Zhang Feng, researcher at the Chinese Academy of Inspection and Quarantine, et al.: Rapid detection of weedwort by a selective ion source modified by a novel molecularly imprinted polymer combined with electrospray ionization mass spectrometry

Achlorazine and atrazine belong to the triazine herbicide, the chemical structure is shown in Table 1, and it is widely
used in the process of crop planting.
However, triazine herbicides are highly toxic, long-term exposure to the human body may cause contact dermatitis, if its residue enters the human body through food, surface runoff and groundwater pollution, it will cause human distortion or induce cancer, so it is of great significance
to develop a method that can effectively detect triazine pesticide residues in food.

Ma Hongyue, Zhang Feifang* from the Engineering Research Center of Pharmaceutical Engineering and Process Chemistry, Ministry of Education, East China University of Science and Technology, and Zhang Feng* from the National Key Laboratory of Market Regulation (Food Quality and Safety) of the Academy of Inspection and Quarantine Sciences, combined molecular imprinting technology with open mass spectrometry to develop a method
for rapid detection of achlorazine and wastamine in food 。 Using iron sheet as solid substrate and cyanochlorazine as virtual template, an open ion source integrating selective enrichment and rapid detection was synthesized in aqueous solution using aniline and methacrylic acid, and the method was experimentally verified that the method was simple to operate, efficient and time-saving, and had high sensitivity, which could realize the rapid detection
of μg/kg level wasmetrazine and atrazine in food.

1.
Scanning electron microscopy characterization

As shown in Figure 1, the surface of the original iron sheet (Figure 1A) is relatively smooth, and after acidification reaction and silicon oxidation modification, Figure 1B is obtained, it can be seen that the galvanized layer is basically corroded, the surface of the iron sheet shows a bumpy form, and the surface is grafted with more spherical groups, which provides a reaction site for the subsequent modification of MIP
.
It can be seen from Fig.
1C and D that the surface of the material is honeycomb shaped, with more pores, which provides conditions
for adsorbing the analyte.

2.
Optimization of extraction conditions and mass spectrometry conditions

In this study, the extraction solvent, extraction time and pH value of the extraction solution were optimized in a mixed solution of 50 μg/kg soymilk powder sample, and the results are shown in
Figure 2.

It can be seen from Figure 2A that compared with acetone and ethyl acetate, acetonitrile is enriched with more targets and higher peak area detected when MIPIS is used as an extraction solvent, so acetonitrile has a better
extraction effect on atamethine and atrazine.
Since achlorazine and atrazine belong to medium-polar pesticides, and ethyl acetate is more suitable for the extraction of some more polar substances, ethyl acetate has a slightly weaker
extraction ability for atamethine and atrazine.
At the same time, acetonitrile has the effect of precipitating proteins, which is conducive to reducing matrix interference in the sample, thereby improving the extraction efficiency; And acetonitrile has highly volatile characteristics, which is more conducive to the blow-drying and reconstitution process
of the extract.
Therefore, acetonitrile was chosen as the extraction solvent
for the experiment.

As shown in Figure 2B, the peak area formed by the quantitative ion of amazette showed an increasing trend in the first 20 min, reached a maximum in 20 min, and within 20~30 min, the macromolecules in the sample matrix interfered with the adsorption site, resulting in a slight decrease in the peak area value, basically reaching dynamic equilibrium
.
Atrazine had the largest peak area value measured at 25 min extraction time, but only 2.
7%
higher than the peak area measured at 20 min extraction time.
Because the extraction experiment was performed in a mixed solution of two targets, in order to unify the extraction time, 20 min was selected as the extraction time of MIPIS
.

The results are shown
in Figure 2C.
Under the condition of pH 7, MIPIS has the best enrichment effect on atrazine and atrazine, and under the condition of pH 3 or 10, MIPIS has poor enrichment effect on asmetrazine and atrazine, which may be due to the instability of azhemetine and atrazine under the condition of strong acid or base, and a decomposition reaction
will occur.
According to the structure of azinenet and atrazine, the hydrogen atom of the amino group and the nitrogen atom of the triazine ring will serve as binding sites to construct the required key interactions, while the carboxyl group in methacrylic acid is both a hydrogen bond acceptor and a donor, so azetinet and atrazine will form a double hydrogen bond
with methacrylic acid, respectively.
At low pH, this may cause the target molecule or hydrogen bond site in methacrylic acid to interact directly with hydrated hydrogen ions because the acidic group and the target are in protonated form, rather than between the target and MIPIS
.
At higher pH, more targets and acidic groups of methacrylic acid also take on ionic form, reducing hydrogen bonding capacity
.
Therefore, higher or lower pH values will lead to a decrease
in the extraction efficiency of MIPIS for achlorphenazine and atrazine.
Therefore, a pH of 7 was chosen as the optimal pH for the extraction solution
.

As shown in Figure 2D, the signal intensity
of the quantitative ions of ammetrazine and atrazine at spray voltages of 2.
8, 3.
0, 3.
3, 3.
5, 3.
8 kV and 4.
0 kV was detected, respectively.
When the spray voltage is less than 2.
8 kV, due to the large surface tension of the spray solvent, an effective spray cannot be formed, so the signal of the target cannot be detected; When the spray voltage is greater than 2.
8 kV, the signal strength increases with the increase of the spray voltage, and when the spray voltage reaches 3.
3 kV, the signal strength reaches a maximum
.
When the spray voltage is greater than 3.
3 kV, the signal intensity gradually decreases, which is because when the spray voltage is too high, the spray solvent wrapped around the target collects at the tip of MIPIS to form large droplets, which are quickly dispersed into small droplets sprayed around the mass spectrometry injection port, and only a small number of small droplets form an effective spray into the mass spectrometry injection port, resulting in a low
signal intensity of the target.
Therefore, 3.
3 kV was chosen as the spray voltage
.

