Pesticides Analysis in Beans by Gas Chromatography Couplet with Tandem Mass Spectrometry

Mizutani G; Bustillos O

Research Article

Ann Agric Crop Sci. 2021; 6(4): 1082.

Pesticides Analysis in Beans by Gas Chromatography Couplet with Tandem Mass Spectrometry

Mizutani G* and Bustillos O

Centro de Química e Meio Ambiente, Instituto de Pesquisas Energéticas e Nucleares, São Paulo, Brazil

*Corresponding author: Mizutani G, Centro de Química e Meio Ambiente, Instituto de Pesquisas Energéticas e Nucleares, São Paulo, Brazil

Received: May 11, 2021; Accepted: June 07, 2021; Published: June 14, 2021

Abstract

Beans are part of the basic diet alimentation for Brazilian population, as they gather proteins, carbohydrates, vitamins, mineral salts, fibers, amino acids and essential nutrients such as iron and calcium, being a complete food that can be compared with the amount of protein that the meat has. Considering the beans world production, in development countries represent almost 50%, being that Myanmar, India and Brazil the top three position. The use of pesticides is widely spread in these countries to reduce agricultural losses due to pests that interfere with grain production. Therefore the risk that could be generated from foods pesticides residues makes their analyses of quantification mandatory. The purpose of this work was to develop an analytical method to quantitatively characterize fungicides pesticides residues, flutriafol, procymidone and tebuconazole that were used to angular spot control, anthracnose, rust and alternaria spot, white mold fungi, present in beans, by means of gas chromatography coupled with a triple quadrupole mass spectrometer. Samples of beans, Phaseolus vulgaris L, types white, black, string and Vigna angularis, type adzuki, had been bought in grains store and supermarkets at metropolitan São Paulo city. The validation of analytical method was explored for sensitivity, selectivity, precision. The extraction procedure was performed in two different forms, QuEChERS, and solid-liquid extraction with low temperature. Through this methodology, reached below the maximum limit allowed by Brazilian law 0.5mgkg^-1 for procymidone and 0.1mgkg^-1 for flutriafol and tebuconazole. Several samples of four types of beans were tested and all of them had procymidone identified and 7% of samples higher than the law limit.

Keywords: Beans; GCMSMS; Pesticides

Introduction

In Brazil, beans are the most popular food for general population, they have nutrients and energy that work in health prevention. In their composition there are proteins, carbohydrates, vitamins, mineral salts, fibers and amino acids [1].

Grains producers have been using several pesticides in this vegetable culture to preserve and improve the crop. The fungicides utilization has a function of preventing plant tissues infection by phytopathogenic fungi, currently we can find other concepts such as chemical compounds that are used to control diseases that are caused by fungi, bacteria and algae, in some cases they do not eliminate fungi, but temporarily inhibit spore germination [2].

The procymidone is using in mold-white fungi in a concentration of 500gkg^-1 and acts to inhibit the growth of micelles, in the protection, cure [3,4].

The flutriafol and tebuconazole are fungicides that doing inhibit the synthesis of sterols that act in the formation and selectivity of the plasma membrane are used to control angular stain, anthracnose, rust and alternaria stain [3,4].

Fruits, vegetables, cereals are the most matrices that could find some pesticides residues in different classes and method development for this compounds are very important [5].

For these pesticides analyses several extraction are being used to aim these analytes. Acetonitrile is the best solvent to extract the samples because it has the best interaction with the analytes [6].

Therefore sample preparation using the QuEChERS multiresidues method, starts with the sample grind to get the most surface area to be in solvent contact, add magnesium sulfate, sodium chloride or sodium acetate, drying agents and clean up processes [7,8].

Other extraction technique used was solid-liquid with low temperature, in this extraction the sample, liquid or solid, in contact with solvent less dense than water and the less melting point less than 20°C negative, after shaking, rest in freezer about 16 hours, the water phase will be freeze and the organic phase will be liquid, this part has to be transfer to a vial and chromatography analyses [9,10].

