Rapid quantitation of NAD⁺/NADH and NADPH/NADP⁺ with mass spectrometry by using calibration constants

NAD⁺/NADH and NADPH/NADP⁺ are important indicators of cellular redox status, but they cannot be reliably calculated from the relative intensities of mass spectrometry signals alone. Establishing accurate redox ratios by mass spectrometry has historically required converting relative signal intensities into absolute concentrations, which is a time-consuming process that limits rapid analysis. Here, we describe a simpler strategy to determine NAD⁺/NADH and NADPH/NADP⁺ by using mass spectrometry. While ionization is strongly influenced by factors such as instrument drift and sample type, we discovered that the relative signal intensities of oxidized and reduced cofactors change at the same rate across experiments performed on the same mass spectrometer. That rate can be experimentally determined and expressed as a calibration constant. Using calibration constants, relative intensities of mass spectrometry signals can be rapidly transformed into accurate redox ratios without the use of authentic standards or isotopically labeled internal standards. We present a metabolomics workflow to measure NAD⁺/NADH and NADPH/NADP⁺ by using calibration constants and compare the results to other methods, including commercial colorimetric assays. Although colorimetric assays are the most widely used, we demonstrate that mass spectrometry quantitation with calibration constants provides more accurate results.