1. The effects of physicochemical properties of ascorbate and its bioavailability
Author: Szőcs Attila
Coordinator: Vancea Szende, lecturer
Department of Physical Chemistry

2. Introduction absence of L-gulonolactone oxidase in humans
more than 70 years of research about ascorbic acid
the most studied molecule in the history
Intravenous administration – the most effective application, considering the bioavailability of vitamin C.
Oral administration – a real challenge even nowadays.

3. Objectives Calcium – ascorbate Liposoluble ? Weaker acidic ?
Better absorption ?
The purpose of this study is to verify this statements and to describe the physical-chemical properties of calcium ascorbate in order to predict the absorption, respectively correlating the absorption with the ascorbic acid concentration in plasma, after oral administration of a dietary supplement.

4. Materials and methods
For all of our purposes we’ve used a dietary supplement, witch contains 90 mg calcium ascorbate and bioflavonoids.

5. pH - Mettler-Toledo MP225 pH meter
Materials and methods
Measurement of solution pH
pH - Mettler-Toledo MP225 pH meter

6. logP
We have determined the partition of calcium ascorbate between 2 different solutions at 2 different pH values and octanol
1. solution at 1,8 pH (0,02 M HCl) and octanol
2. solution at 7,4 pH (100 ml 0,1 M KH2PO4 + 78,2 ml NaOH 0,1 M) and octanol
260 nm - 1,8 pH
282 nm - 7,4 pH

7. pKa
The dissociation constant has been determined using 5 buffer solutions with different pH values: 1,8; 3,2; 4,05; 8; 10.
The concentrations of ionized/nonionized forms have been measured with an UV spectrophotometric method.

8. Plasma concentration 1 volunteer
540 mg of calcium ascorbate with bioflavonoids (6 capsules) in a single dose.
Subsequent blood samples: a jeune, 1/2, 1, 2, 3, 4, 6, 8 hours.
Ascorbic acid plasma concentration has been determined by HPLC-MS method.
The measured Cmax and tmax values were compared with data collected from available publications.
HPLC-MS

9. HPLC conditions: C18 column, mobile phase: 0,1% formic acid+ methanol, injection volume: 2 µL
tR = 1.3 min ascorbic acid

10. ESI negative ionization
Mass spectrum: m/z 175 (M-H) fragmentation to m/z 115

11. Results
The pH value measured for the dietary supplement’s solution is 5,3 compared to ascorbic acid solution pH value that is about 2,5.
Calculated logP values for
calcium ascorbate:
-0,449 for the solution at 1,8 pH
-0,29 for the solution at 7,4 pH.
(not real)
Ascorbic acid’s logP = -2,048

12. The calculated pKa dissociation constant = 4,17
wich corresponds with the known ascorbic acids 4,2 pKa value.

13. Measured pharmacokinetic parameters
Tmax = 4 hours
Cmax = 12 µg/mL
Sample preparation: protein precipitation with methanol, dilution with acetic acid 0,1%
Concentration range: 5-40 µg/ml
Conc. µg/mL
hour

14. Conclusions
The measured pH value supports that calcium-ascorbate is less acidic than ascorbic acid, wich confirms that it can be recommended for patients with a sensitive stomach, acid upset or diarheea.
The higher logP value indicates a better absorption compared to ascorbic acid, and this can be correlated with the measured concentrations.
The 4 hour tmax value corresponds with the ascorbic acids tmax value presented in the available publications ( 2-4 hours).
The determined 12 µg/mL Cmax value is higher than the expected 7-10 µg/mL maximum concentration presented in the literature.
These findings suggests that calcium ascorbate supplemented with bioflavonoids can be an effective alternative to oral administration of vitamin C.
We also concluded that a lot of methods based on the reversible redox reaction of AA oxidation/DHA reduction developed in the past are without true specificity, considering the interferences with other reducing agents. At present HPLC-MS methods are the most accurate methods for measuring AA and DHA concentrations.
To describe the pharmacokinetic parameters further studies are necessary involving more volunteers.

15. Thank you for your attention!