Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD⁺ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD⁺ and NADH, have been limited by the compound’s properties. We achieved highly sensitive simultaneous measurement of NAD⁺ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope ¹³C₅-NAD⁺ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Nicotinamide adenine dinucleotide (NAD) is a critical cofactor in mitochondrial energy production. The NADH/NAD⁺ ratio, reflecting the balance between NADH (reduced) and NAD^{+ (}oxidized⁾, is a key marker for the severity of mitochondrial diseases. We recently developed a streamlined LC-MS/MS method for the precise measurement of NADH and NAD⁺. Utilizing this technique, we quantified NADH and NAD⁺ levels in fibroblasts derived from pediatric patients and in a Leigh syndrome mouse model in which mitochondrial respiratory chain complex I subunit Ndufs4 is knocked out (KO). In patient-derived fibroblasts, NAD⁺ levels did not differ significantly from those of healthy controls (p = 0.79); however, NADH levels were significantly elevated (p = 0.04), indicating increased NADH reductive stress. This increase, observed despite comparable total NAD(H) levels between the groups, was attributed to elevated NADH levels. Similarly, in the mouse model, NADH levels were significantly increased in the KO group (p = 0.002), further suggesting that NADH elevation drives reductive stress. This precise method for NADH measurement is expected to outperform conventional assays, such as those for lactate, providing a simpler and more reliable means of assessing disease progression.