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1270 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005
LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1271
Table 1. Materials and their sources used in the
collaborative study
Materiala
Source
Red wine
Natural colorantb
Strawberry concentratec
Raspberry concentratec
Elderberry concentrate
Cyanidin-3-glucoside
chlorided
Polyphenols Laboratories As
(Sandnes, Norway)
1272 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005
Moisture Determination
A. Principle
Monomeric anthocyanin pigments reversibly change color
with a change in pH; the colored oxonium form exists at
pH 1.0, and the colorless hemiketal form predominates at
pH 4.5. The difference in the absorbance of the pigments at
520 nm is proportional to the pigment concentration. Results
are expressed on a cyanidin-3-glucoside basis. Degraded
anthocyanins in the polymeric form are resistant to color
change regardless of pH and are not included in the
measurements because they absorb at pH 4.5 as well as
pH 1.0.
B. Apparatus
(a) pH meter.Standardized with pH 4.0 and
7.0 standard buffer solutions.
(b) Visible spectrophotometer.Performance of the
spectrophotometer at 520 nm should be verified with
reference standards for wavelength accuracy, photometric
accuracy, photometric linearity, and stray light.
(c) Glass
or
disposable
cuvets
for
spectrophotometer.1 cm pathlength.
(d) Volumetric flasks.50 mL.
C. Reagents
pH Differential Method
First Action 2005
Table 2005.02 Interlaboratory study results for the determination of total monomeric anthocyanin pigment content
by the pH differential method
Mean, mg/La
src
RSDr, %d
sRe
RSDR, %d
rf
Rg
HorRat
13.6
10 (1)
0.57
4.16
1.09
8.00
1.59
3.05
0.74
Red wine
201.6
11 (0)
5.29
2.62
15.99
7.93
14.81
44.76
1.10
Natural colorant
640.8
11 (0)
11.97
1.87
36.52
5.70
33.52
102.25
0.94
Strawberry juice
63.6
10 (1)
2.43
3.82
6.44
10.12
6.81
18.03
1.18
Raspberry juice
336.7
11 (0)
10.80
3.21
17.62
5.23
30.24
49.32
0.79
Elderberry juice
3006.8
10 (0)
31.78
1.06
191.84
6.38
88.97
537.15
1.33
44.8
11 (0)
0.53
1.19
1.20
2.69
1.49
3.37
0.3
Material
Cranberry juice cocktail
Standard
a
a = Number of laboratories retained after removal of outliers; (b) = number of laboratories removed as outliers.
r = Repeatability value.
R = Reproducibility value.
LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1273
1274 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005
Table 2. Total monomeric anthocyanin (mg/L, cyanidin-3-glucoside equivalents) levels of all Youden pair samples
analyzed by collaborators
Cranberry juice
cocktail
Red wine
Elderberry juice
Standard
Xa
Yb
Xa
Yb
Xa
Yb
Xa
Yb
Xa
Yb
Xa
Yb
Xa
Yb
15.0
13.8
218.8
210.9
693.3
672.8
69.0
66.1
364.2
356.7
3113.5
2948.2
46.6
44.5
Collaborator
14.2
13.8
225.0
215.8
688.0
664.6
72.8
70.3
356.1
346.5
3193.7
3030.8
46.3
44.1
16.3c
12.0c
220.0
225.0
724.7
673.0
78.5
69.3
367.4
347.8
1527.9d
2187.6d
47.9
45.8
12.5
13.7
184.0
192.0
623.7
640.8
57.7
56.5
321.9
332.7
