Analysis of Organochlorine Pesticides in a Soil Sample by a Modified QuEChERS Approach Using Ammonium Formate

Podsumowanie

The present protocol describes the utilization of ammonium formate for phase partitioning in QuEChERS, together with gas chromatography-mass spectrometry, to successfully determine organochlorine pesticide residues in a soil sample.

Streszczenie

Currently, the QuEChERS method represents the most widely used sample preparation protocol worldwide for analyzing pesticide residues in a broad variety of matrices both in official and non-official laboratories. The QuEChERS method using ammonium formate has previously proven to be advantageous compared to the original and the two official versions. On the one hand, the simple addition of 0.5 g of ammonium formate per gram of sample is sufficient to induce phase separation and achieve good analytical performance. On the other hand, ammonium formate reduces the need for maintenance in routine analyses. Here, a modified QuEChERS method using ammonium formate was applied for the simultaneous analysis of organochlorine pesticide (OCP) residues in agricultural soil. Specifically, 10 g of the sample was hydrated with 10 mL of water and then extracted with 10 mL of acetonitrile. Next, phase separation was carried out using 5 g of ammonium formate. After centrifugation, the supernatant was subjected to a dispersive solid-phase extraction clean-up step with anhydrous magnesium sulfate, primary-secondary amine, and octadecylsilane. Gas chromatography-mass spectrometry was used as the analytical technique. The QuEChERS method using ammonium formate is demonstrated as a successful alternative for extracting OCP residues from a soil sample.

Wprowadzenie

The need to increase food production has led to the intensive and widespread use of pesticides worldwide over the last few decades. Pesticides are applied to the crops to protect them from pests and increase crop yields, but their residues usually end up in the soil environment, especially in agricultural areas¹. Furthermore, some pesticides, such as organochlorine pesticides (OCPs), have a very stable structure, so their residues do not decompose easily and persist in the soil for a long time². Generally, the soil has a high capacity to accumulate pesticide residues, especially when it has a high content of organic matter³. As a result, the soil is one of the environmental compartments most contaminated by pesticide residues. As an example, one of the complete studies to date found that 83% of 317 agricultural soils from across the European Union were contaminated with one or more pesticide residues⁴.

Soil pollution by pesticide residues may affect non-target species, soil function, and consumer health through the food chain because of the high toxicity of the residues^5,6. Consequently, the evaluation of pesticide residues in soils is essential to assess their potential negative effects on the environment and human health, particularly in developing countries due to a lack of strict regulations on the use of pesticides⁷. This makes pesticide multi-residue analysis increasingly important. However, the rapid and accurate analysis of pesticide residues in soils is a difficult challenge due to the large number of interfering substances, as well as the low concentration level and the diverse physicochemical properties of these analytes⁴.

Of all the pesticide residue analysis methods, the QuEChERS method has become the quickest, easiest, cheapest, most effective, robust, and safest option⁸. The QuEChERS method involves two steps. In the first step, a microscale extraction based on partitioning via salting-out between an aqueous and an acetonitrile layer is performed. In the second step, a cleaning process is carried out employing a dispersive solid phase extraction (dSPE); this technique uses small amounts of several combinations of porous sorbents to remove matrix-interfering components and overcomes the disadvantages of conventional SPE⁹. Hence, the QuEChERS is an environmentally friendly approach with little solvent/chemical going to waste that provides very accurate results and minimizes potential sources of random and systematic errors. In fact, it has been successfully applied for the high-throughput routine analysis of hundreds of pesticides, with strong applicability in almost all types of environmental, agri-food, and biological samples^8,10. This work aims to apply and validate a new modification of the QuEChERS method that was previously developed and coupled to GC-MS to analyze OCPs in agricultural soil.

Protokół

1. Preparation of the stock solutions

NOTE: It is recommended to wear nitrile gloves, a lab coat, and safety glasses during the entire protocol.

1. Prepare a stock solution in acetone at 400 mg/L from a commercial mix of OCPs (see Table of Materials) at 2,000 mg/L in hexane:toluene (1:1) in a 25 mL volumetric flask. Table 1 shows each of the selected OCPs.
2. Prepare the subsequent stock solutions in acetone at concentrations of 50 mg/L, 1 mg/L, and 0.08 mg/L in 10 mL volumetric flasks, and store them in amber glass vials at −18 °C.
   NOTE: The same solutions can be used throughout the work, but it is important to store them under these conditions just after each use.
3. Prepare the stock solutions in acetone at concentrations of 20 mg/L and 0.4 mg/L from a commercial standard of 4,4'-DDE-d8 at 100 mg/L in acetone in 10 mL volumetric flasks, and store in amber glass vials at −18 °C. Use 4,4'-DDE-d8 as an internal standard (IS).

