Nicotinamide adenine dinucleotide (NAD) is a key metabolic intermediate found in all cells and involved in numerous cellular functions. Perturbances in the NAD metabolome are linked to various diseases such as diabetes and schizophrenia, and to congenital malformations and recurrent miscarriage. Mouse models are central to the investigation of these and other NAD-related conditions because mice can be readily genetically modified and treated with diets with altered concentrations of NAD precursors. Simultaneous quantification of as many metabolites of the NAD metabolome as possible is required to understand which pathways are affected in these disease conditions and what are the functional consequences.

Here, we report the development of a fit-for-purpose method to simultaneously quantify 26 NAD-related metabolites and creatinine in mouse plasma, whole blood, and liver tissue using ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). The included metabolites represent dietary precursors, intermediates, enzymatic cofactors, and excretion products. Sample preparation was optimized for each matrix and included 21 isotope-labeled internal standards. The method reached adequate precision and accuracy for the intended context of use of exploratory pathway-related biomarker discovery in mouse models. The method was tested by determining metabolite concentrations in mice fed a special diet with defined precursor content.