BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO.

2, 2003 367

FOOD COMPOSITION AND ADDITIVES

Determination of Total Vitamin C in Fruit Juices and Related

Products by Liquid Chromatography: Interlaboratory Study
ALLAN R. BRAUSE
Analytical Chemical Services of Columbia Inc., 9110 Red Branch Rd, Columbia, MD 21045
DAVID C. WOOLLARD1
AgriQuality NZ Ltd., PO Box 41, Auckland, New Zealand
HARVEY E. INDYK
NZMP-Fonterra, PO Box 7, Waitoa, New Zealand
Collaborators: J. Acar; K. Adadevoh; G. Cherix; B. Durst; T. Eisele; E. Elkins; J. Foos; S. Hammack; D. Hammond;
F. Hesford; C. Hischenhuber; V. Hong; C.J. Huang; S. Kirksey; L. Kline; D. Kruger; M.J. Lawson; A. Lea; G. Martin;
A. Parkih; J. Weiss; E. Wilhelmsen; B. Woodward; R. Wrolstad; L. Zygmunt

A interlaboratory study was conducted to evaluate

a liquid chromatographic (LC) procedure for the
determination of total vitamin C in foods at levels
of 560 mg/100 g. Emphasis was placed on fruit
juices, although selected foods were also included
in the study. Following dissolution of sample in water, endogenous dehydroascorbic acid was converted to ascorbic acid by precolumn reduction with
dithiothreitol at neutral pH. Total ascorbate was determined by C18 reversed-phase LC with a phosphate eluent at pH 2.5, incorporating dithiothreitol to
maintain vitamin C in the reduced form, and UV detection at 254 nm. Seven types of fruit juices and
foods were tested by 19 collaborators in 7 countries. Three duplicate juices and foods met the criteria for Youden pairs and yielded repeatability relative standard deviation of 5.8014.66%.
Reproducibility relative standard deviation ranged
from 6.36 to 35.54% (n = 10) with HORRAT values of
0.824.04. The LC method is suitable for routine use
in fruit products and foods containing >5 mg/100 g
vitamin C and is recommended for further validation
by AOAC INTERNATIONAL and International Fruit
Juice Union.

he importance of vitamin C in human health is well understood, particularly as an antioxidant and in collagen
synthesis (1, 2). The recommended daily allowance
(RDA) for vitamin C is determined as 60 mg/day, sufficient to
prevent scurvy and maintain a stable body pool of
1500 mg (3), much of which comes from fruits and vegetables (4). Vitamin C action is supplied by L-ascorbic acid and

Received July 9, 2002. Accepted by SG September 18, 2002.

1
Author to whom correspondence should be addressed; e-mail:
woollardd@agriquality.co.nz.

its oxidized form, dehydroascorbic acid, each with equivalent

molar activity, with total vitamin C defined as the sum of both
forms.
To support physiological and pharmacological studies, it is
important to have reliable analytical data, which can be problematic in biological materials (5), and methods for the determination of vitamin C have been well-reviewed (3, 68). Food
matrixes are equally difficult to determine, particularly vegetables, where the analyte is in a bound form known as
ascorbigen. The lability of L-ascorbic acid is a critical factor in
analytical procedures with dehydroascorbate, which is particularly unstable, and can lead to irreversible conversion to inactive diketogulonic acid and other carboxylic acids (9). In
some nutritionally supplemented food and feed products, vitamin C esters, notably ascorbyl 5-palmitate or ascorbyl
2-phosphate, can be used to enhance stability (10), although in
natural products and supplemented fruit juices, these compounds are absent.
A test method must be able to assay ascorbic acid and
dehydroascorbic acid without interference from their synthetic
diastereoisomers
isoascorbic
acid
and
dehydroisoascorbic acid or other organic acids. Isoascorbic
acid (also called D-ascorbic acid or erythorbic acid) is legally
used as an antioxidant food additive but has poor antiscorbutic
activity (<5%); therefore, its differentiation is a prerequisite to
any reliable vitamin C method (8, 11). With only 2 active
forms, the determination of vitamin C is seemingly a simple
analytical challenge compared with other vitamins. However,
poor correlation between laboratories is commonly attributable to differences in method specificity for the vitamin C
congeners, analyte instability to elevated pH, autocatalysis
during extraction, and detection sensitivity limitations (3).
The emergence of liquid chromatographic (LC) methods
has been rapid, and many are preferred for water-soluble vitamin analysis (12), although recent developments in capillary
electrophoresis indicate potential advantages for vitamin C
testing (3, 13, 14). Prior to chromatography, vitamin C is usu-

