# Bioavailability of Liposomal Vitamin C in Powder Form: A Randomized, Double-Blind, Cross-Over Trial
## 2 Materials and Methods
### Clinical Study
#### 2.5.5 Methodology  
In total, 10 subjects were enrolled in this study. The participants were instructed to limit their intake of vitamin C-rich foods (primarily, raw fruits and vegetables), vitamin C-containing supplements, and pure vitamin C for a period of 72 h before and during the day of this study. On the day the experiment began, the participants followed a standardized, vitamin C-deficient diet provided by a catering company. The procedure commenced after an overnight fast of at least 8 h. Then, the volunteers orally took a single dose of 1000 mg of non-liposomal vitamin C (powder in capsules) or the liposomal formulation (powder in capsules). After a 14-day washout period, the participants were switched from their original treatment to the alternative one (liposomal or non-liposomal vitamin C formulation).  
The process of preparing samples for analysis was carried out in the AronPharma laboratory (Gdansk, Poland). Venous blood was collected from each participant immediately before the administration of the studied substance (T0) and 30 min and 1, 2, 3, 4, 6, 8, 10, and 24 h post-administration. All the samples were collected in pre-labeled K\({}_{2}\)EDTA vacutainers from the forearm vein using an indwelling cannula, and the heparin-lock technique was applied to prevent blood clotting inside them. Before the blood sample was drawn, 0.5 mL of blood was discarded to prevent the saline-diluted blood and heparin from interfering with the analysis. The cannula was removed after 24 h or earlier as needed or if blocked. The vacutainer tubes were positioned vertically in a stack and were kept on ice. The blood samples collected from all the participants from all time points were first centrifuged at 2800\(\times\)\(g\) for 8 min at 4 \({}^{\circ}\)C to isolate the plasma. Centrifugation was carried out within 30 min of the collection of all the samples at each time point. The resulting supernatant was immediately added to an equal volume of 10% (\(w/v\)) metaphorshoric acid (MPA) in 2 mmol/L of ethylenediaminetetraacetic acid disodium salt. The samples were left on ice for 5 min and centrifuged (16,000\(\times\)\(g\), 10 min, 4 \({}^{\circ}\)C) after adding 5 mmol/L of tris(2-carboxyl)phosphine hydrochloride. Each sample was filtered with a 0.22 \(\upmu\)m polyethersulfone membrane, and the ascorbic acid level was analyzed on the same day by ultra-performance liquid chromatography (UPLC) according to the protocol described in Section 2.4.3, \"Evaluation of vitamin C concentration\". The samples from the clinical trials were programmed for isocratic elution at a flow rate of 0.1 mL/min with 1% organic modifier and a 0.5 min re-equilibration time. The method for determining the concentration of ascorbic acid in the blood samples was validated at the R&D Department of AronPharma. The standard curve was developed by using a peak-areas linear regression equation from six ascorbic acid standards made up in MPA/EDTA and plasma. Each sample run included a plasma quality control sample. The samples were coded for the analytical procedure.