Improved HPLC Assay for Measuring Serum Vitamin C with 1-Methyluric Acid Used as an Electrochemically Active Internal Standard
2005; American Association for Clinical Chemistry; Volume: 51; Issue: 6 Linguagem: Inglês
10.1373/clinchem.2004.046904
ISSN1530-8561
AutoresLeslie F. McCoy, M Bridgette Bowen, Mary Xu, Huiping Chen, Rosemary L. Schleicher,
Tópico(s)Electrochemical Analysis and Applications
ResumoThe National Health and Nutrition Examination Survey (NHANES) laboratory at CDC has used a modification of methods (1)(2) with electrochemical detection for measurement of serum vitamin C for the past 9 years. The assay is relatively rapid, easy to perform, and gives good precision. Quality-control (QC) materials have been kept at −70 °C for more than 10 years without substantial degradation. A drawback of the method is the lack of an internal standard to correct for analyte degradation, procedural errors, and detector drift. Significant vitamin C degradation is intermittently encountered during the analytical process. Oxidation of ascorbic acid (AA) is accelerated by exposure to air, heat, light, and traces of copper and iron (3) and may be introduced through contact with unexpected sources, such as consumable supplies (4). Detector drift is a characteristic of electrochemical detection and has been noted by others performing the serum AA assay (5). Our original HPLC assay used partition of largely un-ionized AA and amperometric detection. A 25-cm C18 reversed-phase column is equilibrated with a mobile phase consisting of monochloroacetic acid (pH 3.0) containing disodium EDTA and sodium octylsulfonate [originally used for ion pairing with catecholamines extracted from adrenal chromaffin cells in a mixture containing AA (1)]. The AA in serum is stabilized by addition of metaphosphoric acid (MPA), reduced by addition of dithiothreitol (DTT), and then oxidized at +650 mV referenced to an Ag/AgCl electrode. The working electrode is a thin-layer detector cell. When serum is treated as indicated, peaks for AA, uric acid (URIC), and DTT are resolved and detected at the applied potential within ∼18 min. The run time for each sample is shortened by injecting the next sample before all peaks from the previous sample have eluted. Several changes suggested themselves to modify this method: (a) Improved …
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