A detection method and application of β-nicotinamide mononucleotide and related substances

Abstract

The present invention belongs to the field of nuclear magnetic quantitative analysis and detection technology, and specifically relates to a detection method and application of β-nicotinamide mononucleotide and related substances. The detection method provided by the present invention can be detected in a "one-pot method" without separation, and can quantify 6 related compounds at the same time, with short detection time and high efficiency. In addition, the nuclear magnetic spectrum signal peaks of different compounds all appear in groups, and a single peak can be judged as a non-test compound, so there will be no false positives. Provide strong technical support for the production and development, quality control, safety testing, market supervision, etc. of subsequent NAD supplement-related products.

Description

Technical Field

[0001] The present invention belongs to the technical field of nuclear magnetic quantitative analysis and detection, and specifically relates to a detection method and application of β-nicotinamide mononucleotide and related substances. Background Art

[0002] β-Nicotinamide mononucleotide (NMN) is the biosynthetic precursor of nicotinamide adenine dinucleotide (NAD, coenzyme I). NAD is a derivative of vitamin PP (niacin and nicotinamide), a coenzyme that plays an important role in glycolysis, gluconeogenesis, tricarboxylic acid cycle, i.e. respiratory chain, and is widely involved in metabolism, aging, cell death, DNA repair, gene expression and other life activities.

[0003] NMN-related substances include the biosynthesis and decomposition metabolites of NMN, which in the present invention refer to nicotinic acid (NA), nicotinamide (NAM), β-nicotinamide riboside (NR), β-nicotinamide adenine dinucleotide (NAD) or coenzyme I for short, and reduced nicotinamide adenine dinucleotide or reduced coenzyme I (NADH) for short.

[0004] Studies have shown that NAD plays an important role in aging and longevity. NA, NAM, NMN, and NR can all supplement NAD through different metabolic pathways. NADH, as the reduced state of NAD, coexists with NAD in a certain proportion and serves as a substrate for various redox reactions to maintain the body's redox homeostasis. Exogenous supplementation of NA, NAM, NMN, NR, NAD, and NADH may increase the level of NAD in the body, thereby producing certain efficacy.

[0005] In order to further study the effects of NAD supplements and control the food safety and quality of related food-derived supplements, the detection technology of NA, NAM, NMN, NR, NAD and NADH in supplements has received more and more attention from the inspection, testing and certification service industry. At present, the focus of food-derived NAD supplement testing is mainly on NMN, and there is no attention to the detection of related substances, making market supervision and quality control of NAD supplement-related products a major problem. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defect in the prior art that NMN and its related substances cannot be detected simultaneously, thereby providing a detection method and application of β-nicotinamide mononucleotide and its related substances.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting β-nicotinamide mononucleotide and related substances thereof, comprising the following steps:

[0009] S1, prepare a heavy water solution of TSP as an internal standard solution;

[0010] S2, using the internal standard solution to prepare different concentrations of NA, NAM, NMN, NR, NAD, and NADH standard working solutions;

[0011] S3, dissolving the sample to be tested by using the internal standard solution, centrifuging, and filtering to obtain a sample solution to be tested;

[0012] S4, setting the operating parameters of the nuclear magnetic resonance test, taking a certain concentration of NA, NAM, NMN, NR, NAD, NADH standard working solution for hydrogen spectrum detection, and determining the characteristic peaks of each component to be tested;

[0013] S5, taking different concentrations of NA, NAM, NMN, NR, NAD, NADH standard working solutions for hydrogen spectrum detection, and drawing standard curves after data processing;

[0014] S6, taking the sample solution to be tested for hydrogen spectrum detection, analyzing the hydrogen spectrum signal peaks and attributing them, and obtaining the concentrations of each component NA, NAM, NMN, NR, NAD, and NADH to be tested in the sample solution according to the above-mentioned standard curve and the determined characteristic peaks, and calculating the content of each component NA, NAM, NMN, NR, NAD, and NADH to be tested in the sample.

[0015] Optionally, in step S4, the characteristic peaks of each component to be tested are determined to be NA, 9.12±0.05ppm; NAM, 7.58±0.05ppm; NMN, 9.47±0.05ppm; NR, 9.62±0.05ppm; NAD, 9.40±0.05ppm; NADH, 6.95±0.05ppm. Affected by the pH of the test solution, the signal peak will drift slightly, generally within ±0.05ppm.

[0016] Optionally, the operating parameters of the nuclear magnetic resonance test in step S4 include acquisition times, relaxation time, pulse width, sampling temperature, pulse angle, and spectrum width.

[0017] Optionally, the number of acquisitions is greater than or equal to 8 times; Optionally, the number of acquisitions is 8-32 times;

[0018] And / or, the relaxation time is greater than or equal to 60s; optionally, the relaxation time is 60-70s.

