LC-FL ―プレカラム自動誘導体化法の適用
රᗜ┴❧ᗣ⏕ά⛉Ꮫ◊✲ᡤᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮◊✲ሗ࿌ ➨ ྕ 㹙ࣀ࣮ࢺ㹛 /&)/ ࢆ⏝࠸ࡓ␆⏘≀୰࠾ࡅࡿ࣐ࢡࣟࣛࢻ⣔ᐤ⏕㥑㝖ࡢ ࢫࢡ࣮ࣜࢽࣥࢢศᯒἲࡢ㛤Ⓨጇᙜᛶホ౯ ̿ࣉ࣒ࣞ࢝ࣛ⮬ືㄏᑟయἲࡢ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 ࣐ࢡࣟࣛࢻ⣔ᐤ⏕㥑㝖ࡣ㸪∵㸪㇜࡞␆⏘≀ࡢ ṧ␃ᇶ‽ࡀタࡅࡽࢀ㸪㏆ᖺ࡛ࡣ㸪࣓࣋ࣝࢡࢳࣥࡀ᳨ ᡤ➼࠾࠸࡚ 2010 ᖺ㸪2011 ᖺ⥆ࡁ 2013 ᖺࡶᇶ‽ ࡧຍᕤရࡢṧ␃ࡀᠱᛕࡉࢀ࡚࠸ࡿ㸬ᡃᅜ࡛ࡶ㣗⫗୰ࡢ 1 ᗣ⛉Ꮫ㒊 2 ⌧රᗜ┴୰☻┴Ẹᒁ ್㉸㐣㐪࡞ࡾ㸪᳨ᰝ௧࠶ࡿ࠸ࡣ᳨ᰝᙉ࡞ࡢᥐ 㸨ูๅㄳồඛ㸸ࠛ652-0032 ⚄ᡞᕷරᗜ༊Ⲩ⏣⏫ 2-1-29 ⨨ࡀࡽࢀࡓ㸬රᗜ┴࠾࠸࡚ࡣ㣗ࡢᏳᏳᚰࡢ☜ಖࡢ රᗜ┴❧ᗣ⏕ά⛉Ꮫ◊✲ᡤ ᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮ ୍⎔ࡋ࡚㸪ື≀⏝་⸆ရࡢ᳨ᰝ㡯┠ࡢᣑࢆ㐍ࡵ࡚࠾ ᗣ⛉Ꮫ㒊 ᚋ⸨ ᧯ ࡾ㸪ᙜ◊✲ࢭࣥࢱ࣮࡛ࡣ㸪࣐ࢡࣟࣛࢻ⣔ᐤ⏕㥑㝖 － 29 － ࢆ᪂ࡓ࡞ᑐ㇟ࡋ᳨࡚ᰝἲࡢ㛤Ⓨྲྀࡾ⤌ࢇ࡛࠸ࡿ㸬␆ 1㸬ヨᩱ ⏘≀୰࣐ࢡࣟࣛࢻ⣔ᐤ⏕㥑㝖ࡢヨ㦂ἲࡋ࡚ࡣ රᗜ┴ෆ࡛ὶ㏻ࡋ࡚࠸ࡓᕷ㈍ࡢ∵㸪㇜࠾ࡼࡧ㭜ࡢ➽⫗ ࠕ࣓࣋ࣝࢡࢳࣥ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ග᳨ฟჾࡁ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ගㄏᑟయࣉ࣒࡛ࣞ࢝ࣛࡢ⮬ື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ࡢ࣐ࢡࣟࣛࢻ ᕼ㔘ࡋ㸪ᶆ‽⁐ᾮ࠾ࡼࡧᶆ‽ΰྜ⁐ᾮࢆㄪ〇ࡋࡓ㸬 ⣔ᐤ⏕㥑㝖ࡢศᯒ࡛Ⰻዲ࡞⤖ᯝࡀᚓࡽࢀࡓࡢ࡛ሗ࿌ ㄏᑟయᛂヨᾮ㸸1-࣓ࢳ࣑ࣝࢲࢰ࣮ࣝ/↓Ỉࢭࢺࢽ ࡍࡿ㸬 ࢺࣜࣝΰᾮ㸦1㸸1,v/v㸧 㸦௨ୗ㸪MI ⁐ᾮࡍࡿ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 ᛂᾮࡍࡿ㸧ࢆ⏝ㄪ〇ࡋࡓ㸬 ᛶホ౯࢞ࢻࣛࣥࡘ࠸࡚ࠖ5)㸦௨ୗ㸪࢞ࢻࣛࣥ ࡑࡢࡢヨ⸆㸸ࢭࢺࢽࢺࣜࣝ㸪࣓ࢱࣀ࣮ࣝࡣග⣧⸆ ࡍࡿ㸧ᚑࡗ࡚ጇᙜᛶホ౯ࢆ⾜ࡗࡓ⤖ᯝࡶేࡏ࡚ሗ࿌ ᕤᴗ(ᰴ)〇㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ⏝ࢆ⏝࠸ࡓ㸬1-࣓ࢳ ࡍࡿ㸬 ࣑ࣝࢲࢰ࣮ࣝࡣග⣧⸆ᕤᴗ(ᰴ)〇ࢆ㸪↓Ỉ◲㓟ࢼࢺ ࣒ࣜ࢘㸪ࢺ࢚ࣜࢳ࣑ࣝࣥ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◊✲ᡤᗣ⛉Ꮫ◊✲ࢭࣥࢱ࣮◊✲ሗ࿌ ➨ ྕ ᅛ┦ᢳฟ⏝࣒࢝ࣛ㸸Oasis HLB㸦60 mg㸪3 mL㸪Waters ᐃ㔞ࢆጉᐖࡍࡿࣆ࣮ࢡࡀ↓࠸ࡇࢆ☜ㄆࡋࡓ㸬 ♫〇㸧ࡣ㸪࠶ࡽࡌࡵࢭࢺࢽࢺࣜࣝ 2 mL ࠾ࡼࡧ 0.1% 7.2 ┿ᗘ㸦ᅇ⋡㸧࠾ࡼࡧ⢭ᗘ ࢺ࢚ࣜࢳ࣑ࣝࣥỈ⁐ᾮ/ࢭࢺࢽࢺࣜࣝΰᾮ㸦7㸸3,v/v㸧 2 mL ࡛ࢥࣥࢹࢩࣙࢽࣥࢢࡋ࡚⏝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 ᐃࢆᐇࡋ 3㸬⨨ ࡓ(n=13)㸬ᚓࡽࢀࡓࢹ࣮ࢱࡽ┿ᗘࢆồࡵ㸪ࡑࡢ⤖ᯝࢆ HPLC-FL㸸(ᰴ)ᓥὠ〇సᡤ〇 LC-20A ୍ඖ㓄⨨ศᩓศᯒࡼࡾ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ࢺ࢚ࣜࢳ࣑ࣝࣥ/ࣜࣥ㓟/Ỉ/࣓ࢱࣀ࣮ࣝΰ ᾮ㸦 0.1:0.1:50:50 㸧 㸹ࢺ࢚ࣜࢳ࣑ࣝࣥ /ࣜࣥ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࠼࡚࣍ࣔࢪࢼࢬᚋ㸪5 A ࢁ⣬ (1) C (2) 㸦ADVANTEC㸧 ࡛ࢁ㐣ࡋ㸪 ᢳฟᾮࢆᚓࡓ㸬 ࡇࡢ࠺ࡕ 1 mL ࢆศྲྀࡋ㸪0.1%ࢺ࢚ࣜࢳ࣑ࣝࣥỈ⁐ᾮ 2 mL ࢆΰᚋ, (3) (4) (5) HLB ࣒࢝ࣛ㈇Ⲵࡋࡓ㸬0.1 %ࢺ࢚ࣜࢳ࣑ࣝࣥ/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⁐ᾮࡋ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‽⁐ᾮࢆᅛࡋ㸪1-࣓ࢳࣝ which eprinomectin (0.025 Pg/mL), moxidectin (0.005 ࣑ࢲࢰ࣮ࣝ/↓Ỉࢭࢺࢽࢺࣜࣝΰᾮ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㸪࢞ࢻࣛࣥᚑ࠸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 ࢭࢵࢺࡋ࡚㸪⧞ࡾ㏉ࡋ 5 ࢭࢵࢺࡢィ 30 µL 1㸬LC-FL ⛣ື┦ࡢ᳨ウ ࡢΰྜ⁐ᾮࢆࢧࣥࣉ࣮ࣝࣝࣉෆಖᣢࡋࡓ᮲௳࡛㸪LC ⛣ື┦ࡣ㸪㏻▱ἲྠᵝࡢ࣓ࢱࣀ࣮ࣝ-Ỉ⣔ࢆ⏝࠸ࡓ㸬 ࡢὀධ๓ࡢಖᣢ㛫㸪ࡍ࡞ࢃࡕᛂ㛫ࡼࡿ⺯ගᙉ ᶆ‽⁐ᾮࡼࡿࢯࢡࣛࢸࢵࢡศᯒ࡛ࡣ㸪⛣ື┦ࡀ ᗘࡢኚࢆㄪࡓ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࠾࠸࡚ࡶᛂ㛫ࡢ ࢀࡶⰋዲ࡞ศ㞳ࣆ࣮ࢡࡀᚓࡽࢀࡓ㸬ࡋࡋ㸪∵⫗ヨᩱ 䠄mV䠅 ῧຍࡋࡓ㝿㸪ヨᩱ⏤᮶ࡢࣆ࣮ࢡࡀศᯒࢆጉᐖࡋࡓ㸬ࡑࡇ 6.0 ࡛㸪ࢢࣛࢪ࢚ࣥࢺኚ᭦ࡋࡓ⤖ᯝ㸪ጉᐖࣆ࣮ࢡࡢᙳ㡪ࡀ A ᅇ㑊࡛ࡁࡓ㸬ࡉࡽ㸪ࢺ࢚ࣜࢳ࣑ࣝࣥ࠾ࡼࡧࣜࣥ㓟ࢆ 4.0 ῧຍࡍࡿࡇ࡛㸪࢚ࣉࣜࣀ࣓ࢡࢳࣥࡢࣆ࣮ࢡᙧ≧ࡣⰋዲ 㸬 ࡛࠶ࡗࡓ 6)㸦Fig.2㸧 2.0 2㸬⺯ගㄏᑟయ᮲௳ࡢ᳨ウ 0.0 ⺯ගㄏᑟయࡣ㸪ᐊ ࡛ᛂࡀ㐍⾜ࡍࡿ 1-࣓ࢳ࣑ࣝ 6.0 ࢲࢰ࣮ࣝࢺࣜࣇࣝ࢜ࣟ㓑㓟↓Ỉ≀ࢆ⏝࠸ࡿ᪉ἲࢆ᥇⏝ ࡋࡓ B 7-10)㸬ᶆ‽ΰྜ⁐ᾮࢆᅛᚋ㸪1-࣓ࢳ࣑ࣝࢲࢰ࣮ 4.0 ࣝ/↓Ỉࢭࢺࢽࢺࣜࣝΰᾮ㸦1㸸1,v/v㸧0.2 mL ࡛⁐ゎࡋ 㸦௨ୗ㸪MI ᶆ‽⁐ᾮࡍࡿ㸧 㸪ࡑࡢ୍㒊ࢆ LC ⏝ࣂ 2.0 Intensity ࣝ⛣ࡋࡓ㸬ูࣂࣝ 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 ࢻࢵࢳᵝࡢ≧ែ࡛㟼⨨ࡋ࡚ᛂࡉࡏࡓ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◊✲ᡤᗣ⛉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 ศẚ㍑ࡋ࡚ 30 ศࡢ⺯ගᙉᗘࡣ⣙ 110%࡛࠶ࡗ ሙྜ 2㸪3 ࠾ࡼࡧ 4 ࢭࢵࢺࡋࡓ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ቑຍࡘࢀ࡚ୖ᪼ࡋࡓ㸦Fig.4㸧 㸬྾⥲㔞⺯ 8 ศタᐃࡋࡓ㸬 ගᙉᗘࡢ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ࢭࢵࢺᩘࡼࡿ⺯ගᙉᗘࡢ TFAA ᛂᾮࡢ 1 ࢭࢵࢺࡢ྾ᘬ㔞ࡣྛ 3 µL ࡎࡘࡋ㸪 㐪࠸ࢆㄪࡓ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ࡢ࣐ࢡࣟࣛࢻ⣔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㏻▱ἲẚ㍑ࡋ㸪 ࢞ࢻࣛࣥࡢᇶ‽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ヨᩱ⁐ᾮࢺ࢚ࣜࢳ࣑ࣝ ྜ≀ᑐࡋ࡚㐺⏝ᛶࢆ᳨ウࡍࡿࡇ᳨࡛ᰝ㡯┠ࡢᣑ ࣥࢆῧຍࡋࡓ 13)㸬 ࢆ㐍ࡵࡿࡇࡀ࡛ࡁ㸪┴ෆࢆὶ㏻ࡍࡿ␆Ỉ⏘≀ࡢᏳᏳ ᚰ☜ಖ㈉⊩࡛ࡁࡿ⪃࠼ࡿ㸬 4㸬ጇᙜᛶホ౯ ᩥ ⊩ ࢞ࢻࣛࣥᚑ࠸ᐇࡋࡓῧຍᅇᐇ㦂㸦┿ᗘ㸧ࡢ ⤖ᯝ㸪୪ࡧ┿ᗘࡽ⟬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ရṧ␃ ⢭ᗘࡣ 2.3%㹼10.8%㸪ᐊෆ⢭ᗘࡣ 7.8%㹼17.5%࡛࡚ ࡍࡿ㎰⸆㸪 㣫ᩱῧຍ≀ཪࡣື≀⏝་⸆ရࡢᡂศ࡛࠶ࡿ ࡢࣃ࣓࣮ࣛࢱ࡛࢞ࢻࣛࣥࡢ┠ᶆ್㸦ῧຍ⃰ᗘ 0.001 ≀㉁ࡢヨ㦂ἲࡘ࠸࡚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⤖ᯝࡽ㸪ᮏἲࡣ࣐ࢡࣟࣛࢻ⣔ᐤ⏕㥑㝖 LC-MS/MS ࡼࡿ␆⏘≀୰ࡢ࣏࢚࣮ࣜࢸࣝ⣔ᢠ⏕ ࡢศᯒἲࡋ࡚ጇᙜ࡛࠶ࡿࡇࢆ☜ㄆ࡛ࡁࡓ㸬 ≀㉁࠾ࡼࡧ࣐ࢡࣟࣛࢻ⣔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➉ⴥỤ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⫗୰ࡢ࣓࣋ࣝࢡࢳࣥ㸪 ࡼ࠺ᐤ⏕㥑㝖ࢆῧຍࡋࡓヨᩱࡘ࠸࡚ศᯒࡋࡓ⤖ᯝ㸪 ࢚ࣉࣜࣀ࣓ࢡࢳࣥ㸪ࢻ࣓ࣛࢡࢳࣥཬࡧࣔ࢟ࢩࢹࢡࢳ ࣆ࣮ࢡ㧗ࡉࡣ࡚ S/NӍ10 ࡛࠶ࡗࡓ㸬ࡲࡓ㸪᳨㔞⥺ࡣ ࣥࡢศᯒ㸬ᮾிᏳ◊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♧ࡍࡇࢆ☜ㄆࡋࡓ㸬 ṧ␃ࡍࡿ㎰⸆➼㛵ࡍࡿヨ㦂ἲࡢጇᙜᛶホ౯࢞ࢻ ࣛࣥࡢ୍㒊ᨵṇࡘ࠸࡚㸪 ᖹᡂ 22 ᖺ 12 ᭶ 24 ᪥㸪 ϫ ⤖ ㄽ 㣗ᏳⓎ 1224 ➨ 1 ྕ (2010) LC-FL ࡼࡿ␆⏘≀୰ࡢ࣐ࢡࣟࣛࢻ⣔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ㄏᑟయᛂ for the Determination of Eprinomectin Marker ࢆ⮬ືࣉ࣒࡛ࣞ࢝ࣛ⾜࠺ࡇࡼࡗ࡚㸪ヨᩱㄏᑟయ Residue in Edible Bovine Tissue㸬J㸬Agric㸬Food ᛂ⁐ᾮࡢΰ᧯స࡞ࡢ↹㞧ࡉࡀゎᾘࡋ㸪ㄏᑟయ Chem㸬 㸪45㸪3501-3506㸦1997㸧 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