2. Sample collection

1. Collect approximately 0.5 kg of the upper 10 cm layer of an agricultural soil in a glass container. The soil object of this study was collected in a traditional agricultural zone of potato crops.
   NOTE: Surface sampling with a spatula was carried out. However, the depth of the soil could influence its physicochemical characteristics. Therefore, if the organic carbon content varies with the depth, it is necessary to take samples at different depths.
2. Take the soil sample to the laboratory, sift it with a 1 mm diameter sieve, and store it until analysis at 4 °C in an amber glass container.
   ​NOTE: The same soil sample can be used throughout the work, but it is important to store it under these conditions just after each use.

3. Sample preparation via the modified QuEChERS method using ammonium formate

NOTE: Figure 1 shows a schematic representation of the modified QuEChERS method.

1. Weigh 10 g of the soil sample in a 50 mL centrifuge tube, and add 50 µL of the IS solution at 20 mg/L to yield 100 µg/kg. For recovery purposes, also add the pesticide solutions prepared in step 1.2 to yield 10 µg/kg, 50 µg/kg, and 200 µg/kg (n = 3 each).
2. Shake the tube using a vortex for 30 s to better integrate the spike into the sample.
3. Add 10 mL of water. Shake the tube using an automated shaker at 10 x g for 5 min.
4. Add 10 mL of acetonitrile. Shake the tube again at 10 x g for 5 min.
5. Add 5 g of ammonium formate (see Table of Materials), shake the tube vigorously for 1 min by hand, and centrifuge at 1,800 x g for 5 min.
6. Transfer 1 mL of the acetonitrile extract to a 2 mL centrifuge tube containing 150 mg of anhydrous MgSO₄, 50 mg of primary-secondary amine (PSA), and 50 mg octadecylsilane (C₁₈) (see Table of Materials) for clean-up purposes by dispersive-solid phase extraction (d-SPE)⁸, vortex for 30 s, and centrifuge at 1,800 x g for 5 min.
7. Transfer 200 µL of the extract to an appropriately labeled autosampler vial with a 300 µL fused insert, and perform an instrumental analysis using a GC-MS system (step 4).
   ​NOTE: Matrix-matched calibration is carried out following the same steps as previously using blank extracts, but 5 mL of the supernatant is cleaned in 15 mL tubes in the d-SPE step (step 3.6) and the spike and IS solutions are not added until step 3.7. Add the calibration standard solutions in the autosampler vials to yield 5 µg/kg, 10 µg/kg, 50 µg/kg, 100 µg/kg, 200 µg/kg, and 400 µg/kg, evaporate to dryness, and add 200 µL of the matrix extracts.

4. Instrumental analysis by GC-MS

1. Perform the GC-MS analyses using a GC-MS system with a single quadrupole mass spectrometer and an electron ionization interface (−70 eV) (see Table of Materials).
2. Set the MS transfer line at 280 °C and the ion source at 230 °C.
3. Use a 5%-phenyl-methylpolysiloxane 30 m x 250 µm x 0.25 µm column (see Table of Materials) and ultrahigh purity He as the carrier gas at a 1.2 mL/min constant flow rate.
4. Maintain the GC oven at 60 °C initially for 2 min, then ramp up the temperature to 160 °C at 25 °C/min, and hold for 1 min. Then, increase the temperature to 175 °C at 15 °C/min, and hold for 3 min. Then, increase to 220 °C at 40 °C/min, and hold for 3 min. Again, increase to 250 °C at 30 °C/min, and hold for 2 min. Finally, take the temperature to 310 °C at 30 °C/min, and hold for 2 min. The total analysis time is 22.125 min.
5. Conduct a full autotune and an air and water check of the MS before each sequence.
   1. Open the MassHunter acquisition software that controls all the parameters of the GC-MS system.
      NOTE: The instrument system includes the MassHunter acquisition software by default.
   2. Open the "View" option on the toolbar, and click on Vacuum control, click on Tune, and click on Autotune. The autotune will end after a few minutes.
   3. Open the "View" option, and click on Instrument control.
   4. Click on Yes, and save the new tune file for the autotune.
   5. Open the "View" option on the toolbar, and click on Vacuum control, click on Tune again, and click on Air & Water check. The air and water check will end after a few seconds.
   6. Open the "View" option, and click on Instrument control.
   7. Click on Yes, and save the new tune file for the air and water check.
6. Perform the injection using an autosampler (see Table of Materials) at 280 °C in the splitless mode, keeping the injection volume 1.5 µL. After 0.75 min of the injection, open the split at a 40 mL/min flow rate.
   NOTE: Between injections, the 10 µL syringe must be washed three times with ethyl acetate and three times with cyclohexane. All the injections are in duplicate.
7. Analyze the analytes in selected ion monitoring (SIM) mode. This is the standard mode used in MS systems with a single quadrupole.
   ​NOTE: Table 1 shows the retention times (min) and the quantification parameters based on using one quantitation and two identification ions for the OCPs and the IS. The quantitative analysis is based on the ratio of the peak area of the quantitation ion to the ion of IS.