[0019] Optionally, other operating parameters of the NMR test can be set according to the instrument model or the selected pulse sequence. Typically, but not limiting, the pulse sequence of the selected nuclear magnetic resonance hydrogen spectrum (PROTON) is the measurement parameters:

[0020] The sampling temperature is set to 298 K, the spectral width is 16 ppm, the pulse width is 9.6 μs, and the pulse angle is 90 degrees. Preferably, the number of acquisitions is 8 times, and the relaxation time is 60 s.

[0021] Optionally, the concentration of each standard working solution in step S2 is 0.1-100 mmol / L;

[0022] Optionally, the concentration of the standard working solution of different concentrations is 0.1mmol / L, 0.2mmol / L, 1mmol / L, 2mmol / L, 10mmol / L, 20mmol / L, 100mmol / L or 50mmol / L.

[0023] Optionally, in step S1, the concentration of TSP in the internal standard solution is above 0.1 mmol / L; preferably, the concentration of TSP in the internal standard solution is 0.1-10 mmol / L.

[0024] Optionally, in step S3, filtration is performed using a 0.22 or 0.45 μm filter membrane; preferably, filtration is performed using a 0.22 μm filter membrane.

[0025] Optionally, the concentration of the standard working solution of a certain concentration in step S4 is 0.1-100 mmol / L.

[0026] Optionally, the standard curve in step S5 takes the concentration of the component to be measured in the standard working solution as the abscissa and takes the ratio of the peak area of ​​the characteristic peak of the component to be measured to the TSP peak area as the ordinate.

[0027] Optionally, the content of the component to be tested is calculated according to the following formula:

[0028]

[0029] X-the content of NA, NAM, NMN, NR, NAD or NADH in the sample, in milligrams per 100 grams (mg / 100g);

[0030] ω-the concentration of NA, NAM, NMN, NR, NAD or NADH in the sample solution to be tested obtained from the standard curve, in millimoles per liter (mmol / L or mM);

[0031] V-total volume of internal standard solution used to dissolve the sample, in milliliters (mL);

[0032] m-the weight of the sample, in milligrams (mg);

[0033] M-relative molecular mass of the component to be measured, in grams per mole (g / mol).

[0034] Optionally, the sample to be tested is a health food, a dietary supplement, a food additive, a health food raw material or a cosmetic.

[0035] Typically, but not limiting, the amount of sample to be taken can be determined during the preparation of the sample solution according to the content of the component to be measured, for example:

[0036] Health food and dietary supplements: Take 20 samples. If it is a capsule preparation, remove the capsule shell. Grind it into powder. According to the concentration of NA, NAM, NMN, NR, NAD and NADH in the sample to be tested, accurately weigh 10 mg (accurate to 0.01 mg). Add 1 mL of TSP heavy water solution to a 2 mL EP centrifuge tube. Vortex or sonicate to fully dissolve it. After centrifugation, take the supernatant and pass it through a 0.22 μm membrane. Transfer it to a 5 mm NMR tube for testing.

[0037] Food additives, health food raw materials (NMN raw materials): accurately weigh 20 mg of sample, put it into a 2 mL EP centrifuge tube, add 1 mL of TSP heavy water solution, vortex or ultrasonicate to fully dissolve it, centrifuge at 4500 r / min, take the supernatant and pass it through a 0.22 μm membrane, and transfer it to a 5 mm NMR tube for testing.

[0038] The technical solution of the present invention has the following advantages:

[0039] The detection method of β-nicotinamide mononucleotide and related substances provided by the present invention comprises the following steps: S1, preparing a heavy water solution of TSP as an internal standard solution; S2, using the internal standard solution to prepare NA, NAM, NMN, NR, NAD, NADH standard working solutions of different concentrations; S3, using the internal standard solution to dissolve the sample to be tested, centrifuge, and filter to obtain the sample solution to be tested; S4, setting the operating parameters of the nuclear magnetic resonance test, taking a certain concentration of NA, NAM, NMN, NR, NAD, NADH standard working solutions for hydrogen spectrum detection, and determining Characteristic peaks of each component to be tested; S5, respectively take different concentrations of NA, NAM, NMN, NR, NAD, NADH standard working solutions for hydrogen spectrum detection, and draw a standard curve after data processing; S6, take the sample solution to be tested for hydrogen spectrum detection, analyze the hydrogen spectrum signal peaks and attribute them, and obtain the concentrations of each component to be tested NA, NAM, NMN, NR, NAD, NADH in the sample solution to be tested according to the above standard curve and the determined characteristic peaks, and calculate the content of each component to be tested NA, NAM, NMN, NR, NAD, NADH in the sample to be tested. The detection method provided by the present invention can be detected by "one pot method" without separation, and 6 related compounds can be quantitatively measured at the same time, with short detection time and high efficiency. In addition, the nuclear magnetic spectra of different compounds all appear in groups, and a single peak can be judged as a non-test compound, so there will be no false positives. Provide strong technical support for the production and development, quality control, safety testing, market supervision, etc. of subsequent NAD supplement-related products.

