Method for measuring nicotinamide mononucleotide (NMN) and related metabolites thereof

Abstract

The present disclosure relates generally to methods and kits for measuring nicotinamide mononucleotides (NMNs) and related metabolites thereof in a biological sample. The disclosure further relates to a method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample. The method uses a liquid chromatography-tandem mass spectrometry (LC-MS / MS) driven method and a dual isotope NMN standard substance. These methods and kits can be used to study nicotinamide adenine dinucleotide (NAD +) metabolism and aging.

Description

Technical Field

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

[0002] It has been reported that aging causes physiological decline in the functions of organs and tissues, which may also lead to aging-related diseases. In recent years, it has been confirmed that a systemic decrease in nicotinamide adenine dinucleotide (NAD+), an essential currency for energy metabolism throughout the body, is one of the important causes of aging. Therefore, nicotinamide mononucleotide (NMN), a key NAD+ intermediate that can enhance NAD+ biosynthesis, has attracted the attention of the scientific community and the public.

[0003] NAD+ is a classical coenzyme for many basic redox reactions and is also a substrate for NAD+-consuming enzymes, including poly(ADP-ribose) polymerases (PARPs), 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 protein 1 (SARM1), and thus plays a key role in a large number of important biological processes including metabolism, DNA damage response, inflammation, cancer, neurodegeneration, and aging. It has been shown that systemic NAD+ reduction is an important cause of age-related tissue dysfunction and diseases. Therefore, methods for supplementing NAD+ intermediates such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have received a great deal of attention from both the scientific community and the public as promising anti-aging interventions.

[0004] NAD+ is synthesized from three major precursors: tryptophan, niacin (NA), and nicotinamide (NAM), and also from two intermediates, NMN and NR. The major NAD+ biosynthetic pathway in mammals starts with NAM and is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). NAMPT converts NAM and 5'-phosphoribosyl pyrophosphate (5'-PRPP) into NMN, which is then converted into NAD+ by NMN adenylyltransferases 1-3 (NMNAT1-3). NR needs to be phosphorylated to NMN by NR kinases 1 and 2 (NRK1 and 2) to enter the major NAD+ biosynthetic pathway. Although the biochemical properties of key NAD+ biosynthetic enzymes have been well characterized, accurate measurement of NAD+-related metabolites such as NMN and NR has been a challenge due to the vulnerability of these compounds to enzymatic degradation, conversion during sample handling, and their complex behavior under different column and extraction conditions. A highly quantitative method for measuring NAD+ and NMN levels in biological samples using high-performance liquid chromatography (HPLC) has been established previously and has been used for mouse and human samples. However, NMN levels are generally much lower than NAD+ levels, and thus the quantification of NMN in biological samples has long been controversial.

[0005] Measuring the levels of NAD+ and its related metabolites has been a major challenge in the field, and thus it is very important to accurately and reliably measure the amounts of NAD+ and its related compounds, especially NMN, in biological samples. To date, due to the lack of optimization of protein removal methods and detection methods for biological samples, it has been difficult to quantify the accurate amount of NMN in biological samples. Therefore, the efficacy of NMN as an anti-aging intervention in preventing and treating age-related diseases cannot be accurately evaluated. The object of the present invention is to provide the most accurate and reliable method for measuring NAD+ and its related compounds in biological samples.

[0006] Some groups have used their own methods using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect and measure NMN in biological samples. However, there has been no comprehensive evaluation of the effects of extraction methods, recovery efficiency, and the complex biochemical environment of the samples (referred to as matrix effects). In this study, these issues were evaluated, and an LC-MS / MS-driven method using a dual-isotope NMN standard (dual-isotope-mediated LC-MS / MS; dimeLC-MS / MS) was successfully developed for accurate 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 I in a sample of a subject, which includes: 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 NMN and the standards by a mass spectrometer, wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference, and wherein the structure of Formula I corresponds to

[0008] .

[0009] The present disclosure further provides a method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, which includes: adding a first stable isotope NMN standard to the sample; pretreating the sample; adding a second stable isotope NMN standard to the sample; measuring NMN and the 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 and the second stable isotope NMN standard have a relative mass difference.

[0010] The present disclosure also relates to a kit for detecting nicotinamide mononucleotide (NMN) in a sample of a subject, which includes: a first stable isotope NMN standard; and a second stable isotope NMN standard, wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference.

[0011] 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to limit the present invention.

[0012] Details of one or more embodiments of the present invention will be set forth in the drawings and the following description. Other features, objects, and advantages of the present invention will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts isotopic compounds for LC-MS / MS analysis. Figure 1A Depicts the structures of stable isotope compounds for LC-MS / MS analysis. Figure 1BDepicts the chromatograms of regular (upper figure) and stable isotope (lower figure) compounds. These regular and isotope compounds were separated by the NMN-2 column developed by Shimadzu. The total ion count (TIC) of the multiple reaction monitoring (MRM) from each compound is shown. Figure 1C Depicts the chromatogram and mass spectrum of regular and isotope NMN. The TIC and transition were obtained from the MRM of regular and isotope NMN as the chromatogram (left figure), and the resulting product ions are shown as the mass spectrum (right figure). Figure 1D Depicts the calibration curves of NMN and other related compounds. The area under the curve (AUC) of each regular compound at different concentration ranges (NMN, NAD+, and NA, 15 - 1500 nM; NAM, 21 - 2100 nM; NR, 0.6 - 60 nM) was measured by LC-MS / MS in the presence of a fixed amount (NMN, NAD+, and NA, 1000 nM; NAM, 2000 nM; NR, 200 nM) of each corresponding isotope compound and normalized to the AUC of each isotope internal standard.

