Vitamin
Joseph
C content
T Vanderslice
and
of foods: sample
Darla
J Higgs
A recent
survey
of foods
that constitute
the
major sources of vitamin
C in the American
diet yielded information on the total content
ofthis vitamin
as well as the amount
ofits two forms, ascorbic
acid and dehydroascorbic
acid (DHAA)
in these foods. Samples
ofindividual
foods showed a surprising
large range ofvitamin
content
even for foods collected
from the
same
regions
of the country
and from the same source.
The
amount
of DHAA
in the different
foods varied
from
10% to
ABSTRACI’
‘
of the
total
the vitamin
man-diet
vitamin
content
studies,
a few foods,
cision.
content.
in a given
when
WORDS
acid,
food
large
range
suggests
food
the major
daily analyses
Am JClin
Nutr
KEY
The
sources
of values
further
that
ofvitamin
are required
for
in hu-
acid,
vitamin
have been a large number
ofstudies
on the total vitamin
content
offoods.
However,
relatively
few ofthese
studies distinguish
between
the two forms of this nutrient,
ascorbic
dehydroascorbic
acid,
to humans
the vitamin
C content
of human
(1-8).
in a given
on growing
conditions,
ferences,
and seasons
or vegetable
are also
source and from the
also been noted (10,
The present
paper
the vitamin
C content
results
conditions
for standards
Extraction
procedures
shown
There
pending
matographic
arc shown
Fig 2.
A flow
C, dehydroascorbic
C (TAA)
and
which
are
comparable
in their
It has long been known
food can vary appreciably
stage
of maturity,
regional
that
dedif-
of the year; variations
within a given fruit
significant
(9). Variations
from a given
same distributor
in a given season have
1 1).
addresses
the problem
of the variability
of
of different
sources and its effect on the
dietary
studies
as generally
was
hydroascorbic
necessary
acid,
reaction
system
postcolumn
with
fluorophores.
is an evolution
from
the classical
by Deutsch
and Weeks (12,
of the ascorbic
acid and deoxidation
orthophenylene
No
evidence
with
HgC12
diamine
of any
, and sub-
to obtain
interference
Printed
for
standard
as
in the
figure
for a food
caption.
extract,
Traces
broccoli,
in
of vitamin
metaphosphoric
and
Weeks
C from
acid/acetic
(12)
was
used.
foods
is
acid/EDTA
Ten
milliliters
added
to the
X g, and
The
variability:
variation
etables
were
centrifugation
the
C content
variation
study
rejected.
on diet
at 48 400
in the
foods.
is tabulated
fruits
is shown
mean
can
In Table
along
with
Handbooks
8-9 and
on the assumption
The agreement
studies
for some
in this laboratory
of two for some
in this
effect
in vitamin
as determined
that
before
filtered.
sample
Food
for I mm
fatty foods, an equal volume
of hexane
aliquot,
vortexed
for I mm, centrifuged
at
between
and
in Table
be of the
order
2 the range
the ranges
8- 1 1 (14).
that any
vegI (10,
of
of values
calculated
These
outliers
the two sets
latter
>
in USA. © 1991 American
2
of data
is reasonable.
These
large
or homemakers
(15), however,
ranges
ofcontent
involved
addressed
might
in day-to-day
this problem
be ofconcern
to dieticians
menu
planning.
and showed
that
Beaton
in a di-
the
acid has ever been observed.
The chromatographic
analysis incorporates
an internal standard,
isoascorbic
acid (IAA),
which is separated
from ascorbic acid and dehydroascorbic
acid.
The oxidation
product
of isoascorbic
acid, dehydroisoascorbic
l991;54:l323S-7S.
compounds
internal
from
diketogulonic
Am J C/in Nuir
3. The
of Dcutsch
48 400
SD
assay developed
column
separation
given
and
for the extraction
from the Agricultural
ranges
were calculated
procedures
The chromatographic
are
the aliquot
and vortexed
x g and filtration. For
found
microfluorometric
13). It involves
the other
useful
of the acid solution
(80 mL glacial acetic acid, 0. 1 mol metsphosphoric
acid, and 0.5 g EDTA/L
ofsolution)
were added per
gram ofsample.
The resulting
mixture
was blended
for 30 s and,
in the absence offat or starch, centrifuged
at 1200 X g for 5 mm
and filtered through 45-zm filters before injection
on the column.
For starchy foods, an equal volume
of n-butanol
was added to
1 1). Note
performed.
Methodology
sequent
chart
in Figure
a factor
Chromatographic
from
is a very
its redox potential
is the same as ascorbic
acid and any appearance of DHIAA
in the chromatographic
trace would indicate
oxidation
of AA as well as IAA is occurring
during extraction
and quantitation.
