Determination Of Pesticide Residues In Vegetables Biology Essay

Published:

In this study, a gas chromatography-mass spectrometry method is successfully developed for the determination of pesticide residues monocrotophos, chlorpyriphos, and endosulfan in cauliflower and capsicum. The samples were extracted with ethyl acetate, cleaned up and purified through solid-phase extraction with Florisil and activated charcoal. Experiments on two fortification concentrations are carried out, and the limits of detection are 0.005, 0.007, and 0.002 mg kg-1 for monocrotophos, chlorpyriphos, and endosulfan respectively. The average recoveries of pesticide residues in cauliflower and capsicum samples are 89.0 to 110.0 %.

Keywords: Monocrotophos; Chlorpyripho; Endosulfan; Chlorpyrifos; Cypermethrin,

1. Introduction

India is produces about 109 million metric tones of vegetables and it is the second largest producer after China, and accounts for 13.4% of world production. Surveys carried out by institutions spread throughout the country indicate that 50-70% of vegetables are contaminated with insecticide residue (1). The use of pesticides to control pest and diseases are a common practice in the fields to increase crop yield. However, these chemicals can reach plant tissues, leaving residues that can be detected in the vegetables. This may become a significant route to human exposure to these toxic compounds. In order to protect consumer's health, maximum residue levels (MRLs) in these vegetables have been established in different countries and internationally by Codex Alimentarius. The high number of pesticides to be monitored in those matrices, along with the typically low concentrations of the MRLs, requires highly sensitive and selective methods. Consequently, sample preparation becomes a key step of the analytical procedure. In recent times, extensive efforts have been made to the development of new sample preparation techniques that save time, labor and solvent consumption to improve the analytical performance of the procedure. Analytical instrument are needed to determine, quantify and confirm pesticide residues in vegetables for both research and regulatory purposes. The pesticides are generally analysed by spectrophotometry [2,3,4], thin layer chromatography (TLC) [5,6,7], high performance liquid chromatography (HPLC) [8,9,10], gas chromatography (GC) [11,12,13, 14] and GC-MS [15,16,17,18,19,20]. The present study describe method of extraction, cleanup and determination of a pesticides by using gas chromatography (GC) equipped with mass detector (MS) for the separation, identification and quantification of monocrotophos, chlorpyriphos and endosulfan on cauliflower and capsicum were developed and validated. Finally, the method was applied to the determination of these pesticides in commercial samples collected from the local markets. Therefore, the purpose of this study was to develop an analysis scheme for determination of these pesticides in cauliflower and capsicum GC-MS.

2. Experimental

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

Chemical and reagents

The organic solvent ethyl acetate and hexane used were HPLC grade and purchased from E Merck. Technical grade pesticide standards were used for standardisations. The standards were stored in a freezer at -5oC. Anhydrous sodium sulphate (AR) from E Merck used for residue extraction was maintained at 300oC overnight and kept in air tight container.

Sample preparation

The fresh cauliflower and capsicum samples were taken for the extraction of pesticide residues. Each vegetable was chopped into small pieces; a representative sample (50gm) was macerated with 5-10gm anhydrous sodium sulphate in blending machine to make fine paste. The macerated sample was extracted with 100 ml of ethyl acetate on mechanical shaker for 1 h; extract was filtered, concentrated up to 5 ml on rotary evaporator and finally injected into GC-MS.

Sample clean up

The clean-up of monocrotophos, chlorpyriphos, and endosulfan was carried out by using column chromatography. Column (60cm x 22mm) was packed with, Florisil and activated charcoal (5:1 w/w) in between the two layers of anhydrous sodium sulphate. Extract was eluted with 125 ml mixture of ethyl acetate and hexane (3:7 v/v). Elute was concentrated to 5 ml on rotary evaporator.

Standard preparation

For preparation of stock solution, standards were dissolved in ethyl acetate and four levels of intermediate standard solution of each pesticide were prepared maintaining the same matrix concentration for the preparation of calibration curve and stored at -4oC in the dark .Working solutions were prepared daily by appropriate dilution with ethyl acetate.

