1
Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)Andrew Aubin Waters Corporation, Milford, MA, USA
IN T RO DU C T IO N
The analysis of fat-soluble vitamins (FSV) formulations, often from oil-filled
and powder-filled capsules, or pressed tablets, can be a challenging task. Most
often, analysis of these formulations employs a normal phase chromatographic
method using traditional normal phase solvents (hexane, tertiary butyl alcohol,
ethyl acetate, dichloromethane, and others) that can be expensive to procure
and dispose. Other analytical chromatographic techniques for these analyses
include reversed phase liquid chromatography, gas chromatography, thin layer
chromatography, and colorimetric techniques. The use of UltraPerformance
Convergence Chromatography™ (UPC2™) in fat-soluble vitamin analysis
provides a single viable technique that is cost-effective, sustainable, and a
green technology alternative that lowers the use of organic solvents, provides
fast analysis times, and maintains chromatographic data quality. A series of FSV
formulations were analyzed using the ACQUITY UPC2 System. The examined
formulations contained vitamin A only, vitamins A + D3, vitamin E, vitamin
D3 only, vitamin K1 only, and vitamin K2 only, as shown in Table 1. Results
from these experiments show that UPC2 has the potential to replace many of the
separation methods in use today as the sole technique with no compromises.
WAT E R S SO LU T IO NS
ACQUITY UPC2 System
ACQUITY UPC2 Column Kit
Empower™ 3 Software
ACQUITY UPC2 PDA Detector
K E Y W O R D S
UPC2, fat-soluble vitamins, vitamin E,
vitamin A, vitamin D3, vitamin K1,
vitamin K2
A P P L I C AT IO N B E N E F I T S■■ Fast analysis of a wide range of fat-soluble
vitamin formulations.
■■ Waters® ACQUITY UPC2™ System was
able to successfully analyze six different
formulations of fat-soluble vitamins.
A single technique was able to quickly
analyze these different formulations.
■■ This system can greatly streamline fat-
soluble vitamin analysis by allowing labs to
use a single technique on a single system to
analyze a wide range of FSV formulations.
■■ Each of the fat-soluble vitamin
formulations were analyzed rapidly,
and components of interest resolved
well from excipient materials.
■■ Isomers of vitamins A, E, and K1 were
successfully resolved from each other.
Active
ingredient(s)
Amount per
capsule/tablet
Inactive ingredients
Vitamin A 10,000 IU A Soy oil, gelatin, glycerin, water
Vitamin A & D3
10,000 IU A 2000 IU D3
Soy oil, gelatin, glycerin, water
Vitamin D3 2000 IU D3 Sunflower oil, gelatin, glycerin, water
Vitamin E 400 IU E Soy oil, gelatin, glycerin, water, FD&C yellow #6 lake, FD&C blue #1 lake, titanium dioxide
Vitamin K1 100 µg Cellulose, CaHPO4, stearic acid, Mg stearate, croscarmellose sodium
Vitamin K2 50 µg Cellulose, Mg stearate, silica
Table 1. Fat-soluble vitamin formulations.
2Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
E X P E R IM E N TA L
System: ACQUITY UPC2
consisting of
ACQUITY UPC2 Binary
Solvent Manager,
Sample Manager,
Convergence Manager,
Column Manager,
and PDA Detector
Columns: ACQUITY UPC2 BEH,
3.0 x 100 mm, 1.7 µm
ACQUITY UPC2
HSS C18 SB,
3.0 x 100 mm, 1.8 µm
ACQUITY UPC2
HSS C18 SB,
2.1 x 150 mm, 1.8 µm
Data system: Empower 3 Software
Separation methods: Details of each
separation method
are included in the
individual results and
discussion sections of
this application note
Sample Preparation
Oil-filled capsules (vitamins A, A + D3, D3) – contents of four individual capsules
were removed and dissolved in 10 mL of iso-octane. No further pre-treatment was
used. The contents of one individual vitamin E capsule was removed and dissolved
in 10 mL of iso-octane. No further pre-treatment was used.
Eight crushed tablets of vitamin K1 were sonicated with iso-octane for
30 minutes. Following settling, an aliquot of the extract was filtered directly
into a sample vial through a 1.0-µm glass fiber filter.
Contents of eight powder-filled vitamin K2 capsules were removed and sonicated
for 30 minutes with iso-octane. Following settling, an aliquot of the extract was
filtered directly into a sample vial through a 1.0-µm glass fiber filter.
