Simultaneous Determination of Water- and Fat-Soluble Vitamins by HPLC

Deanna C. Hurum, Phillip DeLand, Brian M. De Borba, Deepali Mohindra, and Jeffrey S. Rohrer Dionex Corporation, Sunnyvale, CA, USA

Objectives
Develop a single-injection method with minimal sample preparation capable of determining nine vitamins frequently added to vitaminenhanced beverages. Determine both water- and fat-soluble vitamins in three consumer products with increasing complexity of sample matrix. Minimize organic solvent use by using a 2 mm column format.

intrOductiOn
Vitamin determination in complex matrixes is inherently difficult due to diverse structural motifs and disparate concentrations that typically require multiple sample preparation and analysis methods to separately determine the individual vitamins. Vitamin-enhanced functional beverages, an example of a consumer product with a complex matrix, are gaining popularity for convenience, health benefits, and improved flavor over tap water. These beverages are enriched with water- and fat-soluble vitamins, including vitamins C, B-complex, and A and E. These beverages also contain multiple sweeteners, flavorings, colors, citrates, and natural extracts, which add to complexity to the matrix.

Water-soluble vitamins determined

Vitamin Name OH Structure OH E C Ascorbic Acid O O OH O NH2 A Retinol Palminate OH Vitamin Name

Fat-soluble vitamins determined

Structure OH

DL-a-Tocopherol Acetate O

O O

B3 (Niacin)

Nicotinamide

N O B3 (Niacin) Nicotinic Acid OH

MethOdOlOgy1
Dionex UltiMate 3000 system equipped with: SRD-3200 Solvent Rack HPG-3200M pump WPS-3000TSL Micro Autosampler TCC-3200 column compartment PDA-3000 detector Mobile Phase A: 0.015% formic acid Mobile Phase B: 17/83 methanol/acetonitrile
+N H Cl-

N O HO B5 Pantothenic Acid OH N H OH O

HO B6 Pyridoxine HCl HO OH H2N O

Gradient: 100% A for 3 min, 0-45% B in 5 min, 45-100% B in 0.1 min, 100% B for 16.9 min. All separations used a flow rate of 0.21 mL/min and a 5 L sample injection volume.

H2N O H2N H3C H3C H3C O B12 Cyanocobalamine NH2 H3C N N N N C + Co N CH3

O NH2 O

Chromeleon 6.8 Chromatography Management Software was used for system control and data processing. Sample Preparation: Fruit-flavored, vitamin-enhanced water samples were prepared by diluting 500 L of beverage sample with 500 L of mobile phase A.

CH3 CH3

results and discussiOn

The Acclaim PAII column was used to separate B-complex vitamins, vitamin C, and fat-soluble vitamins in a single injection. All vitamins of interest elute within 25 minutes. Citric acid and sodium citrate were added to the standard mixture to simulate the matrix of the beverages.

CH3

H3C O O P O-

NH

O N HO H O H

NH2 CH3

O H H

CH3

HO

O O O N NH2 N N N N H N H

OH OH O

B9

Folic Acid

Simultaneous Determination of Water- and Fat-Soluble Vitamins by HPLC

Column:

Acclaim Polar Advantage II, 2.1 150 mm Eluents: A: 0.015% Formic Acid B: 17/83 Methanol/Acetonitrile Flow Rate: 0.21 mL/min Temperature: 40 C Inj. Volume: 5 L 475 4

Gradient:

2 mAU 1

100% A for 3 min, 045% B in 5 min, 45100% B in 0.1 min, 100% B for 16.9 min 5 min of equilibration Detection: PDA, 210 nm and 350 nm (inset) Sample: Vitamin Standards in mobile phase A Peaks: 1. Pyridoxine HCl 4.0 g/mL 2. Ascorbic Acid 210 3. Nicotinic Acid 42 4. Nicotinamide 41 5. Citric Acid 240 5. Sodium Citrate 130 6. D-Pantothenic Acid 21 7. Cyanocobalamine 4.0 8. Folic Acid 4.1 9. DL--Tocopherol Acetate 3.8 10. Retinol Palmitate < 0.4 2.0 9 0.1 19 20 10 25 25
a

linearity and precisiOn OF standard injectiOns

Correlation coefficients for standard curves ranged from 0.9985 to 0.9997. Peak Area RSDs for standards ranged between 0.28 and 3.47.

saMple analysis
Three samples differing in matrix were analyzed using the proposed method. Brand A: an artificially sweetened fruit flavored vitamin-enhanced water. Brand B: a sugar sweetened fruit flavored vitamin-enhanced water. Brand C: a sugar sweetened, caffeinated, vitamin-enhanced water with additional natural extracts.