This paper developed a method to characterize and quantify the fungicides that is present in vegetable with high protein content, beans specifically, that used a gas chromatography with mass spectrometry triple quadrupole TANDEM.

Methods and Materials

Materials and equipment

The following materials were used in this work:

• Falcon tube 10mL and 50mL;

• Analytical standards, flutriafol, procymidone, tebuconazole;

• Acetronitrile HPLC grade, isopropyl alcohol HPLC grade and ultrapure water;

• Vortex;

• Low temperature freezer -20°C;

• Blender;

• Gas chromatography coupled with Tandem mass spectrometry Bruker-EVOQ.

Method

Extraction: For extraction it was used a two different methods: QuEChERS, the most analytical extraction for pesticides, was used sample crushed, 3g, extraction salt, magnesium sulfate, 4g, and sodium acetate, 1g, with organic solvent, 4mL acetonitrile and 1mL isopropyl alcohol, shake 1 minute, centrifuge transfer 1mL to and clean up vial 150mg magnesium sulfate and 50mg PSA, transfer to 2mL vial and chromatography analyses, the second method solidliquid with low temperature, was used a sample crushed, 3g, in contact with organic solvent less dense than water, 4mL acetonitrile and 1mL isopropyl alcohol, and fusion point less than 20°C negative, the organic phase was liquid, transfer to 2mL vial and chromatography analyses, flowchart should be observe in Figure 1.

Figure 1: Extraction flowchart.

    
    
    Figure 1: Extraction flowchart.

Analytical method: For analytical method it was used an instrumental analytical, gas chromatograph coupled with Tandem mass spectrometry equipment. It was developed method to separate the analytes. This method had a good sensitivity, accuracy and precision to fungicides compounds. All parameters of this analytical method can be found in Table 1 and 2.

Table 1: Chromatography method.




  
    Auto sampler
    1μL
  
  
    Injector
    250°C
  
  
    Flow
    1mLmin-1 splitless 0.80min
  
  
    Carrier Gas
    Helium
  
  
    Column
    5MS - 30m x 0.25mm x 0.25μm
  
  
    Oven
    
    
  
  
    Rate
    Temperature
    Hold
  
  
    Initial
    100°C
    0min
  
  
    20°C min-1
    220°C
    3min
  
  
    40°C min-1
    280°C
    4min



Table 1: Chromatography method.

Table 2: Mass spectrometry method.




  
    Source
    250°C
  
  
    Transfer line
    230°C
  
  
    MS mode
    MRM - Multiple Reaction Monitoring
  
  
    Diuron
    161 > 90 (10eV)
  
  
    Flutriafol
    219 > 164.70 (10eV)
  
  
    Procymidone
    96 > 67 (10eV)
  
  
    Tebuconazole
    250 > 125 (10eV)



Table 2: Mass spectrometry method.

Validation

The analytical method validation has been processed with efficiency; the following parameters were evaluated to suitability guarantee.

These are selectivity, linearity, work range, detection limit, quantitation limit, recovery and accuracy. All validation steps add compound in matrix and follow the INMETRO DOQ-CGCRE-008, Brazilian validation document [11].

Selectivity

An analytical method to quantify the aim analyte in presence with other analytes or interference material was established. To identify the selectivity, standard injection had been done, to evaluate, retention time, fragmentation and column analytes separation. Full scan mode with extraction ions and four multiple reaction monitoring, MRM were done, these are shown in Figure 2.

Figure 2: Full scan, left and MRM, right, analyses.

    
    
    Figure 2: Full scan, left and MRM, right, analyses.

Linearity and work range

Linearity is an analytical procedure that can produce results proportionality to a sample analyte concentration. Work range is the range between the highest and lowest sample concentration, (10ugkg^-1, 25ugkg^-1, 50ugkg^-1, 75ugkg^-1, 100ugkg^-1, 250ugkg^-1, 500ugkg^-1) that it has the method with an acceptable precision, accuracy and linearity. In Figure 3 it can see the linearity for four analytical compounds.

Figure 3: Calibration curve.

    
    
    Figure 3: Calibration curve.

