Nghiên cứu phát triển kỹ thuật QuEchERS GC MS 3 SIM để phân tích đồng thời dư lượng hoá chất bảo vệ thực vật trong đất tt tiếng anh

Thus, the level of pesticide residues in the agricultural soil needs to be monitored as farmers switch to organic farming green and safe agriculture Currently, pesticide residues in soil

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

Pham Tuan Linh

STUDY AND DEVELOPMENT OF QuEChERS GC/MS 3 SIM TECHNIQUE TO ANALYSE MULTI PESTICIDE RESIDUES

This thesis is completed at: Graduate University Science and Technology – Vietnam Academy of Science and Technology

Science instructor 1: Assoc.Prof.Dr Vu Duc Loi

Science instructor 2: Assoc.Prof.Dr Nguyen Hong Khanh

Reviewer 1:

Reviewer 2:

Reviewer 3:

The thesis will be presented to the scientific council

at Graduate University Science and Technology – Vietnam Academy

of Science and Technology

at … … h , date …… month …… , 2019

The thesis can be found at:

- Library of Graduate University Science and Technology

- National library of Vietnam

INTRODUCTION

1 The urgency of the thesis

Nowadays, more than1500 different kinds of pesticides have been used, classified based on chemical structure (chlorinated, phosphorus, Carbamate, Pyrethroid ) or by application (insecticide, fungicide, herbicide )

In Vietnam, more and more different pesticides are used in agriculture (increased from 189 substances in 2003 to 437 substances

in 2010)

Pesticides have been known to be contaminants that persist for a long time in the environment So over time, there will be residues of many different pesticide in the soil

Pesticides accumulated in the soil can be transferred to humans via the food chain, being potentially harmful to human health Thus, the level of pesticide residues in the agricultural soil needs to be monitored as farmers switch to organic farming (green and safe agriculture)

Currently, pesticide residues in soil is determined separately for each substance group, with different procedures, so increasing costs, extend the analysis time and analysis process becomes more complicated

Therefore, the project "Study and development of QuEChERS GC / MS 3 SIM technique to analyze multi pesticide residues in soil" was carried out, in order to reduce the analysis time, minimize the number of analytical steps, use fewer reagents in smaller amounts and provide high recovery

2 Scope of thesis

Develop a rapid and simple method for analysis of multi-residue pesticides, including organophosphate, organochlorine, carbamate, and pyrethroid compounds in soil, with sample preparation based on QuEChERS technique and determination by GC-MS

3 Main contents of thesis

- Survey and select optimal conditions to analyze multi pesticides on

GC / MS system: injection mode, temperature program, parameters for mass spectrometry

- Investigate optimization of processing samples including extraction, cleaning and enrichment: solvent and extraction time, influencing factors, cleaning agents

- Verification of analytical methods: determination of linear range, calibration curve, detection limit and quantitative limit, recovery coefficient and repeatability of the method

- Apply the optimal procedure to analyze 30 soil samples and compare results with 04 laboratories at Vietnam and Korea

4 New contributions of the thesis

- This is the first study in Vietnam to apply the QuEChERS method for multi-residues pesticides analysis in the soil

- d-SPE has been applied instead of soxhlet extraction, so the sample preparation time has been reduced from 24 h to 25 minutes, consuming only 15 ml solvent (the popular methods consume at least 300ml of solvent)

- With a single run, 103 pesticides in soil have been analyzed (the latest publication, only 42 pesticides in the soil were quantified)

- In quantitative step, only GC/MS is used (in publications, GC/MS/MS is analytical method usually selected)

OVERVIEW CHAPTER 1.

Pesticides (herbicides, fungicides or insecticides) are environmental pollutants often found in soil, water, atmosphere and agricultural products, and may exist in harmful levels, posing an environmental threat Even low levels of pesticide can cause adverse effects on humans, plants, animals and ecosystems The application

of pesticides has increased appreciably during the past few decades, resulting in a potential risk for the human health Over 95% of sprayed pesticides reach a destination other (usually soil environment) than their target So, the determination of pesticide residues in soil has been rising in demand

The analysis of pesticide residues in soil consists of sample preparation and the instrumental determination The aim of the sample preparation is to isolate the trace amounts of analytes from a large quantity of complex matrices and eliminate the interferences from the soil matrix as much as possible Typical sample preparation steps include the homogenization, extraction, and clean-up

