A method for simultaneously determining the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food

Abstract

The present invention belongs to the technical field of analysis and detection, and discloses a method for simultaneously determining the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food. The detection principle of the present invention is: NMN and NAD+ in food are ultrasonically extracted with 5% methanol aqueous solution, determined by liquid chromatography triple quadrupole mass spectrometry, quantitatively determined by external standard method, and qualitatively determined by retention time and ion fragment abundance ratio. The present invention can simultaneously and rapidly detect the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food, and the method has high sensitivity, good linear range, good repeatability, and accurate and reliable results. The method can be applied to the detection of the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in different matrix foods, used for product quality control, and meet the needs of the domestic market for the supervision of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food. Background Art

[0002] NMN is the abbreviation of nicotinamide mononucleotide. It exists naturally in living cells and is a precursor of NAD+ (also called oxidized coenzyme I, full name nicotinamide adenine dinucleotide) and a derivative of vitamin B3. NAD+ is the product of the conversion of NMN and AMP in the body. NAD+ exists in cells and participates in thousands of biocatalytic reactions, participates in the synthesis of 95% of the human body's energy, and is an indispensable coenzyme in the human body. In a variety of cellular metabolic reactions, nicotinamide adenine dinucleotide (NAD) molecules play an important role and are an important support for cells to maintain vitality.

[0003] At present, there are many products with NMN added that are sold in the domestic market through cross-border e-commerce. At the same time, there are also a large number of domestic manufacturers producing and developing such foods with NMN as the main raw material. However, nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD+) have not yet obtained drug, health food, food additive and new food raw material licenses in my country. That is, in my country, neither NMN nor NAD+ can be produced and sold as food. On January 13, 2021, the Food Operation Department of the State Administration for Market Regulation issued the letter of the Municipal Supervision and Food Administration (Department) No. (2021) 4 "Letter on the Investigation of Illegal Operation of "Immortality Drugs", clarifying that NMN cannot be produced and sold as food in my country. Since the State Administration for Market Regulation issued the letter to investigate NMN, although there are still foods with NMN as a selling point on the market, the number has decreased. However, foods with NAD+ as a selling point have grown rapidly and are showing a trend of flooding. On January 24, 2022, the National Medical Products Administration announced that NMN had been approved for registration as a new cosmetic raw material, which means that NMN raw materials can be used as a cosmetic ingredient in Chinese cosmetics.

[0004] NMN exists in two isomers, α-NMN and β-NMN, and most drug molecules have different biological activities due to their isomeric characteristics. In order to reduce the differences in efficacy and adverse reactions caused by the isomeric characteristics of drugs, the separation of isomers is a major trend in modern drug research. The β isomer in NMN has biological activity, while the α isomer does not. However, chemically synthesized NMN may contain a certain amount of α isomer. In order to better evaluate its active ingredient content, the separation study of NMN isomers is particularly important.

[0005] At present, there are no national standards, local standards, industry standards or group standards for the detection of nicotinamide mononucleotide content in food. NMN and NAD+ are highly polar, non-volatile, easily soluble in water, and insoluble in organic solvents. There are currently few reports on their quantitative analysis and detection. The detection methods mainly include the use of triple quadrupole liquid-mass spectrometers, nuclear magnetic resonance instruments, and high-performance liquid chromatography. Among them, the nuclear magnetic resonance method cannot be widely used due to the high price of the instrument and the high requirements for the instrument operating environment. The liquid chromatography method also has too many restrictions on the use of the method due to the low detection limit and insufficient sensitivity. In addition, the main object of the detection methods reported in the existing literature is β-NMN, NAD+ is also mentioned in the literature, and the detection of α-NMN has not been reported in the literature. Therefore, in order to meet the needs of the domestic market for the supervision of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food and better ensure food safety, it is necessary to develop a simple and rapid detection method that can simultaneously detect α-NMN and β-NMN and NAD+ in food. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for simultaneously determining the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food.

[0007] The purpose of the present invention is achieved through the following solutions:

[0008] A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food, comprising the following steps:

[0009] (1) Sample pretreatment: weigh the sample into a centrifuge tube, add the extractant, perform ultrasonic extraction, centrifuge, and take the supernatant to make up the volume;

[0010] (2) Preparation of standard solution:

[0011] Preparation of standard stock solution: Accurately weigh β-NMN, NAD+ standard and α-NMN, dissolve them in water and make up to volume to obtain standard stock solutions of β-NMN, NAD+ and α-NMN;

[0012] Preparation of mixed standard intermediate solution: Accurately pipette β-NMN and NAD+ standard stock solutions, dilute to volume with water to obtain β-NMN and NAD+ standard intermediate solutions, then accurately pipette α-NMN standard stock solution, β-NMN, and NAD+ standard intermediate solution, dilute to volume with methanol solution to obtain α-NMN, β-NMN and NAD+ mixed standard intermediate solution.

[0013] Preparation of mixed standard series working solution: Accurately pipette different volumes of mixed standard intermediate solution, and dilute with methanol solution to mixed standard series working solution of 10ng / mL, 20ng / mL, 50ng / mL, 100ng / mL, 200ng / mL, 500ng / mL, and 1000ng / mL for α-NMN, β-NMN, and NAD+, respectively;

[0014] (3) After activating the HLB solid phase extraction column (reverse polar adsorption) and the MAX solid phase extraction column (mixed anion exchange adsorption) with methanol and water respectively, the HLB column is added with the sample solution extracted in step (1) to directly collect the purified liquid; the MAX column is first added with the sample solution extracted in step (1), then rinsed with 5% methanol aqueous solution, and then eluted with potassium dihydrogen phosphate solution, the eluate is collected and fixed to volume with 5% methanol aqueous solution; the two purified liquids are passed through an organic filter membrane to obtain a sample solution to be tested, which is subjected to liquid chromatography tandem mass spectrometry for determination;

[0015] (4) The mixed standard series working solutions of different concentrations in step (2) are detected by liquid chromatography-mass spectrometry to obtain a standard working curve; under the same conditions, the sample solution to be tested prepared in step (3) is detected by liquid chromatography-mass spectrometry, and the retention time and ion fragment abundance ratio are used for qualitative analysis. The concentration of the mixed standard series working solution is used as the horizontal axis and the peak area is used as the vertical axis to draw a standard working curve, and then the peak areas of α-NMN, β-NMN and NAD+ in the sample solution to be tested are calculated and substituted into the standard working curve to obtain the contents of α-NMN, β-NMN and NAD+ in the sample solution to be tested, and then the contents of α-NMN, β-NMN and NAD+ in the sample solution to be tested are calculated according to the mass of the sample represented by the sample solution to be tested.

[0016] In step (1), the sample may be a sample with a simple matrix or a sample with a complex matrix. The sample with a simple matrix may be a nutritionally fortified food such as nicotinamide mononucleotide capsules and tablets; the sample with a complex matrix may be a tomato, vegetable, fruit, Chinese herbal medicine, or a health food with other complex matrices added.

[0017] In step (1), the extractant is at least one of water, methanol and ethanol; the concentration of methanol is 5% to 100%; the concentration of ethanol is 5% to 100%; the methanol ultrasonic extraction time is 10 to 60 minutes;

[0018] Preferably, in step (1), 5% methanol aqueous solution is finally selected as the extraction solvent and ultrasonic extraction is performed for 30 minutes;

[0019] In step (1), 100 to 200 mL of extracting solution is added to every 0.5 to 1 g of a sample with a high NMN or NAD+ content (such as a nutritionally fortified food such as a capsule) for ultrasonic extraction; 10 to 30 mL of extracting solution is added to every 2 to 5 g of a sample with a low NMN or NAD+ content (such as fruits, vegetables, Chinese medicinal materials, etc.) for ultrasonic extraction;

[0020] In step (2), the use of methanol solution for volume determination or dilution refers to selecting 5% methanol aqueous solution for volume determination or dilution;

[0021] In step (3), an HLB solid phase extraction cartridge is selected for purifying samples with relatively simple matrices, and a MAX solid phase extraction cartridge is selected for purifying samples with complex matrices;

[0022] When used to purify a sample with a simple matrix, the HLB solid phase extraction column is activated with methanol and water respectively, and the sample solution extracted in step (1) is added to directly collect the purified liquid; when used to purify a sample with a complex matrix, the MAX solid phase extraction column is activated with methanol and water, and the sample solution extracted in step (1) is first added, and then 5% methanol aqueous solution is added for elution, and then potassium dihydrogen phosphate solution is used for elution, and the eluate is collected and fixed to volume with 5% methanol aqueous solution;

[0023] The concentration of the potassium dihydrogen phosphate solution is 0.5 mol / L and the pH is 3;

[0024] The mass spectrometry detection parameters are:

[0025] Table 1 Mass spectrometry detection parameters of target compounds

[0026]

[0027] Note: * indicates quantitative ion.