5. Data acquisition

1. Open the MassHunter acquisition software that controls all the parameters of the GC-MS system.
2. Open the "Sequence" option on the toolbar, and edit the sequence, including the sample name, the vial number, the number of injections, the instrumental method, and the name of the file to be generated. Add as many rows as necessary.
3. Click on OK, and save the new sequence.
4. Open the "Sequence" option on the toolbar again, and click on Run Sequence in the dropdown menu. A new window will open to confirm the injection method and the folder where the samples will be saved. Click on Run Sequence again, and the injection will begin.

Wyniki

The full validation of the analytical method was performed in terms of linearity, matrix effects, recovery, and repeatability.

Matrix-matched calibration curves with spiked blank samples at six concentration levels (5 µg/kg, 10 µg/kg, 50 µg/kg, 100 µg/kg, 200 µg/kg, and 400 µg/kg) were used for the linearity assessment. The determination coefficients (R²) were higher than or equal to 0.99 for all the OCPs. The lowest calibration level (LCL) was set at 5...

Dyskusje

The original⁹ and the two official versions^13,14 of the QuEChERS method use magnesium sulfate together with sodium chloride, acetate, or citrate salts to promote acetonitrile/water mixture separation during extraction. However, these salts tend to be deposited as solids on the surfaces in the mass spectrometry (MS) source, which causes the need for increased maintenance of liquid chromatography (LC)-MS-based methods. In terms of overcoming...

Materiały

Name	Company	Catalog Number	Comments
15 mL disposable glass conical centrifuge tubes	PYREX	99502-15
2 mL centrifuge tubes	Eppendorf	30120094
50 mL centrifuge tubes with screw caps	VWR	21008-169
5977B mass-selective detector	Agilent Technologies	1617R019
7820A gas chromatography system	Agilent Technologies	16162016
Acetone	Supelco	1006582500
Acetonitrile	VWR	83642320
Ammonium formate	VWR	21254260
Automatic shaker KS 3000 i control	IKA	3940000
Balance	Sartorius Lab Instruments Gmbh & Co	ENTRIS224I-1S
Bondesil-C18, 40 µm	Agilent Technologies	12213012
Bondesil-PSA, 40 µm	Agilent Technologies	12213024
Cyclohexane	VWR	85385320
EPA TCL pesticides mix	Sigma Aldrich	48913
Ethyl acetate	Supelco	1036492500
G4567A automatic sampler	Agilent Technologies	19490057
HP-5ms Ultra Inert (5%-phenyl)-methylpolysiloxane 30 m x 250 µm x 0.25 µm column	Agilent Technologies	19091S-433UI
Magnesium sulfate monohydrate	Sigma Aldrich	434183-1KG
Mega Star 3.R centrifuge	VWR	521-1752
Milli-Q gradient A10	Millipore	RR400Q101
p,p'-DDE-d8	Dr Ehrenstorfer	DRE-XA12041100AC
Pipette tips 2 - 200 µL	BRAND	732008
Pipette tips 5 mL	BRAND	702595
Pipette tips 50 - 1000 uL	BRAND	732012
Pippette Transferpette S variabel 10 - 100 µL	BRAND	704774
Pippette Transferpette S variabel 100 - 1000 µL	BRAND	704780
Pippette Transferpette S variabel 20 - 200 µL	BRAND	704778
Pippette Transferpette S variabel 500 - 5000 µL	BRAND	704782
Vials with fused-in insert	Sigma Aldrich	29398-U
OCPs		CAS registry number
α-BHC		319-84-6
β-BHC		319-85-7
Lindane		58-89-9
δ-BHC		319-86-8
Heptachlor		76-44-8
Aldrin		309-00-2
Heptachlor epoxide		1024-57-3
α-Endosulfan		959-98-8
4,4'-DDE-d8 (IS)		93952-19-3
4,4'-DDE		72-55-9
Dieldrin		60-57-1
Endrin		72-20-8
β-Endosulfan		33213-65-9
4,4'-DDD		72-54-8
Endosulfan sulfate		1031-07-8
4,4'-DDT		50-29-3
Endrin ketone		53494-70-5
Methoxychlor		72-43-5