CN117607305A

CN117607305A - Method for rapidly detecting and quantifying amino acid and biogenic amine in seasoning

Info

Publication number: CN117607305A
Application number: CN202311655792.1A
Authority: CN
Inventors: 薛长湖; 王立昊; 渠文慧; 温运启; 李若姝; 刘红英
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-02-27
Anticipated expiration: 2043-12-05
Also published as: CN117607305B

Abstract

The invention discloses a rapid detection and quantification method for amino acid and biogenic amine in a seasoning, and belongs to the technical field of food detection. According to the invention, pretreatment is carried out according to the characteristics of a sample to be detected to obtain a sample solution; filtering the sample solution by a filter membrane; taking a sample to be detected, an acetonitrile solution containing PITC and an acetonitrile solution containing triethylamine, and carrying out light-shielding, room temperature and intense vortex vibration to carry out a derivatization reaction; HPLC analysis was performed again. The invention can carry out qualitative and quantitative detection on 21 amino acids, various biogenic amines and taurine, and the detection time is far lower than that in the prior art. The method is used for preprocessing the characteristics of the sample, overcomes the interference of PITC (peptide-based acid) derivative on the mechanical effects of high salt, high sugar and the like, has lower cost, is favorable for being popularized in the detection of free amino acid, biogenic amine and taurine of various condiments, and can solve the problem of amino acid detection in various condiments, in particular to high-freshness condiments such as soy sauce and the like.

Description

Method for rapidly detecting and quantifying amino acid and biogenic amine in seasoning

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a rapid detection and quantification method for amino acid and biogenic amine in a condiment.

Background

The traditional flavoring with delicate flavor in China contains various nutritional ingredients, such as soy sauce, oil consumption, table vinegar, cooking wine, fish sauce, flavor stranguria and other novel flavoring materials, and is an important source of nutritional ingredients such as amino acid, biogenic amine and the like besides greatly enriching the flavor of food. The amino acid nitrogen and the content of free amino acid are important indexes (such as classification of national standard soy sauce grade, etc.) for identifying and measuring the flavor, quality and nutrition of the seasoning. The free amino acid is an important flavor substance in the delicious seasoning represented by soy sauce, and glutamic acid and aspartic acid serve as the delicious amino acid to provide the delicious taste in the seasoning.

Biogenic amines are small molecular organic chemical substances containing nitrogen in seasonings generated along with the processes of fermentation, storage and the like, and biogenic amines represented by cadaverine and putrescine are often accompanied by unpleasant odors. The kind and content of the seasoning are closely related to the quality of the seasoning.

The existing determination methods of free amino acid and biogenic amine mainly comprise TLC color development, hitachi amino acid automatic analyzer determination, HPLC method, capillary electrophoresis method, gas chromatography analysis and the like. Among the derivatization methods used in the pre-column are phenyl isothiocyanate (phenyl isothiocyanate, PITC), dansyl chloride (dimethylaminonaphthalene sul-vinyl chloride, dansyl-Cl), phthalaldehyde (1, 2-phthalic dicarboxaldehyde, OPA), fluorenylmethyl chloroformate, 6-aminoquinolinyl-N-hydroxy-succinimidyl formate. The HPLC method using ninhydrin as post-column derivatization reagent is accurate, reliable and good in repeatability, but has low sensitivity. Dansyl-Cl can react with amino acids and primary and secondary amino groups of biogenic amines, but the reaction is slower and derivatizing agents can be responsible for the assay results. The amino acid analysis method using PITC as the pre-column derivatization agent has high and stable reaction speed, and can react with primary and secondary amino groups.

At present, the determination of free amino acid and biogenic amine in seasoning mainly depends on a Hitachi amino acid automatic analyzer to perform qualitative and quantitative determination of free amino acid, the detection time is long, and the determination of a single sample is completed in about 150 minutes. The method has the advantages of high energy consumption, high cost and low sensitivity.

Disclosure of Invention

The invention aims to provide a rapid detection and quantification method for amino acid and biogenic amine in a seasoning, so as to make up for the defects of the prior art.

