JP7627461B2 - Method for measuring the concentration of nicotinamide mononucleotide - Google Patents

Description

The present invention relates to a method for measuring the concentration of nicotinamide mononucleotide.

Nicotinamide mononucleotide (NMN) is the main precursor for the biosynthesis of nicotinamide adenine dinucleotide (NAD ⁺ ) in mammals. It is known that administration of NMN promotes the biosynthesis of NAD ⁺ in mammals such as mice, and provides treatment for various age-related diseases (Non-Patent Documents 1 and 2).
NAD ⁺ is a coenzyme that has been known for a long time and plays an important role in the reduction reaction in cells of various organisms. In mammals, many reports suggest that NAD ^{+ is related to many biological pathways, such as metabolism, cancer, stress response, inflammation, and aging (Non-Patent Document 3). In addition, NAD+ decreases} with age in many tissues, such as the pancreas, adipose tissue, skeletal muscle, liver, skin, and brain, and it has been reported that the decrease in NAD ⁺ amount leads to the development of age-related diseases, such as cancer, heart disease, type II diabetes, obesity, hypertension, and age-related macular degeneration (Non-Patent Document 1).
The main pathway for the biosynthesis of NAD ⁺ is the conversion of nicotinamide (Nic), an amide form of vitamin _B3 , and 5'-phosphoribosyl-pyrophosphate (PEPP) to NMN by nicotinamide phosphoribosyltransferase (NAMPT), which then undergoes the addition of adenine by adenyltransferase to produce NAD ⁺ . In order to promote the biosynthesis of NAD ⁺ , many attempts have been made to administer NMN, the main precursor of NAD ⁺ , and the application of NMN in the treatment of various pathologies, including age-related diseases, has been attracting attention.
Indeed, long-term administration of NMN has been shown to alleviate age-related physiological decline in mice, and has been shown to suppress age-related weight gain, improve energy metabolism, improve physical function, improve insulin sensitivity, improve plasma lipid profile, and improve eye function without obvious toxicity or adverse events (Non-Patent Document 1).

Thus, it has been reported that administration of NMN provides various anti-aging effects in mammals such as mice. Furthermore, in recent reports, the safety of administering NMN to human subjects has also been reported (Non-Patent Document 4), and further medical applications of NMN are expected in the future medical field.
However, since NMN is rapidly metabolized in the body and converted into other metabolic products such as NAD ⁺ , there is no precedent for directly detecting NMN in blood. The above report also states that NMN could not be directly measured because it is rapidly metabolized in blood (Non-Patent Document 4). Detection of NMN in blood requires pretreatment for measurement, and it was difficult to detect NMN, which is quickly metabolized in blood.
Furthermore, it is expected that adding NMN to foods and cosmetics will have an anti-aging effect, but because these products contain a large number of ingredients, it has been difficult to analyze NMN in such complex mixtures.
Therefore, there is a strong demand for the development of methods to detect and quantify NMN in mixtures containing multiple contaminants, such as biological samples, foods, and cosmetics.

Mills, et al., Cell Metabolism 24, 795-806, December 13, 2016 Yoshino et al., Cell Metabolism 14, 528-536, October 5, 2011 Yoshino et al., Sirtuins: Methods and Protocols, Methods in Molecular Biology, vol. 1077, 203-215, 2013 Irie et al., Endocrine Journal 67 (2), 153-160, 2020

One of the problems that the present invention aims to solve is to provide a method for measuring the concentration of NMN in a mixture containing impurities.

As a result of extensive research into the above-mentioned problems, the inventors succeeded in separating NMN from a sample and measuring its concentration using liquid chromatography, thus completing the present invention.

