Development and Validation of a HPLC Method for NAD: Application to Stability Studies in Buffered Solutions and Dry Test Strips

Abstract

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for nicotinamide adenine dinucleotide (NAD) has been developed and validated. The method was then applied to stability studies of NAD in aqueous buffered solutions and commercial dry test strips (Optium Plus™, Abbott Laboratories) for assaying blood glucose. NAD was resolved from its decomposition products on a XTerra C18 column (150 mm × 4.6 mm, 5 µm) using a mobile phase composed of a mixture of methanol and 20 mM potassium phosphate buffer pH 8.0 (5:95, v/v), at a flow rate of 1 mL/min with UV detection at 260 nm. The chief decomposition products were identified as ADP-ribose and nicotinamide, the amount of which depended upon the identity of the buffer. The solid state stability of NAD formulated in Optium Plus™ test strips containing BES buffer at pH 7.0 was determined to be acceptable; >80% residual NAD after 3 months at 50°C.