Improve Pigment Removal and Decrease Matrix Interferences for Pesticide Determination in Oranges Using Supel QuE Z-Sep/C18 QuEChERS Sorbent

By: Olga Shimelis, Emily Barrey, and Jennifer Claus, Reporter US Volume 30.3


Although pesticides have proven to be invaluable in crop control, the same toxicity, stability, and mobility that enables them to effectively kill insects causes detrimental effects in humans and the environment1. Worldwide pesticide use in modern agriculture has led to the manifestation of pesticide residues in the majority of fruits and vegetables. Because of the deleterious effects associated with pesticides, authorities have applied maximum residue limits (MRLs) on many types of produce for environmental and consumer health protection2. Implementation of these MRLs has increased pesticide residue analysis globally.

The current importance of pesticide residue analysis has caused scientists to seek robust, inexpensive, and straightforward methods that produce quality results, for an extensive range of analytes and matrices at low detection limits. The development of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) sample preparation approach by Anastassiades and Lehotay3,4,5, a dispersive SPE method utilizing bulk SPE material for sample cleanup, provides a method fitting the aforementioned criteria. Recently the use of Z-Sep (zirconia-coated silica) in combination with C18 was shown to enhance sample cleanup for complex matrices6. The comparison of Supel™ QuE Z- Sep/C18, PSA/C18, and PSA QuEChERS sorbents, in terms of color removal and analyte recovery, is described in this article for the cleanup of oranges prior to pesticide analysis.


An orange was homogenized with the rind, and 4 replicate samples of 10 grams were weighed into separate 50 mL centrifuge tubes. Three samples were spiked at 50 ng/g and the fourth was left unspiked. Ten mL of acetonitrile was added to each tube and the samples were shaken for 1 minute. Contents of citrate extraction tubes were added to each sample and the tubes were shaken immediately for an additional minute. The tubes were then centrifuged at 3200 rpm for 5 minutes. Separate 0.7 mL aliquots of the acetonitrile layer from each sample were then transferred into separate cleanup tubes, containing Z-Sep/C18, PSA/C18, or PSA sorbent. The resulting 12 tubes were shaken for 1 minute and centrifuged at 5000 rpm for 5 minutes. A 0.2 mL aliquot of the supernatant in each tube was then transferred into an empty 1.5 mL centrifuge tube. To each tube, 0.2 mL of water was added. Tubes were centrifuged again at 5000 rpm for 2 minutes. The resulting supernatant was then transferred to sample vials for analysis. Calibration standards were prepared in acetonitrile:water (1:1), and were analyzed using LC-MS/MS, along with the 12 prepared samples.

LC -MS/MS MRM Transition Chromatograms of a Spiked Orange Extract after Z-Sep/C18 Cleanup

Figure 1. LC -MS/MS MRM Transition Chromatograms of a Spiked Orange Extract after Z-Sep/C18 Cleanup

HPLC Conditions
Ascentis® Express C18, 5 cm x 2.1 mm I.D., 2.7 µm particles, (Product No. 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 ºC; detector: MS,/MS, ESI positive


  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)

Results and Discussion

The chromatogram in Figure 1 illustrates the LC-MS/MS analysis of 38 pesticides extracted from a spiked orange sample following sample cleanup with Z-Sep/C18 sorbent. In this experiment, use of an Ascentis Express C18 column yielded the necessary peak efficiencies to obtain sufficient resolution and elution of all 38 analytes in under 10 minutes; minimizing analysis time and increasing throughput. The visual comparison of extracts shown in Figure 2 illustrates that more thorough removal of color is achieved with Z-Sep/C18 sorbent, as opposed to PSA sorbent. In addition to reducing matrix interferences, the improved cleanup by Z-Sep/C18 sorbent can decrease column and instrument fouling, leading to an extended HPLC column life while reducing instrument downtime.

Visual Comparison of Extracts Cleaned using Z-Sep/ C18 Sorbent (left) and PSA Sorbent (right)

Figure 2. Visual Comparison of Extracts Cleaned using Z-Sep/ C18 Sorbent (left) and PSA Sorbent (right)


The bar chart in Figure 3 provides a visual comparison of selected analyte recovery after using Z-Sep/C18, PSA/C18, and PSA sorbents. Overall, the three methods produced similar recovery values for the majority of the pesticides tested. The Z-Sep/C18 sorbent provided recoveries superior for two of the pesticides tested, anilazine and naled, which were not detectable with PSA/C18 or PSA sorbents due to matrix effects. For a few pesticides, sethoxidim, 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.

Average Recovery of Selected Pesticides from Spiked Oranges (n=3)

Figure 3. Average Recovery of Selected Pesticides from Spiked Oranges (n=3)


Comparison of the color removal and analyte recovery for pesticide residue analysis in orange extracts, after the extracts underwent cleanup with Z-Sep/C18, PSA/C18, or PSA QuEChERS sorbent, was performed. For a number of the pesticides analyzed, results showed the Z-Sep/C18 sorbent was superior to that of PSA/C18 and PSA sorbents. Results for the remaining pesticides were similar to those observed with both PSA/C18 and PSA sorbents, indicating that Z-Sep does not adversely bind any of the tested analytes. Decreased matrix effects were also observed with Z-Sep/C18 sorbent as compared to PSA/C18 and PSA sorbents. Additionally, Z-Sep/C18 sorbent provided improved color removal over PSA sorbent. These observations support the fact that Supel™ QuE Z-Sep/C18 has the potential to be a direct replacement for C18 and PSA phases in current methods.

Legal Information

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC
Supel is a trademark of Sigma-Aldrich Co. LLC




  1. Fenik, J.; Tankiewicz, M.; Biziuk, M. Properties and determination of pesticides in fruits and vegetables. TrAC Trend Anal. Chem. 2011, 30, 814-826.
  2. USDA, Foreign Agricultural Service, Maximum Residue Limit Database. http://, (accessed 8-Mar-2012).
  3. Koesukwiwat, U.; Lehotay, S. J.; Miao, S.; Leepipatpiboon, N. High throughput analysis of 150 pesticides in fruits and vegetables using QuEChERS and lowpressure gas chromatography-time-of-flight mass spectrometry. J. Chromatogr A. 2010, 1217, 6692-6703.
  4. 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.
  5. Lehotay, S. J. Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) Approach for the Determination of Pesticide Residues. Proceedings AOAC Annual Meeting, St. Louis, MO USA, 2004.
  6. Shimelis, O. Analysis of Pesticides in Fruits and Vegetables Using Z-Sep and Z-Sep+ SPE Sorbents in QuEChERS Method for Sample Cleanup. Pittcon 2012 Oral Presentation, Orlando, FL USA, 2012.
  7. 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.
  8. AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate.


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