豬肉、豬內臟、雞肉及雞內臟中殘留四環黴素類抗生素之檢測

Detection of tetracycline antibiotic residues in pork,pig viscera, chicken meat and chicken viscera

洪珮菱(Pei-Ling Hung)

指導教授 ： 駱秋英

國立臺灣大學/生物資源暨農學院/畜產學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01147

摘要

本研究探討利用紙錠擴散法（paper disk diffusion method）、微生物受體分析法（microbial receptor assay, Charm II）、酵素連結免疫分析法（enzyme-linked immunosorbent assay, ELSIA）及高效液相層析法（high performance liquid chromatography, HPLC）進行市售畜禽產品中包括羥四環黴素（oxytetracycline, OTC）、四環黴素（tetracycline, TC）、脫氧羥四環黴素（doxycycline, DC）及氯四環黴素（chlortetracycline, CTC）之多重殘留檢測。經紙錠擴散法、Charm II、ELISA及HPLC法檢測為陽性者，再利用液相層析串聯質譜法（liquid chromatography tandem mass spectrometry, LC/MS/MS）進行確認。
LC/MS/MS法使用之層析管柱為Oasis® HLB（內徑 2.1 × 20 mm，15 μm），以含 0.3 % 蟻酸（formic acid）之水溶液及含 0.3 % 蟻酸之乙腈（acetonitrile）溶液為移動相，以梯度模式變換移動相比例，流速為 0.3 mL/min。質譜儀的電噴灑模式為ESI＋，根據四環黴素類抗生素之母離子可能斷裂的離子片段，挑選分子結構、斷裂特異性且強度較大的特徵離子片段做為子離子，利用子離子掃瞄質譜圖（product ion spectrum）確認待測物之斷裂離子，OTC、TC、DC及CTC之母離子質荷比（m/z）分別為461、445、445及479，OTC主要子離子片段之質荷比為426及443，TC為410、427及428，DC為428，CTC為444及462。藉由多重反應偵測模式（multiple reaction monitoring, MRM）同步定性及定量，OTC、TC、DC及CTC所得波峰之滯留時間分別為 4.96, 4.99, 5.10 及 5.05分鐘，將其子離子片段之質荷比以不同濃度（10 ~ 100 ppb）及測得面積繪製標準曲線，相關係數在 0.97 ~ 0.99之間。
比較各檢測方法，Charm II之檢測時間最短，紙錠擴散法花費最低，紙錠擴散法及ELISA法較容易操作；HPLC法及LC/MS/MS法則可對樣品所含抗生素之種類及殘留量正確地定性及定量。由於紙錠擴散法的檢出極限（method detection limit, MDL）於OTC及TC皆為50 ppb，DC及CTC皆為10 ppb；Charm II的MDL之總四環黴素類抗生素為50 ppb；Charm II的MDL之總四環黴素類抗生素為0.5 ~ 6 ppb；HPLC法則OTC及TC皆為5 ppb，DC及CTC皆為10 ppb。而目前衛生署公告的「動物用藥殘留標準｣，畜禽產品中四環黴素類抗生素之殘留總量在肌肉為 0.2 ppm，肝為0.6 ppm，腎為1.2 ppm；因此，本研究中所使用之紙錠擴散法、Charm II及ELISA篩檢法，或HPLC及LC/MS/MS確認法，均適用於此類產品中四環黴素類抗生素之分析。
將各縣市衛生局於 2003年3月~ 2005年4月間至轄區超市、量販店及傳統市場等地抽驗畜禽產品，包括豬肉 55 件、豬內臟 50 件、雞肉 58 件及雞內臟 46 件，共 209 件樣品，進行四環黴素類抗生素之殘留分析，檢驗結果有2件烏骨雞肉檢出殘留DC，不符衛生法規之「動物用藥殘留標準」。

關鍵字

四環黴素類抗生素 ； 高效液相層析儀 ； 高效液相層析串連質譜儀

並列摘要

Residues of oxytetracycline (OTC), tetracycline (TC), doxycycline (DC), and chlortetracycline (CTC) in livestock products were analyzed by paper disk diffusion method, microbial receptor assay (Charm II), enzyme-linked immunosorbent assay (ELSIA), and high performance liquid chromatography (HPLC). The positive samples with paper disk diffusion method, Charm II, ELISA, and HPLC were further confirmed with liquid chromatography tandem mass spectrometry (LC/MS/MS).
LC/MS/MS method was carried out by Oasis® HLB column (2.1 × 20 mm, 15 μm) with mobile phases of 0.3 % formic acid and 0.3 % formic acid in acetonitrile using a linear gradient elution at a flow-rate of 0.3 ml/min. The ionization mode of mass spectrometry was set up with electrospray in positive ion mode, and parent ions of OTC, TC, DC, and CTC were m/z 461, 445, 445, and 479, respectively. The daughter ions of m/z 426 and 443 for OTC, m/z 410, 427, and 428 for TC, m/z 428 for DC, and m/z 444 and 462 for CTC were chosen for the confirmation by product ion spectrum. They were both qualified and quantified by multiple reaction monitoring (MRM) simultaneously. The retention times of OTC, TC, DC, and CTC were 4.96, 4.99, 5.10, and 5.05 min, respectively. Standard curves were plotted by daughter ions against areas from 1 to 100 ppb, and the correlation coefficients (R2) were 0.97 to 0.99.
Compare with each analytical method, the analysis time of Charm II was the shortest, paper disk diffusion method cost minimum and was easily operated, HPLC, ELISA, and LC/MS/MS methods can qualitate and quantitate residual antibiotics concentrations correctly. Method detection limits (MDLs) for OTC and TC with paper disk diffusion method were both 50 ppb, and 10 ppb for DC and CTC. The MDL for total tetracycline antibiotics (TCs) with Charm II was 50 ppb. The MDLs with HPLC were 5 ppb for OTC and TC, and 10 ppb for DC and CTC. The maximum residues limits (MRLs) set up by Department of Health (DOH) of total TCs residues in muscle, liver, and kidney were 0.2, 0.6, and 1.2 ppm, respectively. The analytical methods used in this study including paper disk diffusion method and Charm II for screening, and HPLC and LC/MS/MS for confirmation were suitable for analysis of tetracycline antibiotics in these products.
Residues of TCs were detected from 209 livestock samples including 55 pork, 50 pig viscera, 58 chicken meat, and 46 chicken viscera collected from the supermarkets, sale stores, and traditional markets by the public health bureau from March, 2003 to April, 2005. The results showed that two black-bone-chicken meat were both found DC.

並列關鍵字

tetracycline antibiotics ； HPLC ； LC/MS/MS

參考文獻

3. 行政院農業委員會。2002。動物用藥品管理法。

4. 行政院農業委員會。2005。動物用藥品使用準則。

5. 行政院農業委員會。2005。含藥物飼料添加物使用規範。

1. 中華藥典。1995。行政院衛生署編印。台北。第四版。

22. 張登欽。1977。抗生素在鴨組織之殘留及其消長。中華民國獸醫學會雜誌。3：41-48。