23.6

24.8

24.5

28.2

24.0

25.2

23.4

25.1

25.2

18.8

23.6

26.5

25.5

31.2

10

11

12

13

14

15

16

17

18

19

26.4

6.2

12.8

50.4f

11.9e

3.4

12.1

16.8

6.0a

5.5a
6.5

12.8

10.3

12.5

11.6

11.8

11.5

11.0

55.6

42.1

48.2

51.0

45.0

57.5

56.4

49.3

56.2

51.0

8.4f

22.3

13.7

10.1

17.8

11.8

19.6

18.1

15.6

18.1

16.0

7.5f

12.3

50.6f

11.0

49.1

21.0
18.7c

59.3

23.9a

21.5

18.0

20.5

69.2c

62.7a

58.6

53.6

51.5

16.3

11.3

12.6

17.2a

5.0

2.4

5.5

7.1

4.9

2.1

NR

NR

5.7

NR

7.5

8.2

11.4a

11.4

7.0a

5.7a
4.3

30.7

26.7

22.4
48.6f

24.2

33.7

26.0

34.4

8.8

9.7

5.1

8.3

6.2

10.4

9.5

3.4a

3.5a
8.8

7.7

10.4

8.5

6.1

13.0c

4.2a

8.7

9.4

8.2

8.8

11.1

7.9

5.2

6.2

Peas

8.0

9.1

7.9

34.7

6.1

36.9e

5.7

3.8a

10.8

7.8

6.7

7.9

10.6

7.3

6.1

6.4

Peas

38.5

36.0

34.4

37.7

37.5

34.3

36.9

29.6

35.8

28.0f

32.2

20.9

33.8

34.9

NR

34.8

27.0

25.6

24.3

15.7

33.3

36.5

40.0

34.8

64.1

32.0

25.7

36.7

Vegetablebased baby Single-strength Single-strength Cranberry juice Cranberry juice

formula
pineapple juice orange juice
concentrate
concentrate

Vegetablebased baby
formula

Invalid data, poor chromatography.

ND = Not detected; NR = not recorded.
Cochran outlier.
Grubbs outlier.
Invalid data, no matched pair.
Invalid data, sample spoilage.

