The effects of physicochemical properties of ascorbate and its bioavailability

Author: Szőcs AttilaCoordinator: Vancea Szende, lecturerDepartment 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.

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 octanol2. 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

230 240 250 260 270 280 290 300 310 320 330 340

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

pH=1.87

nm

Ab

s

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

- MRM (175.0 -> 115.0) CCpl_2504a_21.d

Acquisition Time (min)

0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2

Cou

nts

1x10

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7*RT=1.276Name=AA

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

- MRM (1.154-1.480 min, 97 scans) (175.0 -> **) CCpl_2504_07.d

Mass-to-Charge (m/z)

110 115 120 125 130 135 140 145 150 155 160 165 170 175 180

Cou

nts 1x10

00.05

0.10.15

0.20.25

0.30.35

0.40.45

0.50.55

0.60.65

0.70.75

0.80.85

0.90.95

11.05

1.11.15

1.21.25

1.31.35

1.41.45

115.0

175.0

ESI negative ionizationMass 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.

230 240 250 260 270 280 290 300 310 320 330 340

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1,8 3,2 4,05 8 10

7

nm

Ab

s

Measured pharmacokineticparameters

Tmax = 4 hours

Cmax = 12 µg/mL

0 1 2 3 4 5 6 7 8 90

2

4

6

8

10

12

14

Sample preparation: protein precipitation with methanol, dilution with acetic acid 0,1%

Concentration range: 5-40 µg/ml

Co

nc

. µ

g/m

L

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.

Thank you for your attention!