CN113295779B - Method for rapidly determining beta-nicotinamide mononucleotide in health-care product - Google Patents
Method for rapidly determining beta-nicotinamide mononucleotide in health-care product Download PDFInfo
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- CN113295779B CN113295779B CN202110374837.2A CN202110374837A CN113295779B CN 113295779 B CN113295779 B CN 113295779B CN 202110374837 A CN202110374837 A CN 202110374837A CN 113295779 B CN113295779 B CN 113295779B
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Abstract
The invention discloses a rapid determination method of beta-nicotinamide mononucleotide in a health product, belonging to the technical field of health product detection. The method comprises the following specific steps: (1) pretreatment of health care products; (2) leaching treatment; (3) purifying the extracting solution and performing on-machine detection; preparing a standard solution; and (5) optimizing the compound. The method is simple in process, simple and convenient in pretreatment, and capable of effectively, quickly and accurately measuring the content of the beta-nicotinamide mononucleotide in the health-care product, has high sensitivity, good selectivity and wide linear range, is suitable for content detection in the health-care product, is also suitable for analyzing nutritional ingredients in vegetables, fruits and traditional Chinese medicines, and is a quick, efficient, economical, practical and accurate measuring method and means for the beta-nicotinamide mononucleotide in health care.
Description
Technical Field
The invention belongs to the technical field of health product detection, and particularly relates to a rapid determination method of beta-nicotinamide mononucleotide in a health product.
Background
Beta-nicotinamide mononucleotide is a precursor substance of a salvage synthesis pathway of nicotinamide adenine dinucleotide in a mammal body, and is an endogenous substance in a human body. The beta-nicotinamide mononucleotide is used as the main component of health product and has certain curative effect in delaying senility, treating senile diseases, regulating insulin secretion and regulating mRNA expression. How to rapidly determine beta-nicotinamide mononucleotide in health care products is a problem which needs to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a method for rapidly determining beta-nicotinamide mononucleotide in a health-care product, so as to solve the problems in the background technology.
The technical scheme adopted by the invention is as follows:
a method for rapidly determining beta-nicotinamide mononucleotide in health products comprises the following specific steps:
(1) Pretreatment of health products
Selecting a health food, randomly taking out a plurality of particles from a package, weighing the mass of each health food, summing, calculating the average value of the mass as the average mass of the health food, putting the health food into a mortar, grinding the health food, sieving the ground health food by a sieve of 100-120 meshes, and filling the health food into a clean plastic package bag for later use;
(2) Leaching treatment
Weighing 0.3-0.5g of processed sample powder, averagely distributing the sample powder in three polytetrafluoroethylene spiral cover centrifuge tubes, respectively adding methanol solution with the same volume and methanol and water with the volume ratio of 1;
(3) Extracting solution purification and on-machine detection
Transferring the three leaching solutions obtained in the step (2) into a C18 solid-phase extraction column sequentially activated by 3-6mL of methanol and 3-6mL of primary water under the action of gravity, collecting purified liquid in a colorimetric tube, taking supernatant in the colorimetric tube by using a 2.5mL injection needle tube, filtering by using a 0.22-micron organic filter head, diluting filtrate according to a certain proportion, and detecting a treated sample by using a liquid chromatography tandem mass spectrometer, wherein if a detected concentration value exceeds a standard curve linear range after the filtrate is diluted, further diluting the filtrate and then loading the diluted filtrate on a machine until the concentration value is within the standard concentration control linear range;
(4) Standard solution preparation
Preparing mother liquor: accurately weighing 25.00mg of a beta-nicotinamide mononucleotide standard substance, metering the volume of acetonitrile into a 25mL volumetric flask, and adjusting the concentration to be 1.0mg/L to serve as a mother solution;
preparing an intermediate stock solution: accurately transferring 100 mu L of 1.0mg/L mother solution, diluting acetonitrile to a constant volume of 100mL volumetric flask, and adjusting the concentration of stock solution to 1 mu g/mL;
preparing a working solution: respectively transferring intermediate stock solutions with different volumes, and preparing mixed standard solutions with the concentrations of 10ng/mL, 100ng/mL, 250ng/mL, 500ng/mL and 1000ng/mL according to the initial proportion of the mobile phase;
(5) Compound optimization
Optimizing mass spectrum conditions: optimizing mass spectrum conditions of the intermediate stock solution of the beta-nicotinamide mononucleotide with the concentration of 1 mu g/mL prepared in the step (4), and enabling the fragments of the parent ions and the daughter ions of the substance to respond to the maximum extent by adjusting ion source parameters including spraying voltage, sheath gas pressure, auxiliary gas pressure, capillary temperature and atomizer temperature, so as to improve the sensitivity of beta-nicotinamide mononucleotide detection;
liquid phase condition optimization: the response value and the sensitivity of the beta-nicotinamide mononucleotide are improved by adjusting the flow rate of a mobile phase, the proportion of a liquid phase organic phase and a liquid phase aqueous phase, elution conditions and the like.
