METHOD FOR SIMULTANEOUS DETERMINATION OF FOLPET AND CYMOXANIL ACTIVE INGREDIENTS (COMBINATION PRODUCTS) IN AQUEOUS MEDIA

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1 METHOD FOR SIMULTANEOUS DETERMINATION OF FOLPET AND CYMOXANIL ACTIVE INGREDIENTS (COMBINATION PRODUCTS) IN AQUEOUS MEDIA Author : S.Y. Pandey and Arun Raithatha Jai Research Foundation Valvada, Gujarat, India JAI RESEARCH FOUNDATION A Global Contract Research Organisation 1

2 Details of Folpet Common Name IUPAC Name : Folpet : N-(trichlorolmethylthio)phalimide; N-(trichloromethanesulphenyl)phalimide CA Name : 2-[(trichloromethyl)thio]-1H-isoindole-1,3(2H)-dione CAS RN : [ ] O EEC No : N-SCCl 3 O JAI RESEARCH FOUNDATION A Global Contract Research Organisation 2

3 Details of Folpet Phys-Chemical Properties Technical : 92-95% Molecular Weight : Molecular Formula : C 9 H 4 Cl 3 NO 2 S Form Melting Point : 177 C S.G./ Density Water Solubility Stability Uses : Colourless crystals : 1.72 (20 C) : 0.8 mg/l : Stable in dry state. Slowly hydrolysed by moisture at room temperature. : Downy mildews, powdery mildews, leaf spot and scrab, black rod on fruits and vegetables JAI RESEARCH FOUNDATION A Global Contract Research Organisation 3

4 Details of Cymoxanil Common Name IUPAC Name CA Name : Cymoxanil : 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea : 2-cyano-N-[(ethylamino)carbonyl]-2- (methoxyimino)acetamide CN Structure : CH 3 CH 2 NHCONHCOC=NOCH 3 CAS RN : [ ] EEC No : JAI RESEARCH FOUNDATION A Global Contract Research Organisation 4

5 Details of Cymoxanil Phys-Chemical Properties Tech : >95% Molecular Weight : Molecular Formula : C 7 H 10 N 4 O 3 Form : Colourless Crystals Melting Point : C S.G./Density Water Solubility Stability Uses : 1.31 (25 C) : 890 mg/kg : DT d (ph 5), 34 h (ph 7), 31 min (ph 9) : Fungicide, Preventive and curative action on fruits and vegetables. JAI RESEARCH FOUNDATION A Global Contract Research Organisation 5

6 HPLC Condition Instrument Column Wave length Flow rate : HPLC [Shimadzu, LC-2010AHT with LC-Solution software) : C-18 [250 x 4.6 mm (i.d.) x 5.0 µm particle size] : 235 nm Injection volume : 20 µl Mobile Phase Auto sampler Retention Time : 1.0 ml/minute : Acetonitrile: Milli-Q Water (70:30, v/v) : 4 ±2 0 C : Cymoxanil : 3.4 minutes (approx.) Folpet : 4.7 minutes (approx.) JAI RESEARCH FOUNDATION A Global Contract Research Organisation 6

7 Chromatogram JAI RESEARCH FOUNDATION A Global Contract Research Organisation

8 Linear Dynamic Range of Folpet Purity Determination N of Injection Peak Area Count Area Count % Variation I II I II I II I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 8

9 Linear Dynamic Curve of Folpet Purity Determination JAI RESEARCH FOUNDATION A Global Contract Research Organisation 9

10 Linear Dynamic Range of Folpet Tap Water N of Injection Peak Area Count Area Count % Variation I 2408 II 2434 I 4779 II 4816 I 9584 II 9631 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 10

11 Linear Dynamic Curve of Folpet Tap Water JAI RESEARCH FOUNDATION A Global Contract Research Organisation 11

12 Linear Dynamic Range of Folpet Reconstituted Water N of Injection Peak Area Count Area Count % Variation I 2482 II 2476 I 4821 II 4751 I 9562 II 9631 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 12

13 Linear Dynamic Curve of Folpet Reconstituted Water JAI RESEARCH FOUNDATION A Global Contract Research Organisation 13

14 Linear Dynamic Range of Folpet Algal Media N of Injection Peak Area Count Area Count % Variation I 2462 II 2451 I 4828 II 4784 I 9583 II 9563 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 14

15 Linear Dynamic Curve of Folpet Algal Media JAI RESEARCH FOUNDATION A Global Contract Research Organisation 15

16 Limit of Detection (LOD) of Folpet in Tap Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I 448 II I 894 II 886 I 1434 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 16

17 Limit of Detection (LOD) of Folpet in Reconstituted Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 848 II 855 I 1335 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 17

18 Limit of Detection (LOD) of Folpet in Algal Media Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 859 II 852 I 1366 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 18

19 Limit of Quantitation (LOQ) of Folpet in Tap Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2361 II 2352 I 2765 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 19

20 Limit of Quantitation (LOQ) of Folpet in Reconstituted Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2202 II 2192 I 2757 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 20

21 Limit of Quantitation (LOQ) of Folpet in Algal Media Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2209 II 2199 I 2720 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 21

22 Precision (% RSD) and Accuracy for Folpet in Tap Water Fortification Level Fortified Control 0.0 LOQ x LOQ 0.61 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 I II R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation % RSD JAI RESEARCH FOUNDATION A Global Contract Research Organisation 22

23 Precision (% RSD) and Accuracy for Folpet in Reconstituted Water Fortification Level Fortified Control 0.0 LOQ x LOQ 0.61 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation JAI RESEARCH FOUNDATION A Global Contract Research Organisation 23 % RSD

