CN117723662A - HPLC separation method for beta-nicotinamide mononucleotide and degrading impurities thereof - Google Patents
HPLC separation method for beta-nicotinamide mononucleotide and degrading impurities thereof Download PDFInfo
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- CN117723662A CN117723662A CN202311715255.1A CN202311715255A CN117723662A CN 117723662 A CN117723662 A CN 117723662A CN 202311715255 A CN202311715255 A CN 202311715255A CN 117723662 A CN117723662 A CN 117723662A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, which comprises the following steps: dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution; dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution; detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector; the chromatographic parameters are: the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the CAD detector acquisition frequency was 10Hz and the mobile phase eluted isocratically. The invention adopts HPLC-UV-CAD to carry out analysis and detection, improves the sensitivity of detecting the 5-phosphoric acid-D-ribose, and can accurately detect the purity of NMN, the content of nicotinamide and 5-phosphoric acid-D-ribose.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof.
Background
beta-Nicotinamide Mononucleotide (NMN), which is a nucleotide with important biological activity and widely exists in various organisms, is one of important precursors of NAD+ (coenzyme I) and NADP+ (coenzyme II). In recent years, physiological health care functions of NMN, in particular, functions related to delaying aging of NMN are widely paid attention to at home and abroad, and the NMN is called as an anti-aging drug and becomes a research hot spot in the fields of medicines, health care products, cosmetics and the like. At present, a large number of NMN synthesis methods are reported, mainly including chemical synthesis, enzyme catalysis and fermentation. Less reports are made on NMN purity and impurity detection methods.
Therefore, it is necessary to provide a method for detecting NMN purity and mainly degrading impurities of 5-phosphate-D-ribose and nicotinamide, and effectively monitoring NMN purity, 5-phosphate-D-ribose and nicotinamide content during NMN production and storage.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide an HPLC separation method of beta-nicotinamide mononucleotide and its degradation impurities, which has the advantages of high separation degree, low detection limit, high sensitivity and accurate detection result.
The invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, which comprises the following steps:
a) Dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution;
dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution;
b) Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector;
the chromatographic parameters are:
the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, and the mobile phase is eluted at equal degree; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate.
Preferably, the mobile phase a: the volume ratio of mobile phase B was 65:35.
Preferably, the chromatographic column has a length of 250mm, an inner diameter of 4.6mm and a particle size of 5 μm.
Preferably, the CAD detector atomizes at 50 ℃ and the carrier gas pressure is 35psi.
Preferably, the sample injection volume is 15 μl; the flow rate was 1.0ml/min.
Preferably, the column temperature of the chromatographic column is 25 ℃; the temperature of the sample tray was controlled at 15 ℃.
Preferably, the acetonitrile is post-column compensated, compensating for a flow rate of 1.3ml/min.
Preferably, the nicotinamide has a degree of separation of 28 or more and 5-phosphate-D-ribose has a degree of separation of 2.7 or more.
Preferably, the degradation impurities include nicotinamide and 5-phosphate-D-ribose;
the quantitative limit concentration of nicotinamide is 0.06465 mug/mL, and the detection limit concentration is 0.03232 mug/mL;
5-phosphate-D-ribose quantitative limit concentration is 3.012 mug/mL, and detection limit concentration is 1.506 mug/mL;
the quantitative limit concentration of the beta-nicotinamide mononucleotide is 0.1287, and the detection limit concentration is 0.06435.
Preferably, the said
The linear range of the beta-nicotinamide mononucleotide is 0.0005 mg/mL-2.5 mg/mL;
the linear range of nicotinamide is 0.000065 mg/mL-0.065 mg/mL;
the linear range of the 5-phosphoric acid-D-ribose is 0.003 mg/mL-0.015 mg/mL.
Compared with the prior art, the invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, which comprises the following steps: a) Dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution; dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution; b) Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector; the chromatographic parameters are: the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, and the mobile phase is eluted at equal degree; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate. According to the invention, HPLC-UV-CAD is used for analysis and detection, acetonitrile is used for post-column compensation, CAD background interference is reduced, sensitivity of detecting 5-phosphoric acid-D-ribose is improved, and NMN purity, nicotinamide and 5-phosphoric acid-D-ribose content can be accurately detected.
Drawings
FIG. 1 is a graph (UV graph) showing the purity of 20220701 batches of NMN;
FIG. 2 is a graph (UV graph) showing the purity of 20220801 batches of NMN;
FIG. 3 is a graph (UV graph) showing the purity of 20220802 batches of NMN;
FIG. 4 is a graph of 20220701 batches of raw material (CAD graph);
FIG. 5 is a graph of 20220801 batches of raw material detection (CAD graph);
FIG. 6 is a graph of 20220802 batches of raw material detection (CAD graph);
FIG. 7 is a graph of NMN high temperature damage sample purity detection (UV graph);
FIG. 8 is a NMN base damage sample purity detection profile (UV map);
FIG. 9 is a NMN illumination damage sample purity detection profile (UV map);
FIG. 10 is a graph of NMN high temperature damage sample detection (CAD graph);
FIG. 11 is a sample detection map of MN illumination damage (CAD graph);
FIG. 12 is a NMN base damage sample detection profile (CAD graph);
FIG. 13 is a graph of purity detection (UV plot) of NMN feedstock after 18 months at 40 ℃/75% RH;
FIG. 14 is a graph of purity detection profile (UV graph) of NMN feedstock after 18 months at 25 ℃/60% RH;
FIG. 15 is a graph (CAD graph) of NMN feedstock after 18 months at 40 ℃/75% RH;
FIG. 16 is a graph (CAD graph) of NMN feedstock after 18 months at 25 ℃/60% RH;
FIG. 17 is a CAD graph of the mixed labeling solution;
FIG. 18 is a UV spectrum of the mixed labeling solution;
FIG. 19 is a linear diagram of 5-phosphate-D-ribose;
FIG. 20 is a linear diagram of nicotinamide;
fig. 21 is a linear diagram of NMN.
