Rep.Nat l.foodres.inst No.72, 45 52 2008 305 8642 2 1 12 Hiroyuki Nakagawa, Mayumi Ohnishi Kameyama, Yuki Sago, Hiroshi Yada, Masayo Kushiro, and Hitoshi Nagashima National Food Research Institute, 2 1 12 Kannon dai, Tsukuba, Ibaraki 305 8642 Aflatoxins (AFs) are the most potent carcinogenic and toxic compounds among many kinds of substances in the nature, and benzo a pyrene (BaP) is known as a hardly degradable compound and also recognized as a carcinogen. Simultaneous detection of AFs (AFB, AFB, AFG, and AFG ) and BaP was performed by liquid chromatography /tandem mass spectrometry (LC/MS /MS) equipped with an atomospheric pressure photo ionization (APPI) probe. Mixtures of AFs and BaP were applied to multi functional columns suggested for the purification on an official method for AFs determination, and both of them were well eluted through an Autoprep MF A1000 column. In the aim of practical usage for food analysis, AFs and BaP were added to olive oil at the concentration of 4.0 g/kg and 40 g/kg, respectively, and analyzed with LC/ MS/MS. When AFs and BaP were purified from olive oil with a sosid phase extraction column Autoprep MF A1000, the mean recovery of BaP was low (12.8 %) in contrast to that of AFs (102.1 125.3%). The cause of low recovery of BaP was its low extraction efficiency from olive oil with the solvent of acetonitrile water (90: 10). If the purification process is improved to increase the extraction efficiency of BaP from olive oil, our method must be useful for the analysis of AFs and BaP contaminated in various foods. AF s Aspergillusflavus Aspergillusparasiticus Aspergillusnomius Aspergil lutamarii AFs AFs BAFB BAFB G AFG GAFG 4 1 AFB AFB 10ppb AFB 2007 10 31 2007 12 18 Corresponding author, Hiroyuki Nakagawa
polycyclic aromatic hydrocarbon spah s 2 4 PAHs, a BaP 1 PAHs PAHs PAH BaP IARC 1 1962 FAO WHO EU 2.0ppb 10ppb 2006 7 BaP 2.0ppb WHO 0.7ppb BaP PAH PAHs BaP PAHs AFs BaP LC/MS/MS AFs AFs BaP PAHs AFs AFs PAHs atomospheric pressure photo ionization, APPI LC/MS/MS AF 4 BG Acrosorganics BG BaP AccuStandard 4AFB AFG 2030 LI BRORLM 20 1mg LC/MS 10ml 20ml Arrow 20ml AFs 20BaP 4 30
AFs 4 BaP AFs BaP 1 ppm 1 ml 500 l 200 l 100 l 10 ml 50 50 AFs BaP 100 50 20 10 ppb 50 ppb 1 ml 400 l 200 l 100 l 10 ml 50 50 AFs BaP 5 2 1 0.5 ppb 2 ppb 1 ml 500 l 10 ml 50 50 AFs BaP 0.2 0.1 ppb LC/MS/MS Agilent 1100 Agilent Technologies APPI 4000QTRAP LC/MS/ MS Applied Biosystems LC A 0.1 B 0.1 ZOR BAX Eclipse Plus C182.1mm 150mm 3.5 micron, Agilent Technologies 0.20 ml 0 2 5 B 2 17 5 95 B 17 22 95 B 22 23 95 5 B 23 33 5 B AFBAFB AFGAFG BaP 15.69 15.21 15.24 14.73 24.31 APPI HPLC 0.02 ml/ 625 V500 10psi30 psi 10psi MRM AFs BaP M H MS/MS HPLC AFs BaP AFs BaP AFs AF AF 3 AFs4 BaP AutoprepMF A1000 Multisep 226 AflaZonRomerLabs Multisep 228 AflaPat 1 AFs BaP 1ppm 1ml 10ml 1ml 90 Autoprep MF A1000 2ml 20ml 3.2ml 84 Multisep 226 AflaZon Multisep 228 AflaPat AFs BaP 100ppb AFs AFs AFs AFs Varian 1.0ml 7 0.5ml/1.0ml /7ml GL 1.0ml