Detection and quantitation limit

Detection limit is the lowest concentration that can be found but not necessarily quantified. For this test all spectra compound has a lowest concentration 10ugkg^-1 detectable.

Quantitation limit is the less sample quantity that can be quantified with precision and accuracy. For quantitation limit, four compounds used 25ugkg^-1. Figure 4 shows a spectrum for detection limit, in the left hand and quantitation limit, in the right hand.

Figure 4: Detection limit, left and Quantitation limit, right.

    
    
    Figure 4: Detection limit, left and Quantitation limit, right.

Recovery

Recovery is estimated by fortified sample analysis with known compound and three different concentrations, low, 25ugkg^-1, medium, 75ugkg^-1 and high, 250ugkg^-1, the recovery range is between 80-110. It was used two different compounds to evaluated recovery, Diuron and Tebuconazole standard in solvent, observe the recovery in Table 3 and Table 4.

Table 3: Diuron recovery.



Table 3: Diuron recovery.

Table 4: Tebuconazole recovery.



Table 4: Tebuconazole recovery.

Accuracy

In the accuracy was not more than 20% and the standard deviation relative was not more than 5%. There are three most common evaluations: repeatability, intermediate precision and reproducibility, usually expressed by standard deviation and coefficient of variation. For this validation it was used repeatability which means that this procedure needs the same requirements, such as operator, preparation conditions, systems, and place. The compounds were added the compounds in the matrix and used different range concentration, low, 25ugkg^-1, medium, 50ugkg^-1 and high 250ugkg^-1 (Table 5-7).

Table 5: Flutriafol low precision.




  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Flutriafol
    White beans
  
  
    1
    23.671
    25
    -5.316
  
  
    2
    23.614
    25
    -5.544
  
  
    3
    23.764
    25
    -4.944
  
  
    4
    26.196
    25
    4.784
  
  
    5
    24.202
    25
    -3.192
  
  
    Average
    24.289
    
    
  
  
    SD
    1.09
    
    
  
  
    RSD(%)
    4.49
    
    
  
  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Flutriafol
    Adzuki beans
  
  
    1
    23.753
    25,000
    -4,988
  
  
    2
    24.216
    25,000
    -3,136
  
  
    3
    23.171
    25,000
    -7,316
  
  
    4
    23.528
    25,000
    -5,888
  
  
    5
    23.103
    25,000
    -7,588
  
  
    Average
    23.554
    
    
  
  
    SD
    0.46
    
    
  
  
    RSD(%)
    1.93



Table 5: Flutriafol low precision.

Table 6: Diuron medium precision.




  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Diuron
    String beans
  
  
    1
    58.517
    50
    17.034
  
  
    2
    58.606
    50
    17.212
  
  
    3
    57.982
    50
    15.964
  
  
    4
    54.312
    50
    8.624
  
  
    5
    56.627
    50
    13.254
  
  
    Average
    57.209
    
    
  
  
    SD
    1.8
    
    
  
  
    RSD(%)
    3.15
    
    
  
  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Diuron
    White beans
  
  
    1
    49.924
    50
    -0.152
  
  
    2
    56.291
    50
    12.582
  
  
    3
    54.79
    50
    9.58
  
  
    4
    55.175
    50
    10.35
  
  
    5
    54.15
    50
    8.3
  
  
    Average
    54.066
    
    
  
  
    SD
    2.44
    
    
  
  
    RSD(%)
    4.52



Table 6: Diuron medium precision.

Table 7: Tebuconazole high precision.




  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Tebuconazole
    White beans
  
  
    1
    228.017
    250
    -8.793
  
  
    2
    244.589
    250
    -2.164
  
  
    3
    229.1
    250
    -8.36
  
  
    4
    247.478
    250
    -1.009
  
  
    5
    234.857
    250
    -6.057
  
  
    Average
    236.808
    
    
  
  
    SD
    8.87
    
    
  
  
    RSD(%)
    3.75
    
    
  
  
    
    Sample
    Concentration
    Referencie Concentration
    Accuracy (%)
  
  
    Tebuconazole
    Adzuki beans
  
  
    1
    248.837
    250
    -0.4652
  
  
    2
    259.327
    250
    3.731
  
  
    3
    239.374
    250
    -4.25
  
  
    4
    231.291
    250
    -7.484
  
  
    5
    255.876
    250
    2.35
  
  
    Average
    246.941
    
    
  
  
    SD
    11.61
    
    
  
  
    RSD(%)
    4.7



Table 7: Tebuconazole high precision.

