混合型离子交换液相色谱-串联质谱法检测鸡蛋中10种氨基糖苷类药物残留

该研究系统地优化了样品前处理过程及仪器分析中影响氨基糖苷残留分析准确度与灵敏度的各主要因素,建立了鸡蛋中10种氨基糖苷类药物(链霉素、双氢链霉素、潮霉素B、卡那霉素、阿米卡星、妥布霉素、安普霉素、大观霉素、新霉素、庆大霉素)残留量的混合型离子交换液相色谱-串联质谱分析方法。样品经10 mmol/L乙酸铵缓冲溶液(含0.4 mmol/L EDTA和50 g/L三氯乙酸)超声提取,调节pH至6~7后,经PRiME HLB固相萃取柱富集净化,采用SIELC Obelisc R色谱柱分离,以乙腈和1.0%(v/v)甲酸水溶液(含1 mmol/L甲酸铵)为流动相进行梯度洗脱,在正离子、多反应监测模式下经串联质谱仪测定,外标法定量。该方法在5~200 μg/L质量浓度范围内线性关系良好,相关系数(r²)均大于0.99;方法的检出限(LOD, S/N≥3)为2~5 μg/kg,定量限(LOQ, S/N≥10)为5~10 μg/kg。在空白鸡蛋中进行LOQ、20 μg/kg、100 μg/kg 3个水平的加标回收实验,方法的平均回收率(n=6)为68.1%~111.3%,相对标准偏差为1.2%~12.3%。利用该方法对市售的20批次鸡蛋样品进行测定,均未检出目标物。本方法简单、灵敏、准确,可实现鸡蛋中10种氨基糖苷类药物残留的批量检测。

Determination of ten aminoglycoside residues in eggs by mixed-mode ion exchange liquid chromatography-tandem mass spectrometry

Aminoglycosides (AGs) are a class of broad spectrum antibiotics that have bactericidal activity against some aerobic gram-positive and gram-negative organisms. AGs have been extensively employed in animal husbandry for the treatment of bacterial infections or growth promotion. Many countries have issued strict maximum residue levels (MRLs) for AGs in many animal-origin foods. Analysis of AGs is quite challenging due to their physicochemical properties. The lack of any notable chromophores or fluorophores makes direct detection using ultraviolet (UV) or fluorescence (FLR) spectroscopy unfeasible. Therefore, AGs must be derivatized before they can be analyzed by UV or FLR detection techniques. However, the sensitivity of such derivatization methods is relatively low. Methods based on chromatographic analysis coupled with tandem mass spectrometric detection are emerging as the most common way of identification and quantification. The retention of AGs on reversed-phase column is poor due to the presence of various amino and hydroxyl groups in their structures. Therefore, ion-pair chromatography has reportedly been used to improve the retention of AGs. However, electrospray ionization-mass spectrometric detection was hampered by using an ion pairing reagent due to the suppression of ionization. In this study, a method based on mixed-mode ion exchange liquid chromatography-tandem mass spectrometry was developed for the determination of ten AGs residues (streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin, and gentamycin) in eggs. The main factors governing the method, such as the type of chromatographic column used, the type and proportion of the mobile phase used, mass spectroscopy parameters, type and volume of the extraction solvent used, pH, and the type of solid phase extraction (SPE) column, were investigated during sample pretreatment and instrument analysis. The residues of AGs in the test samples were extracted by ultrasonication with 10 mmol/L ammonium acetate buffer solution (comprising 0.4 mmol/L EDTA and 50 g/L trichloroacetic acid). After adjusting the pH, the AG residues in the sample were purified and enriched using a PRiME HLB SPE column. The target analytes were separated on a SIELC Obelisc R column (150 mm×2.1mm, 5 μm), the column temperature being 40 ℃, the flow rate being 0.3 mL/min, and the injection volume being 5 μL. Gradient elution was carried out with acetonitrile and 1.0%(v/v) formic acid aqueous solution (including 1 mmol/L ammonium formate) as the mobile phases. The detection was performed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in multiple reaction monitoring (MRM) mode. The retention times and ionic ratios were used for qualitative analysis, and the peak areas were used for quantitative analysis by the external standard method. Good correlation coefficients exceeding 0.99 were observed for all the AGs in the concentration range of 5-200 μg/L under the optimum conditions. The limits of detection (LODs, S/N ≥ 3) and limits of quantification (LOQs, S/N≥10) for the ten AGs were 2-5 μg/kg and 5-10 μg/kg, respectively. The recoveries ranged from 68.1% to 111.3% (n=6) at three levels (LODs, 20 μg/kg, and 100 μg/kg) in spiked blank egg samples, and the relative standard deviations were 1.2%-12.3%. The matrix effects of the analytes were between 0.3% and 94.3% after purification on the PRiME HLB column. The applicability of the method was validated by analyzing egg samples purchased from local markets. Overall, the method of mixed-mode ion exchange liquid chromatography-tandem mass spectrometry has proven to be a reliable and powerful technique for the simultaneous quantification and confirmation of ten AGs without using an ion pair reagent. Moreover, the clean-up step only required a kind of PRiME HLB sorbent cartridge. The relative parameter data of established method were consistent with GB/T 27404-2008. With simple pretreatment, rapid determination and high sensitivity, the method can be used in the determination of AGs in eggs.