**Abstract**

GC-MS/MS method has been developed and validated for the determination and quantification of 35 multi-class pesticide residues in grape samples. Pesticides are selected from different families including organochlorines, organophosphorus, carbamates, pyrethroids, triazines, triazoles, pyrazoles, etc. The QuEChERS-dSPE (dispersive solid-phase extraction) method was used for the extraction of residues of pesticide. An extra cleanup step was included with the help of a primary secondary amine (PSA) and graphitized carbon black (GCB). Recoveries ranged from 70 to 100% with 14% relative standard deviation (RSD). Other parameters such as precision, recoveries, limit of detection (LOD), limit of quantification (LOQ ), and linearity were also studied. Finally, the proposed analytical method was successfully employed for the determination of residues of pesticide in grape samples.

**Keywords:** residues of pesticide, QuEChERS-dSPE, GC-MS/MS

### **1. Introduction**

In India, a large quantity of pesticides is used for the cultivation of grapes mainly for the management of various diseases and pests. Due to the stringent rules set by the various developed countries on food safety standards and the regulations on quality parameters, we find that the residues of the pesticides in food are gaining a lot of attention. Keeping in view the problem of residues of pesticides, the present study was conducted on grape (*Vitis vinifera* L.) of Bijapur District for the qualitative and quantitative analysis of pesticides by GC-MS/MS (gas chromatography coupled to mass spectrometry).

In recent years, the production and marketing of food have gained topmost priority. This in turn has given rise for the implementation of better agricultural practices and has also prompted a substantial increase in the importance given to pesticide residues and related aspects. It is important to analyze large numbers of samples for residues of pesticide in the food due to their control and regulatory issues. Analytical procedures for pesticide residues are usually time-consuming and costly. For this reason multiresidue methods have been devised and regularly applied in regulating pesticide monitoring programmes [1, 2].

There is a difficulty in developing a method for the residue analysis mainly due to wider nature of polarity, volatility and solubility of different pesticides [3]. In relation with different pesticide classes, various methodologies using gas chromatography with numerous detectors, like thermal conductivity detector (TCD), nitrogen-phosphorus detector (NPD), electron capture detector (ECD) and flame photometric detector (FPD), have been implemented [4]. Further several methods have been developed for accurate quantification of residues of pesticides in various consumable food products or commodities. All these seem to be much complicated because of the use of large quantity of inert gases which are quite costly and consuming [5, 6]. Therefore, there is a need to develop new methods in the preparation of the sample and the requisite quantification parameters.

QuEChERS which is a novel quick, easy, cheap, effective, rugged, and safe method for preparation of samples in pesticide residue analysis [7] was used. QuEChERS methodology has been devised in the year 2003 for the multiresidue analysis of pesticides in different matrices, and now it is a universally accepted method. In this procedure extraction was performed with acetonitrile solvent initially and then partitioning step was carried out using salt mixture. A small amount of extract was further cleaned by using dispersive solid-phase extraction (dSPE) method. Finally, extract was used for the determination of pesticide residues using GC-MS/MS. This method has several advantages; firstly, sample throughput is very high; secondly, it does not use chlorinated solvents; and thirdly, a very small quantity of solvents is needed which in turn provides a very high recovery percentage for broad-spectrum volatility and polarity range of pesticide molecules. Even though this method was developed recently, it has been widely accepted by the international community of pesticide residue analysts. There have been several publications on this topic often replacing the original method with newer and better ones [8–12].

Chromatographic system (gas chromatography or liquid chromatography) attached to mass spectrometry (MS/MS) determination provides us with a method for identifying and quantifying several pesticides in different food matrices [13]. Simple extraction procedure along with very limited cleanup technologies has been employed as a result of the use of more sensitive and selective MS/MS detection. Martinez Vidal et al. used gas chromatography-mass spectroscopy (GC-MS/MS) with ethyl acetate for extraction of 130 multi-class pesticides [14]. Pihlström et al. slightly modified GC-MS/MS procedure [15]. Hetherton et al. reported the use of LC-MS/MS and acetonitrile extraction for the analysis of 73 pesticides in lettuce and oranges [16]. Pang et al. used both liquid chromatography and gas chromatography attached to mass spectrometry for the simultaneous determination of 336 pesticides in vegetables and fruits [17, 18] and 440 pesticide residues in wine, fruit juice, and honey using solid-phase extraction (SPE) cleanup [7].

