超高效液相色譜-串聯質譜法檢測蜂蜜中九種氨基糖苷類藥物殘留

馬凱，蔡芳葉，黃永橋，吳新文，楊昌彪，李占彬*

1(貴州省分析測試研究院，貴州 貴陽， 550000)2(貴州醫科大學 神奇民族醫藥學院，貴州 貴陽， 550000)

Detectionofnineaminoglycosidesresiduesinhoneybyultra-performanceliquidchromatography-tandemmassspectrometry

MA Kai1,CAI Fangye2,HUANG Yongqiao1,WU Xinwen1,YANG Changbiao1,LI Zhanbin1*

1(Guizhou Academy of Testing and Analysis, Guiyang 550000, China)2(Shenqi Ethnic Medicine College of Guizhou Medical University, Guiyang 550000, China)

ABSTRACTUltra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous detection of aminoglycosides (neomycin, amikacin, dihydrostreptomycin, streptomycin, hygromyxin B, kananmycin, gendamicin, tobramycin, spectinomycin). The residues of aminoglycosides in the honey were extracted with TCA buffer, enriched and cleaned up with HLB solid phase extraction column, and eluted with aqueous elution. And the ion-pairing reagent was added to the final extract and used to optimize the chromatography conditions. Moreover, the extracts were separated on reversed-phase chromatographic column using acetonitrile-water as mobile phase by gradient elution, and detected by liquid chromatography-mass spectrometry under multiple reaction monitoring mode via positive ion mode. All the analytes were calibrated by the external method. The results showed that the method has a good linear relationship in the range of 20-1 000 ng/mL. Moreover, the quantification limit of neomycin, dihydrostreptomycin, streptomycin, kanamycin, and tobramycin is 5 μg/kg; the quantification limit of amikacin, hygromycin B, gentamicin, and spectinomycin is 10 μg/kg. Within the linear range, three concentrations of addition experiments were carried out in the blank honey samples, the sample recovery rates ranged from 72.6%-109.4%, and the relative standard deviations were 2.13%-8.23%. The method developed here has the advantages of easy operation, high sensitivity and good filter effect. By avoiding the use of ion-pairing reagent in the mobile phase, the method could effectively reduce the pollution of mass spectrometer and better meet the detection requirements of the aminoglycoside residues in honey.

Keywordsaminoglycosides; honey; ultra performance liquid chromatograph-tandem mass spectrometry

氨基糖苷類抗生素(aminoglycosides, AGs)是一類含有氨基糖苷健的抗生素，具有廣譜抗菌性，臨床應用廣泛，主要用于治療革蘭氏隱性菌、綠膿桿菌的感染，目前已有數十個天然抗生素或半合成品種[1-2]。隨著養殖業的發展，氨基糖苷類抗生素因其高效、低價，常常被用來治療或者添加到飼料中預防各種動物疾病[3-5]，是使用較為普遍的抗生素種類之一，但AGs同時具有一定的毒副作用，主要表現在腎毒性、耳毒性、神經肌肉毒性等[6]。在我國，AGs在養蜂業中被廣泛使用，主要是防治蜜蜂幼蟲病[7-9]。但是由于蜂農不科學的使用，可能導致蜂蜜及其蜂產品中AGs的殘留，消費者直接食用這類殘留超標的食品會帶來潛在的危害。美國FDA、歐盟委員會和日本厚生勞動省等相關管理機構對動物源性食品中常用的幾種AGs的殘留限量標準作出了規定[10-12]。我國在新頒布實行的GB 31650—2019中對AGs在動物源性食品中也有明確的限量要求[13]。

目前，針對AGs殘留的檢測方法主要有微生物法、酶聯免疫法、液相色譜法、氣相色譜-質譜法和液相色譜-串聯質譜法等[14-16]。液相色譜-串聯質譜法因其高通量、定性定量準確、靈敏度高，而被廣泛應用于此類抗生素的檢測。由于AGs極性較強，在反相色譜柱上保留很弱，其分離檢測仍然是此類藥物殘留檢測的難點之一。本研究以新霉素、阿米卡星、雙氫鏈霉素、鏈……