9


• test mixture 4: t0, 2’-deoxyguanosine (2dG), 3’- deoxyguanosine (3dG)


• test mixture 5: t0, uracil (Ur), sodium p-toluenesulfonate (SPTS)


• test mixture 6: t0, uracil, N,N,N- trimethylphenylammonium chloride (TMPAC)


• test mixture 7: t0, theobromine (Tb), theophylline (Tp)


The chemical structures of the test compounds used in this study, together with their physiochemical properties are given in Table 2.


Chromatographic parameters investigation


All the standard stock solutions for the individual test probes were prepared in 80:20 acetonitrile: water at 1mg/mL. Two test mixtures were then prepared: a basic test mixture, comprising uracil, adenosine, uridine, cytosine and cytidine; an acid test mixture, comprising salicylamide, salicylic acid, aspirin and 3,4-dihydroxyphenylacetic acid (dhpa).


Results


Characterisation tests Hydrophobic Selectivity The degree of hydrophobic interaction between the stationary phase and the test compounds is a useful characteristic in both RPLC [13] and HILIC. It can be measured


from the selectivity for a methylene group, α (CH2). Ikegami et al. obtained α (CH2) from a comparison of k uridine (retention factor for


uridine) and k 5-methyluridine (retention factor for 5-methyluridine), which differs by a


CH2. Uridine and 5-methyluridine were chosen as the α (CH2) probe pair since they are polar enough to afford retention in HILIC


[13]. Figure 1 shows chromatograms for four representative columns.


From Figure 1 it can be seen that uridine is more retained than 5-methyluridine, which reflects the fact that uridine is more hydrophilic than 5MU.


Hydrophilic Separation The degree of hydrophilic interaction between the stationary phase and the test compounds was assessed using the


selectivity for an hydroxy group, α (OH). In this study α (OH) was obtained from a comparison of k uridine and k 2’-


deoxyuridine. Figure 2 shows chromatograms for four representative columns, where it can be seen that uridine is


Column Name Syncronis HILIC (5 µm)


Hypersil GOLD HILIC (5 µm) Hypersil GOLD Silica (5 µm) Hypersil GOLD Silica (1.9 µm) Syncronis Silica (5 µm) Accucore HILIC (2.6 µm)


Acclaim Mixed Mode HILIC-1 (5 µm) Acclaim HILIC-10 (3 µm) Acclaim Trinity P1 (3 µm) Experimental HILIC (3 µm)


α (CH2) 1.477


1.330 1.291 1.253 1.302 1.473 1.000 1.117 1.226 1.530


α (OH) 2.090


1.931 1.697 1.579 1.518 1.942 1.000 1.521 1.828 2.182


k uridine


5.053 2.278 1.377 1.340 3.152 3.753 0.112 1.836 0.869 3.513


Table 3: Separation factors for methylene α (CH2) and hydroxy α (OH) groups and retention factor for uridine. Figure 3: Chromatograms for α (AX) test. Analyte: 1) toluene; 2) uracil; 3) sodium p-toleuenesulfonate, SPTS.


Figure 2: Chromatograms for α (OH) test. Analyte: 1) toluene; 2) 2’-deoxyuridine; 3) uridine.


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