[0014] Figure 2 depicts the modulation of matrix effect and the measurement of NMN in mouse plasma samples. Figure 2A Depicts 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 0, 50, 100, or 500 nM were combined with PCA extracts from mouse plasma at concentrations of 1%, 10%, or 50%. Error bars represent the SEM obtained from three injections into LC-MS / MS. Figure 2B Depicts the modulation of matrix effect by the internal standard method using stable isotope compounds. Regular compounds were added to mouse plasma at a final concentration of 1 μM. The recovery efficiency was calculated by the external standard method (exSTD) or the internal standard method (iSTD). The mean was calculated from three independent experiments. Data are represented as mean ± SEM. Figure 2C Depicts the comparison of HPLC-based and LC-MS / MS-based methods for NMN measurement. Mouse plasma was spiked with a specified concentration of NMN. The same extract from the NMN-spiked plasma was measured by HPLC (y-axis) and LC-MS / MS (x-axis). Figure 2D Depicts the NMN level in mouse plasma after IP injection of NMN. C57BL / 6J mice (n = 10) at 3 to 4 months of age were given NMN by IP injection at 100 mg / kg. Plasma was extracted with PCA and NMN was measured by both HPLC-based and LC-MS / MS-based methods. Statistical analysis was performed using repeated measures one-way ANOVA and Bonferroni multiple comparison post hoc test. ** indicates p < 0.01.

[0015] Figure 3 depicts the dual-isotope-mediated LC-MS / MS method (dimeLC-MS / MS) for NMN measurement. Figure 3A The structure of NMN (M+14) with stable isotopes is depicted. Figure 3B The schematic process of dimeLC-MS / MS for NMN measurement is depicted. Figure 3C The chromatogram, mass spectrum, and calibration curve of NMN (M+14) are depicted. The TIC and transitions are obtained from the MRM of NMN (M+14) and plotted in the chromatogram (left panel), and the corresponding product ions are shown as the mass spectrum (middle panel). After normalizing the AUC of NMN (M+14) to the AUC of NMN (M+5), the calibration curve of NMN (M+14) is plotted. Figure 3D The recovery efficiency of spiked NMN (M+14) and conventional NMN in dimeLC-MS / MS is depicted. NMN (M+14) and conventional NMN at specified concentrations are added to mouse plasma. Each plasma sample is extracted by PCA and analyzed by dimeLC-MS / MS. The recovery efficiency is calculated by the iSTD method. Results are obtained from three independent experiments. Figure 3E The NMN levels in whole blood or plasma of mice after IP injection of NMN are depicted. C57BL / 6J mice at 3 to 4 months of age (n = 10) are given NMN (100 mg / kg) by IP injection. Each sample is extracted by PCA and NMN is measured by dimeLC-MS / MS. For each sample type (whole blood or plasma), the control data are shown in the left column and the 100 mg / kg NMN-treated data are shown in the right column. Two-way ANOVA is performed to compare the results with the Bonferroni multiple comparison post hoc test. Data are expressed as mean ± SEM. * indicates p < 0.05.

[0016] Figure 4 depicts a comparison of the PCA and MeOH extraction methods for NMN and other metabolites. Figure 4A The recovery efficiency of spiked NMN (M+14) and conventional NMN after PCA and MeOH-chloroform extraction is depicted. NMN (M+14) and conventional NMN at specified concentrations are added to mouse plasma. After extracting the plasma samples with PCA or MeOH-chloroform, each extract is analyzed by dimeLC-MS / MS. The recovery efficiency is calculated by the iSTD method. The mean values are obtained from three independent experiments. For each spiked NMN concentration, the PCA results are shown in the left column and the MeOH results are shown in the right column. Two-way ANOVA is performed to compare the results with the Bonferroni multiple comparison post hoc test. Data are expressed as mean ± SEM. ** indicates p < 0.005; *** indicates p < 0.001. Figure 4BDepicts the comparison of PCA and MeOH extraction methods for primary metabolites in plasma. Plasma samples were extracted by PCA or MeOH-chloroform extraction, and 96 primary metabolites were analyzed using the Primary Metabolites Method Package Ver. 2. The average area ratios from three independent experiments were plotted in a volcano plot. Figure 4C Depicts the area ratios of 10 representative compounds shown by PCA and MeOH-chloroform extraction methods. Unpaired Student's t-tests were performed to compare the results. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01.

[0017] Figure 5 depicts the accurate quantification of NMN taken up by AML12 cells. Figure 5A Depicts the dose-dependent increase in NMN (M+14) taken up by AML12 cells 1 hour after adding NMN to the medium. NMN (N+14) at the specified concentration was added to the medium of AML12 cells. One hour after adding NMN, the absolute amounts of regular NMN (left panel, left series), representing the endogenous NMN pool, and NMN (N+14) (left panel, right series), representing the NMN directly transported into AML12 cells, were quantified by dimeLC-MS / MS. The ratio of the 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 multiple comparison post-tests. Data are represented as mean ± SEM. * indicates p < 0.05; ** indicates p < 0.01. Figure 5B Depicts the time course of NMN taken up by AML12 cells. AML12 cells were treated with 200 µM NMN (M+14) for 2 hours. At the time points of 10, 30, 60, and 120 minutes after NMN addition, the absolute amounts of regular NMN (endo-NMN, top data) and NMN (N+14) (M+14 NMN, bottom data) were quantified by dimeLC-MS / MS. Results were obtained from three independent experiments. Data are represented as mean ± SEM.

[0018] Figure 6Depicts the increase of NMN in the plasma of mice after oral gavage with 300 mg / kg NMN. 5-6-month-old (young, n = 9) or 24-25-month-old (old, n = 7) C57BL / 6J male mice were orally administered NMN at 300 mg / kg. Blood was collected from the tail vein at the indicated time points. Plasma was extracted using PCA and analyzed by dimeLC-MS / MS. In the bottom panel, for each time point after oral gavage, data for young mice are shown on the left and data for old mice are shown on the right. Statistical analysis was performed using two-way ANOVA with repeated measures and Bonferroni multiple comparison post hoc tests. * indicates p < 0.05 between young and old mice at the 5-minute time point. # indicates p < 0.05 between 0 and 5-minute time points in young mice. Detailed Description

[0019] Nicotinamide adenine dinucleotide (NAD + ) is a crucial metabolite in fundamental biological phenomena including aging. Nicotinamide mononucleotide (NMN) is a key NAD + intermediate that has been widely tested as an effective NAD + -enhancing compound in mice and humans. However, accurately measuring NMN in biological samples has long been a challenge in this field. Here, a precise quantification method for measuring NMN by mass spectrometry (MS) using a dual-isotope NMN standard was established. In this method, the matrix effect of biological samples was appropriately adjusted, and the fate of NMN could be traced during sample processing. This method can accurately quantify the NMN level in mouse plasma and confirm the rapid and direct uptake of NMN into cultured cells.