30.3

19.6

25.6

23.1

18.6

20.0

26.3

23.1

26.6

24.0

27.4

21.3

23.5

24.2

26.4

26.0

47.6c

25.1

25.1

32.0c

24.8

23.5

23.5

Synthetic
orange
juice

Lab

Synthetic
orange
juice

Table 1. Raw data submitted by participating laboratories

16.0

NR

9.3

9.0

4.4a

13.3

11.3

13.2e

14.3

11.0

12.4

13.5

14.5

12.0

13.7

14.4

13.4

12.5

11.8

Pears

18.5

14.2e

11.6

17.9

11.3a

14.6

20.3

NR

17.8

17.0

10.9

9.7

10.3

17.9

16.1

21.3

19.5

12.7

17.2

Pears

55.6

51.1

38.3

41.9

32.0d

45.5

46.8

41.0

45.1

46.0

38.4

46.5

49.5

40.6

46.3

63.8d

44.7

62.4

42.4

Fortified
apple juice

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

NDb

Unfortified
apple juice

368
BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 369

ally extracted into metaphosphoric or trichloroacetic acid, either of which functions to stabilize ascorbate (15). Most LC
separations are based on ion-exchange (16) or ion-pair reversed-phase chromatography (17). Although many methods
exploit the weak-anion exchange capability of amine-bonded
silica (10, 18), reversed-phase techniques with C18 functionality have inherent advantages and are frequently used with both
silica and polymeric styrene-divinyl benzene supports (3, 11).
Despite the widespread use of LC for determination of
vitamin C in foods, there is no official regulatory LC method
currently available in the 17th Edition of Official Methods of
Analysis (19). Of the 4 current AOAC Methods, 967.21 and
985.33 are based on visual end point indophenol titration and
require no sophisticated equipment. However, such methods
cannot estimate dehydroascorbic acid, lack sensitivity, and are
subject to interferences from coreductants and colored pigments. Methods 967.22 and 984.26 use manual and
semiautomated techniques to assay total vitamin C as
dehydroascorbic acid following catalytic oxidation with activated charcoal and reaction with o-phenylenediamine to the
fluorescent quinoxaline derivative. These latter methods take
advantage of the sensitivity and selectivity of fluorescence detection, and this principle has also been used in a number of
LC approaches for total vitamin C with precolumn oxidation (20, 21). With various combinations of pre- or
postcolumn oxidation schemes, the contributions of ascorbic
acid and dehydroascorbic acid have been individually determined (16, 2229), with dehydroascorbate measured
fluorimetrically, and ascorbate by either UV or electrochemical detection.
Unlike most organic acids, ascorbate absorbs well in the
mid-UV range and may be detected without interference. The
use of direct UV detection for total vitamin C in LC-based
methods, therefore, offers a simple alternative to electrochemistry or fluorescence. Although inherently less sensitive, the
high vitamin C content of fruit juices, vegetable products, and
supplemented foods mitigates against this limitation of UV
detection. Because dehydroascorbic acid has no useful
chromophore, UV detection schemes accordingly require
vitamin C to be in the ascorbate form. Depending on mobile
phase pH, the optimum detection wavelength can be assigned
by using photodiode array detectors (30), although a compromise wavelength of 254 nm is commonly used (10, 25).
Much of the early LC development work for vitamin C
content in foods was performed to establish a reliable nutritional database (4, 10, 11, 16, 26, 31). There have been a number of LC-UV studies for the determination of vitamin C in
fruits and vegetables (22, 25, 30, 3235), often concurrent
with other organic acids. Metaphosphoric acid and
trichloroacetic acid are regularly used both to precipitate protein and to stabilize ascorbic acid. However, the presence of
the weakly retained metaphosphoric acid has occasionally
been reported to interfere with ascorbate if retention is inadequate. Dithiothreitol is frequently used to convert
dehydroascorbate to the reduced form, although the efficiency
of this step is pH-dependent. Other reductants, including
mercaptoethanol (36), homocysteine (11), and cysteine (37),

have been used, but dithiothreitol remains the most common

for LC and capillary electrophoresis applications (3, 14).
In 1994, the National Food Processors Association
conducted an unpublished study for vitamin C in fruit juices,
based on AOAC Official Method 986.13 for organic acids,
which had been developed previously (38). It involved the
separation of organic acids on a C18 column by using
ion-suppression techniques at pH 2.4 with UV detection at
214 nm. This method has now been optimized for vitamin C
analysis, primarily through use of dithiothreitol reduction and
stabilization of the reduced ascorbate form during both sample
preparation and chromatographic analysis. Because processed
juices commonly contain significant dehydroascorbate,
dithiothreitol is incorporated with standards, sample extracts,
and mobile phase. This study reports the results of the method
performance trial, primarily focused on processed fruit juices
and selected foods.
Interlaboratory Study
Fourteen samples with vitamin C concentrations ranging
from 1 to 60 mg/100 g were dispatched to 22 collaborators in
the United States, Canada, Switzerland, the United Kingdom,
France, Austria, and Turkey. Frozen samples were dispatched
in dry ice, with random 5-digit numeric coding, by overnight
air courier in closed light-proof containers under dry ice.
Upon receipt, participants were instructed to store the samples
in a freezer (20C) prior to analysis. Participants were also
requested to perform the determinations (once only) at the earliest convenience, but no specific date was given. All laboratories tested within 4 weeks of receipt. Only data >2 mg/100 g
(20 ppm) were to be recorded.
A practice solution of vitamin C (containing 100 mg ascorbic acid) in nitrogen-flushed amber vials was also included for
each participant to optimize the chromatography. In particular, the use of tandem columns was permitted if retention was
insufficient.
A Youden pair design was intended to give estimates of
within-laboratory precision without compromising data by
matrix identification. However, because it proved difficult to
obtain pairs of samples with concentration differences <5% (a
requirement of the Youden pair statistical model), most of the
samples were subsequently treated statistically as individual
matrixes. Reproducibility is the major parameter obtained
from interlaboratory studies; therefore, loss of repeatability
data was not deemed a disadvantage. The first matrix pair (A
& B) consisted of laboratory-prepared orange juices with
known concentrations of vitamin C to determine method recoveries. All other samples were commercially obtained and
well-mixed before subsampling into nitrogen-flushed polypropylene bottles with sealed tamper-evident caps. These
were stored under dry ice prior to dispatch. Although focused
on fruit juices, namely single-strength orange/pineapple juices
(E & F), concentrated cranberry juices (G & H), and supplemented/natural apple juices (M & N), vegetable-based baby
foods (C & D), canned pears (K & L), and samples of pack-

