of 2,4, 6-tri-tert-butylphenol as the blank control group, and the corresponding trimethylamine concentration was found to be 0.045mg/mL on the standard curve of FIG. 1. Since the total volume of the test solution was 13mL, the mass of trimethylamine in the test solution was 0.045mg/mL 13 ml=0.585 mg in the absence of reaction, and all of the trimethylamine was derived from the 10mL aqueous extract obtained in the step (1), so that the concentration of trimethylamine in the aqueous extract was 0.0585mg/mL.
Trimethylamine concentration x=nv in aquatic products [ (V) 1 +V 2 )/V 3 ]X=0.045 mg/mL 13mL [ (90ml+10ml)/10 mL can be calculated as per M]20 g=29.25 mg/100g, namely, each 100g of mackerel contains 29.25mg of trimethylamine, the fresh index of the mackerel is that the trimethylamine content is less than 39mg/100g, so the mackerel is in a fresh state.
Verification example 1
The trimethylamine concentration of the remaining 90mL aqueous extract of test example 2 was determined by headspace-gas chromatography with reference to HJ 1042-2019 for the measurement process parameters, and the headspace-gas chromatography test procedure was as follows.
Accurately sucking 2ml of water extract into a 20ml headspace bottle, sealing by a gland, and injecting 5.0ml of 50% sodium hydroxide solution by a syringe for later use.
The temperature program of the headspace gas chromatograph was set to be maintained at 40 ℃ for 3min, then at 30 ℃/min to 220 ℃ and at 220 ℃ for 1min.
Setting the sample inlet temperature of a headspace-gas chromatograph to 220 ℃, the detector temperature to 220 ℃, and the split ratio to be 2:1, air flow rate 400ml/min, hydrogen flow rate 40ml/min, and nitrogen flow rate 35ml/min.
Headspace sample injection: and (5) balancing at 40 ℃ for 30min, and then carrying out sample injection measurement.
And (3) carrying out qualitative analysis by referring to retention time of trimethylamine in a standard chromatogram in the ambient air, and carrying out quantitative analysis by adopting an external standard method. And drawing a calibration curve, and calculating the concentration of trimethylamine in the sample solution by using the calibration curve. The final trimethylamine concentration was measured to be 0.0574mg/mL, and the deviation of 0.0585mg/mL obtained in example 2 from the test value was 1.9%, indicating that the trimethylamine concentration measured by absorbance method was accurate.
The concentration X=0.0574 mg/mL (90 mL+10 mL)/20 g=28.7 mg/100g of trimethylamine in the aquatic product can be calculated, namely 28.7mg of trimethylamine is contained in every 100g of mackerel, and the fresh index of the sea fish is that the trimethylamine content is less than 39mg/100g, so that the mackerel is in a fresh state.
Example 3
In this example, grass carp meat was used as a sample, and 13mL of a test solution was obtained under the same test conditions as in example 2, and the absorbance of the test solution at 452nm was measured to be 0.57 by using a spectrophotometer, and the corresponding trimethylamine concentration was found to be 0.0125mg/mL on the standard curve of FIG. 1. Since the total volume of the test solution was 13mL, the mass of trimethylamine in the test solution was 0.0125mg/mL 13 ml=0.1625 mg in the absence of reaction, and all of the trimethylamine was derived from the 10mL aqueous extract obtained in the step (1), so that the concentration of trimethylamine in the aqueous extract was 0.01625mg/mL.
Trimethylamine concentration x=nv in aquatic products [ (V) 1 +V 2 )/V 3 ]X=0.0125 mg/mL 13mL [ (90ml+10ml)/10 mL can be calculated per M]20 g=8.125 mg/100g, namely, each 100g of grass carp meat contains 8.125mg of trimethylamine, and the fresh index of the freshwater fish is that the trimethylamine content is not higher than 6mg/100g, so that the grass carp is in an stale state.
Verification example 2
Test example 3 the trimethylamine concentration of the remaining 90mL aqueous extract was determined by headspace-gas chromatography with reference to HJ 1042-2019 for the measurement process parameters, and the headspace-gas chromatography test procedure was as follows.
Accurately sucking 2ml of water extract into a 20ml headspace bottle, sealing by a gland, and injecting 5.0ml of 50% sodium hydroxide solution by a syringe for later use.
The temperature program of the headspace gas chromatograph was set to be maintained at 40 ℃ for 3min, then at 30 ℃/min to 220 ℃ and at 220 ℃ for 1min.
