Measurement method for nicotinamide mononucleotide (NMN) and its related metabolites

Abstract

The present disclosure is generally directed to methods and kits for measuring nicotinamide mononucleotide (NMN) and its related metabolites in biological samples. The disclosure is further directed to methods for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample. The methods use liquid chromatography-tandem mass spectrometry (LC-MS / MS)-driven techniques and two isotopic NMN standards. These methods and kits can be used to study nicotinamide adenine dinucleotide (NAD+) metabolism and aging.

Description

[Technical Field]

[0001] This disclosure provides methods and kits for measuring nicotinamide mononucleotide (NMN) and its related metabolites in biological samples. The methods use liquid chromatography-tandem mass spectrometry (LC-MS / MS) and two isotopic NMN standards. These methods and kits can be used to study nicotinamide adenine dinucleotide (NAD+) metabolism and aging. [Background technology]

[0002] It has been reported that aging causes physiological declines in organ and tissue function, which may also lead to aging-related diseases. In recent years, it has been demonstrated that a systemic decline in nicotinamide adenine dinucleotide (NAD+), an essential "currency" for energy metabolism, is a significant contributor to aging. Therefore, nicotinamide mononucleotide (NMN), a key NAD+ intermediate that can enhance NAD+ biosynthesis, has attracted attention from both the scientific community and the general public.

[0003] NAD+ is a classical coenzyme for many fundamental redox reactions and a substrate for NAD+-consuming enzymes, including poly (ADP-ribose) polymerase (PARP), sirtuins, cyclic ADP-ribose hydrolase or Cluster of Differentiation 38 (CD38) or ADP-ribosyl cyclase 2 (CD157), and Sterile Alpha and Toll / Interleukin Receptor 1 (TIR) ​​Motif Containing 1 (SARM1), thus playing a crucial role in numerous important biological processes, including metabolism, DNA damage response, inflammation, cancer, neurodegeneration, and aging. Systemic NAD+ depletion has been demonstrated to be an important contributor to age-related tissue dysfunction and disease. Therefore, supplementation of NAD+ intermediates, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), has attracted significant attention from both the scientific community and the general public as a promising anti-aging treatment.

[0004] NAD+ is synthesized from three main precursors: tryptophan, nicotinic acid (NA), and nicotinamide (NAM), as well as two intermediates: NMN and NR. The primary biosynthetic pathway of NAD+ in mammals begins with NAM, catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). NAMPT converts NAM and 5'-phosphoribosylpyrophosphate (5'-PRPP) to NMN, which is then converted to NAD+ by NMN adenylyltransferase 1-3 (NMNAT1-3). NR must be phosphorylated to NMN by NR kinases 1 and 2 (NRK1 and 2) to enter the primary biosynthetic pathway of NAD+. Although the biochemical characteristics of key NAD+ biosynthetic enzymes have been well characterized, precise measurement of NAD+-related metabolites, such as NMN and NR, has been challenging due to the susceptibility of these compounds to enzymatic degradation, their changes during sample processing, and their complex behavior under different column and extraction conditions. Highly quantitative techniques for measuring NAD+ and NMN levels in biological samples using high-performance liquid chromatography (HPLC) have previously been established and applied to mouse and human samples. However, because NMN levels are typically much lower than NAD+ levels, quantifying NMN in biological samples has been controversial for many years.

[0005] Measuring the levels of NAD+ and its related metabolites has been a major challenge in the field, and therefore, it is extremely important to accurately and reliably measure the amount of NAD+ and its related compounds, especially NMN, in biological samples. Until now, quantifying the precise amount of NMN in biological samples has been difficult because protein removal and detection methods have not been optimized for biological samples. Therefore, the effectiveness of NMN as an anti-aging treatment for preventing and treating age-related diseases could not be accurately evaluated. The object of the present invention is to provide a highly accurate and reliable technique for measuring NAD+ and its related compounds in biological samples.

[0006] Several groups have used proprietary techniques based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect and measure NMN in biological samples. However, a comprehensive evaluation of the extraction method, recovery efficiency, and the influence of the sample's complex biochemical context, known as the matrix effect, has not been performed. In this study, we investigated and evaluated these issues and successfully developed an LC-MS / MS-driven technique using a dual-isotope NMN standard (dimeLC-MS / MS) for precise and reliable quantification of NMN in biological samples. Summary of the Invention

[0007] The present disclosure provides a method for detecting nicotinamide mononucleotide (NMN) having the structure of Formula 1 in a sample from a subject, comprising adding a first stable isotope NMN standard to the sample, pretreating the sample, adding a second stable isotope NMN standard to the sample, and detecting the NMN and the standards with a mass spectrometer, wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard, and the structure of Formula 1 is [ka] provides a method equivalent to

[0008] The present disclosure further provides a method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, the method comprising: adding a first stable isotope NMN standard to the sample; pre-treating the sample; adding a second stable isotope NMN standard to the sample; measuring the NMN and the standards; and calculating the recovery efficiency of the first stable isotope NMN standard in the sample based on the measured compounds in the sample, wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard.

[0009] The present disclosure is also directed to a kit for detecting nicotinamide mononucleotide (NMN) in a sample from a subject, the kit comprising a first stable isotope NMN standard and a second stable isotope NMN standard, the first stable isotope NMN standard having a relative mass difference relative to the second stable isotope NMN standard.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although the present invention can be practiced using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned in this application are incorporated by reference in their entirety. In the case of conflicts, the present specification, including definitions, will control. It should be noted that the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0011] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0012] Figures 1A-D depict the isotopic compounds used in LC-MS / MS analysis. [Figure 1A] Figure showing the structure of stable isotope compounds used in LC-MS / MS analysis. [Figure 1B] Chromatograms of a normal compound (top) and a stable isotope compound (bottom). These normal compounds and isotope compounds were separated using an NMN-2 column developed by Shimadzu Corporation. The total ion count (TIC) obtained from multiple reaction monitoring (MRM) of each compound is shown. [Figure 1C]Chromatograms and mass spectra of normal NMN and isotopic NMN. TICs and transitions were obtained as chromatograms from MRM of normal NMN and isotopic NMN (left figures), and the resulting product ions are shown as mass spectra (right figures). [Figure 1D] Calibration curves for NMN and other related compounds. The area under the curves (AUC) of each common compound at different concentrations (15-1500 nM for NMN, NAD+, and NA, 21-2100 nM for NAM, and 0.6-60 nM for NR) were measured by LC-MS / MS in the presence of a fixed amount of the corresponding isotopic compound (1000 nM for NMN, NAD+, and NA, 2000 nM for NAM, and 200 nM for NR) and normalized to the AUC of the respective isotopic internal standard.

