228 Vol. 56, No. 5 7 27 5 29 * Rapid Determination of Seven Fungicides in Citrus Fruits Naoki Yoshioka*, Sachiko Hayashi and Tadaaki Inada Hyogo Prefectural Institute of Public Health and Consumer Sciences: 2 1 29 Arata-cho, Hyogo-ku, Kobe 652 0032, Japan; * Corresponding author A rapid and simple determination method of seven fungicides, thiabendazole TBZ, pyrimethanil PYR, o-phenylphenol OPP, fludioxonil FLD, azoxystrobin AZX, imazalil IMZ and diphenyl DP in citrus fruits by LC-MS and HPLC-FL was developed. The seven fungicides were extracted with acetonitrile from citrus fruits and cleaned up with Z-Sep/C18 cartridges. The LC separation was performed on a phenyl-hexyl column with methanol acetonitrile 10 mmol/l ammonium formate 10 : 35 : 55 as a mobile phase. The recoveries from citrus fruits fortified with the compounds at the MRLs and at 0.1 μg/g ranged from 85.4 to 106.3 and from 75.8 to 99.7, respectively. The quantitation limits S/N 10 were 0.03 0.07 μg/g. Received May 29, 2015 Key words citrus fruit; fungicide; - LC- MS; HPLC-FL 4 4 23 25 Fig. 1 1 HPLC-UV HPLC-FL 4 APPI -LC- MS 2 3 7 LC-MS HPLC-FL * Naoki_Yoshioka@pref.hyogo.lg.jp 652 0032 2 1 29 1 2 OPP AZX IMZ DP Fig 1 Chemical structures of the fungicides
October 2015 7 229 TBZ PYRFLD DP 2,000 μg/ml 500 μg/ml 50 μg/ml 30 7- -3-1- -2-200 μg/ml 1 : 10.5 μg/ml Z-Sep/C18 Z-Sep: 20 mg, C18: 50 mg/ 2 ml Z-Sep 75 mg/2 ml -LH 0.45 μm, PTFE, 4 mm Inertsil Amide 2.1 mm 250 mm, 5 μm Ascentis Express F5 2.1 mm 100 mm, 2.7 μm, Waters XBridge Phenyl 3.0 mm 150 mm, 3.5 μm XBridge C18 4.6 mm 150 mm, 3.5 μm 3 LC-MS Agilent 1200 Series LC 6210 TOF- MSD HPLC LC-10A RF-10A XL MK-K58 IKA T 25 digital ULTRA-TURRAX SR-2W 8700 SCT15B 4 4 1 LC-MS Waters XBridge Phenyl 3.0 mm 150 mm, 3.5 μm 10 mmol/l 10 : 35 : 55 0.35 ml/min 40 1 μl ESI, 2,000 V 40 psi 13 L/min 350 1 9.5 175 275 V Positive 9.5 11.5 175 275 V Negative 11.5 25 175 250 V Positive m/z 40 1,050 121.0509 922.0098 Positive, 112.9856 966.0007 Negative 4 2 HPLC Waters XBridge Phenyl 4.6 mm 150 mm, 3.5 μm 10 mmol/l 10 : 35 : 55 0.8 ml/min 40 10 μl 0 15 290 nm 345 nm 15 20.5 413 nm 478 nm 20.5 25 254 nm 315 nm 5 10 g 50 ml 30 200 μg/ml 50 μl 5 mol/l 1 ml 10 ml 1 4 g 1 g3 1,600 g 5 0.7 ml Z-Sep/C18 1 5,500 g 3 0.4 ml 0.4 ml LC-MS TBZ, PYR, FLD, AZX, IMZ HPLC-FL OPP, DP 1 OPP DP LC-MS ESI * 1 OPP DP HPLC-FL LC-MS TBZ C18 3 HILIC PFP pentafluorophenylpropyl, F5 C18 7 Table 1 175 V 250 V 275 V * 1 106 p. 124 2013.
230 Vol. 56, No. 5 Table 1 Optimized MS parameters of the fungicides 1.0 9.5 min 9.5 11.5 min 11.5 25.0 min TBZ PYR FLD AZX IMZ Target Qualifier Target Qualifier Target Qualifier Target Qualifier Target Qualifier Ion mode Positive Positive Negative Positive Positive Fragmentor voltage 175 275 175 275 175 275 175 250 175 250 V Elemental composition C 10 H 8 N 3 S C 9 H 7 N 2 S C 12 H 14 N 3 C 6 H 7 N 2 C 12 H 5 N 2 O 2 F 2 C 11 H 4 N 2 O C 22 H 18 N 3 O 5 C 21 H 14 N 3 O 4 C 14 H 15 N 2 OCl 2 C 7 H 5 Cl 2 Calculated mass 202.0433 175.0324 200.1182 107.0604 247.0325 180.0329 404.1241 372.0979 297.0556 158.9763 Extracted ion 202.03 175.02 200.11 107.05 247.02 180.02 404.11 372.09 297.05 158.97 range for quantitation 202.05 175.04 200.13 107.07 247.04 180.04 404.13 372.11 297.07 158.99 Table 2 Effect of ph of the extraction solvents on recoveries of the fungicides fortified at 1 μg/g ph Recovery TBZ PYR OPP FLD AZX IMZ DP ph 4 72.8 1.4 88.5 2.6 94.9 0.5 100.3 0.4 98.3 2.2 98.1 3.0 92.0 1.3 ph 7 88.3 0.4 85.7 0.5 94.8 2.7 99.1 0.5 97.3 4.2 93.4 1.9 91.0 1.6 ph 11 89.0 0.9 86.6 1.2 94.3 3.5 98.2 0.8 97.0 3.5 91.0 3.4 91.0 1.9 n 3 OPP 0.05 10 μg/ml DP 0.01 10 μg/ml 0.005 10 μg/ml r 2 0.999 2. QuEChERS 4 pk a 7 ph 10 g1 μg/g 5 mol/l ph 4 ph 7 ph 11 5 Table 2 ph 4 TBZ73 85 ph ph 3 ph 4 ph 7 ph 11 ph 7 ph 11 ph ph ph 11 5 mol/l 1 ml 3 SCX 3 5 * 2 2Z-Sep/C18 Z-Sep 2 Z-Sep Z-Sep TBZ IMZ 40 60 Z-Sep/C18 4 TBZ 2-2-2-7 3-2- -7-30 7- -3-1- -2- * 2 106 p. 102 2013.
