보고서 정보
주관연구기관 |
농림수산검역검사본부 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2012-02 |
과제시작연도 |
2011 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
등록번호 |
TRKO201400001102 |
과제고유번호 |
1545003109 |
사업명 |
2011 수의과학기술개발연구 |
DB 구축일자 |
2014-08-16
|
키워드 |
Anthelmintics.LC-MS/MS.meat.milk.egg.anthelmintic drugs.Residues.QuEChERS.milks.Avermectin drugs.multi-residues.food products of animal origin.
|
DOI |
https://doi.org/10.23000/TRKO201400001102 |
초록
▼
5. 최종 연구결과 요약
가. 축산물내 구충제의 계열별 질량 분석법 개발
○ LC-MS/MS 기기분석조건 확립
- 분석대상물질 (37종) : Abamectin, ivermectin, moxidectin, doramectin, eprinomectin, emamectin, albendazole, thiabendazole, triclabendazole, oxfendazole, flubendazole, fenbendazole, mebendazole, febantel, oxibendazole, praziquantel, mor
5. 최종 연구결과 요약
가. 축산물내 구충제의 계열별 질량 분석법 개발
○ LC-MS/MS 기기분석조건 확립
- 분석대상물질 (37종) : Abamectin, ivermectin, moxidectin, doramectin, eprinomectin, emamectin, albendazole, thiabendazole, triclabendazole, oxfendazole, flubendazole, fenbendazole, mebendazole, febantel, oxibendazole, praziquantel, morantel, pyrantel, levamisole, valnemuline, closantel, rafoxanide, fluazurone, clorsulon 등
- 내부표준물질 (9종) : Albendazole-d3, fenbendazole-d3, flubendazole-d3, 5-OH-mebendazole-d3, thiabendazole-d4, oxibendazole-d7, febantel-d6, salicylanilide, selamectin
- 이동상 용매 선정 및 이동상 분석조건 확립
ㆍ 구충제 37종 동시분석을 위한 이동상 및 gradient program 사용
ㆍ 20 mM ammonium formate와 0.1% formic acid 포함한 증류수와 0.2% formic acid를 포함한 methanol의 시간대 비율을 조절하는 gradient program을 사용
ㆍ컬럼 : Agilent Eclipse plus C18 (2.1x100 mm, 1.8 μm), 유속 : 0.25 mL/min
- 최적의 MS/MS 분석조건 확립
ㆍGas temperature : 350 ℃ ㆍ Gas flow : 13 L/min
ㆍNebulizer : 45 psi ㆍCapillary : 4000 V
- 물질별 최적의 확인ㆍ정량을 위한 정량, 정성이온 선별 및 MRM (multiple reaction monitoring) 분석조건 확립
○ 공시시료별 최적의 시료 전처리법 확립
- 시료 균질화 buffer pH 테스트 : MaIlvain buffer(pH2-6)와 물(pH7) 테스트한 결과 근육, 알은 MaIlvain buffer(pH2), 간장, 신장, 우유는 물(pH7)이 가장 좋은 결과를 나타냄
- 추출용매 테스트 : acetonitrile, 1% acetic acid 포함한 acetonitrile, acetonitrile:methanol, ethyl acetate, acetonitrile:ethyl acetate 용매로 추출능 비교실험 결과 간장, 신장은 1% acetic acid 포함한 acetonitrile 로, 근육, 알, 우유의 경우는 acetonitrile:methanol 로 추출한 결과가 가장 좋았음.