[0013] Figures 2A-D depict the adjustment for matrix effects and measurement of NMN in mouse plasma samples. [Figure 2A] Figure 1 shows the matrix effect of mouse plasma on the AUC of NMN, NAD+, NAM, and NA. Standard solutions of NMN, NAD+, NAM, and NA at concentrations of zero, 50, 100, or 500 nM were combined with PCA extract from mouse plasma at concentrations of 1%, 10%, or 50%. Error bars indicate the standard error of the mean (SEM) obtained from triplicate injections into LC-MS / MS. [Figure 2B] Figure showing the adjustment for matrix effects by the internal standard method using stable isotope compounds. Typical compounds were spiked into mouse plasma at a final concentration of 1 μM. Recovery efficiencies were calculated using the external standard method (exSTD) or the internal standard method (iSTD). Averages were calculated from three independent experiments. Data are presented as mean ± SEM. [Figure 2C] Figure 1 shows a comparison of HPLC-based and LC-MS / MS-based techniques for measuring NMN. Mouse plasma was spiked with NMN at the indicated concentrations. The same extract from the spiked plasma was measured by HPLC (y-axis) and LC-MS / MS (x-axis). [Figure 2D]Figure showing NMN levels in mouse plasma after intraperitoneal injection of NMN. 3-4 month old C57BL / 6J mice (n=10) were given 100 mg / kg NMN via intraperitoneal injection. Plasma was extracted by PCA, and NMN was measured by both HPLC-based and LC-MS / MS-based techniques. Statistical analysis was performed by repeated-measures one-way ANOVA with Bonferroni's multiple comparisons post hoc test. ** indicates p<0.01.

[0014] Figures 3A-E show the double isotope-mediated LC-MS / MS technique (dimeLC-MS / MS) for measuring NMN. [Figure 3A] A diagram showing the structure of NMN(M+14) with stable isotopes. [Figure 3B] Figure 1 shows a schematic flow of dimeLC-MS / MS for NMN measurement. [Figure 3C] Chromatogram, mass spectrum, and calibration curve for NMN (M+14). The TIC and transitions obtained from the MRM of NMN (M+14) were plotted on the chromatogram (left), and the corresponding product ions are shown as mass spectra (middle). The calibration curve for NMN (M+14) was plotted after normalizing its AUC to that of NMN (M+5). [Figure 3D] Figure showing the recovery efficiency of contaminated NMN (M+14) and regular NMN in dimeLC-MS / MS. NMN (M+14) and regular NMN at the indicated concentrations were spiked into mouse plasma. Each plasma sample was extracted by PCA and analyzed by dimeLC-MS / MS. Recovery efficiency was calculated using the iSTD method. Results were obtained from three independent experiments. [Figure 3E]Figure showing NMN levels in whole blood or plasma of mice after intraperitoneal injection of NMN. Three- to four-month-old C57BL / 6J mice (n=10) were given NMN (100 mg / kg) intraperitoneally. Each sample was extracted by PCA and measured for NMN by dimeLC-MS / MS. For each sample type (whole blood or plasma), control data are shown in the left bar, and 100 mg / kg NMN-treated data are shown in the right bar. Two-way ANOVA was performed and results were compared with Bonferroni's multiple comparison post-hoc test. Data are shown as mean ± SEM. * indicates p<0.05.

[0015] Figures 4A-C show a comparison of PCA and MeOH extraction methods for NMN and other metabolites. [Figure 4A] Figure 1 shows the recovery efficiency of spiked NMN (M+14) and regular NMN after PCA extraction and MeOH / chloroform extraction. Mouse plasma was spiked with NMN (M+14) and regular NMN at the indicated concentrations. Plasma samples were extracted using PCA or MeOH / chloroform, and each extract was analyzed by dimeLC-MS / MS. Recovery efficiency was calculated using the iSTD method. Averages were obtained from three independent experiments. PCA results for spiked NMN concentrations are shown in the left columns, and MeOH results are shown in the right columns. Two-way ANOVA was performed and results were compared using Bonferroni's multiple comparison post-hoc test. Data are presented as mean ± SEM. ** indicates p<0.005, *** indicates p<0.001. [Figure 4B] Comparison of PCA and MeOH extraction methods for primary metabolites in plasma. Plasma samples were extracted by PCA or MeOH / chloroform, and 96 primary metabolites were analyzed using the Primary Metabolite Method Package Ver. 2. The average area ratios obtained from three independent experiments were plotted on a Volcano Plot. [Figure 4C]Area ratios of 10 representative compounds are shown for PCA extraction and MeOH / chloroform extraction. An unpaired Student's t-test was performed to compare the results. Data are shown as mean ± SEM. * indicates p<0.05, ** indicates p<0.01.

[0016] Figures 5A and B show precise quantification of NMN uptake into AML12 cells. [Figure 5A] This figure shows the dose-dependent increase in NMN (M+14) absorption into AML12 cells 1 hour after adding NMN to the culture medium. NMN (N+14) was added to the culture medium of AML12 cells at the indicated concentrations. One hour after adding NMN, the absolute amounts of regular NMN (left panel, left side of the figure), representing the endogenous NMN pool, and NMN (N+14) (right panel, left side of the figure), representing NMN directly transported into AML12 cells, were quantified using dimeLC-MS / MS. The ratio of transported NMN (M+14) to the endogenous NMN pool (regular NMN) was calculated (right panel). Results were obtained from three independent experiments. One-way ANOVA was performed with Bonferroni's multiple comparison post-hoc test. Data are shown as mean ± SEM. * indicates p<0.05, ** indicates p<0.01. [Figure 5B] Figure showing the time course of NMN absorption into AML12 cells. AML12 cells were treated with 200 μM NMN (M+14) for 2 hours. The absolute amounts of normal NMN (endo-NMN, upper data) and NMN (N+14) (M+14 NMN, lower data) were quantified by dimeLC-MS / MS at 10, 30, 60, and 120 minutes after NMN addition. Results were obtained from three independent experiments. Data are shown as mean ± SEM.

