රᗜ┴❧೺ᗣ⏕ά⛉Ꮫ◊✲ᡤ೺ᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮◊✲ሗ࿌ ➨ ྕ 㹙ࣀ࣮ࢺ㹛 /&)/ ࢆ⏝࠸ࡓ␆⏘≀୰࡟࠾ࡅࡿ࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖๣ࡢ ࢫࢡ࣮ࣜࢽࣥࢢศᯒἲࡢ㛤Ⓨ࡜ጇᙜᛶホ౯ ̿ࣉ࣒ࣞ࢝ࣛ⮬ືㄏᑟయ໬ἲࡢ㐺⏝̿ ᚋ⸨ ᧯ 1㸨 ᯘ ᖾᏊ 1 ᭹㒊 ᾴᏊ 1 ᯇᒸ ᬛ㑳 2 ୕ᶫ 㝯ኵ 1 Development and Validation of an Analytical Screening Method using Liquid Chromatography with Fluorescence Detection for Macrolide Anti-parasitic Agents in Livestock Products 㸫Application of Automated Precolumn Derivatization㸫 Misao GOTOU1㸨㸪Sachiko HAYASHI1㸪Ryoko HATTORI1㸪Tomofumi MATSUOKA 2 and Takao MITSUHASHI1 Life Science Division㸪Public Health Science Research Center㸪Hyogo Prefectural Institute of Public Health and Consumer Sciences㸪2-1-29㸪Arata-cho㸪Hyogo-ku㸪Kobe 652-0032㸪Japan㸪 2 Naka-Harima District Administration Office㸪Hyogo Prefecture 1 This report describes validation of a simple analytical screening method for residual macrolide anti-parasitic agents (abamectin, eprinomectin, doramectin, ivermectin, and moxidectin) in livestock products by liquid chromatography with fluorescence detection. The analysis was carried out by automated precolumn derivatization at room temperature using a fluorescent indicator derivatized with trifluoroacetic anhydride and 1-methylimidazole. The evaluation was performed by adding each macrolide at its Japanese maximum residue limit (MRL㸸0.01–0.1 Pg/g) to cattle, swine, and chicken muscles in accordance with the Ministry of Health, Labour and