[0040] The detection method of β-nicotinamide mononucleotide and related substances provided by the present invention can further reduce the signal-to-noise ratio and improve the accuracy of the results by limiting the operating parameters of the nuclear magnetic resonance test, especially limiting the relaxation time and the number of acquisitions. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 It is a graph showing the change trend of the peak area ratio of the characteristic peak of NMN to the internal standard peak and the relaxation time in the embodiment of the present invention;

[0043] Figure 2 It is a trend diagram of the peak area ratio between the characteristic peak and the internal standard peak of NMN and the number of acquisitions in the embodiment of the present invention;

[0044] Figure 3 is the H NMR spectrum of NMN in the embodiment of the present invention;

[0045] Figure 4 is the H NMR spectrum of NAD in the embodiment of the present invention;

[0046] Figure 5 is the H NMR spectrum of NADH in the embodiment of the present invention;

[0047] Figure 6 is the H NMR spectrum of NR in the embodiment of the present invention;

[0048] Figure 7 is the H NMR spectrum of NA in the embodiment of the present invention;

[0049] Figure 8 is the H NMR spectrum of NAM in the embodiment of the present invention;

[0050] Fig. 9 is the H NMR spectrum of TSP in the embodiment of the present invention;

[0051] Fig.10 is the H NMR spectrum of MLA in an embodiment of the present invention;

[0052] Fig.11 is the H NMR spectrum of SCA in an embodiment of the present invention;

[0053] Fig.12 is the H NMR spectrum of the 10 mM standard working solution in the embodiments of the present invention;

[0054] Fig.13 is a standard working curve obtained in an embodiment of the present invention;

[0055] Fig.14 is the H NMR spectrum of sample 1 in the embodiment of the present invention;

[0056] Fig.15 is the H NMR spectrum of sample 2 in the embodiment of the present invention;

[0057] Fig.16 is the H NMR spectrum of sample 3 in the embodiment of the present invention;

[0058] Fig.17 is the H NMR spectrum of sample 4 in the embodiment of the present invention;

[0059] Fig.18 is the H NMR spectrum of sample 5 in the embodiment of the present invention;

[0060] Fig.19 It is the H NMR spectrum of sample 6 in the embodiment of the present invention. DETAILED DESCRIPTION

[0061] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0062] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0063] Example

[0064] This embodiment provides a method for detecting β-nicotinamide mononucleotide and related substances, comprising the following steps:

[0065] (I) The detection principle is: use a heavy water solution of 2,2,3,3-deuterated trimethylsilyl propionate (TSP) as an internal standard solution to fully dissolve the substance to be tested, and use nuclear magnetic resonance spectroscopy (NMR) to detect the heavy water solution of the sample, using TSP as the chemical shift zero point (δ=0.00ppm) and the standard sample and the characteristic peak integral reference of the component to be tested, and quantification by external standard method.

[0066] (II) Scope of application: It is suitable for quantitative determination of niacin (NA), nicotinamide (NAM), β-nicotinamide riboside (NR), β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH) in food additives, health food raw materials, health foods and dietary nutritional supplements by nuclear magnetic resonance spectroscopy (NMR).

[0067] (III) Reagents and Materials

[0068] (1) Heavy water (D2O): Deuterium substitution degree 99.9%, brand: Cambridge Isotope Laboratories, Inc.

[0069] (2) Sodium 2,2,3,3-deuterated trimethylsilyl propionate (TSP): 98 atom% D, brand: Cambridge Isotope Laboratories, Inc.

[0070] (3) β-Nicotinamide mononucleotide (NMN) standard: β-Nicotinamide mononucleotide, CAS No.: 1094-61-7, 99%, brand: Macklin, product number: B886299.

[0071] (4) β-Nicotinamide adenine dinucleotide (NAD) standard: β-Nicotinamide adenine dinucleotide, CAS No.: 53-84-9, >98.0%, brand: Macklin, product number: N1511.

[0072] (5) β-Nicotinamide riboside (NR) standard: Nicotinamide riboside chloride (NRC), CAS No.: 23111-00-4, ≥98%, brand: Macklin, product number: N889451.

