3.1.1. Pre-Column Derivatization

Pre-column derivatization of kanamycin changes its polarity, which optimizes its applicability for being analyzed through conventional RPLC. For example, CNBF was used as a pre-column derivatization reagen<sup>t</sup> in kanamycin analysis in different kinds of soil samples with a UV detector at 245 nm with the reaction scheme as presented in Table 4 [31]. CNBF was able to react with primary and secondary amines in alkali condition, producing stable N-substituted-2, 6-dinitro-4-(trifluoromethyl)-benzamine derivative [49]. Unlike FOMC-Cl, CNBF does not need to be removed after derivatization. The analytical column was a kromasil C18 ODS column (250 × 4.6 mm, 5 μm). The SPE column was an AccuBOND ODS-C18 (3 mL/200 mg). Linearity range was 0.01–10.0 mg/kg, and LOD was 0.006 mg/kg. The HPLC-UV Chromatogram of CNBF-kanamycin A derivative is shown in Figure 3 [31].



*Molecules* **2019**, *24*, 1902



**UPLC-MS**/**MS TQD**

kanamycin sulfate

**SPE**, Disposable pipet extraction (DPX), 5 mL tips containing 50 mg WCX sorbent

IPLC

Reversed-phase column, Waters BEH C18 (50 × 2.1 mm, 1.7 μm)

20 mM HFBA in 5% ACN in H2O-20 mM HFBA in ACN, gradient, 3min. Tobramycin as IS

84–92 - 0.005 μg/g [42]

bovine kidney, liver, muscle





*Molecules* **2019**, *24*, 1902 **Figure 3.** The HPLC-UV Chromatogram of CNBF-kanamycin A derivative. (A) The retention time of CNBF-kanamycin A derivative was 2.71 min without TFA in the mobile phase. The derivative could not be separated completely with interference. (B) The 0.1% TFA could improve separation efficiency. A perfect separation of CNBF-kanamycin A derivative was obtained with retention time of 9.58 min. (C) Blank soil sample.

PIC could react easily with primary or secondary amines, forming the stable *<sup>N</sup>*-aryl-*N*-phenyl urea derivative, which was detected by UV at 242 nm. In Patel's study, a corresponding derivative through reaction of KANA with PIC (5 mg/mL in ACN) was formed in the presence of TEA for 10 min, followed by the RPLC method. The derivatives were separated on a Phenomenex C18 column (250 × 4.6 mm, 5 μm). Linearity range was 5–15 μg/mL. LOD was 0.597 μg/mL. The reaction scheme of PIC with kanamycin is presented in Table 4. The HPLC-UV Chromatogram of the kanamycin-PIC derivative is shown in Figure 4 [48].

**Figure 4.** The HPLC-UV Chromatogram of the kanamycin-PIC derivative. (a) Blank; (b) Kanamycin-PIC derivative, 10 mg/mL showing retention time at 8.5 min.

NITC was used as a pre-column derivatization reagen<sup>t</sup> to detect kanamycin A in human plasma by UV at 230 nm. The mixture containing kanamycin A was reacted in pyridine for 1 h. Methylamine was added to eliminate the remnant NITC after derivatization. The stationary phase was a Purospher STAR RP-18 column (55 × 4 mm, 3 μm). Linearity range was 1.2–40 μg/mL, and LOD was 0.3 μg/mL. The reaction scheme of NITC with kanamycin is presented in Table 4. The HPLC-UV Chromatogram of the kanamycin-NITC derivative is shown in Figure 5 [13].

**Figure 5.** HPLC-UV chromatogram of the kanamycin-NITC derivative. (A) Separation of kanamycin A from kanamycin B, each at 40 μg/mL; (B) Determination of kanamycin A in commercial capsule sample. Peaks: 1, kanamycin A-NITC derivative; 2, acenaphthene (IS), 3, kanamycin B-NITC derivative.

FMOC-Cl was commonly used as a pre-column derivatization reagen<sup>t</sup> of kanamycin, and the following detection was conducted by fluorescence. Kanamycin in human plasma reacted with FMOC-Cl in borate buffer solution (pH 8.5) for 30 min at room temperature, then separated by an Eclipse XDB C8 column (150 × 4.6 mm, 5 μm). LOD was 0.01 μg/mL, fluorescence wavelength was set at excitation of 268 nm and emission 318 nm. The reaction mechanism is shown in Table 4. The HPLC-FL Chromatogram of the kanamycin-FMOC derivative is shown in Figure 6 [12]. Similarly, pre-column FMOC-Cl derivatization of kanamycin was performed in swine tissue. The sample tissue was purified with the MCX SPE column. The derivatives were separated on a Waters symmetry C18 column (150 × 3.9 mm, 5 μm). LOD was 0.03 mg/kg for muscle, 0.06 mg/kg for liver and 0.18 mg/kg for kidney. The fluorescence measurements were set as excitation wavelength at 260 nm and emission wavelength at 315 nm. LOQ was 0.025 μg/mL, which was far lower than that reported by other researchers [36]. Another FMOC-Cl derivatization was prepared in wastewater and soil using a Diamonsil C18 column (250 × 4.6 mm, 5 μm). This is the first reported analysis that reduced the kanamycin derivative with the DLLME-SFO procedure. The fluorescence was measured at excitation wavelength 265 nm and emission wavelength 315 nm [23].

**Figure 6.** The HPLC-FL Chromatogram of the kanamycin-FMOC derivative. Kanamycin extracted from plasma from the same person 1.5 h after oral administration of 0.75 g of the drug. Peak 1, kanamycin-FMOC derivative.

OPA is a widely used derivatization reagen<sup>t</sup> that introduces chromophores in HPLC methods using UV or fluorescence detection. A typical example is a pre-column derivatization of kanamycin with OPA in animal feeds; the reaction scheme is presented in Table 4 [35]. Oasis MCX SPE was used

for cleanup. Chromatographic separation was implemented on a XTerra C18 column (250 × 4.6 mm, 5 μm). LOD was 5 g/ton in animal feeds with fluorescence measurement at excitation wavelength of 230 nm and emission wavelength of 389 nm. The HPLC-FL Chromatogram of the kanamycin-OPA pre-column derivative is shown in Figure 7 [35].

**Figure 7.** The HPLC-FL Chromatogram of kanamycin-OPA pre-column derivative. Peak 1: kanamycin-OPA derivative, with kanamycin in poultry feeds at levels of 10 mg/g, 40 mg/g, 80 mg/g, and 200 mg/g.

Although the pre-column derivatization methods can avoid using ion pair reagen<sup>t</sup> (IPR), IPR is still needed under certain conditions. The derivatization of kanamycin using borate complexation is an example of this [9]; with reaction scheme is shown in Table 4. The HPLC-UV chromatogram of the kanamycin A-borate derivative is shown in Figure 8 [9]. Borate ion was obtained by dissolving borax in water. After borate complexation formation, the derivatives were analyzed with a XBridge C18 column (250 × 4.6 mm, 5 μm), using sodium octanesulphonate as IPR, and with UV detection at 205 nm. Baseline separation from kanamycins B, C, and D were achieved.

**Figure 8.** The HPLC-UV chromatogram of kanamycin A borate complexation. Chromatogram obtained after injection of kanamycin A solution (1 g/L) spiked with kanamycins B, C, and D, and paromamine (0.1 g/<sup>L</sup> each).