Welfare Guidelines. Among the samples tested, recovery of the five macrolides ranged from 84.3% to 88.1%. Regarding the relative standard deviation (RSD) of precision, repeatability ranged from 2.3% to 10.8%, and intermediate precision ranged from 7.8% to 17.5%; hence, the results fulfilled the requirements of the Guidelines. For all samples tested, the limit of quantitation (LOQ) was confirmed to be 0.005 Pg/g. Thus, this method has been proven to be applicable to screening analysis for five aforementioned macrolide anti-parasitic agents in the three kinds of livestock products examined. Ϩ ࡣࡌࡵ࡟ 㥑⹸๣࡜ࡋ࡚㸪ୡ⏺୰࡛ᗈࡃ⏝࠸ࡽࢀ࡚࠾ࡾ㸪㣗⫗࠾ࡼ ࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖๣ࡣ㸪∵㸪㇜࡞࡝␆⏘≀ࡢ ṧ␃ᇶ‽ࡀタࡅࡽࢀ㸪㏆ᖺ࡛ࡣ㸪࢖࣓࣋ࣝࢡࢳࣥࡀ᳨␿ ᡤ➼࡟࠾࠸࡚ 2010 ᖺ㸪2011 ᖺ࡟⥆ࡁ 2013 ᖺ࡟ࡶᇶ‽ ࡧຍᕤရ࡬ࡢṧ␃ࡀᠱᛕࡉࢀ࡚࠸ࡿ㸬ᡃᅜ࡛ࡶ㣗⫗୰ࡢ 1 ೺ᗣ⛉Ꮫ㒊 2 ⌧රᗜ┴୰᧛☻┴Ẹᒁ ್㉸㐣㐪཯࡜࡞ࡾ㸪᳨ᰝ࿨௧࠶ࡿ࠸ࡣ᳨ᰝᙉ໬࡞࡝ࡢᥐ 㸨ูๅㄳồඛ㸸ࠛ652-0032 ⚄ᡞᕷරᗜ༊Ⲩ⏣⏫ 2-1-29 ⨨ࡀ࡜ࡽࢀࡓ㸬රᗜ┴࡟࠾࠸࡚ࡣ㣗ࡢᏳ඲Ᏻᚰࡢ☜ಖࡢ රᗜ┴❧೺ᗣ⏕ά⛉Ꮫ◊✲ᡤ ೺ᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮ ୍⎔࡜ࡋ࡚㸪ື≀⏝་⸆ရࡢ᳨ᰝ㡯┠ࡢᣑ඘ࢆ㐍ࡵ࡚࠾ ೺ᗣ⛉Ꮫ㒊 ᚋ⸨ ᧯ ࡾ㸪ᙜ◊✲ࢭࣥࢱ࣮࡛ࡣ㸪࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖๣ － 29 － ࢆ᪂ࡓ࡞ᑐ㇟࡜ࡋ᳨࡚ᰝἲࡢ㛤Ⓨ࡟ྲྀࡾ⤌ࢇ࡛࠸ࡿ㸬␆ 1㸬ヨᩱ ⏘≀୰࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖๣ࡢヨ㦂ἲ࡜ࡋ࡚ࡣ රᗜ┴ෆ࡛ὶ㏻ࡋ࡚࠸ࡓᕷ㈍ࡢ∵㸪㇜࠾ࡼࡧ㭜ࡢ➽⫗ ࠕ࢖࣓࣋ࣝࢡࢳࣥ㸪࢚ࣉࣜࣀ࣓ࢡࢳࣥ㸪ࢻ࣓ࣛࢡࢳࣥཬ 㒊఩ࢆ⏝࠸ࡓ㸬 ࡧࣔ࢟ࢩࢹࢡࢳࣥヨ㦂ἲ㸦␆Ỉ⏘≀㸧 ࠖ1)ࡀཌ⏕ປാ┬࠿ ࡽ㏻▱ࡉࢀ࡚࠸ࡿ㸬ࡑࡢヨ㦂ἲࡣ㸪⺯ග᳨ฟჾ௜ࡁ㧗㏿ 2㸬ヨ⸆࠾ࡼࡧヨᾮ ᾮయࢡ࣐ࣟࢺࢢࣛࣇ㸦LC-FL㸧ࢆ⏝࠸㧗ឤᗘศᯒࡀྍ⬟ ᐤ⏕⹸㥑㝖๣ᶆ‽ရ㸸࢚ࣉࣜࣀ࣓ࢡࢳࣥ㸦࢚ࣉࣜࣀ࣓ ࡞᪉ἲ࡛࠶ࡿࡀ㸪⺯ගㄏᑟయ໬࡞࡝๓ฎ⌮ࡢ᧯సࡀ↹㞧 ࢡࢳࣥ B1a㸧㸪 ࢔ࣂ࣓ࢡࢳࣥ 㸦࢔࣓࣋ࣝࢡࢳࣥ B1a㸹94.0%㸪 ࡜࠸ࡗࡓၥ㢟Ⅼࡀ࠶ࡿ 2-4)㸬 ࢔࣓࣋ࣝࢡࢳࣥ B1b㸹6.0% 㸧ࡣᯘ⣧⸆ᕤᴗ(ᰴ)〇㸪ࣔ࢟ ࡑࡇ࡛ᡃࠎࡣ㸪LC-FL ࢆ⏝࠸㸪ࡼࡾ⡆౽࡛㎿㏿࡞ࢫࢡ ࢩࢹࢡࢳࣥ㸪ࢻ࣓ࣛࢡࢳࣥࡣ Fluka ♫〇㸪࢖࣓࣋ࣝࢡࢳ ࣮ࣜࢽࣥࢢศᯒἲࡢ☜❧ࢆ┠ⓗ࡟㸪๓ฎ⌮࡟ᅛ┦ᢳฟἲ ࣥࡣ Dr Ehrenstorfer ♫〇ࢆ⏝࠸ࡓ㸬ྛᐤ⏕⹸㥑㝖๣ࡢ ࡢ᥇⏝࡜⺯ගㄏᑟయ໬࡟ࣉ࣒࡛ࣞ࢝ࣛࡢ⮬ື໬ࡢ㐺⏝ࢆ ᵓ㐀ᘧࢆ Fig.1 ࡟♧ࡍ㸬ྛᶆ‽ရ ⣙ 20 mg ࢆ⢭⛗ᚋ㸪 ᳨ウࡋࡓ㸬ࡑࡢ⤖ᯝ㸪∵㸪㇜࠾ࡼࡧ㭜ࡢ➽⫗࡟࠾࠸࡚㸪 ࣓ࢱࣀ࣮ࣝ࡟⁐ゎࡋ㸪඲㔞ࢆ 100 mL ࡜ࡋࡓ㸦ᶆ‽ཎᾮ㸸 5 ✀㢮㸦࢔ࣂ࣓ࢡࢳࣥ㸪࢖࣓࣋ࣝࢡࢳࣥ㸪࢚ࣉࣜࣀ࣓ࢡ ྛ 0.20 mg / mL㸧㸬ࡇࢀࡽᶆ‽ཎᾮࢆ࣓ࢱࣀ࣮࡛ࣝ㐺ᐅ ࢳࣥ㸪ࢻ࣓ࣛࢡࢳࣥ㸪ࣔ࢟ࢩࢹࢡࢳࣥ㸧ࡢ࣐ࢡࣟࣛ࢖ࢻ ᕼ㔘ࡋ㸪ᶆ‽⁐ᾮ࠾ࡼࡧᶆ‽ΰྜ⁐ᾮࢆㄪ〇ࡋࡓ㸬 ⣔ᐤ⏕⹸㥑㝖๣ࡢศᯒ࡛Ⰻዲ࡞⤖ᯝࡀᚓࡽࢀࡓࡢ࡛ሗ࿌ ㄏᑟయ໬཯ᛂヨᾮ㸸1-࣓ࢳࣝ࢖࣑ࢲࢰ࣮ࣝ/↓Ỉ࢔ࢭࢺࢽ ࡍࡿ㸬 ࢺࣜࣝΰᾮ㸦1㸸1,v/v㸧㸦௨ୗ㸪MI ⁐ᾮ࡜ࡍࡿ㸧࠾ࡼࡧ ࡲࡓ㸪ศᯒἲࡢಙ㢗ᛶࢆ☜ಖࡍࡿࡓࡵ㸪ཌ⏕ປാ┬ࡀ ࢺࣜࣇࣝ࢜ࣟ㓑㓟↓Ỉ≀/࢔ࢭࢺࢽࢺࣜࣝΰᾮ㸦2㸸3,v/v㸧 ♧ࡋࡓࠕ㣗ရ୰࡟ṧ␃ࡍࡿ㎰⸆➼࡟㛵ࡍࡿヨ㦂ἲࡢጇᙜ 㸦௨ୗ㸪TFAA ཯ᛂᾮ࡜ࡍࡿ㸧ࢆ⏝᫬ㄪ〇ࡋࡓ㸬 ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟ࡘ࠸࡚ࠖ5)㸦௨ୗ㸪࢞࢖ࢻࣛ࢖ࣥ ࡑࡢ௚ࡢヨ⸆㸸࢔ࢭࢺࢽࢺࣜࣝ㸪࣓ࢱࣀ࣮ࣝࡣ࿴ග⣧⸆ ࡜ࡍࡿ㸧࡟ᚑࡗ࡚ጇᙜᛶホ౯ࢆ⾜ࡗࡓ⤖ᯝࡶేࡏ࡚ሗ࿌ ᕤᴗ(ᰴ)〇㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ⏝ࢆ⏝࠸ࡓ㸬1-࣓ࢳ ࡍࡿ㸬 ࣝ࢖࣑ࢲࢰ࣮ࣝࡣ࿴ග⣧⸆ᕤᴗ(ᰴ)〇ࢆ㸪↓Ỉ◲㓟ࢼࢺ ࣒ࣜ࢘㸪ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ㸪ࢺࣜࣇࣝ࢜ࣟ㓑㓟↓Ỉ≀ࡣ ϩ ᪉ ἲ 㻴㻻㻻㻯㻴㻟㻺㻻㻯㻴㻟㻻㻯㻴㻟㻴㻟㻯㻻㻻㻴㻻㻻㻴㻻㻴㻻㻯㻴㻟㻻㻴㻟㻯㻴㻟㻯 Moxidectin 㻯㻴㻟㻻㻻㻴㻴㻻㻴㻴㻟㻯㻻㻻㻴㻻㻴㻯㻴㻟 Abermectin B1a 㻴㻻㻴㻴㻟㻯㻻㻯㻴㻟㻻㻯㻴㻟㻻㻻㻴㻟㻯㻴㻴㻟㻯㻻㻯㻴㻟㻻㻻㻻㻴㻯㻴㻟㻻㻴㻻㻻㻻㻻㻻㻻㻯㻴㻟㻻㻴㻴㻟㻯㻯㻴㻟㻻㻴㻟㻯㻴㻟㻯㻯㻴㻟㻯㻴㻟㻻㻯㻴㻟㻻㻯㻴㻟㻻㻻㻻㻴 Eprinomectin B1a 㻻㻻㻻㻯㻴㻟㻻㻴㻴㻻㻴㻻㻯㻴㻟㻻㻻㻴㻻㻯㻴㻟㻯㻴㻟㻴㻯㻞㻴㻡㻻㻴㻴㻻㻯㻴㻟㻻㻴㻻㻯㻴㻟㻴㻺㻻㻴㻴㻟㻯㻻㻯㻴㻟㻻㻴㻴㻟㻯㻻㻴㻟㻯㻯㻴㻟㻯㻴㻟㻻㻻㻴㻟㻯 ࿴ග⣧⸆ᕤᴗ(ᰴ)〇≉⣭ࢆ⏝࠸ࡓ㸬㻻㻴㻻㻯㻴㻟㻻㻻㻴㻯㻴㻟㻯㻞㻴㻡㻴㻯㻴㻟㻻㻴 Doramectin 㻯㻴㻟 Ivermectin Fig.1 Chemical structures of Eprinomectin B1a㸪Moxidectin㸪Abermectin B1a㸪Doramectin and Ivermectin － 30 －㻯㻴㻟㻯㻞㻴㻡 රᗜ┴❧೺ᗣ⏕ά⛉Ꮫ◊✲ᡤ೺ᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮◊✲ሗ࿌ ➨ ྕ ᅛ┦ᢳฟ⏝࣒࢝ࣛ㸸Oasis HLB㸦60 mg㸪3 mL㸪Waters ᐃ㔞ࢆጉᐖࡍࡿࣆ࣮ࢡࡀ↓࠸ࡇ࡜ࢆ☜ㄆࡋࡓ㸬 ♫〇㸧ࡣ㸪࠶ࡽ࠿ࡌࡵ࢔ࢭࢺࢽࢺࣜࣝ 2 mL ࠾ࡼࡧ 0.1% 7.2 ┿ᗘ㸦ᅇ཰⋡㸧࠾ࡼࡧ⢭ᗘ ࢺ࢚ࣜࢳࣝ࢔࣑ࣥỈ⁐ᾮ/࢔ࢭࢺࢽࢺࣜࣝΰᾮ㸦7㸸3,v/v㸧 2 mL ࡛ࢥࣥࢹ࢕ࢩࣙࢽࣥࢢࡋ࡚౑⏝ࡋࡓ㸬 ᇶ‽್┦ᙜࡢᐤ⏕⹸㥑㝖๣ࢆῧຍࡋࡓྛ㣗⫗ヨᩱ࡟ࡘ ࠸࡚㸪ศᯒ⪅㸯ྡࡀ㸯᪥࡟㸰ヨ⾜ࢆィ㸲᪥㛫࠾ࡼࡧศᯒ ⪅㸯ྡࡀ㸯᪥࡟㸳ヨ⾜ࢆ㸯᪥㛫㸪ࡑࢀࡒࢀ ᐃࢆᐇ᪋ࡋ 3㸬⿦⨨ ࡓ(n=13)㸬ᚓࡽࢀࡓࢹ࣮ࢱ࠿ࡽ┿ᗘࢆồࡵ㸪ࡑࡢ⤖ᯝࢆ HPLC-FL㸸(ᰴ)ᓥὠ〇సᡤ〇 LC-20A ୍ඖ㓄⨨ศᩓศᯒ࡟ࡼࡾゎᯒࡋ㸪ే⾜⢭ᗘ࠾ࡼࡧᐊෆ⢭ ࣍ ࣔ ࢪ ࢼ ࢖ ࢨ ࣮ 㸸 IKA ࢪ ࣕ ࣃ ࣥ ( ᰴ ) 〇 ULTRA ᗘࢆ⟬ฟࡋࡓ㸬 TURRAX T25 digital ࢚ࣂ࣏࣮ࣞࢱ࣮㸸᪥ᮏࣅࣗࢵࣄ(ᰴ)♫〇 Rotavapor R-3 (mV) P9 2.5 4㸬 ᐃ᮲௳ ࣒࢝ࣛ㸸Mightysil RP-18 GPϩ(4.6 mm150 mm㸪5µ A m㸧 ⛣ື┦㸸‫ׇ‬㸹ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ/ࣜࣥ㓟/Ỉ/࣓ࢱࣀ࣮ࣝΰ ᾮ㸦 0.1:0.1:50:50 㸧 ‫׈‬㸹ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ /ࣜࣥ㓟/࣓ࢱࣀ࣮ࣝΰᾮ 0.0 㸦 0.1:0.1:100 㸧 2.5 ࢢࣛࢪ࢚ࣥࢺ㸦‫ׇ‬㸩‫׈‬㸧᮲௳㸸‫ ׈‬80% (0 min.)Ѝ80% (6 min.)Ѝ95% (12 min.)Ѝ95% (22 min.)Ѝ80% (28 min.) P9 PLQ PLQ B Intensity ࣒࢝ࣛ ᗘ㸸40Υ㸪ὶ㏿㸸1.0 mL / min.㸪ὀධ㔞㸸30µL㸪 ᐃἼ㛗㸦ບ㉳Ἴ㛗/⺯ගἼ㛗㸧㸸365 nm / 475 nm 5㸬ヨ㦂⁐ᾮࡢㄪ〇 0.0 ヨᩱ 2.0 g ࡟࢔ࢭࢺࢽࢺࣜࣝ 20 mL ࡜↓Ỉ◲㓟ࢼࢺࣜ 2.5 P9 ࣒࢘ 5 g ࢆຍ࠼࡚࣍ࣔࢪࢼ࢖ࢬᚋ㸪5 A ࢁ⣬ (1) C (2) 㸦ADVANTEC㸧 ࡛ࢁ㐣ࡋ㸪 ᢳฟᾮࢆᚓࡓ㸬 ࡇࡢ࠺ࡕ 1 mL ࢆศྲྀࡋ㸪0.1%ࢺ࢚ࣜࢳࣝ࢔࣑ࣥỈ⁐ᾮ 2 mL ࢆΰ࿴ᚋ, (3) (4) (5) HLB ࣒࢝ࣛ࡟㈇Ⲵࡋࡓ㸬0.1 %ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ/࢔ࢭࢺ ࢽࢺࣜࣝΰᾮ㸦7㸸3,v/v㸧1 mL ࡛Ὑίᚋ㸪࢔ࢭࢺࢽࢺࣜ 0.0 ࣝ 1 mL ࢆ⏝࠸ࡓ⁐ฟᾮࢆ⃰⦰஝ᅛࡋ㸪MI ⁐ᾮ 0.2 mL 0 ࡛⁐ゎࡋࡓ㸬ࡇࡢ⁐ゎᾮ࡜ TFAA ཯ᛂᾮࢆ➼㔞ΰྜࡋࡓ 5 10 15 20 25 PLQ (min.) Retention Time ࡶࡢࢆヨ㦂⁐ᾮ࡜ࡋ㸪LC-FL ࡛ศᯒࡋࡓ㸬 Fig.2 Typical chromatograms of macrolide antiparasitic agents 6㸬᳨㔞⥺ (A) Cattle muscle blank; (B) cattle muscle samples to ᳨㔞⥺⏝ࡢᶆ‽⁐ᾮࡣ㸪ᶆ‽⁐ᾮࢆ஝ᅛࡋ㸪1-࣓ࢳࣝ which eprinomectin (0.025 Pg/mL), moxidectin (0.005 ࢖࣑ࢲࢰ࣮ࣝ/↓Ỉ࢔ࢭࢺࢽࢺࣜࣝΰᾮ㸦1㸸1,v/v㸧0.2 