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CHI TIẾT
Nghiên cứu ứng dụng phương pháp LC-MS/MS xác định dư lượng một số kháng sinh nhóm macrolid có trong nước thải ao, hồ vùng nuôi trồng thủy sản
Simultaneous determination of azithromycin, clarithromycin, erythromycin and roxithromycin in aquaculture wastewater by LC-MS/MS
Trần Văn Duy Thái, Đỗ Châu Minh Vĩnh Thọ
Số: 528 - Tháng 4/2020 - Trang 45-50

Sự đề kháng kháng sinh ngày càng gia tăng đang là vấn đề bức xúc và được quan tâm trên toàn cầu. Việc phát minh kháng sinh mới ra đời ngày càng ít, ngược lại sự đề kháng các kháng sinh phổ rộng, tác dụng mạnh như aminosid, quinolon, macrolid,… ngày càng phổ biến gây ảnh hưởng nghiêm trọng đến vấn đề sức khỏe con người. Có nhiều nguyên nhân dẫn đến việc đề kháng kháng sinh. Tuy nhiên, nguồn gây đề kháng kháng sinh nghiêm trọng nhất từ việc sử dụng kháng sinh không kiểm soát, quản lý trong nuôi trồng thủy hải sản, chăn nuôi thú y dẫn đến việc tồn dư kháng sinh trong nước thải vượt quá mức cho phép ảnh hưởng xấu đến hiệu quả điều trị bệnh, sức khỏe cộng đồng và môi trường. Sự lây lan của các chủng vi khuẩn kháng thuốc ngày càng trở nên đáng báo động. Trên thế giới đã phát hiện rất nhiều chủng vi khuẩn kháng thuốc, siêu kháng thuốc. Chính vì vậy, để tăng cường kiểm soát dư lượng kháng sinh, các nước phát triển đã có những qui định rất chặt chẽ và kiểm soát nghiêm ngặt. Do có phổ kháng khuẩn rộng và khả năng phân bố tốt vào các mô trong cơ thể nên kháng sinh nhóm aminosid được dùng rất phổ biến trong nuôi trồng thủy sản. Hiện nay, đã có một số nghiên cứu về quy trình phân tích dư lượng nhóm macrolid trong sản phẩm nuôi trồng thủy sản bằng phương pháp sắc ký lỏng. Tuy nhiên, chưa có công bố về quy trình phân tích đồng thời azithromycin, clarithromycin, erythromycin, roxithromycin bằng phương pháp LC-MS/MS có trong mẫu nước thải ao, hồ vùng nuôi trồng thủy sản tại Việt Nam.

Đối tượng nghiên cứu

Gồm 4 kháng sinh: Azithromycin (AZI), clarithromycin (CLAR), erythromycin (ERY), roxithromycin (ROXI).

Phương pháp nghiên cứu

Hỗn hợp chuẩn AZI, CLAR, ERY, ROXI và nội chuẩn AMI được pha trong hỗn hợp dung môi MeOH : nước acid formic 0,1 % (30:70, tt/tt) ở nồng độ khoảng 200 ppb và bơm trực tiếp vào hệ thống khối phổ Xevo TQD, sử dụng chế độ Auto tune trong phần mềm Masslynx 4.1 để tối ưu hóa điều kiện khối phổ để thu được ion mẹ và các phân mảnh con ở chế độ MRM (multi-reaction montoring) có cường độ tín hiệu tối ưu của các kháng sinh cần phân tích với các thông số khối phổ cần khảo sát: ES+ hay ES-, thế mao quản, thế cone, tốc độ dòng khí phun (source gas flow), nhiệt độ buồng ion hóa, nhiệt độ khí bay hơi (desolvation temp), tốc độ dòng khí bay hơi (desolvation gas flow), năng lượng va đập (collision energy).

Kết luận

Nghiên cứu đã xây dựng thành công quy trình định tính, định lượng đồng thời dư lượng kháng sinh AZI, CLAR, ERY và ROXI có trong nước thải bằng phương pháp LC-MS/MS. Quy trình định lượng này đã được thẩm định đạt yêu cầu theo hướng dẫn của AOAC, EC-657/2002. Quy trình phân tích có tính chọn lọc, chính xác, tin cậy cao và được ứng dụng thực tiễn để kiểm tra trên 30 mẫu nước thải thu thập tại tỉnh Bạc liêu và Cà mau. Kết quả phát hiện dư lượng kháng sinh ERY (0,8 – 8,5 ppb) tồn dư ở cả 30 mẫu, không phát hiện ROXI và có 20 % mẫu có dư lượng kháng sinh AZI (1,3 - 3,95 ppb) và 13,3 % có dư lượng CLAR (0,9 - 3,2 ppb). Trong đó, mẫu BL-3 có dư lượng AZI và ERY ở mức nồng độ cao lần lượt là 3,95 ppb và 8,5 ppb.

A high selectivity, sensitivity and reliable reversed-phase (LC-MS/MS) method was successfully validated for simultaneous quantitative and qualitative analysis of azithromycin, clarithromycin, erythromycin and roxithromycin in aquaculture waste water with following optimized conditions: Column - Kinetex Phenyl-Hexyl (2.1 mm x 50 mm x 1.7 µm); Mobile phase acetonitrile-methanol-aqueous formic acid 0.1 %, in gradient mode; Injection volume - 10 μL. Electrospray ionization source was operated in positive mode and multi reaction monitoring (MRM) mode using the transition of m/z 749.65 ® 158.23, m/z 748.65 ® 158.10, 716.59 ® 158.10, m/z 837.52 ® 158.11 and m/z 586.33 ® 162.98 for quantification of AZI, CLAR, ERY, ROXI and AMI (IS), respectively. The method was validated in obsevance of AOAC/2012 and EC-647/2002 guidelines showed to meet well the requirements of wide linearity range (0.05-200 ppb) and high correlation coefficient (R2 > 0.993), quite good accuracy (recovery rate of 86.8 - 105.5 %), intra-day precision (RSD < 4.12 %), typical LOD, LOQ values (0.01 0.05 ppb and 0.04 0.13 ppb, respectively) for the four mentioned antibiotics. By this validated method, 30 collected samples were analyzed. Erythromycin residues were found in all the wastewater samples (100 %). Particularly, BL-3 sample contains ERY and AZI at high levels.

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