3.3.2. HILIC-MS/MS

HILIC shows a similar separation to normal phase liquid chromatography (NPLC), but it can also use water and volatile bu ffering solution as the mobile phases of RPLC, which are compatible with MS. Therefore, this technique can be applied to separate strong polar and hydrophilic chemical compounds [47].

Kanamycin is extremely hydrophilic because it has many amino and hydroxyl groups, so it has good solubility in the aqueous mobile phases of HILIC [58]. There is no need to use IPR in the mobile phase of HILIC, so it will cause less ion suppression and is fully compatible with MS systems. HILIC can provide higher sensitivity because the organic solvent-rich mobile phase is more volatile and can enhance desolvation and ionization e fficiency of the ESI source [47].

## 3.3.3. ZIC-HILIC-MS/MS Method

In recent years, HILIC-coupled mass spectrometry has been successfully applied to the separation of AGs. The application of HILIC to quantify kanamycin and other 5 AGs in human serum was reported [37], with a zwitterionic ZIC-HILIC column (100 × 2.1 mm). LOQ of the method was 100 ng/mL for kanamycin [37].

Another application was reported in kidney and muscle tissues using a ZIC-HILIC column (100 × 2.1 mm, 5 μm) [44]. The LOQ of kanamycin was low—50 ng/g. It was observed that the high sorption a ffinity of kanamycin to polar surfaces required only polypropylene during sample preparation and storage, thus glass was avoided [44].

Kanamycin together with six other AGs was determined in veal muscle, and a ZIC-HILIC column (50 × 2.1 mm, 5 μm) was applied [22]. The ZIC-HILIC column (50 × 2.1 mm, 3.5 μm) was also used to determine kanamycin in honey, milk and pork samples [27].

Kumar et al. compared six kinds of HILIC stationary phases, including bare silica (anionic), amino phenol (cationic), amide (neutral), and zwitter ionic (ZIC) materials [39]. They concluded that the ZIC phase o ffered the best result, which might be attributed to the ZIC phase providing interaction with both the electropositive amino and the electronegative hydroxyl. The zwitterionic ZIC-HILIC column (150 × 2.1 mm, 3.5 μm) was used to determine Kanamycin A disulphate dihydrate in honey matrix. Amikacin was selected as the internal standard. The linearity range was 70–2000 μg/L. LOD and LOQ were 8 μg/<sup>L</sup> and 27 μg/L, respectively [39]. The year after that research, the above-mentioned method was improved and applied to the honey and kidney sample analysis for kanamycin, and validated according to Commission Decision 2002/657/EC. The CC α were 50 μg/kg for honey and 2733 μg/kg for kidney. LOQs were 41 μg/kg for honey and 85 μg/kg for kidney, respectively. The linearity was narrowed down to 70–495 μg/kg for honey and 200–4375 μg/kg for kidney [40].

In another similar study, kanamycin was detected in muscle, kidney (cattle and pig) and cow's milk using ZIC-HILIC column (100 × 2.1 mm, 5 μm) [29], and the internal standard tobramycin was used. The CC α ranges from 118 μg/kg to 2829 μg/kg, and the CCβ range from 153 μg/kg to 3401 μg/kg [29].

The usage of a new ZIC-HILIC column Obelisc R (100 × 2.1 mm, 5 μm) was also reported when detecting kanamycin in honey, milk and liver [41]. Obelisc R is a mixed-mode zwitterionic-type LiSC stationary phase, which has a similar structure to ZIC-HILIC column. However, Obelisc R is better than ZIC-HILIC because it has better sensitivity for AGs. The CC α ranges from 3 μg/kg to 793 μg/kg, and CCβ ranges from 5 μg/kg to 881 μg/kg [41].

#### 3.3.4. Other HILIC-MS/MS Methods

The HILIC column CAPCELL PAK ST (150 × 2.0 mm, 4 μm) was applied in separation of 15 AGs residues including kanamycin in animal tissues, milk and eggs [43]. Measurement was carried out through a Thermo electron TSQ Quantum MS. The CCβ of kanamycin ranges from 17.4 μg/kg to 21.9 μg/kg, which was lower than the MRL defined by EU, USA and other countries [43].

In another analysis, kanamycin was separated through an Atlantis HILIC column (150 × 2.1 mm, 3 μm) [14], using apramycin as the internal standard. The calibration range was 100–2500 ng/mL for kanamycin in human plasma [14].

A new Click TE-Cys HILIC column (150 × 3 mm, 3 μm) was used to separate kanamycin in milk sample [30]. The LOD and LOQ were 6.1 μg/kg and 19.4 μg/kg, respectively, and the calibration range was 40 ng/mL to 4000 ng/mL [30].

The Phenomenex Kinetex HILIC column (100 × 2.1 mm, 1.7 μm) was applied to analyze kanamycin residues in different kinds of milk [28]. The LOD and LOQ were 13.6 μg/kg and 45.5 μg/kg, respectively, and the calibration range was 45.5 μg/k to 250 μg/kg kanamycin in milk [28].

Waters HSS T3 column (50 × 2.1 mm, 1.8 μm) was used to analyze kanamycin in serum, gentamicin as IS solution. The LOD and LOQ were 0.5 μg/mL and 2.5 μg/mL, respectively [16]. The LOD and LOQ were further expanded to 0.3 μg/mL and 5.0 μg/mL, respectively, and tested in dried blood spots (DBSs) samples in another study [18].
