Analysis of Pesticides in Oranges by QuEChERS...
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Food & Beverage Although pesticides have proven to be invaluable in crop production, the same toxicity, stability and mobility that enables them to effectively kill insects causes detrimental effects in humans. The importance of pesticide residue analysis led to the development of the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) sample preparation approach by Anastassiades and Lehotay, a dispersive SPE method utilizing bulk SPE material for sample cleanup. 1,2 Agencies in both the U.S.A. and Europe have subsequently established methods. 3,4 Several materials are available as QuEChERS cleanup sorbents, each effective for the removal of specific interference types. These sorbents are summarized in Table 1. They can be used individually or in combinations best suited to the sample matrix. Z-Sep (the Supelco® product name for zirconia on silica) is a new sorbent that enhances sample cleanup for complex matrices. Two versions are offered: • Z-Sep/C18, a blend of two separate sorbents (zirconia on silica and C18 on silica), is recommended as a cleanup sorbent to remove pigments from extracts of samples containing less than 15% fat • Z-Sep+, a hybrid material (zirconia and C18 are on the same silica particles), is recommended as a cleanup sorbent to remove fats from extracts of samples containing greater than 15% fat Table 1. QuEChERS Cleanup Sorbents and Uses Sorbent For the Removal of: Zirconia on silica Pigments PSA on silica Sugars, organic acids, fatty acids and polar pigments C18 on silica Fats Carbon Pigments Experimental One unspiked and three spiked replicate samples of pureed orange (homogenized with rind) were prepared. Following extraction, each of the four extracts was split into three aliquots. Each aliquot was then processed using a different QuEChERS cleanup sorbent (Z-Sep/C18, PSA/C18, or PSA), resulting in a total of twelve extracts. Calibration standards were prepared in acetonitrile:water (1:1) and were analyzed using LC-MS/MS along with the twelve extracts. Complete sample preparation and cleanup procedures for a spiked replicate using Z-Sep/C18 sorbent can be found in Figure 1. Using Supel™ QuE Z-Sep/C18 QuEChERS and an Ascentis® Express C18 HPLC Column Increased Pigment Removal with Analysis of Pesticides in Oranges Figure 1. LC-MS/MS MRM Transition Chromatograms of a Spiked Orange Extract sample/matrix: 10 g of pureed oranges (homogenized with rind); spike at 50 ppb (add 16.75 µL of a custom made pesticide mix, each analyte at 30 µg/mL) extraction: Add 10 mL acetonitrile; shake for 1 minute; add contents of a Supel QuE citrate extraction tube (55227-U); shake immediately for 1 minute; centrifuge at 3200 rpm for 5 minutes; transfer 0.7 mL of the acetonitrile layer into a Supel QuE Z-Sep/C18 cleanup tube (55284-U); shake for 1 minute; centrifuge at 5000 rpm for 5 minutes; transfer 0.2 mL of the supernatant into an empty 1.5 mL centrifuge tube; add 0.2 mL of water; centrifuge at 5000 rpm for 2 minutes column: Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 µm particles (53822-U) mobile phase: (A) 10 mM ammonium acetate in water (B) 10 mM ammonium acetate in acetonitrile gradient: Hold at 30% B for 1 min; 30% to 80% B in 2 min; hold at 80% B for 4 min; hold at 100% B for 3 min; hold at 30% B for 3 min flow rate: 0.3 mL/min pressure: 2730 psi column temp.: 30 °C detector: MS/MS, ESI positive 2 4 6 8 10 Min 1. Methomyl (0.60 min) 2. Trichlorfon (1.22 min) 3. Carbendazim (1.94 min) 4. Aldicarb (2.70 min) 5. Parathion-methyl (5.87 min) 6. Methabenzthiazuron (6.16 min) 7. Naled (6.37 min) 8. Methidathion (6.38 min) 9. Clethodim (6.42 min) 10. Phosmet (6.50 min) 11. Ametryn (6.58 min) 12. Sethoxydim (6.61 min) 13. Anilazine (6.78 min) 14. Fenxehamid (6.94 min) 15. Mecarbam (7.00 min) 16. Oryzalin (7.06 min) 17. Diflunezuron (7.18 min) 18. Fenoxycarb (7.19 min) 19. Iprobenfos (7.21 min) 20. Quinalphos (7.23 min) 21. Edifenphos (7.28 min) 22. Etrimfos (7.33 min) 23. Fenthion (7.34 min) 24. Fenitrothion (7.36 min) 25. Diazinon (7.37 min) 26. Tolclofos (7.52 min) 27. Phorate (7.53 min) 28. Chlorpyrifos-methyl (7.67 min) 29. EPN (7.68 min) 30. Terbufos (8.14 min) 31. Ethion (8.36 min) 32. Lufenuron (8.40 min) 33. Spiromesifen (8.74 min) 34. Octhilinone (8.74 min) 35. Pyraclostrobin (8.75 min) 36. Carbophenothion (8.83 min) 37. Flufenoxuron (9.02 min) 38. Fenpyroximate (9.10 min)
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Transcript of Analysis of Pesticides in Oranges by QuEChERS...
