**4.2 Recovery experiments of spiked samples**

Usually, the extraction and cleanup procedure removes the matrix co-extractives then separates all of the analytes from the matrix. The same does not holds good in most of the matrices during the pesticide residue analysis. As a result, the actual recovery experiments were performed on grape samples. The separated peaks with their tM (retention times) are summarized in **Table 1**. Using the linear regression equation recoveries of individual pesticides with different levels of spiking along with replicates were calculated in grape matrix. **Table 1** gives the average recoveries for all spiked pesticide standards at each spiked level in grape samples. All the tested 35 pesticides displayed a recovery range between 70 and 100% which is quite acceptable. RSD (relative standard deviation) was used to express the reproducibility, and most of the RSD values were found to be less than 14%. Recovery study is conducted using a control sample and at least two fortification levels with three replications. The formula for arriving percentage recovery for method validation is as follows:

*Gas Chromatography - Derivatization, Sample Preparation, Application*

Typical S/N acceptance criteria: LOD—3:1 and LOQ—10:1.

It was determined by elimination of noise at the retention time of the compound, which is performed by fixing two transitions of MS/MS for individual molecule of analyte by considering the adequate precursor and product ions.

Detection limit (LOD) in the chromatogram was calculated by using peak signal of the analyte molecule concentration to the three times background noise in the chromatogram. The quantification limit (LOQ ) in the chromatogram was set as the lowest concentration with very good recovery range (65–100%) and precision (RSD ≤ 20%). The ion ratio (Q/q) was used for the criterion of confirmation in positive samples. The Q/q is the ratio of the intensity quantification (Q ) and

All the 35 certified reference materials of pesticides were determined in a single

for both pure solvent-based and matrix-matched samples. The recovery of all of the compounds was found to be 70–100% with RSD below 14%. **Figure 2** shows chromatograms of typical pesticide peak shapes. The grape sample extracts were slightly yellow because of the co-extraction of carotenoids in the ethyl acetate. It was found that a two-step homogenization procedure significantly increased the precision of analysis. It was also observed that the use of high-speed homogenization process

values (correlation coefficients) of the calibration curve were >0.99

chromatographic window of 22 min run time (**Figure 1**). For most of the com-

**3.2 Selectivity**

**3.3 Sensitivity**

confirmation transition (q) (**Table 1**).

**4. Results and discussion**

**4.1 Method validation**

pounds, the R<sup>2</sup>

**102**

**Figure 1.**

*Total ion chromatogram of 35 certified reference standards.*


In QuEChERS method the use of acetonitrile has several advantages, mainly addition of salt separates it from water without using nonpolar solvents, mutual compatibility with dispersive solid-phase extraction and very good separation/ matching with gas and liquid chromatography. The anhydrous MgSO4 tends to form lumps. Shaking the tubes of the centrifuge on adding the salt mixture for 1 min or more, it was observed that the formation of lumps was eliminated. Next, adding the salt to all of the samples, it was also found that the one-minute extractions of the entire batch could be run parallelly. Dispersive solid-phase extraction with primary secondary amine eliminated the color pigments, acidic components and sugars [10, 11]. Apart from this, sugars, lipids and waxes were removed by freezing which helped in increasing the efficiency of GC analysis [12].
