3.1.2. Post-Column Derivatization

Post-column derivatization requires more complicated instruments [47] and is confined by reaction time and the solvent system. However, the chemical reaction does not need to be complete since it is repeatable, and long-term stability of the derivative is not a concern [47].

OPA could be used as both pre-column [35] and post-column [19] derivatization agent. Post-column derivatization of kanamycin using OPA was achieved after RPLC with a C8 TSK ODS 120T (150×4.6 mm, 5 μm) or Hypersil ODS column (150 × 3.2 mm, 5 μm). Both columns led to good results. The HPLC-FL chromatogram of the kanamycin-OPA post-column derivative is shown in Figure 9 [19]. LOD was 0.2 mg/<sup>L</sup> in pig feeds, detected with fluorescence measurement at excitation wavelength of 355 nm and emission wavelength of 415 nm.

**Figure 9.** The HPLC-FL chromatogram of the kanamycin-OPA post-column derivative. Peak 1: kanamycin-OPA derivative, with kanamycin in swine feed at a level of 120 mg/kg.

#### *3.2. ELSD and PED-Ion Pair Liquid Chromatography*

In ion-pair liquid chromatography (IPLC) methods, the ion pairing reagen<sup>t</sup> (IPR) is used as a mobile phase additive, which interacts with the RPLC stationary phase [47] and allows separating of the ionic and highly polar compounds on RP-HPLC columns. Alkyl sulfonates compounds like octanesulfonate could be used as IPR [10]. Meanwhile, volatile TFA and heptafluorobutyric acid (HFBA) [50,52] could also be used as IPR when coupled with MS detection. Since the high potency of IPR (>20 mM) is harmful to the column packing material, it is ideal to minimize the potency so as to achieve appropriate retention and peak shape [47].

In the IPLC method, an extra buffer system is required to maintain a stable pH of the mobile phase [47]. Ammonium acetate and phosphate are the most frequently used buffer solutions. Phosphate buffer is compatible with UV but not with an MS or ELSD detector. Meanwhile, ammonium acetate buffer is incompatible with UV but compatible with an MS detector [47].

#### 3.2.1. Evaporative Light Scattering Detection (ELSD)

ELSD is increasingly being applied in IPLC for compounds without chromophores, because it eliminates the necessity of derivatization [50]. For HPLC applications in the analysis of kanamycin with ELSD detection, refer to Table 2. Some applications of the IPLC-ELSD methods are discussed hereinafter.

The separation of kanamycins A, B, and sulfate were validated through a novel IPLC-ELSD method without the derivatization step. Chromatographic separations were carried out with a Spherisorb ODS-2C18 column (250 × 4.6 mm, 5 μm) using 11.6 mM HFBA as IPR. The LODs were 0.20 μg/mL for kanamycin A, 1.4 μg/mL for kanamycin B and 2.3 μg/mL for kanamycin sulfates [50]. Another example of the IPLC-ELSD method was determination of kanamycin B and tobramycin impurities with HFBA as IPR. Kanamycin was separated on an Agilent SB-Aq C18 column (150 × 4.6 mm, 5 μm) after sample extraction on a weak acidic cation-exchange resin CD180 [52].

HILIC is a very important alternative approach for the separation of kanamycin. A new HILIC-coupled ELSD method was applied for kanamycin detection. In this research, a HILIC column Click TE-Cys (150 × 4.6 mm, 5 μm) was applied for selective separation of kanamycin. High buffer potency (≥50 mM) and low pH (2.7 or 3.0) are required for the mobile phase to improve peak shape and selectivity [51].

#### 3.2.2. Pulsed Electrochemical Detection (PED)

HPLC together with pulsed electrochemical detector (PED) has been adopted in US Pharmacopoeia [50]. Analysis of kanamycin A and its related substances using IPLC coupled with PED has been reported [10,53]. For IPLC-ELSD applications in the analysis of kanamycin, refer to Table 2.

In Adams' work, octanesulfonate was selected as the IPR. To improve the sensitivity of PED detection, 0.5 M NaOH was added in the post-column e ffluent to adjust the pH to 13. The packing materials of column PLRP-S (250 × 4.6 mm, 8 μm) was poly (styrene-divinylbenzene). Eight components including kanamycin B and D were separated, and the method was applied to commercial samples [53].

Manyanga improved Adams' work [53] and applied the method to silica-based columns Platinum EPS (150 × 4.6 mm, 3 μm). The amount of salt in the mobile phase was reduced to improve stability, with the use of IPR of octanesulfonate remaining [10]. This method indicated better selectivity and sensitivity.

Nevertheless, the PED method has some disadvantages [54]. First, experience is important for repeatable quantitative results. Second, long equilibration time is required after washing of the electrodes of the electrochemical cell. Therefore, the PED method demands further improvement.

## *3.3. Liquid Chromatography-Mass Spectrometry*

LC-MS/MS is a common analytical method in antibiotics residue analysis [33]. Applications of MS with RPLC, IPLC, HILIC or ZIC-HILIC in the analysis of kanamycin are discussed below; refer to Table 3. Mass spectral acquisition was performed in positive-ion mode by applying multiple reactions monitoring (MRM) using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) to detect kanamycin in this review. Kanamycin B produced [M + H]+ ions at *m*/*z* 484, which is the precursor ion (Q1). The most abundant product ion (Q3) from the fragmentation was at *m*/*z* 324, and the relatively abundant product ions were *m*/*z* 205 and *m*/*z* 163. The three transition Q3 fragments of kanamycin were 163 for KANA1, and 324 or 205 for KANA2, respectively. The MS/MS spectra of kanamycin B was shown in Figure 10, and the fragmentation pathway of kanamycin B was shown in Figure 11 [55].

**Figure 10.** MS/MS spectra of [M + H]+ ions of kanamycin B at *m*/*z* 484.

**Figure 11.** Summary of the fragmentation pathway of kanamycin B reference substances.