[0017] [Figure 6]Figure showing the increase in NMN in mouse plasma after oral gavage of 300 mg / kg NMN. Male C57BL / 6J mice aged 5–6 months (young, n=9) and 24–25 months (old, n=7) were orally administered 300 mg / kg NMN. Blood was collected from the tail vein at the indicated time points. Plasma was extracted by PCA and analyzed by dimeLC-MS / MS. In the bottom panel, data from young mice are shown on the left and data from old mice are shown on the right for each time point after oral gavage. Statistical analysis was performed using a two-way repeated measures ANOVA with Bonferroni's multiple comparison post-hoc test. * indicates a p<0.05 between young and old mice at the 5-minute time point. # indicates a p<0.05 between the 0-minute and 5-minute time points in young mice. DETAILED DESCRIPTION OF THE INVENTION

[0018] Nicotinamide adenine dinucleotide (NAD + ) is an essential metabolite for fundamental biological phenomena, including aging. Nicotinamide mononucleotide (NMN) is an effective NAD metabolite in mice and humans. + Important NAD that has been extensively tested as a booster compound + It is an intermediate. However, precise measurement of NMN in biological samples has long been a challenging task in the field. In this study, a precise quantitative method for measuring NMN was established using mass spectrometry (MS) with two isotopic NMN standards. This method appropriately adjusted for the matrix effect of biological samples and enabled tracking of the pathway of NMN during sample processing. This method enabled precise quantification of NMN levels in mouse plasma, confirming the rapid and direct absorption of NMN into cultured cells.

[0019] In particular, LC-MS / MS via these two isotopes (dimeLC-MS / MS) + Other NAD as a reliable standard technique for biology + It can be easily extended to related metabolites.

[0020] The unique features and advantages over existing techniques are as follows:

[0021] 1) To evaluate extraction efficiency, two stable isotope-labeled NMNs are used. By adding a known amount of the first stable isotope-labeled NMN to a biological sample, the amount of NMN degraded during the extraction process can be assessed, confirming extraction efficiency. The second stable isotope-labeled NMN is then added to the extract and used as a normalization standard to quantify the first stable isotope-labeled NMN. This technique allows for precise quantification of NMN levels in different biological samples and for evaluating differences in sample measurement accuracy between various techniques. The actual amount of NMN present in a biological sample can be determined by quantifying endogenous NMN and the first stable isotope-labeled NMN.

[0022] 2) The combination of acid extraction and LC-MS also ensures the accuracy of this technique. Typically, organic solvents are used to extract metabolites from biological samples for LC-MS analysis because inorganic salts are harmful to the interface chamber of a mass spectrometer. In HPLC-based measurements, a relatively large amount of sample extract is required to detect NMN due to the low sensitivity of UV detectors. However, in this technique, most of the inorganic salts can be removed by neutralization and centrifugation, and with the advancement of technology, the sensitivity of mass spectrometry has now become much better, allowing the detection of NMN (or other NAD+-related metabolites) with just a small injection. Therefore, strong acid-mediated extraction can be used for LC-MS.

[0023] The present disclosure provides a method for detecting nicotinamide mononucleotide (NMN) having the structure of Formula 1 in a sample from a subject, comprising adding a first stable isotope NMN standard to the sample, pretreating the sample, adding a second stable isotope NMN standard to the sample, and detecting the NMN and the standards with a mass spectrometer, wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard, and the structure of Formula 1 is [ka] This is intended for a method equivalent to the above.

[0024] The method can further include separating compounds in the sample by liquid chromatography prior to the detecting step. The detecting step can include ionizing the NMN and the standard from the sample to generate NMN ions, first stable isotope NMN standard ions, and second stable isotope NMN standard ions that are detectable by mass spectrometry, and measuring the amounts of the NMN ions, first stable isotope NMN standard ions, and second stable isotope NMN standard ions by mass spectrometry.

[0025] The mass spectrometry may be tandem mass spectrometry.

[0026] The NMN ions detectable by mass spectrometry can include precursor ions having mass-to-charge ratios between 335.20±0.5 and 349.10±0.5 and fragment ions selected from the group of ions having mass-to-charge ratios (i) between 123.05±0.5 and 128.10±0.5 and (ii) between 80.00±0.5 and 84.05±0.5.

[0027] The NMN ions detectable by mass spectrometry can include a precursor ion having a mass-to-charge ratio of 335.20±0.5 and fragment ions selected from the group of ions having mass-to-charge ratios of 123.05±0.5 and 80.05±0.5.

[0028] The first stable isotope NMN standard ion detectable by mass spectrometry can include a precursor ion having a mass-to-charge ratio of 349.10±0.5 and a fragment ion selected from a group of ions having mass-to-charge ratios of 128.10±0.5 and 84.05±0.5.

[0029] The second stable isotope NMN standard ion detectable by mass spectrometry can include a precursor ion having a mass-to-charge ratio of 340.10±0.5 and a fragment ion selected from the group of ions having mass-to-charge ratios of 123.05±0.5 and 80.00±0.5.

[0030] Detecting the NMN and the standard can include determining the amount of NMN based on the amount of the NMN ions, the first stable isotope NMN standard ions, or the second stable isotope NMN standard ions. Preferably, detecting the NMN and the standard can include determining the amount of NMN based on the amount of the NMN ions, the first stable isotope NMN standard ions, and the second stable isotope NMN standard ions.

[0031] The nicotinamide group, ribose, or phosphate group of the second stable isotope NMN standard can be substituted with a stable isotope atom.

[0032] The ribose of the second stable isotope NMN standard can be substituted with a stable isotope atom. 13 Any of the carbon atoms in the ribose moiety of the second stable isotope NMN standard can be substituted with carbon-13 ( 13 Preferably, two or more of the carbon atoms in the ribose moiety of the second stable isotope NMN standard are substituted with carbon-13 ( 13 C) It is assumed to be substituted by an atom.

[0033] In particular, the second stable isotope NMN standard [ka] It can have the following structure.

[0034] The nicotinamide group, ribose, or phosphate group of the first stable isotope NMN standard can be substituted with a stable isotope atom. Preferably, the nicotinamide group, ribose, and phosphate group of the first stable isotope NMN standard are substituted with a stable isotope atom.

[0035] The nicotinamide group of the first stable isotope NMN standard is converted to carbon-13 ( 13 Any of the carbon atoms of the nicotinamide group of the first stable isotope NMN standard can be substituted with carbon-13 ( 13 C) can be substituted for atoms.

[0036] The ribose portion of the first stable isotope NMN standard is carbon-13( 13 Any of the carbon atoms in the ribose moiety of the first stable isotope NMN standard can be substituted with carbon-13 ( 13 Preferably, all of the carbon atoms in the ribose moiety of the first stable isotope NMN standard can be substituted with carbon-13 (C) atoms. 13 C) It is assumed to be substituted by an atom.

[0037] The phosphate group of the first stable isotope NMN standard is converted to oxygen 18( 18 Preferably, two of the oxygen atoms in the phosphate group of the first stable isotope NMN standard can be replaced with oxygen-18 (O) atoms. 18 O) atom is substituted.

[0038] In addition, the nitrogen atom of the first or second stable isotope NMN standard is changed to nitrogen-15( 15 Preferably, the nitrogen of the nicotinamide group of the first stable isotope NMN standard is substituted with nitrogen 15 ( 15 N) atoms are substituted.

