Method for measuring the concentration of nicotinamide mononucleotide

Abstract

To provide a method of measuring the concentration of nicotinamide mononucleotide contained in a mixture.SOLUTION: A method of measuring the concentration of nicotinamide mononucleotide is disclosed, comprising isolating nicotinamide mononucleotide in a sample by liquid chromatography and measuring the concentration thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring the concentration of nicotinamide mononucleotide. [Background technology]

[0002] Nicotinamide mononucleotide (NMN) is the primary regulator of nicotinamide adenine dinucleotide (NAD) in mammals. + ) is the main precursor for the biosynthesis of NAD in mammals such as mice. + It is known that the biosynthesis of β-lactamase is promoted, which leads to the treatment of various age-related diseases (Non-Patent Documents 1 and 2). NAD + is a well-known coenzyme that plays an important role in the reduction reactions in the cells of various organisms. + Many reports suggest that NAD is involved in many biological pathways, such as metabolism, cancer, stress response, inflammation, and aging (Non-Patent Document 3). + With age, NAD decreases in many tissues, including the pancreas, adipose tissue, skeletal muscle, liver, skin, and brain. + It has also been widely reported that a decrease in the amount of retinal pigment epithelium leads to the onset of age-related diseases such as cancer, heart disease, type II diabetes, obesity, hypertension, and age-related macular degeneration (Non-Patent Document 1). NAD + The main pathway for the biosynthesis of vitamin B3 is the conversion of nicotinamide (Nic) and 5'-phosphoribosyl-pyrophosphate (PEPP), which are amide forms of vitamin B3, to NMN by nicotinamide phosphoribosyltransferase (NAMPT), which then converts NMN to NAD by the addition of adenine by adenyltransferase. + There is a pathway where NAD is produced. + To promote the biosynthesis of NAD + There have been many attempts to administer NMN, the main precursor of aging, and the application of NMN in the treatment of various medical conditions, including age-related diseases, has attracted attention. In fact, long-term administration of NMN has been shown to alleviate age-related physiological decline in mice. Furthermore, long-term administration of NMN 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).

[0003] Thus, it has been reported that administration of NMN provides various anti-aging effects in mammals such as mice. Furthermore, recent reports have also shown the safety of administering NMN to human subjects (Non-Patent Document 4), and further medical applications of NMN are expected in the future medical field. However, NMN is rapidly metabolized in the body and converted to NAD + There is no precedent for directly detecting NMN in blood because NMN is converted into other metabolic products such as . The above report also states that NMN could not be measured directly because it is quickly metabolized in blood (Non-Patent Document 4). Detecting NMN in blood requires pretreatment for measurement, and it was difficult to detect NMN because it 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. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mills, et al., Cell Metabolism 24, 795-806, December 13, 2016 [Non-Patent Document 2] Yoshino et al., Cell Metabolism 14, 528-536, October 5, 2011 [Non-Patent Document 3] Yoshino et al., Sirtuins: Methods and Protocols, Methods in Molecular Biology, vol. 1077, 203-215, 2013 [Non-Patent Document 4] Irie et al., Endocrine Journal 67 (2), 153-160, 2020 Summary of the Invention [Problem to be solved by the invention]

[0005] One of the problems that the present invention aims to solve is to provide a method for measuring the NMN concentration in a mixture containing impurities. [Means for solving the problem]

[0006] As a result of intensive research into the above-mentioned problems, the inventors succeeded in separating NMN in a sample and measuring its concentration using liquid chromatography, thereby completing the present invention.

[0007] 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 proportion 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 claim 7, wherein the mass spectrometry is tandem mass spectrometry. Effect of the Invention

[0008] The method of the present invention allows the measurement of NMN concentration in a sample containing impurities. In one embodiment, the method of the present invention allows the separation, detection, and quantification of NMN in a sample containing impurities by using reverse phase liquid chromatography. [Brief description of the drawings]

[0009] [Figure 1] 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. [Diagram 2] Identification of NMN and camphorsulfonic acid components by mass spectrometry. [Diagram 3] Calibration curve for NMN quantification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described in further detail.

[0011] The term "sample" as used herein refers to any sample in which NMN is 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 essences, creams, shampoos, and hair growth agents. Foods are not particularly limited as long as they contain or are mixed with NMN, and examples of such foods include vegetables, beef, beer, bread, seasonings such as soy sauce, supplements, and nutritional drinks.

[0012] The method of the present invention includes a step of separating NMN from other impurities. Liquid chromatography, preferably reverse phase liquid chromatography, is used in the separation step.

[0013] In the method according to the present invention, the column of liquid chromatography is selected to have a moderate interaction with NMN. Examples of the interaction 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, a column that does not excessively adsorb NMN is preferred. In another embodiment, the column is a silica gel column containing a chemically modified group. Examples of chemically modified 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.

