**2.3 Residue extraction and cleanup step**

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


*quantification.*

**101**

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

polypropylene tubes; (3) add 10 ± 0.1 ml acetonitrile (1% acetic acid) to the polypropylene tubes; (4) homogenize the sample at 3000–5000 rpm for 2–3 min. Add 1.5 g sodium acetate and 6 g MgSO4 (anhydrous), and mix it by shaking gently and centrifuging at 3000 rpm for 3 min to separate the organic layer; (5) 1 ml of extract is then taken in a separate dSPE (dispersive solid-phase extraction) tube, and 50 mg PSA and 140 mg magnesium sulfate are then added to it; (6) extracts were then centrifuged at 5000 rpm for 1 min; (7) 1 ml of the supernatant was then transferred to a small glass tube and the solvent was then evaporated using turbo evaporator which was set at 45°C and 20 psi inert nitrogen gas flow; and (8) the final step was the reconstitution of the sample with a 1 ml of ethyl acetate for analysis and confirmation of residues by gas chromatography-mass spectrometry

**GC-MS/MS analysis**. A gas chromatography (Agilent 6890N) with mass spectrometer (Waters, Boston, USA) and an auto-sampler (Agilent 7683) with electron ionization (EI+) mode were used. Separation of analytes was carried out using HP-5MS column (30 m, 0.25 μm internal diameter, 0.25 μm film thickness) (J&W Scientific). The oven temperature was increased as follows: 50°C (1 min), 25°C/min up to 150°C and increased to 10°C/min to 280°C (4 min hold). Split-less injection of 1 μl was carried out with an injector temperature maintained at 280°C and hold time of 1 min. The carrier gas that was used was helium (99.999%) at flow rate of 1.3 ml/min. The interface temperature was maintained

Electron ionization (EI+) mode was selected (4 min solvent delay); the source temperature was set at 250°C. The gas argon (purity 99.99%) was used as collision gas which is used to collide with ions after ionization. The dwell time per channel was between 0.05 and 0.1 s. QuantLynx was used to process the data obtained from calibration of CRMs and also from grape fruit

Heptacosa (perfluorotributylamine) was used to calibrate the mass spectrometer. **Table 1** explains the particular ions of quantification for the MRM mode and retention times (tM) for the residue analysis of individual

In this method, for the fulfillment of validation criterion, single-laboratory

Five calibration levels (1 and 200 ng/mL) were used for constructing the calibration curve by using pure solvent and matrix. The concentration of a pesticide residue can be calculated based upon the calibration curve. The prerequisite for this method is that the peak area should fall within the linear range of the curve. Then the concentration can be calculated on basis of the slope of the calibration curve

Y = mX + C (1)

where Y = peak area, X = concentration, m = slope of the curve and

approach was used. The following validation parameters were used.

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

(GC-MS/MS) (24).

at 250°C.

extract.

substances.

**3.1 Linearity**

C = constant.

**3. Validation study**

using the regression equation:

#### **Table 1.**

*Experimental condition of the optimized GC-MS/MS method parameters, retention time (min), MRM, average recovery (%) and RSD (in parenthesis) of grape samples (n = 4) at two concentration levels.*

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

polypropylene tubes; (3) add 10 ± 0.1 ml acetonitrile (1% acetic acid) to the polypropylene tubes; (4) homogenize the sample at 3000–5000 rpm for 2–3 min. Add 1.5 g sodium acetate and 6 g MgSO4 (anhydrous), and mix it by shaking gently and centrifuging at 3000 rpm for 3 min to separate the organic layer; (5) 1 ml of extract is then taken in a separate dSPE (dispersive solid-phase extraction) tube, and 50 mg PSA and 140 mg magnesium sulfate are then added to it; (6) extracts were then centrifuged at 5000 rpm for 1 min; (7) 1 ml of the supernatant was then transferred to a small glass tube and the solvent was then evaporated using turbo evaporator which was set at 45°C and 20 psi inert nitrogen gas flow; and (8) the final step was the reconstitution of the sample with a 1 ml of ethyl acetate for analysis and confirmation of residues by gas chromatography-mass spectrometry (GC-MS/MS) (24).

**GC-MS/MS analysis**. A gas chromatography (Agilent 6890N) with mass spectrometer (Waters, Boston, USA) and an auto-sampler (Agilent 7683) with electron ionization (EI+) mode were used. Separation of analytes was carried out using HP-5MS column (30 m, 0.25 μm internal diameter, 0.25 μm film thickness) (J&W Scientific). The oven temperature was increased as follows: 50°C (1 min), 25°C/min up to 150°C and increased to 10°C/min to 280°C (4 min hold). Split-less injection of 1 μl was carried out with an injector temperature maintained at 280°C and hold time of 1 min. The carrier gas that was used was helium (99.999%) at flow rate of 1.3 ml/min. The interface temperature was maintained at 250°C.

Electron ionization (EI+) mode was selected (4 min solvent delay); the source temperature was set at 250°C. The gas argon (purity 99.99%) was used as collision gas which is used to collide with ions after ionization. The dwell time per channel was between 0.05 and 0.1 s. QuantLynx was used to process the data obtained from calibration of CRMs and also from grape fruit extract.

Heptacosa (perfluorotributylamine) was used to calibrate the mass spectrometer. **Table 1** explains the particular ions of quantification for the MRM mode and retention times (tM) for the residue analysis of individual substances.