[0039] The hydrogen atoms of the first or second stable isotope NMN standard are replaced with deuterium ( 2 H).

[0040] In particular, the first stable isotope NMN standard [ka] It has the following structure.

[0041] The sample pretreatment may include removing soluble proteins from the sample, which may be removed by treating the sample with an acid, which may be perchloric acid (PCA).

[0042] The sample pretreatment may include adding a reagent that forms an ion pair upon reaction with perchloric acid (PCA). The pretreatment may include removing the formed ion pair. The reagent may include potassium hydroxide, potassium borate, potassium formate, potassium acetate, potassium citrate, potassium carbonate, ammonium sulfate, ammonium chloride, rubidium sulfate, cesium hydroxide, or thallium acetate.

[0043] The sample can be whole blood, plasma, tissue, or cultured cells. The subject can be a human.

[0044] The present disclosure is also directed to a method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, the method comprising: adding a first stable isotope NMN standard to the sample; pretreating the sample; adding a second stable isotope NMN standard to the sample; measuring the concentrations of NMN, the first stable isotope NMN standard, and the second stable isotope NMN standard; and using the concentration of the first stable isotope NMN standard in the sample to calculate the recovery efficiency based on the measured concentration of the second stable isotope NMN standard in the sample, wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard. The method may further comprise determining the effectiveness of the pretreatment based on the calculated recovery efficiency.

[0045] The present disclosure also relates to a kit for detecting nicotinamide mononucleotide (NMN) in a sample from a subject, the kit comprising a first stable isotope NMN standard and a second stable isotope NMN standard, wherein the first stable isotope NMN standard has a relative mass difference from the second stable isotope NMN standard. The ribose of the second stable isotope NMN standard can be substituted with a stable isotope atom. The nicotinamide group, ribose, and phosphate group of the first stable isotope NMN standard can be substituted with a stable isotope atom.

[0046] Isotopes are two or more atoms that have the same atomic number (the number of protons in the nucleus) and position in the periodic table (and therefore belong to the same chemical element), but have different numbers of neutrons in their nuclei, and therefore different nucleon numbers (mass numbers). There are two types of isotopes: radioactive isotopes and stable isotopes or elements. Isotopes that do not emit radiation are called stable isotopes.

[0047] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0048] While the present invention has been described in connection with its detailed description, it should be understood that the foregoing description is intended to illustrate but not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Example 1 Column and isotope preparation

[0049] There are several steps that can lead to inaccurate measurements for quantifying NMN and NR. For example, differences in sample collection and extraction techniques significantly affect the measurement of NAD+ intermediates. While precise measurement of NAD+ levels is possible in HPLC using a C18 column, porous graphite carbon columns and hydrophilic columns work much more effectively to detect and measure NMN. In mass spectrometry, the ionization efficiency of NMN and its related metabolites varies significantly and depends on the biochemical context of sample extraction. This is called the matrix effect. Therefore, all of these issues must be addressed to achieve precise quantitative measurement of NMN and its related metabolites.

[0050] A column capable of robust, stable, and reproducible retention of NMN has been developed. This column, Prototype NMN-2, features octadecyl functional groups on fully end-capped silica particles and a nonmetallic column surface to prevent adsorption of certain compounds. As a result, it offers improved performance for separating and detecting low-concentration compounds, particularly phosphate-containing compounds such as NMN and NAD+. Next, to adjust for the matrix effect of biological extracts, stable isotope compounds were synthesized for NAD+ (M+5), NMN (M+5), NR (M+10), NAM (M+5), and NA (M+4) (Figure 1A). These isotope compounds had the same retention time and relative mass differences compared to the standard compounds (Figure 1B and C). Normalization of the standard curves for each standard compound to its corresponding isotope revealed precise linearity, although slight deviations from linearity were observed for high-concentration NR (Figure 1D). Example 2 : Quantification of NMN in mouse plasma samples after NMN administration

[0051] When HPLC-based techniques were developed for the measurement of NAD+ and NMN, the use of perchloric acid (PCA), a very strong acid, was crucial for efficient extraction of NAD+ and NMN from biological samples such as plasma with minimal loss. Mass spectrometry-based measurements also require the ability to recognize and adjust for matrix effects. We investigated the matrix effects of PCA-extracted mouse plasma by spiking plasma extracts at 1%, 10%, and 50% concentrations of various NAD+, NMN, NAM, and NA using common compounds (Figure 2A). Interestingly, the area under the curve (AUC) for NAD+ increased, whereas the AUC for NMN decreased. For example, the AUC for NAD+ at 500 nM in a 50% concentration of mouse plasma extract increased by 185.6%, whereas the AUC for NMN at 500 nM in a 50% concentration of mouse plasma extract decreased by only 57.0%. This suggests that mouse plasma extracts have opposite matrix effects on NAD+ and NMN. Similar to NMN, mouse plasma extracts also reduced the AUC for NAM and NA (Fig. 2A).

[0052] To adjust for such matrix effects, a fixed amount (1 μM) of each isotopic compound was added to mouse plasma extracts before PCA extraction. Also, 1 μM of each normal compound was spiked into the same plasma sample before PCA extraction. After extraction, the levels of each spiked normal compound were quantified using a standard curve with each normal compound (external standard method, exSTD) or by normalizing to each internal isotopic standard (1 μM) and then comparing to a standard curve also normalized to the internal isotopic standard (iSTD). Recovery rates were then calculated (compared to the original 1 μM of each spiked normal compound). If matrix effects are properly adjusted by normalizing to the internal isotopic standard, the expected recovery rate should be 100%. The recovery efficiencies of NMN, NAD+, NAM, and NA reflected varying matrix effects when measured by exSTD (Figure 2B, left). However, as expected, after adjusting for matrix effects by iSTD, the values ​​showed nearly 100% recovery (Figure 2B, right). These results strongly suggest that adjustment for each matrix effect by normalization to each internal isotope standard is crucial for the accuracy of the measurements. On the other hand, although the recovery of NR was only 89.1% even after adjustment, adjustment for the matrix effect significantly improved the accuracy of the measurements (Figure 2B). This is because PCA extraction affected the elution pattern of NR, resulting in a change in the ratio between normal NR and isotopic NR. At present, the reason for the change in the elution pattern of NR is still unknown.

