*2.3. Solid-Phase Extraction*

In many cases, solid-phase extraction (SPE) have been extensively used to extract and concentrate trace organic materials from samples [24–26]. According to packing materials, the solid phase extraction can be classified into four types: Bonded silica gel particle, high polymer material, adsorptive packing material, and mix-mode and specialized column. In this review, the sorbents used for kanamycin analysis mostly belong to the bonded silica gel type, except for molecularly imprinted polymers (MIPs) [27,28] and Chromabond HR-XC [29], which are a high polymer type sorbent.

According to di fferent retention mechanisms, the SPE sorbents used in this review could be further classified into reversed phases sorbents, ion exchange phases sorbents (cation exchanger and anion exchanger), and normal phases sorbents, as shown in Figure 2. The SPE sorbents included in this review are reversed phase sorbents (ODS-C18, Sep-pak tC18, Oasis HLB), strong cation exchanger (Oasis MCX, Chromabond HR-XC), and weak cation exchanger (WCX, CBA, CBX).

**Figure 2.** The classification and choice of solid-phase sorbents. (A) Strong cation exchanger; (B) Weak cation exchanger; (C) Strong anion exchanger; (D) Weak anion exchanger; (E) Hydrophilic-lipophilic balanced co-polymer-reversed phased retention.

The reversed phase sorbent Sep-pak tC18 [30] and ODS-C18 [31] was able to extract the non-polar compound from the aqueous sample. The porous silica particles surface bonded with C18 or other hydrophobic alkyl groups. Because of its hydrophobic distribution mechanism, it has strong retention with hydrophobic compounds, but weak retention with hydrophilic compounds. Before use, the cartridge must be preconditioned with a water-soluble organic solvent to solvate the alkyl chains, and then equilibrated with water. It must then be loaded with aqueous samples, followed by eluting with water. A drawback is that before loading the sample, the sorbent must be kept wet, otherwise it will result in low analyte recovery or poor reproducibility. The AccuBOND ODS-C18 cartridge was used for cleanup in soil samples with a kanamycin recovery range from 72.3% to 92.5% [31].

The HLB cartridge has both hydrophilic and lipophilic functional groups, which is a new hydrophilic-lipophilic balanced wettable reversed-phase sorbent [32]. It can overcome the limitations of traditional reversed phase sorbents. Firstly, it is wettable with water, so it has good recovery and reproducibility even the cartridge runs dry during processing. Secondly, it is available for a wide range of compounds including both polar and non-polar chemicals. In muscle, kidney, liver, honey and milk samples, kanamycin was extracted through two consecutive Oasis HLB cartridges (3 mL/60 mg) with a recovery range from 71% to 104% [33].

Ion exchange sorbents (MCX, WCX, MAX, WAX) were found to extract ionizable compounds from the aqueous sample. Because of the ion exchange and hydrophobic distribution mechanisms, the ion exchange sorbents have a strong retention to ionic compounds that have the opposite electric charge of the sorbent carrier, but very weak retention to other compounds [34].

The MCX cartridge is a mixed-mode reversed-phase strong cation exchanger with a pKa of less than 1.0; its sulfonic acid groups have high selectivity to alkaline compounds. Prior to use, it was preconditioned with MeOH, followed by water, then loaded with the extracted sample. Kanamycin is a weak alkaline compound with a pKa of 7.2. At pH lower than 5, the kanamycin was essentially charged and absorbed in the cation cartridge; thus, the sample was extracted with strong acid of 0.1 mol/L HCl [35], 10% TCA [36] or 9% FA [37] aqueous solution prior to loading. At pH higher than 9.0, the kanamycin was neutralised, and the elution could take place. Thus, ammonium methanol solution (1–25%, pH 9.2) was applied to elute kanamycin from the sorbent.

The MCX cartridge was used to extract samples in animal feeds [35], swine tissue [36] and human serum [37] with a kanamycin recovery of 98.4–106% [35] and 80.7% to 91.3% [36], respectively.

The Chromabond HR-X cartridge was styrene-divinylbenzene copolymer based strong cation exchanger. Its surface bonded to benzenesulfonic acid groups [38]. Thus, its retention mechanism was similar to the Oasis MCX sorbent. It was used for cleanup in muscle, kidney and milk samples, with kanamycin recovery ranging from 95% to 102% [29].

