J. Biochem. Biophys. Methods 70 (2007) 767 – 772
www.elsevier.com/locate/jbbm
Reduction of dehydroascorbic acid at low pH
Luka Wechtersbach, Blaž Cigić ⁎
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
Received 23 November 2006; received in revised form 23 April 2007; accepted 29 April 2007
Abstract
Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the
routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic
acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely
determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the
stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is
evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same
as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in
the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method,
over those currently applied in a routine assay of vitamin C, performed on large number of samples.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Dehydroascorbic acid; Ascorbic acid; Vitamin C; Tris [2-carboxyethyl] phosphine; Reduction; Dithiothreitol
1. Introduction
Total vitamin C in biological samples comprises ascorbic
acid (AA) and dehydroascorbic acid (DHAA). Ingested AA and
DHAA are absorbed through the gastrointestinal tract [1] and
can be interchanged enzymatically in vivo [2]. The concentration of DHAA in plasma is usually low but is increased in states
of oxidative stress [3,4]. In plants, which are the main sources of
vitamin C in human nutrition, higher amounts of DHAA are
found as a result of stress during the growth or of processing
prior to consumption [5].
Ascorbate-oxidase, dissolved oxygen, copper and iron ions,
hydroxide ions and quinones increase the rate of AA oxidation
to DHAA [5–7]. This results in a time dependent decrease of
AA concentration in samples of blood plasma [8,9], drinking
water [10] and food products [5]. AA can also be oxidised
Abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; DTNB,
5,5′-Dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; MPA, metaphosphoric
acid; TCEP, Tris [2-carboxyethyl] phosphine.
⁎ Corresponding author. Tel.: +386 1 423 11 61; fax: +386 1 256 62 96.
E-mail address: [email protected] (B. Cigić).
0165-022X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2007.04.007
during sample preparation, yielding DHAA as a result of the
experimental procedure [9,11,12]. The low stability of AA and
DHAA at mildly acidic and neutral pH is the major factor that
limits the applicability of the DHAA/AA ratio as an indicator of
physiological stress. The amount of AA oxidised to DHAA can
nevertheless be minimized by sample preparation and storage in
acidic solution, preferably metaphosphoric acid (MPA), which
has been used in vitamin C analysis for decades [13]. DHAA,
like AA, is also more stable in MPA. However, the irreversible
decay at higher pH is faster and results in formation of the open
chain product, diketogulonic acid [14]. The concentration of
DHAA affects the rate of decay [14].
Chromatographic analysis is the most common method for
determining AA and DHAA in biological samples [15]. Due to
the low absorbance of DHAA above 210 nm and lack of
response on electrochemical detectors, reduction of DHAA to
AA is usually employed to determine total vitamin C. The
concentration of DHAA is then calculated by subtracting the
AA determined in the absence of reducing agent.
Reduction is performed by sulphydryl compounds, including
homocysteine, dimercaptopropanol, dimercaptoethanol, glutathione, cysteine and dithiothreitol (DTT) [15], or by phosphorus
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L. Wechtersbach, B. Cigić / J. Biochem. Biophys. Methods 70 (2007) 767–772
based tris-[2-carboxyethyl] phosphine (TCEP) [16]. Thiolcontaining species are efficient only at mildly acidic and neutral
pH. DHAA containing samples, acidified with MPA, are therefore neutralised prior to reduction [8,16,17]. This step complicates the analysis of DHAA and can lead to experimental
errors, since both AA [18] and DHAA [14,19] are unstable at
neutral pH. TCEP is functional over a much wider pH range
[20] and has been applied for DHAA reduction at pH 4.3 [16],
which is the lowest pH reported for the reduction step in vitamin
C analysis. The benefit of TCEP, in comparison to thiols such as
DTT, is its significantly higher stability in the presence of Fe3+
ions [20]. Both reducing agents are nevertheless stoichiometrically oxidised by Cu2+ ions [21,22].
The objective of this study was to test the effectiveness of
TCEP in reducing DHAA at pH below 2, where both forms of
vitamin C are relatively stable. The kinetics of the reduction,
recoveries of AA from DHAA, effect of Cu2+ ions on reducing
agent stability and on the reduction of DHAA and the stability
of the product, were all evaluated.
