Abstract
Vitamin C (VC) is necessary for development of normal connective tissue and human health. VC HPLC separations have remained nearly the same for 25 yrs. Recent advances in column technology have permitted some improvements in the methodology.ObjectiveTo improve serum VC separation, sensitivity and reproducibility w/o ion‐pairing agents or gradients.MethodsSeveral HPLC columns, possible internal standards and two buffers at different concentrations were examined. Both UV and electrochemical (EC) detection was used.ResultsMost columns offered poor retention and peak shape of VC. Phenomenex Synergi Hydro‐RP column provided good retention (k=1.3) symmetry (A=1.1) and tailing (T=1.15). Tris(2‐carboxyethyl)phosphine (TCEP) was used as an alternate reducing agent to dithiothreitol. Hypoxanthine, selected as an internal standard, eluted isocratically at k=3.2 with UV absorbance at 245nm but no EC.DiscussionThe HPLC method modifications yield good retention and peak shape. TCEP provides a cost effective and superior reducing agent. Efficiency and reproducibility were improved by incorporation of an internal standard. The method is amenable to short narrow bore columns.
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