[0073] (6) Nicotinamide (NAM) standard: Nicotinamide, CAS No.: 98-92-0, ≥ 99.5%, brand: Macklin, product number: N814696.

[0074] (7) Nicotinic acid (NA) standard: Nicotinic acid, CAS No.: 59-67-6, 99%, brand: Macklin, product number: N814565.

[0075] (8) Reduced coenzyme I (NADH) standard: reduced coenzyme I disodium salt, CAS No.: 606-68-8, 98%, brand: Macklin, product standard: N814671.

[0076] (IV) Instruments and Equipment

[0077] (1) Nuclear magnetic resonance spectrometer (Agilent Technologies 600MHz DD2) equipped with OneNMR5mm broadband two-in-one liquid probe; 7510 autosampler.

[0078] (2) Analytical balance (METTLER TOLEDO XS104): sensitivity 0.01 mg.

[0079] (3) Vortex mixer (AS ONE TRIO TM-1N).

[0080] (4) Adjustable pipette (Eppendorf): 20-100 μL; 100-1000 μL.

[0081] (5) Desktop high-speed microcentrifuge (Dalong D2012plus): 2ml×12, 500r / min-15000r / min.

[0082] (6) Ultrasonic instrument (Kunshan Shumei KQ5200DE): 200W; 10L; 40KHz general type.

[0083] (7) NMR tube (NORELL): 5 mm.

[0084] (V) Analysis steps

[0085] 1. Preparation of TSP heavy water solution (internal standard solution)

[0086] Use a brown volumetric flask to accurately weigh 68.91 mg of TSP solid powder, dissolve it in heavy water and make up to 100 mL to obtain a 4 mmol / L TSP heavy water solution, and store it at room temperature away from light.

[0087] 2. Preparation of standard intermediate solution

[0088] Before testing on the machine, weigh 12.21 mg NAM, 29.07 mg NRC, 12.31 mg NA, 33.42 mg NMN, 33.17 mg NAD, and 70.94 mg NADH respectively into a 2 mL EP centrifuge tube, add 1 mL 4 mmol / L TSP heavy water solution, vortex to fully mix, and obtain the following concentrations of standard intermediate solutions:

[0089] NAM: 100mmol / L;

[0090] NR: 100mmol / L;

[0091] NA: 100mmol / L;

[0092] NMN: 100mmol / L;

[0093] NAD: 50mmol / L;

[0094] NADH: 100mmol / L.

[0095] 3. Preparation of standard working solution

[0096] The following concentrations of NA, NAM, NMN, NR, NADH, and NAD standard working solutions were prepared in 2 mL EP centrifuge tubes by stepwise dilution. The prepared test solutions were mixed evenly by vortexing and transferred to 5 mm NMR tubes for testing.

[0097] 20mmol / L: 200μL 100mmol / L standard intermediate solution + 800μL TSP heavy water solution;

[0098] 10mmol / L: 500μL 20mmol / L standard working solution + 500μL TSP heavy water solution;

[0099] 2mmol / L: 200μL 10mmol / L standard working solution + 800μL TSP heavy water solution;

[0100] 1mmol / L: 500μL 2mmol / L standard working solution + 500μL TSP heavy water solution;

[0101] 0.2mmol / L: 200μL 1mmol / L standard working solution + 800μL TSP heavy water solution;

[0102] 0.1mmol / L: 500μL 0.2mmol / L standard working solution + 500μL TSP heavy water solution;

[0103] 20mmol / L of NAD: 400μL 50mmol / L standard intermediate solution + 600μL TSP heavy water solution.

[0104] 4. Preparation of sample solution

[0105] Health food or dietary supplement: Take 20 samples. If the sample is a capsule preparation, remove the capsule shell. Grind it into powder. According to the concentration of NA, NAM, NMN, NR, NAD, and NADH in the sample, accurately weigh 10 mg (accurate to 0.01 mg). Add 1 mL of TSP heavy water solution to a 2 mL EP centrifuge tube. Vortex or sonicate to fully dissolve it. Vortexing is used in this embodiment. After centrifugation, take the supernatant and filter it through a 0.22 μm membrane, then transfer it to a 5 mm NMR tube for testing.

[0106] Food additives or health food raw materials (NMN raw materials): accurately weigh 20 mg of sample, add 1 mL of TSP heavy water solution into a 2 mL EP centrifuge tube, vortex or ultrasonicate to fully dissolve it. In this embodiment, ultrasonication is used. After centrifugation, take the supernatant and pass it through a 0.22 μm membrane, and then transfer it to a 5 mm NMR tube for testing.