[0028] The chromatographic conditions are: using a Waters HSS T3 (2.1 mm×100 mm×1.8 μm) chromatographic column; column temperature 30° C.; mobile phase A is 5 mmol / L ammonium acetate containing 0.1% (v / v) formic acid water, mobile phase B is methanol, gradient injection; flow rate 0.2 mL / min, the gradient elution conditions are shown in Table 2:

[0029] Table 2 Mobile phase gradient elution conditions

[0030]

[0031]

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] The present invention establishes a detection method for rapidly, economically, accurately and simultaneously determining nicotinamide mononucleotide α and β isomers and nicotinamide adenine dinucleotide by using a liquid chromatography-tandem triple quadrupole mass spectrometer. The optimized pretreatment detection method of the present invention can simultaneously and rapidly detect the contents of nicotinamide mononucleotide isomers and nicotinamide adenine dinucleotide in food, and the method has high sensitivity, good linear range, good repeatability, and accurate and reliable results. The method can be applied to food component detection and isomer analysis of nicotinamide mononucleotide in conventional mass production processes, and provides an effective separation and detection means for further research on the substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 3 is the total ion current chromatogram of the mixed standard of α-NMN, β-NMN and NAD+, where Figure A is the total ion current chromatogram of the mixed standard of α-NMN and β-NMN, and Figure B is the total ion current chromatogram of the mixed standard of NAD+.

[0035] Figure 2 The extraction efficiency of NMN by different solvents.

[0036] Figure 3 Effect of different ultrasonic extraction time on extraction efficiency DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] The present invention intends to establish a detection method that utilizes liquid chromatography tandem with a triple quadrupole mass spectrometer to rapidly, economically, accurately, and simultaneously determine nicotinamide mononucleotide α and β isomers and nicotinamide adenine dinucleotide.

[0039] 1Optimize the best mass spectrometry conditions.

[0040] In the present invention, NMN and NAD+ standards are prepared into reference solutions with a concentration of 1000ng / mL, and a needle pump flow injection continuous injection method is used to perform full mass spectrometry scanning detection to obtain a primary mass spectrum of the tested compound, find the corresponding parent ions of the two, and then perform a secondary mass spectrometry scan to obtain the corresponding daughter ion information. Then optimize the parameters such as the collision energy and declustering voltage of the two to optimize the parent ion and characteristic fragment ion intensity of the two, and finally determine the qualitative and quantitative ion pairs of NMN and NAD+ and the best mass spectrum parameters.

[0041] In the electrospray ion source positive ion (ESI+) mode, the parent ion of NMN was 335.0 [M + H] +, and the response signal was good. The daughter ion was further scanned to obtain the secondary mass spectrometry fragment ions of NMN. The optimized ion pairs were consistent with the literature reports (REDEUIL K, VULCANO J, PRENCIPE FP, et al. First quantification of nicotinamide riboside with B3 vitamers and coenzymes secreted in human milk by liquid chromatography-tandem-mass spectrometry [J]. Journal of Chromatography B, 2019, 1 110-1 111: 74-80.), which can meet the accurate qualitative and quantitative detection of NMN. According to the literature report on NAD+ (Liu Xiaoqian, Yang Hong, Zhao Jingyuan, Meng Chenxiaoning, Li Chun, Zhang Dong, Chen Liangmian, Yan Yu, Guo Zhongyuan, Wang Zhimin, Yi Hong, UPLC-MS / MS determination of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in Dendrobium officinale and its close relatives, Chinese Journal of Traditional Chinese Medicine, 2021, 46(16).), there is only one pair of ions 664 / 136 in ESI+ mode and the response is very low. In this method, after changing to negative ion mode (ESI-), its parent ion 662.0[MH]- was found, and its daughter ion was scanned to find three secondary mass spectrometry fragment ions with good response. The mass spectrometer has the function of switching positive and negative ions for monitoring, and can perform positive and negative ion scanning simultaneously in the same needle sample operation. The mass spectrometry detection parameters of NMN and NAD+ are shown in Table 1.