2. Experimentals
2.1. Materials
Ascorbic acid, Tris [2-carboxyethyl] phosphine hydrochloride, dithiothreitol, 5,5-dithiobis(2-nitrobenzoic acid), ascorbate-oxidase and CuSO4·5H2O were obtained from Sigma and
dehydroascorbic acid from Aldrich. All other chemicals were of
analytical grade and obtained from Sigma. Water used for all
experiments was purified using a Milli-Q system from Millipore
(resistivity N 18 MΩ cm). Autosampler vials, 1.5 ml ABZ, were
from Supelco. All chemicals were dissolved in purified water
and filtered through 0.45 μm cellulose acetate filter prior to use.
Citrate and acetate buffers were made by titrating the acids with
NaOH.
2.2. Chromatographic analysis of AA
Samples were analysed by HPLC (Marathon-XT autosampler,
Knauer isocratic Pump (K-1001), X-Act degassing unit from Jour
research, Knauer UV–VIS detector, Wellchrom interface box and
personal computer running EuroChrom 2002 software).
Ascorbic acid was analysed on a Synergy Hydro-RP 80
(4 μm, 250 × 4.60 mm, Phenomenex) chromatographic column
equilibrated with 2.5 mM H2SO4 in Milli-Q water at a flow rate
of 1 ml/min. 20 μl samples were injected on the column. AA,
eluted after 4.9 min, was detected at 250 nm.
2.3. Kinetics of DHAA reduction
DHA reduction in citrate buffers was followed spectrophotometrically (Hewlett-Packard HP-8453 UV–VIS detector and
HP-89090A temperature controller) at 25 °C. 800 μl of citrate
buffer (125 mM) was mixed with 100 μl of reducing agent (10
or 100 mM) in a UV semi-micro cuvette (Brand, Germany). The
reaction was started by adding 100 μl of 2 mM DHAA.
Absorbance was recorded at 250 nm.
The kinetics of DHAA reduction in MPA and HCl were
followed chromatographically. 800 μl of 2% MPA (w/v) or
0,125 M HCl was mixed with 100 μl of TCEP (10 or 100 mM)
in the autosampler vial. Reaction was started by adding 100 μl
of DHAA (2 mM or 200 μM). The vial was sealed, stirred by
vortexing and placed on the autosampler tray. The time interval
from adding DHA to injection was defined as the time of
reduction, since DHA and TCEP were separated on the column.
Data were analysed by TableCurve 2D v5.01 software
(Systat Software Inc.). Kinetic data were fitted to a single first
order reaction.
y ¼ a þ bð1 ekt Þ
ð1Þ
Parameter a is the signal at t = 0, b the signal of ascorbic acid
formed at t → ∞, and k is the rate constant (min− 1).
2.4. Recoveries of DHAA reduction to AA
2.4.1. Enzymatically prepared DHAA
DHAA was prepared enzymatically as follows: 780 μl of
125 mM phosphate buffer pH 6.5 was mixed with 20 μl of
ascorbate-oxidase (125 U/ml) and 100 μl of 2 mM AA.
Reaction was complete in 5 min, as confirmed by absorbance at
250 nm. The enzyme was denatured by acidification to pH 2 and
reduction started by adding 100 μl of 100 mM TCEP dissolved
in 1 M HCl. After 3 h, 850 μl of solution was transferred into the
autosampler vial, diluted with 150 μl of purified water and
analysed chromatographically to give the yield of reduced
DHAA.
2.4.2. Commercially available DHAA
Samples were prepared in microcentrifuge tubes in the same
way as in Section 2.3 and left at room temperature for 3 h to allow
complete reduction. 850 μl of each solution was then transferred
to the autosampler vial. When reduction was performed in citrate
and acetate buffers at pH 5, 150 μl of 10% MPA was added to
acidify the mixture prior to chromatographic analysis. For
reduction in citrate pH 7, 150 μl of 2 M HCl was used for
acidification. When reduction was performed in HCl and MPA,
150 μl of purified water was added. All samples were prepared in
triplicate and analysed chromatographically for AA content.
