Ascorbic acid is one of the compounds found in a number of commercially important native plants fruits e.g. Kakadu plum, wild lime and bush tomato. High performance liquid chromatography (HPLC) was used to determine ascorbic acid in these native plant fruits. Ascorbic acid degradation of both standards and plant extracts was observed during HPLC sequence runs. These losses were considerable even though factors such as light, temperature and water activity, which accelerate the loss of ascorbic acid, were eliminated. Several concentrations of sodium metabisulphite were added to both standards and plant extracts to evaluate the effect on the rate of ascorbic acid degradation. A concentration of 500 μg/mL was the most effective but did not eliminate the problem. To correct for any loss still occurring, the rate constant k for ascorbic acid degradation was calculated and used to extrapolate back to the original ascorbic acid concentration. The k value was also found to vary for the different plants studied. For example the k value without added sodium metabisulphite for Kakadu plum, wild lime and Kakadu plum intermediate raw material were 0.00532, 0.02710 and 0.04429 respectively. With the addition of 500 μg/mL sodium metabisulphite the k value decreased to 0.00005, 0.00915 and 0.00586 respectively

Caffin, N.

Kerven, G.

McDonald, J.

Sommano, S.

University of Queensland eSpace

© All Rights Reserved
*Corresponding author. 
Email: s.sommano1@uq.edu.au
International Food Research Journal 18(3): 1017-1020 (2011)
*Sommano, S., Caffin, N., McDonald, J. and Kerven, G.
School of Agriculture and Food Sciences, The University of Queensland, 
Brisbane,QLD, 4067, Australia
Measurement of Ascorbic acid in Australian native plants
Abstract: Ascorbic acid is one of the compounds found in a number of commercially important native plants 
fruits e.g. Kakadu plum, wild lime and bush tomato.  High performance liquid chromatography (HPLC) was 
used to determine ascorbic acid in these native plant fruits.  Ascorbic acid degradation of both standards and 
plant extracts was observed during HPLC sequence runs.  These losses were considerable even though factors 
such as light, temperature and water activity, which accelerate the loss of ascorbic acid, were eliminated.  Several 
concentrations of sodium metabisulphite were added to both standards and plant extracts to evaluate the effect 
on the rate of ascorbic acid degradation.  A concentration of 500 µg/mL was the most effective but did not 
eliminate the problem.  To correct for any loss still occurring, the rate constant k for ascorbic acid degradation 
was calculated and used to extrapolate back to the original ascorbic acid concentration.  The k value was also 
found to vary for the different plants studied.  For example the k value without added sodium metabisulphite 
for Kakadu plum, wild lime and Kakadu plum intermediate raw material were 0.00532, 0.02710 and 0.04429 
respectively.  With the addition of 500 µg/mL sodium metabisulphite the k value decreased to 0.00005, 0.00915 
and 0.00586 respectively.
Keywords: Bush food, high-performance liquid chromatography (HPLC), dissolved oxygen, sodium 
metabisulphite  
Introduction
Ascorbic acid (AA) is water soluble vitamin which 
also known to be one of the more labile vitamins. 
Fruits and vegetables are the primary dietary source 
of ascorbic acid.  In the past ascorbic acid was named 
to be anti-scorbutic factor because many studies 
illustrated its function to prevent scurvy in animals 
(Asard et al., 2004).  Recently, the role of ascorbic 
acid as an in vivo antioxidant has obtained much 
attention.  As a function of free-radical scavenger, 
ascorbic acid can react against in vivo peroxidations, 
including quenching of singlet oxygen species.  
Kakadu plum (Terminalia  spp.) is one of the 
famous native fruits found in the Northern Territory 
and Western Australia (Ahmed and Johnson, 2000). 
It is pale green olive-sized fruit with a stone that 
clings to the fruit flesh as in a mango.  It also has been 
accepted that Kakadu plum have a high vitamin C 
content which ranges from 406–5320 mg/100 g edible 
portion (Brand Miller et al., 1993) and as such the fruit 
has been recommended for further marketing studies 
and the optimisation of vitamin C production.
Wild lime (Microcitrus spp.) is newly named due 
to their very small juvenile leaves and their minute 
flowers (Birmingham, 1998).  The fruit is round to 
oblate in shape and approximately 2 cm in diameter, 
weighing from 1-3 g.  The skin is light yellow-green 
on maturity and contains a large oil gland.  The natural 
distribution of this specie is the semi-arid regions of 
eastern Australia, from Rockhamton to Longreach in 
Queensland, south to Dubbo in central New South 
Wales and west to Quorm, in the flinders ranges of 
South Australia.  Wild lime contains relatively high 
