sign in sign up
<<
Home , Coumaphos, Ethion, Ethoprophos, Monocrotophos, Phenthoate, Phorate

Method for Analysis of Organophosphorous Pesticides in Produce using Acetonitrile Extraction, Dual-Layer -Aminopropylsilica Cleanup and GC-NPD Detection

•Olga Shimelis, Katherine Stenerson, Michael Ye, Carmen T. Santasania, and Ed Mauney Supelco, Div. of Sigma-Aldrich, Bellefonte, PA 16823 USA

www.sigma-aldrich.com

T410102 Abstract

Organophosphorous 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 contained 35% toluene and 65% acetonitrile for optimum recovery. Solvent exchange into ethyl acetate provided sample extracts with minimal background for GC-nitrogen- phosphorous detection (NPD). Using matrix-matched standards for quantitation, average recoveries for most organophosphorous pesticides, spiked at 10 ng/g, were in the 60-100% range with average standard deviations of 5% in cabbage and apples, 8% in mushroom, and 12% in green onions. 2 Experimental

Extraction method was based on Shimelis et al. in J. Chromat. A 1165(1-2),18-25. Sample preparation • 10 g homogenized food sample – green onions, apples, Napa cabbage, button mushrooms o • 10 mL acetonitrile and 4 g MgSO4 (heated at 550 C), 1 g NaCl – mixed, centrifuged

• Supernatant mixed with 1 g MgSO4, mixed, centrifuged • 5 mL of the resulting sample was evaporated down to 1 mL for SPE loading 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

3 addition of ethyl acetate Spiking levels • All produce was spiked at 10 ng/g. Calibration • Matrix-matched standards were used for calibration.

4 Figure 1. Examples of Structures of Organophosphorous Pesticides

O S

N O P O NO2 O P O

O O O Methyl Dichrotophos

S S O O P S S P O O P S O O S

5 Results

Optimization 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% . 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 Table 1. Optimization 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% 0% 80% 90%

NOTE: All samples were evaporated to dryness after SPE and reconstituted in hexane- acetone 1:1. Analysis was done by GC-MS.

Result: 10 mL elution volume was chosen.

7 Table 2. Optimization 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%

NOTE: 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.

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. 8 Figure 2. Analysis of Green Onion Extract, GC-NPD vs. GC-MS/SIM

3 5 GC-NPD 1 2 6 4

1. Ethoprophos 2. CIPC 3. 4. 4 5. 6. Thiometon GC-MS/SIM

2 5

3 6 1

9 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 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 enhance- ment 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 Table 3. Recovery Data; Matrix-Matched vs. Solvent Standards

Avg. % Recovery CabbageOnions (%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%) NOTE: All samples analyzed by GC-NPD

Result: Some matrix enhancement effect observed; matrix-matched standards used for quantitation.

12 Figure 3. GC-NPD Analysis of Spiked Mushroom Extract

39,40 38 34 44,45 46,47 35 42 33 3637 43 41

56 34 36 38 57 Time (min) 53

19,20 58 9 10 11 17 14,15 61 59 62 12 18 22 55 60 2 8 25,26 31,32 48 49 5254 16 29 63 3 21 23 28 6 13 30 51 7 27 50 24 1 5

4

10 20 30 40 50 Time (min)

13 Peak List for Figure 3

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

14 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 Table 4. Recovery Data; Spiking Level of 10 ng/g (%RSD, n=3)

Cabbage Mushrooms Onions Apples Avg. Avg. Avg. Avg. % Re cove ry % Re cove ry % Recovery % Re cove ry 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%) 70 (7%) 77 (11%) 68 (7%) 77 (2%) Trichlorphan 13 (24%) 61 (28%) 62 (20%) 47 (27%) Omethoate 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 16 Diazinon 64 (3%) 78 (9%) 59 (10%) 77 (4%) 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 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. • 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. • CUSTOM-MADE dSPE extraction tubes with required salts (from Supelco) saved analysis time in sample preparation.

19 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/MgSO4 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 matrix- dependent; in onion and cabbage samples the recoveries were lower than in apple and button mushroom samples.

20