PO-CON1508E
Simultaneous Analysis of Water- and
Fat-Soluble Vitamins in Beverages Using
an ODS-Modified Metal-Doped Column
Pittcon 2015
1550-3P
Kenichiro Tanaka, William Hedgepeth
Shimadzu Scientific Instruments, Inc., Columbia, Maryland
Simultaneous Analysis of Water- and Fat-Soluble Vitamins in
Beverages Using an ODS-Modified Metal-Doped Column
Introduction
Vitamins play vital roles in the metabolic activities of the
body. However, because they all cannot be synthesized in
sufficient quantities in the body, they must be ingested
either in our meals or by other means, such as tablets.
Vitamins can be classified into two groups, water-soluble
and fat-soluble, and their analysis is generally conducted
by high-performance liquid chromatography. The
water-soluble type consists mainly of highly polar basic
components which exhibit weak retention in analysis by
reversed-phase liquid chromatography. Since they exhibit
weak retention, the analysis is typically conducted using
ion-pair reagents. That makes it difficult to apply gradient
elution. On the other hand, the fat-soluble type consists
of hydrophobic components. They are usually analyzed by
reversed-phase liquid chromatography with organic
solvent mobile phases or normal-phase liquid
chromatography.
In this study, a Shim-pack MAqC-ODS I was used to
analyze water- and fat-soluble vitamins in a single
method. This column contains ODS-modified and
metal-doped stationary phase, so it was able to retain the
weakly-retained components without ion-pairing
reagents. In addition, by using a 100% organic solvent
mobile phase after the water-soluble vitamins were
eluted, fat-soluble vitamins were also able to be analyzed.
Materials and Method
Reagents and standards
Reagents: Ascorbic acid, thiamine hydrochloride,
riboflavin, nicotinic acid, nicotinamide, D-pantothenic acid
hemicalcium salt, pyridoxine hydrochloride, biotin, folic
acid, cyanocobalamine, retinol, retinyl acetate, retinyl
palmitate, α-tocopherol, DL-α-tocopherol acetate,
ergocalciferol, cholecalciferol, phytonadione,
menaquinone, phosphoric acid, sodium phosphate
monobasic, sodium hydroxide, ethanol, and chloroform
were purchased from Sigma-Aldrich. Water was made in
house using a Millipore Milli-Q Advantage A10 Ultrapure
Water Purification System. Acetonitrile was purchased from
Honeywell.
Stock solutions: Ascorbic acid, thiamine hydrochloride,
nicotinic acid, nicotinamide, D-pantothenic acid
hemicalcium salt, pyridoxine hydrochloride, and
cyanocobalamine were separately dissolved in water.
Riboflavin, biotin, and folic acid were separately dissolved
in 20 mmol/L sodium hydroxide aqueous solution. Retinol,
α-tocopherol, DL-α-tocopherol acetate, ergocalciferol, and
cholecalciferol were separately dissolved in ethanol. Retinyl
acetate, retinyl palmitate, phytonadione, and menaquinone
were dissolved in chloroform. They were all made to 1000
mg/L.
Standard solutions: The stock solution of water-soluble
vitamins (ascorbic acid, thiamine hydrochloride, nicotinic
acid, nicotinamide, D-pantothenic acid hemicalcium salt,
pyridoxine hydrochloride, cyanocobalamine, riboflavin,
biotin, and folic acid) was mixed equally and diluted to 50
mg/L and transferred to a 1.5 mL vial for analysis. The
stock solution of fat-soluble vitamins (retinol, α-tocopherol,
DL-α-tocopherol acetate, ergocalciferol, cholecalciferol,
retinyl acetate, retinyl palmitate, phytonadione, and
menaquinone) was mixed equally and diluted to 50 mg/L
and transferred to a 1.5 mL vial for analysis.
Sample solution: Vitamin-fortified water was purchased
at a local supermarket. The sample was directly transferred
to a 1.5 mL vial for analysis.
Methods
Samples were injected to a Shimadzu Prominence-i
integrated system (PDA model). Water- and fat- soluble
vitamins were separated on the Shim-pack MAqC-ODS I
column and detected by the PDA detector incorporated in
the integrated system. Table1 shows analytical conditions.