That is, embodiments of the present invention include the following.
[1] A method for measuring a nicotinamide mononucleotide concentration, comprising a step of separating nicotinamide mononucleotide in a sample by reverse phase liquid chromatography.
[2] The method according to item 1, wherein the step uses a silica gel column selected from an octadecylsilyl group-bonded silica gel column and a pentafluorophenyl group-bonded silica gel column.
[3] The method according to item 2, wherein a pentafluorophenyl group-bonded silica gel column is used in the step.
[4] The method according to any one of items 1 to 3, wherein in the step, a mixture of water containing 0.1% (v/v) formic acid and acetonitrile is used as a mobile phase.
[5] The method according to item 4, wherein the mobile phase uses a gradient in which the ratio of acetonitrile gradually increases in the range of 0 to 40% (v/v).
[6] The method according to any one of items 1 to 5, wherein the sample is selected from blood, urine, cosmetics, and food.
[7] The method according to any one of items 1 to 6, further comprising a step of quantifying the nicotinamide mononucleotide separated in the step of separating nicotinamide mononucleotide in the sample by mass spectrometry.
[8] The method according to item 7, wherein the mass spectrometry is tandem mass spectrometry.

The method of the present invention allows for the measurement of NMN concentration in a sample containing contaminants. In one embodiment, the method of the present invention allows for the separation, detection, and quantification of NMN in a sample containing contaminants by using reverse phase liquid chromatography.

Separation behavior of NMN on various columns. Mass chromatograms of MRM (335.05>123.2) are shown for (a) Discovery HS F5, (b) ODS4, (c) SIL100A, and (d) Diol columns, respectively. Identification of NMN and camphorsulfonic acid components in mass spectrometry. Calibration curve for NMN quantification.

The present invention is described in further detail below.

As used herein, a "sample" refers to any sample containing NMN mixed with other impurities. In one embodiment, the sample contains multiple impurities. Examples of samples include, but are not limited to, biological samples (e.g., body fluids such as blood and urine, tissues, and cell extracts), cosmetics, and foods. In a preferred embodiment, blood or urine is used as the sample, and more preferably human blood. In another preferred embodiment, cosmetics are used as the sample. Cosmetics are not particularly limited as long as they contain NMN, and examples of such cosmetics include lotions, beauty serums, creams, shampoos, and hair growth agents. Foods are not particularly limited as long as they contain or contain NMN, and examples of such foods include vegetables, beef, beer, bread, seasonings such as soy sauce, supplements, and nutritional drinks.

The method according to the present invention includes a step of separating NMN from other contaminants. The separation step uses liquid chromatography, preferably reverse phase liquid chromatography.

In the method according to the present invention, a column for liquid chromatography is selected that has a suitable interaction with NMN. Examples of interactions between NMN and the column include adsorption, hydrophilic interaction, hydrophobic interaction, dipole-dipole interaction, π-π interaction, ionic bond, and hydrogen bond. Examples of preferred interactions include hydrophobic interaction, dipole-dipole interaction, π-π interaction, or any combination thereof.
In one embodiment, the column is preferred to be one that does not excessively adsorb NMN. In another embodiment, the column is a silica gel column containing a chemical modification group. Examples of chemical modification groups of silica gel columns include, but are not limited to, octadecylsilyl groups, octyl groups, triacontyl groups, pentafluorophenyl groups, phenyl groups, diol groups, amino groups, amide groups, cyano groups, etc. In a preferred embodiment, the silica gel column is an octadecylsilyl group-bonded silica gel column or a pentafluorophenyl group-bonded silica gel column. In a further preferred embodiment, the silica gel column is a pentafluorophenyl group-bonded silica gel column. The silica gel column is preferably end-capped to reduce the effect of non-chemically modified silanols.

As used herein, "octadecylsilyl group-bonded silica gel column" refers to a silica gel column in which the silicon on the column surface has been chemically modified with octadecylsilyl groups. As used herein, "pentafluorophenyl group-bonded silica gel column" refers to a silica gel column in which the silicon on the column surface has been chemically modified with pentafluorophenyl groups.

In the method according to the present invention, the mobile phase used in the liquid chromatography is arbitrarily selected depending on the interaction between the NMN and the column used. The mobile phase used in the liquid chromatography may be water, an organic solvent, or a mixture of water and an organic solvent. Examples of organic solvents include, but are not limited to, acetonitrile, methanol, and 2-propanol. In a preferred embodiment, the mobile phase used in the liquid chromatography is a mixture of water and acetonitrile.