Setting the sample inlet temperature of a headspace-gas chromatograph to 220 ℃, the detector temperature to 220 ℃, and the split ratio to be 2:1, air flow rate 400ml/min, hydrogen flow rate 40ml/min, and nitrogen flow rate 35ml/min.
Headspace sample injection: and (5) balancing at 40 ℃ for 30min, and then carrying out sample injection measurement.
And (3) carrying out qualitative analysis by referring to retention time of trimethylamine in a standard chromatogram in the ambient air, and carrying out quantitative analysis by adopting an external standard method. And drawing a calibration curve, and calculating the concentration of trimethylamine in the sample solution by using the calibration curve. The final trimethylamine concentration was 0.0161mg/mL, and the 0.01625mg/mL deviation from the test value obtained in example 3 was 0.9%, indicating that the trimethylamine concentration was accurate by absorbance.
Summarizing: from a combination of the above examples and the verification examples, it was found that the trimethylamine concentration measured after the interference of ammonia, methylamine and dimethylamine was removed was accurate. The detection interference of other amines in the aquatic products on the concentration of the trimethylamine is not obvious, so the method can be used for determining the concentration of the trimethylamine in the aquatic products, and the result is accurate.
Comparative example 1
The comparative example 1 and example 2 used substantially the same technical scheme to detect trimethylamine content in mackerel meat, except that the addition of maleic anhydride solution and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate to the extract was omitted and 1mL toluene was added instead, as follows.
Taking 20g of mackerel meat as in example 2, mashing, adding 30mL of water, stirring for 10min, centrifuging, and collecting supernatant. The remaining solid residue was repeatedly added with 30mL of water twice, shaken and centrifuged to obtain a supernatant, and the three supernatants were combined and added with 90mL of water three times. 10mL of an aqueous solution of trichloroacetic acid having a concentration of 0.2g/mL was added to the combined 90mL of the supernatant, the mixture was shaken, the protein in the supernatant was completely precipitated with trichloroacetic acid, and the mixture was centrifuged to obtain about 100mL of a total supernatant of the upper layer, which was an aqueous extract containing ammonia, methylamine, dimethylamine and trimethylamine, followed by adding the reagent as follows.
(1) Taking 10mL of water extract, adding 1mL of formaldehyde solution with the concentration of 0.1g/mL, stirring for 1min, wherein the solvent is water; adding 10mL of toluene, oscillating, separating the solution into a toluene layer and a water layer, obtaining the toluene layer, wherein the toluene layer contains substances such as methylamine, dimethylamine, trimethylamine and the like, adding anhydrous sodium sulfate, and drying to obtain an extract;
(2) Adding 1mL of toluene to the extract;
(3) And finally, adding 2mL of 2,4, 6-tri-tert-butylphenol solution with the concentration of 0.01g/mL into the extract, taking toluene as a solvent, and carrying out oscillation reaction for 1min to obtain the test solution.
The absorbance of the test solution at 452nm was 1.09, and the corresponding trimethylamine concentration was found to be 0.065mg/mL on the standard curve of FIG. 1. Since the total volume of the test solution was 13mL, the mass of trimethylamine in the test solution was 0.065mg/mL 13 ml=0.845 mg in the absence of reaction, and all of the trimethylamine was derived from the 10mL aqueous extract obtained in the step (1), so that the concentration of trimethylamine in the aqueous extract was 0.0845mg/mL.
Trimethylamine concentration x=nv in aquatic products [ (V) 1 +V 2 )/V 3 ]X=0.065 mg/mL 13mL [ (90ml+10ml)/10 mL can be calculated as./M]20 g=42.25 mg/100g, i.e. 42.25mg of trimethylamine is contained in every 100g of mackerel, whereas the concentration of trimethylamine contained in the mackerel measured in example 2 is 29.25mg/100g, test example 1 measured and calculated to give the concentration of trimethylamine contained in the mackerel of 28.7mg/100g, it was found that the concentration of trimethylamine contained in the mackerel measured in comparative example 1 was inaccurate, and the result was large, which is that methylamine and dimethylamine contained in the mackerel were not reacted by other reagents, methylamine and dimethylamine were directly complexed with 2,4, 6-tri-t-butylphenol, and the complexing product increased the result of absorbance of the test solution.
Comparative example 2
The comparative example 2 and example 2 adopt the same technical scheme to detect the trimethylamine content in mackerel, the only difference is that the step (2) is that the maleic anhydride solution is added into the extract liquor and the 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluene sulfonate is not contained, and the concrete steps are as follows.