[0053] Plasma samples spiked with different concentrations of NMN were then measured using a mass spectrometry-based method, adjusting for matrix effects using iSTD, and a previously developed HPLC-based method (Yoshino, J. & Imai, S. Methods Mol. Biol. 1077, 203-215, 2013). Remarkably, the values ​​obtained were nearly identical for both mass spectrometry-based and HPLC-based measurements (Figure 2C). This confirms the importance of PCA extraction and the precision of both methods. Furthermore, NMN concentrations in mouse plasma were measured 5 minutes after intraperitoneal (IP) injection of 100 mg / kg NMN. The NMN values ​​measured by both techniques were compared. Interestingly, after NMN administration, both values ​​were in good agreement, with concentrations ranging from 2–3 μM to 20 μM among individual mice (Figure 2D). These results confirm the extremely rapid absorption of NMN into the blood circulation, as reported in the original report. Furthermore, the HPLC-based and mass spectrometry-based techniques yielded very different values ​​for endogenous NMN levels in mouse plasma (before NMN administration). The values ​​for endogenous NMN levels in mouse plasma obtained by the HPLC-based technique were much higher than those obtained by mass spectrometry. The reason for this discrepancy is currently under investigation. Example 3 : Absolute quantification of NMN levels in biological samples

[0054] The nearly 100% recovery efficiency of NMN (Figure 2B) strongly suggests that this particular sample collection and extraction procedure did not degrade or alter NMN to related compounds such as NAM and NR. However, NMN is generally susceptible to enzymatic degradation or alteration during sample collection and extraction. Therefore, it is crucial to be able to monitor and control such NMN degradation and alteration processes throughout sample collection and extraction. Therefore, we monitored these processes using another isotope of NMN, namely NMN(M+14) (Figure 3A). By adding NMN(M+14) immediately after collection of the biological sample, we were able to monitor the pathway of NMN throughout the entire sample handling process (Figure 3B). After PCA extraction, NMN(M+5) was also added to adjust for matrix effects (Figure 3B). NMN(M+14) has the same retention time and a relative mass difference from normal NMN, and its measurement showed precise linearity (Figure 3C). By using these two different isotopic NMN compounds and assessing the ratio of NMN (M+14) to NMN (M+5), we were able to calculate the precise concentration of NMN (M+14) and, therefore, the precise recovery efficiency of PCA extraction. To demonstrate this, we spiked mouse plasma with 2.5 μM NMN (M+14) before PCA extraction, and then added 500 nM NMN (M+5) after extraction to adjust for matrix effects (Figure 3D). We then investigated the recovery efficiency of 2.5 μM NMN (M+14) and 1 μM, 10 μM, and 40 μM regular NMN in PCA extraction. Again, the recovery efficiency was nearly 100% (95.3%–99.1%) for both NMN (M+14) and regular NMN (Figure 3D). This clearly supports the importance of PCA extraction.

[0055] To further demonstrate the advantages of this two-isotope technique, we measured NMN concentrations in two different biological samples, mouse whole blood and plasma, which may have different matrix effects. In both samples, NMN concentrations were quantified 5 min after intraperitoneal injection of 100 mg / kg NMN. 2.5 μM NMN (M+14) was added to mouse whole blood or plasma before PCA extraction, and 500 nM NMN (M+5) was added to each extract. Again, after adjusting for the matrix effects of whole blood and plasma, the extraction efficiency of NMN (M+14) approached 100% for both whole blood and plasma samples obtained from control and NMN-treated mice (Figure 3E, left). While NMN concentrations in whole blood extracts did not differ significantly between control and NMN-treated mice, NMN concentrations in plasma extracts showed a significant increase in NMN-treated mice (Figure 3E, right). Again, there was a large variability in plasma NMN concentrations after intraperitoneal injection. These results strongly suggest that although degradation and changes of NMN were minimal during sample processing in both whole blood and plasma, plasma samples separated immediately after blood collection should be used to clearly detect increases in NMN after NMN administration. In summary, this technique using two isotopic NMN standards is highly advantageous for precisely monitoring the pathways of NMN and assessing the extraction efficiency and absolute concentration of NMN in different types of biological samples. Example 4 : Comparison of PCA extraction method and MeOH extraction method

[0056] Methanol (MeOH) is commonly used in mass spectrometry-driven metabolomic analyses to extract as many metabolites as possible. However, it remains unclear whether this MeOH-based extraction method is suitable for measuring NMN and its related metabolites. Using our dual-isotope mediated LC-MS / MS (dimeLC-MS / MS) technique, we compared the recovery efficiencies between PCA and MeOH. Again, PCA extraction yielded nearly 100% recovery, whereas MeOH extraction yielded only about 70% recovery for 1 μM, 10 μM, and 40 μM NMN (M+14) and normal NMN (Figure 4A). This suggests that MeOH-based extraction methods are not suitable for precise measurement of NMN.

[0057] Next, we investigated whether similar differences existed for other metabolites between the PCA and MeOH extraction methods. For this evaluation, mouse plasma samples were extracted using either PCA or MeOH / chloroform, spiked with 100 μM 2-morpholinoethanesulfonic acid (MES) as an internal standard. Metabolite analysis was performed using the Primary Metabolite Method Package Ver. 2, which can detect and analyze 96 primary metabolites (see Materials and Methods). Interestingly, the metabolites fell into two groups: those better extracted by PCA and those better extracted by MeOH (Figure 4B). NAM, NA, nucleosides (cytosine, thymidine, and uridine), and cyclic nucleotides (cAMP and cCMP) belonged to the PCA group, while amino acids (serine and lysine) and ornithine belonged to the MeOH group (Figure 4C). These results strongly suggest that the use of PCA extraction methods rather than MeOH-based extraction methods is crucial for accurate measurement of NAD precursors and intermediates in biological samples. Example 5 : Quantitative measurement of direct uptake of NMN into cells

[0058] Finally, we quantitatively measured the direct uptake of NMN into cultured cells using the dimeLC-MS / MS technique. The mouse hepatocyte cell line AML12 was selected because it has been reported that Slc12a8, a recently discovered NMN transporter, is relatively well expressed in AML12 cells. NMN (M+14) was added to the medium at a final concentration of 100 μM or 300 μM, and then attomoles (10 atmole) were measured at 1 hour after NMN addition. -18 Absolute levels of regular NMN and NMN(M+14) were calculated by measuring the AUC in units of (mole) / cell. During calculation, all measured AUCs for regular NMN and NMN(M+14) were normalized to NMN(M+5). Interestingly, the endogenous NMN pool (regular NMN) was approximately 5 attomoles / cell, and was not significantly affected by treating cells with 100 μM or 300 μM NMN for 1 hour (Figure 5A, left). However, dose-dependent direct absorption of NMN(M+14) was clearly detected (Figure 5A, left), and the extent of this direct absorption reached up to 30% of the endogenous NMN pool (Figure 5A, right). Next, we examined the time course of NMN absorption by adding 200 μM NMN(M+14) to the culture medium. NMN was rapidly transported into the cells at 10 min, and NMN levels steadily increased to approximately 10% of the endogenous NMN pool over 2 hours (Figure 5B). On the other hand, the endogenous NMN pool remained largely unchanged, implying that NMN was immediately available for intracellular use (Figure 5B). These results clearly demonstrate that the dimeLC-MS / MS technique allows for precise quantitative measurement of NMN absorption and confirm that NMN can be directly transported into cells without being degraded to NAM or converted to NR. Example 6 Measurement of plasma levels of NMN, nicotinamide (NAM), nicotinic acid (NA), and nicotinamide riboside (NR) after oral administration of NMN to young and old mice.

