## 3 Results and Discussion
To point out the physico-chemical differences between 2\(R\)- and 25-LP2, the HPLC method developed in this study aimed at finding the best conditions for the chromatographic separation of the two 2\(R\)/\(S\)-LP2 diastereoisomers on an achiral column, also obtaining a good peak shape and optimal retention, according to the recommendations by the International Conference on Harmonization (ICH) [25]. Reverse-phase chromatography (RPC) has become the predominant technique of analytical chromatography due to numerous advantages. The operative retention mechanisms operating in RPC permit excellent separations due to the possibility of varying pH and allow the use of organic modifiers and additives [31]. However, the analysis of basic compounds, such as LP2 diastereoisomers, poses difficulties in RPC analysis. In fact, preliminary analysis of LP-2 diastereoisomers in RPC provided large peaks with strong tailing that resulted in almost complete overlapping of the 2\(R\)/\(S\)-LP2 peaks. In RPC, the problem of poor column efficiency and tailing peaks is attributed to mixed mechanisms involving the residual silanol groups (pKa 7-9) present in the C18 phase (40-50% in a typical C18-endcapped column). Hydrophobic interactions play a fundamental role, but a secondary mechanism involving ion exchange between analytes and the residual silanol groups can take place, as well. The slower sorption-desorption kinetics of silanol ion-exchange sites is responsible for tailing and poor efficiency (kinetic tailing) [32-34]. For these reasons, the effect of pH and the use of additives were evaluated as important factors to improve the separations and peak symmetry of 2\(R\)/\(S\)-LP2 diastereoisomers. In order to suppress kinetic tailing due to the interaction between the basic nitrogen of LP2 and the residual silanol groups, mobile phases with a pH in the range of 2.5-3.5 were assessed. At this acidic pH, both the silanol groups and basic nitrogen are protonated, resulting in a weakening of their mutual interaction. To improve the peak symmetry, a tertiary amine, namely, 0.3% triethylamine (TEA), was used as an additive, as such bases have been proven to shield the silanol groups at an acidic pH [35,36], avoiding any residual interaction. However, a major drawback of working at a low mobile-phase pH is that analytes exhibit low retention because basic molecules in the protonated form are more hydrophilic and have lower interaction with the lipophilic C18 stationary phase, with loss of selectivity, especially for very similar molecules, such as LP2 diastereoisomers [37]. For these reasons, the pH has been appropriately adjusted using trifluoroacetic acid, as this acid is a good ion-pairing reagent capable of increasing the retention of basic analytes, compensating for the loss of retention due to the low pH. Perfluorinated acids not only neutralize the positively charged nitrogen groups of the analytes by decreasing their hydrophilicity but are also able to improve the affinity for the hydrophobic stationary phase, thus increasing the retention. To further improve the resolution of the 2\(R\)- and 25-LP2 peaks, the behavior of the solvent eluents methanol (MeOH) and acetonitrile (MeCN) was also carefully evaluated. MeOH is mainly a proton-donor solvent with relatively high dipolar properties. MeCN possesses the largest dipole moment among the modifiers but is characterized by weak properties as a proton acceptor and much weaker properties as a proton donor. It has been demonstrated that, in reverse phase analysis, analytes can interact with the stationary phase by means of solvation complexes; therefore, the solvent composition is crucial to obtain a good separation. [PERSON] et al. [38] reported that the hydrogen-bonding capacity for different solvent compositions of MeCN and MeOH was the following: methanol \(\cong\) water \(>\) methanol-water complex \(>>\) acetonitrile-water complex \(\cong\) acetonitrile.