Development and implementation of an HPLC ECD method for analysis of vitamin C in plasma using single column and automatic alternating dual column regeneration
Objectives Vitamin C (l ascorbic acid) is a water soluble micronutrient necessary for human life. Inadequate intake can lead to the fatal disease scurvy. Measurement of vitamin C is used to assess nutritional status and to monitor supplementation. The goal of this study was to develop a chromatographic method for the quantitation of vitamin C in human plasma. Design and methods Samples were prepared by protein precipitation, addition of internal standard, and reduction with dithiothreitol. Separation of ascorbic acid was accomplished by isocratic elution on a reverse phase column; concentration was determined by coulometry. The method was validated through studies of assay linearity, sensitivity, imprecision, accuracy, analytical specificity, and carryover. Results The new assay was developed using a single pump/single analytical column HPLC system. Results correlated well with our previously used spectrophotometric method. The analytical measurement range was 1.0 2500 umol/L. The injection to injection time was 13 min. Subsequently, to increase method throughput and shorten turnaround time, a dual LC pump system with a 2 position/10 port switching valve capable of performing automatic alternating column regeneration was validated and implemented. The injection to injection time was reduced 2 fold to 6 min. The method was linear to 5000 umol/L; limit of quantification was 1.9 umol/L. Total imprecision was less than 5% Conclusions We have developed a robust method suitable for routine clinical measurement of vitamin C in plasma specimens. The method incorporates a simplified sample preparation and a stable, non endogenous internal standard to specifically quantify vitamin C. Faster throughput was achieved by employing an automatic alternating column regeneration system.
Practical laboratory medicine