A diagram
of the system is shown in Figure 1 and the chro-
procedure
bioavailability
IAA
pre-
Introduction
acid
is also separated
purposes.
C are from
for the necessary
l99l;54:l323S-7S.
Ascorbic
acid (DHIAA),
quantitation
I From
the Nutrient
Composition
Laboratory,
Agricultural
Research
Service, US Deportment
of Agriculture,
Beltsville,
MD.
2 Address
reprint requests
to IT Vanderslice,
Building
161, Room
202,
10300
Baltimore
Society for Clinical
Avenue,
Nutrition
Barc-east,
Beltsville,
MD 20705.
l323S
Downloaded from ajcn.nutrition.org by guest on October 6, 2014
20%
,2
VANDERSLICE
l324S
AND
HIGGS
Standards
SampI
Broccoli
Extract
Vial
FIG 1. Schematic
of chromatographic
system.
Columns,
2 PLRP-S
versified
diet
ation
vitamin
in intake
C comes
from
one
from
source
many
sources
is balanced
and
by the
the vari-
variation
in
intake
from other sources
and, on the average,
percentage
of people
on a normal
diversified
an overwhelming
diet would
receive
adequate
the recommended
intakes
dietary
deviation
ofthe
those
foods
whose
observed
ranges.
The observed
in human
nutrient
large
dietary
variability
procedures
and
are
more
in the data
lead
to bias
8-1 1 (14)
can
lead
while
that
the
Block
et al (personal
plus
pill,
can
they
communication,
in Handbooks
just
or a vitamin
mg vitamin
the repletion
oranges,
iron.
orange
The
C but,
study,
of vitamin
the
amount
from
Table
tamin
tigator
the
different
shown
in column
4 of Table
3 are
the
Extraction
a
Flow
Chart
into six
phase
a vitamin
pill contained
The
upon
analysis,
contained
107.5
the object was to feed the subjects
the
necessary
actual
sources
C
nom-
different
sources.
for the
intake
is shown
subjects
of vitamin
in the
In the
values from
and would
to receive
C, however,
second
column
of
3 and was calculated
from our analysis
of the actual viC content
of the food. It is clear that although
the inveswould
an identical
have
C/d.
values
other
completed
juice,
vitamin
C from
of food
100 mg vitamin
the
During
the repletion
study each of the
C from only one source,
either
raw
pill plus
amounts
extract.
example,
study
in which
the subjects
were divided
first half of the study
involved
a depletion
phase.
then
in the raw
in. For
1990)
traces ofstandards
and broccoli
standard
to broccoli.
received
have
dose
believed
of vitamin
substantially
that
all study
groups
were
C, in fact,
several
groups
more
than
the
vitamin-pill
IAA
The repletion
study lasted for 28 d. If the average values for
the vitamin
C content
of the food for the 28-d period are used,
the actual intake of the nutrient
is shown in the column
3. If
the actual
daily analysis
of the food were used to obtain
intake,
would
as to swamp
be interested
FIG 2. Chromatographic
initially added as internal
of
cook-
of foods
in content
absence
of any analysis,
one would assume
the
Handbooks
8-9 and 8-1 1 (14) to be representative
serve
and
than
be so large
of the
minH
F-6
6
sources
handling
to the data
variation
might
received
vitamin
cooked
broccoli,
inally
100
mg. During
equal
k- min-.j
however,
the
storage,
careful
respect
that
investigator
a repletion
six groups
broccoli,
with
end
problems,
studies,
controlled
more
the large
to variances
effects
human-diet
groups.
The
The
lower
collection,
carefully
can
cause
in such
the food
household.
8-9 and
is at the
does
Often
be in a normal
food
In addition,
content
studies.
C are limited
vitamin
ing
nutrient.
allowances
(16) are normally
set at twice
the standard
above the mean,
which should
further
compensate
for
receiving
would
group.
FIG 3. Block
diagram
ofextraction
of vitamin
C from
foods.
intakes.
Downloaded from ajcn.nutrition.org by guest on October 6, 2014
(Polymer
Lab), 250 x 4.6 mm at 4 #{176}C;
mobile phase, 0.2 mol phosphate/
L, pH 2.14, 0.5 mL/min;
postcolumn
oxidation,
2.5 X l0 mol HgCl2/
L, 0.5 mL/min,
reaction coil 32 cm; postcolumn
fluorescence,
3 X l0
mol o-phenylene-diamine/L,
0.5 mL/min;
heating bath, 70 #{176}C;
heating
coil, 45.7 m; cooling bath, 20 #{176}C;
cooling coil, 1.5 rn; detection,
fluorescence,
LS-40 (Perkin Elmer), cx 350 nm, em 430 nm.