[0020] Specifically, this dual-isotope-mediated LC-MS / MS( d ouble i sotope- me diated LC-MS / MS, dimeLC-MS / MS) can be easily extended as a reliable standard method for NAD + biology to other NAD + -related metabolites.

[0021] The unique features and advantages over existing methods are as follows:

[0022] 1) The extraction efficiency is evaluated using NMN labeled with two stable isotopes. By adding a known amount of primary NMN labeled with a stable isotope to a biological sample, the amount of NMN decomposed during the extraction process can be evaluated, and the extraction efficiency can be confirmed. Then, by adding secondary stable isotope-labeled NMN to the extract and using it as a normalization reference, the primary NMN labeled with a stable isotope can be quantified. This method allows for the accurate quantification of NMN levels in different biological samples and for the evaluation of differences in the accuracy of sample measurements between methods. The actual amount of NMN present in a biological sample can be measured by quantifying endogenous NMN and primary stable isotope-labeled NMN.

[0023] 2) The combination of acid extraction and LC-MS also ensures the accuracy of this method. Generally, since inorganic salts are harmful to the interface compartment of the mass spectrometer, organic solvents are used to extract metabolites from biological samples for LC-MS analysis. In the case of HPLC-based measurements, due to the lower sensitivity of the UV detector, a relatively large amount of sample extract is required to detect NMN. However, in this method, most of the inorganic salts can be removed by neutralization and centrifugation, and NMN (or other NAD+-related metabolites) can be injected into the mass spectrometer in a small volume for detection because its sensitivity is now much better due to technological development. Therefore, strong acid-mediated extraction can be used for LC-MS.

[0024] The present disclosure relates to a method for detecting nicotinamide mononucleotide (NMN) having the structure of Formula I in a sample of 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 by a mass spectrometer, wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference, and wherein the structure of Formula I corresponds to

[0025] .

[0026] The method may further comprise separating the compounds in the sample by liquid chromatography before detection. The detection may comprise: ionizing the NMN and the standards 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 determining the amounts of the NMN ions, the first stable isotope NMN standard ions, and the second stable isotope NMN standard ions by mass spectrometry.

[0027] The mass spectrometry may be tandem mass spectrometry.

[0028] The NMN ions detectable by mass spectrometry may include precursor ions with a mass-to-charge ratio of 335.20 ± 0.5 to 349.10 ± 0.5 and fragment ions selected from the group of ions with a mass-to-charge ratio of (i) 123.05 ± 0.5 to 128.10 ± 0.5 and (ii) 80.00 ± 0.5 to 84.05 ± 0.5.

[0029] The NMN ions detectable by mass spectrometry may include a precursor ion with a mass-to-charge ratio of 335.20 ± 0.5 and fragment ions selected from the group of ions with a mass-to-charge ratio of 123.05 ± 0.5 and 80.05 ± 0.5.

[0030] The first stable isotope NMN standard ions detectable by mass spectrometry may include a precursor ion with a mass-to-charge ratio of 349.10 ± 0.5 and fragment ions selected from the group of ions with a mass-to-charge ratio of 128.10 ± 0.5 and 84.05 ± 0.5.

[0031] The second stable isotope NMN standard ions detectable by mass spectrometry may include a precursor ion with a mass-to-charge ratio of 340.10 ± 0.5 and fragment ions selected from the group of ions with a mass-to-charge ratio of 123.05 ± 0.5 and 80.00 ± 0.5.

[0032] Detecting NMN and the standards may include determining the amount of NMN based on the amount of NMN ions, the first stable isotope NMN standard ions, or the second stable isotope NMN standard ions. Preferably, detecting NMN and the standards may include determining the amount of NMN based on the amount of NMN ions, the first stable isotope NMN standard ions, and the second stable isotope NMN standard ions.

[0033] The nicotinamide group, ribose, or phosphate group of the second stable isotope NMN standard may be substituted by stable isotope atoms.

[0034] The ribose of the second stable isotope NMN standard may be substituted by stable isotope atoms. The ribose of the second stable isotope NMN standard may be substituted by carbon-13 ( 13 C) atoms. Any carbon atom in the ribose moiety of the second stable isotope NMN standard may be substituted by carbon-13 ( 13 C) atoms. Preferably, more than one carbon atom in the ribose moiety of the second stable isotope NMN standard is substituted by carbon-13 ( 13 C) atoms.

[0035] In particular, the second stable isotope NMN standard may have the following structure:

[0036] .

[0037] The nicotinamide group, ribose, or phosphate group in the first stable isotope NMN standard can be substituted by stable isotope atoms. Preferably, the nicotinamide group, ribose, and phosphate group in the first stable isotope NMN standard are substituted by stable isotope atoms.

[0038] The nicotinamide group of the first stable isotope NMN standard can be substituted by carbon-13 ( 13 C) atoms. Any carbon atom in the nicotinamide group of the first stable isotope NMN standard can be substituted by carbon-13 ( 13 C) atoms.

[0039] The ribose moiety of the first stable isotope NMN standard can be substituted by carbon-13 ( 13 C) atoms. Any carbon atom in the ribose moiety of the first stable isotope NMN standard can be substituted by carbon-13 ( 13 C) atoms. Preferably, all carbon atoms in the ribose moiety of the first stable isotope NMN standard are substituted by carbon-13 ( 13 C) atoms.

[0040] The phosphate group of the first stable isotope NMN standard can be substituted by oxygen-18 ( 18 O) atoms. Preferably, 2 oxygen atoms in the phosphate group of the first stable isotope NMN standard are substituted by oxygen-18 ( 18 O) atoms.