[0042] Table 1 Mass spectrometry detection parameters of target compounds

[0043]

[0044] Note: * indicates quantitative ion.

[0045] 2. Optimize the most suitable chromatographic conditions such as mobile phase and chromatographic column.

[0046] Since α-NMN and β-NMN have the same mass-to-charge ratio, their mass spectrometry parameters are the same and can only be separated by liquid chromatography conditions. α-NMN, β-NMN and NAD+ standards were prepared into mixed reference solutions with concentrations of 1000ng / mL, respectively, to explore universal chromatographic separation conditions suitable for all target samples. For ionizable samples, formic acid has the effect of improving peak shape. Therefore, in the present invention, in order to obtain good peak shape and ionization efficiency, 0.1% (v / v) formic acid water-methanol, 0.1% (v / v) formic acid water-acetonitrile, 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid water-methanol, and 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid water-acetonitrile were compared. The results showed that when other conditions were the same and methanol was used as the mobile phase, the signals of NMN and NAD+ were significantly stronger than acetonitrile, and the retention time was later. The addition of ammonium acetate to the aqueous phase can not only increase the response value, but also improve the peak shape. After comprehensive consideration, 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid in water-methanol was selected as the mobile phase for the test.

[0047] Under the mobile phase of 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid water-methanol, the peak shape and separation of the BEH C18 column, HSST3 column and Hilic column were compared. The retention capacity of the target substance of C18 and Hilic columns was worse than that of the T3 column. Under the same chromatographic conditions, the retention time of T3 was later. And by optimizing the mobile phase gradient, only the T3 column can completely separate the two isomers of NMN. The optimized chromatographic conditions are to use a Waters HSS T3 (2.1mm×100mm×1.8μm) column; column temperature 30℃; mobile phase A is 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid water, mobile phase B is methanol, gradient injection; flow rate 0.2mL / min, gradient elution conditions are shown in Table 2.

[0048] Table 2 Mobile phase gradient elution conditions

[0049]

[0050] Under the optimized chromatographic and mass spectrometric conditions, the total ion current chromatogram of the mixed standard of α-NMN, β-NMN (A) and NAD+ (B) is shown in Figure 1 . Figure 1 The retention time of α-NMN in A is 1.43min, and the half-peak width is 0.06min; the retention time of β-NMN is 1.95min, and the half-peak width is 0.05min. The separation calculation formula in the Chinese Pharmacopoeia is:

[0051]

[0052] Where: is the retention time of the latter of two adjacent chromatographic peaks; W is the retention time of the previous peak between two adjacent chromatographic peaks; 1，h / 2 , W 2，h / 2 are the half-peak widths of the two adjacent chromatographic peaks. It can be concluded that the separation degree of α-NMN and β-NMN is 5.56>1.5, which meets the requirements of matrix separation, achieves complete separation of α and β isomers, and can accurately quantify the two isomers.

[0053] 3. Optimize a pre-treatment method for detecting NMN and NAD+ in different foods.

[0054] In view of the physical properties of NMN and NAD+ that are easily soluble in water but difficult to dissolve in organic solvents, the present invention compares the extraction effects of ultrasonic extraction for 30 minutes using pure water, 5% methanol, 10% methanol, 50% methanol, 75% methanol, 100% methanol, 5% ethanol, 10% ethanol, 50% ethanol, 75% ethanol, and 100% ethanol as extraction agents.

[0055] Take a commercially available nicotinamide mononucleotide capsule health product as a sample, remove its capsule shell and take 0.5g of its contents into a 200mL brown volumetric flask, add 150mL of extraction solution, vortex, mix, ultrasonic extraction for 30min, and dilute to the scale with extraction solution. Accurately transfer 100μL of the sample solution to a 50mL volumetric flask and dilute to the scale with 5% methanol aqueous solution. The diluted sample solution is passed through a 0.22μm organic filter membrane (nylon 66) and then subjected to liquid chromatography tandem mass spectrometry. The optimized mass spectrometry and chromatographic conditions are selected for determination. The extraction efficiency of NMN by different solvents is as follows. Figure 2 As shown, the extraction rate of NMN was the highest when 5% methanol aqueous solution was used as the extraction solvent.

