Method Migration of a Normal Phase HPLC Method for Tocopherols in Dietary Supplements

Normal phase HPLC is a valuable technique for the separation of polar and hydrophilic compounds and for isomer separation. Normal phase HPLC requires a polar stationary phase combined with non or moderately polar mobile phases. Normal phase HPLC is frequently used for the analysis of compounds that have limited water solubility. These compounds can be extracted directly into non-polar solvents and analyzed making the sample preparation compatible with normal phase. In contrast, for reversed phase (RP) separation these compounds require additional, often time-consuming sample preparation steps including evaporation and reconstitution into a solvent that is compatible with reversed phase separations.

Normal phase and reversed phase chromatography can often be performed on the same HPLC system however some system modifications may be required to convert systems in the standard configuration to the normal phase configuration. This is due to the incompatibility of materials of construction with normal phase solvents.
Specifically, hardware modifications (e.g., check valves, tubing, pump seals) may be required. Additionally, a solvent changeover process is needed when switching between modes to avoid solvent immiscibility issues.

Vitamin E is a name assigned to a class of fat-soluble compounds that exhibit distinctive antioxidant properties. There are four tocopherols (alpha, beta, gamma, delta) that display vitamin E activity. Dietary supplements from natural sources contain a distribution of these four tocopherols, however alpha-tocopherol is the only form that is recognized to meet human nutrition requirements.

A normal phase method for the determination of tocopherols in dietary supplements was developed. The method was successfully run on two legacy HPLC systems (System 1=Waters Alliance e2695 System; System 2=System Y from a different vendor). The method was then successfully migrated to a modern system, the Alliance iS HPLC System. The criteria for successful method migration was defined as the ability to meet the established system suitability requirements of the method. Dietary supplement samples were prepared and analyzed on the Alliance iS HPLC System and the results compared against the label claims.