Determination of Soluble Vitamins in Beverages

Functional beverages are vitamin-enhanced waters that are popular with consumers because of convenience, perceived health benefits, and improved flavor over tap water. These beverages, enriched with vitamin C, B-complex vitamins, and vitamins A and E, are promoted as offering the benefits of increased energy from B vitamins and antioxidant value from vitamins A, C, and E. Sales of these beverages are expected to increase to 4.4 billion liters per year by 2011.¹

Because the U.S. Food and Drug Administration (FDA) regulates how the nutritional content is listed on these beverages, vitamin assay methods are needed to support product labeling.² Determination of vitamins in foods is inherently difficult, and deviation of the determined amounts from the labeled values has been observed.³ Analysis of these waters is challenging due to the presence of both water- and fat-soluble vitamins. Proprietary formulations of vitamins that remain soluble and shelf-stable are used to enrich these beverages. Additionally, gums, preservatives, and other additives are used to emulsify and stabilize the drink.

Traditional determinations require that multiple methods be used to quantify the various vitamins added to the beverage. Water-soluble vitamins are often determined with reversed phase-high performance liquid chromatography (HPLC) using an aqueous mobile phase, while the fat-soluble vitamins use organic solvent mobile phases in both reversed- and normal-phase HPLC methods.⁴ Simultaneous determination of both types of vitamins poses a challenge due to the difference in solubility limits of the two classes of vitamins and the many different biologically equivalent compounds that can be added but are listed as a single vitamin. For example, niacin is available as nicotinic acid and nicotinamide, both of which are biologically active and referred to as niacin in product labeling.

The simultaneous determination of a wide range of vitamins increases the complexity of an analytical method. Their structures range from small unconjugated organic acids that are minimally ultraviolet (UV) active, such as pantothenic acid (vitamin B₅), to large complexes that absorb at different wavelengths, such as cyanocobalamine (vitamin B₁₂). Due to the chemical diversity of vitamins, multiple detection wavelengths are needed to optimize the method sensitivity.

click for large version

Figure 1. Separation of Vitamin Standards on the Acclaim PolarAdvantage II Column.

HPLC Method

Described below is a single method with simple sample preparation that can determine many common vitamins added to functional waters.⁵ By using a silica-based polar-embedded column compatible with 100% aqueous mobile phases over a wide pH range (1.5-10), a single gradient method can be used to quantify both water- and fat-soluble vitamins with stable retention times, excellent peak shapes, and high efficiencies for basic and acidic compounds.

Column: Acclaim PolarAdvantage II 3 µm, 2.1 x 150 mm

Gradient: Mobile phase A: 0.015% formic acid in deionized (DI) water Mobile phase B: 17/83 methanol/acetonitrile 100% A for three min, 0–45% B in five min, 45–100% B in 0.1 min, 100% B for 16.9, five min of equilibration at 100% A prior to injection

Flow Rate: 0.21 mL/min

Temperature: 40°C

Inj. Volume: 5 µL

Detection: Photodiode array detector; 210, 280, and 350 nm

Three fruit-flavored vitamin-enhanced waters were analyzed for vitamin content. Samples were diluted 1:1 with 0.015% formic acid prior to analysis.

Water-Soluble Vitamin Standards: Vitamin standards of pyridoxine HCl, nicotinic acid, nicotinamide, D-pantothenic acid, and cyanocobalamine were prepared by weighing 10 mg to 20 mg of the vitamin powder and adding DI water to a total of 10 g to 20 g to form a stock solution of one mg/mL for each individual vitamin. Water-soluble vitamin stock solutions were stored at -20°C when not in use. Working standards containing vitamins in 0.015% formic acid (mobile phase A) were prepared from these stocks on the day of use.