Due to the low concentration levels of pesticide in soil, a technique strong enough to extract bound residues is necessary The most common of these techniques are mechanical agitation by shaking, sonication, microwave energy, and liquid-solid extraction (e.g.: Soxhlet extraction; accelerated solvent extraction, pressurized liquid extraction, and, supercritical fluid extraction) The most popular clean-up methods are based on the solid phase extraction technique using florisil cartridges These established methods are effective, yet time consuming (taking as long as 1,5day), complicated and expensive

As a result, the development of new analytical methods for the determination of multi residue pesticides in soil samples is currently a high-interest research area

Many innovations have occurred in analytical methods for the extraction of organic compounds from different matrices that reduce the analysis time, minimize the number of analytical steps, use fewer reagents in smaller amounts and provide high recovery In

2003, Michelangelo Anastassiades developed a method for the class, multi-residue extraction of pesticides in fruits and vegetables This method was called QuEChERS, which stands for Quick, Easy, Cheap, Rugged and Safe, and it is based on dispersive solid phase extraction (d-SPE) In d-SPE, pesticides are extracted with an aqueous miscible solvent with a high amount of salt, in order to induce liquid phase separation

multi-The QuEChERS method is particularly popular for the determination of wide range of chemical residues, mostly pesticides

in various food matrices, because of its simplicity, low cost, and high efficiency with a minimal number of steps QuEChERS approach is very flexible and it serves as a template for modification depending

on the analyte properties, matrix composition, equipment and analytical technique available in the laboratory

Unfortunately, the application of the d-SPE technique in the analysis of pesticides in agricultural soils is very rare, with a limited number of pesticides analyzed

Currently, in Vietnam, QuEChERS method has only been applied for pesticide analysing in food and medicinal plant sample The level of pesticide residues in soil, still determined by GC/ECD or GC/NPD, with Shoxlet extraction

EXPERIMENT CHAPTER 2.

Materials and apparatus

2.1

Stock standard solutions of 2.00 mg/g of each pesticide were prepared in MeCN, stored in −20◦C Intermediate mixture standard solution (10 mg/kg) was prepared by diluting the stock standard solutions with MeCN Gas chromatography mass spectrometry system MS-QP 2010 (Shimadzu), DB-5MS capillary column

2.2.2 Survey and select optimal conditions to analyze multi pesticides on GC / MS system

- Survey the injection temperature and injection speed, sample volume and carrier gas

- Survey temperature program

- Select ion mass for quantitative

2.2.3 Investigate optimization of processing samples

- Solvent and extraction time,

- Influencing factors: pH, ionic strength, organic,

linear range, calibration curve, detection limit and quantitative limit, recovery coefficient and repeatability of the method

Analysis of real samples

2.4

Apply the optimal procedure to analyze 30 soil samples (were collected in Nam Dinh, Nghe An, and Hanoi with different pH, ion exchange and organic matter characteristics) and compare results with 04 laboratories at Vietnam and Korea

RESULTS AND DISCUSSION CHAPTER 3.

Survey and select optimal conditions to analyze multi 3.1

pesticides on GC / MS system

3.1.1 Survey the injection temperature and injection speed

With the slow injection mode, the sensitivity of pesticides surveyed was 50% lower than that of the fast and medium mode In addition, the stability is not high, most % RSD> 30%

The sensitivity has been significantly improved, and there is not so much difference between the fast and medium sample injection modes However, the fast injection mode is more stable,% RSD is <10% at all survey temperatures

The result also show that the sensitivity of the substances is relatively uniform at different temperatures and reaches a maximum

at 2600C (except for permethrine at 2100C) However, at 2600C and

in the fast injection mode the highest stability with relative standard deviation% RSD is around 2-4%

From the research results show that the optimal injection port temperature for pesticide analysis is 260oC and the sample injection speed is set to "Fast"

3.1.2 Sample injection volume and carrier gas speed

The effect of carrier gas velocity complies with the van Deemter equation According to the calculation results, the sample injection volume is suitable for MeCN from 1.0 to 1.2 L From the theoretical calculation, combined with the carrying gas speed, we conducted experiments to find the sample injection volume of 1.0 L and the carrier gas rate of 1.7 mL / minute, which is suitable for the analysis of pesticides

3.1.3 Temperature

program

06 temperature programs

have been run The results

show that program No 6

has the best separation

Specifically, the initial

temperature of 60 ° C,

hold for 1 minute; increased 20oC/min to 180oC; increased 10oC/min to 190oC; increased 3oC/min to 240oC; increased 10oC/min to 300oC, and held for 5 minutes