m/z m/z AFB 312 313 285 76 35 AFB 314 315 287 131 39 AFG 328 329 243 111 39 AFG 330 331 313 101 39 BaP 252 253 248 96 105 DP:Declusteringpotential V CE:Collisionenergy V DP CE Agilent Technologies LC/MS/MS AFs Papachristou BaP AFs BaP AFs4 BaP AFs 50ml 35mm 110mm 2.5g AFs4 0.1ppm BaP 1 ppm 100 l AFs4 4.0 g/kg BaP 40 g/kg 30 90 10 10ml KM ShakerV DX 300rpm 30 15ml 16.5mm 125mm3000 g 10 Varian 1.0 ml GL 50 50 100 l Agilent LC/MS/MS 3 Agilent 3 2.5g AFs BaP 100 l 2 AFs BaP M H LC/MS/MS AFs4 BaP 1 AFs 4 BaP 0.1 100 ppb AFBAFG 0.5 100 ppb AFBAFG 1.0 100 ppb BaP 2.0 100 ppb 0.990 S/N 3 S/N 10 AFB 0.2ppb 0.5ppb AFB 0.5 ppb 1.0 ppb AFG 0.2 ppb 0.5 ppb AFG 0.5 ppb 1.0 ppb BaP 1.0 ppb 2.0 ppb AFs BaP AFs BaP 100ppb 3 AutoprepMF A1000 Multisep 226 AflaZonMultisep 228
49 表２ 多機能カラム Autoprep MF A1000 における AFs と BaP の流出パターン a 濃度 ppb フラクション番号 1 AFB1 95.5 AFB2 96.7 AFG1 AFG2 BaP 102 97.9 19.4 2 100 104 105 95.7 60.4 3 107 107 105 97.5 88.2 4 105 107 105 106 108 95.9 109 96.9 106 96.5 5 99.3 6 101 7 106 98.1 101 102 100 99.1 104 96.4 a AFs と BaP を各 100ppb ずつ含む 90 アセトニトリルを負荷し 1 ml ずつ分取した 図２ LC/MS/MS による AFs と BaP の同時分析の MS/MS クロマトグラム AflaPat に負荷したところ AFs はいずれのカラム ラムのベッドボリュームが異なるので充. 剤の材質だ においてもフラクション 1 本目から 95.5 105 ppb 負 けに依存するとは限らないが AFs と BaP の同時精 荷濃度の 95.5 105 相当 の流出が確認された 製には Autoprep MF A1000 を用いるのが適当である これに対して BaP は AutoprepMF A1000 ではフラク と判断した 表 2 に Autoprep MF A1000 における AFs ション 3 本目以降で 88.2 104ppb 負荷濃度の 88.2 および BaP の流出パターンを示す 104 相当 Multisep226 ではフラクション 6 本目 以降で 105 145ppb 負荷濃度の 105 145 相当 3 オリーブ油への添加回収試験 の流出が確認された 一方 Multisep228 ではフラク オリーブ油への添加回収試験は最も良好な流出パタ ション 7 本目まで BaP の溶出が確認されず 流出速 ーンを示した Autoprep MF A1000 を用いて精製を行っ 度も遅かったため AFs と BaP の同時精製には不適 た 抽出はアセトニトリル 水混合液 90 10 を用 当であると判断した データ省略 以上より 各カ いて行った 表 2 の結果より BaP はフラクション 4
AFB AFB AFG AFG BaP Spiked levelg/kg 4.0 4.0 4.0 4.0 40 Mean Recovery %n 3 110.9 113.7 102.1 125.3 12.8 Relative standard deviation RSD 10.9 13.2 15.3 10.6 2.1 AFs BaP 4 4 MS/MS 2 3 3 AFBAFBAFGAFG 110.9113.7102.1125.3 RSD 10.913.215.310.6 BaP 12.8 2.1 2.5g 100 l AFs BaP AFs BaP LC/MS/MS AFs APPI LC/MS Takino PAHs APPI LC/MS/MS Moriwaki PAHs PAHs PAHs GC/MS GC MS/MS LC/MS/MS APPI LC/MS/MS PAHs LC/MS/MS GC/MS /MS OH COOH LC/MS/MS APPI LC/MS/MS AFs 4 BaP PAHs LC/MS/MS PAHs APPI LC/MS/MS Moriwaki BaP 1.0 ppb Moriwaki BaP M H M AFs BaP M H BaP M LC 0.1 M H AFs BaP Autoprep MF A1000 AFs BaP AFs AF Autoprep MF A1000 102.1 125.3 BaP 12.8AFs AFs BaP 2 BaP BaP Autoprep MF A1000 90 10 Autoprep MF A1000
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