R E SU LT S A N D D IS C U S S IO N
Vitamin A
This formulation of vitamin A was labeled as derived from fish liver oil and
contained soy oil, gelatin, glycerin, and water as inactive ingredients. Two
primary forms of vitamin A palmitate (cis and trans isomers, 1.325 and 1.394
minutes, respectively) were noted and resolved well from the small excipient
peaks, as shown in Figure 1, which elute in the range of 2.0 to 2.5 minutes. This
separation was accomplished using a gradient of carbon dioxide and methanol
(containing 0.2% formic acid) 97:3 to 90:10 over 3 minutes with an Active Back
Pressure Regulator (ABPR) setting of 2176 psi. Further details are contained in
Table 2. Using this separation method, vitamin A acetate, palmitate, and retinol
were easily resolved, as seen in Figure 2.
1.32
51.
394
AU
0.00
0.10
0.20
0.30
0.40
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
trans-vitamin A palmitate
cis-vitamin A palmitate
Figure 1. UPC2 separation of the components of a vitamin A capsule.
3Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Column ACQUITY UPC2 HSS C18 SB, 3.0 x 100 mm, 1.8 µm
Flow rate 2.0 mL/min
Gradient 97:3 to 90:10 over 3 minutes
Mobile phase A/B CO2 and methanol containing 0.2% formic acid
Detection UV at 320 nm, compensated (500 to 600 nm)
Injection volume 1 µL
ABPR pressure 2176 psi
Column temp. 50 °C
Table 2. Separation method details of vitamin A.
0.57
80.
609
1.32
31.
392
1.45
0
AU
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
Vitamin A acetate
Vitamin A palmitate
Retinol
Figure 2. Separation of vitamin A acetate, vitamin A palmitate, and retinol.
4Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Vitamin A + D3
Similar to the previous example, this formulation of vitamins A + D was also labeled as derived from fish
liver oil and contained soy oil, gelatin, glycerin, and water as inactive ingredients. Again, two forms of
vitamin A palmitate (cis and trans isomers, 2.626 and 2.851 minutes, respectively) were noted before the
bulk of excipient peaks. To fully resolve vitamin D3 (cholecalciferol, 6.862 minutes) from the major excipient
materials and a number of other compounds contained in the formulation, shown in Figure 3, it was necessary
to use a longer column that provided enough separation efficiency to accomplish this goal. The system provided
enough sensitivity to easily detect the vitamin D3 peak, as shown in Figure 3 inset.
2.62
62.
851
6.86
2
AU
0.00
0.25
0.50
0.75
1.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
cis-vitamin A palmitate
trans-vitamin A palmitate
cholecalciferol
6.86
2
AU
0.000
0.006
0.012
0.018
0.024
Minutes
4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00
cholecalciferol
Figure 3. UPC2 separation of the components of a vitamin A + D3 capsule.
5Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Vitamin D3
Using identical separation conditions as those used for vitamins A + D3, as shown in Table 3, vitamin D3
(cholecalciferol, 6.867 minutes) was easily resolved from the capsule excipient material, which was labeled
as primarily sunflower oil, shown in Figure 4 and Table 3.
This separation was accomplished using a gradient of carbon dioxide and methanol (containing 0.2% formic
acid), 99:1 to 90:10 over 10 minutes. Further details are outlined in Table 3.
Column ACQUITY UPC2 HSS C18 SB, 2.1 x 150 mm, 1.8 µm
Flow rate 1.0 mL/min
Gradient 99:1 to 90:10 over 10 minutes
Mobile phase A/B CO2 and methanol containing 0.2% formic acid
Detection UV at 263 nm, compensated (500 to 600 nm)
Injection volume 1 µL
ABPR pressure 2176 psi
Column temp. 50 °C
Table 3. Separation method details of vitamin A + D3 and D3 only.
6.86
7
AU
0.000
0.012
0.024
0.036
0.048
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
cholecalciferol
Figure 4. UPC2 separation of the components of a vitamin D3 capsule..
6Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Vitamin E
A very rapid gradient analysis (~ 90 second run time) that easily provided baseline resolution of the four
tocopherol isomers (d-alpha, d-beta, d-gamma, d-delta) was developed for the vitamin E capsule, shown in
Figure 5. This separation was accomplished using a gradient of carbon dioxide and methanol, 98:2 to 95:5
over 1.5 minutes. Further details are shown in Table 4.