1.61
12.94

3.08

1.87
5.14

10.15

Determination of Vitamin C in Fruit Juices and

Selected Foods Using LC

10.27

23.05

14.46

2.49

4.68

2.50

14.91

2.17

METHOD
20.61

16.70

13.08

2.88
10.23

1.95
18.03

21.25

12.11

0.82
2.09

4.04
5.27

6.36

35.54

7.63
3.99
11.09
2.72

R
r
RSDR, %
SR

aged peas (I & J) were included in the study to demonstrate the

acceptability of the method to related food samples.
Following removal of invalid data, outliers identified by
the Cochran test for extremes of repeatability (where possible)
and the Grubbs test for extremes of reproducibility were omitted from estimates of precision. The means, within (sr) and between (sR) laboratory standard deviations, repeatability (r) and
reproducibility (R) values, relative standard deviations (RSDr
and RSDR), and HORRAT values were determined according
to AOAC guidelines (39).

4.62

3.63

1.83

1.67
9.53

3.65

5.33
14.66
4.67

0.7

6.46

5.80
1.42

0.75

RSDr, %c
Src

1.88

(Applicable to the determination of ascorbic acid in fruit

juices and selected foods at 560 mg/100 g.)
Principle
Vitamin C is maintained in its reduced form with
dithiothreitol,
which
also
converts
endogenous
dehydroascorbic acid to ascorbic acid. Fruit juices are assayed
directly or diluted with water; semisolid matrixes are dispersed in water and filtered. Total vitamin C is determined as
ascorbic acid with LCUV. Separation is on a C18 reversed-phase column with a phosphate-buffered mobile phase
(pH 2.5) and detection at 254 nm.

Number of laboratoriess or tests retained after invalid data and outliers were eliminated.
Number of laboratoriess or tests removed as outliers (in parentheses).
Reported for Youden pairs only (difference in means <5%).
b

44.98
16(3)
16(3)
Fortified apple juice
M

15.74
17(0)

12.68
17(0)

17(0)
Pears
L

17(0)
Pears
K

8.12

31.88
30(2)

32(2)

15(1)

Peas
I and J

16(1)

Cranberry juice concentrate

G and H

17.19
16(0)
16(0)
Orange juice, single-strength
F

53.35
16(0)
16(0)
Orange/pineapple juice, single-strength
E

5.29

14(2)
14(2)
Infant formula
D

11.76

13(0)
13(0)
Infant formula
C

24.56
38(2)
18(1)
A and B

Synthetic orange juice

No. of
testsa,b
Material

No. of
labsa,b

Mean,
mg/100 g

Apparatus

ID

Table 2. Statistical analysis of vitamin C in fruit juices and related products by liquid chromatography