When the color of the sugar coating of the health-care product is not white, after high-speed homogeneous extraction in an ice bath environment, 100-200mg of graphitized carbon black needs to be added into a centrifugal tube, vortex is carried out for 3-5min, the centrifugal tube is taken out, then centrifugation is carried out in a refrigerated centrifuge at the rotating speed of 8000-10000r/min for 2-3min, and finally, the supernatant is taken out for purification treatment, and then the subsequent treatment is carried out according to the step (3).
The liquid chromatography conditions in step (3) were as follows:
and (3) chromatographic column: syncronis C18 (100X 2.1mm,1.7 μm);
sample injection amount: 10 mu L of the solution;
column temperature: 35 ℃;
flow rate: 0.2min/mL;
mobile phase: the phase A is an organic phase containing 100% acetonitrile; phase D was an aqueous phase containing 0.1% formic acid.
The mass spectrum conditions in the step (3) are as follows:
an ion source: electrospray ionization ESI;
the scanning mode comprises the following steps: positive ion scan ESI (+);
the detection mode comprises the following steps: multiple Reaction Monitoring (MRM).
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the method for determining the content of the beta-nicotinamide mononucleotide in the health-care product has the advantages of simple process, simple and convenient pretreatment, and capability of effectively, quickly and accurately determining the content of the beta-nicotinamide mononucleotide in the health-care product, has high sensitivity, good selectivity and wide linear range, is suitable for content detection in the health-care product, is also suitable for analyzing nutrient components in vegetables, fruits and traditional Chinese medicines, and is a quick, efficient, economic, practical and accurate determination method and means for the beta-nicotinamide mononucleotide in the health-care product. The method can effectively and quickly determine the content of the beta-nicotinamide mononucleotide in the health-care product, is simple to operate and is convenient for mass test.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following examples further illustrate the present invention in detail.
Example 1:
a method for rapidly determining beta-nicotinamide mononucleotide in a health product comprises the following specific steps:
(1) Pretreatment of health products
Selecting a health food, randomly taking out a plurality of particles from a package, weighing the mass of each health food, summing, calculating the average value of the mass as the average mass of the health food, putting the health food into a mortar, grinding the health food, sieving by a 120-mesh sieve, and filling into a clean plastic package bag for later use;
(2) Leaching treatment
Weighing 0.3g of processed sample powder, evenly distributing the sample powder in three polytetrafluoroethylene spiral cover centrifuge tubes, respectively adding methanol solution with the same volume, methanol with the volume ratio of 1 and water into three test tubes as extracting solution, carrying out high-speed homogenization in an ice bath environment at the rotating speed of 25000r/min, leaching for 3min, taking out the centrifuge tubes, centrifuging the centrifuge tubes at the rotating speed of 10000r/min for 2min, and finally taking out supernate for purification treatment;
(3) Extracting solution purification and on-machine detection
Transferring the three leaching solutions obtained in the step (2) into a C18 solid-phase extraction column which is sequentially activated by 3mL of methanol and 3mL of primary water under the action of gravity, collecting the purified liquid in a colorimetric tube, taking the supernatant in the colorimetric tube by using a 2.5mL injection needle tube, filtering by using a 0.22-micron organic filter head, diluting the filtrate according to a certain proportion, performing on-machine detection on the processed sample by using a liquid chromatography tandem mass spectrometer, and further diluting and then loading on a machine until the concentration value exceeds the linear range of a standard curve after the filtrate is diluted;
(4) Standard solution preparation
Preparing mother liquor: accurately weighing 25.00mg of beta-nicotinamide mononucleotide standard substance, metering the acetonitrile to a 25mL volumetric flask, and adjusting the concentration to be 1.0mg/L to serve as mother solution;
preparing an intermediate stock solution: accurately transferring 100 mu L of 1.0mg/L mother solution, diluting acetonitrile to a constant volume of 100mL volumetric flask, and adjusting the concentration of stock solution to 1 mu g/mL;
preparing a working solution: respectively transferring intermediate stock solutions with different volumes, and preparing mixed standard solutions with the concentrations of 10ng/mL, 100ng/mL, 250ng/mL, 500ng/mL and 1000ng/mL according to the initial proportion of the mobile phase;
(5) Compound optimization
Optimizing mass spectrum conditions: optimizing mass spectrum conditions of the intermediate stock solution of the beta-nicotinamide mononucleotide with the concentration of 1 mu g/mL prepared in the step (4), and enabling the parent ion fragments and the daughter ion fragments of the substance to respond to the maximum extent by adjusting plasma source parameters including spraying voltage (V), sheath gas pressure (Arb), auxiliary gas pressure (Arb), capillary temperature (DEG C) and atomizer temperature (DEG C), so that the sensitivity of beta-nicotinamide mononucleotide detection is improved;
liquid phase condition optimization: the response value and the sensitivity of the beta-nicotinamide mononucleotide are improved by adjusting the flow rate of a mobile phase, the proportion of a liquid phase organic phase and a liquid phase aqueous phase, elution conditions and the like.