[0059] The results of this experiment are shown graphically in FIG.

[0060] Using this mass spectrometry-driven technology, named dimeLC-MS / MS, we successfully measured changes in plasma levels of NMN, nicotinamide (NAM), nicotinic acid (NA), and nicotinamide riboside (NR) after oral administration of 300 mg / kg of NMN to young and old mice.

[0061] Although a clear increase in NMN was detectable at 5 minutes in both young and old mice, the increase was much smaller in the old mice, and the increase in the old mice did not reach statistical significance.

[0062] On the other hand, NAM showed very similar and significant increases over 15 min in both young and old mice, whereas NA and NR showed no significant changes.

[0063] These results strongly suggest that 1) NMN can be rapidly transported from the intestine to the blood circulation (within 5 minutes), 2) the main degradation product of NMN is NAM, and no conversion of NMN to NR occurs within 15 minutes, and 3) NMN absorption is significantly reduced in aged mice.

[0064] These results also provide strong evidence that this technique, dimeLC-MS / MS, can be meaningfully applied to biological samples such as plasma. Example 7 Overview of biological applications

[0065] Quantitative measurement of NAD+ intermediates, especially NMN, has long been a significant challenge in the field of NAD+ biology. One reason is that NMN can easily be degraded or converted to other related metabolites, such as NAM and NR, during collection and processing of biological samples. Blood is perhaps the most challenging sample to handle because it hosts the important activities of CD38 and CD73 (ecto-5'-nucleotidases), both of which can utilize NMN as a substrate. Another reason is that the behavior of NMN in the column is highly complex, likely due to the bipartite nature of its charge, and subtle differences in extraction and column conditions significantly affect the reliability and accuracy of NMN detection. To overcome these challenges, an HPLC-driven technique was previously developed to measure NMN levels in biological samples. This method was used to evaluate the pharmacokinetics of NMN after oral administration in mice, demonstrating that absorption of NMN from the intestine into the circulation occurs within 2–3 minutes and transport into tissues occurs within 10–30 minutes. This surprising research result led to the discovery of the NMN transporter Slc12a8 20. However, due to the difficulty in precisely measuring NMN, intense debate arose as to whether NMN could be directly transported into cells or the blood circulation. In this study, these difficulties were overcome and a precise and reliable LC-MS / MS technique (dimeLC-MS / MS) using two isotopic NMN standards was successfully developed.

[0066] The advantages of dimeLC-MS / MS are twofold. First, we confirmed that the recovery efficiency of NMN and other NAD+-related metabolites was approximately 70% with MeOH-chloroform extraction, whereas immediate PCA extraction achieved nearly 100% recovery. Therefore, in this method, matrix effects were adjusted by adding an isotopic NMN standard, i.e., NMN (M+5), to the biological sample even after extraction. Adjusting for matrix effects is crucial because the AUC of NMN decreased by approximately 40% in mouse plasma extracts. The dimeLC-MS / MS method can appropriately adjust for matrix effects and quantify the NMN levels in biological sample extracts. Second, adding a second isotopic standard, i.e., NMN (M+14), to the biological sample immediately after sample collection allowed us to accurately track the NMN pathway during sample processing. Furthermore, by calculating the ratio of NMN(M+14) to NMN(M+5), the exact concentration of NMN(M+14) can be calculated, which allows for accurate calculation of the recovery efficiency of any extraction method. Therefore, the use of two isotopic NMN standards, NMN(M+14) and NMN(M+5), in mass spectrometry-driven techniques significantly increases the accuracy and reliability of NMN measurements in biological samples.

[0067] Two important biological results were obtained during the development of this method.

[0068] First, 5 minutes after intraperitoneal injection of NMN, a significant increase in plasma NMN levels was detected, whereas no increase in NMN levels was detected in whole blood extracts. Therefore, it is crucial to assess plasma NMN levels rather than whole blood NMN levels, and it is clear that NMN can be rapidly transported into the systemic circulation. Importantly, at least after NMN administration, the results obtained by the previously developed HPLC-driven technique were very similar to those obtained by the LC-MS / MS-driven technique after appropriate adjustment for matrix effects. This convincingly corroborates the results from the two independent techniques.

[0069] Second, using this dimeLC-MS / MS technique, we found that NMN was directly transported into AML12 cells within 10 minutes, and that the level of the transported NMN increased by up to 30% of the endogenous NMN pool. This clearly indicates that a significant amount of NMN can be directly transported into cells without being degraded to NAM or converted to NR. Similar results were previously obtained in primary hepatocytes, where this direct transport of NMN was almost completely abolished in hepatocytes lacking Slc12a8. While the previous study was unable to calculate the absolute amount of transported NMN, this study allowed us to precisely quantify the absolute levels of the endogenous NMN pool and the NMN transported into cells. The fact that NMN transported up to 30% of the endogenous NMN pool without appreciable changes in the endogenous pool strongly implies that NMN must be converted to NAD+ or other metabolites immediately upon transport into cells. Therefore, dimeLC-MS / MS will also be effective in investigating the precise dynamics and pathways of NMN and NAD+-related metabolites in cells or tissues.

