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Detection method of beta-nicotinamide mononucleotide

A single nucleotide and detection method technology, which is applied in the detection field of beta-nicotinamide mononucleotide, can solve the problems of low separation degree and short retention time, and achieves high separation degree, good linear relationship, and simple and convenient operation. Effect

Pending Publication Date: 2022-06-24
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the retention time of NMN under the above conditions is still short, usually within 3 minutes, it is easy to overlap with other substances, and the resolution is low

Method used

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  • Detection method of beta-nicotinamide mononucleotide
  • Detection method of beta-nicotinamide mononucleotide
  • Detection method of beta-nicotinamide mononucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 chromatographic condition selection

[0038] (1) NMN wavelength scan

[0039] The pure NMN solution (control sample solution) was subjected to wavelength scanning using a Shimadzu LC-16 HPLC system, see figure 1 . NMN is detectable at 190-280 nm, preferably 266 nm.

[0040] (2) Column selection

[0041] Experiments were carried out using reversed C18 chromatographic column, sulfonic acid strong cation chromatographic column and forward amino-bonded silica gel chromatographic column. The results showed that the retention time of NMN on the first two chromatographic columns was too short for less than 2 minutes, and it was not easy to separate from impurities. The retention time on the forward amino-bonded silica gel column is more than 5 minutes, and it can be effectively separated from impurities, so the forward amino-bonded silica gel column is selected. The separation effect of the reversed C18 chromatographic column is shown in Comparative Example 1....

Embodiment 2

[0056] Example 2 Methodology establishment

[0057] (1) Exclusivity

[0058] Use pure water and impurity-containing NMN solution (control sample) to inject samples, record the chromatogram, the chromatogram shows that the solvent water has no interference, and the resolution of NMN and other impurities meets the requirements.

[0059] (2) Linear range

[0060]Accurately weigh 250.18 mg of NMN standard sample (control sample), put it into a 50 ml volumetric flask, and make up to a constant volume to prepare a standard solution. The standard solution was precisely measured and prepared into samples of different concentrations for detection. The data results are shown in Table 4. With the concentration as the abscissa and the corresponding peak area as the ordinate, a linear standard curve was established. The concentration of NMN is lower than 5μg / ml, higher than 2000μg / ml, the deviation is too large, and the linear relationship between the concentration of 5-1000μg / ml is bet...

Embodiment 3

[0072] NMN samples were detected using HPLC under the following conditions:

[0073] Detection instrument: Shimadzu LC-16 high performance liquid phase system.

[0074] Chromatographic column: Amino-bonded silica gel column, Welch Ultimate XB-NH2, 150×4.6mm, 5 μm.

[0075] Mobile Phase Preparation: Prepare 1M KH 2 PO 4 solution, using H 3 PO 4 or KOH to adjust pH to 5.5. Dilute 200 times to make 5mM KH 2 PO 4 solution, take 900ml, add 100ml methanol, mix well and degas.

[0076] Detection wavelength: 266nm.

[0077] Detection flow rate: 1.0ml / min.

[0078] Loading sample:

[0079] Control sample: The concentration of the NMN control sample is known to be 98.5%, 23.76 mg of NMN is accurately weighed and placed in a 50 ml volumetric flask, and the volume is adjusted to prepare a control sample.

[0080] Test sample: NMN crystallization dry sample, accurately weigh 25.84mg into a 50ml volumetric flask, and prepare the test sample with constant volume.

[0081] Injecti...

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Abstract

The invention discloses a method for detecting beta-nicotinamide mononucleotide. The method comprises the following steps: (1) chromatographic conditions: a chromatographic column: a filler is amino bonded silica gel; the mobile phase is obtained by mixing an inorganic phase and an organic phase; the detection wavelength is 190 to 280 nm; the detection flow rate is 0.8 to 1.2 ml / min; the column temperature is 25-35 DEG C; (2) preparing a sample to be tested and a control sample: weighing the beta-nicotinamide mononucleotide standard sample as the control sample, weighing the beta-nicotinamide mononucleotide crystal sample as the sample to be tested, and dissolving the sample to a constant volume by using a mobile phase; and (3) determination method: filtering the control sample solution and the test sample solution, injecting into a high performance liquid chromatograph, recording a chromatogram, and calculating the peak area. The detection method is not easy to coincide with other substances, and the separation degree is high; the linear relation is good in a wide range, and the method is suitable for accurate quantification of the NMN; direct sample loading is achieved, operation is easy and convenient, and time is saved; and the cost is low.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting beta-nicotinamide mononucleotide. Background technique [0002] β-nicotinamide mononucleotide, referred to as NMN, molecular weight 334.22, molecular formula C 11 H 15 N 2 O 8 P, the structure is as follows: [0003] NMN can be used as a precursor to synthesize coenzyme I (nicotinamide adenine dinucleotide, NAD), which is essential for human metabolism. Studies have shown that it plays an important role in human cell repair and slowing down aging. In recent years, top journals such as Nature and Cell have published a number of articles on the efficacy of NMN. Harvard experiments have confirmed that supplementation with NMN can prolong mammalian lifespan by more than 30%. Several universities and companies such as Japan and the United States have carried out clinical trials one after another. NMN is regarded as an "elixir of life" and is highly sought af...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/34
CPCG01N30/88G01N30/34G01N2030/8813
Inventor 刘珊珊秦远超侯健秦永发尹延明杨顺清乔春鑫
Owner ANHUI GSH BIO TECH CO LTD
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