**3. Validation study**

In this method, for the fulfillment of validation criterion, single-laboratory approach was used. The following validation parameters were used.

## **3.1 Linearity**

*Gas Chromatography - Derivatization, Sample Preparation, Application*

**Pre. ion**

**Prod. ion**

 DEET 7.06 119 65 21 90 (7) 95 (12) 0.001 Propiconazole 7.65 69 41 6 88 (14) 92 (11) 0.01 Phorate 7.85 260 75 5 69 (9) 75 (12) 0.002 Carbofuran 8.35 164 149 8 78 (4) 84 (7) 0.002 Atrazine 8.45 215 58 8 95 (7) 99 (5) 0.005 Lindane 9.16 184 145 10 90 (5) 95 (8) 0.001 Diazinon 9.74 179 137 17 79 (16) 87 (12) 0.0005 Chlorothalonil 9.95 266 133 26 84 (16) 86 (9) 0.004 Metalaxyl 10.37 206 59 8 74 (9) 76 (13) 0.002 Fenitrothion 10.64 125 79 11 89 (9) 91 (4) 0.002 Malathion 10.70 173 99 10 91 (7) 89 (11) 0.003 Aldrin 11.54 263 193 22 91 (7) 89 (11) 0.003 Fenthion 11.99 278 109 12 80 (11) 92 (15) 0.005 Chlorpyrifos 12.05 197 169 16 93 (8) 96 (7) 0.0005 Parathion 12.39 291 109 10 83 (6) 95 (12) 0.003 Triadimefon 12.77 208 181 6 88 (9) 91 (10) 0.006 Pendimethalin 13.39 252 162 16 95 (3) 98 (13) 0.005 Captan 13.95 79 51 20 72 (10) 79 (8) 0.002 Phenthoate 14.19 274 121 16 88 (4) 90 (6) 0.0005 2,4-DDT 14.61 146 118 7 70 (15) 82 (10) 0.00001

**MRM CE Fortification levels** 

14.95 241 170 25 70 (12) 82 (9) 0.004

17.41 241 170 25 70 (14) 87 (9) 0.005

20.17 163 127 6 91 (11) 95 (8) 0.005

 Butachlor 15.29 176 146 20 94 (8) 99 (13) 0.001 Profenofos 15.76 337 267 8 73 (11) 78 (6) 0.005 2,4-DDD 16.34 235 165 16 95 (11) 98 (9) 0.00001 Endrin 16.85 263 193 22 83 (6) 86 (5) 0.005 Chlorfenapyr 17.15 247 75 17 76 (5) 78 (13) 0.02

 Quinalphos 17.87 235 165 15 98 (9) 100 (5) 0.003 Ethion 17.89 231 129 18 91 (14) 96 (10) 0.0001 Triazophos 18.72 161 77 19 72 (5) 79 (10) 0.005 Iprodione 18.91 314 245 10 87 (7) 90 (4) 0.02 Beta-cyfluthrin 19.63 165 127 5 96 (15) 99 (8) 0.01

34 Fenvalerate 20.72 167 125 8 89 (9) 94 (3) 0.005 35 Deltamethrin 21.73 181 152 18 70 (15) 78 (11) 0.008 *tM, retention time; MRM, multiple reaction monitoring; CE, collision energy; LOD, limit of detection; LOQ, limit of* 

*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.*

**(mg/kg)**

**0.02 0.05**

**LOD (mg/ kg)**

**tM (min)**

**Sl. no.**

21 Alpha-

27 Beta-

33 Alpha-

*quantification.*

**Table 1.**

endosulfan

endosulfan

cypermethrin

**Reference standards**

**100**

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 using the regression equation:

$$\mathbf{Y} = \mathbf{mX} \mathbf{+C} \tag{1}$$

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