- Quechers clean-up kit : C18, PSA, MgSO4 포함한 kit와 PSA, MgSO4 비교한 결과 PSA, MgSO4 kit 가 더 좋은 결과를 보였음
- 시료별 최종분석법 확립 : 시료 2g에 pH 2 MaIlvain buffer(근육, 알) 혹은 물(간장, 신장, 우유) 2ml과 NaCl 1g 넣고 섞어준 후에 ACN:MeOH(근육, 알, 우유) 혹은 1% acetic acid 포함한 ACN(간장, 신장) 10ml로 추출 후 추출액 15ml clean-up kit에 넣고 정제 후 농축하고, 농축잔사를 DMSO로 녹인 후 여과하여 분석
○ 분석법의 유효성 확인(Method validation)
- Modified Queschers 방법의 시료별로 구충제 잔류허용기준(MRL) 중 최소농도 기준치의 0.5x, 1x, 2x MRL 농도로 분석법 검증 실시
- 구충제의 0.5x, 1x, 2x MRL 농도별 평균 회수율
ㆍ 근육(2-amino flubendazole 제외 34종) : 55.3~118.5 %, 59.8~128.5%, 55.8~121.1 %
ㆍ 간장(avermection 계열제외 26종) : 53.0~134.7 %, 51.2~127.4%, 46.5~130.9 %
ㆍ 신장 : 55.2~127.4 %, 48.1~129.4%, 49.3~136.3 %
ㆍ 우유 : 56.8~139.0 %, 65.4~137.1%, 66.0~137.3 %
ㆍ 알 : 51.3~126.6 %, 53.4~133.3%, 50.8~126.4 %
- 변이계수는 모두 15 % 이내로 국제기준을 충족하였음
- 시료별 매질효과를 제거하기 위해서 matrix curve를 이용하여 정량
- 각 물질별 정량한계는 시료별로 0.1-100 ng/g 수준으로 잔류허용기준 이하로, 정량 가능함
나. 축산물내 기준 설정 대상 구충제의 잔류실태 조사
◦ 잔류조사 시료채취 품목 및 건수
- 품목 : 소, 돼지, 닭의 간장 (구충제 target tissue는 간장임), 우유
- 계획건수 : 소 90건, 돼지 90건, 닭 90건, 우유 45건을 지역별로 균등하게 배분하였음
◦ 잔류조사 결과
- 소 : 90건 검사결과 3건에서 ivermection이 10.5 ~ 45.6 ppb 농도로 검출되었으나, 잔류 허용기준 (0.1 mg/kg) 이하이고, 1건에서 축산물에 잔류허용기준이 없는 chlorfluazuron이 10.8 ppb 농도로 검출
- 돼지 : 100건 검사결과 19건에서 febantel/fenbendazole/oxfendazole(oxfendazole sulfone)이 0.004~0.9 mg/kg 농도로 검출되었으며, 이 중 1건(경북)에서는 잔류허용기준을 초과하여, 잔류원인조사 중임
- 닭 : 90건 조사결과, 검출내역없음
- 우유 : 55건 조사결과, 검출내역없음
Abstract
▼
Title : Simultaneous determination of anthelmintic drug residues in porcine liver a modified QuEChERS-type method and LC-MS/MS
Several anti-parasitic drugs are licensed for treating helminth infections in food-producing animals.
These can be broadly classified as nematicides, flukicides and e
Title : Simultaneous determination of anthelmintic drug residues in porcine liver a modified QuEChERS-type method and LC-MS/MS
Several anti-parasitic drugs are licensed for treating helminth infections in food-producing animals.
These can be broadly classified as nematicides, flukicides and endectocides. Only a limited number of anthelmintics have a maximum residue limit (MRL) in Korea. To monitor anthelmintic drugs in domestic and imported bovine milk, the confirmation method has been developed for the determination of 34 drug residues (Abamectin, albendazole, bithionol, clorsulon, closantel, doramectin, emamectin, febantel, fenbendazole, fluazurone, ivermectin, levamisole, morantel, niclosamide, nitroxynil, oxfendazole-sulfone, oxfendazole, oxyclozanide, rafoxanide, thiabendazole, albendazole-2-amino-sulphone, albendazole-sulphone, albendazole-sulphoxide, triclabendazole, keto-triclabendazole, 5-hydroxy-thiabendazole, oxibendazole, amino-flubendazole, flubendazole, mebendazole, eprinomectin, moxidectin, praziquantel and pyrantel) using liquid chromatography tandem mass spectrometry. The 2g samples were fortified with internal standard and added 1g NaCl and 2ml water. The samples were added 10ml acetonitrile and shaked on a mechanical shaker for 15min. After centrifuging, the extracts were transferred to a dispersive-SPE cleanup kit containing MgSO4(900mg) and PSA(150mg) and then vortexed for 30 sec and centrifuged for 10min at 4000rpm. The supernatant was transferred to a glass tube containing DMSO 200ul and concentrated under nitrogen to 200ul. The concentrates were analyzed by liquid chromatography system, using 10mM ammonium formate and 0.1% formic acid in water and 0.2 % formic acid in methanol gradient on a C18 reversed phase column. Mass spectral acquisition was achieved in an electrospray negative ion mode for 8 analytes (bithionol, closantel, niclosamide, keto-triclabendazole, oxyclozanide, rafoxanide, clorsulon and nitroxynil) and positive ion mode for 26 analytes by applying multiple reaction monitoring of 2 or 3 fragment ion transitions. Good linear relationship (R2>0.99) was observed within the concentration of 10~200ng/g and the interference effect of matrix on the ionization was effectively eliminated. Average recoveries at fortification level 5, 10, 15 ng/g in milk were 40.0-115.4%. The percent relative standard deviation for the described method was less than 15.0%. The limits of quantification ranged from 5 to 10ng/g.