1.59

BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

HORRAT

370

(a) LC system.Automated or manual equipped with

pump for continuous delivery at 0.51.0 mL/min and accurate
injection device for 50 mL. A UV detector with stable baseline
is set at 254 nm. The use of a photodiode array detector is recommended. Data collection is by integrator or PC.
(b) Chromatography column.Any suitable 5 mm
monomeric or polymeric silica-based C18 reversed-phase column (nominally 250 4 mm). Tandem columns may be used
if required.
(c) Solvent and sample clarification apparatus.With
0.45 mm hydrophilic membrane.
Reagents
Water should be analytical LC grade with resistivity 18 MW.
(a) Dithiothreitol.Aldrich Chemical Co. (Milwaukee,
WI; 15,046.0), or equivalent.
(b) Mobile phase.KH2PO4 (0.5%, w/v), pH 2.5, with
dithiothreitol (0.1%, w/v): Weigh 5.0 g KH2PO4 in 1 L volumetric flask and add ca 950 mL water. Add 1.0 g
dithiothreitol, stir until dissolved, and adjust pH to 2.5 with
concentrated phosphoric acid. Dilute to 1 L with water, and
filter through 0.45 mm membrane.
(c) Vitamin C standards (1050 mg/mL).Dissolve
5.0 mg ascorbic acid in 100 mL water to give 50 mg/mL. Dilute 2, 4, 6, and 8 mL to 10 mL with water to give
1040 mg/mL, respectively. Add ca 10 mg dithiothreitol to
each standard. Prepare fresh each day.

BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 371

ing the single-level standard corresponding most closely to the

unknown:
Vitamin C =

sample response standard concentration(mg / mL) dilution factor

standard response weight(or volume) used 10

Collaborators Comments

Figure 1. Typical chromatogram of supplemented

food product.

Preparation of Test Solutions

(a) Fruit juices.Dilute juice samples so that ascorbic acid
concentration is in response range of standards (e.g., because
orange juice usually contains >30 mg/100 mL ascorbic acid, dilute 1 mL to 10 mL with water). Record dilution factor and immediately add ca 1 mg dithiothreitol for each 1 mL filtrate.
(b) Fruit and processed foods.In a mixer, blend 10 g
food with sufficient water so that ascorbic acid concentration
is in response range of standards. Record dilution factor and
immediately add ca 1 mg dithiothreitol for each 1 mL filtrate.
Filter through fluted 18.5 cm cellulose filter paper (Fisher Scientific Co., Pittsburgh, PA; 09-790-14F, or equivalent). If
sample contains proteinaceous material, mix equal parts of extract with 5% trichloroacetic acid solution before filtration.
Extracts difficult to filter may be clarified by centrifugation.
Allow extracts to stand for at least 2 h, filter through
0.45 mm membrane, and proceed to determination step.
Determination
Set up instrumentation and software according to manufacturers instructions. Set detector to 254 nm with appropriate
sensitivity (usually 0.1 aufs). Set flow rate to 0.5 mL/min, or
other, suitable to avoid excessive system pressure, and run
water through the column for ca 30 min, followed by mobile
phase for 1 h to equilibrate column.
Inject 50 mL of each of the 5 calibration standards and ensure stable retention times. On a 25 cm column, ascorbic acid
elution time is typically ca 10 min. Measure peak response
(height or area), plot manually or electronically against concentration, and ensure a linear response.
Inject 50 mL of each test solution. Repeat samples that do
not lie within the calibration range with appropriate dilution.
If photodiode array detector is available, confirm spectral
identity and peak purity of putative ascorbic acid peak.
Calculations
The concentration of vitamin C (mg/100 g or mg/100 mL)
can be interpolated directly from the calibration regression using automated data reduction software, from a manually constructed calibration curve, or from the following equation us-