Instrumentation

GC-MS analysis was performed with a Varian 3800 gas chromatograph with electronic flow control (EFC) and fitted with a Saturn 2200 ion-trap mass spectrometer (Varian Instruments, Sunnyvale, CA, USA). Samples were injected into a Varian 8200 autosampler SPI / 1079 split / splitless programmed-temperature injector using a 10µl syringe operated in the large volume injection technique. The glass liner was equipped with a plug of carbofrit (Resteck, Bellefonte, PA, USA). A fused-silica untreated capillary column 2 m30.25 mm I.D. from Supelco (Bellefonte, PA, USA) was used as a guard column connected to a Rapid-MS [wall-coated open tubular (WCOT) fused-silica CP-Sil 8 CB low bleed of 10m 30.53 mm I.D., 0.25 mm film thickness] analytical column from Varian Instruments (Sunnyvale, CA, USA) for high speed analysis. The mass spectrometer was operated in electron impact (EI) ionization mode. The computer that controlled the system also held a GC-MS library specially created for the target analytes under our experimental conditions. The mass spectrometer was calibrated weekly with perfluoro-tributylamine. Helium (99.999%) at a flow-rate of 1 ml min-1 was used as carrier and collision gas.

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

2.6 Instrumental conditions

Sample aliquots of 10 ml were injected into the GC operating at a syringe injection flow-rate of 10 ml s-1. The initial injector temperature of 700C was held for 0.5 min and then increased at 1000C min-1 to 3100C, which was held for 10 min. After injection the column temperature, initially 700C, was held for 3.5 min, then increased at 500C min-1 to 1500C, then increased at 30C min-1 to 2350C and finally raised to 3000C at 500C min-1 and held for 3 min. The ion-trap mass spectrometer was operated in EI-MS mode. The transfer line, manifold and trap temperatures were 280, 50 and 2000C, respectively. The analysis was performed with a filament-multiplier delay of 4.75 min to prevent instrument damage. The automatic gain control (AGC) was activated with an AGC-target of 5000 counts. The emission current for the ionisation filament was set at 80 mA, generating electrons with an energy of 70 eV. The axial modulation amplitude voltage was 4.0 V.

3. Results and discussion

Validation of the method

In order to check the feasibility of the GC-MS method for the analysis of pesticide residues in fresh vegetable sample extracts, it was validated using cauliflower and capsicum extracts.

Identification and confirmation of target analytes

The identification of the pesticides was based on the retention time windows (RTW) that are defined as the retention time average 63 S.Ds of the retention time when 10 blank samples spiked at the second calibration level of each compound were analysed. The confirmation of a previously identified compound was done by comparing the GC-MS spectra obtained in the sample with another stored as reference spectrum in the same experimental conditions. The reference spectra were obtained daily by injecting a blank cauliflower and capsicum sample spiked at the concentration of the second calibration point.

3.3 Identification and quantification

The compound was identified by comparing its retention time with respect to technical grade reference standard. The quantitative determination was carried out with the help of a calibration curve drawn from chromatographic experiments with standard solution. For quantification an external calibration curve with four different concentrations of each pesticide, with matrix matching were made. The standard solutions for the calibration curves were prepared in control matrix because samples may possess co-extractants in the matrix which may affect the peak area of the unknown samples.

3.4 Limit of detection and limit of quantification

The limit of detection (LoD) was calculated from the peak intensity at 0.01mg kg-1 and blank in recovery tests. LoD was defined as S/N>4 so that it is in the linear range of the standard calibration. The LoD of monocrotophos, chlorpyriphos, and endosulfan was 0.005, 0.007, and 0.002 mg kg-1 respectively. LoQ was obtained for monocrotophos, chlorpyriphos, and endosulfan was 0.015, 0.021and 0.006 mg kg-1 respectively (table 1). Linear calibration curves were found between peak areas and analyte concentration in the whole range of studies. The linear regression (y = a + bx) parameters for method calibration were taken (table 2). The correlation coefficient of analytical curves were near 0.99, with linearity for each compound, which allows the quantitation of these compounds by the method external standardization.