When the color of the sugar coating of the health-care product is not white, after high-speed homogeneous leaching in an ice bath environment, 100mg of graphitized carbon black needs to be added into a centrifugal tube, the centrifugal tube is vortexed for 3min, the centrifugal tube is taken out, then the centrifugal tube is centrifuged for 2min at the rotating speed of 10000r/min in a refrigerated centrifuge, finally the supernatant is taken for purification treatment, and then the treatment is carried out according to the step (3).
The liquid chromatography conditions in step (3) were as follows:
a chromatographic column: syncronis C18 (100X 2.1mm,1.7 μm);
sample introduction amount: 10 mu L of the solution;
column temperature: 35 ℃;
flow rate: 0.2min/mL;
mobile phase: the phase A is an organic phase containing 100% acetonitrile; phase D was an aqueous phase containing 0.1% formic acid, and the gradient elution conditions are shown in table 1:
TABLE 1 gradient elution conditions
| Time/min | A phase/%) | D phase/%) |
| 0.00 | 10 | 90 |
| 0.50 | 10 | 90 |
| 3.00 | 90 | 10 |
| 4.00 | 90 | 10 |
| 5.00 | 10 | 90 |
| 6.00 | 10 | 90 |
The mass spectrum conditions in the step (3) are as follows:
an ion source: electrospray ionization ESI;
scanning mode, ion information and collision energy: positive ion scan ESI (+) with ion information and collision energy as shown in table 2:
TABLE 2 measured substance ion information and Collision energy
The detection mode comprises the following steps: multiple Reaction Monitoring (MRM).
The mass spectrometry ion source parameters are shown in table 3:
table 3 mass spectrometry ion source parameters
| Name (R) | Numerical value |
| Spray voltage (V) | 3500 |
| Sheath gas pressure (Arb) | 30 |
| Auxiliary air pressure (Arb) | 10 |
| Capillary temperature (. Degree. C.) | 270 |
| Atomizer temperature (. Degree. C.) | 300 |
Results of the experiment
Through optimization, verification and repeated tests on liquid phase conditions and mass spectrum conditions, the information and the correlation coefficient of the beta-nicotinamide mononucleotide standard curve are shown in a table 4:
TABLE 4 Standard curves and correlation coefficients for the Compounds
The same health food is extracted by three different concentration extraction processes of methanol solution, mixed solution of methanol and water with volume ratio of 1 and pure water, and other operation processes are the same, and the detection results are shown in table 5:
TABLE 5 comparative experimental results of health products under different technologies
Experiments show that under the pure water extraction process and under the condition of an optimal compound-optimized liquid chromatography tandem mass spectrometry instrument (LC-MS-MS), the health care product has the highest content of beta-nicotinamide mononucleotide and the best recovery rate. Therefore, the method is simple in process, simple and convenient in pretreatment, and capable of effectively, quickly and accurately measuring the content of the beta-nicotinamide mononucleotide in the health-care product, and meanwhile, the method is high in sensitivity, good in selectivity, wide in linear range, suitable for content detection in the health-care product and analysis of nutritional ingredients in vegetables, fruits and traditional Chinese medicines, and is a quick, efficient, economical, practical and accurate measuring method and means for the beta-nicotinamide mononucleotide in health care.
