兵庫県立健康生活科学研究所健康科学研究センター研究報告 第 6 号 2015 [ノート] 輸入水産食品中の使用禁止抗菌剤, マラカイトグリーンの LC/TOF-MS による分析法の検討 服部 涼子 * 後藤 操 赤松 成基 吉岡 直樹 川元 達彦 稲田 忠明 Studies on the determination of Malachite green, a forbiddance antimicrobial agent in imported aquatic foods by using LC/TOF-MS Ryoko HATTORI * , Misao GOTOU, Shigeki AKAMATSU, Naoki YOSHIOKA, Tatsuhiko KAWAMOTO and Tadaaki INADA Life Science Division, Public Health Science Research Center, Hyogo Prefectural Institute of Public Health and Consumer Sciences, 2-1-29, Arata-cho, Hyogo-ku, Kobe 652-0032, Japan In order to measure the residues of malachite green (MG) and its metabolites leucomalachite green (LMG) in aquatic foods, we have studied the determination by applying a d-SPE (dispersive solid phase extraction) used in QuEChERS methods using liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). MG and LMG were extracted and dehydrated by citrate-phosphate buffer (pH3.0) , acetonitrile, anhydrous magnesium sulfate, and sodium chloride under shaded conditions. The extracts were cleaned up by the d-SPE containing PSA and anhydrous magnesium sulfate, and then, purified by strongly acidic cation exchange (SCX) solid columns. We evaluated the analytical values corrected by stable isotope labeling, MG-d5 and LMG-d5 (surrogates). Compared with the official method, the sample preparation was quicker and simpler. In addition, there was no need to use dichloromethane concerned about its residues in environment and hexane cause of low recovery rate. The limits of quantification by this method were 0.002~0.006μg/g for MG, 0.002~0.004μg/g for LMG. This result showed that the method is suitable for the detection of low concentration levels of these analytes. The recovery rate corrected by surrogates, the variation coefficient, and the recovery rate of surrogates generally conformed to the guideline of the Japanese Ministry of Health, Labour and Welfare. Ⅰ はじめに 1. 実験目的 マラカイトグリーン(以下, MG とする.)は工業用合 成色素であり,繊維や皮製品の染料として用いられてい る.また,MG は還元性を有し,抗菌活性があるため, 観賞魚の白癬症等の治療薬としても使用されている.し 健康科学部 *別刷請求先:〒652-0032 神戸市兵庫区荒田町 2-1-29 兵庫県立健康生活科学研究所 健康科学研究センター 健康科学部 服部 涼子 かし,発がん性を有する 1 ) ことから,日本では全ての養 殖水産動物への使用が禁止されており,食品衛生法の規 格基準 2 ) において「不検出」となっている.2005年に中 国産のうなぎ蒲焼で基準値超過が発生して以降,違反が 相次いでおり,違反発生時には早急に対応ができる迅速 な検査体制が求められている. 2. MG及びその代謝物の安定性 MG及びその代謝物である 3 ) ロイコマラカイトグリー ン(以下, LMGとする.)は,厚生労働省告示 2 ) (以下, 公定法とする.)において同時分析法が示されている.し かし, MGは光分解性を有し,不安定な性質があるため, ともに安定して回収率が得ることができないことがある. − 19 −
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[平成27年3月30日受理]
兵庫県立健康生活科学研究所健康科学研究センター研究報告 第 6 号 2015
-19-
[ノート]
輸入水産食品中の使用禁止抗菌剤, マラカイトグリーンの
LC/TOF-MSによる分析法の検討
服部 涼子* 後藤 操 赤松 成基 吉岡 直樹 川元 達彦 稲田 忠明
Studies on the determination of Malachite green, a forbiddance
antimicrobial agent in imported aquatic foods by using LC/TOF-MS
Life Science Division, Public Health Science Research Center, Hyogo Prefectural Institute of Public Health and Consumer Sciences, 2-1-29, Arata-cho, Hyogo-ku, Kobe 652-0032, Japan
In order to measure the residues of malachite green (MG) and its metabolites leucomalachite green (LMG) in aquatic foods, we have studied the determination by applying a d-SPE (dispersive solid phase extraction) used in QuEChERS methods using liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). MG and LMG were extracted and dehydrated by citrate-phosphate buffer (pH3.0), acetonitrile, anhydrous magnesium sulfate, and sodium chloride under shaded conditions. The extracts were cleaned up by the d-SPE containing PSA and anhydrous magnesium sulfate, and then, purified by strongly acidic cation exchange (SCX) solid columns. We evaluated the analytical values corrected by stable isotope labeling, MG-d5 and LMG-d5 (surrogates). Compared with the official method, the sample preparation was quicker and simpler. In addition, there was no need to use dichloromethane concerned about its residues in environment and hexane cause of low recovery rate. The limits of quantification by this method were 0.002~0.006µg/g for MG, 0.002~0.004µg/g for LMG. This result showed that the method is suitable for the detection of low concentration levels of these analytes. The recovery rate corrected by surrogates, the variation coefficient, and the recovery rate of surrogates generally conformed to the guideline of the Japanese Ministry of Health, Labour and Welfare.