mL Pg/mL), abamectin (0.0025 Pg/mL), doramectin (0.0025 ࡛⁐ゎᚋ㸪TFAA ཯ᛂᾮ࡜➼㔞ΰྜࡋసᡂࡋࡓ㸬 Pg/mL), and ivermectin (0.0025 Pg/mL) was added; and (C) standard solutions: eprinomectin (0.025 Pg/mL), 7㸬ጇᙜᛶホ౯ moxidectin (0.005 Pg/mL), abamectin (0.0025 Pg/mL), ጇᙜᛶࢆホ౯ࡍࡿࡓࡵ㸪࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ࠸㸪௨ୗࡢ doramectin (0.0025 Pg/mL), and ivermectin (0.0025 㡯┠࡟ࡘ࠸࡚ヨ㦂ࢆ⾜ࡗࡓ㸬ྛᐤ⏕⹸㥑㝖๣ࡣ㸪㣗ရヨ Pg/mL) ᩱ࡟ᑐࡋ㸪ࡑࢀࡒࢀᇶ‽್┦ᙜ࡜࡞ࡿࡼ࠺ῧຍࡋࡓ㸬 The peaks represent (1) eprinomectin, (2) moxidectin, 7.1 㑅ᢥᛶ (3) abamectin, (4) doramectin, and (5) ivermectin. ᐤ⏕⹸㥑㝖๣ࢆྵ᭷ࡋ࡚࠸࡞࠸ࣈࣛࣥࢡヨᩱࢆ ᐃࡋ㸪－ 31 － Ϫ ⤖ᯝ࠾ࡼࡧ⪃ᐹ ⥆྾ᘬࢆ 1 ࢭࢵࢺ࡜ࡋ࡚㸪⧞ࡾ㏉ࡋ 5 ࢭࢵࢺࡢィ 30 µL 1㸬LC-FL ⛣ື┦ࡢ᳨ウ ࡢΰྜ⁐ᾮࢆࢧࣥࣉ࣮ࣝࣝࣉෆ࡟ಖᣢࡋࡓ᮲௳࡛㸪LC ⛣ື┦ࡣ㸪㏻▱ἲ࡜ྠᵝࡢ࣓ࢱࣀ࣮ࣝ-Ỉ⣔ࢆ⏝࠸ࡓ㸬 ࡬ࡢὀධ๓ࡢಖᣢ᫬㛫㸪ࡍ࡞ࢃࡕ཯ᛂ᫬㛫࡟ࡼࡿ⺯ගᙉ ᶆ‽⁐ᾮ࡟ࡼࡿ࢔࢖ࢯࢡࣛࢸ࢕ࢵࢡศᯒ࡛ࡣ㸪⛣ື┦ࡀ ᗘࡢኚ໬ࢆㄪ࡭ࡓ㸬ࡑࡢ⤖ᯝ㸪Fig.3 ࡟♧ࡍࡼ࠺࡟㸪⺯ ࣓ࢱࣀ࣮ࣝ/Ỉ㸦93㸸7㸧࠾ࡼࡧ㸦95㸸5㸧ࡢ᮲௳࡛࠸ࡎ ගᙉᗘࡣ㸪࠸ࡎࢀࡢᐤ⏕⹸㥑㝖๣࡟࠾࠸࡚ࡶ཯ᛂ᫬㛫ࡢ ࢀࡶⰋዲ࡞ศ㞳ࣆ࣮ࢡࡀᚓࡽࢀࡓ㸬ࡋ࠿ࡋ㸪∵⫗ヨᩱ࡟ 䠄mV䠅 ῧຍࡋࡓ㝿㸪ヨᩱ⏤᮶ࡢࣆ࣮ࢡࡀศᯒࢆጉᐖࡋࡓ㸬ࡑࡇ 6.0 ࡛㸪ࢢࣛࢪ࢚ࣥࢺ࡟ኚ᭦ࡋࡓ⤖ᯝ㸪ጉᐖࣆ࣮ࢡࡢᙳ㡪ࡀ A ᅇ㑊࡛ࡁࡓ㸬ࡉࡽ࡟㸪ࢺ࢚ࣜࢳࣝ࢔࣑ࣥ࠾ࡼࡧࣜࣥ㓟ࢆ 4.0 ῧຍࡍࡿࡇ࡜࡛㸪࢚ࣉࣜࣀ࣓ࢡࢳࣥࡢࣆ࣮ࢡᙧ≧ࡣⰋዲ 㸬 ࡛࠶ࡗࡓ 6)㸦Fig.2㸧 2.0 2㸬⺯ගㄏᑟయ໬᮲௳ࡢ᳨ウ 0.0 ⺯ගㄏᑟయ໬ࡣ㸪ᐊ ࡛཯ᛂࡀ㐍⾜ࡍࡿ 1-࣓ࢳࣝ࢖࣑ 6.0 ࢲࢰ࣮ࣝ࡜ࢺࣜࣇࣝ࢜ࣟ㓑㓟↓Ỉ≀ࢆ⏝࠸ࡿ᪉ἲࢆ᥇⏝ ࡋࡓ B 7-10)㸬ᶆ‽ΰྜ⁐ᾮࢆ஝ᅛᚋ㸪1-࣓ࢳࣝ࢖࣑ࢲࢰ࣮ 4.0 ࣝ/↓Ỉ࢔ࢭࢺࢽࢺࣜࣝΰᾮ㸦1㸸1,v/v㸧0.2 mL ࡛⁐ゎࡋ 㸦௨ୗ㸪MI ᶆ‽⁐ᾮ࡜ࡍࡿ㸧㸪ࡑࡢ୍㒊ࢆ LC ⏝ࣂ࢖࢔ 2.0 Intensity ࣝ࡟⛣ࡋࡓ㸬ูࣂ࢖࢔ࣝ࡟ TFAA ཯ᛂᾮࢆྲྀࡾ㸪LC ࡢ ࣮࢜ࢺࢧࣥࣉࣛࡢࣉࣟࢢ࣑ࣛࣥࢢࢆ⏝࠸࡚୧ᾮࢆ⮬ື࡛ ΰྜࡍࡿࣉ࣒ࣞ࢝ࣛㄏᑟయ໬ 11)ࢆ⾜ࡗࡓ㸬᭱኱ᐜ㔞 50 0.0 6.0 µL ࡢ LC ࡢࢧࣥࣉ࣮ࣝࣝࣉෆ࡟㸪MI ᶆ‽⁐ᾮ࡜ TFAA C ཯ᛂᾮࢆ஺஫࡟➼㔞ࢆ㐃⥆྾ᘬࡋ࡚ಖᣢࡋ㸪୧ᾮࢆࢧࣥ 4.0 ࢻ࢖ࢵࢳᵝࡢ≧ែ࡛㟼⨨ࡋ࡚཯ᛂࡉࡏࡓ㸬 ཯ᛂ᮲௳࡜ࡋ࡚㸪᭱ึ࡟཯ᛂ᫬㛫࡟ࡘ࠸᳨࡚ウࡋࡓ㸬 2.0 MI ᶆ‽⁐ᾮࢆ 3 µL㸪⥆࠸࡚ TFAA ཯ᛂᾮࢆ 3 µL ࡢ㐃 0.0 (mV) 6.0 