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

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.

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.

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

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

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

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.

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

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

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

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

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

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

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.

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