As the high-freshness seasoning generally contains flavor substances such as higher salt content, amino acid, nucleotide and the like, the matrix effect is obvious in the chromatographic measurement process, and the matrix effects of different high-freshness seasonings are different, so that the chromatographic measurement process is easy to interfere. At present, high-freshness seasonings such as soy sauce, fish sauce and the like are freshly analyzed by adopting a high performance liquid chromatography method.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

a rapid detection and quantification method for amino acid and biogenic amine in seasoning, which comprises the following steps:

(1) Pretreating a sample to be tested to obtain a sample solution;

(2) Filtering the sample solution by a filter membrane;

(3) Taking a sample to be detected, an acetonitrile solution containing PITC and an acetonitrile solution containing triethylamine, and carrying out light-shielding, room temperature and intense vortex vibration to carry out a derivatization reaction;

(4) After stopping the reaction, avoiding light, carrying out room temperature and intense vortex vibration, centrifuging, and taking the supernatant as a liquid to be detected for later use;

(5) And (3) performing high performance liquid chromatography detection on the liquid to be detected, and analyzing a chromatogram.

Further, in the step (1), if the seasoning to be detected is a high-flavor seasoning, the acid treatment is first performed: adding a proper amount of sample to be measured into 5% perchloric acid solution with four volumes, sufficiently oscillating to make the solution uniform, centrifuging, and taking supernatant; the flavoring agent with high delicate flavor is flavoring agent with high macromolecular protein content, such as soy sauce, fish sauce, etc.

Further, the centrifugation conditions are 8000-12000rpm, 5-20min.

Further, in the step (1), if the solution of the sample to be tested is deep (such as a flavoring agent with color generated by fermentation, such as soy sauce, etc.), the decolorizing treatment is needed in the pretreatment, specifically: adding active carbon, fully oscillating, adsorbing at room temperature, and standing for 1h.

Further, in the step (1), if the sample to be tested is a solid sample, the pretreatment may be a solid phase extraction method.

Further, in the step (2), filtration is performed with a 0.45 μm filter.

Further, in the step (3), 200 microliters of a sample to be tested, 100 microliters of an acetonitrile solution containing 6-12 microliters of PITC and 100 microliters of an acetonitrile solution containing 10% triethylamine which are prepared at present are taken; or taking 100 microliters of a sample to be tested, 150 microliters of an acetonitrile solution containing 6-12 microliters of PITC per milliliter and 150 microliters of an acetonitrile solution containing 10% triethylamine; vortex shaking for 15-60min.

Further, in the step (4), normal hexane is added to stop the reaction, the reaction is protected from light and is subjected to room temperature and intense vortex vibration for 20-40min, centrifugation is carried out for 10-15min at 8000-12000rpm, and the supernatant is taken out for standby.

Further, in the step (5), the chromatographic conditions are:

c18 chromatographic column with detection wavelength of 254nm and temperature of 36.5 ℃; the mobile phase is: phase A: 97%0.1mol/L sodium acetate 3% acetonitrile; and B phase: the gradient elution time of 75% acetonitrile in water was:

or (b)

Time	A	B
			5	100	0
14	94	6
			18	84	16
25	72	28
			33	50	50
35	0	100
			38	100	0
40	100	0

Compared with the prior art, the invention has the advantages that:

the invention can carry out qualitative and quantitative detection on 21 amino acids, various biogenic amines and taurine, and the detection time is far lower than that in the prior art. The method is used for preprocessing the characteristics of the sample, overcomes the interference of PITC (peptide-based acid) derivative on the mechanical effects of high salt, high sugar and the like, has lower cost, is favorable for being popularized in the detection of free amino acid, biogenic amine and taurine of various condiments, and can solve the problem of amino acid detection in various condiments, in particular to high-freshness condiments such as soy sauce and the like.

Drawings

FIG. 1 is an HPLC detection spectrum of 21 amino acid standards in example 1.

FIG. 2 is an HPLC detection spectrum of biogenic amine standard in example 2.

FIG. 3 is an HPLC detection spectrum of taurine standard in example 3.

FIG. 4 is an HPLC detection spectrum of soy sauce in comparative example 1.

FIG. 5 is an HPLC detection spectrum of soy sauce in example 4.

FIG. 6 is an HPLC detection spectrum of fish gravy in comparative example 2.

FIG. 7 is an HPLC detection spectrum of fish gravy in example 5.

FIG. 8 is an HPLC detection spectrum of cooking wine in example 6.

FIG. 9 is a HPLC detection spectrum of rice vinegar in example 7.

FIG. 10 is an HPLC detection spectrum of rice wine in example 8.