Four laboratories reported that the samples were at room

temperature or warm upon arrival. Laboratory 12 requested a
new set of samples. Where there was any suggestion of spoilage, the data were removed as invalid. Possible fermentation of
2 orange juice and 2 cranberry juice samples was considered
sufficient by the participants to adversely affect their results,
and were therefore not subjected to statistical evaluation.
Laboratory 8 reported initial chromatographic difficulties
and repeated 3 samples. Laboratory 10 suggested that a
solid-phase extraction cleanup was required to remove
nonpolar compounds and to prevent carryover of late-eluting
peaks. Laboratories 10, 11, and 16 reported the use of 20 mL
injection volumes rather than 50 mL. Laboratory 11 clarified
extracts by using centrifugation at 40 000 g rather than filtration, while Laboratory 12 commented on the slow filtration
of some samples. Laboratories 13 and 15 suggested
predissolution of dithiothreitol rather than adding as a solid.
Laboratory 16 suggested using a closed container during reduction of vitamin C with dithiothreitol, possibly with N2
flushing, and suggested that the column be selected to give an
adequate separation of ascorbic acid and isoascorbic acid.
Only one collaborator used a dual-column configuration,
because most single-column systems provided sufficient retention and resolution of target analyte. The use of dual columns,
however, was considered an advantage in the separation of
ascorbic acid from the potential adulterant, isoascorbic acid.
Advice from a collaborator stated that the weight of ascorbic acid used in the standard solution was too low for accurate
measurement.
Results and Discussion
The interlaboratory trial protocol aims to assess the
interlaboratory capability of a previously intralaboratory validated analytical method, primarily involving an estimation of
precision parameters following removal of discordant values.
A total of 22 participants were invited to participate in the
study, 19 of which submitted data. Although laboratory ranking analysis indicated that Laboratory 4 had significant positive bias (p = 0.05), the total data set was not removed as invalid. However, poor chromatography reported by this
participant for 2 sets of samples (baby food and orange juice)
justified their removal from statistical analysis. Similarly,
Laboratory 15 showed a borderline negative bias, and
one sample (peas) was removed from further consideration.
Of the total raw data set listed in Table 1, 6 failed to report for
paired samples and 11 were compromised either by prior sample spoilage or poor chromatography and were therefore eliminated as invalid data prior to statistical analysis.

372

BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

Because of its low vitamin C content (<0.5 mg/100 g), raw

data for sample N (unfortified apple juice) are not reported
(only 3 laboratories reported estimates), and this material was
used to define the lower limits of detection of the method.
All collaborators returned acceptable standard calibration parameters with linear regression coefficients (r) typically >0.998.
All valid data sets were submitted to the AOAC harmonized statistical protocol. Cochran (p = 0.025, 1-tail) and
Grubbs (single and double, p = 0.025, 2-tail) tests were used to
determine outliers. Three Cochran outliers and 2 Grubbs outliers were observed among the Youden pairs. Five Grubbs outliers were removed from 7 unpaired matrixes, where only an estimate of reproducibility precision was available. The total
rejection rate was therefore 10 of 209 data points, which is below the rejection level considered acceptable in interlaboratory
studies (39). Table 2 lists the results of the statistical analysis for
means, precision, and HORRAT values following outlier removal.
RSDR values estimated for all samples containing vitamin C at 553 mg/100 g ranged overall from 6.4 to 35.5%
(mean, 17.1%). Because observed variance was reasonably
consistent across most samples and concentrations, the mean
RSDR may be considered to represent overall method precision. Limiting HORRAT values of 0.52.0 demonstrates acceptable method precision (39), and in this study, a mean
HORRAT value of 2.28 (range, 0.824.04) exceeded these
guidelines. However, known exceptions to these general
guideline values include unstable analytes, and the lability of
ascorbate is likely to contribute to poorer precision values. Indeed, a similar recently approved European standard method
for foods, EN 14130 (40), also yielded a comparably elevated
mean HORRAT value of 2.97 (range, 2.863.48).
RSDr values were available for only 3 paired matrixes, but
the overall mean RSDr:RSDR value measured 0.57, which
complies with the accepted guideline of 0.50.7 for the relationship between repeatability and reproducibility precision.
The method protocol allowed for a wide range of LC system configurations with respect to analytical column and detector, and a typical chromatogram is illustrated in Figure 1.
Although most laboratories reported using a single
250 mm column, 3 participants used a 300 mm column, and
one laboratory used a 150 mm column in tandem with a
250 mm column to increase resolution. Five laboratories used
the Supelcosil (Supelco, Bellefonte, PA) LC-18 column.
Samples A and B were laboratory-made synthetic orange juices supplemented to 25.0 mg/100 mL and
25.5 mg/mL, respectively. Overall means for these samples
were 25.34 and 25.38 mg/100 mL, which indicate recoveries
of 101.4 and 99.5%, respectively.
Despite the availability of electrochemical or fluorescence
detection techniques, UV detection is the most facile detection
strategy, with lmax of ascorbate at 243 nm, although spectral
characteristics are pH-dependent (pKa of ascorbate, 4.17). The
mid-UV detection of ascorbate minimizes the potential for interference from several organic acids, ubiquitous in fruit juices
and other foods, which absorb in the low-UV region (<220 nm).
Nevertheless, use of photodiode array detection is considered