Food & Beverage
Although pesticides have proven to be invaluable in crop production, the same toxicity, stability and mobility that enables them to effectively kill insects causes detrimental effects in humans. The importance of pesticide residue analysis led to the development of the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) sample preparation approach by Anastassiades and Lehotay, a dispersive SPE method utilizing bulk SPE material for sample cleanup.1,2 Agencies in both the U.S.A. and Europe have subsequently established methods.3,4
Several materials are available as QuEChERS cleanup sorbents, each effective for the removal of specific interference types. These sorbents are summarized in Table 1. They can be used individually or in combinations best suited to the sample matrix. Z-Sep (the Supelco® product name for zirconia on silica) is a new sorbent that enhances sample cleanup for complex matrices. Two versions are offered:
•Z-Sep/C18, a blend of two separate sorbents (zirconia on silica and C18 on silica), is recommended as a cleanup sorbent to remove pigments from extracts of samples containing less than 15% fat
•Z-Sep+, a hybrid material (zirconia and C18 are on the same silica particles), is recommended as a cleanup sorbent to remove fats from extracts of samples containing greater than 15% fat
Table 1. QuEChERS Cleanup Sorbents and Uses
Sorbent For the Removal of:Zirconia on silica Pigments
PSA on silicaSugars, organic acids, fatty acids and polar pigments
C18 on silica Fats
Carbon Pigments
ExperimentalOne unspiked and three spiked replicate samples of pureed orange (homogenized with rind) were prepared. Following extraction, each of the four extracts was split into three aliquots. Each aliquot was then processed using a different QuEChERS cleanup sorbent (Z-Sep/C18, PSA/C18, or PSA), resulting in a total of twelve extracts. Calibration standards were prepared in acetonitrile:water (1:1) and were analyzed using LC-MS/MS along with the twelve extracts. Complete sample preparation and cleanup procedures for a spiked replicate using Z-Sep/C18 sorbent can be found in Figure 1.
Using Supel™ QuE Z-Sep/C18 QuEChERS and an Ascentis® Express C18 HPLC Column
Increased Pigment Removal with Analysis of Pesticides in Oranges
Figure 1. LC-MS/MS MRM Transition Chromatograms of a Spiked Orange Extract sample/matrix: 10 g of pureed oranges (homogenized with rind); spike at
50 ppb (add 16.75 µL of a custom made pesticide mix, each analyte at 30 µg/mL)
extraction: Add 10 mL acetonitrile; shake for 1 minute; add contents of a Supel QuE citrate extraction tube (55227-U); shake immediately for 1 minute; centrifuge at 3200 rpm for 5 minutes; transfer 0.7 mL of the acetonitrile layer into a Supel QuE Z-Sep/C18 cleanup tube (55284-U); shake for 1 minute; centrifuge at 5000 rpm for 5 minutes; transfer 0.2 mL of the supernatant into an empty 1.5 mL centrifuge tube; add 0.2 mL of water; centrifuge at 5000 rpm for 2 minutes
column: Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 µm particles (53822-U) mobile phase: (A) 10 mM ammonium acetate in water
(B) 10 mM ammonium acetate in acetonitrile gradient: Hold at 30% B for 1 min; 30% to 80% B in 2 min; hold at 80% B
for 4 min; hold at 100% B for 3 min; hold at 30% B for 3 min flow rate: 0.3 mL/min pressure: 2730 psi column temp.: 30 °C detector: MS/MS, ESI positive
2 4 6 8 10Min
1. Methomyl (0.60 min)2. Trichlorfon (1.22 min)3. Carbendazim (1.94 min)4. Aldicarb (2.70 min)5. Parathion-methyl (5.87 min)6. Methabenzthiazuron (6.16 min)7. Naled (6.37 min)8. Methidathion (6.38 min)9. Clethodim (6.42 min)
10. Phosmet (6.50 min)11. Ametryn (6.58 min)12. Sethoxydim (6.61 min)13. Anilazine (6.78 min)14. Fenxehamid (6.94 min)15. Mecarbam (7.00 min)16. Oryzalin (7.06 min)
17. Diflunezuron (7.18 min)18. Fenoxycarb (7.19 min)19. Iprobenfos (7.21 min)20. Quinalphos (7.23 min)21. Edifenphos (7.28 min)22. Etrimfos (7.33 min)23. Fenthion (7.34 min)24. Fenitrothion (7.36 min)25. Diazinon (7.37 min)26. Tolclofos (7.52 min)27. Phorate (7.53 min)28. Chlorpyrifos-methyl (7.67 min)
29. EPN (7.68 min)30. Terbufos (8.14 min)31. Ethion (8.36 min)32. Lufenuron (8.40 min)33. Spiromesifen (8.74 min)34. Octhilinone (8.74 min)35. Pyraclostrobin (8.75 min)36. Carbophenothion (8.83 min)37. Flufenoxuron (9.02 min)38. Fenpyroximate (9.10 min)
Food & Beverage
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©2012 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Solutions within is a trademark of Sigma-Aldrich Co. LLC. CHROMASOLV is a registered trademark of Sigma-Aldrich Laborchemikalien GmbH. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC. ENVI-Carb and Supel are trademarks of Sigma-Aldrich Co. LLC. Supelco brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.