[0014] As used herein, the term "octadecylsilyl group-bonded silica gel column" refers to a silica gel column in which silicon on the column surface is chemically modified with octadecylsilyl groups. As used herein, the term "pentafluorophenyl group-bonded silica gel column" refers to a silica gel column in which silicon on the column surface is chemically modified with pentafluorophenyl groups.

[0015] In the method according to the present invention, the mobile phase used in liquid chromatography is arbitrarily selected according to the interaction between NMN and the column used. The mobile phase used in liquid chromatography can be water, an organic solvent, or a mixed solvent 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 liquid chromatography is a mixed solvent of water and acetonitrile.

[0016] 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 utilizes a gradient in which the ratio 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 utilizes a gradient in which the ratio 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 utilizes a gradient in which the ratio 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 utilizes a gradient in which the ratio of acetonitrile gradually increases in the range of 0 to 40% (v / v).

[0017] In some 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.

[0018] In one embodiment, the volatile acid contained in the mobile phase of liquid chromatography may be, for example, in the range of 0.01 to 1% (v / v) relative to the mobile phase. In another embodiment, the volatile acid contained in the mobile phase may be 1% (v / v) or more. In a preferred embodiment, the volatile acid contained in the mobile phase is 0.1% (v / v) relative to water.

[0019] The method according to the present invention may include a step of pretreating the sample before the step of separating NMN from other impurities 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 a blood sample, the mixing ratio of chloroform, methanol, and water is particularly preferably 1:1:0.8 (v / v / v).

[0020] The solvent used in the pretreatment of the sample can be any organic solvent or a mixture of organic solvent and water, including chloroform, methanol, acetonitrile, acetone, hexane, or any combination thereof.

[0021] 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 at a certain ratio and used to measure the NMN concentration.

[0022] In the method of the present invention, the NMN concentration in the sample is measured by quantifying the NMN separated from other impurities in the sample by liquid chromatography using any quantitative method known in the art.In one embodiment, the quantitative determination is performed using relative quantitative determination by mass spectrometry.When performing relative quantitative determination, for example, a calibration curve is created from mass spectrometry intensity using a NMN solution of known concentration, and the concentration of NMN in the actual sample is calculated.In another embodiment, the quantitative determination is performed using absolute quantitative determination by mass spectrometry.When performing absolute quantitative determination, a 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 intensity.

[0023] In the method according to the present invention, any mass spectrometry method known in the art may be used as the mass spectrometry. In an 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 an 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.

[0024] 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 up, and multiple reaction monitoring (MRM) is used to detect ions that can pass through these two mass filters, enabling specific quantification of trace amounts of target components contained in a high matrix.

[0025] In one embodiment, the concentration of NMN contained in a biological sample collected from a mammalian subject is measured. In one embodiment, the concentration of NMN contained in a blood or urine sample collected from a human subject is measured. In a preferred embodiment, a blood sample is collected from a mammalian subject who has been administered or ingested NMN, and the concentration of NMN contained in the blood is measured. In one embodiment, the mammalian subject has a temporary higher NMN concentration in the blood than normal 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 is administered NMN from food, such as vegetables, beef, beer, bread, seasonings such as soy sauce, supplements, or nutritional drinks. As a model of human blood administered or ingested with NMN, a commercially available human plasma sample to which NMN has been added may be used.

[0026] In one embodiment, the concentration of NMN contained in a cosmetic sample is measured. The cosmetic containing NMN is stored for a certain period of time and used. In one embodiment, the NMN-containing cosmetic is stored at different temperatures for a period of 1 month to 3 months, and then each sample is sampled and the concentration of NMN contained in the cosmetic is measured.

[0027] The present invention is not limited to the above-mentioned embodiments and the following examples. In addition, the above-mentioned embodiments can be arbitrarily combined as long as they are combinable and do not cause any contradiction, and the combined embodiments are also included within the scope of the present invention. EXAMPLES

[0028] Example 1: Column screening First, we investigated columns to be used for the measurement of NMN. As reverse-phase silica gel columns, we used 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.), and as normal-phase silica gel columns, we used 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.). 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℃ Flow rate: 0.25mL / min (HSF5), 0.3mL / min (other columns) Injection volume: 2μL Mobile phase A: Water containing 0.1% (v / v) formic acid Mobile phase B: Acetonitrile Gradient (HSF5, ODS-4): [Table 1] Gradient (SIL-100A, Diol): [Table 2] The conditions for mass spectrometry are as follows: Polarity: Positive and Negative Electrospray voltage: -3.0kV Desolvation tube (DL): 250℃ Heat block temperature: 400℃ Atomization gas (N2) flow rate: 2.0 L / min ー1 Drying gas (N2) flow rate: 10.0 L / min -1 CID gas (argon) flow rate: 0.19Mpa Detection voltage: 1.80kV Residual time: 5ms Stopping time: 5ms