amount of ascorbic acid (0-82 mg/100 g edible 
portion) portion (Brand Miller et al., 1993).  
Oxygen, temperature, light, metal catalysts, pH 
and the presence of ascorbic acid oxidase lead to the 
degradation of ascorbic acid.  At low pH, ascorbic 
acid is stable in the fully protonate from.  Maximal 
stability usually performs at pH about 4. 
Metal catalysts can increase degradation rate 
compare to uncatalyzed oxidation.  The loss of 
vitamin C while cooking depends on the degree of 
heating and how much surface area exposed to the 
oxygen.  However, in the low water activity condition, 
the crystalline L-ascorbic acid remains highly stable 
even in the presence of oxygen.
Champion et al. (2004) stated that ascorbic acid is 
oxidized when reacting with oxygen in the presence 
of air or with oxidant substance or with particular 
enzyme.  Jung et al. (1995) studied the effect of singlet 
oxygen quencher on the destruction of ascorbic acid. 
They found that the oxidation of ascorbic acid was 
greatly reduced by the addition of 0.2 mM sodium 
azide even with the acceleration of ribloflavin.  The 
result shown that 0.2 mM sodium azide inhibited 
almost 81% of photooxidation after 12 min light 
storage.  Sulpher dioxide and it’s satls (sodium and 
potassium salts) are used in the production of wine. 
Sulpher dioxide (SO
2
) is added in to a bottle of wine 
1018 Sommano, S., Caffin, N., McDonald, J. and Kerven, G. 
International Food Research Journal 18(3): 1017-1020
in according to inhibit microorganisms and bind with 
the dissolve oxygen. Excess amount of dissolved 
oxygen combines directly with ethanol and higher 
alcohol to form aldehydes which is accumulated and 
gives wine a flat and stale aroma and flavour (Rotter, 
2001).
High performance liquid chromatography (HPLC) 
was used to determine ascorbic acid in these native 
plant fruits.  The problem due to the use of a sequence 
HPLC run is that ascorbic acid still degrades even 
with the temperature and light control. For example, 
when 60 samples (including replications) are running 
on HPLC in sequence and the retention time to make 
a complete elution is set at 30 min, the ascorbic acid 
content of the last sample may decrease to almost 
half of the first one. However there is no reported 
work examining this problem of loss of ascorbic acid 
during HPLC sequence runs.
Sodium metabisulphite (Na
2
S
2
O
5
) does not 
interfere with ascorbic acid elution but is able to 
retard the oxidation of ascorbic acid. Since each plant 
has a different matrix, ascorbic acid might react with 
some compound in the matrix to make it less or more 
stable.
This study determines the effect of an oxygen 
quencher added to native food samples containing 
ascorbic acid. Sodium metabisulphite was chosen 
because it does not interfere with ascorbic acid 
elution but is able to retard the oxidation of ascorbic 
acid.  The rate of degradation of ascorbic acid in each 
type of raw material was also studied.  Since each 
sample has a different food matrix, ascorbic acid may 
react with some compound in the matrix to make it 
less or more stable.
Material and Methods
Raw materials 
Kakadu plum which was supplied by Robins 
Foods , Braeside VIC , Australia were thinly sliced 
and placed in the plastic trays. The fruits were 
prefrozen overnight before transferring to freeze 
drier.  As well as wild lime (Robins Foods , Braeside 
VIC , Australia), the whole fruits were halved and 
placed in the plastic trays.  Dried fruits were grinded 
to powder, stored in the containers in – 80°C freezer.
Kakadu plum filtrate which was used as an 
intermediate material for commercial sauces.  It was 
prepared by boiling 12 kg of the plum in 150 kg of 
water for 30 min.  The stone of the fruit could be 
removed from the fresh in this stage.  The filtrate was 
stored in cool room (4°C)  prior to the extraction. 
The amount of oxygen quencher (sodium 
metabisulphite) on standard stability test
The stock standard 1000 µg/mL of ascorbic acid 
(Sigma-Aldrich, NSW, Australia) was prepared by 
dissolving 100 mg of ascorbic acid in 100 mL volume 
metric flask with nitrogen spiked deionize water. 
Working standards were also prepared by diluting 
down the stock standard 0.5 mL to 10 mL volumetric 
flask. Sodium metabisulphite (Sigma-Aldrich, NSW, 
Australia) at concentrations of 0, 5.0 and 10 mg/100 
mL were added in the working standards.  Each of 
the standards was transferred to three vials and the 
ascorbic acid contents were detected by HPLC at 
time 0, 24 and 48 hr. 
The test was also confirmed by using raw material 
sample (Kakadu plum filtrate). Approximately 15 
g of the filtrate was mixed with 10 mL of 0.05 N 
phosphoric acid (H
3
PO
4
) on a rotary shaker for min. 
The mixture was then centrifuged at 5,000 rpm for 15 
min at 4ºC.  The effect of Na
2
S
2
O
5 
was then tested on 