[0107] 5. Instrument parameters

[0108] The pulse sequence of nuclear magnetic resonance hydrogen spectrum (PROTON) was selected, and the measurement parameters were:

[0109] The sampling temperature was set to 298K, the spectral width (Spectral Width) to 16ppm, the pulse width (Observe Pulse) to 9.6μs, and the pulse angle (Observe Degrees) to 90 degrees.

[0110] (1) Setting of relaxation time parameters

[0111] NMR detection requires a sufficiently long relaxation delay (d1) to allow the protons excited by the NMR pulse to completely recover to the ground state before the next NMR pulse excitation. The present invention uses 10s as the interval for setting the relaxation time parameter and investigates the change trend of the peak area ratio between the characteristic peak of NMN and the internal standard peak under the conditions of setting the relaxation time parameter from 30s to 70s. Figure 1 As shown, when the relaxation time is above 60 s, the peak area ratio and the first-order derivative of the relaxation time infinitely approach 0. In order to improve the test efficiency, 60 s is selected as the instrument parameter setting value of the relaxation time of the present invention.

[0112] (2) Setting of scanning acquisition parameters

[0113] The number of scans requested of NMR is closely related to the signal-to-noise ratio of the test results. The more scans requested, the lower the signal-to-noise ratio and the longer the test time. The present invention investigates the trend of the peak area ratio between the characteristic peak of NMN and the internal standard peak when the number of scans requested is set to 2, 4, 8, 16, and 32, respectively. Figure 2 As shown, when the scanning acquisition number is set to 8 or more, the peak area ratio and the first-order derivative of the relaxation time infinitely approach 0. In order to improve the test efficiency, 8 is selected as the instrument parameter setting value of the scanning acquisition number of the present invention.

[0114] 6. Data processing

[0115] The MestReNova software was used to process the collected NMR wave signals, and the NMR software Vnmrj was used to automatically adjust the phase and baseline correction. The internal standard TSP signal peak was taken as the zero point of the chemical shift, and the TSP and the characteristic peaks of each component to be measured were integrated.

[0116] 7. Qualitative analysis

[0117] Using a standard intermediate solution of appropriate concentration (there is no special requirement for the concentration as long as it is above the detection limit of the instrument, for example, it can be 0.1-100 mmol / L, 10 mmol / L in this embodiment), hydrogen spectrum detection is performed on the components to be tested NMN, NAD, NADH, NR, NA, and NAM, respectively, and their hydrogen spectrum signals are collected. The spectra are analyzed respectively, the chemical shift, splitting and ratio of the hydrogen signal peaks are summarized, and the hydrogen spectrum signal peaks are analyzed and attributed.

[0118] (1) The NMR spectrum of NMN is as follows Figure 3 As shown, 1H NMR(600MHz,D2O)δ9.47(s,1H),9.30(d,J=6.3Hz,1H),8.99(dt,J=8.2,1.5Hz,1H),8.31(dd,J=8.1,6.3Hz,1H),6.23(d,J=5.4Hz,1H),4.65 (t,J=2.6Hz,1H), 4.57(t,J=5.2Hz,1H), 4.45(dd,J=5.1,2.6Hz,1H), 4.31(ddd,J=11.9,4.4,2.4Hz,1H), 4.16(ddd,J=11.9,5.1,2.1Hz,1H).

[0119] (2) The NMR spectrum of NAD is as follows Figure 4 As shown, 1 H NMR(600MHz,D2O)δ9.40(s,1H),9.29(d,J=6.3Hz,1H),8.95(dt,J=7.9,1.6Hz,1H),8.59( s,1H),8.39(s,1H),8.31(dd,J=8.0,6.3Hz,1H),6.19(d,J=5.5Hz,1H),6.13(d,J=5.4Hz,1 H), 4.74 (t, J = 5.3Hz, 1H), 4.62 (t, J = 2.6Hz, 1H), 4.58 (t, J = 5.3Hz, 1H), 4.53 (t, J = 4.5Hz, 1H), 4.49 (dd, J = 5.1, 2.7Hz, 1H), 4.44-4.38 (m, 2H), 4.32-4.26 (m, 2H), 4.26-4.20 (m, 1H).

[0120] (3) The NMR spectrum of NADH is as follows Figure 5 As shown, 1 H NMR(600MHz,D2O)δ8.46(s,1H),8.16(s,1H),6.95(s,1H),6.11(d,J=5.4Hz,1H), 6.00(d,J=7.9Hz,1H),4.79-4.75(m,2H),4.71(t,J=5.2Hz,1H),4.53(t,J=4.6Hz ,1H),4.40(d,J=3.8Hz,1H),4.32–4.26(m,1H),4.25(d,J=5.6Hz,2H),4.23-4.18 (m,1H),4.13-4.08(m,3H),2.78(d,J=18.0Hz,1H),2.69(dd,J=18.1,3.2Hz,1H).