Fig. 2 Effects of weight composition ratios between PSA and
C18 (n=5)
: correction values of MG by MG-d5, : correction values of LMG by LMG-d5, PSA/C18(0 mg:300 mg)(a), PSA/C18(100 mg:200 mg)(b), PSA/C18(150 mg:150 mg)(c), PSA/C18(200 mg:100 mg)(d), PSA/C18(300 mg:0 mg)(e)
兵庫県立健康生活科学研究所健康科学研究センター研究報告 第 6 号 2015
-23-
EIC(329.2012) Frag=300V
EIC(331.2169) Frag=100V
Counts vs. Acquisition Time (min)
(d)
(a)
(b)
(c)
EIC(329.2012) Frag=300V
EIC(331.2169) Frag=100V
13.236
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Fig.3 Chromatograms of extract of MG standard solution
(0.0004µg/mL) (a), extract of MG blank solution of giant tiger prawns (b), extract of LMG standard solution (0.0004µg/mL) (c), extract of LMG blank solution of giant tiger prawns (d)
Fig.4 Mass spectra of MG and LMG standard solution (0.1 µg/mL) target ion for MG (a), qualifier ion for MG (b), target ion for LMG (c),qualifier ion for LMG (d)
a) 200 µl of standard solution (0.02 µg/mL) was added to 2 g sampleb) correction value by surrogate, ( ): CV%, n=3c) LOD: Limits of Detection defined by S/N≧3d) LOQ: Limits of Quantification defined by S/N≧10
a) 200 µl of standard solution (0.02 µg/mL) was added to 2 g sampleb) correction value by surrogate, ( ): CV%, n=3c) LOD: Limits of Detection defined by S/N≧3d) LOQ: Limits of Quantification defined by S/N≧10
3) Jun, B. L., Hee, Y. K., Young, M. J., Young, S., Sung, M. W., Mi, S. P., Hyun, S. L., Soon, K. L. and Meehye, K. : Determination of malachiete green and crystal violet in processed fish products. Food Additives and Contaminants, 27, 953-961 (2010)
10) Maria, V., Bienvenida, G., Juan, F. G., Natividad, R. M. and Antonio, M.: Muticlass detection and quantitation of antibiotics and veterinary drugs in shrimps by fast liquid chromatography time-of-flight mass spectrometry. Talanta, 85, 1419-1427 (2011)
11) Juliana, C. H. and Jonas, A. R. P. : A simple method for the determination of malachite green and leucomalachite green residues in fish by a modified QuEChERS extraction and LC/MS/MS. Journal of AOAC International, 95, 913-922 (2012)
12) Noelia, L., Robert, R., José, L. M. V. and Antonia, G. F. :Analysis of triphenylmethane dyes in seafood products : a review of extraction methods and determination by liquid chromatography coupled to mass spectrometry. Analytical Methods, 5, 3434-3449 (2013)
14) Michelangelo, A. and Steven, J. L:Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “Dispersive Solid-Phase Extraction” for the determination of pesticide residues in produce. Journal of AOAC International, 86, 412-431 (2003)