the supernatant (ascorbic acid extract).
The amount of sodium metabisulphite was chosen 
regarding to the ability to delay the degradation of 
ascorbic acid. 
Ascorbic acid stability test on the raw material 
samples
The dried fruits (0.5 and 1.0 g for Kakadu plum 
and wild lime respectively) were extracted with 15 
mL of extracting solution.
The mixture was then mixed for 30 min at high 
speed in a rotary shaker and centrifuged at 5,000 rpm 
for 15 min.  The supernatant was transferred to 100 
mL volumetric flask.  The first sample was diluted 
without the addition of sodium metabisulphite. 
The second one was with the addition of x µg/mL 
of sodium metabisulphite (the amount was chosen 
according to the previous standard test).  Each samples 
was spiked with 50 µg/mL of ascorbic acid,purified 
by passing through a C
18
 cartridge, preconditioned by 
flushing with methanol followed by deionize water, 
and a 0.45-µm Milipore filter.  The ascorbic acid was 
detected at three time interval 0, 5, 20 hrs.  Taking 
into account that each replicated sample took 30 min 
cycle to run on HPLC, four replications of each of the 
samples were , thear for, analysed each days.
HPLC analysis
The prevail C
18
 column (150 x 4.6 mm) was used 
with 25 mM KH
2
PO
4
 pH 2.25 as mobile phase (as 
per procedure developed at Analytical services, The 
University of Queensland, QLD, Australia).  Ascorbic 
acid standards were prepared at the concentrations of 
0, 250, 500 and 1,000 µg/mL.  The detection of the 
ascorbic acid was performed at 235 nm using UV 
Measurement of Ascorbic acid in Australian native plants 1019
International Food Research Journal 18(3): 1017-1020
detector.
Results and Discussion
The amount of singlet oxygen quencher (sodium 
metabisulphite) on standard stability test
The results (Table 1 and 2) showed that 
sodium metabisulphite could beneficially retard 
the degradation of ascorbic acid by removing the 
oxygen.  The following equation explains how 
sodium metabisulphit (sulpher dioxide salt) quenches 
with oxygen (Rotter 2001):
                      SO
3
2- + O
2 
 2SO
4
2-
At 48 hr and the presence of sodium 
metabisulphite, % loss decreased from about 95% 
to 20% (Table 1).  Addition of 500 µg/mL sodium 
metabisulphite into the standard solution was more 
effective inminimizing the degradation than 1000 µg/
mL.  Similar result was obtained with plant material 
samples (Table 1).  Therefore, sodium metabisulphite 
at concentration of 500 µg/mL was chosen for the 
subsequence tests. 
Ascorbic acid stability test on the raw material 
samples
The result demonstrated that the % loss of ascorbic 
acid weredifferent for the three sample tested.  Kakadu 
plum (dried fruit) exhibited the lowest % loss (13%). 
Kakadu plum filtrate, nonetheless, gave the highest 
% loss (60%) (Table 2).  This may be explained by 
the fact that ascorbic acid is more stable in the sample 
with lower aw (Uddin et al., 2001).
The result in Table 3 also supported the use of 
singlet oxygen quencher. Using 500 µg/mL sodium 
metabisulphite in dried Kakadu plum extract 
decreased the  loss from 13% to 0.1% in 20 hrs 
and more than haft of the % loss decreased in wild 
lime.  Even in the highly water containing sample 
(Kakadu plum filtrate), the loss reduced from about 
60% to 10%. Some of the standard deviation values 
are quite high but they are consistent across time for 
each sample. The method may need some attention to 
improve accuracy.
Figure 2 represents the relation between ascorbic 
acid and time of measurement for dried Kakadu 
plum.  Graphs demonstrate the first order relationship 
and thus the equation for first–order kinetics upon 
integration of a general reaction rate expression 
for degradation kinetic (Nisha et al., 2004) can be 
expressed as:
                              ln c/c
0
 = -kt
To gain advantage from this relationship, the 
rate constant k and half life for each sample were 
calculated in order to extrapolate back to the initial 
Table 1. The effect of sodium metabisulphite (Na
2
S
2
O
5
) 
on stability of standard ascorbic acid (50 µg/mL) and 
fruit material (Kakadu plum filtrate)
1) Ascorbic acid standard 50 µg/mL
[Na
2
S
2
O
5
]
Time (hr) 0 500 µg/mL 100 µg/mL
0 51.5± 0.1 51.1± 0.05 51.3± 0.07
18 32.1 ± 0.2 46.8 ± 0.1 45.9 ± 0.3
24 25.7 ± 0.4 45.8 ± 0.1 44.4 ± 0.3
48 3.2 ± 0.33 40.7 ± 0.3 37.6 ± 1.06
% loss 93.69% 20.41% 26.76%
2) Kakadu plum filtrate extracts
[Na
2
S
2
O
5
]
Time (hr) 0 500 µg/mL 100 µg/mL
0 10.5± 0.2 11.2 ± 0.3 10.0 ± 0.1
24 1.9 ± 0.6 9.2 ± 0.3 8.04 ± 0.4
48 1.4 ± 0.1 7.4 ± 0.4 6.2 ± 0.6
% loss 87.14% 33.69% 37.91%
Values are mean of triplicate samples ± SD.  
Table 2.  Loss (%) of ascorbic acid in dried Kakadu plum, dried 
wild lime and Kakadu plum filtrate with and without the 
addition of sodium metabisulphite
Time 
(min)
Dried Kakaduplum Dried wild lime Kakadu plum filtrate
 
