Olga Shimelis, Katherine Stenerson, Michael Ye, Carmen T. Santasania, and Ed Mauney. Supelco, Div. of Sigma-Aldrich, Bellefonte, PA USA

Transcription

1 Method for Analysis of rganophosphorous Pesticides in Produce using Acetonitrile Extraction, Dual-Layer Carbon-Aminopropylsilica Cleanup and GC-NPD Detection lga Shimelis, Katherine Stenerson, Michael Ye, Carmen T. Santasania, and Ed Mauney Supelco, Div. of Sigma-Aldrich, Bellefonte, PA USA T

2 Abstract 2 rganophosphorous compounds belong to a large class of pesticides used in agriculture since they are more amenable to environmental degradation in comparison to organochlorine or organonitrogen compounds. Because of their potential health effects, organophosphorous pesticides are of particular concern on produce imported from areas in which they are commonly used. We present here a method for analysis of 65 organophosphorous pesticides from spiked cabbage, green onions, button mushrooms and apples. The extraction was achieved using acetonitrile, with the addition of sodium chloride and magnesium sulfate to induce a salting out effect. A dual layer primary-secondary amine (PSA)/graphitized carbon black (GCB) SPE tube was used for extract cleanup, and the elution solvent contained 35% toluene and 65% acetonitrile for optimum recovery. Solvent exchange into ethyl acetate provided sample extracts with minimal background for GC-nitrogenphosphorous detection (NPD). Using matrix-matched standards for quantitation, average recoveries for most organophosphorous pesticides, spiked at 10 ng/g, were in the % range with average standard deviations of 5% in cabbage and apples, 8% in mushroom, and 12% in green onions.

3 Experimental Extraction method was based on Shimelis et al. in J. Chromat. A 1165(1-2), Sample preparation 10 g homogenized food sample green onions, apples, Napa cabbage, button mushrooms 10 ml acetonitrile and 4 g MgS 4 (heated at 550 o C), 1 g NaCl mixed, centrifuged Supernatant mixed with 1 g MgS 4, mixed, centrifuged 5 ml of the resulting sample was evaporated down to 1 ml for SPE loading 3 SPE method Condition: 5 ml acetone:toluene (65:35) Load: 1 ml of the extract in acetonitrile Elution: 10 ml acetone:toluene (65:35) Elution fraction evaporated to 0.5 ml and volume was adjusted to 1 ml by addition of ethyl acetate

4 Spiking levels All produce was spiked at 10 ng/g. Calibration Matrix-matched standards were used for calibration. 4

5 Figure 1. Examples of Structures of rganophosphorous Pesticides S N P N 2 P Dichrotophos Methyl Parathion Ethoprophos P S S S P S S P Ethion S 5

6 Results ptimization of SPE Protocol to Increase Recoveries Evaluated: Changing the elution solvent volume; 5 ml, 10 ml, 20 ml Changing composition of SPE elution solvent: 25% vs. 35% toluene Evaporation to dryness vs evaporation to 0.5 ml Quantitation using matrix-matched vs. solvent standards All samples for optimization were loaded in pure acetonitrile without matrix. 6

7 Table 1. ptimization of Elution Solvent Volume: Recovery Evaluation for Representative Pesticides Pesticides 5 ml 10 ml 20 ml Dichrotophos 66% 99% 96% Ethoprophos 9% 32% 48% Methyl parathion 35% 62% 65% Ethion 71% 96% 94% Coumaphos 0% 80% 90% NTE: All samples were evaporated to dryness after SPE and reconstituted in hexaneacetone 1:1. Analysis was done by GC-MS. Result: 10 ml elution volume was chosen. 7

8 Table 2. ptimization of Elution Solvent Composition: Recovery Evaluation for Representative Pesticides Pesticides 25% toluene 35% toluene Dichrotophos 74% 92% Ethoprophos 78% 112% Methyl parathion 65% 85% Ethion 73% 79% Coumaphos 78% 86% NTE: All samples were eluted using 10 ml solvent and evaporated to 0.5 ml after SPE, diluted with ethyl acetate to 1 ml. Analysis was done by GC-MS. 8 Result: 1. 35% toluene / 65% acetone gave better recoveries and was used as elution solvent. 2. Evaporation down to 0.5 ml and not to complete dryness gave better recoveries for lighter compounds.