2.5. Stability of reducing agents
In order to determine the effect of Cu2+ ions on the stability
of the reducing agents, TCEP and DTT were incubated in the
presence of Cu2+ ions. 940 μl of buffer (50 mM citrate pH 5,
50 mM acetate pH 5 or 2% MPA) was mixed with 50 μl of
20 mM reducing agent and 10 μl of CuSO4 (10 mM or 1 mM)
and stored at 25 °C.
The concentration of remaining DTT and TCEP was determined with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as described [20]. After predetermined time intervals, 30 μl of the
reaction mixture was added to 970 μl of 500 μM DTNB in
50 mM Tris, pH 9. The increase of absorbance at 412 nm was
read after 1 min. All experiments were performed in triplicate.
L. Wechtersbach, B. Cigić / J. Biochem. Biophys. Methods 70 (2007) 767–772
Table 2
Yields of AA from reduction of DHAA
2.6. Stability of ascorbic acid
Long-term stability of AA formed by reduction of DHAA
was determined in the presence of Cu2+ ions. 100 μl of DHAA
(2 mM or 200 μM) was diluted with 790 μl of the appropriate
buffer (25 mM acetate pH 5, 25 mM citrate pH 5 or 2% MPA)
and 10 μl of 10 mM CuSO4 in autosampler vials. 100 μl of
reducing agent (100 mM or 10 mM) was immediately added to
start reduction. Vials were maintained at 25 °C.
For reduction with 10 mM TCEP, 100 μl of DHAA (2 mM or
200 μM) was added to a mixture of the appropriate buffer
(765 μl), 1 M NaOH (25 μl) and 10 mM CuSO4 (10 μl). Direct
contact between NaOH and DHAA was avoided in order to
prevent local hydrolysis of DHAA with concentrated base. The
concentration of AA was determined chromatographically after
4 h, then daily for 4 days and after a week.
3. Results
3.1. Kinetics of DHAA reduction with DTT and TCEP
Rates of reduction of DHAA by TCEP and DTT were
determined as a function of pH (Table 1). The rate of reduction
in neutral and mildly acidic solutions was determined by
following absorbance at 250 nm. 250 nm is the isosbestic point,
independent of pH, where protonated and ionised forms of AA
have the same molar extinction coefficient [23]. Kinetics were
best fitted as a single first order reaction (Eq. (1)).
Reduction was 99% complete with both TCEP and DTT
within 3 min (Table 1). Reduction at pH 7 is faster with DTT
than with TCEP. At pH 5 the rate is slower, especially with DTT,
where 99% of AA is formed in 15 min. At lower pH values,
DTT is less effective [16] and is practically inactive at pH 3,
whereas rate of reduction with TCEP at this pH is still higher
than the reduction with DTT at pH 5 (Table 1).
Slower reaction rates in MPA and HCl allowed chromatographic analysis of the AA formed (Table 1). TCEP is effective
in 0.1 M HCl (pH ≈ 1), as well as in 1.6% MPA (pH 1.9) in
which samples for vitamin C analysis are usually prepared. 99%
of AA is formed in MPA in ≈ 20 min and ≈ 45 min in HCl. The
reaction being pseudo-first order, the rate constants are independent of DHAA concentration when TCEP is in large excess
Table 1
Time required to obtain 99% of AA from DHAA and the related rate constants
under different conditions of reduction
Reduction conditions
Citrate pH 7
Citrate pH 7
Citrate pH 5
Citrate pH 5
Citrate pH 3
Citrate pH 3
MPA pH 1.9
MPA pH 1.9
MPA pH 1.9
MPA pH 1.9
HCl pH ≈ 1
200 μM DHAA
200 μM DHAA
200 μM DHAA
200 μM DHAA
200 μM DHAA
200 μM DHAA
200 μM DHAA
20 μM DHAA
20 μM DHAA
200 μM DHAA
200 μM DHAA
10 mM DTT
10 mM TCEP
10 mM TCEP
10 mM DTT
10 mM TCEP
10 mM DTT
10 mM TCEP
10 mM TCEP
1 mM TCEP
1 mM TCEP
10 mM TCEP
769
t (min) 99%
k (min− 1)
1.6
2.4
6.9
15
13
N1440
17
18
170
180
48
2.8
1.9
0.66
0.31
0.36
b0.0032
0.27
0.26
0.027
0.025
0.096
10 mM
10 mM
1 mM
1 mM
reducing agent reducing agent reducing agent reducing agent
200 μM
DHAA a
Citrate
100 ± 0.3
pH 7 b, c
Acetate
98 ± 2.1
pH 5 b, c
Acetate
99 ± 1.8
pH 5 c, d
MPA
100 ± 1.0
pH 1.9 b, c
HCl
99 ± 2.3
pH ≈ 1 b, c
20 μM
DHAA a
200 μM
DHAA a
20 μM
DHAA a
99 ± 0.6
97 ± 0.9
91 ± 1.1
96 ± 1.6
98 ± 2.3
96 ± 1.0
99 ± 0.9
100 ± 0.5
99 ± 0.7
96 ± 2.0
99 ± 1.0
98 ± 0.5
n.d.
