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Abstract
A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 μg/mL. The inter- and intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.
References
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Figure 1.
Chemical structure of ascorbic acid and dehydroascorbic acid as depicted through a redox reaction
Figure 2.
Recovered ascorbic acid contents in standard solutions stored at −70°C (■), −4°C (▲), 25°C (◆) and light shielded at 25°C (●)
Figure 3.
Chromatogram of ascorbic acid and internal standards in human plasma. HPLC conditions: mobile phase, 5 mM HTAB + 50 mM KH2PO4; column, C18 column (symmetry® 4.6 μm × 280 mm, 5 μm particle size, Waters, USA); flow rate, 1.2 mL/min; UV detection wavelength, 254 and 265 nm.
Table 1.
Intra- and inter-day precision and accuracy (n=5) of ascorbic acid QC samples in human plasma
Table 2.
Intra- and inter-day recovery of ascorbic acid in human blank plasma QC samples (n=5)
| Concentration (μg/mL) | Recovery (%, Mean±S.D) | |
|---|---|---|
| Intra-day | Inter-day | |
| 1 | 106.00±3.20 | 98.98±6.52 |
| 2 | 108.30±4.39 | 110.05±12.84 |
| 50 | 118.41±3.44 | 109.41±8.93 |
| 80 | 100.89±2.69 | 104.01±2.72 |
Table 3.
Dehydroascorbic acid concentrations
| Free AA (μg/mL) | Total AA (μg/mL) | DHAA |
|---|---|---|
| AA 50 μg/mL | AA 50 μg/mL + 2 mM DTT | Total AA – free AA |
| 37.06 | 44.24 | 7.18 |
Table 4.
Pharmacokinetic parameters for ascorbic acid and dehydroascorbic acid in human plasma (mean±SD) obtained after administration of vitamin C 4,000 mg
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