October 2015 7 231 LC-MS ESI HPLC 2-2- IMZ DP 2-7 30 DP 5 TBZ, PYR, OPP, FLD, AZX 10 μg/g IMZ 5 μg/g DP 70 μg/g 5Table 3 0.1 μg/g Table 4 Fig. 2 LC-MSFig. 3 HPLC-FL 87.0 98.3, 89.6 106.1, 85.4 106.3 RSD 1.3 5.7, 3.1 4.7, 1.8 5.5 0.1 μg/g 87.9 97.9, 82.8 97.0, 75.8 99.7 RSD 1.7 5.0, 1.2 5.0, 1.9 6.4 S/N 10 FLD 0.03 μg/g PYR 0.04 μg/g, TBZ, AZX, IMZ 0.05 μg/g, OPP 0.06 μg/g DP 0.07 μg/g 7 TBZ, PYR, FLD, AZX IMZ LC-MS OPP DP HPLC-FL IMZ: 5 μg/g, DP: 70 μg/g 10 μg/g85.4 106.3 0.1 μg/g75.8 99.7 0.03 0.07 μg/g 109 2015 5 Table 3 Recoveries of the fungicides from citrus fruits fortified at the MRLs Lemon Orange Grapefruit Fortification level μg/g Recovery RSD Recovery RSD Recovery RSD TBZ 10 94.4 1.3 92.9 3.8 85.4 5.2 PYR 10 92.6 1.5 89.6 3.7 100.8 4.5 OPP 10 98.3 2.5 106.1 4.7 106.3 2.7 FLD 10 96.9 4.2 102.3 4.1 103.0 3.5 AZX 10 87.0 5.7 92.6 3.2 94.1 5.5 IMZ 5 91.0 1.3 93.6 3.1 95.1 4.8 DP 70 87.8 3.3 98.5 3.1 99.9 1.8 n 5 Table 4 Recoveries of the fungicides from citrus fruits fortified at 0.1 μg/g Lemon Orange Grapefruit Fortification level μg/g Recovery RSD Recovery RSD Recovery RSD TBZ 0.1 88.0 1.8 91.2 1.8 75.8 2.3 PYR 0.1 94.9 4.9 82.8 4.1 95.6 2.6 OPP 0.1 96.4 1.8 92.5 5.0 91.3 1.9 FLD 0.1 94.1 4.1 97.0 1.2 97.1 3.3 AZX 0.1 97.9 4.3 90.6 3.7 93.8 6.4 IMZ 0.1 87.9 1.7 94.8 3.4 92.9 5.0 DP 0.1 90.5 5.0 93.2 4.0 99.7 2.2 n 5
232 Vol. 56, No. 5 Fig 2 Typical extracted ion chromatograms obtained from A lemon fortified with 1 TBZ, 2 PYR, 3 FLD, 4 AZX, 5 IMZ at 0.1 μg/g, B blank lemon sample, and C 0.05 μg/ml standard solution by LC-MS Fig 3 Typical chromatograms obtained from A lemon fortified with 1 OPP and 2 DP at 0.1 μg/g, B blank lemon sample, and C 0.05 μg/ml standard solution by HPLC-FL 1 2003, p. 112 126. 2 Yoshioka, N., Akiyama, Y., Teranishi, K. Rapid simultaneous determination of o-phenylphenol, diphenyl, thiabendazole, imazalil and its major metabolite in citrus fruits by liquid chromatography-mass spectrometry using atmospheric pressure photoionization. J. Chromatogr. A, 1022, 145 150 2004. 3 Ozawa, H., Hirokado, M., Shimamura, Y., Kimura, K., Saito, K. A simple and systematic determination of fungicides in citrus fruits, bananas and fruit juice concentrates by solid phase extraction method. Tokyo-Toritsu Eisei Kenkyusho Kenkyu Nenpo Annual Report of Tokyo Metropolitan Research Laboratory of Public Health, 52, 78 83 2001. 4 Anastassiades, M., Lehotay, S. J., Stajnbaher, 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., 86, 412 431 2003. 5 Takahashi, K., Horie, M., Hirose, Y. Determination of diphenyl and o-phenylphenol in agricultural products by GC/MS. Shokuhin Eiseigaku Zasshi J. Food Hyg. Soc. Jpn., 49, 403 406 2008.