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.
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 µmol/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 µmol/L; limit of quantification was 1.9 µmol/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.