The above description is only exemplary of the invention, and any modification, equivalent replacement, and improvement made within the spirit and principle of the invention should be included in the protection scope of the invention.
Claims (3)
1. A method for rapidly determining beta-nicotinamide mononucleotide in health products is characterized by comprising the following steps: the method comprises the following specific steps:
(1) Pretreatment of health products
Selecting a health food, randomly taking out a plurality of particles from a package, weighing the mass of each health food, summing, calculating the average value of the mass as the average mass of the health food, putting the health food into a mortar, grinding the health food, sieving the ground health food by a sieve of 100-120 meshes, and filling the health food into a clean plastic package bag for later use;
(2) Leaching treatment
Weighing 0.3-0.5g of processed sample powder, evenly distributing the sample powder in three polytetrafluoroethylene spiral cover centrifuge tubes, respectively adding methanol solution with the same volume, methanol and water with the volume ratio of 1;
(3) Extracting solution purification and on-machine detection
Transferring the three leaching solutions obtained in the step (2) into a C18 solid-phase extraction column which is sequentially activated by 3-6mL of methanol and 3-6mL of primary water under the action of gravity, collecting purified liquid in a colorimetric tube, taking supernatant in the colorimetric tube by using a 2.5mL injection needle tube, filtering by using a 0.22-micron organic filter head, diluting filtrate according to a certain proportion, performing on-machine detection on a processed sample by using a liquid chromatography tandem mass spectrometer, and further diluting and then loading on a machine until a concentration value is detected to be beyond a standard curve linear range after the filtrate is diluted;
wherein the liquid chromatography conditions are as follows:
a chromatographic column: syncronis C18 with specification of 100 × 2.1mm,1.7 μm;
sample injection amount: 10. mu.L;
column temperature: 35. DEG C;
flow rate: 0.2min/mL;
mobile phase: the phase A is an organic phase containing 100% acetonitrile; phase D is an aqueous phase containing 0.1% formic acid; the gradient elution conditions were:
(4) Standard solution preparation
Preparing mother liquor: accurately weighing 25.00mg of a beta-nicotinamide mononucleotide standard substance, metering the volume of acetonitrile into a 25mL volumetric flask, and adjusting the concentration to be 1.0mg/L to serve as a mother solution;
preparing an intermediate stock solution: accurately transferring 100 mu L of 1.0mg/L mother solution, fixing the volume of acetonitrile into a 100mL volumetric flask, and adjusting the concentration of a stock solution to 1 mu g/mL;
preparing a working solution: respectively transferring intermediate stock solutions with different volumes, and preparing mixed standard solutions with the concentrations of 10ng/mL, 100ng/mL, 250ng/mL, 500ng/mL and 1000ng/mL according to the initial proportion of the mobile phase;
(5) Compound optimization
Optimizing mass spectrum conditions: optimizing mass spectrum conditions of the intermediate stock solution of the beta-nicotinamide mononucleotide with the concentration of 1 mu g/mL prepared in the step (4), and enabling the parent ion and the daughter ion fragments of the substance to respond to the maximum extent by adjusting ion source parameters including spraying voltage, sheath gas pressure, auxiliary gas pressure, capillary temperature and atomizer temperature, so as to improve the sensitivity of beta-nicotinamide mononucleotide detection;
optimizing liquid phase conditions: the response value and the sensitivity of the beta-nicotinamide mononucleotide are improved by adjusting the flow rate of a mobile phase, the proportion of a liquid-phase organic phase and a liquid-phase aqueous phase and elution conditions.
2. The method for rapidly determining beta-nicotinamide mononucleotide (beta-nicotinamide mononucleotide) in health care product according to claim 1, wherein the method comprises the following steps: when the color of the sugar coating of the health-care product is not white, after high-speed homogeneous extraction in an ice bath environment, 100-200mg of graphitized carbon black needs to be added into a centrifugal tube, vortex is carried out for 3-5min, the centrifugal tube is taken out, then centrifugation is carried out in a refrigerated centrifuge at the rotating speed of 8000-10000r/min for 2-3min, and finally, the supernatant is taken out for purification treatment, and then the subsequent treatment is carried out according to the step (3).
3. The method for rapidly determining β -nicotinamide mononucleotide (β -nicotinamide mononucleotide) in health product according to claim 1, comprising the steps of: the mass spectrum conditions in the step (3) are as follows:
an ion source: electrospray ionization ESI;
the scanning mode is as follows: positive ion scan ESI +;
the detection mode is as follows: multiple reactions monitor MRM.
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