Taking 20g of mackerel meat as in example 2, mashing, adding 30mL of water, stirring for 10min, centrifuging, and collecting supernatant. The remaining solid residue was repeatedly added with 30mL of water twice, shaken and centrifuged to obtain a supernatant, and the three supernatants were combined and added with 90mL of water three times. 10mL of an aqueous solution of trichloroacetic acid having a concentration of 0.2g/mL was added to the combined 90mL of the supernatant, the mixture was shaken, the protein in the supernatant was completely precipitated with trichloroacetic acid, and the mixture was centrifuged to obtain about 100mL of a total supernatant of the upper layer, which was an aqueous extract containing ammonia, methylamine, dimethylamine and trimethylamine, followed by adding the reagent as follows.
(1) Taking 10mL of water extract, adding 1mL of formaldehyde solution with the concentration of 0.1g/mL, stirring for 1min, wherein the solvent is water; adding 10mL of toluene, oscillating, separating the solution into a toluene layer and a water layer, obtaining the toluene layer, wherein the toluene layer contains substances such as methylamine, dimethylamine, trimethylamine and the like, adding anhydrous sodium sulfate, and drying to obtain an extract;
(2) 1mL of maleic anhydride solution with the concentration of 0.1g/mL is added into the extract, and the solvent is toluene;
(3) And finally, adding 2mL of 2,4, 6-tri-tert-butylphenol solution with the concentration of 0.01g/mL into the extract, taking toluene as a solvent, and carrying out oscillation reaction for 1min to obtain the test solution.
The absorbance of the test solution at 452nm wavelength was 1.07 as measured by a spectrophotometer, and the corresponding trimethylamine concentration was found to be 0.062mg/mL on the standard curve of FIG. 1. Since the total volume of the test solution is 13mL, the mass of trimethylamine in the test solution is 0.062mg/mL 13 ml= 0.806mg without reaction, and the total mass of the trimethylamine is derived from 10mL of the aqueous extract obtained in the step (1), so that the concentration of trimethylamine in the aqueous extract is 0.0806mg/mL.
Trimethylamine concentration x=nv in aquatic products [ (V) 1 +V 2 )/V 3 ]X=0.062 mg/mL 13mL [ (90ml+10ml)/10 mL can be calculated]20 g=40.3 mg/100g, i.e. 40.3mg of trimethylamine is contained per 100g of mackerel, whereas example 2 shows that the mackerel contains trimethylamine at a concentration of 29.25mg/100g, test example 1 shows that the mackerel contains trimethylamine at a concentration of 28.7mg/100g, and comparative example 2 shows that the mackerel contains trimethylamine at an inaccurate concentration, because maleic anhydride and methylamine react slowly with dimethylamine without 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, the reaction rate is low, so that the mackerel contains methylamine and dimethylamine can complex with 2,4, 6-tri-t-butylphenol, and the absorbance of the test solution is increased by the complex product, resulting in a larger concentration of trimethylamine.
By combining the above examples, the verification examples and the comparison examples, when the concentration of trimethylamine in the aquatic products is tested, formaldehyde is added into the sample liquid first, so that the interference effect of ammonia can be eliminated, and then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluene sulfonate are added into the sample liquid, so that maleic anhydride, methylamine and dimethylamine generate amidated products, and the interference of methylamine and dimethylamine is eliminated. And finally, adding 2,4, 6-tri-tert-butylphenol into the sample liquid, wherein the 2,4, 6-tri-tert-butylphenol can be complexed with trimethylamine to generate yellow 2,4, 6-tri-tert-butylphenol trimethylamine, and detecting absorbance according to the yellow 2,4, 6-tri-tert-butylphenol trimethylamine, so as to calculate the concentration of the trimethylamine in the sample. Verification of trimethylamine concentration determination by headspace-gas chromatography, the scheme for trimethylamine concentration test of the examples was adopted, and the obtained results were accurate.
The above is merely a preferred embodiment of the present application, the protective scope of which is not limited to the examples described above, it should be pointed out that modifications and adaptations to those skilled in the art without departing from the principles of the present application should and are considered to fall within the protective scope of the present application.