[0056] In order to select the optimal extraction time and extraction efficiency, the extraction efficiency of different ultrasonic extraction times (10min, 20min, 30min, 40min, 50min, 60min) was compared with 5% methanol aqueous solution as the extraction solvent at (200W, 40kHz). The results showed that the NMN content gradually increased with the increase of ultrasonic time within 0-30min, and the content decreased after 30-40min and remained basically stable. Figure 3 Based on the above results, the extraction method of ultrasonic extraction for 30 min using 5% methanol aqueous solution as the extraction solvent was considered.

[0057] Considering that different types of food have different matrices, and the influence of matrix effect on the accuracy of liquid chromatography tandem mass spectrometry measurement results cannot be ignored, the samples need to be purified. In order to achieve the best purification effect, a solid phase extraction column is selected to purify the sample. According to the molecular structure of NMN and NAD+ and its weakly acidic physicochemical properties, the present invention selects HLB solid phase extraction column (reverse polar adsorption) and MAX solid phase extraction column (mixed anion exchange adsorption) as purification columns, and the purification effect is compared in the experiment.

[0058] Prepare standard mixed solutions of α-NMN, β-NMN and NAD+ with concentrations of about 500ng / mL, respectively. After activating the HLB column (60mg, 3mL) and MAX column (60mg, 3mL) with 3mL methanol and 3mL water, add 2mL of mixed standard solution respectively, collect the purified solution, rinse the extraction column with 1mL 5% methanol aqueous solution, collect and combine the purified solution, and make it up to 4mL with 5% methanol aqueous solution. The purified solution obtained above was passed through a 0.22μm organic filter membrane and then subjected to liquid chromatography tandem mass spectrometry, and the results are shown in Table 3. It can be seen from the results that the HLB column has basically no retention for the three target substances α-NMN, β-NMN and NAD+, and the recovery rate is between 90% and 110%, so it can be used to remove weak polar impurities in the sample solution; and the MAX column can completely retain the three target substances on the solid column due to its high selectivity and strong retention capacity for weakly acidic compounds. According to the optimal pH value for compound isoelectric point optimization and the phosphate groups on the molecular structure of NMN and NAD+, 0.5mol / L potassium dihydrogen phosphate solution with pH=3.0 was selected as the eluent. 2mL of the mixed standard solution was transferred to the activated MAX column, 2mL of 5% methanol aqueous solution was added for elution, and then 2mL of 0.5mol / L potassium dihydrogen phosphate solution with pH=3 was used for elution. The eluate was collected and fixed to 4mL with 5% methanol aqueous solution, and then passed through a 0.22μm organic filter membrane for liquid chromatography tandem mass spectrometry. The results are shown in Table 3. The recovery rates of the three target substances α-NMN, β-NMN and NAD+ were all between 90% and 110%, and they were basically completely eluted. Based on the above results, the present invention selects two purification methods: direct purification by HLB solid phase extraction column and purification by elution by MAX solid phase extraction column + 0.5mol / L pH=3 potassium dihydrogen phosphate solution. Among them, HLB column direct purification can be used for samples with relatively simple matrices to remove weak polar impurities in the samples that may interfere with the separation and analysis; while MAX solid phase extraction column + 0.5 mol / L pH=3 potassium dihydrogen phosphate solution elution purification is used for the selective purification of NMN and NAD+ in samples with complex matrices.

[0059] Table 3 Purification and recovery effects of HLB columns and MAX columns on α-NMN, β-NMN and NAD+

[0060]

[0061]

[0062] Example 1

[0063] 1. Detection method for the content of nicotinamide mononucleotide isomers and nicotinamide adenine dinucleotide in food 1.1 Principle of the detection method:

[0064] NMN and NAD+ in food were extracted with 5% methanol aqueous solution by ultrasonic method, determined by liquid chromatography triple quadrupole mass spectrometry, quantified by external standard method, and qualitatively determined by retention and ion fragment abundance ratio time.