Figure 2. Chromatogram of ascorbic and isoascorbic

acid standards.

expedient to confirm both the identity and peak purity of putative ascorbic acid in poorly characterized food samples.
The polar nature of reduced vitamin C results in relatively
poor retention on hydrophobic reversed-phase functionalities,
which is partly moderated through use of a low-pH eluent. Although ion-pair techniques may be used to further improve retention, the opportunity for interference remains. In reality, although samples with low concentrations of vitamin C cannot be
unequivocally assayed with UV detection, for fruit, juices, and
selected foods with high (>5 mg/100 mL) natural and/or added
ascorbate content, the described method provided reliable data.
Although none of the samples in this study contained
isoascorbic acid (erythorbic acid), a biologically inactive
stereoisomer of ascorbic acid, its use in foods can cause potential overestimation of the vitamin C content of supplemented
food products. These isomers cannot be distinguished by traditional nonchromatographic methods because they have
identical chemical properties. Separation of these isomers,
however, is a desirable attribute, and although resolution is not
complete with all C18 columns, the current method will identify the presence of isoascorbic acid (Figure 2).
The reduction of endogenous dehydroascorbate and stabilization of reduced ascorbic acid with dithiothreitol is a popular approach in vitamin C analysis techniques, and was used in
the method reported here. Because ascorbic acid is also vulnerable to on-column oxidation during chromatography,
dithiothreitol was included in the mobile phase at a concentration of 0.1%, thereby reversing potential auto-oxidation, a
technique previously used for fruit juices (30). It has also been
reported that, in contrast to the typically acidic ascorbate extraction conditions frequently described, maintenance of a
near neutral solution pH during precolumn reduction is significant in optimizing the reduction efficiency of dithiothreitol,
under which conditions ascorbate exists predominantly in the
monoanion form. Stoichiometry and kinetic considerations
require both excess reductant and adequate reaction time during precolumn reduction, thereby resulting in quantitative
conversion. Further, the reduced ascorbate is stable for many
weeks under refrigeration in the dark.
It is commonly assumed that ascorbic acid and
dehydroascorbic acid have equal equimolar antiascorbutic ac-

BRAUSE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 373

tivities, so that the concentrations of each can be summed to

assess the total vitamin C status of foods (3, 8). In reality,
dehydroascorbic acid has a somewhat lower activity
(approximately 80%), but adjustments for this difference are
not necessary. However, if the recent estimations of
dehydroascorbic acid activities in rats (approximately 10%)
can be verified in humans, then there are implications for future nutritional assessments (41).

Frank Hesford, Swiss Federation Research Station,

Wadenswil, Switzerland
Claudia Hischenhuber, Nestle, Geneva, Switzerland
Gilles Martin, Eurofins Labs, Nantes, France
Josef Weiss, Hohere Bunidesleh und Versuchanstalt fur
Wein Klosterneuberg, Austria