24 Precision (% RSD) and Accuracy for Folpet in Algal Media Fortification Level Fortified Control 0.0 LOQ x LOQ 0.61 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation JAI RESEARCH FOUNDATION A Global Contract Research Organisation 24 % RSD

25 Linear Dynamic Range of Cymoxanil Purity Determination N of Injection Peak Area Count Area Count % Variation I II I II I II I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 25

26 Linear Dynamic Curve of Cymoxanil Purity Determination JAI RESEARCH FOUNDATION A Global Contract Research Organisation 26

27 Linear Dynamic Range of Cymoxanil Tap Water N of Injection Peak Area Count Area Count % Variation I 1856 II 1882 I 3066 II 3086 I 5920 II 5908 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 27

28 Linear Dynamic Curve of Cymoxanil Tap Water JAI RESEARCH FOUNDATION A Global Contract Research Organisation 28

29 Linear Dynamic Range of Cymoxanil Reconstituted Water N of Injection Peak Area Count Area Count % Variation I 1891 II 1856 I 3173 II 3143 I 6257 II 6197 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 29

30 Linear Dynamic Curve of Cymoxanil Reconstituted Water JAI RESEARCH FOUNDATION A Global Contract Research Organisation 30

31 Linear Dynamic Range of Cymoxanil Algal Media N of Injection Peak Area Count Area Count % Variation I 1852 II 1873 I 3063 II 3034 I 5902 II 5941 I II I II I II JAI RESEARCH FOUNDATION A Global Contract Research Organisation 31

32 Linear Dynamic Curve of Cymoxanil Algal Media JAI RESEARCH FOUNDATION A Global Contract Research Organisation 32

33 Limit of Detection (LOD) of Cymoxanil in Tap Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 703 II 691 I 1321 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 33

34 Limit of Detection (LOD) of Cymoxanil in Reconstituted Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 725 II 640 I 1245 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 34

35 Limit of Detection (LOD) of Cymoxanil in Algal Media Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 698 II 706 I 1300 II LOD N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 35

36 Limit of Quantitation (LOQ) of Cymoxanil in Tap Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2152 II 2128 I 2823 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 36

37 Limit of Quantitation (LOQ) of Cymoxanil in Reconstituted Water Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2167 II 2184 I 2844 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 37

38 Limit of Quantitation (LOQ) of Cymoxanil in Algal Media Solution N of Injection Peak Area Count Area Mean Noise Signal to Noise Ratio (S/N) Remark I II I 2126 II 2158 I 2778 II LOQ N of Injection of Blank Total Area of Noise in Blank (a) N of Noise in Blank (b) Average = a/b I II Average Noise Area of Blank JAI RESEARCH FOUNDATION A Global Contract Research Organisation 38

39 Precision (% RSD) and Accuracy for Cymoxanil in Tap Water Fortification Level Fortified Control 0.0 LOQ x LOQ 0.41 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation JAI RESEARCH FOUNDATION A Global Contract Research Organisation 39 % RSD

40 Precision (% RSD) and Accuracy for Cymoxanil in Reconstituted Water Fortification Level Fortified Control 0.0 LOQ x LOQ 0.41 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation JAI RESEARCH FOUNDATION A Global Contract Research Organisation 40 % RSD

41 Precision (% RSD) and Accuracy for Cymoxanil in Algal Media Fortification Level Fortified Control 0.0 LOQ x LOQ 0.41 Replication Injection Peak Area of Sample (Y) Recovered Recovery (%) R1 I - ND - R2 I - ND - R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 I II I II I II I II I II I II I II I II I II I II Mean Mean Recovery (%) Standard Deviation JAI RESEARCH FOUNDATION A Global Contract Research Organisation 41 % RSD

42 Summary - Folpet Parameters Results Test Media Tap Water Reconstituted Water Algal Medium Specificity (Non-analyte Interference) No interference No interference No interference Linear (LDR) Range Intercept (a) Slope of the line (b) Correlation Coefficient (r) 0.05 to to to Limit of Detection (LOD) [mg/l] Limit of Quantitation (LOQ) [mg/l] Fortification Level Precision (% RSD) LOQ % RSD [Horwitz equation] x LOQ % RSD [Horwitz equation] Accuracy (Recovery %) LOQ x LOQ JAI RESEARCH FOUNDATION A Global Contract Research Organisation 42

43 Summary - Cymoxanil Parameters Test Media Specificity (Non-analyte Interference) Linear (LDR) Range Intercept (a) Slope of the line (b) Correlation Coefficient (r) Tap Water No interference 0.03 to Results Reconstituted Water No interference 0.03 to Algal Medium No interference 0.03 to Limit of Detection (LOD) [mg/l] Limit of Quantitation (LOQ) [mg/l] Fortification Level Precision (% RSD) LOQ % RSD [Horwitz equation] x LOQ % RSD [Horwitz equation] Accuracy (Recovery %) LOQ x LOQ JAI RESEARCH FOUNDATION A Global Contract Research Organisation 43

44 Conclusion From the results of the validation it is concluded that the method is sensitive and precise for the analysis of active ingredient content concentrations of Folpet, Cymoxanil and Fosetyl-Al in test media. The results of validation criteria are within the guidelines (SANCO/3029/99 rev.4 and U.S. EPA OPPTS ) specified limit. JAI RESEARCH FOUNDATION A Global Contract Research Organisation 44

45 JAI RESEARCH FOUNDATION A Global Contract Research Organisation 45