[0065] 1.2 Preparation of standard solution

[0066] (1) Preparation of standard stock solutions: Accurately weigh 20 mg of β-NMN and NAD+ standards and 100 μg of α-NMN, dissolve them in first-grade water and transfer them to a 10 mL brown volumetric flask to the mark to obtain β-NMN, NAD+ and α-NMN standard stock solutions with concentrations of 2000 μg / mL, 2000 μg / mL and 10 μg / mL, respectively.

[0067] (2) Preparation of mixed standard intermediate solution: Accurately pipette 50 μL of β-NMN and NAD+ standard stock solutions, dilute to 10 mL brown volumetric flasks with first-grade water, and obtain β-NMN and NAD+ standard intermediate solutions with a concentration of 10 μg / mL. Then accurately pipette 1 mL of α-NMN standard stock solution and 1 mL of β-NMN and NAD+ standard intermediate solutions, and dilute to 10 mL brown volumetric flasks with 5% methanol aqueous solution, and obtain α-NMN, β-NMN and NAD+ mixed standard intermediate solutions with a concentration of 1 μg / mL, respectively.

[0068] (3) Preparation of mixed standard series working solutions: Accurately pipette different volumes of mixed standard intermediate solutions and dilute them with 5% methanol aqueous solution to mixed standard series working solutions with concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL for α-NMN, β-NMN, and NAD+, respectively.

[0069] 1.3 Sample pretreatment

[0070] (1) Extraction

[0071] A. Select a commercially available nicotinamide mononucleotide capsule, remove its capsule shell, take 0.5g of its contents into a 200mL brown volumetric flask, add 150mL 5% methanol aqueous solution, vortex, mix, ultrasonically extract for 30min, and dilute to scale with 5% methanol aqueous solution. Take 20mL of the sample solution and centrifuge at 8000r / min for 5min, accurately transfer 100μL of the supernatant to a 50mL brown volumetric flask, and dilute to scale with 5% methanol aqueous solution.

[0072] B. Select fresh commercially available tomatoes as samples, homogenize them, weigh 5 g into a centrifuge tube, add 30 mL of 5% methanol aqueous solution, vortex, mix, ultrasonically extract for 30 min, centrifuge at 8000 r / min for 5 min, and dilute the supernatant to 50 mL with 5% methanol aqueous solution.

[0073] (2) Purification

[0074] A. After the HLB solid phase extraction column (60 mg, 3 mL) was activated and balanced with 3 mL of methanol and 3 mL of primary water, about 6 mL of each of the above extraction solutions was taken and passed through the column, the first 3 mL was discarded, and the purified liquid at the back was collected and passed through a 0.22 μm organic filter membrane for liquid chromatography tandem mass spectrometry.

[0075] B. After MAX solid phase extraction cartridge (60 mg, 3 mL) was activated and balanced with 3 mL methanol and 3 mL primary water, 2 mL of the above extraction solution was taken and passed through the cartridge, then rinsed with 2 mL 5% methanol aqueous solution, and eluted with 2 mL 0.5 mol / L potassium dihydrogen phosphate solution with pH = 3. The eluate was collected and made up to 4 mL with 5% methanol aqueous solution, and then passed through a 0.22 μm organic filter membrane and subjected to liquid chromatography tandem mass spectrometry.

[0076] 1.4 Liquid chromatography-tandem mass spectrometry conditions

[0077] (1) Liquid chromatography conditions

[0078] Waters ACQUITY HSS T3 chromatographic column (2.1 mm × 100 mm × 1.8 μm); injection volume 5 μL; flow rate: 0.2 mL / min; column temperature 30°C; gradient elution: eluent A was 5 mmol / L ammonium acetate containing 0.1% (v / v) formic acid water, eluent B was acetonitrile, and the elution program was shown in Table 2.

[0079] (2) Mass spectrometry conditions

[0080] Curtain gas flow rate: 30 L / min; Nebulizer gas flow rate (GS1): 50 L / min; Auxiliary heating gas flow rate (GS2): 50 L / min; Collision gas (CAD): medium intensity (medium); Auxiliary heating gas temperature: 500 °C; Spray voltage: 5000 V (ESI+) / -4500 V (ESI-); Scan mode: Multiple reaction monitoring mode (MRM). The mass spectrometry detection parameters of the components to be measured are detailed in Table 1.