6.0 D Intensity 4.0 4.0 2.0 2.0 0.0 0.0 0 10 20 30 40 50 䠄ǍL䠅 Sample solutions 0 10 20 30 (min.) Holding Time Fig.4 Effects of sample and reagent solution volumes on fluorescence intensity in derivatization (A) 2 PL each of sample and reagent, (B) 3 PL each of sample and reagent, (C) 4 PL each of sample and reagent, Fig.3 Effect of pre-injection holding time on fluorescence and (D) 5 PL each of sample and reagent intensity in precolumn autoderivatization (ʆ) abamectin (0.0125 Pg/mL), (Ō) moxidectin (0.005 (ʆ) abamectin (0.0125 Pg/mL), (Ō) moxidectin (0.005 Pg/mL), (×) doramectin (0.0125 Pg/mL), (‫ )ۍ‬eprinomectin (0.0125 Pg/mL), and (ŏ) ivermectin (0.0125 Pg/mL) － 32 － Pg/mL), (×) doramectin (0.0125 Pg/mL), (‫ )ۍ‬eprinomectin (0.0125 Pg/mL), and (ŏ) ivermectin (0.0125 Pg/mL) රᗜ┴❧೺ᗣ⏕ά⛉Ꮫ◊✲ᡤ೺ᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮◊✲ሗ࿌ ➨ ྕ 3 µL /1 ࢭࢵࢺࡢሙྜ 3㸪4 ࠾ࡼࡧ 6 ࢭࢵࢺ㸪ྛ 4 µL /1 ⤒㐣࡟ࡘࢀ࡚ୖ᪼ࡋ㸪0-5 ศ࡟࠾ࡅࡿ⺯ගᙉᗘࡢୖ᪼ࡣ ᛴ⃭࡛࠶ࡗࡓ㸬⺯ගᙉᗘࡢୖ᪼ࡣ 8 ศ௨㝆࡛ࡺࡿࡸ࠿࡜ ࢭࢵࢺࡢሙྜ 2㸪3 ࠾ࡼࡧ 5 ࢭࢵࢺ㸪ྛ 5 µL /1 ࢭࢵࢺࡢ ࡞ࡾ㸪8 ศ࡜ẚ㍑ࡋ࡚ 30 ศࡢ⺯ගᙉᗘࡣ⣙ 110%࡛࠶ࡗ ሙྜ 2㸪3 ࠾ࡼࡧ 4 ࢭࢵࢺ࡜ࡋࡓ㸬࡞࠾㸪཯ᛂ᫬㛫ࡣ඲ ࡓ㸬ࡇࡢഴྥࡣ㸪ヨ⸆ࡢΰྜẚࡣ␗࡞ࡿࡀྠヨ⸆ࢆ⏝࠸ ࡚ 8 ศ࡛⤫୍ࡋࡓ㸬1 ࢭࢵࢺࡢ୧ᾮࡢ྾ᘬ㔞ࡀྛ 3㸪4㸪 7)ࡢሗ࿌࡜ྠᵝ࡛࠶ࡗࡓ㸬ᮏἲ࡟࠾ 5 µL ࡢሙྜ㸪⺯ගᙉᗘࡣ㸪࠸ࡎࢀࡢᐤ⏕⹸㥑㝖๣࡟࠾࠸ ࠸࡚ࡣ㸪㎿㏿࡞ࢫࢡ࣮ࣜࢽࣥࢢࢆ┠ⓗ࡜ࡋ㸪཯ᛂ᫬㛫ࡣ ࡚ࡶᐜ㔞ቑຍ࡟ࡘࢀ࡚ୖ᪼ࡋࡓ㸦Fig.4㸧㸬྾཰⥲㔞࡜⺯ 8 ศ࡟タᐃࡋࡓ㸬 ගᙉᗘ࡜ࡢ㛵ಀ࡛ホ౯ࡍࡿ࡜㸪ྛ 3 µL ࡎࡘ྾ᘬࡋࡓሙ ᐊ ࡛⾜ࡗࡓ▼஭ࡽ ࡲࡓ㸪MI ᶆ‽⁐ᾮ࡜ TFAA ཯ᛂᾮࡢΰྜ࡟࠾ࡅࡿ㸪 ྜࡀ᭱ࡶⰋዲ࡛࠶ࡗࡓ㸬ࡼࡗ࡚㸪MI ヨᩱ⁐ᾮ࠾ࡼࡧ ୧⁐ᾮࡢ 1 ࢭࢵࢺࡢ྾ᘬ㔞࡜ࢭࢵࢺᩘ࡟ࡼࡿ⺯ගᙉᗘࡢ TFAA ཯ᛂᾮࡢ 1 ࢭࢵࢺࡢ྾ᘬ㔞ࡣྛ 3 µL ࡎࡘ࡜ࡋ㸪㐪࠸ࢆㄪ࡭ࡓ㸬⤌ࡳྜࢃࡏࡣ㸪MI ᶆ‽⁐ᾮ࡜ TFAA ཯ ⧞ࡾ㏉ࡋ 5 ࢭࢵࢺ྾ᘬࡋࡓ⤌ࡳྜࢃࡏ㸦⥲㔞 30 