## Alternative Organic Solvents in RP-HPLC
### Ethanol
#### 2.1.2 Pharmaceutical Applications
Xbridge BEH Shield RP18 (50 \(\times\) 4.6 mm i.d., 2.5 \(\upmu\)m); Chromolith (SpeedROD RP18, 50 \(\times\) 4.6 mm i.d., 2 \(\upmu\)m); YMC-ODS-AQ (50 \(\times\) 4.6 mm i.d., 3 \(\upmu\)m); Hypersil GOLD (50 \(\times\) 4.6 mm i.d., 1.9 \(\upmu\)m). Isoeluotropic mobile phases: EtOH/formic acid (pH 2.5, 25 mM) in proportions 40/60 (\(v/v\)) for YMC column; 38/62 (\(v/v\)) for Xbridge and Hypersil columns; and 35/65 (\(v/v\)) for Chromolith column. HETP: height equivalent to a theoretical plate. Similar to Reference [42]. of total error, and was used to analyze the main degradation products of dextromethorphan in a pharmaceutical product (Figure 2). The greenness of this method was also checked using the analytical Eco-scale tool. Another application still in the field of pharmaceutical quality control, but this time dealing with multiproduct analysis, was recently published [54]. In this study, a specific and robust RP-HPLC method was successfully developed through QbD approach for the simultaneous separation of 16 active pharmaceutical ingredients (among them artemether without chromophore) using a gradient elution with EtOH as an organic modifier. Throughout the study, acetonitrile and ethanol-based mobile phases were investigated and compared. Optimal conditions could be found only with an ethanol-based mobile phase showing different selectivity between the two organic solvents (Figure 3). As shown in Figure 3, at 210 nm, a relatively high baseline drift was noticed in gradient mode using EtOH due to its high UV cut-off, however, artemether--a compound without chromophore in its structure--can still be detected. Figure 3: Separation of 16 active pharmaceutical ingredients. Xbridge BEH Shield RP18 column (50 \(\times\) 4.6 mm i.d., 2.5 \(\upmu\)m). Gradient elution of ethanol from 5% to 80% (slope of 2.57%/min) with acetate buffer 20 mM at pH 4.85. Flow rate: 0.8 mL/min. Temperature: 33.7 \({}^{\circ}\)C. Detection at 210 nm. Peak identification: 1 = paracetamol; 2 = doxylamine; 3 = ondansetron; 4 = melatonin; 5 = quinine; 6 = metopimazine; 7 = domperidone; 8 = pravastatin; 9 = ketoprofen; 10 = rousvastatin; 11 = midazolam; 12 = diclofenac; 13 = atorvastatinatin; 14 = fluvastatin; 15 = artemether; and 16 = simvastatin. Similar to Reference [54]. Figure 2: Reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of a mixture of dextromethorphan (DXM), its impurities, and excipients. Acquity BEH C18 column (50 \(\times\) 2.1 mm i.d, 1.7 \(\upmu\)m). Gradient elution of ethanol (slope of 3.1%/min) with 10 mM ammonium formate adjusted at pH 4.7 with formic acid. Flow rate: 0.23 mL/min. Temperature: 38.0 \({}^{\circ}\)C. Detection at 280 nm. Similar to Reference [44]. In the field of bioanalysis, there are also some articles dealing with the use of EtOH as a green organic modifier in the mobile phase for the chromatographic analysis of pharmaceutical compounds [55; 56]. For example, a HPLC assay was developed and validated by [PERSON] et al. [56] for the determination of capecitabine in human plasma using a C18 reversed-phase analytical column and a mobile phase composed of formic acid solution (pH 3):EtOH (55:45, \(v/v\)). All these studies, using EtOH as organic modifier in RP-HPLC for pharmaceutical analysis, were carried out in the last decade, and deal with quality control and bioanalysis. This highlights anincreasing use of EtOH for chromatographic analysis at different stages of the pharmaceutical product lifecycle. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Compounds** & **Matrix** & **Column** & **Mobile Phase** & **Fiber Rate** & **Temperature** / **CO** & **Detection** & **Ref** \\ \hline ProInInisotope & Tables & \begin{tabular}{c} Phenomenence CIS \\ (105 \(\times\) 46 mm, 5 mm) \\ \end{tabular}  & EOH/\(\#\)\(wether\) (020, \(\mathrm{\SIUnitSymbolMicro m}\)) & 0.8 & 50 & UV (254 mm) & [(9)] \\ \hline Diltsame & Typical formulations & \begin{tabular}{c} (150 mm) \\ (250 \(\times\) 46 mm, 5 mm) \\ \end{tabular}