n.d.
n.d.
n.d. not determined.
a
Commercially available DHAA (purity ≥80%).
b
Reduction was performed with TCEP.
c
Values shown are expressed as percentages of maximal yield determined by
chromatographic analysis.
d
Reduction was performed with DTT.
(10 mM). At 1 mM TCEP, rates are ≈ 10 fold lower, at both
initial concentrations of DHAA (Table 1).
3.2. Recoveries of DHAA reduction to AA
DHAA, prepared by enzymatic oxidation of AA by ascorbateoxidase at pH 6.5, was reduced effectively with TCEP at pH 2.
Recovery of AA under these conditions was (98 ± 2) %.
When the efficiency of reducing agents was tested over a
wider pH range, commercially available DHAA was used.
Selection of DHAA source was based on the fact that ascorbateoxidase is active at slightly acidic and neutral pH [24] and could
interfere with the reduction process when performed under
these conditions. The yield of AA is independent of
concentration and type of reducing agent, concentration of
DHAA and pH (Table 2). The lower yield in citrate pH 7 can be
ascribed to the low stability of DHAA at this pH [19].
3.3. Stability of TCEP and DTT in the presence of Cu2+ ions
Copper ions have a significant effect on the rate of TCEP
oxidation in acetate buffer. 1 mM TCEP incubated with 100 μM
Cu2+ is oxidised within 5 h and in 10 μM Cu2+ in 2 days.
Oxidation of TCEP in citrate, which forms strong complexes
with Cu2+, is slower than in acetate buffer, especially at lower
concentrations of remaining TCEP, where the initial rate of
reaction is decreased (Fig. 1).
Cu2+ ions do not significantly affect the stability of TCEP
dissolved in MPA. Only 3 % of 1 mM TCEP is oxidised within
2 days, even in the presence 100 μM Cu2+ (Fig. 1). The rate of
copper catalysed TCEP oxidation in MPA is at least one order of
magnitude lower than in either acetate or citrate buffers at pH 5.
TCEP is completely stable in the absence of Cu2+ ions for
2 days in all three buffers tested (results not shown).
DTT is quite stable in acetate buffer pH 5, even in the
presence of copper ions. 1 mM DTT, incubated with 100 μM
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L. Wechtersbach, B. Cigić / J. Biochem. Biophys. Methods 70 (2007) 767–772
Cu2+, is completely oxidised only after 35 h. The rate of
oxidation is further decreased in the presence of 10 μM Cu2+,
where only 15% of DTT is degraded in 2 days (Fig. 1).
3.4. Stability of AA formed by reduction of DHAA in the
presence of Cu2+ ions
Concentrations of copper ions similar to those present in
biological samples [25] significantly affect the stability of
TCEP and DTT at pH 5, the typical conditions for reduction of
DHAA (Fig. 1). Vitamin C analysis is a routine assay, usually
performed on large number of samples that cannot be analysed
immediately following preparation. Depletion of reducing
agent, used at relatively low concentrations [16], could therefore lead to oxidation of AA in samples and underestimation of
total vitamin C. Copper ions indeed affect the yield of AA
formed by reduction of DHAA with TCEP, and its stability
(Fig. 2).