FIG. 11 is an HPLC detection spectrum of various amino acid standards in example 9.

FIG. 12 is an HPLC detection spectrum of various amino acid standards in example 10.

FIG. 13 is an HPLC detection spectrum of various amino acid standards in comparative example 3.

Detailed Description

The technical scheme of the invention is further described and illustrated below by combining with the embodiment.

In the following examples, samples were subjected to different pretreatment according to the conditions for condiments of different texture components. The method is characterized in that aiming at the characteristics of high salt (soy sauce and fish sauce), high sugar (honey and mirin), high concentration ethanol (cooking wine and rice wine) and high concentration acetic acid (brewed vinegar) in different seasonings, the method for quantitatively determining amino acid and biogenic amine by using PITC as a pre-column derivatization agent and adopting an HPLC method for pretreatment of various seasonings is constructed.

Example 1

A method for determining a standard of 21 amino acids comprising the steps of:

(1) 10mg of each of the 20 amino acids and histidine was weighed out, and dissolved in ultrapure water using a 10mL volumetric flask. The mixed standard with the concentration of 1mg/mL is prepared.

(2) Filtering the mixed standard solution with a 0.25 micron filter membrane;

(3) Taking 200 microliters of mixed standard solution, 100 microliters of acetonitrile solution containing 12 microliters of PITC (PITC) and 100 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex oscillation in a dark and room temperature environment for carrying out a derivatization reaction for 30min;

(4) After stopping the reaction, 600 microliters of normal hexane is added, and the derivatization is stopped by intense vortex oscillation for 10min under the conditions of light shielding and room temperature; centrifuging at 10000xg for 5min, taking the supernatant as the liquid to be detected for standby;

(5) And (3) performing high performance liquid chromatography detection on the liquid to be detected, wherein the phase A is 0.1mol/L sodium acetate solution containing 3% acetonitrile, and the phase B is chromatographic pure acetonitrile.

The liquid phase system is Hitachi 1140 type high performance liquid chromatography which is determined by adopting Agilent XDB C18 mm chromatographic column. The schedule is as follows:

as shown in the detection result figure 1, it can be seen that the peaks of the 21 amino acid standard products can be completely separated, and accurate detection can be obtained.

Example 2:

the detection method of the biogenic amine standard substance comprises the following steps:

(1) Cadaverine, putrescine, histamine, octopamine standard 10mg were weighed separately and dissolved in ultrapure water using a 10mL volumetric flask. Preparing a mixed standard with the concentration of 1 mg/mL;

(2) Filtering the sample solution by a filter membrane;

(4) After stopping the reaction, 600. Mu.l of n-hexane was added and the derivatization was stopped by vigorous vortexing for 10min at room temperature in the absence of light. Centrifuging at 10000xg for 5min, taking the supernatant as the liquid to be detected for standby;

The liquid phase system is Hitachi 1140 high performance liquid chromatography measured by using an Osaka Cao SG 120C 18 mm chromatographic column. The schedule is the same as in example 1.

As shown in fig. 2, the biogenic amine has a peak time later than the amino acid due to hydrophobicity, which is shown in the sequence from left to right: histamine, octopamine, derivative peaks, putrescine, cadaverine. It is shown that biogenic amine can be successfully detected by the method, and the peak is not overlapped.

Example 3

The detection method of the taurine standard substance comprises the following steps:

(1) Taurine 5mg was weighed and dissolved in ultrapure water using a 5mL volumetric flask. Preparing a standard substance solution with the concentration of 1 mg/mL;

(2) Filtering the sample solution by a filter membrane;

(3) Taking 200 microliters of taurine standard solution, 100 microliters of acetonitrile solution containing 12 microliters of PITC (PITC) and 100 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex oscillation in a dark and room temperature environment for carrying out a derivatization reaction for 30min;

(5) And (3) performing high performance liquid chromatography detection on the liquid to be detected, wherein the phase A is 0.1mol/L sodium acetate solution containing 3% acetonitrile, and the phase B is chromatographic pure acetonitrile. The liquid phase system is Hitachi 1140 high performance liquid chromatography measured by Agilent XDB C18 mm chromatographic column. The time schedule is as follows,

as shown in FIG. 3, the peak time of taurine was 20.92min, indicating that taurine could be detected smoothly.