TABLE
1
Sample
and
VITAMIN
market
variability
of vitamin
C content
of some
C CONTENT
OF
FOODS
1325S
food sources
Vitamin
Markett
Ascorbic
acid
C content5
Dehydroascorbic
acid
Total
mg/lOOg
Banana
Beans,
snap,
Broccoli,
fresh
fresh
fresh
Cabbage,
fresh
Grapefruit
1
2
3
4
fresh
4.5
9.5
15.3
15.0
10.0
89.0
117.0±
104.2
119.5±
123.2
121.2
139.1
145.9±
148.2
2
2
2
2
1
1 d old
±
1.0
7.7
± 2.6
12 ±
±
±
0.4
2.5
2.5 ± 0.6
3.3 ± 0.6
12 ±
I
19 ±
3
±
0.0
2.7
± 0.6
18 ±
1
±
0.0
2.0 ± 0.0
12 ±
0
2.0
7.7
97 ±
2
125±10
1 13 ±
129±
131 ±
131 ±
4
5
3
5
±
±
±
±
9.7
3.6
5.4
3.1
4.4
±
8.0
±
2.1
3.0
± 0.6
8.4±0.9
9.1 ± 0.6
9.1 ±0.7
7.6 ± 1.0
10.7 ± 1.2
9.1
± 0.7
13.4±0.6
15.2 ± 0.5
139
3
±
8
159±
163 ±
2
3
59 ±
3
54.0 ±
77.3±
67.7 ±
72.3 ±
60.7 ±
2
65.3
±
3
67.0
±
1
2
3
50.0 ±
47.0 ±
60.0±
4.6
3.7 ± 0.6
54 ±
5
2.6
2.3
49 ±
3
1.0
5.3±0.6
65±
1
1
1
1
3
23.0 ±
8.7±
13.7 ±
27
0.0
1.2
1.2
5.6 ± 0.6
4.3±0.6
7.3 ± 0.6
4
29 ±
13±
21 ±
31
I
l
1
2
3
4
4
4
3
3
66.0
56.3
52.1
59.7
78.1
44.0
54.7
3.0
5.0
1.2
6.0±0.0
± 1.0
83±
1
4.9
5.6
± 0.6
73 ±
5
5.8
5.6
± 0.6
78 ±
6
4.0
5.7 ± 0.6
66 ±
4
4.9
4.3
± 0.6
70 ±
5
5.6
6.7
± 1.2
74 ±
6
± 0.6
(red)
Orange
California
California
California
California
California
navel
navel
navel
navel
navel
Florida
Florida
(w/skin)
Russet
Idaho
Maine
Red-skinned
Potato (w/skin)
Bag 1
2
3
Spinach (fresh)
±
1.0
6.7
± 2.5
73 ±
3
±
4.2
12.0
± 0.0
68 ±
4
52
2
± 0.3
63 ±
±
1.6
±
2.1
±
±
1.4
3.0
±
-
3.2
±
3
78 ±
1
9.0 ± 1.7
53 ±
3
2.5
8.3
± 1.2
63 ±
3
9.0 ±
6.7 ±
0.0
4.3
± 0.6
13 ±
1
0.6
4.3
± 0.6
11 ±
1
7.7±
1.2
5.3±0.6
13±
1
1
-
Potato
3
3
3
3
23.7
±
0.6
3.3 ± 0.6
27 ±
3
3
3
1
2
6.7 ±
6.3±
6.7 ±
67.7 ±
22.5 ±
0.6
4.3
± 0.6
11 ±
1
0.6
0.6
4.3±0.6
4.3 ± 0.6
11 ±
11 ±
1
I
3.8
2.3
± 0.6
70 ±
4
3.5
2.7
±
25
4
3
49.3±10.0
3
43.3
±
3
3
16
17.3 ±
15.7 ±
Idaho
1.2
±
1.3±1.1
51±10
0.4
3.2
47
I .2
4.0
± 0.0
2 1 ±
I
0.6
4.3
± 0.6
20 ±
I
±
0.6
±
I
Tomato
Homegrown
Store-bought
Cherry
S
2
SD.
t Markets
:j:Grapefruit
§ Average
1 , 2, and 3 are local
in poor condition.
of two measurements.
supermarket
For all other
sources
samples,
whereas
market
n = 3.
4 is a food wholesaler.