9 Figure 2. Analysis of Green nion Extract, GC-NPD vs. GC-MS/SIM GC-NPD 4 GC-MS/SIM 1. Ethoprophos 2. CIPC 3. Dicrotophos 4. Monocrotophos 5. Phorate 6. Thiometon

10 Advantage of a Selective Detector A nitrogen phosphorous detector (NPD) was chosen for this analysis due to its selectivity for the compounds of interest. Analysis was initially attempted using a single-quadrupole GC-MS in selected ion mode (SIM), however matrix interference resulted in elevated recoveries and difficulties in peak identification. Figure 2 shows a comparison between NPD and GC-MS-SIM of the same elution range of pesticides from the extract of an onion sample. Even in SIM, the GC-MS system was not able to resolve the peaks of interest from the matrix. A more selective mass detector such as GC-MS/MS would be warranted in this case. However, due to the high expense of this type of instrumentation, it is not available to many laboratories. 10

11 Matrix Enhancement Effect Compared to plain solvent, pesticide response is enhanced when injected in a complex sample matrix. Complex sample matrices can contain compounds which mask GC inlet and column active sites, thus reducing adsorption and degradation of the pesticides. Erroneously high recovery results can result if matrix samples are quantified against standards prepared in solvent. Matrix-matched standards are used to compensate for matrix enhancement effect. The effect is less pronounced with non MS detectors (such as NPD and FPD), however coeluting peaks cannot be accurately quantitated on these detectors. 11

12 Table 3. Recovery Data; Matrix-Matched vs. Solvent Standards Avg. % Recovery Cabbage nions (%RSD n=3) Solvent Matrix-matched Solvent Matrix-matched Dicrotophos 134 (5%) 65 (6%) 157 (10%) 63 (11%) Ethoprophos 85 (6%) 63 (9%) 75 (9%) 66 (11%) Methyl parathion 66 (7%) 65 (2%) 94 (13%) 66 (11%) Ethion 82 (2%) 73 (1%) 92 (5%) 79 (6%) Coumaphos 65 (8%) 72 (8%) 78 (1%) 69 (8%) NTE: All samples analyzed by GC-NPD Result: Some matrix enhancement effect observed; matrix-matched standards used for quantitation. 12

13 Figure 3. GC-NPD Analysis of Spiked Mushroom Extract ,40 44,45 46, Time (min) , , ,26 31, Time (min) 13

14 Peak List for Figure 3 1. Methamidophos 22. Isazofos 43. Isophenfos-methyl 2. Dichlorvos 23. Kitazine 44. Phosfolan 3. Phosdrin 24. Formothion 45. Isophenfos 4. Trichlorphan 25. Dichlofenthion 46. Quinalphos 5. methoate 26. Phosphamidon, isomer #2 47. Phenthoate 6. Ethoprophos 27. Chlorpyrifos-methyl 48. Methidathion 7. CIPC 28. Methyl parathion 49. Bromophos-ethyl 8. Dicrotophos 29. Ronnel 50. Iodophenfos 9. Sulfotepp 30. Paraoxon-ethyl 51. Profenfos 10. Cadusafos 31. Pirmiphos-methyl 52. Fensulfothion 11. Monocrotophos 32. Fenitrothion 53. Ethion 12. Phorate 33. Malathion 54. Famphur 13. Thiometon 34. Chloropyrifos 55. Edinphos 14. Dimethoate 35. Fenthion 56. Pyridaphenthion 15. Demeton-ethyl 36. Parathion 57. Phosmet 16. Terbufos 37. Isocarbophos 58. EPN 17. Dyfonate 38. Trichloronate 59. Phosalone 18. Propetamphos 39. Fosthiazate, isomer #1 60. Azinphos-methyl 19. Phosphamidon, isomer #1 40. Bromophos-methyl 61. Azinphos-ethyl 20. Diazinon 41. Fosthiazate, isomer #2 62. Coumaphos 21. Disulfoton 42. Pirmiphos-ethyl 63. Temephos 14

