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

Anal Biochem. 2006 May 15;352(2):282-5. doi: 10.1016/j.ab.2006.02.017. Epub 2006 Mar 9.


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.

MeSH terms

  • Adenosine Diphosphate Ribose / analysis
  • Adenosine Monophosphate / analysis
  • Animals
  • Chromatography, Liquid / methods
  • Erythrocytes / chemistry
  • HL-60 Cells
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • NAD / analogs & derivatives
  • NAD / analysis*
  • Niacin / analysis
  • Niacinamide / analysis
  • Nicotinamide Mononucleotide / analogs & derivatives
  • Nicotinamide Mononucleotide / analysis
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • NAD
  • nicotinamide arabinoside adenine dinucleotide
  • Nicotinamide Mononucleotide
  • Adenosine Diphosphate Ribose
  • Niacinamide
  • Niacin
  • nicotinate mononucleotide
  • Adenosine Monophosphate