In the method according to the present invention, when a mixed solvent is used in the mobile phase, the combination of the solvents and the mixing ratio thereof are arbitrarily changed depending on the combination with the column used. In one embodiment, when the mobile phase in the reversed-phase liquid chromatography is a mixed solvent of water and an organic solvent, the mobile phase uses a gradient in which the proportion of the organic solvent gradually increases in the range of 0 to 100% (v/v). For example, when the mobile phase in the reversed-phase liquid chromatography is a mixed solvent of water and acetonitrile, the mobile phase uses a gradient in which the proportion of acetonitrile gradually increases in the range of 0 to 100% (v/v). In a preferred embodiment, the mobile phase in the reversed-phase liquid chromatography uses a gradient in which the proportion of acetonitrile gradually increases in the range of 0 to 50% (v/v). In a more preferred embodiment, the mobile phase in the reversed-phase liquid chromatography uses a gradient in which the proportion of acetonitrile gradually increases in the range of 0 to 40% (v/v).

In certain embodiments, the mobile phase in liquid chromatography may include a volatile acid. Examples of volatile acids include, but are not limited to, tetrafluoroacetic acid, formic acid, and acetic acid. In a preferred embodiment, the volatile acid is formic acid. In a preferred embodiment, the mobile phase in liquid chromatography is a mixed solvent of water containing formic acid and acetonitrile.

In one embodiment, the volatile acid in the mobile phase of the liquid chromatography may be, for example, in the range of 0.01-1% (v/v) of the mobile phase. In another embodiment, the volatile acid in the mobile phase may be 1% (v/v) or more. In a preferred embodiment, the volatile acid in the mobile phase is 0.1% (v/v) of water.

The method according to the present invention may include a step of pretreating the sample before the step of separating NMN from other contaminants in the sample. Examples of sample pretreatment include, but are not limited to, any method known in the art for removing proteins, lipids, nucleic acids, metals, precipitation, ions, etc. in the sample. In an embodiment, when a biological sample is used, protein removal and lipid removal may be performed as sample pretreatment. An example of biological sample pretreatment is, for example, a method of extracting proteins and lipids in a biological sample using a mixed solvent of chloroform, methanol, and water, known in the art as the Bligh and Dyer method. In an embodiment, the mixing ratio of chloroform, methanol, and water used in the pretreatment of the biological sample may be 1:2:1 (v/v/v). When a biological sample is used, the mixing ratio of chloroform, methanol, and water used in the pretreatment may be appropriately changed depending on the sample. In the mixed solvent of chloroform, methanol, and water, the ratio of methanol to chloroform may be any ratio between 0.5 and 3. In the mixed solvent of chloroform, methanol, and water, the ratio of water to chloroform can be any ratio between 0.5 and 3. For measuring the NMN concentration in blood samples, the mixture ratio of chloroform, methanol, and water is particularly preferably 1:1:0.8 (v/v/v).

The solvent used in the sample pretreatment can be any organic solvent or a mixture of organic solvent and water. Organic solvents include chloroform, methanol, acetonitrile, acetone, hexane, or any combination thereof.

In one embodiment, a step of separating NMN from other contaminants in a sample can be performed without pretreatment of the sample. For example, the sample can be diluted with water in a certain ratio and used to measure the NMN concentration.

In the method according to the present invention, the concentration of NMN in a sample is measured by quantifying NMN separated from other impurities in the sample by liquid chromatography using any quantification method known in the art. In one embodiment, the quantification is performed using relative quantification by mass spectrometry. When performing relative quantification, for example, a calibration curve is created from mass spectrometry intensities using an NMN solution of known concentration, and the concentration of NMN in the actual sample is calculated. In another embodiment, the quantification is performed using absolute quantification by mass spectrometry. When performing absolute quantification, stable isotope-labeled NMN is added to the sample at a specific concentration, and mass spectrometry is performed simultaneously with the NMN in the sample, and the concentration of NMN in the sample is calculated from the ratio of mass spectrometry intensities.

In the method according to the present invention, any mass spectrometry method known in the art may be used as the mass spectrometry. In one embodiment, the molecules separated by liquid chromatography are ionized and mass spectrometry is performed. In a preferred embodiment, the mass spectrometry utilizes ionization based on electrospray ionization (ESI). The detection unit of the mass spectrometry may be a detection device known in the art. In one embodiment, the detection unit of the mass spectrometry is a tandem mass spectrometer such as a triple quadrupole mass spectrometer, a quadrupole orbitrap mass spectrometer, or a quadrupole time-of-flight mass spectrometer. The detection unit of the mass spectrometry is preferably a triple quadrupole mass spectrometer.