15 Run Conditions for Figure 3 column: SLB-5ms, 30 m x 0.25 mm I.D., 0.25 µm oven: 110 C (2 min.), 2.5 C/min. to 205 C, 10 C/min. to 310 C (10 min.) inj.: 250 C det.: NPD, 310 C carrier gas: helium, 0.9 ml/min., constant flow injection: 2.0 µl, splitless liner: 4 mm I.D., dual tapered 15

16 Table 4. Recovery Data; Spiking Level of 10 ng/g (%RSD, n=3) Cabbage Mushrooms nions Apples 16 Avg. % Recovery Avg. % Recovery Avg. % Recovery Avg. % Recovery Methamidophos 76 (10%) 70 (5%) 70 (45%) 58 (3%) Dichlorvos masked by matrix 89 (7%) 57 (8%) 76 (6%) Phosdrin 58 (6%) 72 (19%) 75 (7%) 74 (3%) Acephate 70 (7%) 77 (11%) 68 (7%) 77 (2%) Trichlorphan 13 (24%) 61 (28%) 62 (20%) 47 (27%) methoate 72 (5%) 79 (7%) 74 (11%) 78 (4%) Ethoprophos 63 (9%) 77 (9%) 66 (11%) 75 (7%) Dibromfos 35 (20%) ND ND ND CIPC 70 (3%) 84 (9%) 76 (5%) 80 (3%) Dicrotophos 65 (6%) 78 (10%) 63 (11%) 74 (7%) Sulfotepp 66 (5%) 60 (7%) 68 (12%) 77 (22%) Cadusafos 41 (26%) 83 (13%) 51 (23%) 68 (6%) Monocrotophos 76 (12%) 89 (6%) 78 (14%) 74 (29%) Phorate 68 (3%) 83 (7%) 66 (11%) 80 (7%) Thiometon 61 (4%) 72 (8%) 58 (12%) 69 (4%) Dimethoate Demeton-ethyl 74 (3%) 91 (5%) 75 (9%) 81 (5%) Terbufos 60 (4%) 84 (11%) 66 (11%) 74 (8%) Dyfonate 64 (5%) 79 (8%) 65 (9%) 72 (5%) Propetamphos 68 (5%) 77 (8%) 70 (13%) 78 (2%) Phosphamidon #1 Diazinon 64 (3%) 78 (9%) 59 (10%) 77 (4%)

17 Table 4. Recovery Data; Spiking Level of 50 ng/g (%RSD, n=3) (contd.) Disulfoton 58 (3%) 73 (7%) 64 (12%) 73 (7%) Isazofos 69 (6%) 79 (8%) 66 (3%) 77 (2%) Kitazine 66 (4%) 84 (7%) 68 (10%) 73 (5%) Formothion 55 (1%) 54 (6%) 51 (18%) 71 (3%) Phosphamidon #2 Dichlofention 65 (2%) 82 (7%) 71 (9%) 78 (3%) Chloropyrifos-methyl 62 (3%) 80 (8%) 68 (9%) 73 (2%) Methyl parathion 65 (2%) 81 (7%) 66 (11%) 77 (6%) Ronnel 60 (8%) 83 (11%) 71 (5%) 75 (4%) Paraoxon-ethyl 57 (2%) 77 (5%) 68 (15%) 84 (1%) Pirmiphos-methyl Fenitrothion 67 (2%) 85 (8%) 76 (16%) 78 (1%) Malathion 67 (2%) 83 (8%) 70 (15%) 81 (1%) Chloropyrifos 66 (3%) 84 (7%) 73 (18%) 76 (2%) Fenthion 66 (3%) 86 (6%) 67 (20%) 77 (4%) Parathion 69 (2%) 86 (5%) 70 (16%) 79 (2%) Isocarbophos 74 (2%) 90 (7%) 74 (12%) 86 (5%) Trichloronate 64 (1%) 82 (7%) 73 (13%) 78 (9%) Bromophos-methyl Fosthiazate #1 67 (3%) 83 (8%) 72 (11%) 83 (5%) Fosthiazate #2 73 (3%) 86 (6%) 76 (8%) 91 (10%) Pirmiphos-ethyl 69 (4%) 86 (8%) 77 (10%) 83 (5%) 17

