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 x 2. 1 mm, 5 pi, m), the column temperature being 40 degrees C, the flow rate being 0. 3 mL/min, and the injection volume being 5 pi, L. 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 mu g, g/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 g/kg and 5-10 pi, mu g/kg, respectively. The recoveries ranged from 68. 1% to 111.3% (n= 6) at three levels (LODs, 20 g/kg, and 100 mu g/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.