mAU
mAU
1
B12 @361 nm
5
0.75
0.5
3
0
1
-0.25
0
-0.5
1.1
1
2
75
3
0.25
Ar
ea
:
Determination of Water-and Fat-Soluble Vitamins in Gummies by
Reversed-Phase Liquid Chromatography
4
-0.75
-1
8.5
9
9.5
10
10.5
min
mAU
8
8.5
9
9.5
10
10.5
11
mAU
Biotin @210 nm
20
50
20
0
10
-10
0
9.8
gelain
23.75%
vitamins
food dyes and others
75.00%
Fig. 1 Ingredients of Gummy
4000
Vitamin
10
10.2
10.4
10.6
min
9.5
min
2.9
9
12
4
Ar
ea
:
100
5
50
0
0
8
80.0%
210
40.0%
D3
265
C, B6, B9, E
8.8
9
min
7.5
8
8.5
9
5 hrs
36 hrs
60 hrs
0.0%
292
A
80.0%
60.0%
324
40.0%
B12
361
20.0%
0.0%
RESULTS
Vitamins were detected at different
wavelengths. Figure 3 shows the
chromatograms of standard and sample
solution of nine fat- and water- soluble
vitamins. Figure 4 presents recoveries of
vitamins in standard and sample solutions.
Fig. 4 recoveries of vitamins in standard and sample solutions
Tab. 3 Determination of water- and fat-soluble vitamins in gummies
Recov
Labeled
Found
RSD
Vitamins
SD
ery
(mg/kg)
(mg/kg)
(%)
(%)
mAU
D3 @254 nm
10
10
5
5
0
0
97
.4
15
ea
:
15
48
2
20
-5
C
4595.588
3919.469
73.52
1.88
85.29
B6
Pantothenic
acid
Folic acid
459.559
41.015
2.25
5.48
8.83
1194.853
760.228
97.24
12.79
63.63
59.743
24.039
1.29
5.37
40.24
B12
1.379
0.119
0.02
17.50
8.66
Biotin
13.787
24.777
2.68
10.81
179.71
D3
2.298
2.391
0.02
0.70
104.05
E
2527.574
1450.034
7.62
0.53
57.37
A
144.761
251.286
15.85
6.31
173.59
-5
19
20
21
22
23
min
24
mAU
18
19
20
21
22
min
23
mAU
38
3
4.1
6
175
E @292 nm
150
8
Ar
ea
:
10
125
100
6
75
4
50
2
25
0
0
-2
-25
20
22
24
26
28
30
32
min
34
mAU
6
23
24
25
26
27
28
29
min
30
mAU
70
A @324 nm
60
50
4
40
2
30
CONCLUSIONS
20
0
10
0
-2
-10
-20
37.5
40
42.5
45
47.5
50
52.5
55
min
57.5
m AU
37.5
40
42.5
45
47.5
50
52.5
55
57.5
min
mAU
50
C @292 nm
40
40
30
30
20
20
10
10
0
1.2
1.4
1.6
1.8
2
2.2
2.4
1
m in
2.6
mAU
1.2
1.4
1.6
1.8
2
2.2
2.6 min
2.4
mAU
B6 @292 nm
25
250
20
200
150
10
100
:2
06
15
50
5
0
0
-50
4.25
4.5
4.75
5
5.25
5.5
6 min
5.75
Pantothenic acid @210 nm
mAU
3.5
4
4.5
5
5.5
6
120
60
100
50
80
40
30
60
20
40
10
20
0
0
7.4
7.6
7.8
8
ACKNOWLEDGEMENTS
min
mAU
70
7.2
Separation and determination of the vitamins
was achieved by RP-LC. For most vitamins,
quantitative results close to the label claim are
determined. The stability of the vitamin
extracts and potential interference by coextracted sugar are being investigated to
improve these results prior to validation of the
final method.
.40
1
All chromatographic separations were
performed under room temperature. The
elution program is listed in table 1. The
-1
flow rate was 1.0 ml min . The detection
was achieved at five different wavelengths.
8.6
100.0%
18
0.0
0.0
13.0
30.0
100.0
100.0
0.0
8.4
20.0%
-10
Solvent B
(Acetonitrile)
%
8.2
60.0%
-10
Fig. 2 Concentration range of vitamins in gummies (mg kg-1)
9.8
100.0%
Ar
Tab. 1 Elution program for the RP-HPLC determination of
Water- and fat- soluble vitamins in Gummies
3000
0
9.6
200
Detection
wavelength(nm)
B5, B8
1
1000
9.4
Fig. 3 Chromatograms of standard and samples of nine fat- and watersoluble vitamins
Tab. 2 Detection wavelengths for water- and fat-soluble vitamins
0
2000
9.2
250
10
20
100.0
100.0
87.0
70.0
0.0
0.0
100.0
9
150
mAU
0.0
3.5
4.0
12.0
12.5
53.0
54.0
min
15
3.7
7
sugars
10.8
20
Ar
ea
0.50%
10.6
Folic acid @292 nm
:6
32
0.75%
10.4
mAU
25
Determination of six water-soluble
vitamins [calcium pantothenate (B5),
pyridoxine (B6), biotin (B8), folic acid
(B9), cyanocobalamin (B12) and ascorbic
acid (C)] and three fat-soluble vitamins
[retinyl palmitate (A), cholecalciferol
(D3) and alpha-tocopheryl acetate (E)] in
gummies was studied by RP-LC with UV
detector. Sample was prepared as follows:
• Gummy was dissolved in the mixture
of water/methanol (v/v=1:1)
• Ethanol was used to precipitate the
gelatin
• Nitrogen gas was used to evaporate
the solvent
• The final sample was injected onto a
C-8 column
Time
(min)
10.2
30
METHODOLOGY
Solvent A (0.025
M Phosphate
buffer, pH=3.5)
%
10
mAU
Ar
ea
Vitamins are essential substances for
human health and growth. Storage,
aging, and processing of foods may
cause vitamin loss. There is a need to
develop a fast and accurate analytical
method for the determination of
vitamins in foods. The determination of
vitamins in sugar-based matrix is usually
a challenging task due to the complexity
of the matrix and instability of vitamins.
Gummies are sugar-based candies which
are fortified with selected vitamins.
Until now, no method has been proposed
for the determination of vitamins in
gummies.
In
this
work,
the
determination of six water-soluble
vitamins and three fat-soluble vitamins
is studied by reverse phase liquid
chromatograph(RP-LC) with ultra-violet
detector at different wavelengths, on a
C-8 column using phosphate buffer and
acetonitrile as mobile phase.
Ar
ea
:
30
7.8
INTRODUCTION
19
1
10
.35
9
40
Changling Qiu & Douglas Raynie
Department of Chemistry and Biochemistry
South Dakota State University
Brookings, SD 57007 USA
[email protected]
min
11.5
min
7
7.2
7.4
7.6
7.8
8
min
1.
2.
3.
4.
5.
The Green Chemistry Laboratory, SDSU
Chemistry and Biochemistry Dept., SDSU
Dr. Brian Logue
Robert Oda
Dionex for funding this project