Conclusions

(1) Sauberlich, H.E. (1991) Ann. Rev. Nutr. 14, 371391

(2) Gershoff, S.N. (1993) Nutr. Rev. 51, 313326
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Raton, FL, pp 223270
(4) Vanderslice, J.T., Higgs, D.J., Hayes, J.M., & Block, G.
(1990) J. Food Comp. Anal. 3, 105118
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(6) Nyyssonen, K., Salonen, J.T., & Parviainen, M.T. (2000) in
Modern Chromatographic Analysis of Vitamins, 3rd Ed.,
Chromatographic Science Series, 84, A.P. De Leenheer,
W.E. Lambert, & J.F. Van Bocxlaer (Eds), Marcel Dekker,
New York, NY, pp 271300
(7) Moser, U., & Bendich, A. (2000) in Handbook of Vitamins,
2nd Ed., L.J. Machlin (Ed.), Marcel Dekker, New York, NY,
pp 195232
(8) Ball, G.F.M. (1998) Bioavailability and Analysis of Vitamins
in Foods, Chapman & Hall, London, UK, pp 517560
(9) Niemela, K. (1987) J. Chromatogr. 399, 235243
(10) Sapers, G.M., Douglas, F.W., Ziolkowski, M.A., Miller,
R.L., & Hicks, K.B. (1990) J. Chromatogr. 503, 431436
(11) Hidiroglou, N., Madere, R., & Behrens, W. (1998) J. Food
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(12) Ball, G.F.M. (1994) Water-Soluble Vitamin Assays in Human
Nutrition, Chapman & Hall, London, UK, pp 274295
(13) Thompson, C.O., & Trenerry, V.C. (1996) Food Chem. 53,
4350
(14) Cancalon, P.F. (2001) J. AOAC Int. 84, 987991
(15) Kim, S.R., Lane, R.H., A-Ghany, M., & Stitt, K.R. (1987) J.
Food Quality 10, 17
(16) Vanderslice, J.T., & Higgs, D.J. (1984) J. Chromatogr. Sci.
22, 485489
(17) Sood, S.P., Sartori, L.E., Wittmer, D.P., & Haney, W.G.
(1976) Anal. Chem. 49, 796798
(18) Dennison, D.B., Brawley, T.G., & Hunter, G.L. (1981) J.
Agric. Food Chem. 29, 927929
(19) Official Methods of Analysis (2000) 17th Ed., AOAC INTERNATIONAL, Gaithersburg, MD
(20) Dodson, K.Y., Young, E.R., & Soliman, A-G.M. (1992) J.
Assoc. Off. Anal. Chem. 75, 887891
(21) Speek, A.J., Schriver, J., & Schreurs, W.H.P. (1984) J. Agric.
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(22) Kall, M.A., & Anderson, C. (1999) J. Chromatogr. B 730,
101111
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162168
(24) Kneifel, W., & Sommer, R. (1985) Z. Lebensm. Unters.
Forsch. 181, 107110

The described LC method is applicable over a range of

vitamin C concentrations; however, a study of reproducibility
indicates that it is moderately less precise at the lower concentration range. Nevertheless, despite a mean HORRAT value
>2.0, the method is considered fit-for-purpose for nutritional
labeling and compositional tables. Although unsuitable for
foods containing low concentration of vitamin C, levels
<5 mg/100 mL are nutritionally insignificant relative to the
RDA of 60 mg.
On the basis of this interlaboratory study, it is recommended that the LC method for the determination of total vitamin C in fruit juices and selected foods at 560 mg/100 g is
suitable for routine industrial use. The method indicates that it
is suitable for further interlaboratory study by AOAC, particularly with high concentration materials such as fruit juice and
fortified infant formula. It is suggested that alternative procedures be used for general food items.
Acknowledgments
The assistance of David Copestake (Fonterra Research Centre,
Palmerston North, New Zealand) is acknowledged. We thank the
following collaborators for their participation in this study:
AOAC Collaborators
Genevieve Cherix, Nestle Quality Assurance Lab, Dublin, OH
Tom Eisele, TreeTop Inc., Selah, WA
Ed Elkins and C.J. Huang, NFPA East, Washington, DC
Jane Foos, ABC Labs, Gainesville, FL
Stacie Hammack and Betsy Woodward, Florida Dept. of
Agriculture, Tallahassee, FL
Sanford Kirksey, Proctor & Gamble, Cincinnati, OH
Linda Kline, Gerber Products, Fremont, MI
Dana Kruger, Krueger Food Labs, Cambridge, MA
Mary Jane Lawson, FDA, Atlanta, GA
Eric Wilhelmsen and Victor Hong, Dole Food Co., San
Jose, CA
Ron Wrolstad and Bob Durst, Oregon State University,
Corvallis, OR
L. Zygmunt, Quaker Oats, Barrington, IL
IFU Collaborators
Jale Acar, Hacettepe University, Ankara, Turkey
Kodjo Adadevoh and Archana Parkih, Analytical Chemistry Services of Columbia, MD
David Hammond and Andrew Lea, RSSL, Reading, UK

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