Analysis of the Purity and Storage

Stability of L-Ascorbic Acid Powders

Application Note

Author Introduction
Linda Lloyd Most previous studies of the storage stability of L-ascorbic acid were done on
Agilent Technologies, Inc. solutions of the acid. However, L-ascorbic acid is usually kept in the dry powder
form, batches of which may contain breakdown products or other impurities arising
from the production process. The presence of these contaminants is suspected to
adversely affect the stability of the acid and/or contribute to color changes in the
stored product over time.
This note describes a trial of the purity and stability of stored L-ascorbic acid
powder samples from four different manufacturers, and assesses degradation
compounds and/or precursors from the production process. Ion suppression
reversed phase HPLC was used, with PLRP-S columns. PLRP-S is a rigid
macroporous styrene/divinylbenzene HPLC phase with outstanding chemical and
physical stability with acidic eluents.
 Materials and Reagents Table 1. Retention times of reference compounds.

Limit of
Samples: L-ascorbic acid powders from different L-ascorbic acid Retention
Sample detection
association time (min)
manufacturers produced from 1971 to 1987 (µmol)
Impurity reference compounds: L-dehydroascorbic acid degradation
oxalic acid 6.00 0.039
(DHAA), 2,3-diketo-L-gulonic acid, L-threonic acid, oxalic acid, product
furfural, 5,6-O-isopropylidene-L-ascorbic acid, D-gulonic acid- degradation
L-threonine 6.30 0.053
1,4-lactone product
2,3-diketo-L-gulonic
precursor 6.60 -
acid
Conditions
Column: PLRP-S 100Å 5 µm, 150 x 4.6 mm (p/n PL1111-3500) D-gulonic acid-1,4-
precursor 6.60 -
Eluent: 0.2 M NaH2PO4, pH 2.14 lactone
Flow rate: 0.5 mL/min L-dehydroascorbic degradation
Detector: UV, 268 and 220 nm 7.40 0.017
acid product
L-ascorbic acid - 8.70

Results and Discussion 5,6-O-isopropylidene-

L-ascorbic acid
precursor 8.70

A plot of manufacturing date against purity is given in Figure degradation

fufural 12.10
1, showing a gradual decline in L-ascorbic acid with age even product
in dry powder formulations. Comparison of the retention times
of sample peaks with those of the reference compounds
(Table 1) did not reveal any sign of degradation products or The samples varied in their degree of whiteness regardless
precursors associated with the manufacture or storage of of the date of production, suggesting that differences in
L-ascorbic acid. The absence of these compounds suggests whiteness were not indicators of purity but may have been
that other degradation routes are present when L-ascorbic be due to differing production methods. However, the aged
acid is stored under near anaerobic conditions of low water samples manufactured in 1971 and 1978 had a brown
activity. appearance, most evident in the 1978 sample. Measurement
of the L-ascorbic acid content of these samples was lower
compared to newer samples, but no impurities were detected.

The oxidative degradation of a solution of L-ascorbic acid was

100
assessed, following the pathway shown in Figure 2.

O
99
C OH COOH
L-ascorbic acid purity (%)

+
H C OH COOH
O2 HO C H
98 O O O
H C OH
C C C OH

HO C O C O C C
H 2O
H
O O
HO C O C O C L-threonic acid oxalic acid
97
H C H C H C OH

HO C H HO C H HO C H O O
C
O 2
CH 2O
H C
H 2O
H C
H 2O
H C OH C OH
96 L-ascorbic acid L-dehydro 2,3-diketo- H C OH HO C H
+
ascorbic acid L-gulonic acid H C OH H C OH

HO C H HO C H

95 C
H 2O
H C
H 2O
H

L-lyxonic acid L-xylonic acid

1988 ‘86 ‘84 ‘82 ‘80 ‘78 ‘76 ‘74 ‘72 ‘70 Figure 2. The proposed oxidative degradation path of L-ascorbic acid.
production date

Figure 1. Graph of L-ascorbic acid purity and production date of powder

samples of L-ascorbic acid.

2
Table 1 shows the minimum limits of detection of the
degradation products L-dehydroascorbic acid, oxalic acid
and L-threonic acid from this assay. It was not possible to
carry out a test for 2,3-diketo-L-gulonic acid because a pure
reference compound was not available.

The complete data set and analysis is available in Kennedy et

al. (1989).

Conclusion
Ion suppression HPLC using PLRP-S columns successfully
elucidated the effects of age on the gradual deterioration of
dry powder L-ascorbic acid, and suggested the presence of
alternative degradation mechanisms when the acid is stored
in dry powder form.

Reference
Kennedy, JF, Rivera, ZS, Lloyd, LL, Warner, FP, White, CA
(1989) The use of reversed-phase HPLC for the determination
of purity and stability on storage of L-ascorbic acid samples.
J. Micronutr. Anal., 5, 281-289.

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This information is subject to change without notice.
© Agilent Technologies, Inc. 2011
Published in UK, May 9, 2011
5990-8162EN