18
Downloaded from ajcn.nutrition.org by guest on October 6, 2014
Cabbage,
1
2
3
4
Cabbage,
1
3
3
3
3
3
3
3
4
4
4
4
4
4
VANDERSLICE
l326S
TABLE 2
Range of vitamin
C values
in some
Sample
fruits
#8
mg/lOOg
Beans,snap,raw
0-2
7-12
11-22
<1
12-19
12-18
Broccoli, raw
77-109
97-163
Cabbage,
31-64
0-10
28-48
1-7
42-83
13-14
21-31
5-6
raw
Cucumber
Grapefruit,
Lettuce
Oranges
California
Florida
red
navel
precision
if overall
This studyt
mg/lOOg
Apple juice
Bananas
HIGGS
TABLE 4
Modification
of study required
to achieve equal
instead of daily averages are used for broccoli5
and vegetables
Handbook
AND
40-74
45
Modification
factor
Sample
Subject
Number
Length
5
52-78
53-63
3.7
2.3
Raw
Cooked
4.0
2.4
of study
Days
man
Raw
Cooked
At 95% confidence
Diet Study.
interval.
Repletion
phase
in first one-half
of Hu-
Potatoes
Idaho
0-30
25-70
25
14-19
6-50
5-44
9-26
Tomatoes
Range obtained
from mean value ± twice the SD found
dard error and number
of samples tabulated
in handbook.
range
of means
the standard
from
Note
that
those
crate
ofcolumn
variation
Thus,
the use ofthe
daily
analysis
down
by a factor
of two
and
factor
of four.
same,
ofcourse,
different
deviations
samples.
in column
The
mean
the
values
but differ
cuts the standard
corresponding
from
from
variation
Let
between
us assume
analysis
TABLE
Average
because
has not
3
vitamin
the
the values
by a
4 are
the
in column
because
of the more careful
foodexample,
broccoli
was cooked
in
the vitamin
C content.)
Thus,
in
moving
from column
2 to 3 in Table
in moving
from
column
3 to 4 the
of four
withincrates.
deviation
3 and
3, bias
variance
within-crate
is eliminated
is reduced
variation
and
by a
is less than
the
crates.
that
the
bias
been
performed.
C intake
based
has
been
eliminated
Column
on different
3 values
and
a daily
would
then
data sets5
Handbook
#8
This data
(average)
This data
(daily)
mg
mg
mg
Broccoli, raw
Broccoli, cooked
Oranges (navel)
Orangejuice
Vitamin
pillt
S
129.7
127.5
132.8
I 12.6
107.5
± 20.1
107.5
± 16.7
107.5
±
± 24.5
107.5
± 20.6
107.5
± 12.9
content
ofvitamin
8.4
± 18.6
107.5
± 15.0
107.5
± 13.9
±
5.9
107.5
±
5.6
107.5
±
5.6
±
1.32
107.5
±
1.3
107.5
±
1.3
SD.
t Actual
C. Nominal
value
100 mg.
could
(1 test
the added
analysis).
expense
ask how
down
In either
case,
of analysis
study by a factor of four ifthe
variability
to the total variance
The
dietary
results
presented
studies
involving
the investigator
or the
expense
is faced
of expanding
lowest contribution
is desired.
from
with
the
sample
here
vitamin
strongly
suggest
C, daily
that,
analysis
in human
of the
food
source of this nutrient
should be done, particularly
when the
source is from one or a few fruits or vegetables.
The variance
due to the variability
in vitamin
C content
for some foods can
be substantial
and can mask other effects in which the investigator
is interested.
B
References
1. Mills MB, Damnon
CM, Roe JH. Ascorbic acid. dehydroascorbic
acid, diketogulonic
acid in fresh and processed
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2. Davidek
I, Velisek I, Domah AMB. Determination
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Z Lebensm
Unters Forsch
1974;! 54:18-22.
3. Rose RC, Nahrwold
DL. Quantitative
analysis ofascorbic
acid and
dehydnoascorbic
acid by high-performance
liquid chromatography.
Anal Biochem
Basis of intake
Sample
one
the variance
Summary
one-half
variance
columns
significantly
2. Bias in the data is evident
preparation
procedures.
(For
plastic
bags to better
preserve
factor
4 are only
3 for raw broccoli.
This is because
the
is much
less than the variation
between
data
four
from stan-
S
and
to reduce
27
Frozen
t Observed
the study would have to be varied
to that obtained
with daily analyses.
The results
are shown
in Table
4. For raw broccoli
either the
number ofsubjects
would have to be increased
by approximately
four or the study would have to be lengthened
by approximately
apply
11-13
l981;ll4:140-5.
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8. Behrens WA, Madere R. A highly sensitive high performance
liquid
chromatography
method
for the estimation
of ascorbic
and dehy-
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Red skinned
Spinach
Fresh
VITAMIN
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