µL㸧࡟ ᛂᾮࡀྛ 2 µL /1 ࢭࢵࢺࡢሙྜ 4㸪 6 ࠾ࡼࡧ 10 ࢭࢵࢺ㸪ྛ タᐃࡋࡓ㸬 Table 1 Results of validation of the five test agents in livestock products Compound Abamectin Doramectin Eprinomectin Ivermectin Moxidectin Mean Intermediate Fortified level recovery 䠄µg/g䠅䠄%䠅䠄RSD %䠅㸦RSD %䠅 cattle muscle 0.01 86.0 4.8 9.2 swine muscle 0.01 85.5 3.3 9.4 chicken muscle 0.01 85.7 3.3 11.2 cattle muscle 0.01 85.1 5.2 8.4 swine muscle 0.01 84.5 3.3 8.3 chicken muscle 0.01 84.5 3.0 11.4 cattle muscle 0.1 86.1 6.7 8.2 swine muscle 0.01 85.7 3.8 13.1 chicken muscle 0.01 88.1 10.8 17.5 cattle muscle 0.01 84.3 5.2 7.8 swine muscle 0.02 84.5 2.4 8.1 chicken muscle 0.01 86.7 2.3 14.4 cattle muscle 0.02 87.0 6.3 10.4 swine muscle 0.01 87.7 2.4 8.1 chicken muscle 0.01 87.2 2.3 11.5 Sample a) n = 13 b) n = 10 － 33 － a) b) Repeatability b) Precision 3㸬๓ฎ⌮ࡢ᳨ウ ࡜࡞ࡗࡓ㸬 ᐤ⏕⹸㥑㝖๣ࢆศᯒࡍࡿ㝿ࡢヨᩱ⁐ᾮࡢ๓ฎ⌮࡛ࡣ㸪 ∵㸪㇜࠾ࡼࡧ㭜⫗࡟ࡘ࠸࡚ 5 ✀㢮ࡢ࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ ᮏἲ࡛ࡣ㸪 ᵝࠎ࡞ᅛ┦ᢳฟ࣒࢝ࣛࡀ⏝࠸ࡽࢀ࡚࠸ࡿ 12-13)㸬 ⏕⹸㥑㝖๣ࡢጇᙜᛶࢆホ౯ࡋࡓ⤖ᯝ㸪ᅇ཰⋡ࡣ 84.3%࠿ ᖜᗈ࠸ᴟᛶ≀㉁࡟฼⏝࡛ࡁ㸪ỗ⏝࣒࢝ࣛࡢ୍ࡘ࡛࠶ࡿ ࡽ 88.1%ࡢ⠊ᅖ࡜࡞ࡾ㸪 ୪⾜⢭ᗘ࠾ࡼࡧᐊෆ⢭ᗘࡶྵࡵ㸪 HLB ࣒࢝ࣛࢆ⏝࠸ࡓ 14)㸬ࡑࡢ⤖ᯝ㸪㏻▱ἲ࡜ẚ㍑ࡋ㸪 ࢞࢖ࢻࣛ࢖ࣥࡢᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓ㸬ᮏἲࡣ㸪3 ✀㢮ࡢ సᴗ᫬㛫ࡣ⣙ 1/3 ௨ୗ࡜㎿㏿࡞ศᯒࡀྍ⬟࡜࡞ࡗࡓ㸬ࡲ 㣗⫗ࢆᑐ㇟࡜ࡋࡓ⡆౽࣭㎿㏿࡞ࢫࢡ࣮ࣜࢽࣥࢢศᯒἲ࡜ ࡓ㸪࢚ࣉࣜࣀ࣓ࢡࢳࣥࡢᅇ཰⋡ࢆྥୖࡉࡏࡿࡓࡵ࡟㸪 ࡋ࡚᭷⏝࡛࠶ࡿ㸬௒ᚋ㸪௒ᅇࡢᑐ㇟௨እࡢ␆⏘≀࠾ࡼࡧ HLB ࣒࢝ࣛ࡟㈇Ⲵࡍࡿ๓ࡢヨᩱ⁐ᾮ࡟ࢺ࢚ࣜࢳࣝ࢔࣑ ໬ྜ≀࡟ᑐࡋ࡚㐺⏝ᛶࢆ᳨ウࡍࡿࡇ࡜᳨࡛ᰝ㡯┠ࡢᣑ඘ ࣥࢆῧຍࡋࡓ 13)㸬 ࢆ㐍ࡵࡿࡇ࡜ࡀ࡛ࡁ㸪┴ෆࢆὶ㏻ࡍࡿ␆Ỉ⏘≀ࡢᏳ඲Ᏻ ᚰ☜ಖ࡟㈉⊩࡛ࡁࡿ࡜⪃࠼ࡿ㸬 4㸬ጇᙜᛶホ౯ ᩥ ⊩ ࢞࢖ࢻࣛ࢖ࣥ࡟ᚑ࠸ᐇ᪋ࡋࡓῧຍᅇ཰ᐇ㦂㸦┿ᗘ㸧ࡢ ⤖ᯝ㸪୪ࡧ࡟┿ᗘ࠿ࡽ⟬ฟࡋࡓే⾜⢭ᗘ࠾ࡼࡧᐊෆ⢭ᗘ ࡢ್ࢆ Table 1 ࡟♧ࡋࡓ㸬┿ᗘࡣ 