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Applicability of Gas and Liquid Chromatography to the Analysis of Metalaxyl-

M and Chlorothalonil Residues

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Vojislava Bursic Sanja Lazić

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Proceedings of the 7th IMT-GT UNINET and The 3rd International PSU-UNS Conferences on Bioscience

Applicability of Gas and Liquid Chromatography to the

Analysis of Metalaxyl-M and Chlorothalonil Residues
Vojislava Bursić1*, Sanja Lazić1, Mira Pucarević2, Dragana Šunjka1, Maja
Đogo3, Slavica Vuković1
1
Department of Environmental and Plant Protection, Faculty of Agriculture, Trg Dositeja Obradovića 8,
Novi Sad
2
EDUCONS University, Vojvode Putnika bb, Sremska Kamenica
3
Faculty of Technical Sciences, Trg Dositeja Obradovića 6, Novi Sad, Serbia
*Corresponding author: e-mail: bursicv@polj.uns.ac.rs, phone: +381-21-4853-346

Introduction

It is known that in the conditions of the contemporary agricultural production over 30 % of the
produce does not find its way to the consumers, whereas without the application of the chemical agents
the loss would be doubled (Bursić et al., 2009). Therefore the pesticides are essential in modern
agricultural practices but, due to their biocide activity and potential risk to consumers, the control of the
presence of pesticide residues in foods is a growing source of concern for the general population (Vidal et
al., 2002). A cucumber is one of the most significant vegetable crops in Serbia. It is mostly affected by
the downy mildew disease and it is treated also with the chlorothalonil and metalaxyl-M. For apolar and
middle polar pesticides the detection of pesticide residues is commonly achieved through the analysis
with gas chromatography (GC) coupled with single quadropole (SQ) and less frequently, triple
quadropole (QQQ) mass spectrometers (MS) (Lesueur et al., 2008). In the literature citation Keramat et
al. (2005) used HPLC (High performance liquid chromatography) coupled with DAD, method for the
determination of metalaxyl residues.

Materials and methods

Chemicals and Apparatus. The analytical standard of metalaxyl-M (98.5%) and chlorothalonil
(99.1%) was used (Dr. Ehrenstorfer GmbH, Germany). The stock standard solutions of fungicide were
prepared in toluene for GC-MS and in methanol for HPLC-DAD. All the solvents were of pesticide
residue analysis grade (Carlo Erba), celit 545 and sodium chloride (J.T. Bacer). The analysis with gas
chromatograph 6890N (Agilent Technologies) equipped with a mass-selective detector 5975B, liquid
autosampler and HP-5MS column was used for GC analysis. The pulsed splitless injection (pressure
pulse 40 psi, pulse time 0.2 min, inlet temperature 300 oC, injection volume 1 µl, syringe size 10 µl) was
done. The oven temperature programme: initial temperature of 70 oC (hold 2 min), rising at 25 oC/min
to 150 oC, then at the rate of 3 oC/min to 200 oC and the rate of 8 oC/min to 280 oC (hold 10 min). The
total run time was 41.87 minutes. Helium was used as a carrier gas at a constant rate of 33.8 cm/sec. MSD
was equipped with a quadrupole analyser operating in the electron ionisation mode. Ion source
temperature was 230 oC, and MS Quad temperature 150 oC. The identification and quantification
of metalaxyl-M and chlorothalonil were performed in the selected ion monitoring mode (SIM) for
ions 85, 109, 124, 130, 132, 133, 146, 160, 206 and 266 (m/z). Agilent 1100 Series HPLC-DAD system
with Hypersil ODS column was used in the analysis. The mobile phase consisted of water:methanol
(25:75 v/v) at 0.6 ml/min, the injection volume 20 µl and the column temperature at 40 oC. The detection
was carried out at 210 nm.

Field trials. Cucumbers were treated with Folio Gold (37.5 metalaxyl-M, 500 chlorothalonil g/kg)
in the dose of 2.5 kg/ha. The samples were collected on the 0, 2, 4, 7 and 14 day after the application.

Extraction. The extraction was done by the modified Navarro (1999) method. The fungicides
were extracted from a 15 g homogenized sample with the mixture of 30 ml of acetone and methylene
chloride with 2 g celit and 1 g sodium chlorid using an Ultra-Turax. After the filtration, extract was
evaporated to dryness and dissolved in toluene for GC-MS and in methanol for HPLC-DAD analysis.

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Proceedings of the 7th IMT-GT UNINET and The 3rd International PSU-UNS Conferences on Bioscience

Analysis. The retention time of metalaxyl-M with, was 9.36 min with GC-MS, and for HPLC-
DAD was 1.39 min, for the chlorothalonil was 8.92 min with GC-MS. There were no interference peaks
in this region of the chromatogram. The quantification was carried out using the calibration curves with 6
levels, of the concentrations ranging from 0.1−1.25 µg/ml prepared in matrix.