[0121] (4) The H NMR spectrum of NR is as follows Figure 6As shown, 1 H NMR(600MHz,D2O)δ9.62(s,1H),9.28(d,J=6.3Hz,1H),8.99(d,J=8.0Hz,1H),8.29(dd,J=8.0,6.3Hz,1H),6.26(d,J=4.5Hz,1H) ,4.53(t,J=4.8Hz,1H), 4.49(q,J=3.6Hz,1H), 4.37(t,J=4.7Hz,1H), 4.06(dd,J=12.9,2.9Hz,1H), 3.91(dd,J=12.9,3.6Hz,1H).

[0122] (5) The H NMR spectrum of NA is as follows Figure 7 As shown, 1 H NMR (600MHz, D2O) δ9.12 (s, 1H), 8.90 (d, J = 8.1, 1H), 8.84 (d, J = 5.8 Hz, 1H), 7.95 (dd, J = 8.1, 5.8 Hz, 1H).

[0123] (6) The H NMR spectrum of NAM is as follows Figure 8 As shown, 1 H NMR (600MHz, D2O) δ8.91 (s, 1H), 8.70 (d, J = 5.0 Hz, 1H), 8.23 ​​(d, J = 8.1 Hz, 1H), 7.58 (dd, J = 8.0, 5.1 Hz, 1H).

[0124] 8. Quantitative Analysis

[0125] (1) Internal standard compound

[0126] Select a suitable internal standard compound and collect the H NMR spectrum signal of the internal standard compound. The principle of selecting the internal standard compound is stability, simple signal, and no overlap with the H NMR signal peak of the target compound. In this technical solution, the H NMR spectra of 2,2,3,3-deuterated trimethylsilyl propionate (TSP), maleic acid (MLA) and succinic acid (SCA) were compared, and TSP was selected as the internal standard for chemical shift positioning. Fig. 9 is the H NMR spectrum of TSP, 1 H NMR (600MHz, D2O) δ0.00 (s, 9H). It can be seen from the figure that the signal peak does not overlap with the signal peak of the chemical component to be measured. TSP can be used as an internal standard for chemical shift positioning and for content calculation.

[0127] Fig.10 is the H NMR spectrum of maleic acid (MLA), 1H NMR (600MHz, D2O) δ6.39 (s, 2H). It can be seen from the figure that NMN, NAD, and NADH have signal peaks in the range of 6.0-6.5ppm, which are easy to be confused; Fig.11 is the H NMR spectrum of succinic acid (SCA), 1 HNMR (600MHz, D2O) δ2.69 (s, 4H). As can be seen from the figure, NADH has a signal peak of 2.69. Therefore, these two compounds were not used as internal standards.

[0128] (2) Quantitative characteristic peaks

[0129] When selecting characteristic peaks, the integrity of the peak shape should be considered. There should be enough chemical shift difference between peaks. The peaks should not overlap with the signal peaks of other targets and should not be interfered by impurities. According to the above principles, the chemical shift δ of the characteristic peaks of NMN, NAD, NADH, NR, NA, and NAM are selected as 9.47ppm, 9.40ppm, 6.95ppm, 9.62ppm, 9.12ppm, and 7.58ppm, respectively.

[0130] Fig.12 This is the nuclear magnetic hydrogen spectrum of the 10mM standard working solution. The quantitative characteristic peaks of each test compound in the figure are: 1. NR, 9.62ppm; 2. NMN, 9.47ppm; 3. NAD, 9.40ppm; 4. NA, 9.12ppm; 5. NAM, 7.58ppm; 6. NADH, 6.95ppm.

[0131] (3) Standard working curve

[0132] Under the instrument parameter settings, hydrogen spectrum detection was performed on the standard working solutions of NA, NAM, NMN, NR, NADH, and NAD. After data processing, the standard working curves of each compound to be tested were drawn with the millimole per liter concentration of the compound in the standard working solution as the horizontal axis and the ratio of the characteristic peak area of ​​the compound to the TSP peak area as the vertical axis. The intercept of each standard curve was set to 0.0.

[0133] (4) Sample measurement

[0134] Under the same instrument conditions, the pre-treated sample was injected for NMR analysis. The chemical shift, splitting and ratio of the hydrogen signal peak of the standard were used for qualitative analysis, and the ratio of the quantitative characteristic peak to the TSP internal standard peak area was used for quantitative analysis by external standard method. The concentration of the component to be measured in the sample solution was calculated according to the linear regression equation.

[0135] If the concentration of the component to be measured in the sample solution exceeds the corresponding linear range, the added volume of TSP heavy water solution should be appropriately increased and the measurement should be repeated.

