NADH Analysis by UPLC-UV

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. The compound is a dinucleotide, because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide. NADH exists in two forms: an oxidized form and reduced form (NAD+ and NADH), respectively. This pair of coenzyme is involved in redox reactions, carrying electrons from one reaction to another (Figure 1). NAD+ accepts electrons from other molecules to become reduced form NADH. NADH passes its protons to an acceptor to become an oxidized from NAD+. This pairs of compounds plus another pair compounds NADP+ and NADPH by accepting and donating their electrons in an electron transport chains play important roles in energy formation, storage and usage during cellular biological process of cells.

The analysis of NADH and NAD+ may be carried out by a number of different methods. The first method is to use UV-VIS spectrophotometric method. In this method, the Absorbance of the assay solution is read at 340 nm. Because NADH has a maximum absorbance at 340 nm and little and no absorbance for NAD+. Based on the reading, we can calculate NADH concentration. By adding a reducing agent to the assay to reduce NAD+ to NADH, followed reading at 340 nm again, one can determine the difference of the two readings, which is considered as NAD+. This method does not require expensive equipment and simple and fast. However, when the sample contains compounds with absorbance at 340 nm, it would over estimate NADH concentration. If the system contains redox compounds, the conversion of NAD+ to NADH will also be interfered.

The second method is using fluorescence spectrophotometric method. This is similar to the UV—VIS Spectrophotometric method but using fluorescence detection. NADH has an excitation wavelength at 340 nm and emission at 460 nm. Setting the fluorescence allows us to reduce the interference of compounds that does not have such fluorescence characteristics. The method is also relatively simple, but it is often interfered by other compounds in the sample.

The third method is to use enzyme coupled assay using dehydrogenase. In this method, a system was set-up containing sufficient dehydrogenase (i.e., alcohol dehydrogenase and substrate (i.e., alcohols): Alcohols + NAD+ ⇌ Aldehydes + NADH + H+ . By monitoring the changes at 340 nm, one can determine the abundance (concentration) of NAD+/NADH in the reaction by comparing to a reference solution with known amount of references NAD+/NADH. This method is sensitive, but it is challenging to produce consistent results.

The fourth method is HPLC (UPLC)-UV method which we used in our recent assay. If the concentration is low or matrix is complex, UPLC/MS/MS method may be used. In the dietary supplement industry, the NADH products are mostly formulated with mg of NADH per dosage. A typical UPLC-UV chromatogram is shown in Figure 2. As can be seen, it is easy to determine NADH content when compared to reference standard NADH. Our validation of this method has indicated that the UPLC-UV method is accurate with average recovery of 100%, and precise with a relative deviation of 2.6% . The method is also specific with little and no interference.