Results and Discussion

The analytical method validations were satisfactory for selectivity, sensitivity, linearity and accuracy. Two different extractions were done, for QuEChERS extraction we made a comparative between clean-up and without clean-up. The results showed that cleanup was very effective to matrix effect and reduced the signal to noise chromatogram protein and showed better results than without cleanup, Table 8. The solid- liquid extraction at low temperature had different results for all types of beans, these results may have occurred due to bad interaction of the contact surface or bad organic solvent interaction (Table 9). Therefore, in this comparative extraction method low temperature was not efficient to these fungicides’ compounds.

Table 8: QuEChERS comparations results.




  
    
    
    Beans
  
  
    Black
    White
    String
    Adzuki
    Unit
  
  
    QuEChERS
    Without cleanup
    0
    0
    0
    0
    μgkg-1
  
  
    0
    0
    0
    0
  
  
    0
    0
    0
    0
  
  
    0
    0
    0
    0
  
  
    0
    0
    0
    0
  
  
    With cleanup
    24.171
    24.162
    23.268
    24.546
    μgkg-1
  
  
    24.423
    24.072
    25.268
    24.233
  
  
    24.28
    23.561
    24.83
    25.963
  
  
    23.422
    23.932
    23.814
    26.6
  
  
    23.823
    25.142
    22.896
    24.627



Table 8: QuEChERS comparations results.

Table 9: Low temperature extraction results.




  
    
    Beans
  
  
    Black
    White
    String
    Adzuki
    Unit
  
  
    Low temperature
    145.117
    77.314
    203.524
    103.577
    μgkg-1
  
  
    143.115
    77.61
    199.542
    102.983
  
  
    43.567
    31.932
    25.024
    0.000
  
  
    40.059
    45.41
    70.903
    30.968
  
  
    35.888
    49.25
    59.902
    0.000



Table 9: Low temperature extraction results.

All samples had showed procymidone as identified and their quantitative results are shown at Table 10. Nevertheless the most procymidone samples results are below the Brazilian law regulation limit 0.5mgkg-1 and that means that it is good for consumption but in 7% in all sample showed a lit bit higher than law regulation limit. Flutriafol and tebucanozole don’t appear in spectrum results or above the detection limit (Figure 5).

Figure 5: Sample spectrum, left without cleanup, middle with cleanup, right low temperature.

    
    
    Figure 5: Sample spectrum, left without cleanup, middle with cleanup, right low temperature.

Table 10: Sample Results.



Table 10: Sample Results.

Conclusion

The methodology developed in this work shows a good agreement due to validation method for linearity, sensibility, accuracy, detection and quantitation limit.

Two different extractions were tested and it was observed that low temperature extraction has a disadvantage, a long time inside low temperature freezer, about 15 hours, to freeze the aqueous phase but was not efficient for these fungicides compounds, but it is not necessarily useless for other pesticides compounds.

In QuEChERS extraction showed that the clean-up extraction was efficient to prevent matrix effect, the signal to noise chromatogram protein is reduced and better results were achieved, but in disadvantage some difficult to buy a extraction salt and a abruptly agitation about 1 minute.

We were analyzed about 30 samples and the fungicides results in the beans stood below the regulation limit, which means that they are good for consumption except for 2 sample in 30, that a lit bit higher than regulation limit, that maybe bought when the store recently receive, this proves that we have a fungicides degradation by time of exposure.

References

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Citation:Mizutani G and Bustillos O. Pesticides Analysis in Beans by Gas Chromatography Couplet with Tandem Mass Spectrometry. Ann Agric Crop Sci. 2021; 6(4): 1082.

