Separation, Identification, and Quantitation of Water-Soluble Vitamins by HPLC - - Chromatography Online
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Separation, Identification, and Quantitation of Water-Soluble Vitamins by HPLC
Mar 1, 2009
By: Karyn M. Usher, Anna Glinko, Michael J. Bozym, Michelle L. Owens
LCGC North America

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The accurate measurement of vitamins in conventional dietary supplement tablets and dietary supplement drinks that are consumed widely by the general public is of utmost importance. Vitamins are necessary for proper body function, and consuming too little or too much can be harmful to an individual's health. This makes it imperative that values reported for the amounts of vitamins in these supplements are measured accurately. Dwyer and colleagues (2) have shown that while analytically validated information on the vitamin content in food is relatively complete, the same information for dietary supplements is not. According to the authors, the differences between label values and actual content of vitamins in dietary supplements have not been studied extensively. Labels are required to reflect minimum content of nutrients according to U.S. regulatory requirements, so the actual nutrient content often is higher than stated. The amount of the overage is dependent upon several factors, including the cost of the vitamin and its stability.


Figure 1
Many methods, including USP methods (3) currently used for the separation and quantitation of water-soluble vitamins use mobile phase additives including ion-pair reagents and EDTA. Ion-pair reagents are used as mobile phase additives to allow successful separation of polar substances on reversed-phase high performance liquid chromatography (HPLC) columns. They improve resolution and peak shape, particularly by reducing tailing. However, their use can result in irreversible changes in column performance due to adsorption of the ion-pair reagent onto the stationary phase (4), resulting in either a change in selectivity of the column or longer equilibration times. Through the elimination of ion-pair reagents, the proposed method avoids this damage to the chromatographic column. In addition, the simplified mobile phase lowers the cost of analysis and avoids interferences with the separation and problems with detection during gradient elution.

Experimental


Table I
Materials: Analytical grade vitamin standards were purchased from Sigma-Aldrich (Milwaukee, Wisconsin). See Figure 1 for structures. Water (EMD Chemicals, Gibbstown, New Jersey) and methanol (Burdick and Jackson, Muskegan, Mchigan) were HPLC grade. Sodium dihydrogen phosphate, ultrapure bioreagent grade, was purchased from J.T.Baker, Phillipsburg, New Jersey, and phosphoric acid, HPLC grade, was purchased from Fisher Scientific, Pittsburgh, Pennsylvania.


Table II
Instrumentation: An Agilent 1200 SL Rapid Resolution liquid chromatograph with a binary pump, autosampler, inline degasser, temperature-controlled column compartment, and an 80 Hz diode-array detector (Agilent Technologies, Inc., Santa Rosa, California) were used for the analysis. The detector flow cell selected for this study was a micro flow cell with a 2-ľL volume. Sample peaks were identified positively by two methods: by matching retention times and by comparison of UV spectra from a spectral database created with this instrument. Spectra also were used to determine the peak purity. Chemstation for LC 3D Systems, Rev. B.03.01, was used for data collection and analysis.

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Source: LCGC North America, 3/1/2009
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