2
Simultaneous Analysis of Water- and Fat-Soluble Vitamins in
Beverages Using an ODS-Modified Metal-Doped Column
Table 1 Analytical conditions
System
Column
Guard Column
Mobile Phase A
Mobile Phase B
Time Program
Flow Rate
Column Temperature
Injection Volume
Detection
:
:
:
:
:
:
:
:
:
:
Prominence-i PDA model (LC-2030 3D)
Shim-pack MAqC-ODS I (150 mm L. x 4.6 mm I.D., 5 μm)
Shim-pack GVP-ODS (10 mm L. x 4.6 mm I.D., 4.6 μm)
10 mmol/L phosphate (sodium) buffer (pH 2.6)
Acetonitrile
0 % (0-2.5 min) – 40 % (10 min) – 100 % (25-45 min) – 0 % (45.01-55 min)
1.2 mL/min
40 °C
10 μL
220 nm
Results
Analysis of standard solution
Fig. 1 shows spectra of water- and fat-soluble vitamins. We set
220 nm as the detector wavelength in this study. Fig. 2 shows
chromatograms of water- and fat-soluble vitamin standard
solutions. The Shim-pack MAqC-ODS I contains an
ODS-modified metal-doped stationary phase, so it was able to
mAU
mAU
300
250
200
200
350
nm
600
283
nm
260
239
300
350
nm
241
200
370
250
300
350
nm
mAU
mAU
100
mAU
125
350
nm
200
Retinol
250
300
350
nm
250
202
nm
214
348
328
300
350
nm
200
250
300
350
0
nm
Ergocalciferol
mAU
mAU
200
350
25
0
Menaquinone
mAU
201
25
291
200
Retinyl acetate
mAU
75
200
250
300
Cholecalciferol
327
300
nm
100
229
250
350
50
246
257
266
0
0
200
228
100
254
259
223
253
260
223
0
300
50
200
100
50
250
Biotin
75
300
200
200
Riboflavin
400
300
379
252
308
337
250
Cyanocobalamine
327
mAU
400
0
0
200
265
350
50
25
100
305
250
276
300
75
200
0
250
nm
mAU
100
200
350
150
300
50
300
175
150
Folic acid
326
600
125
200
60
100
50
200
250
300
350
α-Tocopherol
75
200
250
300
245
257
266
30
328
10
0
350
nm
α-Tocopherol acetate
255
260
20
290
284
25
0
nm
40
50
315
0
294
50
340
50
257
100
223
150
100
226
150
255
200
mAU
700
100
250
Pyridoxine
400
Pantothenic acid
100
200
500
nm
150
nm
200
0
mAU
0
350
250
100
350
300
Nicotinamide
300
50
300
250
300
200
250
200
nm
400
100
200
350
350
500
150
300
mAU
mAU
196
mAU
200
250
Nicotinic acid
Thiamine
Ascorbic acid
0
200
362
300
258
264
250
267
200
223
nm
50
0
201
350
100
202
212
300
0
362
250
0
50
266
278
200
395
333
0
150
100
236
100
50
346
261
215
206
25
200
100
330
50
150
290
300
150
265
246
200
75
300
250
328
400
301
mAU
250
100
229
mAU
243
mAU
retain the weakly-retained components without ion-pairing
reagents. In addition, by using a 100% organic solvent mobile
phase after the water-soluble vitamins were eluted, fat-soluble
vitamins were also able to be analyzed. All the water- and
fat-soluble vitamins were well separated in a single method.
0
200
250
300
350
nm
200
phytonadione
250
300
350
nm
Retinyl palmitate
Fig.1 Spectra of analytes
3
Simultaneous Analysis of Water- and Fat-Soluble Vitamins in
Beverages Using an ODS-Modified Metal-Doped Column
550000
uV
9
500000
450000
400000
7
350000
300000
8
250000
200000
3
150000
1 2
100000
50000
4
5
10
6
0
2.0
550000
3.0
4.0
5.0
6.0
7.0
8.0
9.0
min
35.0
min
uV
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
uV
125000
13
100000
15
14
75000
1617
12
50000
25000
18
11
19
0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
min
■ Peaks
1. Ascorbic acid (Vitamin C)
2. Thiamine (Vitamin B1)
3. Nicotinic acid (Vitamin B3)
4. Nicotinamide (Vitamin B3)
5. Pyridoxine (Vitamin B6)
6. Pantothenic acid (Vitamin B5)
7. Folic acid (Vitamin B9)
8. Cyanocobalamine (Vitamin B12)
9. Riboflavin (Vitamin B2)
10. Biotin (Vitamin B7)
11. Retinol (Vitamin A)
12. Retinyl acetate (Vitamin A acetate)
13. Menaquinone (Vitamin K2)
14. Ergocalciferol (Vitamin D2)
15. Cholecalciferol (Vitamin D3)
16. α-Tocopherol (Vitamin E)
17. α-Tocopherol acetate (Vitamin E acetate)
18. Phytonadione (Vitamin K1)
19. Retinyl palmitate (Vitamin A palmitate)
Fig.2 Chromatogram of standard solutions
4
Simultaneous Analysis of Water- and Fat-Soluble Vitamins in
Beverages Using an ODS-Modified Metal-Doped Column
Analysis of sample solution
Fig. 3 shows a chromatogram of vitamin fortified water.
All the water- and fat-soluble vitamins were able to
detected in a single run. It was difficult to detect retinyl
palmitate at 220 nm because of the low concentration,
but it was able to be detected at 330 nm, which is a
wavelength of maximum absorption.
uV
1
250000
225000
200000
5000
175000
uV
19
2500
150000
0
125000
330 nm
220 nm
-2500
37.5
100000
40.0 min
75000
17
50000
5 6
25000
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
min
■ Peaks
1. Ascorbic acid (Vitamin C)
5. Pyridoxine (Vitamin B6)
6. Pantothenic acid (Vitamin B5)
17. α-Tocopherol acetate (Vitamin E acetate)
19. Retinyl palmitate (Vitamin A palmitate)
Fig.3 Chromatogram of vitamin fortified water
Conclusions
• 10 water-soluble vitamins and 9 fat-soluble vitamins were successfully analyzed on a Shim-pack MAqC-ODS I in a single
method.
• Water- and fat- soluble vitamins in a vitamin fortified water were successfully analyzed in a single run.
Reference
Shimadzu application news No. L459: Simultaneous Analysis of Water-Soluble Vitamins by a Newly Developed
“Shim-pack MAqC-ODS I” Column
First Edition: March, 2015
www.shimadzu.com/an/
For Research Use Only. Not for use in diagnostic procedures.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.
The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject
to change without notice.
© Shimadzu Corporation, 2015