Claims (6)
1. A method for detecting trimethylamine concentration, the method comprising:
making a standard curve of trimethylamine concentration corresponding to absorbance: preparing a plurality of groups of trimethylamine standard solutions with different concentrations, and respectively adding 2,4, 6-tri-tert-butylphenol; 2,4, 6-tri-tert-butylphenol solution is used as a blank standard control group, and the absorbance of a plurality of groups of trimethylamine standard solutions with different concentrations relative to the blank standard control group is tested to obtain a plurality of relative absorbance values; preparing the standard curve according to the relation that a plurality of concentration values of a plurality of groups of trimethylamine standard solutions are in one-to-one correspondence with a plurality of relative absorbance values;
trimethylamine concentration in test aquatic products: firstly, extracting trimethylamine in an aquatic product to obtain an extracting solution; obtaining a plurality of extracting solutions, adding formaldehyde to fully react ammonia in the extracting solutions, then adding maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate to fully react methylamine and dimethylamine in the extracting solutions, and finally adding 2,4, 6-tri-tert-butylphenol to react with trimethylamine in the extracting solutions to obtain test solutions; taking a 2,4, 6-tri-tert-butylphenol solution as a blank test control group, testing the relative absorbance of the test solution, finding out the trimethylamine concentration corresponding to the measured relative absorbance on the standard curve, and calculating the trimethylamine concentration contained in the water product according to the found trimethylamine concentration;
the extraction of trimethylamine in aquatic products comprises the following steps: mashing aquatic product with mass of M, and adding volume of V 1 Stirring, centrifuging to obtain volume V 1 Supernatant, adding V to the supernatant 2 To precipitate protein in the clear liquid, centrifuging, and collecting the solution with volume of V 3 As the extract, the extract contains trimethylamine;
the mass of formaldehyde added is 0.001M [ V ] 3 /（V 1 +V 2 ）]10 to 70 times of the total number of the components; the mass of maleic anhydride added is 0.001M [ V ] 3 /（V 1 +V 2 ）]10 to 70 times of the total number of the components; the mass of the added 2,4, 6-tri-tert-butylphenol is 0.001M [ V ] 3 /（V 1 +V 2 ）]5 to 15 times of the total weight of the steel sheet.
2. The method for detecting the concentration of trimethylamine according to claim 1, wherein when a standard curve of the concentration of trimethylamine corresponding to absorbance is prepared, a plurality of groups of trimethylamine standard solutions in a concentration range of 0.002-0.2 mg/mL are prepared, 2,4, 6-tri-tert-butylphenol is added into each group of trimethylamine standard solutions, the mass of each group of added 2,4, 6-tri-tert-butylphenol is the same, and the molar amount of each group of added 2,4, 6-tri-tert-butylphenol is larger than the molar amount of trimethylamine in each group of trimethylamine standard solutions; calculating according to an unreacted state, wherein the concentration of the added 2,4, 6-tri-tert-butylphenol in the trimethylamine standard solution is 1-3 mg/mL; the concentration of 2,4, 6-tri-tert-butylphenol in the blank standard control group is the same as the concentration of 2,4, 6-tri-tert-butylphenol in the trimethylamine standard solution calculated in an unreacted state.
3. The method for detecting a trimethylamine concentration according to claim 2, wherein the plurality of sets of trimethylamine standard solutions in the concentration range of 0.002 to 0.02mg/mL are respectively 0.002mg/mL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL, 0.010mg/mL, 0.012mg/mL, 0.014mg/mL, 0.016mg/mL, 0.018mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.10mg/mL, 0.11mg/mL, 0.12mg/mL, 0.13mg/mL, 0.14mg/mL, 0.15mg/mL, 0.16mg/mL, 0.17mg/mL, 0.18mg/mL, 0.19mg/mL, and a trimethylamine concentration range of 0.20 mg/mL.
4. The method for detecting the concentration of trimethylamine according to claim 1, wherein when the concentration of trimethylamine in an aquatic product is tested, the formaldehyde is added to fully react ammonia in the extracting solution to obtain a primary reaction solution, and toluene is added to the primary reaction solution to extract to obtain an extracting solution; and then maleic anhydride and 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate are added into the extract liquid to completely react methylamine and dimethylamine in the extract liquid, thus obtaining a secondary reaction liquid.
5. The method for detecting the concentration of trimethylamine according to claim 4, wherein 2,4, 6-tri-tert-butylphenol and trimethylamine are added into the secondary reaction solution to react, thereby obtaining the test solution; the concentration of the 2,4, 6-tri-tert-butylphenol in the blank test control group is the same as the concentration of the 2,4, 6-tri-tert-butylphenol in the test solution calculated according to the unreacted state; testing the relative absorbance of the test solution, and finding out that the trimethylamine concentration corresponding to the relative absorbance is N on the standard curve, wherein the volume of the test solution is V, and the trimethylamine concentration X=NV [ (V) in the aquatic product 1 +V 2 ）/V 3 ]/M。
6. The method for detecting the concentration of trimethylamine according to claim 1, wherein the relative absorbance of the trimethylamine standard solution and the test solution is measured as the absorbance of the test solution at 452 nm.
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