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Twenty-one samples of commercial annatto formulations have been analyzed for color content (as
bixin or norbixin) using a developed HPLC-PDA method and the results compared with those
obtained using UV-vis spectrophotometric methods. HPLC-PDA provided superior qualitative
and quantitative data, particularly with respect to the determination of colored degradation
compounds. Two samples of norbixin of known production history were subjected to detailed HPLC
analysis to identify possible differences in their colored and degradation component profiles. The
samples differed significantly in their all-trans- and di-cis-norbixin isomer contents, which was
indicative of their respective production histories.
Keywords: Annatto; Bixa orellana; bixin; norbixin; coloring materials; additives; HPLC; photodiode
array; spectrophotometry
INTRODUCTION
Scotter et al.
type
form
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
S18
S19
S20
S21
bixin
bixin
bixin
bixin
bixin
norbixin
norbixin
norbixin
norbixin
norbixin
bixin
norbixin
bixin
bixin
norbixin
bixin
norbixin
norbixin
bixin
norbixin
bixin
dry powdera
dry powdera
crystalline
crystalline
oil suspension/solution
solution
granules
granulesb
powderb
granules
encapsulated dry powder
aqueous solution
water dispersible liquid
crystalline
granules
crystalline
aqueous solution
aqueous solution
oil solution
spray-dried on maltodextrin carrier
oil suspension/solution
a Essentially the same product processed at different facilities. b Same type of norbixin but ground to different degrees. c N/A, information
not available.
282.6
dilution vol (mL)
100
1000
sample wt (g)
Smith method
(bixin %)
McKeown method
(pigment %)
HPLC method
(bixin %)a
S1
S2
S3b
S3c
S4
S5
S13
S14
S16b
S16c
S19
S21
9.0
47.2
78.5
38.9
30.3
3.2
0.2
77.4
66.6
17.2
0.8
4.0
12.6
42.9
79.0
39.1
34.4
3.7
na
89.0
68.4
20.1
0.5
4.7
9.2
51.0
nae
31.6
34.5
1.8d
0.08
na
na
16.8
0.25d
3.8
Smith method
(norbixin %)
HPLC method
(norbixin %)d
S6
S7
S8
S9
S10
S11a
S12
S15
S17
S18
S20
2.5
37.7
41.4
40.1
24.9
0.7
0.6
35.0
3.3, (4.8)c
b, (1.0)c
16.4, (10.2)c
1.5
43.6
47.3
30.2
25.9
7.3
3.3
32.6
5.9
1.7
9.5
Scotter et al.
retention
time (min)
max
(nm)
dmax (nm)
from trans-
REL(I)
(%)
isomera,i
(tentative)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
3.3
3.6
5.3
6.4
6.8
7.0
7.4
7.7
8.1
8.9
9.6
10.2
12.1
18.3
19.8
396
458
448
465
454
454
448
445
442
445
459
459
459
452b
412b
c
7
17
0
11
11
17
20
23
20
6
6
6
c
c
c
d
9
9
13
25
d
19
24
d
11
23
54
c
c
trans C17
mono-cis-h
di-cistrans-e
di-cisdi-cisdi-cistri-cis- ?
tri-cis- ?
tri-cis- ?
mono-(9)-cis-e
mono-cis-f
mono-cis-g
unknown
unknown
9-cis
11b
all-trans
4
mono-cise
13
di-cise
5c
di-cise
6c
di-cise
3
49500
84.1
900
1.5
225
0.4
8000
13.6
245
0.3
13400
80.7
1520
9.1
381
2.3
61
0.2
1134
6.8
108
0.7
a As given in Table 4. b Not possible to resolve from peak 12. c Peaks 5 and 6 poorly resolved.
Arbitrary assignment, see Table 4.
Figure 2. Comparison of spectra obtained from commercial norbixin sample S20 (1) and a 9-cis-norbixin standard (2) in 0.1 M
NaOH.
Scotter et al.
Figure 3. HPLC-PDA chromatograms of commercial norbixin samples S17 (a) and S18 (b). Peak numbers correspond to those
given in Table 4.
Figure 4. HPLC-PDA spectra of the major norbixin isomers in commercial sample S18: peak 4 (s); peak 6 (- -); peak 11
(- - - -); peak 13 (- - -). Peak numbers correspond to those given in Table 4.
Scotter et al.
matography with photodiode-array detection. Food Addit.
Contam. 1994, 11 (3), 301-315.
Smith, P. R.; Blake, C. J.; Porter, D. C. Determination of added
natural colours in foods. III. Annatto. Leatherhead Food R.
A. Research Report 431. 1983.
Zechmeister, L. Cis-trans isomeric carotenoid pigments. Prog.
Chem. Nat.Prod. 1960, 18, 232-238.