[0029] The chromatogram shows the intensity of the mass spectrometry when each column was used (Figure 1). When a pentafluorophenyl group-bonded silica gel column (Discovery (registered trademark) HS F51) and an octadecylsilyl group-bonded silica gel column (Inertsil (registered trademark) ODS-4), which are reverse phase silica gel columns, were used, a sharp elution peak of NMN was obtained. In particular, when a pentafluorophenyl group-bonded silica gel column was used, NMN in the eluate was observed with high intensity, and it was found that the column has high separation ability for separating NMN. On the other hand, when an unmodified silica gel column (Inertsil (registered trademark) SIL-100A), which is a normal phase silica gel column, was used, the elution peak of NMN was broad. Furthermore, when a diol group-bonded silica gel column (Inertsil (registered trademark) Diol), which is a normal phase silica gel column with strong polarity, was used, NMN was not detected in the eluate. These results showed that a reverse phase silica gel column is preferable for measuring NMN, and a pentafluorophenyl group-bonded silica gel column is particularly preferable. In the following measurements of NMN, a pentafluorophenyl group-bonded silica gel column was used.

[0030] 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 blood samples after NMN administration, samples of human plasma to which NMN had been added were 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 safe lock 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 2 μM of NMN aqueous solution was added to this. The amount of NMN aqueous solution added was determined by analysis of an unadded sample, and was an amount that would result in a final concentration of NMN in the analysis 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. 600μL of the supernatant of each solution was taken into an Eppendorf tube, and chloroform (600μL) and water (480μL) were added to a new tube. The solution in the tube was centrifuged at 16000Xg for 5 minutes at 4°C. 720μL of the upper layer of each tube was taken to obtain two types of analysis samples, a human plasma sample with NMN and a human plasma sample without NMN.

[0031] Mass spectrometry analysis of NMN in blood samples The sample prepared above was 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 software Labsolutions Ver. 5.93 (Shimadzu Co.). Mass spectrometry detected NMN and the internal standard 10-camphorsulfonic acid (Figure 2).

[0032] Creating a calibration curve For the NMN aqueous solutions of 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, or 5.0 μM, the measured intensity in mass spectrometry was obtained as a peak area value. Similarly, for the internal standard 10-camphorsulfonic acid, the measured intensity in mass spectrometry was obtained as a peak area value for each of the aqueous solutions of 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 μM. 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 the internal standard 10-camphorsulfonic acid to obtain the relative peak area value, which is shown in the table below. [Table 3] Based on the above table, a calibration curve was created showing the correlation between relative peak area values ​​and NMN concentration (Figure 3).

[0033] Quantitative Analysis of NMN in Blood Samples Next, liquid chromatography and mass spectrometry were performed under the same conditions as above for the NMN-free human plasma sample and NMN-added human plasma sample prepared above, and the NMN concentration in each sample was quantified. The NMN concentration was calculated from the relative peak area value in mass spectrometry using the calibration curve created above. Furthermore, the NMN addition recovery rate by liquid chromatography was calculated from the quantified NMN concentration. The NMN addition recovery rate can be calculated using the following formula: Spiked recovery (%) = (spiked sample concentration value - unspiked sample concentration value) / spiked concentration x 100 Each sample was tested three times (n=3). The results of quantification of NMN concentration and recovery rate of NMN are shown in the table below. [Table 4] * < 0.001 mM (on-column amount: 2 fmol) In the case of human plasma samples not containing NMN, NMN was not detected by mass spectrometry. On the other hand, in the human plasma samples containing NMN at a final concentration of 1 μM, the average NMN concentration quantified after recovery was 0.79 μM (relative standard deviation 0.13). In other words, 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 shows a recovery rate of NMN that can be applied to the measurement of NMN in blood samples.

[0034] Example 3: Detection of NMN in cosmetic samples The NMN concentration was measured for cosmetic A, which is labeled as containing NMN. 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 for 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 the standard addition method. As a standard sample in the standard addition method, 50 μL of a solution obtained by diluting cosmetic A 1000 times with water was added to 50 μL of a 2.0 μM NMN aqueous solution (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 values ​​and calculated NMN concentrations for each sample are shown in the table below. It was shown that cosmetic A before dilution contained NMN at a concentration of 0.04% (v / v). [Table 5] [Industrial Applicability]

[0035] The method for detecting and measuring NMN concentration of the present invention shows sufficient quantitative performance in the analysis of blood samples, and the method enables the measurement of NMN concentration in other samples as well. The method can be applied to various fields, such as elucidating the pharmacokinetics of NMN and quantifying NMN contained in foods and cosmetics.

Claims

1. 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. 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. 3. The method of claim 2, wherein the mobile phase utilizes an increasing gradient of acetonitrile ranging from 0 to 40% (v / v).

4. The method according to any one of claims 1 to 3, wherein the sample is selected from blood, urine, cosmetics, and food.

5. The method according to any one of claims 1 to 4, further comprising a step of quantifying the separated nicotinamide mononucleotide in the step of separating nicotinamide mononucleotide in the sample by mass spectrometry.

6. The method of claim 5 , wherein the mass spectrometry is tandem mass spectrometry.

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