In analysis using a triple quadrupole mass spectrometer, a combination (MRM transition) of a mass filter (Q1) that allows ions with a specific mass (precursor ions) to pass and a mass filter (Q3) that allows fragments generated by gas collision-induced dissociation (CID) to pass is set, and multiple reaction monitoring (MRM) is used, which allows specific quantification of trace amounts of target components contained in a high matrix by detecting ions that can pass through these two mass filters.

In one embodiment, the concentration of NMN in a biological sample taken from a mammalian subject is measured. In one embodiment, the concentration of NMN in a blood or urine sample taken from a human subject is measured. In a preferred embodiment, a blood sample is taken from a mammalian subject who has been administered or ingested NMN, and the concentration of NMN in the blood is measured. In one embodiment, the mammalian subject has a temporary higher-than-normal concentration of NMN in the blood due to the administration of NMN. In one embodiment, the mammalian subject is administered NMN using a method of administering a drug that is commonly used in the art, such as oral administration, subcutaneous injection, or intravenous injection. Alternatively, the mammalian subject ingests NMN from food, such as vegetables, beef, beer, bread, seasonings such as soy sauce, supplements, or nutritional drinks. A commercially available human plasma sample to which NMN has been added may be used as a model of human blood administered or ingested with NMN.

In one embodiment, the concentration of NMN contained in a cosmetic sample is measured. The cosmetic containing NMN is stored and used for a certain period of time. In one embodiment, the NMN-containing cosmetic is stored at different temperatures for a period of one to three months, and then each sample is sampled and the concentration of NMN contained in the cosmetic is measured.

The present invention is not limited to the above-mentioned embodiments and the following examples. Furthermore, the above-mentioned embodiments can be combined in any manner as long as they are combinable and do not cause any contradiction, and such combined embodiments are also included within the scope of the present invention.

勾配（ＳＩＬ－１００Ａ、Ｄｉｏｌ）：

また、質量分析の条件は以下の通りである。
極性：陽性および陰性
エレクトロスプレー電圧：－３．０ｋＶ
脱溶媒管（ＤＬ）：２５０℃
ヒートブロック温度：４００℃
霧化ガス（Ｎ_２）流速：２．０Ｌ分^ー１
乾燥ガス（Ｎ_２）流速：１０．０Ｌ分^－１
ＣＩＤガス（アルゴン）流速：０．１９Ｍｐａ
検出電圧：１．８０ｋＶ
残留時間：５ｍ秒
停止時間：５ｍ秒 Example 1: Column screening First, columns for use in the measurement of NMN were examined. As reverse-phase silica gel columns, a pentafluorophenyl group-bonded silica gel column (Discovery® HS F5 (2.1x150 mm, 3 mm), Sigma-Aldrich Co.) and an octadecylsilyl group-bonded silica gel column (Inertsil® ODS-4 (2.1x150 mm, 3 mm), GL Sciences Co.) were used, and as normal-phase silica gel columns, a silica gel column without chemically modified groups (Inertsil® SIL-100A (2.1x150 mm, 3 mm), GL Sciences Co.) and a diol group-bonded silica gel column (Inertsil® Diol (2.1x150 mm, 3 mm), GL Sciences Co.) were used. For each column, 10 μL of NMN preparation (confirmed to be 99.5% or more pure by HPLC) was subjected to liquid chromatography and mass spectrometry (UHPLC Nexera + LCMS-8060, Shimadzu Co.).
The conditions for liquid chromatography are as follows.
Column temperature: 40°C
Flow rate: 0.25 mL/min (HSF5), 0.3 mL/min (other columns)
Injection volume: 2 μL
Mobile phase A: Water containing 0.1% (v/v) formic acid Mobile phase B: Acetonitrile Gradient (HSF5, ODS-4):

Gradient (SIL-100A, Diol):

The conditions for mass spectrometry are as follows:
Polarity: positive and negative Electrospray voltage: -3.0 kV
Desolvation tube (DL): 250°C
Heat block temperature: 400°C
Atomization gas ( _N2 ) flow rate: 2.0 L min ^-1
Drying gas (N ₂ ) flow rate: 10.0 L min ⁻¹
CID gas (argon) flow rate: 0.19 MPa
Detection voltage: 1.80 kV
Remaining time: 5ms Stopping time: 5ms

