DETERMINATION OF IMAZALIL AND BENZIMIDAZOLE RESIDUES IN PROCESSED

DETERMINATION OF IMAZALIL AND BENZIMIDAZOLE RESIDUES IN PROCESSED FRUIT PRODUCTS BY LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY Luciana Bolzoni, Anna Sannino, Mirella Bandini SSICA – Stazione Sperimentale per l’Industria delle Conserve Alimentari Experimental Station for Preserved Food Industry Viale F. Tanara, 31/A 43100 Parma, Italy tel. 0521 7951 e-mail: ssicapr@rsadvnet. it Introduction Imazalil and benzimidazoles are systemic fungicides very important in monitoring programs because they are used either as preharvest or postharvest treatment for the control of a wide range of fruit and vegetable pests. Several LC methods have been reported for the determination of carbendazim (MBC), thiophanate methyl (TM) and thiabendazole (TBZ) including one described by A. Sannino (1, 2). A LC-mass spectrometric method was described for the determination of benzimidazole residues in fruits and vegetables (3). GC methods reported in the literature for Imazalil are not always easy to reproduce (4, 5). LC methods with UV or photodiode -array detection did not reach quantitation limits low enough to be used for the analysis of baby foods or organic products (6). A LC-mass spectrometric method was reported for imazalil in oranges (7). In this work extraction and clean up were the same as those of above-mentioned method (1). The instrumental determination was obtained by liquid chromatography-atmospheric pressure ionization-tandem mass spectrometry with electrospray (ES) interface. The results of Imazalil recoveries from peach and citrus juice are shown in the table. Mean results at 3 fortification levels were higher than 72% with coefficients of variation lower than 14%. The quantitation limit was 0. 001 mg/kg for carbendazim and thiabendazole; 0. 005 mg/kg for imazalil and 0. 010 mg/kg for thiophanate methyl. The following mass chromatograms show imazalil transitions (297 159 and 299 161) for both unfortified and fortified ( 0. 005 mg/kg ) lemon juices. S/N=17. 6 UNFORTIFIED Material and methods FORTIFIED WITH IMAZALIL AT 5 g/Kg Reagents and standards Solvents suitable for residues and HPLC analysis and Extrelut 20 columns were obtained from Merck (Darmstad, Germany). Fungicide standards included d 3 MBC were obtained from S. I. Ehrenstorfer (Augsburg, Germany) through Lab. Service (Bologna Italy). m/z 297 m/z 159 LEMON JUICE Apparatus -Homogeniser Ultra-Turrax T 25 -Vacuum rotary evaporator Buchi Rotavapor Model R 110 (Buchi, Flawil, Switzerland) -Liquid chromatograph HP series 1100 (Agilent Technologies, Germany) -Zorbax Eclipse XDB-C 8 column 4. 6 mm ID x 150 mm (5µm) (Agilent Technologies, Germany) -Mass spectrometer triple quadrupole API 2000 Applied Biosystems/MDS Sciex (CA, USA) S/N=15. 8 m/z 299 m/z 161 Homogeneus sample Mass Spectrometry API 2000 was used with Ion source Turbo Ion spray (ESI). General parameter: Ion polarity positive; Curtain gas 35 psi; Collision gas 5 units; Ion spray voltage 5500 V; Gas 1 pressure 40 psi; gas 2 pressure 40 psi; Temperature gas 2 300ºC; Resolution MS 1 unit; Resolution MS 2 unit; Dwell time 500 ms. Parameter of transition 192 160 Carbendazim FP 375 V; EP -8 V; DP 34 V; CE 22 V; CXP 7 V. Parameter of transition 195 163 d 3 Carbendazim FP 375 V; EP -8 V; DP 34 V; CE 22 V; CXP 7 V Parameter of transition 202 131 Thiabendazole FP 390 V; EP -10 V; DP 32 V; CE 45 V; CXP 22 V Parameter of transition 297 159 Imazalil FP 375 V; EP -7 V; DP 28 V; CE 34 V; CXP 22 V Parameter of transition 299 161 Imazalil FP 370 V; EP -10 V; DP 28 V; CE 35 V; CXP 15 Parameter of transition 343 151 Thiphanate methyl FP 385 V; EP -11 V; DP 18 V; CE 25 V; CXP 25 V Extraction with HCL 0. 02 N/Me. OH Centrifugation-filtration adjusting p. H to 7. 5 Partitioning into CH 2 Cl 2 on Extrelut 20 Liquid chromatography and tandem mass spectrometry allowed higher sensitivity to be obtained with lower quantitation limits as compared to previously published methods. Another important advantage of this technique is its selectivity allowing analysis of complex matrixes difficult to be analyzed by HPLC and conventional detectors. The other mass chromatograms were obtained for thiophanate methyl (transition 343 151) from both unfortified and fortified (0. 010 mg/kg) orange juices. By the previous method TM quantitation was precluded in citrus samples because of a peak interfering with the active compound. UNFORTIFIED WITH TM 10 g/Kg ORANGE JUICE S/N= 29 Analysis of methanolic extract (1 g sample/ml) m/z 343 m/z 151 by HPLC/MS/MS Table. Recoveries % of Imazalil, (C. V. %)* Spike level, mg/kg 0. 005 0. 50 Peach juice 82. 3 (6. 9) 85. 5 (11. 0) 81. 7 (5. 9) Orange juice 72. 7 (13. 7) 82. 3 (6. 6) 79. 4 (9. 0) Conclusions *Each value is the mean of 5 -6 determinations The proposed method allows simultaneous determination of carbendazim (MBC), thiophanate methyl (TM) and thiabendazole (TBZ) and imazalil in fruit juices with high selectivity and sensitivity. Results and Discussion References Good linearity (r 2 > 0. 995) was obtained in the range 0. 005 -2. 5 mg /l for the 4 pesticides ( 6 concentration levels). Tests were performed to determine whethere was a difference between the response of matrix-matched standards and standards in solvent. For the extracts from fresh products containing 1 g sample/ ml no significant differences were observed. However, we used d 3 MBC as internal standard added at the beginning of sample preparation because allowed us to better control the whole procedure. (1) A. Sannino, (1995), Food Chemistry 52, 57 -61 (2) M. Hiemstra, J. A. Josten, and A. De Kok, (1995), Journal of AOAC International 78, 1267 -1273 (3) A. R. Fernandez-Alba, A. Tejedor, A. Aguera, (2000), Journal of A. O. A. C. International, 83, 748 -755 (4) Y. Yamazaki, T. Ninimiya, (1996) Journal of A. O. A. C. International 79, 787 -790 (5) M. L. Lopez, M. Riba, (1999), J. Agric. Food Chem, 47, 3228 -3236 (6) T. Ohura, A. Yamamoto, S. Kodama, A. Matsunaga, (1998), J. of the Food Hygienic Society of Japan 39, 426 -430 (7) M. Fernandez, Y. Pico and J. Manes, (2001), Food and Contaminants, 18, 615 -624