Validation and Stability Study as well as Quality Control of an Assay for Plasma Vitamin C Assessment Using High Performance Liquid Chromatography at Ramathibodi Hospital
Keywords:
Vitamin C, Ascorbic acid, Hogh performance liquid chromatography, Stability methodAbstract
Background: L-ascorbic acid content in plasma is widely accepted as an indicator for vitamin C (VC) status. We set up a method using high performance liquid chromatography (HPLC) to improve the accuracy of plasma VC assessment at Ramathibodi Hospital. The objectives of this study were to validate the method and to determine the stability of VC in blood samples.
Subjects and methods: Heparinized 225-mL plasma was extracted from healthy volunteers. The levels of VC were quantified using a HPLC system with UV detector at the wavelength of 245 nm. For stability analysis, aliquots of the extracts, which were preserved with 10% meta-phosphoric acid and stored at -70oC, were analyzed until the day 15th after collection.
Results: Using our method. the percentage of accuracy was between 101.14 and 105.90%. Percentage of precision was 0.47%, on the same day, and 2.34 %, on a different day. Limit of detection (LOD) and limit of quantification (LOQ) were 0.07 and 0.23 mg/mL, respectively. The linearity for VC concentrations, ranging from 1 - 40 mg/mL, had the correlation coefficient of 0.9997. The stability analysis in this study showed that VC levels remained stable at -70oC for 2 weeks.
Conclusion: We demonstrated that our method provided the good accuracy and precision as well as acceptable LOD and LOQ for VC analysis with a small volume of plasma. Plasma kept for 2 weeks at -70oC could be analyzed for levels of VC. Therefore, this method is suitable for vitamin C assessment in patients, especially padiatric patiants, and researches
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