A Scientific Method for Quantifying NMN in Biological Samples
1. IntroductionSupplementation of nicotinamide mononucleotide (NMN) to upregulate the level of nicotinamide adenine dinucleotide (NAD+) has been unveiled to be a promising anti-aging intervention. However, it is still a serious challenge to accurately quantify NAD+ intermediates, NMN in particular. This study is powered to introduce a novel method, double isotope-mediated LC-MS/MS methodology (dimeLC-MS/MS), for the precise quantification of NMN in biological samples.
2. Factors affecting the accurate detection of NMNNMN is hard to be accurately detected due to its vulnerability to enzymatic degradation, conversion in sample processing, its complex behaviors in different column and extraction conditions, as well as matrix effect.
Specifically, NMN has the properties of high polarity and low volatility, which is easy to dissolve in water but difficult to dissolve in organic solvents. These properties greatly restrict the application of many conventional quantitative analysis methods.
Biological samples such as blood carries significant activities of CD38 and CD73 (ecto-5’-nucleotidase), both of which could use NMN as a substrate.
The behavior of NMN in the column is very complex probably because of the bipartite nature of its charges so that subtle differences in extraction and column conditions significantly affect the reliable and accurate detection of NMN.
3. Coping strategies of dimeLC-MS/MS to reduce the impact of impact factorsTo avoid the interference of above-mentioned factors, a prototype column NMN-2 is applied. This column contains C18-based high-purity silica particles which are more capable of binding hydrophilic compounds than carbon particles, improving the ability of separation.
Perchloric acid (PCA) is employed since it can efficiently extract NAD+ and NMN from biological samples such as plasma with minimal losses.
To adjust for matrix effects, a fixed amount (1 μM) of each isotopic compound is added to biological samples prior to the PCA extraction.
4. The advantages of dimeLC-MS/MSDouble isotopic NMN standards, NMN (M + 14) and NMN (M + 5), in the LC-MS/MS-driven methodology can precisely trace the fate of NMN during sample processing, which significantly increases the accuracy and the reliability of NMN measurement in biological samples. Besides, dimeLC-MS/MS can evaluate the extraction efficiency and the absolute concentrations of NMN in different types of biological samples.
5. ConclusionThis new LC-MS/MS-driven methodology with double isotopic NMN standards can accurately and reliably measure NMN in biological samples. It can be used for future studies on NMN intake.
6. ReferenceUnno, Junya et al. “Absolute quantification of nicotinamide mononucleotide in biological samples by double isotope-mediated liquid chromatography-tandem mass spectrometry (dimeLC-MS/MS).” npj aging vol. 10,1 2. 2 Jan. 2024, doi:10.1038/s41514-023-00133-1
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