7 6 8 10 15

50

A mobile phase blank has been subtracted from the chromatogram displayed to remove baseline shifts due the gradient.
25991

Minutes

Figure 1. Separation of vitamins on the Acclaim Polar Advantage II column.

vitaMin detectiOn Wavelength OptiMizatiOn

With the exception of Vitamin A, as retinol palmitate, 210 nm allows detection of each vitamin of interest. However, detection of these compounds at 210 nm is nonspecific. Some vitamins are more easily quantified at longer wavelengths due to reduced interferences. These include: Vitamin B6, as pyridoxine HCl; 280 nm Folic Acid; 280 nm Vitamin C absorbs strongly at 254 nm, however 210 nm is preferred to simplify data collection. Vitamin A absorbs at 350 nm.

table 1. linearity, lOd, and lOQ of vitamins determined

analyte Pyridoxine HCl Pyridoxine HCl Ascorbic Acid Nicotinic Acid Nicotinamide D-Pantothenic Acid Cyanocobalamine Folic Acid DL--Tocopherol Acetate Retinol Palmitate detection Wavelength (nm) 210 280 210 210 210 210 210 280 210 350 correlation coefficient (r2) 0.9994 0.9997 0.9992 0.9995 0.9995 0.9993 0.9995 0.9987 0.9985 0.9996 range (g/ml) 1.020 1.020 15300 5.0100 5.0100 5.0100 1.020 0.252.5 1.225 0.6312 lOd (ng/ml) 20 20 5000 84 35 213 14 20 83 125 lOQ (ng/ml) 60 60 5000 250 100 640 40 60 250 400 retention time precision (rsd)* n=7 0.10 0.10 0.07 0.07 0.08 0.23 0.09 0.16 0.14 0.17 peak area precision (rsd)* n=7 0.28 0.75 3.47 0.40 0.54 0.75 0.83 0.97 0.77 0.54

*Analyte concentrations for precision injections: Pyridine HCl (5 g/mL), Ascorbic Acid (77 g/mL), Nicotinic Acid, Nicotinamide, and Pantothenic Acid (25 g/mL each), Cyanocobalamine (5 g/mL), Folic Acid (1.3 g/mL), DL--Tocopherol Acetate (6.2 mg/mL), and Retinol Palmitate (1.3 g/mL).

Column: Eluents:

750

2 mAU 1 A 1 B 20
a

4 5

Acclaim Polar Advantage II, 2.1 150 mm A: 0.015% Formic Acid B: 17/83 Methanol/Acetonitrile Flow Rate: 0.21 mL/min Temperature: 40 C Inj. Volume: 5 L Gradient: 100% A for 3 min, 045% B in 5 min, 45100% B in 0.1 min, 100% B for 16.9 min 5 min of equilibration Detection: PDA, 210 nm (A) and 280 nm (B) Sample: Brand A fruit-flavored water 1:1 dilution with eluent A Peaks: 1. Pyridoxine HCl (280 nm) 3.1 g/mL 2. Ascorbic Acid 67 3. Nicotinamide 19 4. Citrates 6 5. D-Pantothenic Acid 12 6. DL--Tocopherol Acetate 8.6

Column: Eluents:

400 2

mAU 3 4 A 1 B 40 0 5 10 Minutes

Acclaim Polar Advantage II, 2.1 150 mm A: 0.015% Formic Acid B: 17/83 Methanol/Acetonitrile Flow Rate: 0.21 mL/min Temperature: 40 C Inj. Volume: 5 L Gradient: 100% A for 3 min, 0-45% B in 5 min, 45100% B in 0.1 min, 100% B for 16.9 min 5 min of equilibration Detection: PDA, 210 nm (A), 280 nm (B), and 350 nm (inset) Sample: Brand C fruit-flavored water 1:1 dilution with eluent A Peaks: 1. Pyridoxine HCl (280 nm) 4.6 g/mL 2. Nicotinamide 10 3. Citrates 5 4. D-Pantothenic Acid 6.0 5. DL--Tocopherol Acetate 5.3 6. Retinol Palmitate (inset) 0.44
6 4 0
a

A mobile phase blank has been subtracted from the chromatogram displayed to remove baseline shifts due the gradient.

10

Minutes

15

20

25
25992

15

20

25

Baseline corrected, 15% shift in signal intensity between A and B

25993

Figure 2. Separation of Brand A, a fruit-flavored, artificially-sweetened, vitaminenhanced water.a

Figure 4. Separation of Brand C, a fruit-flavored, sugar-sweetened, vitaminenhanced water with added natural extracts and caffeine.*

Column: Eluents:

425

2 7 3

Acclaim Polar Advantage II, 2.1 150 mm A: 0.015% Formic Acid B: 17/83 Methanol/Acetonitrile Flow Rate: 0.21 mL/min Temperature: 40 C Inj. Volume: 5 L Gradient: 100% A for 3 min, 0-45% B in 5 min, 45-100% B in 0.1 min, 100% B for 17 min 5 minutes of equilibration Detection: PDA, 210 nm (A) and 280 nm (B) Sample: Brand B fruit flavored water 1:1 dilution with eluent A Peaks: 1. Pyridoxine HCl 3.2 g/mL 2. Ascorbic Acid 76 3. Nicotinamide 9.4 4. Citrates 5. D-Pantothenic Acid 5.1 6. Folic Acid 0.27 7. DL--tocopherol acetate 15