Validation. For the recovery the samples of untreated cucumbers were spiked with the appropriate
volumes of standard solvent concentration ranged from 0.1-1.25 µg/ml. The detection limit (LOD) was
determined as the lowest concentration giving a response of three times the average of baseline. The limit
of quantification (LOQ) was calculated from LOD. The linearity in the response was studied with a
standard solution prepared in the matrix extract in the concentration ranging from 0.1 to 1.25 µg/ml.

Results and Dscussion

Analytical method. The obtained validation parameters are arranged in a table:

Average
LOD LOQ CV
Fungicide Chromatography (mg/kg) (mg/kg)
R2 Equation (y) Recovery
(%)
(%)
GC-MS 0.01 0.030 0.997 y=0.966x+0.0354 97.0 5.80
Metalaxyl-M
HPLC-DAD 0.017 0.047 0.983 y=0.626x+0.0051 99.0 7.08
Chlorothalonil GC-MS 0.01 0.030 0.998 y=0.972x+0.0421 94.0 7.70

By the application of 0.2 kg metalaxyl/ha the residue values ranged from 0.14 mg/kg for 1-3 days
after the application, below 0.04 mg/kg for 5-8 days and 0.05 mg/kg after 14 days (INCHEM, 1982). By
applying 0.19 and 0.08 mg/kg of metalaxyl Talebi (2002) established the decrease of metalaxyl residues
in cucumber below the MRLs of 0.5 mg/kg after 7 days. Our analysis results show the residue decrease
up to 14 days, ranging from 0.11 to 0.05 mg/kg, and on 14th day there was an increase to 0.13 mg/kg. The
chlorothalonil residue analysis in cucumbers carried by Valvedere et al. (1993) reported 0.15 mg/kg
residues on the 1st day, 0.09mg/kg on 3rd after the treatment and 0.02 mg/kg on 14th. In our results the
residue content ranged from 0.26-0.13 mg/kg and on the 14th day it was 0.04 mg/kg.

Conclusion

GC-MS is suitable for the analysis of metalaxyl-M and chlorothalonil residues, but the HPLC-
DAD analysis showed no linearity for the chlorothalonil determination. The T-test showed no significant
difference between various chromatography techniques used for the metalaxyl-M residue determination,
at the 95% confidence level. In the Republic of Serbia the PHI (pre-harvest interval) for metalaxyl-M and
chlorothaonil is 7 days and after that period the obtained metalaxyl-M and chlorothalonil was under the
Serbian MRLs, of 0.5 for metalaxyl-M and 1.0 mg/kg for chlorothalonil.

Reference

Bursić V., Lazić S., vuković S., Šunjka D. and Pucarević M. Diseppearance of azoxzstrobin in cucumber.
Contemporary Agriculture. 3-4, 81-86.
IPCS INCHEM, 590. Metalaxyl, Pesticide residues in food, 1982.
Keramat J., Talebi Kh., Mosaffa L., Determination of the variations of metalaxyl residues in cucumbers
grown under modern and traditional conditions, Journal of Science and Technology of Agriculture
and Natural Resources, 9, 1, 205-214, 2005.
Lesueur C., Knittl P., Gartner M., Mentler A., Fuerhacker M., Analysis of 140 pesticide from
conventional farming foodstuff sample after extraction with the modified QuECheRS method, Food
Control, 19, 906-914, 2008.
Navarro, S., Barba, A., Oliva, J., Navarro, G., Pardo, F., Evolution of residual levels of six pesticides
during elaboration of red wines. Effect of wine-making procedures in their dissappearance, Journal
of Agriculural and Food Chemistry, 47, 264-270, 1999.

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 Proceedings of the 7th IMT-GT UNINET and The 3rd International PSU-UNS Conferences on Bioscience

Talebi, K., Effects of two different formulations of metalaxyl on their residues in cucumber, Meded
Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet., 67 (2), 79-83, 2002.
Valvedere-Garcia A., Gonzales-Prades E., Aguilera-Del Real A., Urena-Amate M.D., Determination and
degradation study of chlorothalonil residues in cucumbers, peppers and cherry tomatoes, Analytica
Chimica Acta, 276, 1, 15-23, 1993.
Vidal M.J.L., Arrebola F.J., Mateu-Sanchez M., Application of gas chromatography-tandem mass
spectrometry to the analzsis of pesticide in fruits and vegetables, Journal of Chromatography A,
959, 203-213, 2002.

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