Vol. 49, No. 5. Analysis of Chlorophenol Compounds Contaminating Food by LC/MS

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1 356 LC/MS Analysis of Chlorophenol Compounds Contaminating Food by LC/MS Rie IH=>> and Masakazu HDG>: Saitama Prefectural Institute of Public Health: Kamiokubo, Sakura-ku, Saitama , Japan; Corresponding author Asimple method using a liquid chromatograph with a mass spectrometer has been developed for the determination of chlorophenols in food. mass spectral acquisition was performed in the negative mode by applying sungle ion monitoring. In LC separation, Cadenza CD-C18 and 10 mmol/l ammonium acetate methanol were used as the column and mobile phase, respectively. Chlorophenols in food were extracted with methanol, and cleaned up with an Oasis HLB cartridge. The quantification limits were 0.2 ng/g for 4-chlorophenol, 2 ng/g for 2,6-dichlorophenol, 1 ng/g for 2,4-dichlorophenol and 0.5 ng/g for 2,4,6-trichlorophenol. The average rocoveries of chlorophenols from food at the spiked level of 20 ng/g were in the range of The relative standard deviations of all examined compounds were within 10. (Received April 21, 2008) Key words: chlorophenol; food; dichlorophenol; / LC/MS; mushroom 1), 2) 1), 2) 2,4- (2,4-DCP) 2) 2- (2-MCP) 4- (4- MCP) 2,4-DCP 2,6- (2,6-DCP) 1 2,4,6- (2,4,6-TCP) kwfjdn:1:basic 1 GC 3) LC 4) GC 5 2,4-DCP 2) 3 LC/MS 1. 2-MCP, 4-MCP, 2,6-DCP 2,4,6-TCP Dr. Ehrenstofer GmbH 2,4-DCP 50 mg

2 October ml 5 10 ml 2-10 mmol/l (1 : 1) 100 ml 2-10 mmol/l (1 : 1) Oasis HLB (200 mg) Waters 2-2. Waters Alliance 2695 Waters Quattro premier Organomation Associates, Inc. N-EVAP 3. LC/MS Table 1, g 10 ml 30 5 Table 1. LC-MS operating conditions Parameter LC parameters Analytical column Mobile phase Settings Cadenza CD-C18 ( mm, 3 mm) A water B methanol C 200 mmol/l ammonium acetate Time (min) A ( ) B ( ) C ( ) Flow rate 0.2 ml/min Column temperature 35 Injection volume 10 ml Run time 25 min AP interface parameters Ionization mode Electrospray ionization (negative mode) Capillary voltage 2.63 kv Source temperature 120 Desolvation temperature 400 Cone gas flow 49 L/hr Desolvation gas flow 900 L/hr MS parameters MS1 resolution 11.0 MS1 ion energy 0.1 MS2 resolution 11.0 MS2 ion energy 3.0 Multiplier 650 V AP atmospheric pressure Table 2. SIM parameters Analyte Time window (min) Monitored scan mass (m/z) Dwell time (s) Cone voltage (V) 2-MCP, 4-MCP b) ,6-DCP, 2,4-DCP ,4,6-TCP MCP 2-chlorophenol; 4-MCP 4-chlorophenol; 2,4-DCP 2,4-dichlorophenol; 2,6-DCP 2,6-dichlorophenol; 2,4,6-TCP 2,4,6-trichlorophenol b) Underlined ions are those used for quantitation; others were used for confirmation.

3 358 3, ml 80 ml 1 mol/l ph 2.0 3, Oasis HLB (200 mg) 5mL 10 5mL ml 500 ml 10 mmol/l 1mL ml LC/MS 1. LC/MS 1.1 MS [M H] MS Selected Ion Monitoring (SIM) 2-MCP ng/g 2,6-DCP 2,6-DCP 5) 1), 2) 2,4-DCP, 2,6-DCP 2,4,6-TCP 2-MCP 2-MCP 4 [M H] 20 MS Table 1, LC ODS mm 100 mm Cadenza CD-C18 Imtact L-column ODS Xbridge C18 Waters Mightysil RP-18 GP Cadenza CD-C18 MS 5, 10, 20 mmol/l 10 mmol/l Table MCP ng/ml ( ng), 2,6-DCP ng/ml (0.1 5ng), 2,4-DCP ng/ml ( ng) 2,4,6-TCP ng/ml ( ng) (r 0.999) MassLynx RMS S/N MCP 1ng/mL 0.2 ng/g 2,6- DCP 10 ng/ml (2 ng/g), 2,4-DCP 5 ng/ml (1 ng/g), 2,4,6-TCP 2.5 ng/ml (0.5 ng/g) 2,6-DCP 1 10 ng/g, 2,4- DCP ng/g, 2,4,6-TCP 1,000 10,000 ng/g 5) 1), 2) 2.3 n 5, GC 1), 3) LC/MS 4) SPME GC 2) GC

