Effect of Short-Chain Organic Acids, Cations and Anions on the Retention of Citicoline Under Hydrophilic Interaction Liquid Chromatography Conditions

The optimization of mobile phase composition in HPLC is crucial for achieving excellent chromatographic performance. Mobile phase additives are very often added to control retention, resolution, peak shape and efficiency. According to the Hofmeister series, additives are classified into chaotropic and kosmotropic agents. When ionizable compounds are analysed by hydrophilic interaction liquid chromatography (HILIC), additives can control the electrostatic interactions and affect the chromatographic parameters. In this study, citicoline, a neurotransmitter, was analysed under HILIC conditions using various additives. Due to the solubility limit in high organic content, the composition of the mobile phase was fixed at a 2/1 (V/V) acetonitrile/water ratio, and the salt concentration set to 25 mM. A total of 21 additives were tested, including short-chain organic acids (formic, acetic, propionic, trifluoroacetic and trichloroacetic acid), cations (lithium, sodium, potassium and ammonium) and anions (acetate, bromide, chloride, dihydrogen citrate, dihydrogen phosphate, nitrate, perchlorate and tetrafluoroborate). With additive-free mobile phases, weak retention of citicoline was observed, which can be explained by the small thickness of the water layer on the surface of the silica stationary phase and electrostatic repulsion between deprotonated silanols and negatively charged citicoline. However, the use of additives improves retention. Short-chain organic acids increased retention, but produce poor peak shape. Cations affected retention in the following order: \({{\varvec{L}}{\varvec{i}}}^{+}<{{\varvec{N}}{\varvec{H}}}_{4}^{+}<{{\varvec{N}}{\varvec{a}}}^{+}<{{\varvec{K}}}^{+}\) ${\mathit{L}\mathit{i}}^{+}<{\mathit{N}\mathit{H}}_4^{+}<{\mathit{N}\mathit{a}}^{+}<{\mathit{K}}^{+}$ , corresponding to the reversed Hofmeister series. The anions trend was: \({{\varvec{C}}{\varvec{H}}}_{3}{\varvec{C}}{{\varvec{O}}{\varvec{O}}}^{-}<{{\varvec{H}}}_{2}{{\varvec{P}}{\varvec{O}}}_{4}^{-}<{{\varvec{B}}{\varvec{r}}}^{-}<{{\varvec{C}}{\varvec{l}}}^{-}<{{\varvec{H}}}_{2}{{\varvec{C}}{\varvec{i}}{\varvec{t}}{\varvec{r}}{\varvec{a}}{\varvec{t}}{\varvec{e}}}^{-}<{{\varvec{N}}{\varvec{O}}}_{3}^{-}<{{\varvec{C}}{\varvec{l}}{\varvec{O}}}_{4}^{-}<{{\varvec{B}}{\varvec{F}}}_{4}^{-}\) ${\mathit{C}\mathit{H}}_3\mathit{C}{\mathit{O}\mathit{O}}^{-}<{\mathit{H}}_2{\mathit{P}\mathit{O}}_4^{-}<{\mathit{B}\mathit{r}}^{-}<{\mathit{C}\mathit{l}}^{-}<{\mathit{H}}_2{\mathit{C}\mathit{i}\mathit{t}\mathit{r}\mathit{a}\mathit{t}\mathit{e}}^{-}<{\mathit{N}\mathit{O}}_3^{-}<{\mathit{C}\mathit{l}\mathit{O}}_4^{-}<{\mathit{B}\mathit{F}}_4^{-}$ , which corresponds to the direct Hofmeister series, except for acetate and dihydrogen phosphate.