3181.1
3093.5
45.0
42.2
13.0
13.1
211.7
195.2
652.1
621.2
65.1
62.6
345.7
325.6
3147.7
2955.7
44.8
43.6
13.2
12.8
209.7
200.6
657.3
634.2
64.5
64.5
353.2
325.1
3208.3
3076.8
45.8
43.3
14.4
15.5
173.9
172.1
598.0
584.5
61.1
58.8
359.7
314.7
3101.5
2931.4
44.8
44.0
13.4
12.9
199.6
200.2
648.8
628.8
60.6
58.0
326.5
322.3
3248.2
3202.2
44.3
41.9
12.4
11.4
202.7
200.7
641.2
625.1
51.9
56.7
308.1
302.3
2941.5
2782.5
44.6
42.8
10
13.4
13.4
182.9
180.5
600.3
563.6
65.8
62.3
331.5
325.6
3019.2
2848.8
46.4
45.4
11
15.0
15.4
213.7
200.8
637.9
622.9
92.7c
66.8c
350.7
322.3
2615.0
2496.5
47.3
44.1
a
b
c
d
10
11
NSD
9.12
9.14
9.19
9.20
9.13
9.13
9.13
9.21
9.14
9.21
9.17
9.18
9.17 (0.03)
0.62
9.17
9.18
9.17
9.17
9.17
9.21
9.14
9.14
9.21
9.21
9.13
9.13
9.54 (0.02)
NSD
9.56
9.56
9.55
9.58
9.56
9.56
9.57
9.57
9.55
9.54
9.49
9.49
Yb
Mean (SD )
0.06
9.52
9.56
9.52
9.52
9.52
9.52
9.53
9.54
Xa
Red wine
0.15 NSD
P-value
9.52
NSD
0.74
12
9.52
9.54
9.55
Yb
Xa
Measurements
NSD
1.63
1.65
1.62
1.63
1.62
1.63
1.73
1.73
1.67
1.67
1.67
1.66
1.66 (0.03)
0.90
1.65
1.65
1.69
1.69
1.67
1.67
1.62
1.60
1.68
1.67
1.65
1.65
Xa
NSD
1.65
1.63
1.62
1.63
1.60
1.59
1.57
1.56
1.56
1.59
1.59
1.56
1.59 (0.03)
0.57
1.62
1.63
1.62
1.60
1.59
1.58
1.56
1.57
1.59
1.59
1.56
1.56
Yb
Natural colorant
NSD
8.18
8.17
8.18
8.17
8.19
8.19
8.18
8.18
8.18
8.18
8.18
8.20
8.18 (0.01)
0.79
8.17
8.17
8.18
8.18
8.19
8.19
8.18
8.18
8.18
8.18
8.18
8.19
Xa
NSD
7.86
7.86
7.87
7.87
7.85
7.86
7.87
7.87
7.87
7.87
7.87
7.87
7.86 (0.01)
0.26
7.86
7.86
7.87
7.86
7.86
7.85
7.86
7.86
7.86
7.86
7.87
7.88
Yb
Strawberry juice
NSD
9.17
9.17
9.16
9.17
9.16
9.16
9.16
9.16
9.18
9.18
9.16
9.17
9.17 (0.01)
0.41
9.17
9.17
9.16
9.16
9.17
9.16
9.17
9.17
9.18
9.18
9.17
9.17
Xa
NSD
8.82
8.82
8.82
8.82
8.81
8.81
8.81
8.82
8.82
8.82
8.82
8.82
8.82 (0.01)
0.3
8.82
8.82
8.81
8.82
8.81
8.81
8.82
8.81
8.80
8.82
8.82
8.82
Yb
Raspberry juice
Table 3. $Brix values for the homogeneity of the test materials (12 measurements from 2 randomly selected vials)
9.99
9.97 10.02
9.94
Yb
0.77 NSD
10.63 (0.28) 10.16 (0.12)
0.19 NSD
10.49 10.51
10.46 10.49
Xa
Elderberry juice
NSD
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.02
0.03
0.03
0.03
0.03 (0.00)
0.30
0.02
0.03
0.03
0.02
0.03
0.02
0.03
0.03
0.03
0.03
0.03
0.03
Xa
0.03
0.02
0.03
0.02
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
NSD
Yb
0.03 (0.00)
0.19
0.03
0.02
0.02
0.03
0.03
0.02
0.02
0.02
0.03
0.03
0.03
0.03
Standardc
LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1275
1276 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005
detected by LC, that absorbed 520 and 280 nm). On the basis
of the absorbance at 520 and 280 nm, the purity values for
standard 1 were 98.2 and 93.8%, respectively. Purity values
for standard 2 were 99.6% (monitored at 520 nm) and 98.9%
(monitored at 280 nm). Purity values for standard 3 were
95.2% (monitored at 520 nm) and 94.1% (monitored at
280 nm). The purities based on molar absorptivity were 74.6,
99.1, and 80.3% for standards 1, 2, and 3, respectively.
