Neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), and Alzheimer's Disease (AD), pose significant healthcare challenges, especially with the aging population. Despite their prevalence, effective therapies for these disorders are currently unavailable. The molecular mechanisms governing these pathologies remain unclear, underscoring the imperative for research in this area. Nicotinamide adenine dinucleotide (NAD+) and its metabolites play important roles in maintaining cellular homeostasis. NAD+ is an essential coenzyme mediating various redox reactions. Particularly, mitochondrial NAD+ plays a critical role in energy production pathways, including the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and oxidative phosphorylation. NAD+ is synthesized from tryptophan, nicotinic acid (NA), and nicotinamide (NAM) through de novo, Preiss–Handler, and salvage pathways, respectively. Of these pathways, NAD+ recycling via the NAM salvage pathway is the most important in mammals [1], [2], [3], [4]. This pathway restores NAD+ levels following irreversible degradation by various NAD+-consuming enzymes, including NAD+ glycohydrolase (NADase) CD38 [5], poly (adenosine diphosphate–ribose) polymerases (PARPs) [6], sirtuins (histone deacetylases) [7], and Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) [8,9] (Fig. 1). These enzymes regulate cellular processes such as cell survival [10], transcription [11], [12], [13], apoptosis [14], [15], [16], [17], [18], DNA repair [19,20], and calcium signaling [21]. Exploring the connections between NAD+ and its metabolite levels and cellular health has revealed therapeutic applications in aging-related conditions [22], [23], [24], [25], [26]. The recently uncovered role of the NAD+ salvage pathway in axon survival [27], [28], [29], [30] is particularly critical given its involvement in neurodegenerative disorders. Therefore, accurate quantification of NAD+ and its metabolites in the salvage pathway is vital for advancing research in aging and neurodegenerative diseases.

Traditional methods for quantifying NAD+ and its metabolites, such as HPLC-UV [31], NMR [32], capillary zone electrophoresis [33], or colorimetric enzymatic assays [34], have limitations in sensitivity, selectivity, and indirect measurement. While the LC-MS/MS approach provides analytical power, it presents technical challenges for measuring NAD+ and its metabolites due to poor retention caused by high polarity and in-source fragmentation issues, yielding interfering isobaric species. For instance, NAD+ and adenosine diphosphate ribose (ADPR) overlap in the cyclic adenosine diphosphate ribose (cADPR) transition, and nicotinamide mononucleotide (NMN) appears in the nicotinamide (NAM) transition. Additionally, significant differences in concentration levels of these substances in actual test samples also pose difficulties for detection. Existing LC-MS/MS assays for cellular or tissue NAD+ and its metabolites measurements have limitations, such as extended run times [35], [36], [37], [38], [39], [40], [41], [42], [43] attributable in part to simultaneous quantitation of numerous NAD+ metabolome or relates, poor chromatographic retention behavior [40,44,45], and unsatisfactory peak shapes [36,40,41,46]. Moreover, these methods typically cover only one to three substances in the NAD+ salvage pathway outlined in this study [35,[37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51]] (Fig. 1). One reported method includes all five analytes but with limited separation for isobaric substances (ADPR and cADPR co-elute) [36]. Therefore, a pressing need exists for a rapid, sensitive, and accurate method to assess NAD+ and its metabolites levels in the NAD+ salvage pathway, enabling high-throughput analysis.

In this study, we present a robust and accurate LC-MS/MS method enabling effective baseline separation of NAD+, NMN, NAM, ADPR, and cADPR within a brief runtime of 5 min. Considering the phosphate group properties of these analytes, we systematically optimized the column and mobile phase, significantly improving sensitivity and peak shape. The method underwent full validation, demonstrating satisfactory performance, and water was confirmed as an effective surrogate matrix for the mice sciatic nerve. The method was applied to quantify NAD+ and its metabolites levels in normal and injured mice sciatic nerve, confirming that cADPR was a sensitive biomarker in the neurodegeneration model.