1. 11 Determination of the Levels of Fungicides on Citrus Fruits By Miseung Borgers Chem 4101, Fall 2008

2. 22 Introduction Fungicides applied on the surface of post- harvest citrus fruits are possible health threats since they can be easily absorbed into the body through peeling and eating with hands. Controlling and monitoring the appropriate levels of fungicides would reduce the possible harmful effects by the ingestion.

3. 3 Maximum residue limits for five fungicides and their structures Benomyl10ppm Carbendazim0.5 ppm Imazalil10 ppm Thiabendazole10 ppm Thiophanate methyl 0.5 ppm (Ref. 1, 3)

4. 44 Method HPLC/MS - Since the fungicides have different polarities, they are retained to the stationary phase by different degrees. This allows separation by HPLC. - High sensitivity proved by the limit of detection (< 0.03 ppm) - No derivatization is needed - Fastness (No additional clean-up for sample preparation) - Highly automated (reduced manual labor and human intervention) (Ref. 2)

5. 5 Procedure Homogenization Extraction LC/MSD system www.thesun.co.uk www.buchi.com ic3db.ou.edu www.hhsc.ac.uk

6. 6 Preparation of standards, controls and samples (1) Homogenization - Blend chopped oranges with ethyl acetate, anhydrous sodium sulphate - For controls, use oranges without post harvest fungicide treatment (2) Extraction - Use sodium sulphate for filtering - Add acetonitrile, Dry - Dissolve in acetonitrile: 2 mM ammonium formate (50:50, v/v) - For standards, controls mixed with fungicide solutions - Standard Concentrations 0.05, 0.5, 5 ppm (Ref. 3)

7. 7 Conditions for HPLC Injection volume: 5 µL Solvents used for mobile phase: methanol, 2 mM ammonium formate in water Gradient elution with flow rate of 1 mL/min Methanol 50 % Methanol 80 % Methanol 30 % (Ref. 3)

8. 8 Conditions for interface and MS APCI Interface - Vaporizing temp. 325 ºC - Nebulizer gas – Nitrogen at 4.0 bar - Drying gas – Nitrogen at 4 L/min at 350 ºC - Capillary voltage –4000 V - Corona current – 4 µA - LOD 0.01 µg/mL except for benomyl at 0.1 µg/mL MS -Use SIM mode, fragmentor voltage 70 V (Ref. 3) Compoundm/z Carbendazim, benomyl 192 Thiabendazole202 Imazalil297 Thiophanate methyl 343

9. 9 Chromatogram A) Standard with concentration of 0.5 ppm, B) Control, C) Sample Peak identification: (1) Carbendazim, (2) thiabendazole, (3) thiophanate methyl, (4) benomyl, and (5) imazalil (Ref. 3)

10. 10 Results and Conclusions HPLC/MS provides a reliable, sensitive analytical tool in determining the levels of fungicides on citrus fruits. By monitoring the levels of fungicides, the possible health threats can be minimized. (Ref.3)

11. 11 Instrument Specifications HP-1100 Series LC/MSD - quadrupole based LC/MSD system - HPLC column- Zorbax Rapid Resolution SB-C 18 (30 x 4.6 mm I.D., 3.5 µm) column with a guard column of LiChrosorb RP-8 (10 x 4.6 mm, 5 µm) - standard API source that can be configured as APCI or ES (Ref. 3) - mass range 2-3000 m/z - mass accuracy ±0.13 u within the calibrated mass range in scan mode - mass axis stability ±0.13 u over 8 hours - scan speed 2500 u/s standard mode 5250 u/s fast scan mode - SIM sensitivity ESI at 400 µL/min APCI at 1000 µL/min - multiple signal acquisition: ability to cycle through four different acquisition modes on a scan-by-scan basis within a single run (Ref. 4)

12. 12 References 1) http://postharvest.ifas.ufl.edu/MRLs/FL%20Citrus%20 MRLs%20-%209.2.08.pdf accessed December 2 nd, 2008 2) http://cvuas.xn--untersuchungsmter-bwnzb.de/pdf/http://cvuas.xn--untersuchungsmter-bwnzb.de/pdf/ poster_israel1999_orgzinn.pdf accessed December 3 rd, 2008 3) Fernández, M.; Rodríguez, R.; Picó, Y.; Mañes, J. J. Chromatogr. A. 2001, 912, 301-310 4) http://www.gentechscientific.com/10156.htm accessedhttp://www.gentechscientific.com/10156.htm December 3 rd, 2008