[0041] In addition, the nitrogen atoms in the first or second stable isotope NMN standard can be substituted by nitrogen-15 ( 15 N) atoms. Preferably, the nitrogen in the nicotinamide group of the first stable isotope NMN standard is substituted by nitrogen-15 ( 15 N) atoms.

[0042] The hydrogen atoms in the first or second stable isotope NMN standard can be substituted by deuterium ( 2 H).

[0043] Specifically, the first stable isotope NMN standard has the following structure:

[0044] .

[0045] The pretreatment of the sample can include removing soluble proteins from the sample. The soluble proteins can be removed by treating the sample with an acid. The acid can be perchloric acid (PCA).

[0046] The pretreatment of the sample may include adding a reagent that forms an ion pair by reacting 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.

[0047] The sample may include whole blood, plasma, tissue, or cultured cells. The subject may be human.

[0048] The present disclosure also relates to a method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, which includes: 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 calculating the recovery efficiency based on the concentration of the second stable isotope NMN standard measured in the sample using the concentration of the first stable isotope NMN standard in the sample, wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference. The method may further include determining the effectiveness of the pretreatment based on the calculated recovery efficiency.

[0049] The present disclosure also relates to a kit for detecting nicotinamide mononucleotide (NMN) in a sample of a subject, which includes: a first stable isotope NMN standard; and a second stable isotope NMN standard, wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference. The ribose of the second stable isotope NMN standard may be replaced by a stable isotope atom. The nicotinamide group, ribose, and phosphate group of the first stable isotope NMN standard may be replaced by stable isotope atoms.

[0050] Isotopes are two or more atoms that have the same atomic number (the number of protons in their nuclei) and position in the periodic table (and thus belong to the same chemical element) and have different mass numbers (nucleon numbers) due to different numbers of neutrons in their nuclei. There are two types of isotopes: radioactive isotopes and stable isotopes or elements. Isotopes that do not emit radioactivity are called stable isotopes.

[0051] The present invention will be further described in the following examples, but it does not limit the scope of the present invention described in the claims.

[0052] Examples

[0053] It should be understood that although the present invention has been described in detail in connection with its description, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0054] Example 1 : Preparation of Columns and Isotope Compounds

[0055] There are multiple steps that can lead to inaccuracies in the quantitative measurement of NMN and NR. For example, differences in sample collection and extraction methods significantly affect the measurement of NAD+ intermediates. Although C18 columns allow for accurate measurement of NAD+ levels in HPLC, porous graphitic carbon columns or hydrophilic columns are better for detecting and measuring NMN. In mass spectrometry, the ionization efficiencies of NMN and its related metabolites are significantly different and depend on the biochemical background of the sample extract (referred to as matrix effect). Therefore, in order to achieve accurate quantitative measurement of NMN and its related metabolites, all these issues need to be improved.

[0056] Columns that allow for strong, stable, and reproducible retention of NMN were developed. The column, Prototype NMN-2, has octadecyl functional residues on fully end-capped silica particles and a non-metallic column surface to prevent the adsorption of specific compounds, thereby improving the ability to separate and detect low-concentration compounds, especially compounds containing phosphate groups such as NMN and NAD+. Next, stable isotope compounds of NAD+(M+5), NMN(M+5), NR(M+10), NAM(M+5), and NA(M+4) were synthesized to modulate the matrix effect of biological extracts ( Figure 1A ). These isotope compounds showed the same retention times and relative mass differences as the conventional compounds ( Figure 1B and Figure 1C ). Standard curves of each conventional compound normalized to each corresponding isotope compound showed accurate linearity, although high concentrations of NR showed a slight deviation from linearity ( Figure 1D ).

[0057] Example 2 : Quantification of NMN in Mouse Plasma Samples after NMN Administration

[0058] When developing HPLC-based methods for the measurement of NAD+ and NMN, it is crucial to use a very strong acid, perchloric acid (PCA), to effectively extract NAD+ and NMN from biological samples such as plasma with minimal loss. For mass spectrometry-based measurements, it is also necessary to understand and modulate the matrix effect. Using conventional compounds, the matrix effect of mouse plasma extracted with PCA was examined by adding 1%, 10%, and 50% plasma extracts to different concentrations of NAD+, NMN, NAM, and NA ( Figure 2A)。Interestingly, the area under the curve (AUC) of NAD+ increased, while that of NMN decreased. For example, 50% mouse plasma extract showed a 185.6% increase in the AUC of NAD+ at 500 nM, while the same concentration of mouse plasma extract inhibited the AUC of NMN by 57.0% at 500 nM, indicating that mouse plasma extract has opposite matrix effects on NAD+ and NMN. Similar to NMN, mouse plasma extract inhibited the AUC of NAM and NA ( Figure 2A ).

[0059] To adjust such matrix effects, a fixed amount (1 µM) of each isotopic compound was added to the mouse plasma samples before PCA extraction. Before PCA extraction, 1 µM of each conventional compound was also spiked into the same plasma samples. After extraction, the levels of each added conventional compound were quantified using a standard curve of each conventional compound (external standard method; exSTD), or after normalization to each internal isotopic standard (1 µM) and comparison with a standard curve that was also normalized to the internal isotopic standard (internal standard method; iSTD). Then, the recovery percentage (compared to the initial 1 µM of each added conventional compound) was calculated. When the matrix effects were appropriately adjusted by normalization to internal isotopic standards, the expected recovery percentage should be 100%. When measured by exSTD values, the recovery efficiencies of NMN, NAD+, NAM, and NA reflected different matrix effects ( Figure 2B , left), but as expected, after adjusting the matrix effects by iSTD, they showed approximately 100% recovery ( Figure 2B , right). These results strongly suggest that adjusting each matrix effect by normalization to each internal isotopic standard is crucial for the accuracy of the measurement. On the other hand, NR showed a recovery of 89.1% even after adjustment, although the adjustment of its matrix effect significantly improved the measurement accuracy ( Figure 2B ). This is because PCA extraction affected the elution pattern of NR, thus changing the ratio between conventional NR and isotopic NR. Currently, the reason for the change in the elution pattern of NR remains unclear.

