## 3 Results and Discussion
Optimal separation of the two LP2 diastereoisomers peaks was obtained using methanol/TEA 0.3% (pH = 3 with TFA acid) as an eluent, while the use of acetonitrile significantly worsened the resolution between the two diastereoisomers. The 2-methoxy groups of LP2 diastereoisomers could be involved in proton-acceptor interactions with -OH groups of water and methanol of the eluent, but such interactions could not occur with acetonitrile, whose weaker proton-donor properties led to poor interactions with the methoxy group. The good resolution obtained with methanol as an eluent could be due to these solvation processes that could take place at different extents for the two LP2 diastereoisomers. The methoxy moiety of the 25-LP2 diastereoisomer could be better solvated compared to the 2\(R\)-LP2 isomer; therefore, 25-LP2 was relatively more hydrophobic and could interact more strongly with C18's stationary phase compared to 2\(R\)-LP2. This different behavior of 2\(R\)- and 25-LP2 could be responsible for the complete resolution obtained using MeOH-based eluents. The effect of column temperature in the range of 20-50 \({}^{\circ}\)C to obtain the best peak shape was also evaluated. As no significant change was recorded, analyses were performed at 22 \({}^{\circ}\)C.  
In summary, the best conditions were obtained using a reversed-phase column, Knauer Eurosphere II 100-3 C18 (150 mm \(\times\) 4.60 mm, 5.0 \(\upmu\)m), using an isocratic mobile phase consisting of 50% MeOH and 50% TEA at 0.3% (pH = 3 with TFA acid) with a flow rate of 1.0 mL/min at 22 \({}^{\circ}\)C. Under these conditions, a baseline resolution of the two LP2 diastereoisomers was obtained with a value of R = 1.80 (USP, Ph. Eur. ), with an acceptable peak shape (tailing factor = 1.18, calculated according to USP and Ph. Eur.). The retention times of 2\(R\)- and 25-LP2 were approximately 9.0-9.5 and 10.0-10.5 min, respectively (Figure 5). Identification of the 2\(R\)/\(S\)-LP2 diastereoisomers was performed by comparing the retention times and UV spectra of the analyzed samples with those of standards. Furthermore, peak purity tests [39; 40] were used to demonstrate the absence of coeluting peaks. Peak purity was determined with OpenLab software (Agilent Technologies) using photodiode-array detector spectra. The calibration curve was constructed using seven calibrators for each diastereoisomer. The method was linear in the range of 60.00-0.54 \(\upmu\)g/mL for 2\(R\)-LP2 and 44.00-0.56 \(\upmu\)g/mL for 25-LP2 (Table 2). Calibration curves were evaluated at the beginning of six consecutive days (\(n=6\)). The equation of the calibration curve gave correlation coefficients higher than 0.999 for both diastereoisomers (Table 2). Figure 5: Chromatograms of 2\(R\)-LP2 and 25-LP2. The limit of detection (LOD) and limit of quantification (LOQ) were established with an S/N ratio of 3:1 and 10:1, respectively (\(n=6\)). The LOD values were 0.18 \(\mu\)g/mL for both 2\(R\)/S-LP2 diastereoisomers, while the LOQ values were 0.54 \(\mu\)g/mL and 0.56 \(\mu\)g/mL for 2\(R\)- and 25-LP2, respectively (Table 2). The precision and accuracy were determined at three concentration levels in the dialysis medium consisting of water/ethanol (80/20 \(v/v\)) spiked with three known amounts within the calibration range by assaying replicates (\(n=6\)) of 2\(R\)-LP2 (60.46, 8.75, and 0.54 \(\mu\)g/mL, Table 3) and 25-LP2 (43.84, 8.75, and 0.54 \(\mu\)g/mL, Table 4), and processed as unknowns. To determine intra- and inter-day precision and accuracy, samples were analyzed in replicates on the same day and on three different days, respectively. Intra- and inter-day precision was reported as the relative standard deviation (RSD%), with an acceptability limit set at \(\leq\)15%. Accuracy was calculated by a comparison of mean assay results with the nominal concentrations, with acceptability limits set at \(\pm\)15%. The intra-day and inter-day recoveries ranged from 96.30 to 104.57% for the two diastereoisomers, while the precision expressed as RSD% was less than 15% in both cases.