As shown in Figure 2E, the elution capacity of amylene and atrazine on MIPIS was investigated as spray solvents by methanol, 0.
2% formic acid-methanol and 0.
2% ammonia-methanol
, respectively.
According to the peak area of each quantitative ion, the spray solvent was the largest amount of ammetine and atrazine eluted by 0.
2% ammonia-methanol, and its values were 1.
19 times and 1.
13 times the desorption amount when the spray solvent was methanol, and 1.
45 times and 1.
39 times
that of the spray solvent was 0.
2% formic acid-methanol.
This is due to the fact that hydrogen bonds are more likely to break under alkaline conditions, making it easier to separate the target from the selective site on MIPIS, thereby eluting more targets
.
Therefore, 0.
2% ammonia-methanol was selected as the spray solvent
for the experiment.

3.
MIPIS material evaluation

Specific evaluation

The adsorption results of MIPIS and NIPIS on ammetrazine, atrazine and sulfonamide for trimethopyrimidine are shown
in Figure 3.
The results showed that the corresponding peak areas of achlorazine and atrazine adsorbed by MIPIS were 1.
6 times and 2.
6 times that of NIPIS, which was due to the specific adsorption site of MIPIS, which had a large adsorption capacity for the target, while NIPIS was disturbed by other macromolecular substances in the sample, so the adsorption capacity of the target would be reduced
.
It can also be seen that MIPIS and NIPIS have small adsorption capacity due to the lack of specific adsorption pores for sulfonamide to trimetoximidine
.

Enrichment efficiency evaluation

The results showed that the enrichment factors of MIPIS for atazine and atrazine were 22.
24±2.
30, 28.
14±3.
75, respectively, and the enrichment factor values of azinenet were slightly lower than those of atrazine
.
From the chemical structure, because the substituent steric resistance on the triazine ring of atrazine is greater, it shows weaker competitiveness when competing with atrazine for specific adsorption sites on MIPIS, so the enrichment value of atrazine is higher
.

4.
Methodological evaluation of MIPIS combined with electrospray ionization mass spectrometry

Examination of matrix effects

The matrix effect has matrix inhibition and matrix promotion effect, and its evaluation criteria are: when the matrix effect is greater than 20%, it is manifested as obvious matrix enhancement effect; When the matrix effect is less than -20%, it is manifested as obvious inhibition; When the matrix effect is between -20%~20%, the matrix effect is not obvious
.
When matrix effects are apparent, accurate quantification using matrix standard curves is required to eliminate matrix effects
.
According to calculations, the matrix effects of azinenet and atrazine were -23.
9% and -8.
7%, respectively, and the results showed that the sample matrix had a matrix inhibitory effect on atrazine, and the matrix effect of atrazine was not obvious, that is, for azine, MIPIS was weak in resistance to matrix interference in soymilk powder samples
.
Therefore, for more accurate quantitative results, matrix standard curves of azamethine and atrazine were used as the quantitative standard
.

Standard curve, LOD and LOQ results

The test was performed according to the method in section 1.
3.
3, and the obtained standard curve results are shown
in Table 3.
Achlorazine and atrazine had good linearity in the range of 0.
2~200 μg/L mass concentration (^R2>0.
998 2).

In addition, the limit of detection (LOD) and limit of quantification (LOQ) of asymenet and atrazine were also determined, and the results were shown in Table 3, the LOD range was between 0.
2~0.
5 μg/kg, and the LOQ was between 0.
6~1.
5 μg/kg, indicating that MIPIS combined with electrospray ionization mass spectrometry method can achieve sensitive detection
of trace asyphenazine and atrazine residue in soy milk powder.

Repeatability and reproducibility results

Repeatability and reproducibility
are evaluated by measuring intraday precision and interday precision.
The repeatability and reproducibility
of MIPIS-combined electrospray ionization mass spectrometry were evaluated by continuous analysis of soybean milk powder spiked samples for 6 times within 1 d and measured for 3 days.
A mixed standard of ataminet and atrazine was added to the blank soy milk powder sample to be tested to prepare a mixed solution with a content of 50 μg/kg, and the RSD
of achlorazine and atrazine was determined.
As shown in Table 3, the results showed that the intraday precision was 12.
10% and 10.
63%, and the intraday precision was 8.
79% and 13.
04%, indicating that MIPIS combined with electrospray ionization mass spectrometry had good repeatability and reproducibility for the detection of atsumazine and atrazine in soy milk powder
.

5.
Material reusability investigation

As shown in Figure 4, the recovery rate of MIPIS gradually decreases with the increase of the number of uses, which may be due to the fact that each elution causes a certain amount of damage to the adsorption site on MIPIS, and the adsorption site will swell in the organic solvent, resulting in a decrease
in the effective adsorption site.
In these five adsorption-elution cycles, the recovery rates of azine~95.
06% and 82.
43%~106.
45%, respectively, proved that MIPIS can be reused at least 5 times
.

6.
Actual sample determination and accuracy investigation

As shown in Table 4, the recovery rate of azamethine and atrazine was in the range of 81.
05%~109.
66%, and the RSD was between 4.
03%~15.
89%, indicating good
accuracy.

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