Analysis of
multi chemical class pesticide residues in Citrus by LC-MS/MS adopting
QuAChERS technique.
2Senior Scientist
, All India Net Work Project on Pesticide Residues, Rajendranagar,
PJTS Agricultural University, Hyderabad – 500 030 , India
3Professor, Dept.
of Chemistry, University college of Science, Osmania
University, Hyderabad – 500 007, India.
4Principal
Scientist (Chemistry) Retired. All India Net Work Project on Pesticidce Residues, Prof. Jayashankar
Telangana State Agricultural University, Rajendranagar, Hyderabad – 500 030, India.
*Corresponding
Author E-mail: knreddy_4@yahoo.com
ABSTRACT:
There are many analytical methods to
estimate the pesticide residues in agriculture produce and food products . The
multi chemical group pesticide residue
analytical method has been validated for the routine analysis of 54 multi
chemical class pesticide residues in Citrus fruit (citrus limetta) and fortified
levels of 10 µgkg-1 to 100 µgkg-1 by Liquid
chromatography coupled with triple quadrupole mass
spectrometry (LC-MS/MS- TQ) in a single run
of 24.01minutes. And the method
was applied for the analysis of different fruits i.e., Mango, bear, Banana,
grapes, pomegranate, guava etc. By this
method volatile and non volatile
pesticides of different chemical class
can be identified and quantified in single stroke in a sample. Samples
were extracted by the adopting QuEChERS method including extraction of sample with acetonitrile and simultaneous liquid partitioning by adding
addition of Sodium chloride and Sodium sulphate followed by simple clean up by adding anhydrous magnesium sulfate (MgSO4)
and primary secondary amine (PSA) based on the principle of dispersive solid
phase extraction(dSPE). By this method the finding obtained were high recoveries, accuracy, repeatability,
reproducibility of results and small quantity of sample and
less volumes of solvents are used by this method. The limit
of detection (LOD) was 5 µg kg-1
and limit of
quantification in citrus fruit
was (LOQ) 10 µg kg-1 found
by this method.
KEYWORDS: Multi chemical
class Pesticide Residues, citrus, LC/MS/MS, QuAChERS
method.
INTRODUCTION:
In the field of agriculture many agricultural chemicals are using for the control of pests, insects and fungal diseases and their toxicological effects of the chemicals on human and animals are exposed daily are of ever increasing concern. Most of the agricultural chemicals are endocrine disruptors (Wissem Mnif
et al., 2011) and manmade chemicals are interfering with the hormones system by blocking or mimicking normal function (Commission of the European Communities, 2001). The long term and frequent application of pesticides on the crops will lead to form an un degradable toxic products that are called residues. These residues remained on vegetable and fruits even after the post harvest stages. The application of agricultural chemicals on crops leads to the human exposure when the agriculture produce is consumed. These pesticides may have been different chemicals class and different activities viz., Insecticides, acaricides, fungicides and herbicides during different stages of the plant and storage. The Pesticide residues have potential adverse effects on vegetables, fruits animals resources and human health (Perez Bendito et al. 1999). Because of their widespread usage, their toxic residues have been reported in various environmental matrices (Kumari B et al 1996, 2002). Therefore it is essential to studies of residues of these chemicals in food products. The citrus limetta is an important crop in south India and utilized in various types of foods preparations viz., vegetarian, non vegetarian diet, curries, pickles and juices etc. Various studies have been done towards the beneficial properties of fruit juices, because they have several components such as phenols, vitamins and flavonoids with anti oxidant properties (Benzie IF et al. 2014). An attempt made to standardize the analytical method in citrus fruit (Citrus limetta i.e., cheeni variety) as the plants is treated with different chemicals class of viz., insecticides, fungicides and herbicides etc. A new analytical method for the analysis of pesticide residues was modified and developed (Anastassiades et el., 2013) to monitor the agriculture produce, and name as quick, easy, cheap, effective rugged and safe (QuEChERS) method. To change matrices an attempt was made to develop the new multi chemical class residue analytical method to monitor the citrus samples for 54 different class of agricultural chemicals at a time. By adopting this multi residue analytical technique the analysis time will reduced to 20 to 25 minutes and preparation of sample has to meet the international standards in pesticide residue analysis (Gontarev M et al 2007). Rapid, simple and robust extraction methods are required in routine analysis of pesticide residue laboratories. The QuEChERS method has several advantages over old methods of pesticide residues, as the recoveries are high (>85%) compared with old traditional methods (Luke, M., et al 1975 and Fillion J et al, 2000). The sample taken for analysis is small about 10 to 20 grams and clean up by dispersive solid phase extraction (dSPE) method and whole laboratory process will be completed 30- 40 minutes. By reducing the analysis time (Hiemstra et al., 2007) the solvent usage is very low volumes without any organo chlorinated solvent. The QuEChERS extraction and cleanup procedure has been following in many residue laboratories because of obtaining high recovery values even in very low concentrations. The QuEChERS analytical procedure involves utilizes methyl cyanide (Acetonitrile) for extraction, followed by liquid partitioning step. The addition of sodium chloride (NaCl) sodium sulphate (Na2SO4) ) to induce partitioning of the Acetonitrile extract from the water in the sample. An aliquot of the extract is then cleaned up by adding Sorbents Magnesium sulphate (MgSO4), Primary Secondary Amine (ethylenediamine N-propyl bonding with silica gel base) and graphitized carbon black (GCB) works with the principle of dSPE (dispersive solid phase extraction), no organo chlorinated solvent were used. The final extract in Acetonitrile is directly injected and analyzed on LC-MS/MS. Primary Secondary Amine (PSA) has been found as the most effective sorbent for removal of various carboxylic acids, carbonyl group compounds etc., and significantly reducing matrix- enhancement effect. On the other hand, graphitized carbon black (GCB) is very useful for removal of coloring substances (i.e. pigments) and sterols. The development of new methods in the analysis of pesticide residues on Liquid chromatography coupled with triple quadrupole mass spectrometry (LC/MS/MS - TQ), and these methods mainly focused on accuracy, repeatability and reproducibility of results and another one important accept is that volatile and non volatile compounds of various chemical classes pesticides with different activities can be easily identified and quantified by LC/MS/MS. The triple quadrupole mass analyzers are powerful detectors with an enhanced selectivity and data acquisition speed these will allows the simultaneous monitoring of a higher number of co-eluting compounds and identification and quantification of pesticide residues in trace analysis of complex matrices. The current analytical procedure was properly validated under the guidelines of European SANCO and the ISO 17025 norms (SANCO 2013). The aim of this study is to develop and validate the analysis of volatile and non volatile multi chemical class pesticides and analyzed in a single injection by adopting best extraction methods with Liquid Chromatography mass spectrometric Detector. These studies were focus on citrus sample preparation, sample extraction following QuEChERS analytical method and instrumental operating parameter are optimized by finding the multiple reaction monitoring (MRM) parameters in LC-MS-MS to identify and quantify about 54 multi chemical group of pesticides of various activity in Citrus fruit in a short period i.e., within 24.01 minutes.|
Triple
Quadruple Mass Spectrometer equipped with ESI (Electron Spray Ionization)
interface |
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Table. 4 Percentage recoveries of
different fortification levels of pesticides in citrus
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Average % recovery at different fortification levels (mean ) |
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LOD: Limit of detection LOQ: Limit of quantification
Analysis of
real citrus samples by adopting
validated method