Grape (*Vitis vinifera* L.) is one of the most important fruit crops cultivated in the subtropical regions of India (60,000 ha). The states, namely Maharashtra, Karnataka, Andhra Pradesh, Tamil Nadu, Punjab, and Haryana, are the grapegrowing regions in India. Amongst them, Maharashtra and Karnataka rank first and second in terms of area and productivity, respectively. Grapes cultivated in Maharashtra and Karnataka are mainly exported to Europe, the Middle East and to some extent West Asia. As a result, a large quantity of pesticides is used in their cultivation. This is mainly due to the presence of heavy insect pest infestation. Excess usage of pesticides often results in the accumulation of pesticides on the fruit and causes various health hazards and is also more prone for rejection in the international market.

This paper explains an effective and simple experimental procedure for extraction of sample by employing QuEChERS (slightly modified) method and the use of gas chromatographic system with mass spectrometric determination for 35 pesticide residues in grape samples.

**99**

*Gas Chromatographic-Mass Spectrometric Detection of Pesticide Residues in Grapes*

a.GC-MS/MS instrument: gas chromatograph (Agilent 6890N) with autosampler and a triple quadrupole mass spectrometer (Quattro Micro RAB120

Waters) detector was used for the analysis of the pesticides studied. MassLynx Solution software was used for the instrument control and data

nitrogen was used for the evaporation of the solvent.

b.Low-volume concentrator: Turbovap (Caliper Life Sciences, USA) with inert

c.Chopper and homogenizer: vegetable chopper was used for chopping, and a homogenizer (Heidolph) was used for proper mixing of the fruit samples.

d.Centrifuge: centrifuge (Sigma 3K 10) was used for both 2 and 50 ml polypro-

e.Weighing balance: weighing balance (Sartorius) was used to weigh the chopped samples and preparation of reference standard reagents.

a.Ethyl acetate and acetic acid (glacial): ethyl acetate and acetic acid (glacial) of sufficient quality for pesticide residue analysis were procured from

b.Sodium acetate and magnesium sulfate: reagent-grade anhydrous sodium acetate and magnesium sulfate were procured from Merck (India).

c.Certified reference materials (CRMs): certified reference materials of pesticides were procured from Sigma-Aldrich/Riedel-de-Haen (Zwijndrecht, The Netherlands). The individual stock solutions of 1000 ppm were prepared in toluene and hexane (1:1), and working standards containing 35 pesticides at

d.Primary secondary amine (PSA): SPE sorbent PSA (40 μm, Bondesil PSA) was

e.Grape samples: grape samples (2 kg each) were collected from the field in

The method of preparation of the sample for multiresidue pesticide analysis in grapes involved the following steps: (1) crush 2 kg grape samples under ambient conditions and then 200 g of sample further homogenized for 2 min for proper mixing; (2) accurately weigh a 10 ± 0.1 g of this sample into each 50 ml

different concentration levels were prepared in ethyl acetate.

purchased from Agilent Technologies (Bangalore, India).

Vijayapura district (Karnataka state).

**2.3 Residue extraction and cleanup step**

*DOI: http://dx.doi.org/10.5772/intechopen.80438*

**2. Experimental**

analysis.

pylene tubes.

Sigma-Aldrich.

**2.2 Reagents**

**2.1 Apparatus**

*Gas Chromatographic-Mass Spectrometric Detection of Pesticide Residues in Grapes DOI: http://dx.doi.org/10.5772/intechopen.80438*