TCEP is a poor reducing agent in the presence of Cu2+ ions
at pH 5. Treating 20 μM DHAA with 1 mM TCEP in acetate for
4 h produced no AA (Fig. 2A). Using 200 μM DHAA, less than
10% of the theoretical yield of AA was obtained after 4 h, all of
which was re-oxidised in 1 day. Stability of AA was improved
by using 10 mM TCEP. At higher initial DHAA concentrations,
more than 20% of the AA produced was degraded only after one
week. At lower initial DHAA concentrations, the AA produced
was less stable and completely degraded within 7 days, even in
10 mM TCEP. AA was a little more stable in citrate buffer pH 5,
however a significant proportion of the AA formed was still
degraded, even at 10 mM TCEP (Fig. 2B).
In the presence of copper ions, TCEP reduced DHAA more
effectively in MPA (Fig. 2C) than in acetate or citrate buffers.
The resulting AA was stable in the presence of 10 mM TCEP at
both initial concentrations of DHAA tested. More than 95% of
the initial yield of AA obtained in the absence of copper ions,
was still present after one week of incubation. At lower TCEP
concentrations, formed AA is less stable.
Fig. 2. Stability of AA produced by reduction under different conditions. AA
produced by reduction of DHAA with TCEP in the presence of 100 μM CuSO4
was determined in acetate buffer pH 5 (A), citrate buffer pH 5 (B) and in MPA
pH 1.9 (C). Initial concentrations were 200 μM DHAA and 10 mM reducing
agent (■), 200 μM DHAA and 1 mM reducing agent (♦), 20 μM DHAA and
10 mM reducing agent (▴) and 20 μM DHAA and 1 mM reducing agent (●).
Results are expressed as percentages of AA initially formed in the absence of
copper ions. Error bars correspond to SD (n = 3). Absence of error bars indicates
that the SD is smaller than dot size.
Ascorbic acid formed by reduction of DHAA is stable in the
absence of Cu2+, when stored in autosampler vials. More than
95% of the AA formed initially, was still present after one week,
if reduction was performed with TCEP in MPA or in both
buffers at pH 5.
4. Discussion
Fig. 1. Effect of Cu2+ on the stability of TCEP and DTT. TCEP (1 mM) was
incubated in acetate buffer pH 5 containing 10 μM Cu2+ (○) and 100 μM Cu2+
(●), in citrate buffer pH 5 containing 100 μM Cu2+ (♦) and in MPA pH 1.9
containing 100 μM Cu2+ (■). The stability of 1 mM DTT was determined in
acetate buffer pH 5 containing 10 μM Cu2+ (▵) and in acetate buffer pH 5 in the
presence of 100 μM Cu2+ (▴). Results are expressed as percentages of initial
concentration of reducing agent determined in the absence of copper ions. Error
bars correspond to SD (n = 3). Absence of error bars indicates that the SD is
smaller than dot size.
TCEP has been shown to reduce DHAA completely in MPA
at pH 1.9 within 20 min (Table 1). Yields of AA are the same as
at higher pH values (Table 2). The time needed for reduction in
MPA is sufficiently short for the procedure to be applied routinely for determining DHAA and total vitamin C.
Reduction at neutral pH is faster (Table 1), however it can be
compromised by the lower stability of DHAA under these
L. Wechtersbach, B. Cigić / J. Biochem. Biophys. Methods 70 (2007) 767–772
conditions [12,14,19], which can lead to smaller yields of AA
formed by reduction (Table 2). The half-life of DHAA at neutral
pH in the presence of bicarbonate is only 2 min [11], which is
comparable to the rate of reduction at this pH (Table 1).
Reduction in MPA has an additional advantage over
reduction at higher pH. Ascorbic acid is a weak acid with
pK1 ≈ 4.2, and is thus in the dissociated form above this pH.
Chromatographic separation of AA is commonly performed on
C18 columns [15], where retention of the ionised form is poor
and ascorbate is eluted with the front. This is a problem when
reduction is performed at pHs around the pK1 of AA. The
problem can be solved by re-acidification and chromatography
on the protonated form [17]. An alternative is chromatography
in a buffer with a pH sufficiently above pK1 [8,26,27], where
retention of ascorbate is improved with the ion-pair reagent.