The chromatogram shows the intensity of the mass spectrometry when each column was used (Figure 1). When the pentafluorophenyl-bonded silica gel column (Discovery® HS F51) and the octadecylsilyl-bonded silica gel column (Inertsil® ODS-4), which are reverse-phase silica gel columns, were used, a sharp elution peak of NMN was obtained. In particular, when the pentafluorophenyl-bonded silica gel column was used, NMN was observed with high intensity in the eluate, indicating that the column has high separation ability for separating NMN. On the other hand, when the normal-phase silica gel column, an unmodified silica gel column (Inertsil® SIL-100A), was used, the elution peak of NMN was broad. Furthermore, when the diol-bonded silica gel column (Inertsil® Diol), which is a normal-phase silica gel column with a strong polarity, was used, NMN was not detected in the eluate. These results indicate that the reverse-phase silica gel column is preferable for measuring NMN, and the pentafluorophenyl-bonded silica gel column is particularly preferable. In the following NMN measurements, a pentafluorophenyl group-bonded silica gel column was used.

Example 2: Detection of NMN in blood samples Using the pentafluorophenyl group-bonded silica gel column, NMN was measured in blood samples. As a model of a blood sample after administration of NMN, a sample in which NMN was added to human plasma was used.
Preparation of blood samples
20 μL of human plasma (Metabolites in human plasma NIST (registered trademark) SRM (registered trademark) 1950, Merck) was added to a 2 mL safelock tube, and 960 μL of methanol cooled to -30°C was added. 20 μL of a stock aqueous solution of 125 μM 10-camphorsulfonic acid was added as an internal standard substance, and a 2 μM aqueous solution of NMN was added to the solution. The amount of the aqueous solution of NMN added was determined by the analysis of an unadded sample, and was an amount that would result in a final concentration of NMN in the analyzed sample of 1 μM. In addition, a solution was prepared by adding 960 μL of methanol cooled to -30°C to 20 μL of human plasma (Metabolites in human plasma NIST (registered trademark) SRM (registered trademark) 1950, Merck) and adding 20 μL of a stock aqueous solution of 125 μM 10-camphorsulfonic acid as an internal standard substance. Each tube was vortexed for 1 minute under ice cooling to mix each plasma sample, and then sonicated for 5 minutes. Each tube was left on ice for 5 minutes. Each tube was centrifuged at 16000Xg for 5 minutes at 4°C. 600 μL of the supernatant of each solution was dispensed into an Eppendorf tube, and chloroform (600 μL) and water (480 μL) were added to a new tube. The solution in the tube was centrifuged at 16000Xg for 5 minutes at 4°C. 720 μL of the upper layer of each tube was dispensed to obtain two types of analysis samples, a human plasma sample with NMN and a human plasma sample without NMN.

Mass spectrometry of NMN in blood samples The samples prepared above were subjected to liquid chromatography and mass spectrometry. The liquid chromatography and mass spectrometry conditions were the same as those described above. Data analysis in mass spectrometry was performed using Labsolutions Ver. 5.93 (Shimadzu Co.) software.
Mass spectrometry detected NMN and the internal standard 10-camphorsulfonic acid (Figure 2).

上記表に基づいて、相対ピーク面積値とＮＭＮ濃度との相関を表す検量線を作成した（図３）。 Preparation of calibration curves For 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, or 5.0 μM NMN aqueous solutions, the measured intensities in mass spectrometry were obtained as peak area values. Similarly, for 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 μM aqueous solutions of 10-camphorsulfonic acid, the internal standard, the measured intensities in mass spectrometry were obtained as peak area values. For each sample, mass spectrometry was performed three times at each concentration (n=3). At each concentration, the peak area value of NMN obtained was divided by the peak area value of 10-camphorsulfonic acid, the internal standard, to obtain the relative peak area value, which is shown in the table below.

Based on the above table, a calibration curve showing the correlation between the relative peak area value and the NMN concentration was prepared (FIG. 3).