Comparative example 1:

a PITC derivatization detection method of soy sauce comprises the following steps:

(1) Filtering commercial soy sauce with a filter membrane;

(2) Taking 200 microliters of soy sauce, 100 microliters of acetonitrile solution containing 12 microliters of PITC per milliliter and 100 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex vibration in a dark and room temperature environment for carrying out a derivatization reaction for 30min;

(3) After stopping the reaction, 600. Mu.l of n-hexane was added and the derivatization was stopped by vigorous vortexing for 10min at room temperature in the absence of light. Centrifuging at 10000xg for 5min, taking the supernatant as the liquid to be detected for standby;

(4) And (3) performing high performance liquid chromatography detection on the liquid to be detected, wherein the phase A is 0.1mol/L sodium acetate solution containing 3% acetonitrile, and the phase B is chromatographic pure acetonitrile. The liquid phase system is Hitachi 1140 high performance liquid chromatography measured by Agilent XDB C18 mm chromatographic column. The schedule is the same as in example 1.

The detection result is shown in fig. 4, the method has no obvious amino acid peak except the derivative peak, and the sample is obviously layered in the derivative process, which means that the method can not detect the amino acid component in the soy sauce.

Example 4

An HPLC detection method of soy sauce, comprising the following steps:

(1) Adding one fifth of active carbon of soy sauce in a container, fully shaking and uniformly mixing with the soy sauce, and standing for 1h at room temperature;

(2) Filtering the solution to obtain clear soy sauce decolorized solution, adding perchloric acid solution with the equal volume concentration of 4%, fully mixing, and then placing in a dark place at 4 ℃ for incubation for 30min;

(3) Centrifuging 10000xg of liquid for 10min, taking supernatant, and filtering to be detected;

(4) Taking 100 microliters of soy sauce solution to be detected, 100 microliters of acetonitrile solution containing 12 microliters of PITC (PITC) and 200 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex vibration in a dark and room temperature environment for carrying out a derivatization reaction for 30min;

(5) After stopping the reaction, 600. Mu.l of n-hexane was added and the derivatization was stopped by vigorous vortexing for 10min at room temperature in the absence of light. Centrifuging at 10000xg for 5min, taking the supernatant as the liquid to be detected for standby;

(6) And (3) performing high performance liquid chromatography detection on the liquid to be detected, wherein the phase A is 0.1mol/L sodium acetate solution containing 3% acetonitrile, and the phase B is chromatographic pure acetonitrile. The liquid phase system is Hitachi 1140 high performance liquid chromatography measured by Agilent XDB C18 mm chromatographic column. The schedule is the same as in example 1.

As shown in FIG. 5, the main body of the soy sauce is composed of sweet amino acid and bitter amino acid, which shows that the nitrogen-containing compound including amino acid in the soy sauce can be successfully derived by the method.

Comparative example 2:

a fish sauce measuring method comprises the following steps:

(1) Filtering commercial fish gravy with a filter membrane;

(2) 200 microliters of fish sauce, 100 microliters of acetonitrile solution containing 12 microliters of PITC (PITC) and 100 microliters of acetonitrile solution containing 10% triethylamine are taken, uniformly mixed, and subjected to intense vortex vibration in a dark and room temperature environment for carrying out a derivatization reaction for 30 minutes;

As a result, as shown in FIG. 6, it is shown that the fish gravy is layered in the derivatization system during the derivatization process, and after the detection is completed, it is found that only the derivatization peak, pro (22.63 min), cys (22.43 min) and other amino acids are incompletely derivatized to generate smaller peaks, which are different from the fact, and it can be judged that the derivatization is failed, namely, the detection of the amino acid and other components in the fish gravy cannot be completed by the method.

Example 5

A fish sauce detection method comprises the following steps:

(1) Adding one-fourth of active carbon in the mass fraction of the fish gravy into a container, fully vibrating and uniformly mixing the active carbon and the fish gravy, and standing for 1h at room temperature;

(2) Filtering the solution with a filter membrane, centrifuging 10000xg for 10min, and collecting supernatant;

(5) And (3) performing high performance liquid chromatography detection on the liquid to be detected, wherein the phase A is 0.1mol/L sodium acetate solution containing 3% acetonitrile, and the phase B is chromatographic pure acetonitrile. The liquid phase system is Hitachi 1140 high performance liquid chromatography measured by Agilent XDB C18 mm chromatographic column. The schedule is the same as in example 1.

