## 4 Discussion  
_Derivatization Mechanism_  
Alizarin shows weak fluorescence by itself, but when it interacts with a boronic acid, a fluorescent boronic ester is formed. The related substances and side products in the OATD-02 synthesis process contained a boronic acid function and they could form fluorescence complexes with alizarin dye (Figure 6). Post-column derivatization with alizarine and fluorescence detection was used, because of fast and efficient generation of fluorescent esters. The derivatization reaction is selective so that derivate products can be easily seen against a complex background. Therefore, the post-column derivatization technique is used with fluorescence detection to increase sensitivity and selectivity in HPLC analysis. The analytical method package for active pharmaceutical ingredient must meet the criteria, which are set out in the guidelines such as the ICH guidelines [1]. Use of boronic acid compounds is increasing recently because of their anticancer, antibacterial, and antiviral activity, as well as their application as sensors and delivery systems. Previously, compounds with boron were thought to be toxic, but now they are widespread as biologically active compounds and pharmaceutical agents [35; 36]. OATD-02, an arginase inhibitor, was selected as a clinical candidate for cancer immunotherapy, because of direct antitumor efficacy in animal models [3]. Its structure contains chromophore groups with a low molar absorption coefficient in the UV and visible light range. The selected compound is polar and non-soluble in most organic solvents (unsuitable for NP-HPLC). However, OATD-02 contains boronic acid, and with forms a fluorescent complex the alizarine. This approach has enabled using HPLC with a fluorescence detector (FLD) and with post-column derivatization. It is important to find good consistency of hydrophobic and silanophilic properties of stationary phases to reach good chromatographic repeatability of results for different compounds. To control the ionization state of the analyzed compounds, the effect of the mobile phase pH on retention was checked. At a lower mobile phase pH, below the pKa of the basic compounds, they were mostly ionized and likely to participate in hydrogen bonding, so they spent less time in hydrophobic interactions with stationary phase and in result have less retention. At a higher pH in sodium acetate, there were both ionized and neutral forms of the compounds, the charged form had less hydrophobic interaction while uncharged form had hydrophobic interaction and in result the broad and asymmetrical peaks were observed. Moreover, the few free silanol groups present in the stationary phase participated in ionic interactions with ionized compounds. At the highest pH in sodium bicarbonate in mobile phase, OATD-02 and other basic impurities were mostly deprotonated and were more retained without tailing peaks. The pH of the mobile phase based on sodium bicarbonate is above 8 and OATD-02 has deprotonated carboxylic group and uncharged amine groups, whereas OAT-9016, which has an amide group instead of a carboxylic group is retained more strongly on the non-polar stationary phase. OAT-9015 contains a hydroxy group instead of tertiary amine which causes the lower retention time due to ionization of the hydroxyl group. OAT-9014, which is an  
Figure 6: Reaction of alizarin with OATD-02 to form a fluorescent complex. due to the steric resistance of three functional groups has short retention time than OATD-02. Compounds with hydroxyl and amines groups could display the ability to interact with stationary phases through hydrogen bonding. These compounds can additionally interact with the silanol groups found on the surface of the silica. Comparison of column selectivity with combined hydrophobic and hydrogen bond capacity, can help improve resolution. Focusing on manipulation of hydrophobic retention and hydrogen bond capacity is useful for analysis of polar compounds.