Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates.
A fast and efficient method for the simultaneous separation of highly polar compounds, in this case nucleosides and nucleotide mono-, di- and triphosphates, using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) is proposed. This new separation method revealed the possibilities of the formation of hydrophilic ion-pairing compounds. Three stationary phases (HILIC XBridge-Amide, HILIC-CoreShell and ZIC-HILIC) were assayed for the separation of 20 target analytes, and a detailed study of the composition of the mobile phase was made using different salts at different concentrations in a organic-rich mobile phase. We report that in order to prevent the adsorption of nucleotides on the LC-MS setup and to enhance their retention on the HILIC stationary phase, a mobile phase containing hexafluoro-2-propanol and different cations should be used. Four cations were evaluated: ammonium, diethylammonium, triethylammonium and tetrabutylammonium. The results revealed the formation of an ionic-association compound between the phosphorylated analytes and the cationic ion-pairing reagents, whose retention increased with the polarity of the cationic ion-pairing reagent. HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100mM hexafluoro-2-propanol and 50mM diethylamine (w(w)pH 9-w(s)pH 10). In a total analysis time of 8min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out.
Departamento de Química Analítica, Nutrición y Bromatología, Universidad de Salamanca, 37008 Salamanca, Spain.
María
M
Mateos-Vivas
Departamento de Química Analítica, Nutrición y Bromatología, Universidad de Salamanca, 37008 Salamanca, Spain. Electronic address: erg@usal.es.
Encarnación
E
Rodríguez-Gonzalo
Departamento de Química Analítica, Nutrición y Bromatología, Universidad de Salamanca, 37008 Salamanca, Spain.
Diego
D
García-Gómez
Departamento de Química Analítica, Nutrición y Bromatología, Universidad de Salamanca, 37008 Salamanca, Spain.
Rita
R
Carabias-Martínez
D000327:Adsorption; D002853:Chromatography, Liquid / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D009705:Nucleosides / Q000302:isolation & purification*; D009711:Nucleotides / Q000302:isolation & purification*; D011122:Polyphosphates; D020005:Propanols; D012997:Solvents; D053719:Tandem Mass Spectrometry / Q000379:methods; D006845:Hydrocarbons, Fluorinated
D009705:Nucleosides; D009711:Nucleotides; D011122:Polyphosphates; D020005:Propanols; D012997:Solvents; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C005692:triphosphoric acid
Hydrophilic interaction chromatography (HILIC); Hydrophilic ion-pairing; Nucleosides; Nucleotide mono-, di- and triphosphates