The results are shown in FIG. 7, which illustrates that the method can determine various amino acids in fish gravy.

Example 6

A method for detecting cooking wine, comprising the steps of:

(1) Taking a proper amount of yellow wine type material wine, centrifuging 10000xg for 10min, and taking supernatant;

(2) Filtering the solution by a filter membrane;

(3) Taking 200 microliters of a solution to be detected, 100 microliters of an acetonitrile solution containing 12 microliters of PITC (PITC) and 100 microliters of an acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out a derivatization reaction for 30min under the conditions of light shielding and room temperature;

As shown in FIG. 8, the yellow wine type cooking wine has less amino acids, and the method proves that the yellow wine type cooking wine contains Gly, ser, aln and other sweet amino acids, has a main flavor of Thr, ala, arg and has partial bitter amino acids.

Example 7

A rice vinegar detection method comprises the following steps:

(1) Taking a proper amount of rice vinegar, centrifuging at 10000xg for 5min, and taking supernatant;

(2) Filtering the sample solution by a filter membrane;

(3) Taking 100 microliters of rice vinegar solution, 100 microliters of acetonitrile solution containing 12 microliters of PITC (PITC) and 200 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex vibration in a dark and room temperature environment for carrying out a derivatization reaction for 30min;

As shown in FIG. 9, the amino acids in rice vinegar are not only small amounts of Ala and Lys amino acids generated in the fermentation process, but also Glu (5.967 min) is added to the rice vinegar to make the flavor of the rice vinegar prominent, so that the rice vinegar can be observed obviously in a spectrogram.

Example 8

A rice wine measuring method comprises the following steps:

(1) Taking a proper amount of rice wine, centrifuging at 10000xg for 5min, and taking supernatant;

(2) Filtering the sample solution by a filter membrane;

(3) Taking 200 microliters of rice wine, 100 microliters of acetonitrile solution containing 12 microliters of PITC per milliliter and 100 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex vibration in a dark and room temperature environment for carrying out a derivatization reaction for 30 minutes;

As shown in FIG. 10, the rice wine produced various amino acids during fermentation, and the rice wine had lower amino acid content and fewer kinds than the flavoring due to the process.

Example 9 implementation by using an Osaka Cadda column as an example

The detection method of the amino acid standard substance comprises the following steps:

(1) Asp, gly, ala, tyr, leu each of the above-mentioned substances was weighed 10mg, and dissolved in ultrapure water using a 10mL volumetric flask. The mixed standard with the concentration of 1mg/mL is prepared.

(2) Filtering the mixed standard solution with a 0.25 micron filter membrane;

chromatographic conditions: the detector was a Hitachi 1440 HPLC with a detection wavelength of 254nm using an Osaka Caida C18 SG120 150mm chromatographic column with a column oven at 36.5 ℃ and phase A: 97%0.1mol/L sodium acetate 3% acetonitrile; and B phase: 75% acetonitrile in water, schedule as follows:

as shown in fig. 11, it can be seen that Asp, gly, ala, tyr, leu can be accurately detected, and the peaks are symmetrical without overlapping.

Example 10:

(1) Ser, his, pro, met, lys each of the above-mentioned substances was weighed 10mg, and dissolved in ultrapure water using a 10mL volumetric flask. The mixed standard with the concentration of 1mg/mL is prepared.

(2) Filtering the mixed standard solution with a 0.25 micron filter membrane;

chromatographic conditions: the detector was a Hitachi 1440 HPLC with a detection wavelength of 254nm using an Osaka Caida C18 SG120 150mm chromatographic column with a column temperature oven of 36.5 ℃ and phase A: 97%0.1mol/L sodium acetate 3% acetonitrile; and B phase: 75% acetonitrile in water, schedule was the same as in example 9.

As shown in fig. 12, it can be seen that Ser, his, pro, met, lys can be accurately detected, and the peaks are symmetrical without overlapping.

Comparative example 3:

the prior method for detecting the amino acid standard comprises the following steps:

(1) Glu was taken as 10mg and dissolved in ultrapure water using a 10mL volumetric flask. The concentration of the product is 1 mg/mL.

