Determination of 13 Aminoglycoside Residues in Milk by Weak Cation Exchange Solid Phase Extraction Coupled with Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

In this study, a method based on weak cation exchange (WCX) solid phase extraction (SPE) coupled with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 13 aminoglycoside (apramycin, amikacin, paromomycin, hygromycin B, ribostamycin, kanamycin, streptomycin, gentamicin C₁, dihydrostreptomycin, tobramycin, spectinomycin, neomycin and micronomicin) residues in milk. Samples were extracted using a mixture of ammonium acetate buffer solution and acetonitrile, and purified using a WCX SPE column. Chromatographic separation was achieved using a SILICA SG80 column with gradient elution using a mobile phase made up of 0.1% formic acid aqueous solution and acetonitrile. Detection was accomplished using an electrospray ionization source (EIS) in the positive ion mode, and quantification was carried out using a matrix-matched external standard method. Excellent linear relationships were observed for the 13 aminoglycosides in the concentration range of 5.0-500.0 ng/mL (R² ≥ 0.9983). The limit of detection (LOD) for hygromycin B, ribostamycin, streptomycin, tobramycin, and dihydrostreptomycin were 3 μg/kg, and the limit of quantification (LOQ) were all 10 μg/kg for these aminoglycosides. For apramycin, amikacin, paromomycin, kanamycin, gentamicin C₁, micronomicin, neomycin and spectinomycin, the LODs and LOQs were 15 and 50 μg/kg, respectively. The recovery rates of milk spiked at 1 ×, 2 × and 5 × LOQ levels were 73.8%–107.4%, with relative standard deviation (RSD) of 1.0%-9.8%. This developed method is sensitive, simple, stable, and suitable for the determination of these 13 aminoglycoside residues in milk.