18 Table 4. Recovery Data; Spiking Level of 50 ng/g (%RSD, n=3) (contd.) Isophenfos-methyl 68 (4%) 85 (7%) 69 (9%) 90 (9%) Phosfolan Isophenfos 76 (6%) 87 (8%) 72 (10%) 86 (3%) Quinalphos Phenthoate 69 (6%) 83 (9%) 74 (9%) 81 (4%) Methidathion 76 (4%) 91 (8%) 68 (17%) 86 (6%) Bromophos-ethyl 63 (10%) 85 (8%) 80 (8%) 84 (9%) Iodofenphos 69 (4%) 88 (6%) 78 (10%) 81 (8%) Profenfos 72 (3%) 92 (7%) 77 (11%) 82 (6%) Fensulfothion 79 (2%) 92 (9%) 72 (8%) 87 (5%) Ethion 73 (1%) 93 (8%) 79 (6%) 88 (4%) Famphur 74 (5%) 90 (7%) 61 (9%) 87 (5%) Edinphos 71 (1%) 90 (8%) 67 (9%) 92 (2%) Pyridaphenthion 76 (2%) 88 (6%) 65 (23%) 83 (1%) Phosmet 74 (5%) 94 (8%) 55 (20%) 87 (4%) EPN 74 (2%) 90 (8%) 73 (19%) 92 (6%) Phosalone 76 (2%) 98 (10%) 72 (9%) 92 (5%) Azinphos-methyl 70 (6%) 94 (6%) 45 (12%) 93 (3%) Azinphos-ethyl 75 (2%) 89 (12%) 67 (11%) 85 (3%) Coumaphos 72 (8%) 94 (12%) 69 (8%) 88 (6%) Temephos 67 (16%) 85 (2%) ND 102 (6%) Avg %RSD 5% 8% 12% 6% 18 Note: Coeluting pesticides have only one recovery value.

19 As illustrated in Figure 3 for mushrooms, using acetonitrile as the extraction solvent did not affect detection using NPD; the acetonitrile was completely evaporated in the final step. Coeluting pesticides were reported as a single recovery for both peaks. Recovery values for some pesticides were matrix dependent (shown in Table 4, e.g. trichlorphan, famphur, temephos). Green onions showed the highest variability in recoveries. CUSTM-MADE dspe extraction tubes with required salts (from Supelco) saved analysis time in sample preparation. 19

20 Conclusion GC analysis coupled with Nitrogen-Phosphorous detection could selectively detect organophosphorous pesticides with low background in comparison to MS detection. The clean-up protocol using acetonitrile/mgs 4 extraction and dual layer carbon/psa SPE cleanup resulted in acceptable recoveries for a majority of the pesticides studied. The recoveries were improved by optimizing the toluene content of the SPE elution solvent to 35% toluene-65% acetone. The recovery of organophosphorous pesticides was found to be matrixdependent; in onion and cabbage samples the recoveries were lower than in apple and button mushroom samples. 20