^＊< 0.001 mM (オンカラム量: 2 fmol)
ＮＭＮ非添加ヒト血漿サンプルについて、質量分析においてＮＭＮは検出されなかった。一方で、ＮＭＮ最終濃度１μＭのＮＭＮ添加ヒト血漿サンプルにおいて、回収後に定量したＮＭＮ濃度の平均値は、０．７９μＭ（相対標準偏差０．１３）であった。すなわち、本発明の方法を用いたＮＭＮの回収率は７９％と算出され、本発明の方法が血液サンプル中のＮＭＮの測定に応用可能なＮＭＮの回収率を示すことが示された。 Quantitative analysis of NMN in blood samples Next, the NMN-free human plasma sample and the NMN-added human plasma sample prepared above were subjected to liquid chromatography and mass spectrometry under the same conditions as above to quantify the NMN concentration in each sample. The NMN concentration was calculated from the relative peak area value in mass spectrometry using the calibration curve prepared above. Furthermore, the NMN recovery rate by liquid chromatography was calculated from the quantified NMN concentration. The NMN recovery rate can be calculated by the following formula:
Spiked recovery (%) = (spiked sample concentration value – unspiked sample concentration value) / spiked concentration x 100
Each sample was tested in triplicate (n=3). The results of the quantification of NMN concentration and the recovery rate of NMN spiked are shown in the table below.

^* < 0.001 mM (on-column amount: 2 fmol)
For the human plasma samples not spiked with NMN, NMN was not detected by mass spectrometry. On the other hand, for the human plasma samples spiked with NMN at a final concentration of 1 μM, the average NMN concentration quantified after recovery was 0.79 μM (relative standard deviation 0.13). That is, the recovery rate of NMN using the method of the present invention was calculated to be 79%, demonstrating that the method of the present invention exhibits a recovery rate of NMN that can be applied to the measurement of NMN in blood samples.

Example 3: Detection of NMN in cosmetic samples Cosmetic A, which is labeled as containing NMN, was subjected to measurement of the NMN concentration.
Water-soluble cosmetic A was diluted 2000 times with water, and liquid chromatography and mass spectrometry were performed using the same equipment, separation conditions, and measurement conditions as those used in the quantitative analysis of NMN in blood samples. The measured intensity in mass spectrometry was obtained as a peak area value. The NMN concentration in cosmetic A was calculated by standard addition method. As a standard sample in the standard addition method, a solution was prepared by adding 50 μL of 2.0 μM NMN aqueous solution to 50 μL of a solution obtained by diluting cosmetic A 1000 times with water (NMN added amount: 100 pmol, sample dilution rate 2000 times), and the measured intensity in mass spectrometry was similarly obtained as a peak area value. The peak area value and calculated NMN concentration in each sample are as shown in the table below. It was shown that NMN was contained in cosmetic A before dilution at a concentration of 0.04% (v/v).

The NMN detection method and concentration measurement method of the present invention exhibit sufficient quantitative performance in the analysis of blood samples, and the method also enables the measurement of NMN concentrations in other samples. The method can also be applied in a variety of fields, such as elucidating the pharmacokinetics of NMN and quantifying NMN contained in foods, cosmetics, etc.

Claims (6)

A method for measuring a nicotinamide mononucleotide concentration, comprising a step of separating nicotinamide mononucleotide in a sample selected from a biological sample, a cosmetic, and a food by reverse phase liquid chromatography,
The sample is pretreated with a mixed solvent of chloroform, methanol, and water;
A method in which a pentafluorophenyl group-bonded silica gel column (with the exception that no C-18 is bonded to said column) is used in the step of reversed-phase liquid chromatography of the obtained pretreated sample . 2. The method according to claim 1 , wherein in the step, a mixture of water and acetonitrile containing 0.1% (v/v) formic acid is used as the mobile phase. 3. The method of claim 2 , wherein the mobile phase utilizes an increasing gradient of acetonitrile ranging from 0 to 40% (v/v). The method according to any one of claims 1 to 3 , wherein the sample is selected from blood, urine, cosmetics, and food. The method according to any one of claims 1 to 4 , further comprising a step of quantifying the nicotinamide mononucleotide separated in the step of separating nicotinamide mononucleotide in the sample by mass spectrometry. The method of claim 5 , wherein the mass spectrometry is tandem mass spectrometry.

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