84.3%㹼88.1%㸪ే⾜ 1) ཌ⏕ປാ┬་⸆㣗ရᒁ㣗ရᏳ඲㒊㛗㏻▱㸸㣗ရ࡟ṧ␃ ⢭ᗘࡣ 2.3%㹼10.8%㸪ᐊෆ⢭ᗘࡣ 7.8%㹼17.5%࡛඲࡚ ࡍࡿ㎰⸆㸪㣫ᩱῧຍ≀ཪࡣື≀⏝་⸆ရࡢᡂศ࡛࠶ࡿ ࡢࣃ࣓࣮ࣛࢱ࡛࢞࢖ࢻࣛ࢖ࣥࡢ┠ᶆ್㸦ῧຍ⃰ᗘ 0.001 ≀㉁ࡢヨ㦂ἲ࡟ࡘ࠸࡚㸪 ᖹᡂ17ᖺ1᭶24 ᪥ (2005)㸪㸫0.01µg/g㸸┿ᗘ 70%㸫120%㸹ే⾜⢭ᗘ㸺25%㸹ᐊෆ 㣗ᏳⓎ➨ 0124001 ྕ㸪ูῧ㸦᭱⤊ᨵṇ㸸ᖹᡂ 25 ᖺ ⢭ᗘ㸺30%㸪ῧຍ⃰ᗘ 0.01㸫0.1 µg/g㸸┿ᗘ 70%㸫 10 ᭶ 1 ᪥㸧 (2013) 120%㸹ే⾜⢭ᗘ㸺15%㸹ᐊෆ⢭ᗘ㸺20%㸧ࢆ‶ࡓࡋ࡚࠸ 2) ᒣ ཱྀ ⍞㤶㸪 ᰠ ᮏ೺ స㸪 ᒣ ཱྀ㈗ ᘯ㸪 ᑿ ⰼ⿱ Ꮥ㸸 ࡓ㸬௨ୖࡢ⤖ᯝ࠿ࡽ㸪ᮏἲࡣ࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖 LC-MS/MS ࡟ࡼࡿ␆⏘≀୰ࡢ࣏࢚࣮ࣜࢸࣝ⣔ᢠ⏕ ๣ࡢศᯒἲ࡜ࡋ࡚ጇᙜ࡛࠶ࡿࡇ࡜ࢆ☜ㄆ࡛ࡁࡓ㸬 ≀㉁࠾ࡼࡧ࣐ࢡࣟࣛ࢖ࢻ⣔㥑⹸⸆ࡢ୍ᩧศᯒ㸬㣗⾨ ᐃ㔞㝈⏺್࡟ࡘ࠸࡚ࡣ㸪௒ᅇ᳨ウࡋࡓᐤ⏕⹸㥑㝖๣ࡢ ㄅ㸪52㸪281-286㸦2011㸧 ᇶ‽್ࡀ 0.01㹼0.1 µg/g ࡛࠶ࡿࡓࡵ㸪㏻▱ἲࡢᐃ㔞㝈⏺ 3) ᆏᮏ⨾✑㸪➉ⴥ࿴Ụ㸪➲ᮏ๛⏕㸪ⲡ㔝཭Ꮚ㸪ᯘὒ㸪 ್ 0.005 µg/g ࡜ྠ⛬ᗘ࡛࠶ࢀࡤᇶ‽್࡟ᑐࡍࡿ㐺ྰࢆุ 㔠஭⠇Ꮚ㸪⚄⏣┿㌶㸪Ọᒣᩄᘅ㸪᳃ㅬ୍㑻㸸LC-FL ᩿࡛ࡁࡿ㸬ࡑࡇ࡛ࣈࣛࣥࢡヨᩱ࡟ 0.005 µg/g ┦ᙜ࡜࡞ࡿ ཬࡧ LC-MS/MS ࡟ࡼࡿ㣗⫗୰ࡢ࢖࣓࣋ࣝࢡࢳࣥ㸪 ࡼ࠺ᐤ⏕⹸㥑㝖๣ࢆῧຍࡋࡓヨᩱ࡟ࡘ࠸࡚ศᯒࡋࡓ⤖ᯝ㸪 ࢚ࣉࣜࣀ࣓ࢡࢳࣥ㸪ࢻ࣓ࣛࢡࢳࣥཬࡧࣔ࢟ࢩࢹࢡࢳ ࣆ࣮ࢡ㧗ࡉࡣ඲࡚ S/NӍ10 ࡛࠶ࡗࡓ㸬ࡲࡓ㸪᳨㔞⥺ࡣ ࣥࡢศᯒ㸬ᮾி೺Ᏻ◊ࢭᖺሗ㸪60㸪139-145㸦2009㸧 30 µL ὀධ࡛࢚ࣉࣜࣀ࣓ࢡࢳࣥࡀ 0.2㹼50 ng/mL㸪ࣔ࢟ 4) బ⸨┤அ㸪▼஭ᩗᏊ㸪బ⸨᫛⏨㸪᪥㧗฼ኵ㸪㛗ᒸⓏ㸸 ࢩࢹࢡࢳࣥࡀ 0.04㹼20 ng/mL㸪࢔ࣂ࣓ࢡࢳࣥࡀ 0.1㹼50 ᕷ㈍∵⫗୰ࣔ࢟ࢩࢹࢡࢳࣥࡢ LC/MS ࡟ࡼࡿศᯒ࠾ ng/mL㸪ࢻ࣓ࣛࢡࢳࣥࡀ 0.2㹼50 ng/mL㸪࢖࣓࣋ࣝࢡࢳ ࡼࡧ⬡⫫㸪➽⫗⤌⧊࡛ࡢṧ␃ᛶ㸬㣗⾨ㄅ㸪44㸪198-202 ࣥࡀ 0.4㹼50 ng/mL ࡢ⃰ᗘ⠊ᅖ࡛┦㛵ಀᩘ 0.999 ௨ୖࡢ 㸦2003㸧 5) ཌ⏕ປാ┬་⸆㣗ရᒁ㣗ရᏳ඲㒊㛗㏻▱㸸㣗ရ୰࡟ Ⰻዲ࡞┤⥺ᛶࢆ♧ࡍࡇ࡜ࢆ☜ㄆࡋࡓ㸬 ṧ␃ࡍࡿ㎰⸆➼࡟㛵ࡍࡿヨ㦂ἲࡢጇᙜᛶホ౯࢞࢖ࢻ ࣛ࢖ࣥࡢ୍㒊ᨵṇ࡟ࡘ࠸࡚㸪 ᖹᡂ 22 ᖺ 12 ᭶ 24 ᪥㸪 ϫ ⤖ ㄽ 㣗ᏳⓎ 1224 ➨ 1 ྕ (2010) LC-FL ࡟ࡼࡿ␆⏘≀୰ࡢ࣐ࢡࣟࣛ࢖ࢻ⣔ᐤ⏕⹸㥑㝖๣ 6) Payne L.D.㸪Mayo V.D.㸪Morneweck L.A.㸪Hicks ࡢࢫࢡ࣮ࣜࢽࣥࢢศᯒ࡟࠾࠸࡚㸪⮬ືࣉ࣒ࣞ࢝ࣛㄏᑟయ M.B.㸪Wehner T.A.㸸HPLC-Fluorescence Method ໬ࢆ㐺⏝ࡋࡓศᯒἲࡢጇᙜᛶࢆホ౯ࡋࡓ㸬ㄏᑟయ໬཯ᛂ for the Determination of 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