[0070] In conclusion, we successfully developed an LC-MS / MS-driven technique using a biisotopic NMN standard, named dimeLC-MS / MS, and demonstrated its precision and reliability in measuring NMN in biological samples. dimeLC-MS / MS accurately measured plasma NMN levels after intraperitoneal injection in mice and provided absolute quantification of the amount of NMN directly transported into cells. The dimeLC-MS / MS method will open up many interesting opportunities for assessing the kinetics of NMN uptake and NAD+ biosynthesis in different metabolic conditions and tissues. Example 8 Materials and Methods Chemicals and Reagents

[0071] NMN was a gift from Mirai Lab Biosciences Inc. (Tokyo, Japan). NAD+ (#N1511), NR (#SMB00907), NAM (#72340), and NA (#N0761) were purchased from Sigma (USA). [C5]-NMN (#C7934), [C9,15N]-NR (#C7990), and [H4]-NA (#C2885) were purchased from Alsachim (France). Other isotopic compounds were custom-synthesized by Alsachim. Mouse experiments

[0072] C57BL / 6J mice were group-housed in an SPF mouse facility under a 12-hour light / 12-hour dark cycle. All mice were fed a normal diet (PicoLab 5053 Rodent Diet 20; Lab Diets) ad libitum. Blood samples were collected from the tail vein using microhematocrit heparinized capillary tubes (Fisher Scientific), and plasma was separated immediately after blood collection. NMN was administered by intraperitoneal (IP) injection. All animal experiments were approved by the Washington University Animal Studies Committee and in accordance with the guidelines of the National Institutes of Health. Perchloric acid (PCA) extraction of mouse plasma samples

[0073] Plasma was isolated from mouse whole blood by centrifugation at 6,500 rpm for 7 minutes at 4°C in a microcentrifuge. The plasma was then mixed with ice-cold 10% PCA solution containing a stable isotope compound as an internal standard and incubated on ice for 15 minutes. After centrifugation at 21,500 × g for 5 minutes at 4°C, the supernatant was separated and neutralized with ice-cold 3 M K2CO3 solution at a 1:3 ratio. The mixture was then incubated on ice for 15 minutes with the lid open. The resulting salt was pelleted by centrifugation at 21,500 × g for 5 minutes at 4°C. The resulting supernatant (final extract) was analyzed using a high-performance liquid chromatography triple quadrupole mass spectrometer system (LC-MS / MS, LCMS-8060, Shimadzu Corporation). The concentrations of NMN and other related metabolites in the extracts were quantified by comparing the area under the curve (AUC) of each to a standard curve (normalized by a standard isotope compound) and back-calculating by normalizing for dilution and plasma volume. MeOH chloroform extraction

[0074] 200 μL of MeOH containing the internal standard was added to 20 μL of mouse plasma and mixed thoroughly for several minutes. 200 μL of chloroform was then added and mixed using a vortex mixer. 80 μL of LCMS-grade pure water was added and mixed thoroughly. The mixture was centrifuged at 21,500 × g for 15 minutes at 4 °C. 200 μL of the upper aqueous phase was transferred to a new tube and completely dried in a speed-vac. Metabolites were dissolved in 100 μL of LCMS-grade pure water and measured by LC-MS / MS (LCMS-8060, Shimadzu Corporation). LC-MS / MS analysis

[0075] LC-MS / MS analysis was performed using a Nexera X2 Ultra HPLC system connected to a triple quadrupole mass spectrometer (LCMS-8060, Shimadzu Corporation). During analysis, samples were held in an autosampler at 4 °C, and 2 μL of each sample was injected onto a Prototype Column NMN-2 (150 × 2.0 mm, 2.2 μm particle size, Shimadzu Corporation). The column temperature was maintained at 21 °C. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% formic acid. Chromatographic separation was performed at a flow rate of 0.2 mL / min using a gradient elution time program consisting of 1% B (0–2 min), 1–38.6% B (2–10 min), 95% B (10.01–12 min), and 1% B (12.01–15 min). NMN and NAD+ typically eluted at 3.5 and 7.5 min, respectively.

[0076] Metabolites were detected by electrospray ionization (ESI) in positive mode using single reaction monitoring, also known as multiple reaction monitoring (MRM). The target cycle time was set to 0.4 seconds, with a 1-millisecond pause between each transition. The dwell time per transition was automatically set to 10–15 milliseconds. Other mass spectrometer parameters were: interface voltage 3.5 kV, nebulizer gas flow rate 3 L / min, heating gas flow rate 10 L / min, drying gas flow rate 10 L / min, interface temperature 300°C, desolvation channel temperature 200°C, heating block temperature 350°C, and collision-induced dissociation gas pressure 270 kPa. All data were processed using LabSolutions software (Shimadzu Corporation). Metabolomic analysis

[0077] Metabolites were extracted with PCA or MeOH / chloroform with the addition of 100 μM 2-morpholinoethanesulfonic acid (MES) as an internal standard. Metabolites were analyzed using an LC-MS / MS (LCMS-8060, Shimadzu) with the Primary Metabolite Method Package Ver. 2 (Shimadzu). Area ratios were calculated by dividing the AUC of each target metabolite by the AUC of the internal standard. Cell culture and drug treatment

[0078] AML12 cells were obtained from ATCC (American Type Culture Collection) and maintained in DMEM / F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin (Life Technologies), 40 ng / mL dexamethasone, 0.005 mg / mL insulin (Insulin-Transferrin-Selenium [ITS-G], #41400045, Gibco), 0.005 mg / mL transferrin, and 5 μg / mL selenium. Dexamethasone and ITS were removed from the AML12 medium between drug treatments. For NMN absorption experiments, 8 × 10 5 AML12 cells were plated in 6 cm dishes and treated with 0.5 μM 78c (CD38 inhibitor) and 50 μM adenosine-5'-(α,β-methylene)diphosphate (AOPCP, CD73 inhibitor) for 16 hours. Then, 2 μM dipyridamole (ENT inhibitor) and 100 nM FK-866 (NAMPT inhibitor) were added to the medium for 1 hour. After treatment with all inhibitors, stable isotope NMN (M+14) was added to the AML12 cells at the indicated concentrations without removing the inhibitors. Cells were harvested with trypsin-EDTA solution and frozen at -30°C until analysis. To measure cellular uptake of NMN, the cell pellet was resuspended in LC / MS-grade pure water containing stable isotope NMN (M+5) and extracted with perchloric acid as described above. statistical analysis

[0079] All data are presented as the mean or mean ± SEM. Statistical significance between control and experimental samples was determined using an unpaired Student's t-test. Statistical significance of differences between multiple NMN samples was analyzed by one-way, two-way, or one-way repeated measures analysis of variance (ANOVA) using Bonferroni's multiple comparison test as a post-hoc test. A p-value of <0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism (Ver. 9.4.1).

[0080] When introducing elements of the present invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are inclusive and mean that there may be additional elements other than the listed elements.

[0081] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0082] Since various changes can be made in the above-described structures and methods without departing from the scope of the invention, all matter contained in the above description and shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

1. 1. A method for detecting nicotinamide mononucleotide (NMN) having the structure of Formula 1 in a sample from a subject, comprising: adding a first stable isotope NMN standard to said sample; pre-treating the sample; adding a second stable isotope NMN standard to the sample; and detecting the NMN and the standard with a mass spectrometer (MS), wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard, and the structure of Formula 1 is 【Transformation 5】 The equivalent method.