4 October GC/MS LC/MS 4), 6), 7), 2 Oasis HLB ph ,4-DCP 2,6-DCP 2,4-DCP 2,6-DCP Marchese 6), 8) (Fig. 1(B)) Fig. 1. SIM chromatograms of mixed standard solution (100 ng/g) (A), a blank mushroom sample (B), a mushroom sample spiked with each chlorophenol at 20 ng/g (C), a sample in which 4-MCP was detected at 2 ng/g, 2,6-DCP at 54 ng/g, 2,4- DCP at 14 ng/g (D). Arrow indicates the retention time of each chlorophenol. 20 ng/g Table ng/g Fig. 1(C) 5. 4-MCP 33 2,6-DCP 67 2,4-DCP 67 2,4,6-TCP EU 3 3 Method validation and quality control procedures for pesticide residues analysis in food and feed, Document No. SANCO/2007/3131, 31/October/2007, Supersedes Document No. SANCO/10232/2006, p. 18.

5 360 Table 3. Recoveries of chlorophenols from mushrooms, bamboo shoots and nozawanas Analyte Fortification concentration (ng/g) Recovery ( ) Mushroom Bamboo shoot Nozawana 4-MCP b) ,6-DCP ,4-DCP ,4,6-TCP MCP 4-chlorophenol; 2,6-DCP 2,6-dichlorophenol; 2,4-DCP 2,4-dichlorophenol; 2,4,6-TCP 2,4,6-trichlorophenol b) Average recovery standard deviation, n MCP (2 ng/g), 2,6-DCP (54 ng/g), 2,4-DCP (14 ng/g) (Fig. 1(D)) 5) ,6-DCP 2,6-DCP LC/MS 1. Oasis HLB 2. LC/MS ESI 10 mmol/l Cadenza CD-C MCP 0.2 ng/g, 2,6-DCP 2ng/g, 2,4-DCP 1ng/g, 2,4,6-TCP 0.5 ng/g 20 ng/g ) Ogiwara, T., Kondo, H., Miyakoda, M., Yamada, Y., Aoyagi, Y., Sato, H., Amakawa, E., Matsumoto, H., Kasuya, Y., Nakazato, M., Yasuda, K. A case study on complaint about glazed adzuki beans contaminated by chlorophenol compounds. Tokyoto Kenko Anzen Kenkyu Senta Kenkyu Nenpo (Annual Report of Tokyo Metropolitan Institute of Public Health), 54, (2003). 2) Tukioka, T. SPME-GC/MS Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 44, J-9 J-11 (2003). 3) Iida, K., Watanabe, S., Ikeda, H. Analysis for residue of chlorophenols in vegetables. Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 19, (1978). 4) Carabias-Mart&inez, R., Rodríguez-Gonzalo, E., Revilla- Ruiz, P. Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry study of background contamination. J. Chromatogr. A, 1137, (2006). 5) Fukaya, T., Ishiguro, Y., Yokoyama, M., Hiroi, S., Muraoka, A. Appearance of 2,6-dichlorophenol in carrot treated with sodium hypochlorite. Nippon Shokuhin Kogyo Gakkaishi (J. Food Sci. Technol.), 40, (1993). 6) Marchese, S., Gentili, A., Perret, D., Sergi, M., Notari, S. Hybrid quadrupole time-of-flight for the determination of chlorophenols in surface water by liquid chromatography-tandem mass spectrometry. Chromatographia, 59, (2004). 7) Carabias-Martínez, R., Rodríguez-Gonzalo, E., Revilla- Ruiz, P. Determination of weakly acidic endocrinedisrupting compounds by liquid chromatographymass spectrometry with post-column base addition. J. Chromatogr. A, 1056, (2004). 8) Marchese, S., Perret, D., Gentili, A., D Ascenzo, G., Faberi, A. Determination of phenoxyacid herbicides and their phenolic metabolites in surface and drinking water. Rapid Commun. Mass Spectrom., 16, (2002).