Title : Simultaneous determination of anthelmintic drug residues in bovine milk using a modified QuEChERS-type method and LC-MS/MS
Several anti-parasitic drugs are licensed for treating helminth infections in food-producing animals. These can be broadly classified as nematicides, flukicides and endectocides. Only a limited number of anthelmintics have a maximum residue limit (MRL) in Korea. To monitor anthelmintic drugs in domestic and imported bovine milk, the confirmation method has been developed for the determination of 34 drug residues (Abamectin, albendazole, bithionol, clorsulon, closantel, doramectin, emamectin, febantel, fenbendazole, fluazurone, ivermectin, levamisole, morantel, niclosamide, nitroxynil, oxfendazole-sulfone, oxfendazole, oxyclozanide, rafoxanide, thiabendazole, albendazole-2-amino-sulphone, albendazole-sulphone, albendazole-sulphoxide, triclabendazole, keto-triclabendazole, 5-hydroxy-thiabendazole, oxibendazole, amino-flubendazole, flubendazole, mebendazole, eprinomectin, moxidectin, praziquantel and pyrantel) using liquid chromatography tandem mass spectrometry. The 2g samples were fortified with internal standard and added 1g NaCl and 2ml water. The samples were added 10ml acetonitrile and shaked on a mechanical shaker for 15min. After centrifuging, the extracts were transferred to a dispersive-SPE cleanup kit containing MgSO4(900mg) and PSA(150mg) and then vortexed for 30 sec and centrifuged for 10min at 4000rpm. The supernatant was transferred to a glass tube containing DMSO 200ul and concentrated under nitrogen to 200ul. The concentrates were analyzed by liquid chromatography system, using 10mM ammonium formate and 0.1% formic acid in water and 0.2 % formic acid in methanol gradient on a C18 reversed phase column. Mass spectral acquisition was achieved in an electrospray negative ion mode for 8 analytes (bithionol, closantel, niclosamide, keto-triclabendazole, oxyclozanide, rafoxanide, clorsulon and nitroxynil) and positive ion mode for 26 analytes by applying multiple reaction monitoring of 2 or 3 fragment ion transitions. Good linear relationship (R2>0.99) was observed within the concentration of 10~200ng/g and the interference effect of matrix on the ionization was effectively eliminated. Average recoveries at fortification level 5, 10, 15 ng/g in milk were 40.0-115.4%. The percent relative standard deviation for the described method was less than 15.0%. The limits of quantification ranged from 5 to 10ng/g.
Title : Multi-residue method for the confirmation of six avermectin residues in food products of animal origin by liquid chromatography tandem mass spectrometry
A confirmation method was developed for the rapid determination abamectin, ivermectin, doramectin, moxidectin, emamectin and eprinomectin residues in various food products of animal origin, such as pork muscle, pork liver, pork kidney, milk and egg. Samples were homogenized, extracted by acetonitrile, cleaned via dispersive-clean up procedure using magnesium sulfate and primary secondary amine(PSA) on based QuEChERS method (quick, easy, cheap, effective, rugged and safe). All the six avermectin residues in different animal-food products were simultaneously separated and determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) within 10 min. Data acquisition under positive ESI-MS/MS was performed by applying multiple reaction monitoring(MRM) for both identification and quantification, and mass spectrometric conditions were optimized to increase selectively and sensitivity. The matrix-matched calibration curves for different matrices, such as pork muscle, pork liver, pork kidney, milk and egg, were constructed and the interference effect of different sample matrices on the ionization was effectively eliminated. The LC-MS/MS method was validated with satisfactory linearity, recovery, precision and stability. Matrix-matched calibration curves of abamectin, ivermectin, doramectin, moxidectin, emamectin and eprinomectin in five different matrices were linear(r2≥0.990) in the range 2.5?2000 ug/kg. The limit of detection and quantification for the six avermectins were in the range 0.1-12.2 and 0.2-40.6 ug/kg, respectively.
Recoveries were 62.9-120.9% with good intra- and inter-day precision of 7.0-14.4 and 8.6-15.0%, respectively. The method was rapid, sensitive and reliable, and can be applied to the quantitative analysis of avermectin residues in different animal food products.
목차 Contents
- 총괄요약표 ... 1
- 연구과제 최종결과보고서 ... 3
- I. 연구배경 및 목표 ... 3
- Ⅱ. 연구방법 및 수행전략 ... 5
- Ⅲ. 연구결과 ... 5
- Ⅳ. 연구결과요약 ... 17
- Ⅴ. 고 찰 ... 17
- Ⅵ. 연구결과 활용실적 및 계획 ... 20
- Ⅶ. 참고문헌 ... 21
- Ⅷ. 영문초록 ... 24
- 끝페이지 ... 26
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