[0136] (5) Presentation of analysis results

[0137] The content of the component to be tested (NA, NAM, NMN, NR, NAD or NADH) in the sample is calculated according to the following formula:

[0138]

[0139] X-the content of NA, NAM, NMN, NR, NAD or NADH in the sample, in milligrams per 100 grams (mg / 100g);

[0140] ω - the concentration of NA, NAM, NMN, NR, NAD or NADH in the sample TSP heavy water solution obtained from the standard curve, in millimoles per liter (mmol / L or mM);

[0141] V-total volume of TSP heavy water solution in which the sample is dissolved, in milliliters (mL);

[0142] 100 - conversion factor for converting the result from milligrams per gram to milligrams per hundred grams of sample;

[0143] m-the weight of the sample, in milligrams (mg);

[0144] M-relative molecular weight of the component to be tested, in grams per mole (g / mol). Among them, NMN 334.22g / mol, NAD 663.43g / mol, NR 255.25g / mol, NAM 122.12g / mol, NA 123.11g / mol, NADH 709.6g / mol.

[0145] The calculation result shall be rounded to two decimal places.

[0146] (6) Precision

[0147] The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0148] (VI) Sample testing

[0149] 1. Standard working curve

[0150] According to the "(V) Analysis Step" of this case, standard working solutions of NA, NAM, NMN, NR, NAD, and NADH were prepared, instrument parameters were set, NMR measurements were performed, and standard working curves for quantification were prepared, such as Fig.13 As shown:

[0151] NMN showed a good linear relationship in the concentration range of 0.1mM to 100mM, and the linear regression equation was y=0.0358x(R 2 =1.0000).

[0152] NAD showed a good linear relationship in the range of 0.1mM to 50mM, and the linear regression equation was y = 0.0330x (R 2 =1.0000).

[0153] NADH showed a good linear relationship in the range of 0.2mM to 100mM, and the linear regression equation was y = 0.0339x (R 2 =0.9999).

[0154] NR showed a good linear relationship in the range of 0.2mM to 100mM, and the linear regression equation was y = 0.0335x (R 2 =1.0000).

[0155] NA showed a good linear relationship in the range of 0.1mM to 100mM, and the linear regression equation was y = 0.0361x (R 2 =1.0000).

[0156] NAM showed a good linear relationship in the range of 0.2mM to 100mM, and the linear regression equation was y = 0.0361x (R 2 =0.9999).

[0157] 2. Measurement results

[0158] (1) The present invention tested 3 commercially available NMN capsules / tablets (samples 1-3) and 2 multivitamin health foods containing NAM (samples 4-5). The samples were pre-treated according to the "(V) analysis step" of the present invention, and the injected test solution was subjected to NMR detection under the same instrument conditions as the standard working curve test. The nuclear magnetic hydrogen spectrum was as follows Figure 14-18 The test result is expressed in units, which are converted to the value units of the nutrition label of the test sample, mg / capsule.

[0159] Table 1. Actual sample test results

[0160]

[0161] (2) The present invention prepares a mixed sample (sample 6) containing six substances to be tested at the same time. The sample is pre-treated according to the "(V) analysis step" of the present invention. The injected test solution is subjected to NMR detection under the same instrument conditions as the standard working curve test. The prepared content and the detected content are shown in Table 2. The nuclear magnetic hydrogen spectrum is as follows Fig.19 shown.

[0162] Table 2 Preparation content and test results of sample 6

[0163] Sample 6 Preparation content (mmol / L) Test content (mmol / L) RSD(%) NA 14.21 14.52 0.22 NAM 12.45 12.80 0.43 NR 6.09 6.02 3.67 NMN 7.48 7.32 2.82 NAD 3.89 3.96 3.13 NADH 3.09 3.06 4.59

[0164] The internal standard compound selected by the present invention is sodium 2,2,3,3-deuterated trimethylsilyl propionate (TSP), which is commonly used as a chemical shift reference reagent for aqueous systems. The present invention proposes to use this compound as an internal standard compound for the first time, and its advantage is that its signal peak position does not overlap with other compounds under normal circumstances. Therefore, the selection of this internal standard can be said to be "killing two birds with one stone", which is not only a chemical shift reference reagent, but also can be used as a detection internal standard.

[0165] The selection of relaxation time in the present invention is the key to ensuring accurate detection results. This is because, in the process of nuclear magnetic resonance spectroscopy detection, each pulse excitation must wait until the previous excitation is completely restored to the ground state before the next excitation, otherwise it will cause a proportional deviation of the signal peak. Therefore, the time setting of d1 is the key to accurate detection results, and in the present invention it must be greater than 60s.