table 2. sample analysis precision, n=3

sample vitamin Pyridoxine HCl Ascorbic Acid Brand A Nicotinamide D-Pantothenic Acid Vitamin E Pyridoxine HCl Ascorbic Acid Brand B Nicotinamide D-Pantothenic Acid Folic Acid Vitamin E peak area precision (rsd) 0.32 6.1 0.65 0.95 0.74 0.89 1.2 0.59 1.7 1.3 1.3 0.47 1.27 0.79 1.21 1.56 amount (g/ml) 3.1 67 19 12 8.6 3.2 72 9.4 5.1 0.25 15 2.2 10 6.0 5.3 0.44

mAU

A 1 B 25 0 5

Pyridoxine HCl Nicotinamide

10 Minutes

15

20

25

Brand C

D-Pantothenic Acid Vitamin E Vitamin A (retinol palmitate)

* Baseline corrected, 17% shift in signal intensity between A and B 26178

Figure 3. Separation of Brand B, a fruit-flavored, artificially-sweetened, vitaminenhanced water.a

Simultaneous Determination of Water- and Fat-Soluble Vitamins by HPLC

vitaMin c, FOlic acid, and precisiOn

Ascorbic acid (AA) exists in solution in equilibrium with the oxidation product dehydroascorbic acid (DHAA). Both compounds are biologically active as Vitamin C. DHAA does not have a strong UV absorption and is difficult to quantify. The oxidation reaction is reversible, and is minimized at low pH or by adding a reducing agent.2 Folic acid, with a lowest pKa of 4.7, precipitates from solution at low pH. Precision of ascorbic acid (vitamin C) is low due to oxidation processes that have been minimized, but not prevented, by lowering the pH while avoiding folic acid precipitation.
sample

table 4. recovery values for vitamins

vitamin amount Found in sample (g/ml) 3.1 66 <LOD 19 12 <LOD <LOD 8.6 <LOD 3.2 72 <LOD 9.4 5.1 <LOD 0.25 15 <LOD 2.2 <LOD <LOD 10 6.0 <LOD <LOD 5.3 0.44 amount added (g/ml) 7.6 250 7.5 7.5 5.0 1.0 0.25 7.9 1.0 7.6 250 7.5 7.5 5.0 1.0 0.25 7.9 1.0 7.6 250 7.5 7.5 5.0 1.0 0.25 7.9 1.0 % recovery

Pyridoxine HCl Ascorbic Acid Nicotinic acid Nicotinamide Brand A D-Pantothenic Acid Cyanocobalamine Folic Acid Vitamin E Retinol Palmitate Pyridoxine HCl Ascorbic Acid Nicotinic Acid Nicotinamide Brand B D-Pantothenic Acid Cyanocobalamine Folic Acid Vitamin E Retinol Palmitate

103 119 99 106 110 93 118 * * 111 118 101 115 102 93 106 * * 101 114 94 100 104 94 115 * *

between-day precision
Between-day peak area precision for analysis of three samples ranged from 0.37% to a maximum of 9.5%.

table 3. interday precision (n=3) for selected vitamins

sample Brand A vitamin Pyridoxine HCl Ascorbic Acid Brand B Nicotinamide Vitamin E Folic Acid Brand C D-Pantothenic Acid Retinol Palmitate between-day average amount (g/ml) 3.2 71 9.5 15 0.24 6.0 0.44 between-day precision (rsd) 1.16 9.3 1.75 0.37 9.52 0.61 4.11 Brand C

Pyridoxine HCl Ascorbic Acid Nicotinic acid Nicotinamide D-Pantothenic Acid Cyanocobalamine Folic Acid Vitamin E Retinol Palmitate
*See text for discussion on recovery values for these vitamins.

accuracy
Accuracy was demonstrated by measuring recoveries for the water- and fat-soluble vitamins, which ranged from 93% to 119% for all three samples analyzed.

cOnclusiOns
This method enables the determination of water- and fat-soluble vitamins with a single injection in <25 min (Figure 1). These results show the suitability of the developed method to determine vitamins in complex matrixes, such as vitamin-enhanced beverages (Figure 3).

reFerences
1. Dionex Corporation. Determination of Water- and Fat-Soluble Vitamins in Functional Waters by HPLC with UV-PDA Detection, Application Note 216, LPN 2145. Sunnyvale, CA, 2009. 2. Margolis, Sam A.; Duewer, David L.; Measurement of Ascorbic Acid in Human Plasma and Serum: Stability, Intralaboratory Repeatability, and Interlaboratory Reproducibility, Clinical Chemistry, 1996, 42, 1257-1262.

Acclaim, Chromeleon, and UltiMate are registered trademarks of Dionex Corporation.

Passion. Power. Productivity.

dionex corporation 1228 Titan Way P.O. Box 3603 Sunnyvale, CA 94088-3603 (408) 737-0700
north america europe asia pacific

U.S./ Canada (847) 295-7500

south america Simultaneous Determination of Water- and Fat-Soluble Vitamins by HPLC

Brazil (55) 11 3731 5140

Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722

Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190 Taiwan (886) 2 8751 6655

www.dionex.com

LPN 2310-01 9/09 2009 Dionex Corporation