[0060] Then, plasma samples spiked with different concentrations of NMN were measured using a mass spectrometry-based method (whose matrix effect was adjusted by iSTD) and a previously developed HPLC-based method (Yoshino, J. & Imai, S. Methods Mol. Biol. 1077, 203-215, 2013). Notably, the measured values based on both mass spectrometry and HPLC were almost the same ( Figure 2C), which confirmed the importance of PCA extraction and the accuracy of both methods. The NMN concentration in mouse plasma was also measured 5 minutes after intraperitoneal (IP) injection of NMN at a dose of 100 mg / kg. The NMN values measured by the two methods were compared. Interestingly, the two values were very consistent after NMN administration, and the concentration was highly variable, ranging from 2 - 3 µM to 20 µM, depending on the individual mouse( Figure 2D ). These results confirmed the very rapid kinetics of NMN uptake into the blood circulation, as originally reported. It should be noted that the HPLC-based and mass spectrometry-based methods gave very different values for the endogenous NMN levels (before NMN administration) in mouse plasma. The HPLC-based measurements gave much higher values for endogenous NMN in mouse plasma compared to those by mass spectrometry. The reason for this difference is currently under investigation.

[0061] Example 3 : Absolute quantification of NMN levels in biological samples

[0062] The recovery efficiency of NMN was approximately 100%( Figure 2B ). This fact strongly indicates that during this specific process of sample collection and extraction, NMN is not degraded or converted into other related compounds such as NAM and NR. However, generally, NMN is vulnerable to enzymatic degradation or conversion during sample collection and extraction. Therefore, it is crucial to be able to monitor and control such NMN degradation and conversion processes throughout sample collection and extraction. Therefore, another isotope of NMN, namely NMN(M + 14), was used to monitor these processes( Figure 3A ). By adding NMN(M + 14) to the biological sample immediately after collection, the fate of NMN during the entire sample processing can be monitored( Figure 3B ). NMN(M + 5) was also added after PCA extraction to adjust the matrix effect( Figure 3B ). NMN(M + 14) showed the same retention time and relative mass difference as conventional NMN, and had accurate linearity in its measurement( Figure 3C ). By using these two different isotope NMN compounds and evaluating the ratio of NMN(M + 14) and NMN(M + 5), the exact concentration of NMN(M + 14) can be calculated, which allows the calculation of the exact recovery efficiency in PCA extraction. To demonstrate this, 2.5 µM of NMN(M + 14) was added to mouse plasma before PCA extraction, and 500 nM of NMN(M + 5) was added after extraction to adjust the matrix effect( Figure 3D ). Then the recovery efficiency of 2.5 µM of NMN(M + 14) and 1, 10, and 40 µM of conventional NMN in PCA extraction was examined. Similarly, the recovery efficiency of both NMN(M + 14) and conventional NMN was almost ~100% (95.3% - 99.1%)(Figure 3D ), which clearly confirms the importance of PCA extraction.

[0063] To further demonstrate the advantages of this dual-isotope method, the NMN concentrations in two different biological samples (mouse whole blood and plasma) that may have different matrix effects were measured. In both samples, the NMN concentration was quantified 5 minutes after IP injection of NMN at a dose of 100 mg / kg. Before PCA extraction, 2.5 µM of NMN (M+14) was added to mouse whole blood or plasma, and 500 nM of NMN (M+5) was added to each extract. Similarly, after adjusting for the matrix effects of whole blood and plasma, the extraction efficiency of NMN (M+14) was close to 100% for both whole blood and plasma samples from control and NMN-administered mice ( Figure 3E , left). Although there was no significant difference in the NMN concentration in the whole blood extract between control and NMN-administered mice, the NMN concentration in the plasma extract was significantly increased in NMN-administered mice ( Figure 3E , right). Similarly, the plasma NMN concentration was highly variable after IP injection. These results strongly suggest that, although minimizing the degradation and conversion of NMN during sample processing for both whole blood and plasma, plasma samples, which are separated immediately after blood collection, should be used to clearly detect the increase in NMN after NMN administration. Overall, this method using dual-isotope NMN standards demonstrated its great advantages in accurately monitoring the fate of NMN and evaluating the extraction efficiency and absolute concentration of NMN in different types of biological samples.

[0064] Example 4 : Comparison of PCA and MeOH extraction methods

[0065] Mass spectrometry-driven metabolomics analysis usually uses methanol (MeOH) to extract as many metabolites as possible. However, it is currently unclear whether this MeOH-based extraction is suitable for the measurement of NMN and its related metabolites. Using this dual-isotope-mediated LC-MS / MS method (dimeLC-MS / MS), the recovery efficiency between PCA and MeOH was compared. Although the recovery efficiency of nearly 100% in PCA extraction was confirmed again, the MeOH extraction showed a ~70% recovery efficiency for both NMN (M+14) and conventional NMN at 1, 10, and 40 µM ( Figure 4A ), indicating that the MeOH-based extraction method is not suitable for accurate NMN measurement.

[0066] Next, we examined whether such differences in other metabolites could be observed between the PCA and MeOH extraction methods. For this assessment, mouse plasma samples were extracted with PCA or MeOH-chloroform and spiked with 100 µM of 2-(N-morpholino)ethanesulfonic acid (MES) as an internal standard. For metabolite analysis, the primary metabolite method package Ver. 2 was used, which can detect and analyze 96 metabolites (see Materials and Methods). Interestingly, the metabolites were divided into two groups: one group of metabolites was better extracted by PCA, and the other group of metabolites was better extracted by MeOH( Figure 4B ). NAM, NA, nucleosides (cytidine, 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 PCA extraction method, rather than the MeOH-based extraction method, is crucial for accurately measuring the precursors and intermediates of NAD+ in biological samples.