3.5 Recovery

Recovery studies were performed to examine the efficacy of extraction and clean up. Untreated cauliflowers and capsicum samples were spiked with known concentration of the pure pesticides standard solution and extraction and clean-up were performed as described earlier. The concentration of each pesticide in the final extracts was calculated (table 3). The average recoveries of pesticide residues in cauliflower and capsicum samples are 89.0 to 110.0 %.

4. Application to the analysis of market samples

In order to test the feasibility of the GC-MS approach for routine analysis of pesticide residues in the market samples of vegetables (cauliflower and capsicum) were analysed for the target compounds. The concentrations of each pesticide in the final extracts of the market were obtained and calculated (table 4). Figure 1 shows TIC of monocrotophos, chlorpyriphos, and endosulfan in actual sample of capsicum and figure 2 shows mass spectra of monocrotophos, chlorpyriphos, and endosulfan.

5. Conclusion

In this study, the operating parameters of GC-MS for the analysis of 3 representative pesticides in cauliflower and capsicum were optimized, and sample preparation method was evaluated. The main conclusions of the study can be summarised as follows: (i) a good separation and high sensitivity was achieved by GC-MS method for all pesticides using a capillary column, (ii) the classical procedure that involves extraction with ethyl acetate, partitioned from the aqueous matrix using anhydrous sodium sulphate a cleanup with florisil and activated charcoal , showed an efficient removal of interferences, providing a simple, rapid and reliable analysis of pesticides in all matrices; (iii) for most of the pesticides assayed the performance characteristics obtained within validation study were acceptable, within the quality control requirements. Applying this method, analysis time is shorter compared to other methods. Thus, high sample throughput can, therefore, be achieved, which is useful in pesticide monitoring programs with a large number of samples to analyse.

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work

Table 1: Molecular formula, retention time, LODs and LOQs of monocrotophos,

chlorpyriphos and endosulfan.

Compound

Molecular formula

RT

(min)

LoDs

(mg kg-1 )

LoQs

(mg kg-1)

Monocrotophos

C7H14NO5P

17.89

0.005

0.015

Chlorpyriphos

C9H11Cl3NO3PS

25.12

0.007

0.021

Endosulfan

C9H6Cl6O3S

26.72

0.002

0.006

Table 2 Quantitation ion, conformation ion and calibration range of monocrotophos,

chlorpyriphos and endosulfan.

Compound

Quantitation ion

Confirmation ion

Calibration range

(mg kg-1 )

Correlation

coefficient

Coefficient

of variation (n = 5)

%

Monocrotophos

127

98

0.02-1.00

0.991

5.8

Chlorpyriphos

97

314

0.02-1.00

0.988

6.0

Endosulfan

373

237

0.02-1.00

0.989

5.8

Table 3: Recovery of pesticides in the spiked samples.

Sample

Compound

Concentration

(mg kg-1)

Recovery

(%)

Coefficient of

variation (n = 5)

%

Cauliflower

Monocrotophos

1.0

89.80

4.58

Cauliflower

Chlorpyrifos

1.0

99.80

3.90

Cauliflower

Endosulfan

1.0

108.20

4.40

Capsicum

Monocrotophos

1.0

91.20

4.25

Capsicum

Chlorpyrifos

1.0

100.30

4.56

Capsicum

Endosulfan

1.0

110.00

4.68

Table 4: Amounts of pesticides residue detected in cauliflower and capsicum samples.

Sample

Monocrotophos

(mg kg-1)

Chlorpyriphos

(mg kg-1)

Endosulfan

(mg kg-1)

Cauliflower

nd

nd

0.002

Cauliflower

0.024

nd

0.003

Cauliflower

nd

0.002

0.001

Cauliflower

0.027

nd

nd

Cauliflower

nd

nd

0.002

Capsicum

nd

0.012

0.002

Capsicum

0.021

nd

0.021

Capsicum

0.024

0.008

0.027

Capsicum

0.018

0.003

nd

Capsicum

0.020

nd

0.026

nd = non detected

Fig. 1 TIC of monocrotophos1, chlorpyriphos2, and endosulfan3 in actual sample of capsicum.

Fig. 2 Mass spectra of monocrotophos, chlorpyriphos, and endosulfan.