with 500 
µg/mL 
Na
2
S
2
O
5
with 500 
µg/mL 
Na
2
S
2
O
5
with 500 
µg/mL 
Na
2
S
2
O
5
0 861.5
±96
661.9
±135
106.9
±13.5
118.6
±15.3
86.1
±9.6
110.3
±16.1
5 842.4
±86.150
661.2
±141.3
96.2
±13.0
113.6
±14.6
73.0
±7.2
106.8
±15.5
20 774.6
±86.0
661.2
±141.4
62.2
±11.3
98.8
±9.8
35.5
±5.6
98.1
±14.4
% loss 13.6 0.1 41.8 16.7 58.8 11.1
Values are mean of triplicate samples ± SD.  
Table 3.  Rate constants (calculated) and half-lives 
of the reaction
Samples Rate Constant k( hour-1) R
2 Half-life
(hours)
Dried Kakadu plum 0.00532 0.999 130.3
DKP + 500 µg/mL 
Na
2
S
2
O
5
0.00005 -0.374 14508.3
Dried wild lime 0.02710 1 25.6
DWL + 500 µg/mL 
Na
2
S
2
O
5
0.00915 0.999 75.7
Kakadu plum filtrate 0.04429 0.998 15.6
KPF + 500 µg/mL 
Na
2
S
2
O
5
0.00586 0.999 118.2
Result shows good suits in most cases (R2 > 0.998).  
1020 Sommano, S., Caffin, N., McDonald, J. and Kerven, G. 
International Food Research Journal 18(3): 1017-1020
amount of ascorbic acid since there was a long gap 
between the first and the last samples on HPLC run. 
The half-lives of dried Kakadu plum, dried wild lime 
and Kakadu plum filtrate increased by approximately 
100, 3, 10 folds respectively (Table 3).  These were 
greater than other food sources such as the half life 
of ascorbic acid was 34.1 hours at 30°C in dried kiwi 
fruits (a
w
 0.8) (Uddin et al., 2001) and 55 hours at 
35°C, in intermediate moist apples (a
w
 0.83) (Singh 
et al., 1983).  
Conclusion
The use of sodium metabisulphite as a singlet 
oxygen quencher is very useful in retarding the rate 
of ascorbic acid degradation. The factors effecting 
degradation are oxygen, light, temperature and a
w
. 
However, during HPLC sequence runs where all of 
those factors are reduced or eliminated, there are still 
some losses. The food matrix could be considered 
one of the factors which might affect ascorbic acid 
degradation.  This study found that the ascorbic acid 
in different type of fruits showed different rates of 
degradation. Thus, they were studied individually. 
Moreover, since the degradation kinetics can be 
expressed as a first order reaction, the rate constant 
k can be calculated for each sample.  Since loss of 
ascorbic acid was still observed during long HPLC 
sequence runs, the k values can be used to extrapolate 
back to the initial ascorbic acid concentration in the 
samples.
Acknowledgements
This work was a part of the research project 
founded by RIRDC, Australia.  The author would like 
to thank Mr. Graham Kervin, school of Lands, Crop 
and Food Sciences, The University of Queensland, 
Australia for his grateful experience on HPLC.  
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-10
0
10
20
30
40
50
0 2 4 6
m
AU
Time (min)
Figure 1. HPLC Chromatogram of stand ascorbic acid 
(RT= 4.9 min) on C
18
 column with H
2
SO
4
 mobile phase 
(see material and methods)
Figure 2. Correlation curve between amount of ascorbic 
acid and time (hr) of Kakadu plum.  ◊-◊ correlation curve 
of dried Kakadu plum (R2 = 0.999)  ○-○ correlation 
curve of dried Kakadu plum plus 500 µg/mL sodium 
metabisulphite


Measurement of ascorbic acid in Australian native plants

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Measurement of ascorbic acid in Australian native plants

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