Hygroscopicity values for standards 1, 2, and 3 at 83%
relative humidity were 22.4, 10.0, and 22.2%, respectively.
There was no uniformity regarding each companys
instructions on how to store and handle the anthocyanin
standards. One datasheet advised storage in darkness at low
temperatures (<5$C) for no more than a few days. Another
firm advised storing the standard at 20$C in a dry and dark
place, and also placing the standard in a desiccator under
vacuum for 48 h before use. Neither company provided an
expiration date, but both recommended using the standard
soon after reception, and to never store the product in solution.
They further stated that the stability of the products was not
always known and very difficult to measure.
The validity and basic principles for determining the
anthocyanin pigment concentration by the pH differential
method have been widely accepted by natural product chemists
for years. A combination of the following factors may account
for the low recovery or purity of the standard: moisture content,
hygroscopicity of anthocyanins, presence of impurities
(polyphenolics and/or polymeric anthocyanins), the possibility
that the molar extinction coefficient of 26 900 L & mol"1 & cm1
is not the true value, measurement of the absorbance at
520 nm rather than at 510 nm, and the minor contribution of
quinoidal and flavylium forms to absorbance at pH 4.5. This
collaborative study has demonstrated that total monomeric
anthocyanin pigment content can be measured with excellent
agreement between laboratories. A further advantage of the
method is that it does not require the purchase of costly
standards, as is the case when anthocyanin content is measured
by LC by the external standard method. The experiments
concerning the moisture content, purity, and hygroscopicity of
anthocyanin standards in this investigation call attention to the
importance of taking these properties into consideration when
experiments are conducted with anthocyanin standards.
Collaborators Comments
Collaborators made the following comments during the
study: (1) several "samples" produced spectra whose
maximum was not 520 nm; (2) "sample" absorbance was
measured with a 2 mm cell; (3) the use of 100 mL volumetric
flasks or serial dilution for the dark juices should be allowed;
and (4) "sample" container arrived cracked.
The statement about analytes having a different maximum
absorbance at a different wavelength is well taken and
discussed above. We believe that the statement to determine
the appropriate dilution factor is sufficient for the analyst to
make the decision about whether to use a 2 mm cell,
Figure 4. Values for total monomeric anthocyanin content, obtained after the thermal shock and during the
long-term stability study of the test samples (X = undiluted member of the Youden pair sample, Y = member of the
Youden pair sample diluted to make !5% difference).
LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005 1277
Table 4. Molar absorptivity (() values of cyanidin-3-glucoside reported in the literature
"vis-max, nm
Ref.
510
26900
10
1% HCl in methanol
530
34300
13
0.1N HCl
520
25740
14
Buffer, pH 1.0
510
24800
15
512
18800
16
Buffer, pH 1.0
510
20000
17
530
2787632678
18
0.1N HCl
510
26300
19
Solvent system
Acknowledgments
Recommendations
On the basis of the results from the collaborative study, the
Study Director recommends that the pH differential method for
determination of total monomeric anthocyanin pigment content
of fruit juices, beverages, natural colorants, and wines be adopted
as a First Action Official Method of AOAC
INTERNATIONAL.
Standard 2b
Standard 3c
% Moistured
12.9
3.5
10.5
Hygroscopicitye, % EMC
22.4
10.0
22.2
74.6
20072
99.1
26672
71.0
19103
93.2
25076
>97
99.3
98.2
99.6
80.3
21606
76.3
20526
>97
95.2
93.8
98.9
94.1
$806.68 for 50 mg
Standard used in the collaborative study; purchased from Polyphenols Laboratories As.
Standards were placed in an 83% relative humidity chamber (saturated potassium bromide solution placed in a 25$C incubator).
Extinction coefficient determined by dissolving cyanidin-3-glucoside chloride in pH 1.0 buffer and measuring the absorbance at 510 nm.
Extinction coefficient determined by dissolving cyanidin-3-glucoside chloride in pH 1.0 buffer and measuring the absorbance at 520 nm,
which is the "max used in the collaborative study.
1278 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 5, 2005
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