3.3.1. IPLC-MS/MS

The IPLC with MS/MS is a powerful tool commonly used in the separation of aminoglycosides [56,57]. The widely used IPRs in kanamycin IPLC-MS-MS analysis were HFBA [17,21,42], TCA [15] and Nonafluoropentanoic acid (NFPA) [20].

In a recent example, kanamycin along with other 12 aminoglycoside antibiotics (AGs) was determined in muscle, kidney, liver, honey, and milk [33]. Volatile HFBA was used as IPR, which was compatible with mass spectrometry and could cause strong retention on the reversed-phase column. Separation was performed using Capcell Pak C18 UG120 column (150 × 2.0 mm, 5 μm). Tobramycin was used as an internal standard (IS). Another rapid qualitative determination of 9 AGs including kanamycin in bovine matrix was realized by IPLC-MS/MS on a Waters BEH C18 (50 × 2.1 mm, 1.7 μm) column, using HFBA as IPR and tobramycin as IS. Since the column material particles were only 1.7 μm ID, the analysis time was shortened to 2.4 min [42]. In another multi-residue study, kanamycin together with 35 other antibiotics were detected in chicken meat on a Betasil phenyl hexyl column (50 × 2.1 mm, 3 μm) [21]. HFBA was chosen as an optimal IPR with minor or no ion suppression effect. For kanamycin detection, LOQ was 25 μg/kg, the decision limit CCα was 121 μg/kg, and detection capability CCβ was 143 μg/kg. Another example was the determination of kanamycin and amikacin in serum using IPLC-MS [17]. IPLC separation was achieved through a water-methanol gradient, containing 0.05% HFBA as IPR, on a Thermo ScientificTM HyPURITYTM C18 column (5.0 × 2.1 mm, 3 μm). Apramycin was used as IS solution [17].

Kanamycin, gentamicin and apramycin were quantified in rat plasma by Cheng et al. [15]. In this research, TCA acted as both a protein precipitator and an IPR, which only existed in the sample but not in the mobile phase; ye<sup>t</sup> the system yielded better sensitivity. The absence of TCA in the mobile phase could reduce the contamination of ion source and result in good reproducibility [15]. The retention of AGs was improved on the Phenomenex Synergi C12 Max-RP column (50 × 2.0 mm, 4 μm), using tobramycin as the internal standard.

In a multi-residue analysis, kanamycin and nine other AGs were detected in bovine milk and bovine, swine and poultry muscle using a Waters X-Terra C18 column (100 × 2.1 mm, 3.5 μm) [20]. NFPA was used as IPR in the mobile phase, which improved kanamycin retention in the C18 column and improved its ionization, enhancing the MS/MS signal. Monitoring and screening was performed by LC-QTOF-MS and then confirmed by the LC-MS/MS method. LOQs for kanamycin were 37.5 ng/g for milk and 25 ng/g for muscle. The LODs for kanamycin was 15 ng/g in milk and muscle [20].