Due to the low stability of AA at high pH and trace amount of
redox active ions, a metal chelator and/or reducing agent are
added to the buffer.
The mobile phase used in chromatographic analysis was
2.5 mM H2SO4. Solvent preparation is very simple and no
filtration is needed prior to application, as is the case for buffers
with higher ionic strength. The number of theoretical plates for
the analyte was 13 000 when 20 μl of sample, prepared by
reduction of 200 μM DHAA with TCEP in MPA, was injected
on the C18 column.
The peak corresponding to AA was distorted, and the yield
vanishingly small, when samples prepared in acetate buffer pH
5 at concentrations higher than 30 mM, were injected directly
onto the C18 column. Such problems are avoided by reduction
in MPA. AA and total vitamin C can be determined in two runs
under the same chromatographic conditions, i.e. running an
aliquot of the sample in MPA (AA) and another in MPA in the
presence of TCEP (total vitamin C). As reduction is performed
in MPA, re-acidification prior to analysis of total vitamin C is
not required. This can significantly reduce the amount of work
and experimental error in routine analysis of vitamin C, when
large number of samples are usually analysed.
One of the major benefits of TCEP, in comparison to thiols
such as DTT, is its significantly higher stability in the presence
of Fe3+ ions [20]. Both reducing agents are nevertheless stoichiometrically oxidised by Cu 2+ ions [21,22]. Copper is
efficiently re-oxidised in acetate and citrate buffers at pH 5,
resulting in depletion of TCEP in solution (Fig. 1). The rate of
TCEP oxidation and/or Cu2+ recycling is significantly lower in
MPA. The sensitivity of TCEP to oxidation by copper ions at
mildly acidic pH can limit its applicability as reducing agent for
some purposes, protein biochemistry for example, when copper
ions are present as contaminants.
TCEP and DTT reduce DHAA effectively at neutral and
mildly acidic pH in the absence of redox active ions (Table 2).
Redox active copper and iron ions significantly affect the
stability of AA in solution. AA in blood plasma samples [16],
where iron is the most important oxidant, can be stabilised by
TCEP at neutral pH. The other widely used reducing agent,
DTT, is not able to stabilise AA in autosampler glass vials and a
significant proportion of the AA can be degraded in 1 day at
room temperature, due solely to metal contaminants in the glass
771
[28]. We have shown that TCEP is a poor reducing agent at
mildly acidic pH in the presence of copper ions. Ascorbic acid,
formed by reduction of DHAA with TCEP, is significantly more
stable in MPA (pH 1.9) than in acetate or citrate buffers at pH 5
(Fig. 2). Reduction of DHAA in MPA with TCEP, and stability
of the AA produced, are therefore both superior to reactions at
mildly acidic pH, when copper ions are present as contaminants.
5. Simplified description of the method and its applications
The reduction of DHAA to AA, which is an obligatory step in
total vitamin C analysis, is currently performed in neutral and
mildly acidic solutions. Samples for vitamin C analysis are
nevertheless prepared in MPA at pH 2 or lower, in order to
stabilise both the ascorbic and dehydroascorbic acids. For reduction, the pH must be raised. This represents additional work
and, above all, exposes both forms of vitamin C to conditions
under which they are less stable. Our results show that reduction
of DHAAwith TCEP in MPA is suitable for routine determination
of DHAA and total vitamin C. The major benefits are that the AA
formed is stable, even in the presence of Cu2+, the rate of
reduction is acceptable, high yields of AA are obtained and the
experimental procedure is simple, with consequently smaller
experimental error, which is the weakness of methods that involve
subtraction of two experimentally determined values. Reduction
with TCEP in MPA can be applied for determining dehydroascorbic acid and total vitamin C in a variety of biological samples
and pharmaceutical products.
Acknowledgments
The authors would like to thank Veronika Abram and Roger
H. Pain for their valuable suggestions and discussion of the
paper. The research was supported in part by project M4-0007.
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