(2) Filtering Glu standard solution with 0.25 micron filter membrane;

(3) Diluting Glu standard solution with ultrapure water to a concentration of 0.04mg/mL;

(4) Taking 200 microliters of Glu standard solution, 100 microliters of acetonitrile solution containing 12 microliters of PITC per milliliter and 100 microliters of acetonitrile solution containing 10% triethylamine, uniformly mixing, and carrying out vigorous vortex oscillation in a dark and room temperature environment for carrying out a derivatization reaction for 30 minutes;

chromatographic conditions: the detector is Hitachi 1440 type HPLC with detection wavelength of 254nm, adopting Agilent C18 SDB-250mm chromatographic column, column temperature box 36.5 ℃, phase A: 97%0.03mol/L sodium acetate; and B phase: 75% acetonitrile in water, schedule as follows:

as shown in FIG. 13, the glutamic acid peak was estimated to be located at the position of FIG. 4 and 793min above, but after the measurement of the samples under a plurality of groups of different concentration gradients, the linear relationship of the peak was poor, and it was difficult to obtain a standard curve for the measurement.

By specific analysis of the above examples, the present method overcomes the influence of matrix effect on the determination of amino acids in savory flavoring in liquid chromatography, which is common in operation, and successfully determines common main tasting amino acids. In the method, amino acid generated by fermentation or exogenously added amino acid can be effectively screened according to the characteristic fingerprint spectrum of the amino acid. Compared with a Hitachi amino acid automatic analyzer, the method has the advantages of lower cost and shorter time, and can be used for measuring the amino acid of the delicious seasoning in a common laboratory.

The present invention has been described in detail with reference to the above embodiments, and the functions and actions of the features in the present invention will be described in order to help those skilled in the art to fully understand the technical solution of the present invention and reproduce it.

Finally, although the description has been described in terms of embodiments, not every embodiment is intended to include only a single embodiment, and such description is for clarity only, as one skilled in the art will recognize that the embodiments of the disclosure may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. A rapid detection and quantification method for amino acid and biogenic amine in seasoning is characterized by comprising the following steps:

(1) Pretreating a sample to be tested to obtain a sample solution;

(2) Filtering the sample solution by a filter membrane;

(3) Taking a sample to be detected, an acetonitrile solution containing PITC and an acetonitrile solution containing triethylamine, and carrying out a derivatization reaction;

(4) After stopping the reaction, centrifuging, and taking the supernatant as a liquid to be detected for later use;

2. The rapid assay quantification method of claim 1, wherein in step (1), if the flavoring to be measured is a high-freshness flavoring, the acid treatment is performed first: adding a proper amount of sample to be measured into perchloric acid solution, sufficiently oscillating to make the solution uniform, centrifuging, and taking supernatant; the high-freshness seasoning comprises a seasoning with high macromolecular protein content.

3. The rapid assay quantification method of claim 2, wherein four volumes of 5% perchloric acid solution is added for acid treatment; the centrifugation condition is 8000-12000rpm, 5-20min.

4. The rapid assay quantification method of claim 1, wherein in the step (1), if the solution of the sample to be assayed is deep, a decolorization treatment is required.

5. The rapid assay quantification method of claim 1, wherein in step (2), filtration is performed using a 0.45 micron filter.

6. The rapid assay quantification method of claim 1, wherein in step (3), 200 microliters of the sample to be tested, 100 microliters of acetonitrile solution containing 6-12 microliters of PITC per milliliter, 100 microliters of 10% triethylamine in acetonitrile solution are taken; or taking 100 microliters of a sample to be tested, 150 microliters of acetonitrile solution containing 6-12 microliters of PITC per milliliter, and 150 microliters of acetonitrile solution of 10% triethylamine; vortex shaking for 15-60min.

7. The rapid detection and quantification method according to claim 1, wherein in the step (4), normal hexane is added to stop the reaction, the reaction is protected from light, room temperature and intense vortex vibration are carried out for 20-40min, centrifugation is carried out for 10-15min at 8000-12000rpm, and the supernatant is taken for later use.

8. The rapid assay quantification method of claim 1, wherein in step (5), the chromatographic conditions are: c18 chromatographic column with detection wavelength of 254nm; the mobile phase is: phase A: 97%0.1mol/L sodium acetate 3% acetonitrile; and B phase: the gradient elution time of 75% acetonitrile in water was:

or (b)

Time A B 5 100 0 14 94 6 18 84 16 25 72 28 33 50 50 35 0 100 38 100 0 40 100 0

。