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Sample	Amount	Reference	Recovery	Sample	Amount	Reference	Recovery	Sample	Amount	Reference	Recovery
1	24.915	25	99.66	1	79.865	75	106.486	1	251.538	250	100.615
2	24.117	25	96.468	2	73.68	75	98.24	2	241.158	250	96.463
3	22.005	25	88.02	3	75.573	75	100.764	3	247.505	250	99.002
4	23.789	25	95.156	4	73.003	75	97.337	4	244.964	250	97.985
5	22.809	25	91.236	5	72.969	75	97.292	5	243.366	250	97.346
6	22.933	25	91.732	6	78.727	75	104.969	6	241.52	250	96.608
7	22.242	25	88.968	7	72.842	75	97.123	7	233.069	250	93.227
8	20.983	25	83.932	8	70.818	75	94.424	8	245.562	250	98.224
9	21.965	25	87.86	9	67.38	75	89.84	9	244.704	250	97.881
10	22.064	25	88.256	10	73.77	75	98.36	10	246.558	250	98.623
Average	22.782			Average	73.862			Average	243.994
SD	1.19			SD	3.6			SD	4.87
RSD(%)	5.21			RSD(%)	4.87			RSD(%)	2

Sample	Amount	Reference	Recovery	Sample	Amount	Reference	Recovery	Sample	Amount	Reference	Recovery
1	27.017	25	108.068	1	73.47	75	97.96	1	236.964	250	94.785
2	22.18	25	88.72	2	69.259	75	92.345	2	252.761	250	101.104
3	22.711	25	90.844	3	78.465	75	104.62	3	252.986	250	101.194
4	22.265	25	89.06	4	74.692	75	99.589	4	238.172	250	95.268
5	27.209	25	108.836	5	82.358	75	109.81	5	248.208	250	99.283
6	27.036	25	108.144	6	75.666	75	100.888	6	252.419	250	100.967
7	26.548	25	106.192	7	75.92	75	101.226	7	252.308	250	100.923
8	26.634	25	106.536	8	71.885	75	958.466	8	254.679	250	101.871
9	24.045	25	96.18	9	83.509	75	111.345	9	240.578	250	96.231
10	21.121	25	84.484	10	78.16	75	104.213	10	240.134	250	96.053
Average	24.676			Average	76.338			Average	246.92
SD	2.44			SD	4.43			SD	7.1
RSD(%)	9.9			RSD(%)	5.81			RSD(%)	2.88

Sample	Feijão				Unit
Sample	Black	White	String	Adzuki	Unit
1	188.562	376.87	39.799	28.038	μg kg-1
2	81.485	47.794	37.58	46.64
3	76.05	53.36	41.263	48.008
4	80.736	47.213	36.133	46.229
5	80.422	37.362	37.039	49.239
6	77.025	29.606	37.458	50.501
7	72.305	28.769	35.849	47.078
8	295.22	40.293	259.189	31.537
9	228.267	39.446	264.227	35.264
10	143.115	40.36	265.558	35.286
11	43.567	39.164	261.514	35.319
12	128.507	39.388	274.735	31.957
13	85.193	39.66	43.386	32.938
14	83.439	216.96	35.667	31.284
15	76.419	235.209	35.978	269.502
16	67.355	252.365	33.286	273.641
17	64.969	214.37	33.251	264.055
18	318.243	198.962	31.454	258.239
19	229.906	203.131	30.278	263.66
20	230.778	183.936	28.689	427.029
21	237.54	296.371	34.841	411.554
22	492.752	286.666	36.896	625.149
23	515.174	267.477	33.919	283.468
24	256.627	267.268	34.278	353.411
25	276.036	260.405	33.076	513.987
26	467.731	286.363	31.281	311.317
27	632.52	378.321	584.215	550.785
28	287.996	670.378	484.459	188.446
29	126.227	188.18	505.939	174.998
30	137.286	618.594	295.423	265.352
31	224.257	415.396	98.444	52.011