Figure 4: Individual standard injections using step 3 with the Diamond Hydride column. Standard injections are (a) ascorbic
acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
Ammonium acetate was chosen as the mobile-phase additive to investigate the effect of ascorbic acid ionization on its retention.
The mobile phase will be at a near neutral pH with this additive so it was deemed suitable for this purpose. Also, ammonium
acetate is an LC–MS-compatible mobile-phase additive and is therefore within the scope of the method goals. Data for the ANP
method step using ammonium acetate in the A and B solvents (step 3) is shown in Figure 4. As the ANP retention mechanism predicts,
ascorbic acid shows far better retention than with 0.1% formic acid as the mobile phase additive. The selectivity between
ascorbic acid, riboflavin, and pyridoxine could be improved by further modification of the gradient. However, the primary
issue with the data from this step is the thiamine retention and peak shape. Even with a lengthy hold time at strong eluting
ANP conditions (70% solvent A), the compound still retains excessively and displays a very broad tailing peak. This is likely
caused by undesirable electrostatic interactions with residual surface silanols, which contribute to thiamine retention. These
silanols are anionic at this mobile-phase pH and therefore have a greater affinity for interaction with the cationic thiamine.
This secondary retention mechanism results in both increased retention and decreased efficiency.
Figure 5: Individual standard injections using step 4 with the Diamond Hydride column. Standard injections are (a) ascorbic
acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
The two mobile-phase pH conditions both have advantages. The formic acid method elutes thiamine in a reasonable time frame
and with good efficiency, and the ammonium acetate method exhibits good ascorbic acid retention. The data then suggest that
a combination of the two approaches could provide the benefits of both methods while avoiding the drawbacks. The gradient
should start out at a moderate pH to retain ascorbic acid, but would need to increase in acidity as well as water content
to avoid the detrimental effects observed with thiamine using only ammonium acetate. For this reason, step 4 was devised which
has formic acid in the A solvent and ammonium acetate in the B solvent. The data using this approach are shown in Figure 5.
Here, the retention for ascorbic acid is improved while the thiamine peak is eluted readily and with good efficiency. Still,
ascorbic acid retention is not as high as would be expected, considering its stronger retention from step 3.
Figure 6: Individual standard injections using step 5 with the Diamond Hydride column. Standard injections are (a) ascorbic
acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
A possible reason for the lower ascorbic acid retention is that the buffer capacity or ionic strength in the B solvent is
not sufficient. Therefore step 5 was investigated in which the concentration of ammonium acetate in the B solvent was increased
sixfold. The A solvent used was the same as in step 4. The results from this further refined method are shown in Figure 6.
Under these new conditions, the pH gradient now behaves as originally predicted. Ascorbic acid shows excellent retention and
thiamine is not retained excessively. The method combines the best features of both mobile-phase pH conditions. The only obstacle
left is to obtain adequate separation between ascorbic acid and riboflavin.
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