[0081] 1.5 Test results

[0082] The test results of α-NMN, β-NMN and NAD+ contents in the above samples are shown in Table 4.

[0083] Table 4 Sample test results

[0084]

[0085] ND means not detected

[0086] 2. Method Validation

[0087] 2.1 Linear range

[0088] The standard curve was drawn with the concentration of the mixed standard series working solution as the horizontal axis and the peak area as the vertical axis. The linear regression equation of α-NMN was y=4256.1x+2855.9, and the correlation coefficient R 2 is 0.9999; the linear regression equation of β-NMN is y=3941.3x–5887.0, and the correlation coefficient R 2 is 0.9998; the linear regression equation of NAD+ is y=565.0x–782.4, and the correlation coefficient R 2 The value was 0.9995. This indicates that the method has a good linear relationship when testing the contents of α-NMN, β-NMN and NAD+ in the range of 10 ng / mL to 1000 ng / mL.

[0089] 2.2 Method detection limit and quantification limit

[0090] The standard solution was serially diluted and analyzed on the analyzer. The concentration when the signal-to-noise ratio was 3 was taken as the detection limit, and the concentration when the signal-to-noise ratio was 10 was taken as the quantification limit. The results are shown in Table 5.

[0091] Table 5 Detection limit and quantification limit

[0092]

[0093] 2.3 Method precision

[0094] Tomato samples were selected for precision experiments on the contents of α-NMN, β-NMN and NAD+. According to the above sample pretreatment method, 12 sample solutions were prepared for testing (6 each for HLB column purification and MAX column purification). α-NMN and NAD+ were not detected in the 12 samples. The average content of β-NMN test results in 6 samples purified by HLB column was 0.424 mg / kg, and the relative standard deviation RSD = 4.8%. The average content of β-NMN test results in 6 samples purified by MAX column was 0.437 mg / kg, and the relative standard deviation RSD = 3.9%. The above results all meet the requirements of the laboratory coefficient of variation of the concentration in Appendix F of "Laboratory Quality Control Specification for Physical and Chemical Testing of Food" GB / T27404-2008, which is ≤11%, and the method has good repeatability.

[0095] 2.4 Method accuracy

[0096] Fresh tomatoes were used as samples to conduct accuracy experiments on the determination methods of α-NMN, β-NMN and NAD+ contents. Appropriate amounts of α-NMN, β-NMN and NAD+ standards were added to the samples, and the above optimized pretreatment detection method was used for parallel determination 6 times. The recovery rates were calculated and the results are shown in Table 6. The recovery rates were all within the range of 80-110%, which met the requirements of Appendix F of "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods" GB / T 27404-2008.

[0097] Table 6 Recovery rate experimental results

[0098]

[0099]

[0100] In summary, the optimized pretreatment detection method of the present invention can simultaneously and rapidly detect the content of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food, and the method has high sensitivity, good linear range, good repeatability, and accurate and reliable results. The method can be applied to the detection of nicotinamide mononucleotide isomers and nicotinamide adenine dinucleotide content in different matrix foods, used for product quality control, and meet the needs of the domestic market for the supervision of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food, characterized in that: The following steps are involved: (1) Sample pretreatment: weigh the sample into a centrifuge tube, add the extractant, perform ultrasonic extraction, centrifuge, and take the supernatant to make up the volume; (2) Preparation of standard solution: Preparation of standard stock solution: Accurately weigh β-NMN, NAD+ standard and α-NMN, dissolve them in water and make up to volume to obtain standard stock solutions of β-NMN, NAD+ and α-NMN; Preparation of mixed standard intermediate solution: accurately pipette β-NMN and NAD+ standard stock solutions, dilute to volume with water to obtain β-NMN and NAD+ standard intermediate solutions, then accurately pipette α-NMN standard stock solution, β-NMN, and NAD+ standard intermediate solution, dilute to volume with methanol solution to obtain α-NMN, β-NMN, and NAD+ mixed standard intermediate solution; Preparation of mixed standard series working solution: Accurately pipette different volumes of mixed standard intermediate solution, and dilute with methanol solution to mixed standard series working solution of 10ng / mL, 20ng / mL, 50ng / mL, 100ng / mL, 200ng / mL, 500ng / mL, and 1000ng / mL for α-NMN, β-NMN, and NAD+, respectively; (3) After activating the HLB solid phase extraction column and the MAX solid phase extraction column with methanol and water respectively, the HLB column is added with the sample solution extracted in step (1) to directly collect the purified liquid; the MAX column is first added with the sample solution extracted in step (1), then rinsed with 5% methanol aqueous solution, and then eluted with potassium dihydrogen phosphate solution, the eluate is collected and fixed to volume with 5% methanol aqueous solution; the two purified liquids are passed through an organic filter membrane to obtain a sample solution to be tested, which is subjected to liquid chromatography tandem mass spectrometry for determination; (4) The mixed standard series working solutions of different concentrations in step (2) are detected by liquid chromatography-mass spectrometry to obtain a standard curve; under the same conditions, the sample solution to be tested prepared in step (3) is detected by liquid chromatography-mass spectrometry, and the retention time and ion fragment abundance ratio are used for qualitative analysis. The concentration of the mixed standard series working solution is used as the horizontal axis and the peak area is used as the vertical axis to draw a standard curve, and then the peak areas of α-NMN, β-NMN and NAD+ in the sample solution to be tested are calculated and substituted into the standard curve to obtain the contents of α-NMN, β-NMN and NAD+ in the sample solution to be tested, and then the contents of α-NMN, β-NMN and NAD+ in the sample solution to be tested are calculated according to the mass of the sample represented by the sample solution to be tested.