[0067] Example 5 : Quantitative measurement of direct incorporation of NMN into cells

[0068] Finally, the dimeLC-MS / MS method was used to quantitatively measure the direct incorporation of NMN into cultured cells. The cell line AML1 of mouse hepatocytes was selected because AML12 cells have been reported to show relatively high expression of Slc12a8, a recently identified NMN transporter. NMN (M+14) was added to the medium at a final concentration of 100 or 300 µM, and then at the time point of 1 hour after NMN addition, the absolute levels of conventional NMN and NMN (M+14) were measured and calculated in attomoles (10 -18 moles) / cell as the unit. During the calculation, the AUCs of all measured conventional NMN and NMN (M+14) were normalized to those of NMN (M+5). Interestingly, the endogenous NMN pool (conventional NMN) was approximately 5 attomoles / cell and was not significantly affected by treating the cells with 100 or 300 µM of NMN for 1 hour( Figure 5A , left). However, a dose-dependent direct uptake of NMN (M+14) was clearly detected( Figure 5A , left), and the extent of this direct uptake was up to 30% of the endogenous NMN pool( Figure 5A , right). Then, the time course of NMN uptake was examined by adding 200 µM of NMN (M+14) to the medium. NMN was rapidly transported into the cells at the 10-minute time point, and the NMN level steadily increased within 2 hours, up to ~10% of the endogenous NMN pool( Figure 5B ). At the same time, the endogenous NMN pool did not change much, suggesting the immediate utilization of intracellular NMN( Figure 5B)。These results clearly demonstrate that the dimeLC-MS / MS method allows for accurate and quantitative measurement of NMN uptake, and also confirm that NMN can be directly transported into cells without degradation to NAM or conversion to NR.

[0069] Example 6. After oral administration of NMN to young and old mice, the levels of NMN, nicotinamide (NAM), nicotinic acid (NA), and nicotinamide riboside (NR) in plasma were measured.

[0070] The results of this experiment are shown graphically in Figure 6 。

[0071] By using this mass spectrometry-driven technique called dimeLS-MS / MS, the changes in the levels of NMN, nicotinamide (NAM), nicotinic acid (NA), and nicotinamide riboside (NR) in plasma were successfully measured after oral administration of NMN to young and old mice at a dose of 30 mg / kg.

[0072] Although an increase in NMN could be clearly detected at the 5-minute time point in both young and old mice, the increase was significantly less in old mice and did not reach statistical significance in old mice.

[0073] On the other hand, NAM showed a very similar significant increase within 15 minutes in both young and old mice, while NA and NR did not show any significant changes at all.

[0074] These results strongly suggest that 1) NMN can be rapidly (within 5 minutes) transported from the intestine to the blood circulation, 2) the main degradation product of NMN is NAM and there is no conversion of NMN to NR within 15 minutes, and 3) NMN uptake is significantly reduced in old mice.

[0075] These results also provide convincing evidence for the wide application of this technique, dimeLC-MS / MS, in biological samples such as plasma.

[0076] Example 7. Summary of Biological Applications

[0077] The quantitative measurement of NAD+ intermediates, especially NMN, has long been a formidable challenge in the field of NAD+ biology. One reason is that NMN can be readily degraded or converted to other related metabolites, such as NAM and NR, when collecting and processing biological samples. Blood is perhaps the most difficult biological sample to handle because blood exhibits significant activities of CD38 and CD73 (ecto-5'-nucleotidase), and both CD38 and CD73 can use NMN as a substrate. Another reason is that the behavior of NMN in columns is very complex, which may be due to its bipartite nature of charge, such that minor differences in extraction and column conditions significantly affect the reliable and accurate detection of NMN. To overcome these problems, HPLC-driven methods have been previously developed to measure NMN levels in biological samples. This method was used to evaluate the pharmacokinetics of NMN after oral administration in mice and showed that NMN uptake from the intestine into the blood circulation occurred within 2 - 3 minutes and was transported to tissues within 10 - 30 minutes. This surprising finding led to the discovery of the NMN transporter Slc12a8. However, the difficulty in accurate NMN measurement has led to a heated debate about whether NMN can be directly transported into cells or the blood circulation. In this study, these difficulties were overcome, and an accurate and reliable LC-MS / MS method (dimeLC-MS / MS) using a dual-isotope NMN standard was successfully developed.

[0078] The advantages of dimeLC-MS / MS are twofold: First, it was confirmed that immediate PCA extraction gave almost 100% recovery efficiency of NMN and other NAD+-related metabolites compared to a recovery efficiency of ~70% by MeOH-chloroform extraction. Thus, in this method, even after extraction, the matrix effect was adjusted by adding the isotopic NMN standard, i.e., NMN(M+5), to the biological sample. Adjusting the matrix effect is crucial because the NMN AUC in mouse plasma extracts was suppressed to ~40%. The dimeLC-MS / MS method allows for proper adjustment of the matrix effect and quantification of the NMN level contained in the extract of the biological sample. Second, adding a second isotopic standard, NMN (M+14), to the biological sample immediately after sample collection allowed for precise tracking of the fate of NMN during sample processing. Furthermore, by calculating the ratio of NMN(M+14) and NMN(M+5), the exact concentration of NMN(M+14) can be calculated, which allows for the calculation of the exact recovery efficiency in any extraction method. Thus, using the dual-isotope NMN standards, NMN(M+14) and NMN(M+5), in the LC-MS / MS-driven method significantly increases the accuracy and reliability of NMN measurement in biological samples.

[0079] During the development of this method, two important biological results were obtained:

[0080] First, 5 minutes after IP injection of NMN, a significant increase in plasma NMN level was detected, while no increase in NMN level was detected in whole blood extracts. Therefore, it is crucial to evaluate plasma NMN level rather than whole blood NMN level, and it is clear that NMN can be rapidly transported into the systemic circulation. Importantly, at least after NMN administration, the previously developed HPLC-driven method gave very similar results to those by LC-MS / MS-driven methods after appropriate adjustment of its matrix effect, providing convincing results proof by two independent methods.

