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.
Yamada
Kazuo
K
Department of Biochemistry, Faculty of Medicine, Shimane University, Izumo 693-8501, Japan. yamadak@med.shimane-u.ac.jp
Hara
Nobumasa
N
Shibata
Tomoko
T
Osago
Harumi
H
Tsuchiya
Mikako
M
D000246:Adenosine Diphosphate Ribose / Q000032:analysis; D000249:Adenosine Monophosphate / Q000032:analysis; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009243:NAD / Q000031:analogs & derivatives / Q000032:analysis*; D009525:Niacin / Q000032:analysis; D009536:Niacinamide / Q000032:analysis; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives / Q000032:analysis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009243:NAD; C053851:nicotinamide arabinoside adenine dinucleotide; D009537:Nicotinamide Mononucleotide; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D009525:Niacin; C002953:nicotinate mononucleotide; D000249:Adenosine Monophosphate