The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high -performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer. We found a good linear response for each NAD(+)-related compound. The limits of quantification for NAD(+) and related compounds range from 0.1 to 1 pmol. The extraction efficiency of NAD(+) and related compounds from mouse erythrocytes is between 84 and 114%. The coefficients of variation for the analyses are all less than 6%. Using our method, we measured, in a single analysis, the amounts of NMN, NAMN, NAD(+), and 5'AMP present in mouse erythrocytes. Additionally, this is the first report of a direct determination of the amounts of NMN and NAMN present in any type of cell. These results indicate that our method sensitively, specifically, and simultaneously measures cellular NAD(+) and related compounds.