Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column

Abstract

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

Capillary electrophoresis coupled to mass spectrometry employing hexafluoro-2-propanol for the determination of nucleosides and nucleotide mono-, di- and tri-phosphates in baby foods

“…Target compounds (Padivitage, Dissanayake, & Armstrong, 2013) were guanosine (G), uridine (U), inosine (I); and mono-diand tri-phosphate of adenosine (AMP, ADP, ATP), cytidine (CMP, CDP, CTP), guanosine (GMP, GDP, GTP), inosine (IMP, IDP, ITP) and uridine (UMP, UDP, UTP), respectively, and have been described in a previous paper (Mateos-Vivas, Rodríguez-Gonzalo, . Stock solutions containing the nucleosides and nucleotides reference standards, at 500 lg mL À1 , were prepared in ultra-high quality (UHQ) water.…”

Liquid chromatography/tandem mass spectrometry method for simultaneous quantification of eight endogenous nucleotides and the intracellular gemcitabine metabolite dFdCTP in human peripheral blood mononuclear cells

“…This technique has generally yielded poor separation and unsatisfactory peak shapes for di-and triphosphorylated analytes, and HILIC columns are not straightforward to optimize. [21][22][23] We describe a sensitive IP LC-MS/MS method for separation and quantification of 8 endogenous nucleotides (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP) and 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP), the active metabolite of gemcitabine, in peripheral blood mononuclear cells (PBMC).The method has been validated for sensitive and accurate determination of all analytes and innovative steps in preparation of standards are described. The water (analytical grade) was produced in our laboratory via a Milli-Q (Millipore Corporation, MA, USA) deionized water system.…”

Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection

“…HILIC has been useful at separating hydrophilic analytes and has been successful for the separation of nucleotides, sugar phosphates and nucleotide sugars [7][8][9]. Recently, separation of nucleotides was achieved with a cyclofructan based column using ammonium acetate buffer in both gradient and isocratic methods [7].…”

“…These include reversed-phase ion-pairing (RP-IP), porous graphite carbon (PGC), hydrophilic interaction liquid chromatography (HILIC) and mixed mode chromatography [1,2,[4][5][6][7][8][9][10]. Guanosine diphosphate (GDP) sugars and their metabolites were separated by ion-pairing chromatography utilizing ODS-columns (C-18) and ion-pairing agents such as tributylamine with acetic acid [4].…”