2.5. LC-MS/MS method for NADmetabolites

Separation of the NAD⁺ metabolites was accomplished using a C-18 Security guard cartridge (2.1 × 4 mm) and an Accucore HILIC column (2.1 × 150 mm, 2.6 μm, Thermo) at 32 °C. Mobile phase A consisted of ammonium acetate [7.5 mM, pH 7.86] and mobile phase B was acetonitrile. The following linear gradient was run for 14.0 min at a flow rate of 0.4 ml/min: 0–1 min 90% B, 1.5 min 72.5% B, 2.5 min 67.5% B, 8.0 min 20% B, 10 min 20% B, 10.1 min 90% B. Calibration curves were prepared in buffered ethanol by a 0.5 serial dilution of standards from 20 μg/ml to 0.156 μg/ml for NAD⁺; 10 μg/ml to 0.078 μg/ml for NADP; 8 μg/ml to 0.0625 μ/ml for NADPH; 5 μg/ml to 0.039 μg/ml for AcCoA; 4 μg/ml to 0.03125 μg/ml for NADH; 2.5 μg/ml to 0.019 μg/ml for NR; 2 μg/ml to 0.015625 μg/ml for NAAD, NMN, NAMN, ADPR; 1 μg/ml to 0.0078 μg/ml NAMN, NA. The injection volume per sample was 10 μL. Samples were kept at 4 °C in the autosampler tray prior to injection.

Relative values for the metabolites were determined using area ratios of the targeted metabolites and the corresponding internal standard using the following heavy standards: C¹³-NAD, C¹³-NADH, C¹³-ADPR, C¹³-AcCoA (Suppl Table 1b). Calibration curves were carried out in standard solutions. Matrix effects were accounted for in each targeted matrix by adjusting calculated levels based on three quality controls (low, middle, high). All studies were carried out with a daily calibration curve and quality controls as well as a pooled sample from the entire study for each matrix.

Data were acquired using a Nexera XR HPLC (Shimadzu) coupled with a QTRAP 5500 (SCIEX) and were analyzed with Analyst 1.6 (SCIEX). The positive ion mode data was obtained using multiple reaction monitoring (MRM). The instrumental source setting for curtain gas, ion spray voltage, temperature, ion source gas 1, ion source gas 2 were 30 psi, 5500 V, 500 C, 65 psi, and 55 psi, respectively. The collision activated dissociation was set to medium and the entrance potential was 10 V. The standards (Supplemental Table 1A) and internal standards (Supplemental Table 1B) were characterized using the MRM ion transitions, declustering potentials (DP), collision energies (CE), and collision cell exit potentials (CXP) listed in Supplemental Table 1.