[0166] In the present invention, the increase in the number of scans can reduce the signal-to-noise ratio, and the number of scans in the present invention needs to be greater than 8.

[0167] The six test compounds in the present invention are all highly water-soluble, so heavy water is selected as the deuterated reagent, which is cheaper and more environmentally friendly than other deuterated organic solvents.

[0168] The characteristic peaks of the target compounds in the present invention should be selected as simple as possible, with less split signal peaks, and no overlap with other target compounds and internal standards. Therefore, the characteristic peak chemical shifts δ of NMN, NAD, NADH, NR, NA, and NAM were selected as 9.47ppm, 9.40ppm, 6.95ppm, 9.62ppm, 9.12ppm, and 7.58ppm, respectively.

[0169] The detection method provided by the present invention can be used for the simultaneous determination of six compounds (NMN, NAD, NADH, NR, NA, NAM) of β-nicotinamide mononucleotide and its metabolic related substances in health foods, dietary supplements, and health food raw materials.

[0170] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A method for detecting β-nicotinamide mononucleotide and related substances, characterized in that: The steps include: S1, prepare a heavy water solution of TSP as an internal standard solution; S2, using the internal standard solution to prepare different concentrations of NA, NAM, NMN, NR, NAD, and NADH standard working solutions; S3, dissolving the sample to be tested by using the internal standard solution, centrifuging, and filtering to obtain a sample solution to be tested; S4, setting the operating parameters of the nuclear magnetic resonance test, taking a certain concentration of NA, NAM, NMN, NR, NAD, NADH standard working solution for hydrogen spectrum detection, and determining the characteristic peaks of each component to be tested; Among them, the characteristic peaks of each component to be tested are NA, 9.12±0.05ppm; NAM, 7.58±0.05ppm; NMN, 9.47±0.05ppm; NR, 9.62±0.05ppm; NAD, 9.40±0.05ppm; NADH, 6.95±0.05ppm; S5, taking different concentrations of NA, NAM, NMN, NR, NAD, NADH standard working solutions for hydrogen spectrum detection, and drawing standard curves after data processing; S6, taking the sample solution to be tested for hydrogen spectrum detection, analyzing the hydrogen spectrum signal peaks and attributing them, and obtaining the concentrations of each component NA, NAM, NMN, NR, NAD, and NADH to be tested in the sample solution according to the above-mentioned standard curve and the determined characteristic peaks, and calculating the content of each component NA, NAM, NMN, NR, NAD, and NADH to be tested in the sample.

2. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 1, characterized in that: The operating parameters of the NMR test in step S4 include acquisition times, relaxation time, pulse width, sampling temperature, pulse angle, and spectrum width.

3. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 2, characterized in that: The number of acquisitions is greater than or equal to 8 times.

4. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 2, characterized in that: The relaxation time is greater than or equal to 60 s.

5. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 2, characterized in that: The concentration of each standard working solution in step S2 is 0.1-100 mmol / L.

6. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 2, characterized in that: In step S1, the concentration of TSP in the internal standard solution is greater than 0.1 mmol / L.

7. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 2, characterized in that: The concentration of the standard working solution in step S4 is 0.1-100 mmol / L.

8. The method for detecting β-nicotinamide mononucleotide and related substances thereof according to any one of claims 2 to 7, characterized in that: The number of acquisitions is 8-32 times; And / or, the relaxation time is 60-70s; And / or, the concentrations of the standard working solutions of different concentrations in step S2 are 0.1mmol / L, 0.2mmol / L, 1mmol / L, 2mmol / L, 10mmol / L, 20mmol / L, 100mmol / L; And / or, the concentration of TSP in the internal standard solution in step S1 is 0.1-10 mmol / L.

9. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 1, characterized in that: The standard curve in step S5 takes the concentration of the component to be measured in the standard working solution as the abscissa and the ratio of the peak area of ​​the characteristic peak of the component to be measured to the TSP peak area as the ordinate.

10. The method for detecting β-nicotinamide mononucleotide and related substances according to claim 1, characterized in that: The content of the component to be tested is calculated according to the following formula: X - The content of NA, NAM, NMN, NR, NAD or NADH in the sample, in milligrams per hundred grams; ω - the concentration of NA, NAM, NMN, NR, NAD or NADH in the sample solution to be tested obtained from the standard curve, in millimoles per liter; V-total volume of internal standard solution used to dissolve the sample, in milliliters; m-the weight of the sample, in milligrams; M-relative molecular mass of the component to be measured, in grams per mole.

11. The method for detecting β-nicotinamide mononucleotide and related substances thereof according to any one of claims 1 to 10, characterized in that: The sample to be tested is a health food, a dietary nutritional supplement, a food additive, a health food raw material or a cosmetic.

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