[0081] Second, by using this dimeLC-MS / MS, it was shown that NMN was directly transported into AML12 cells within 10 minutes, and it was also shown that the level of transported NMN increased up to ~30% of the endogenous NMN pool, clearly indicating that a significant amount of NMN can be directly transported into cells without being degraded to NAM or converted to NR. Similar results were obtained previously in primary hepatocytes, and in Slcl2a8-deficient hepatocytes, this direct transport of NMN was almost completely eliminated. Although the absolute amount of transported NMN could not be calculated in previous studies, the absolute levels of the endogenous NMN pool and NMN transported into cells could be accurately quantified in this study. The fact that the endogenous NMN pool did not change much even when NMN was transported up to 30% of its endogenous pool strongly implies that NMN must be immediately converted to NAD+ or other metabolites once it is transported into cells. Therefore, dimeLC-MS / MS will also be powerful in examining the precise kinetics and fate of NMN and NAD+-related metabolites in cells or tissues.

[0082] In summary, an LC-MS / MS-driven method using a dual-isotope NMN standard, namely dimeLC-MS / MS, was successfully developed and its accuracy and reliability in measuring NMN in biological samples were demonstrated. dimeLC-MS / MS accurately measured the plasma NMN level after IP injection in mice and performed absolute quantification of the amount of NMN directly transported into cells. The dimeLC-MS / MS method will open up many interesting opportunities for evaluating the kinetics of NMN uptake and NAD+ biosynthesis under different metabolic conditions and in different tissues.

[0083] Example 8. Materials and Methods

[0084] Chemicals and Reagents

[0085] NMN was a gift from Mirai Lab Biosciences Ltd. (Tokyo, Japan). NAD+ (#N1511), NR (#SMB00907), NAM (#72340), and NA (#N0761) were purchased from Sigma (USA). [13C5]-NMN (#C7934), [13C9, 15N]-NR (#C7990), and [2H4]-NA (#C2885) were purchased from Alsachim (France). Other isotopic compounds were custom-synthesized at Alsachim.

[0086] Mouse Experiments

[0087] C57BL / 6J mice were group-housed in an SPF mouse facility under a 12-hour light / 12-hour dark cycle. All mice had free access to a regular chow diet (PicoLab 5053 Rodent Diet 20; LabDiets). Blood samples were collected from the tail vein using a microhematocrit heparinized capillary (Fisher Scientific), and plasma was separated immediately after blood collection. NMN administration was performed by intraperitoneal (IP) injection. All animal studies were approved by the University of Washington Animal Studies Committee and were in compliance with NIH guidelines.

[0088] Perchloric Acid (PCA) Extraction of Mouse Plasma Samples

[0089] Plasma was separated from mouse whole blood by centrifugation at 6,500 rpm for 7 minutes at 4°C in a microcentrifuge tube. Then, mouse plasma was mixed with ice-cold 10% PCA solution containing stable isotope compounds as internal standards and incubated on ice for 15 minutes. After centrifugation at 21,500 x g for 5 minutes at 4°C, the supernatant was separated and neutralized with ice-cold 3M K2CO3 solution in a 1:3 ratio. Then, the mixture was incubated on ice for 15 minutes with the lid open. The resulting salts were precipitated by centrifugation at 21,500 x g for 5 minutes at 4°C. The resulting supernatant (final extract) was measured using a high-performance liquid chromatography triple quadrupole mass spectrometry system (LC-MS / MS, LCMS-8060, Shimadzu). The concentrations of NMN and other related metabolites in the extract were quantified based on the area under each curve (AUC) compared to a standard curve (normalized by standard isotope compounds), and were back-calculated by normalization of the dilution rate and plasma volume.

[0090] MeOH-Chloroform Extraction

[0091] To 20 μL of mouse plasma, 200 μL of MeOH containing internal standard was added and mixed well for several minutes. Then, 200 μL of chloroform was added and mixed with a vortex mixer. 80 μL of LCMS-grade water was added and mixed well. The mixture was centrifuged at 4 °C at 21,500 x g for 15 minutes. 200 μL of the upper aqueous phase was transferred to a new tube and then dried using a speed-vac. The metabolites were dissolved in 100 μL of LCMS-grade water and measured using LC-MS / MS (LCMS-8060, Shimadzu).

[0092] LC-MS / MS Analysis

[0093] LC-MS / MS analysis was performed using a Nexera X2 Ultra HPLC system coupled to a triple quadrupole mass spectrometer (LCMS-8060, Shimadzu). Samples were kept in an autosampler at 4 °C during analysis, and 2 μL of each sample was injected into a prototype column NMN-2 (150 mm x 2.0 mm, particle size 2.2 μm, Shimadzu). The column temperature was maintained at 21 °C. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% formic acid. Using a flow rate of 0.2 mL / min, chromatographic separation was achieved with the following gradient elution time program: 1% B (0 - 2 minutes), 1 - 38.6% B (2 - 10 minutes), 95% B (10.01 - 12 minutes), and 1% B (12.01 - 15 minutes). NMN and NAD+ typically eluted at 3.5 minutes and at 7.5 minutes, respectively.

[0094] After electrospray ionization (ESI) in the positive mode, metabolites were detected by single reaction monitoring (also known as multiple reaction monitoring (MRM)). The target cycle time was set to 0.4 seconds, with a 1 ms pause between each transition. The dwell time for each transition was automatically set to 10 - 15 ms. Other parameters of the mass spectrometer were set as follows: interface voltage was 3.5 kV, nebulizing gas flow was 3 L / min, heating gas flow was 10 L / min, drying gas flow was 10 L / min, interface temperature was 300 °C, desolvation line temperature was 200 °C, heating block temperature was 350 °C, and collision-induced dissociation gas pressure was 270 kPa. All data were processed using LabSolutions software (Shimadzu).

[0095] Metabolomics Analysis

[0096] Metabolites were extracted using PCA or MeOH-chloroform extraction, and 100 μM 2-(N-morpholino)ethanesulfonic acid (MES) was added as an internal standard. Metabolites were analyzed using the primary metabolite method package ver. 2 (Shimadzu) and LC-MS / MS (LCMS-8060, Shimadzu). The area ratio of each metabolite was obtained by dividing the AUC of the target metabolite by the AUC of the internal standard.