The WCX cartridge is a mixed-mode reversed-phase weak cation exchanger with pKa of about 5.0. Its carboxyl groups have high selectivity to strong alkaline compounds. Prior to use, it was preconditioned with MeOH, followed by water, then loaded with the extracted sample. At pH over 6.5, this sorbent was essentially charged to retain kanamycin, so the PH of extracted sample was adjusted to 6.5~7.5 with NaOH and HCl prior to loading. At pH lower than 3.0, the charge on the sorbent was neutralised, and the elution could take place. So, ammonium formate bu ffer solution (pH 3) [39–41] orformic acid 10% [42], 40% methanol solution [30] were applied to elute kanamycin from the sorbent.

The WCX cartridge (Accell plus CM) was used for cleanup in honey and kidney samples, with kanamycin recovery range from 82% to 105% [40]. The Taurus WCX cartridge was used in honey, milk and liver samples, with a kanamycin recovery range from 58% to 96% [41]. Consecutive SPE cleanup using Sep-pak tC18 and Oasis WCX were applied in milk sample, with a reduced matrix effect and improved absolute kanamycin recoveries from 69.9% to 77.9% [30]. Lehotay et al. used DPX SPE (conducted in a pipet tip rather than a cartridge or centrifuge tube) with 5 mL tips (10 per row) containing 50 mg WCX sorbent for the cleanup of bovine kidney, liver, and muscle samples. The recovery of kanamycin was from 82% to 94% at a spiking level of 0.1 μg/g [42].

The carboxylic acid (CBA) cartridge was a weak cation exchanger with pKa of about 4.8, similar to the Oasis WCX cartridge. Ammonium acetate (pH 7.0) was chosen as the conditioning solution. The pH of the extracts was adjusted to pH 7.5. The 2% FA in methanol was applied to elute kanamycin from the sorbent. It was used to purify the extracts in muscle, liver, kidney, milk and egg samples [43].

The carboxypropyl (CBX) cartridge was a weak cation exchanger, similar to the Oasis WCX cartridge. The pH of the tissue extract was adjusted to pH 7.0, and then passed slowly through the CBX column. The column was washed with water and then eluted with 5 mL of acetic acid-water-methanolmixture (1:1:8) to ge<sup>t</sup> kanamycin; final recoveries range from 81.1% to 104% [44].

Recently, novel sorbents such as molecularly imprinted polymers (MIPs) have emerged [28,45,46]; they are synthetic materials that provide complementary binding sites to specifically capture the target analyte kanamycin. Thus, they are ideal for selective extraction and to reduce the matrix effect. MISPE-Aminoglycoside cartridges (50 mg, 3 mL) were used for extraction and clean-up processes for honey, pork and milk samples, achieving kanamycin recoveries within 78.2–97% and 70–106%, respectively [27,28]. The matrix effect results were both lower than 15%, showing that this method provided very clean extracts [27,28].

## **3. Liquid Chromatography Methods**

HPLC is a conventional analytical method because of its low demand for instruments, and has been widely used in the analysis of kanamycin in different samples [36]. Depending on the retention mechanisms, the chromatographic columns used in this review were mainly divided into three types: Reversed-phase (RP) column, mixed-mode column, and hydrophilic interaction chromatography (HILIC) column. Each column type is marked in Tables 1–3. The different detectors coupled with HPLC mainly include UV/Fluorescence, Evaporative Light Scattering Detector (ELSD)/Pulsed Electrochemical Detection (PED), and Mass Spectrometry. The following content will be unfolded mainly on the basis of the classification above.

#### *3.1. UV and Fluorescence-Reserved Phase Liquid Chromatography after Derivatization*

Kanamycin is very polar and lacks chromophore or fluorophore, which makes it difficult to separate using traditionally reverse phase liquid chromatography (RPLC) recruiting UV or fluorescence monitoring. To overcome this problem, researchers have employed many different pre-column or post-column derivatization agents [47].

Derivatization of kanamycin is mainly focused on modifying its primary amine functions. The commonly used pre-column derivatization reagents include Phenylisocyanate (PIC) [48], 4-chloro-3,5-dinitrobenzotrifluoride(CNBF) [31], 1-naphthyl isothiocyanate (NITC) [13] and 9-fluorenylmethyl chloroformate (FMOC-Cl) [23]. Another reagen<sup>t</sup> *o*-phthaldialdehyde (OPA) [35] can also be employed both in pre-column and post-column derivatization. Table 1 shows HPLC applications in the analysis of kanamycin with UV and fluorescence detection.