2. 10. The method of claim 1, further comprising separating compounds in the sample by liquid chromatography (LC) prior to detecting the NMN and standards by MS.

3. detecting the NMN and standard by MS; ionizing the NMN and the standard from the sample to generate NMN ions, first stable isotope NMN standard ions, or second stable isotope NMN standard ions detectable by mass spectrometry; and measuring the amount of the NMN ions, first stable isotope NMN standard ions, or second stable isotope NMN standard ions by mass spectrometry; 3. The method of claim 1 or 2, comprising:

4. detecting the NMN and standard by MS; ionizing the NMN and the standard from the sample to generate NMN ions, first stable isotope NMN standard ions, and second stable isotope NMN standard ions detectable by mass spectrometry; and measuring the amounts of the NMN ions, first stable isotope NMN standard ions, and second stable isotope NMN standard ions by mass spectrometry; 4. The method of claim 3, comprising:

5. The method according to any one of claims 1 to 4, wherein the mass spectrometry is tandem mass spectrometry (MS / MS).

6. 6. The method of claim 3, wherein the NMN ions detectable by mass spectrometry comprise precursor ions having mass-to-charge ratios between 335.20±0.5 and 349.10±0.5 and fragment ions selected from the group of ions having mass-to-charge ratios (i) between 123.05±0.5 and 128.10±0.5 and (ii) between 80.00±0.5 and 84.05±0.

5.

7. 7. The method of claim 3, wherein the NMN ions detectable by mass spectrometry comprise a precursor ion having a mass-to-charge ratio of 335.20±0.5 and fragment ions selected from the group of ions having mass-to-charge ratios of 123.05±0.5 and 80.05±0.

5.

8. 8. The method of claim 3, wherein the first stable isotope NMN standard ion detectable by mass spectrometry comprises a precursor ion having a mass-to-charge ratio of 349.10±0.5 and a fragment ion selected from the group of ions having mass-to-charge ratios of 128.10±0.5 and 84.05±0.

5.

9. 8. The method of claim 3, wherein the second stable isotope NMN standard ions detectable by mass spectrometry comprise a precursor ion having a mass-to-charge ratio of 340.10±0.5 and a fragment ion selected from the group of ions having mass-to-charge ratios of 123.05±0.5 and 80.00±0.

5.

10. 10. The method of any of claims 1 to 9, wherein detecting the NMN and standard by MS comprises determining the amount of the NMN based on the amount of the NMN ion, the first stable isotope NMN standard ion, or the second stable isotope NMN standard ion.

11. 11. The method of claim 10, wherein detecting the NMN and standard by MS comprises determining the amount of the NMN based on the amounts of the NMN ions, the first stable isotope NMN standard ions, and the second stable isotope NMN standard ions.

12. 12. The method of any one of claims 1 to 11, wherein a nicotinamide group, a ribose, or a phosphate group of the second stable isotope NMN standard is substituted with a stable isotope atom.

13. 13. The method of claim 12, wherein the ribose of the second stable isotope NMN standard is substituted with the stable isotope atom.

14. The ribose of the second stable isotope NMN standard has at least one carbon-13 ( １３ The method according to claim 12 or 13, wherein the C) atom is substituted.

15. One carbon atom of the ribose of the second stable isotope NMN standard is carbon-13 ( １３ The method of claim 14, wherein the C) atom is substituted.

16. 16. The method of any one of claims 1 to 15, wherein a nicotinamide group, a ribose, or a phosphate group of the first stable isotope NMN standard is substituted with a stable isotope atom.

17. 17. The method of claim 16, wherein the nicotinamide group, ribose, and phosphate group of the first stable isotope NMN standard are substituted with stable isotope atoms.

18. The nicotinamide group of the first stable isotope NMN standard is carbon-13 ( １３ The method according to claim 16 or 17, wherein the C) atom is substituted.

19. The ribose of the first stable isotope NMN standard is carbon-13 ( １３ The method according to any one of claims 16 to 18, wherein the substituted C) atom is a C) atom.

20. Two or more of the carbon atoms of the ribose of the first stable isotope NMN standard are carbon-13 ( １３ The method according to any one of claims 16 to 19, wherein the substituted C) atom is a C) atom.

21. The phosphate group of the first stable isotope NMN standard is oxygen-18 ( １８ The method according to any one of claims 16 to 20, wherein the aryl group is substituted with an O) atom.

22. The method of any one of claims 1 to 21, wherein pre-treating the sample comprises removing soluble proteins from the sample.

23. 23. The method of claim 22, wherein the soluble proteins are removed by treating the sample with acid.

24. 24. The method of claim 23, wherein the acid is perchloric acid (PCA).

25. The method according to any one of claims 22 to 24, wherein pre-treating the sample comprises adding an agent that forms an ion pair by reaction with perchloric acid (PCA).

26. 26. The method of claim 25, wherein pre-treating the sample comprises removing the formed ion pairs.

27. 27. The method of claim 25 or 26, wherein the agent comprises potassium hydroxide, potassium borate, potassium formate, potassium acetate, potassium citrate, potassium carbonate, ammonium sulfate, ammonium chloride, rubidium sulfate, cesium hydroxide, or thallium acetate.

28. The method according to any one of claims 1 to 27, wherein the sample is selected from whole blood, plasma, tissue, or cultured cells.

29. The method of any one of claims 1 to 28, wherein the subject is a human.

30. the first stable isotope NMN standard 【Transformation 6】 The method according to any one of claims 1 to 29, having the structure:

31. the second stable isotope NMN standard 【Transformation 7】 The method according to any one of claims 1 to 30, having the structure:

32. 1. A method for calculating recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, comprising: adding a first stable isotope NMN standard to said sample; pre-treating the sample; adding a second stable isotope NMN standard to the sample; measuring the concentrations of the NMN and the first and second stable isotope NMN standards; and Calculating the recovery efficiency of the first stable isotope NMN standard in the sample based on the compounds measured in the sample, wherein the first stable isotope NMN standard has a relative mass difference with respect to the second stable isotope NMN standard.

33. 33. The method of claim 32, further comprising determining the effectiveness of the pretreatment based on the recovery efficiency.

34. 1. A kit for detecting nicotinamide mononucleotide (NMN) in a sample from a subject, comprising: a first stable isotope NMN standard; and A kit comprising a second stable isotope NMN standard, said first stable isotope NMN standard having a relative mass difference with respect to said second stable isotope NMN standard.

35. 35. The kit of claim 34, wherein the ribose of the second stable isotope NMN standard is substituted with a stable isotope atom.

36. 36. The kit of claim 34 or 35, wherein the nicotinamide group, ribose, and phosphate group of the first stable isotope NMN standard are substituted with stable isotope atoms.

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