Analysis of Fat-Soluble Vitamin Capsules using ... · 1 Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2) Andrew Aubin Waters Corporation,

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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.


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.


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.


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.



Gradient 99:1 to 90:10 over 10 minutes

Mobile phase A/B CO2 and methanol containing 0.2% formic acid




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..


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



Gradient 98:2 to 95:5 over 1.5 minutes

Mobile phase A/B CO2 and methanol



Column temp. 50 °C

Table 4. Separation method details of vitamin E.


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.



Isocratic 99% A and 1% B

Mobile phase A/B CO2 and methanol/acetonitrile 1:1




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.


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


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.



Isocratic 95% A and 5% B

Mobile phase A/B CO2 and methanol




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

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Analysis of Fat-Soluble Vitamin Capsules using ... · 1 Analysis of Fat-Soluble Vitamin Capsules using UltraPerformance Convergence Chromatography (UPC2) Andrew Aubin Waters Corporation,