[0097] Cell Culture and Drug Treatment

[0098] AML12 cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM / F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin (Life Technologies), 40 ng / mL dexamethasone, 0.005 mg / mL insulin, 0.005 mg / mL transferrin, and 5 μg / mL selenium (insulin-transferrin-selenium [ITS-G], #41400045, Gibco). During drug treatment, dexamethasone and ITS were removed from the AML12 medium. In the NMN uptake experiment, 8 x 10 5 AML12 cells were plated in 6 cm culture dishes and treated with 0.5 μM 78c (CD38 inhibitor) and 50 μM adenosine-5'-(α,β-methylene)diphosphate (AOPCP, CD73 inhibitor) for 16 h. Then, 2 μM dipyridamole (ENT inhibitor) and 100 nM FK-866 (NAMPT inhibitor) were added to the medium for 1 h. After treating the cells with all inhibitors, the indicated concentration of stable isotope NMN (M+14) was added to the AML12 cells without removing the inhibitors. Cells were harvested with trypsin-EDTA solution and frozen at -30 °C until analysis. To measure the NMN incorporated into the cells, the cell pellet was resuspended in LC / MS-grade water containing stable isotope NMN (M+5) and then extracted with perchloric acid as described above.

[0099] Statistical Analysis

[0100] All data are presented as mean or mean ± SEM. Statistical significance between control and experimental samples was determined by unpaired Student's t-test. Statistical significance of differences among multiple NMN samples was analyzed by one-way, two-way, or repeated measures one-way ANOVA, with Bonferroni multiple comparison test as the post hoc test. A p-value < 0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism (Ver 9.4.1).

[0101] When introducing elements of the present invention or its preferred embodiments, the articles "a", "an", "the", and "said" are intended to mean that there is one or more than one element. The terms "comprising", "including", and "having" are intended to be inclusive and mean additional elements in addition to the listed elements.

[0102] In view of the foregoing, it will be seen that several objects of the present invention are achieved and other advantageous results are obtained.

[0103] Since various changes can be made in the above compositions and methods without departing from the scope of the present invention, it is intended that all matter contained in the above description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

Claims

1. A method for detecting nicotinamide mononucleotide (NMN) having the structure of Formula I in a sample of 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 by a mass spectrometer (MS), wherein the first stable isotope NMN standard and the second stable isotope NMN standard have a relative mass difference, and wherein the structure of Formula I corresponds to 。 2. The method according to claim 1, further comprising separating the compounds in the sample by liquid chromatography (LC) before detecting the NMN and the standards by MS.

3. The method according to claim 1 or 2, wherein detecting the NMN and the standards by MS comprises: ionizing the NMN and the standards from the sample to generate NMN ions, first stable isotope NMN standard ions, or second stable isotope NMN standard ions that are detectable by mass spectrometry; and determining the amounts of the NMN ions, first stable isotope NMN standard ions, or second stable isotope NMN standard ions by mass spectrometry.

4. The method according to claim 3, wherein detecting the NMN and the standards by MS comprises: ionizing the NMN and the standards 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 determining the amounts of the NMN ions, first stable isotope NMN standard ions, and second stable isotope NMN standard ions by mass spectrometry.

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

6. The method according to any one of claims 3 to 5, wherein the NMN ions detectable by mass spectrometry comprise precursor ions having a mass-to-charge ratio of 335.20 ± 0.5 to 349.10 ± 0.5 and fragment ions selected from the group consisting of ions having a mass-to-charge ratio of (i) 123.05 ± 0.5 to 128.10 ± 0.5 and (ii) 80.00 ± 0.5 to 84.05 ± 0.

5.

7. The method according to any one of claims 3 to 6, wherein the NMN ions detectable by mass spectrometry comprise precursor ions having a mass-to-charge ratio of 335.20 ± 0.5 and fragment ions selected from the group consisting of ions having a mass-to-charge ratio of 123.05 ± 0.5 and 80.05 ± 0.

5.

8. The method according to any one of claims 3 to 7, wherein the first stable isotope NMN standard ions detectable by mass spectrometry comprise precursor ions having a mass-to-charge ratio of 349.10 ± 0.5 and fragment ions selected from the group consisting of ions having a mass-to-charge ratio of 128.10 ± 0.5 and 84.05 ± 0.

5.

9. The method according to any one of claims 3 to 7, wherein the second stable isotope NMN standard ions detectable by mass spectrometry comprise precursor ions with a mass-to-charge ratio of 340.10 ± 0.5 and fragment ions selected from the group consisting of ions with a mass-to-charge ratio of 123.05 ± 0.5 and 80.00 ± 0.

5.

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

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

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

13. The method according to claim 12, wherein the ribose of the second stable isotope NMN standard is substituted by the stable isotope atoms.

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

15. The method according to claim 14, wherein the carbon atoms of the ribose of the second stable isotope NMN standard are replaced by carbon-13 ( 13 C) atoms.

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

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

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

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

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

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

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

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

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

25. The method according to any one of claims 22 to 24, wherein pretreating the sample comprises adding a reagent that forms an ion pair by reacting with perchloric acid (PCA).

26. The method according to claim 25, wherein pretreating the sample comprises removing the formed ion pair.

27. The method according to claim 25 or 26, wherein the reagent 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 according to any one of claims 1 to 28, wherein the subject is human.

30. The method according to any one of claims 1 to 29, wherein the first stable isotope NMN standard has the structure: 。 31. The method according to any one of claims 1 to 30, wherein the second stable isotope NMN standard has the structure: 。 32. A method for calculating the recovery efficiency of nicotinamide mononucleotide (NMN) in a sample, 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 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 from the second stable isotope NMN standard.

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

34. A kit for detecting nicotinamide mononucleotide (NMN) in a sample of a subject, 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.

35. The kit according to claim 34, wherein the ribose of the second stable isotope NMN standard is substituted by stable isotope atoms.

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

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