0.48
9
0.69
5
0.76
3
0.89
3
AU
0.00
0.26
0.52
0.78
1.04
Minutes
0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20
α-tocopherol
β-tocopherol
γ-tocopherol
δ-tocopherol
Figure 5. UPC2 separation of the components of a vitamin E capsule.
Column ACQUITY UPC2 BEH, 3.0 x 100 mm, 1.7 µm
Flow rate 2.5 mL/min
Detection UV at 293 nm, compensated (500 to 600 nm)
Gradient 98:2 to 95:5 over 1.5 minutes
Mobile phase A/B CO2 and methanol
Injection volume 1 µL
ABPR pressure 1885 psi
Column temp. 50 °C
Table 4. Separation method details of vitamin E.
7Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Vitamin K1
The vitamin K1 tablets generated two fully resolved (Rs > 2.0), distinct peaks with a simple isocratic
method consisting of 99% CO2 and 1% methanol/acetonitrile 1:1, shown in Figure 6. UV spectra (collected
simultaneously as the UV at 246 nm channel) of both peaks were similar, indicating that the peaks were
related, as displayed in Figure 7. Although not confirmed (individual standards of each of the isomers were not
available at time of analysis), it is likely that the two peaks are stereoisomers of phylloquinone (vitamin K1).
Further details are shown in Table 5.
Column ACQUITY UPC2 HSS C18 SB, 2.1 x 150 mm, 1.8 µm
Flow rate 1.5 mL/min
Isocratic 99% A and 1% B
Mobile phase A/B CO2 and methanol/acetonitrile 1:1
Detection UV at 248 nm, compensated (300 to 400 nm)
Injection volume 2 µL
ABPR pressure 1885 psi
Column temp. 50 °C
2.06
2
2.22
6
AU
0.0000
0.0026
0.0052
0.0078
0.0104
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
cis-phylloquinone
trans-phylloquinone
Figure 6. UPC2 separation of the components of a vitamin K1 tablet.
Table 5. Separation method details of vitamin K1.
8Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Figure 7. UV spectra of vitamin K peaks observed at 2.064 and 2.227 minutes.
2.064 Peak 1 246.4
317.6
393.6
AU
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
2.227 Peak 2 246.4
324.7
391.2
AU
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
nm
210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00 310.00 320.00 330.00 340.00 350.00 360.00 370.00 380.00 390.00
trans-phylloquinone
cis-phylloquinone
9Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2)
Vitamin K2
Vitamin K2 consists of menaquinone (MK) forms MK-3 through MK-14. The various forms of vitamin K2 have
side chain lengths comprised of a variable number of unsaturated isoprenoid units. This tablet formulation
showed one predominant peak and several smaller ones, as seen in Figure 8, using an isocratic separation of
95:5 CO2 /methanol, and was identified as MK-7 (data not shown). This result is consistent with the capsule
label claim, which indicated that this formulation should have contained predominantly MK-7. Further method
details are shown in Table 6.
1.388
AU
0.000
0.002
0.004
0.006
0.008
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
menaquinone-7 (MK7)
Figure 8. UPC2 separation of the components of a vitamin K2 capsule.
Column ACQUITY UPC2 HSS C18 SB, 3.0 x 100 mm, 1.8 µm
Flow rate 3.0 mL/min
Isocratic 95% A and 5% B
Mobile phase A/B CO2 and methanol
Detection UV at 248 nm, compensated (500 to 600 nm)
Injection volume 1 µL
ABPR pressure 1885 psi
Column temp. 50 °C
Table 6. Separation method details of vitamin K2.
Waters Corporation34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
CO N C LU S IO NS■■ Waters’ ACQUITY UPC2 System was able to successfully
analyze six different formulations of fat-soluble vitamins.
■■ Each of the FSV formulations were analyzed rapidly with
components of interest resolved from excipient materials.
■■ Isomers of vitamins A, E, and K1 were successfully resolved
from each other.
■■ This system can greatly streamline FSV analysis by enabling
laboratories to use a single technique on a single system
to analyze a wide range of FSV formulations.
Waters is a registered trademark of Waters Corporation. ACQUITY UPC2, UltraPerformance Convergence Chromatography, UPC2, Empower, and T he Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2012 Waters Corporation. Produced in the U.S.A. June 2012 720004394EN AG-PDF