OQY11410-513821 / T412073
1082
Featured and Related ProductsDescription Qty. Cat. No.Supel™ QuE QuEChERS ProductsAcetate Extraction Tube, 12 mL 50 55234-UCitrate Extraction Tube, 12 mL 50 55227-UZ-Sep/C18 Cleanup Tube, 2 mL 100 55284-UZ-Sep+ Cleanup Tube, 12 mL 50 55296-UPSA/C18/ENVI-Carb™ Cleanup Tube, 12 mL 50 55286-UPSA/C18 Cleanup Tube, 12 mL 50 55229-UPSA/C18 Cleanup Tube, 2 mL 100 55288-UPSA/ENVI-Carb Cleanup Tube 1, 12 mL 50 55230-UPSA Cleanup Tube, 12 mL 50 55228-UPSA Cleanup Tube, 2 mL 100 55287-UEmpty Centrifuge Tube, 50 mL 50 55248-UAscentis Express HPLC Columns, 2.7 µm particlesC18, 5 cm x 2.1 mm I.D. 1 53822-UC18, 15 cm x 4.6 mm I.D. 1 53829-URP-Amide, 15 cm x 4.6 mm I.D. 1 53931-UPhenyl-Hexyl, 15 cm x 4.6 mm I.D. 1 53353-UF5, 15 cm x 4.6 mm I.D. 1 53591-UAnalytical Reagents and SolventsAmmonium acetate, for HPLC, >99.0% 17836Acetonitrile, LC-MS CHROMASOLV®, >99.9% 34967
Results and DiscussionThe visual comparison of extracts shown in Figure 2 illustrates more thorough removal of color (pigment compounds) when using Z-Sep/C18 sorbent as opposed to PSA sorbent. In addition to reducing matrix interferences, the improved cleanup can decrease column and instrument fouling, leading to extended LC column lifetime and reduced instrument downtime. The use of an Ascentis® Express C18 column yielded the necessary peak efficiencies to obtain sufficient resolution and elution of all 38 analytes in less than 10 minutes, minimizing analysis time and, thus, increasing throughput.
Figure 2. Visual Comparison of Extracts after Cleanup
Z-Sep/C18 PSA
More color (pigment) removed
Overall, the three cleanup sorbents produced similar recovery values (an average of 92%) among the majority of pesticides tested, indicating that the Z-Sep material does not adversely bind any of the tested analytes. The Z-Sep/C18 sorbent provided recoveries superior to that of PSA/C18 and PSA sorbents for two of the pesticides tested (anilazine and naled). For a few pesticides (sethoxydim, clethodim and edifenphos), matrix effects showed increased ion suppression in samples cleaned with PSA sorbent. In addition, carbophenothion exhibited more matrix enhancement with the PSA sorbent than with the Z-Sep/C18 sorbent.
References1. Anastassiades, M; Lehotay, S. J.; Stajhbaher, D.; Schenck, F. J. Fast and easy
multiresidue method employing acetonitrile extraction/partitioning and “Dispersive Solid-Phase Extraction” for the determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412-431.
2. Koesukwiwat, U.; Lehotay, S. J.; Miao, S.; Leepipatpiboon, N. High throughput analysis of 150 pesticides in fruits and vegetables using QuEChERS and low-pressure gas chromatography-time-of-flight mass spectrometry. J. Chromatogr A. 2010, 1217, 6692-6703.
3. AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate.
4. EN15662:2008, Foods of plant origin – Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and cleanup by dispersive SPE – QuEChERS-method.
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