## 3 Results and Discussion  
### Chromatographic Method Development  
The purpose of the following investigation was to suggest a reliable analytical method that would meet the chromatographic requirements, i.e., satisfactory peak shape (Tf = 1 \(\pm\) 0.2), effective separation (Rs \(\geq\) 1.5) and short analysis time (t\({}_{\text{R}}\) < 20 min). In addition to the two APIs (ACA and DIP), special emphasis was also given to the adequate separation of salicylic acid (SAL), which is a degradation product of ACA and is considered an indicator of its hydrolysis. In order to select the optimal stationary phase, different types of reversed-phase columns (phenyl, C8, C18, -CN and -NH2) were tested, under similar analytical conditions. In Table S1, a summary of all the experimental investigations is presented. In general, the main problem that had to be addressed was the insufficient separation of ACA and SAL, which mainly co-elute immediately after the solvent front, while the DIP peak was usually delayed. Reducing the organic-mobile phase ratio, increased the elution time of both peaks but did not separate them sufficiently, while DIP was delayed further. In most cases, also, the acidic pH of the mobile phase gave a triple peak in SAL, broken and with the solvent front, while ACA was wide and broken. Comparatively better behavior was observed in C18 type columns, of different dimensions (150 and 250 mm). Of these, Supelco Discovery(r) HS (25 cm \(\times\) 4.6 mm, 5 \(\upmu\)m) provided the best separations, with sharp peaks, short analysis time and isocratic elution. Acetonitrile was used as the organic solvent in the mobile phase instead of methanol mainly because it improved the peak shape for DIP (Tf MeOH = 1.5 and Tf\({}_{\text{ACN}}\) = 1.05) and gave low backpressure. Another notable observation was that very small changes in the concentration of the organic solvent (ACN) in the mobile phase significantly affected the retention time of DIP and its elution order, compared to ACA and SAL (Figure 2). The optimum mobile phase was a mixture of 65:35 \(v/v\) of two phases (A): aqueous solution at pH 2.4 with H\({}_{2}\)O and Formic acid 0.2%, and (B) acetonitrile-H\({}_{2}\)O 90:10 \(v/v\), in isocratic elution. To select the optimal pH value of the mobile phase and in order to prevent hydrolysis of ACA, it had to be acidic [33]. Different acids such as formic, acetic or trifluoroacetic as well as phosphate buffers were tested. Based on the pKa values of the analytes (Table S2) [34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45] and using phosphate buffer, three pH (2.8, 3.5 and 4.25) were tested. The two APIs, which are acids (pKa\({}_{\text{(SAL)}}\) 2.98 and pKa\({}_{\text{(ACA)}}\) 3.5) at pH values higher than their pKa, are ionized and their retention time is reduced. DIP, on the other hand, as a base (pKa 3.54), at pH \(>\) pKa, due to its non-ionization, is eluted at tr \(>\) 20 min. Therefore, lower pH values were considered more appropriate since they delay the elution of the two acids and accelerate that of dipyridamole (20 mM NaH\({}_{2}\)PO\({}_{4}\) at pH = 2.8 with phosphoric acid). Figure 2: Effect of acetonitrile on the retention time (t\({}_{\text{R}}\)) of the analytes. Six different ratios were tested, three times each, in terms of reproducibility. Compared to phosphate buffer and the other acidifying agents, formic acid gave the best chromatogram (Table 2) and was chosen for pH adjustment. Specifically, phosphate showed more noise in the baseline, while the other two acids (formic or trifluoroacetic) gave worse tailing factor values (Tf \(>\) 1.1) for DIP. Formic acid was added only to the aqueous phase, as it did not improve the result when added to the organic phase. It was studied at three concentration levels (0.05%, 0.1%, and 0.2%) and 0.2% was selected as optimal due to the sharpest peaks. The choice of the appropriate diluent in the final samples was based on the solubility of the APIs, their stability (especially of ACA) and the resulting chromatograms.