Figure 3.9 Standard chromatogram at different temperature program

50 100 150 200 250 300 350

0 5 10 15 20 25 30 35

Program 1 Program 2 Program 3 Program 4 Program 5 Program 6

Terbufos & Quintozene

Diazinon, Etrimfos & BHC-delta

Chlorpyrifos, Fenthion & Parathion

Fludioxonil & Isoprothiolane

pp’-DDE, Oxadiazone & op-DDD

pp’-DDD, op-DDT & Ethion

Figure 3.10 Resolution of some pesticides at different temperature programs

3.1.4 Select ion mass for quantitative

The selection of a main fragment for quantitation is based on the following priority criteria:

 Main fragment of pesticide (M +: Mother ion);

 If there is no mother ion, select the segment with the highest signal Priority should be given to the fragment having m / z greater than 100 to avoid the effect of fragments formed by solvent;

 Avoid using identical forming fragments of pesticides with adjacent elution times

On the basis of the resulting chromatograms, the main and fragments have been established

Investigate optimization of processing samples

3.2

3.2.1 Selection of extraction solvent

Table 3.6 shows that MeCN has the best recovery and repeatability among the researched solvents and is used as the solvent for sample extraction

Table 3.6 Recovery, standard deviation of pesticide at

different extraction solvents

3.2.2 Selection of extraction time

Extraction time of 1, 3, 5, 10 and 20 minutes using QuEChERS technique has been surveyed The results show that the extraction time 3-5 minute is most appropriate (Table 3.8)

Table 3.8 Results of the effect on sample extraction time

Extraction time

Recovery (%) 47-114 69-110 69-111 67-119 63-111

Pesticides with recovery < 50% 1 0 0 0 0

Pesticides with recovery 60-70% 11 1 2 4 7 Pesticides with recovery >70% 86 102 101 99 96

3.2.3 Effect of absorbents

Interferences have maximum absorption in the wavelength range of 190 - 230 nm, including fats ((max = 205 - 233 nm), sugar compounds ((max = 190 nm); and triaglycerol ((max = 210 nm)

Figure 3.14 Efficient removal of interferences by adsorbents

(a) florisil; (b) C18; (c) PSA; and (d) GCB

Experimental results (Figure 3.14) show that florisil, C18, PSA with content of 20 mg / mL and GCB with content of 10 mg / L are all capable of cleaning

Figure 3.16 The soil sample chromatogram is purified by various

absorbents

However, when observing the baseline as well as the intensity

of impurities in the chromatogram (Figure 3.16), we can see:

- For florisil: baseline rise to 150,000 and decrease to baseline until 18 minutes Impurities are very much with high intensity

- For C18 and GCB: baseline rises to 100,000 and after 11 minutes decreases to baseline

- For PSA: the baseline only rises to 60,000 to the baseline (50,000) after 8 minutes

Therefore, absorbents PSA (500mg) and GCB (10mg) were selected for the cleaning of pesticides in soil samples

3.2.4 Effect of sample matrix

- Effect of pH:

pH can affect the chemical and physical properties of pesticides as well as the efficiency of sample extraction With agriculture soil pH ranges from 5.5 to 8.5

Experimental results show that pH from 5 to 9 has no significant effect on the extraction process The recovery is not much different and is in the range of 75 - 110% with % RSD ranging from

1 - 18%

- The influence of ionic strength

The ionic level of the mixture, making it easier to extract pesticides However, some metals with the ability to form complexes such as Co, Cd and Cu will reduce the ability to extract some highly polarized pesticides

Studies with Cu content of 1000 mg / kg showed no effect on the extraction of pesticides in soil The recovery ranges from 69 - 110% with % RSD from 1 - 17%

- Influence of sample size and organic matter content:

Studies conducted with coarse-grained (<2 mm), grained (<0.05 mm) and organic matter content (10%) showed that the sample was crushed through a 2 mm sieve and organic matter content not exceeding 10% will not affect sample extraction performance

Develop analytical process

3.3

3.3.1 Sample preparation procedure

Sample preparation is summarized in Figure 3.18

Figure 3.18 Sample preparation diagram

3.3.2 Analytical procedure on the GC/MS system

a) GC setting

Injection port temperature: 260 oC; Injection volume: 1,0

L; Air flow rate: 1.7 mL / minute;

150mg MgSO 4 , 50mg PSA, 10mg GCB Shake 30 second

Centrifuge for 5 min at 4.000 rpm