2. A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (1), the sample is a sample with a simple matrix or a sample with a complex matrix. The sample with a simple matrix is ​​nicotinamide mononucleotide capsules and tablets; the sample with a complex matrix is ​​tomatoes, vegetables, fruits, Chinese medicinal materials, and health foods with other complex matrices added.

3. A method for simultaneously determining the contents of α, β isomers of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (1), the extractant is at least one of water, methanol and ethanol; the concentration of methanol is 5% to 100%; the concentration of ethanol is 5% to 100%; and the methanol ultrasonic extraction time is 10 to 60 minutes.

4. A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (1), 5% methanol aqueous solution is selected as the extraction solvent and ultrasonic extraction is performed for 30 minutes.

5. A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (1), 100 to 200 mL of extracting solution is added for every 0.5 to 1 g of capsules, tablets or nutritionally fortified foods with high NMN or NAD+ content for ultrasonic extraction; and 10 to 30 mL of extracting solution is added for every 2 to 5 g of fruits, vegetables or Chinese medicinal materials with low NMN or NAD+ content for ultrasonic extraction.

6. A method for simultaneously determining the contents of α, β isomers of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (3), an HLB solid phase extraction cartridge is selected for purifying samples with relatively simple matrices, and a MAX solid phase extraction cartridge is selected for purifying samples with complex matrices.

7. A method for simultaneously determining the contents of α, β isomers of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food according to claim 6, characterized in that: When used to purify samples with simple matrices, the HLB solid phase extraction column is activated with methanol and water respectively, and the sample solution extracted in step (1) is added thereto and the purified liquid is directly collected; when used to purify samples with complex matrices, the MAX solid phase extraction column is activated with methanol and water, and the sample solution extracted in step (1) is first added thereto, and then a 5% methanol aqueous solution is added for elution, and then eluted with potassium dihydrogen phosphate solution, and the eluate is collected and fixed to volume with a 5% methanol aqueous solution.

8. A method for simultaneously determining the contents of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: In step (3), the concentration of the potassium dihydrogen phosphate solution is 0.5 mol / L and the pH is 3.

9. A method for simultaneously determining the contents of α, β isomers of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: The mass spectrometry detection parameters are: Table 1 Mass spectrometry detection parameters of target compounds Note: * indicates quantitative ion.

10. A method for simultaneously determining the contents of α, β isomers of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in food according to claim 1, characterized in that: The chromatographic conditions are as follows: a Waters HSS T3 chromatographic column; a column temperature of 30°C; mobile phase A is 5mmol / L ammonium acetate containing 0.1% (v / v) formic acid in water; mobile phase B is methanol, gradient injection; flow rate is 0.2mL / min, and the gradient elution conditions are as shown in Table 2: Table 2 Mobile phase gradient elution conditions

Images