FIG. 22 is a C18 column assay of comparative example 1.
FIG. 23 is an amino column test pattern of comparative example 2.
FIG. 24 is a chart of different mobile phases of comparative example 3.
FIG. 25 is a graph showing the different elution gradients of comparative example 4.
FIG. 26 is a map of the method of comparative example 5 (CN 114487218A).
Detailed Description
The invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the specification. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and they are intended to be within the scope of the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention is used for detecting NMN which is synthesized by catalyzing with immobilized enzyme and transferase by taking D-ribose and nicotinamide as raw materials. NMN and main degradation impurities 5-phosphoric acid-D-ribose and nicotinamide thereof have large polarity, and the problems of weak compound retention, poor peak shape, poor separation degree of each peak and the like exist in the process of separating and eluting by adopting a conventional C18 column. And an amino column is adopted, so that the detection background noise is large, and the detection sensitivity is low. According to the invention, a Ultimate HILIC Amide column and an acetonitrile-ammonium formate (formic acid) mobile phase system are adopted for isocratic analysis elution, so that the target object retention is enhanced, the peak shapes of NMN, 5-phosphoric acid-D-ribose and nicotinamide are good, and the separation degree of each peak meets the detection requirement.
The inventor finds that no chromophore exists in the 5-phosphoric acid-D-ribose structure, no absorption exists under an ultraviolet detector, and the response is weak under nicotinamide CAD, the invention creatively adopts HPLC-UV-CAD combination for analysis and detection, and simultaneously adopts acetonitrile for post-column compensation, thereby reducing CAD background interference and improving the sensitivity of detecting 5-phosphoric acid-D-ribose, and the invention can accurately detect NMN purity, nicotinamide and 5-phosphoric acid-D-ribose content.
The invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, which comprises the following steps:
a) Dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution;
dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution;
b) Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector;
the chromatographic parameters are:
the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, and the mobile phase is eluted at equal degree; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate.
Degradation impurities according to the invention include nicotinamide and 5-phosphate-D-ribose.
According to the HPLC separation method of the beta-nicotinamide mononucleotide and the degraded impurities thereof, firstly, a sample to be detected is dissolved by adopting 65% acetonitrile, so as to obtain a sample solution.
And (3) dissolving a sample to be tested by adopting 65% acetonitrile, and filtering by using a 0.45 mu m organic phase filter membrane to obtain a sample solution.
The liquid to be tested in the invention comprises, but is not limited to, NMN which is synthesized by catalyzing immobilized enzyme and transferase by taking D-ribose and nicotinamide as raw materials. Also comprises high temperature, alkali and illumination to destroy NMN and main degradation impurity in the sample.
And dissolving the beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution.
Dissolving the beta-nicotinamide mononucleotide by adopting 65% acetonitrile, and filtering by using a 0.45 mu m organic phase filter membrane to obtain a beta-nicotinamide mononucleotide reference substance solution;
dissolving nicotinamide by adopting 65% acetonitrile, and filtering by using a 0.45 mu m organic phase filter membrane to obtain a nicotinamide reference substance solution;
the 5-phosphate-D-ribose was dissolved in 65% acetonitrile and filtered through a 0.45 μm organic phase filter to give a 5-phosphate-D-ribose reference solution.
Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts a combination of an ultraviolet detector and a CAD detector.
In the invention, NMN purity and nicotinamide content are calculated according to a correction peak area normalization method after blank solvent peaks are subtracted in a UV chromatogram. In the CAD chromatogram, the content of the 5-phosphoric acid-D-ribose is calculated by adopting an external standard method.
The chromatographic parameters are:
the chromatographic column is Ultimate HILIC Amide column; the chromatographic column has a length of 250mm, an inner diameter of 4.6mm and a particle size of 5 μm. The column temperature of the chromatographic column is 25 ℃; the invention adopts acetonitrile post-column compensation, and the compensation flow rate is 1.3ml/min.
The invention adopts the ultra-pure silica gel as the hydrophilic chromatographic column of the matrix, and the bonding functional group is the carbamoyl functional group, so the separation effect is good.
The detector is a UV-CAD combined detector, and the post-column compensation mode is adopted to improve the detection sensitivity.
The ultraviolet detector wavelength was 261nm.
The CAD detector acquisition frequency was 10Hz, the atomization temperature was 50℃and the carrier gas pressure was 35psi.
The mobile phase is eluted isocratically; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate.
In some preferred embodiments, the mobile phase a: the volume ratio of mobile phase B was 65:35.
In some preferred embodiments, the sample volume is 15 μl;
in some preferred embodiments, the flow rate is 1.0ml/min.
In some preferred embodiments, the sample tray is temperature controlled to 15 ℃.
The quantitative limit concentration of the beta-nicotinamide mononucleotide is 0.1287, and the detection limit concentration is 0.06435.
The quantitative limit concentration of nicotinamide is 0.06465 mug/mL, and the detection limit concentration is 0.03232 mug/mL;
the quantitative limit concentration of the 5-phosphoric acid-D-ribose is 3.012 mug/mL, and the detection limit concentration is 1.506 mug/mL.
As can be seen from the above, the detection limit of the invention is low, the quantitative limit is low, and the sensitivity is high.
The linear range of the beta-nicotinamide mononucleotide is 0.0005 mg/mL-2.5 mg/mL;
the standard curve of NMN is y= 182.2557x-0.0929, and the correlation coefficient R 2 ＝1.0000。
The linear range of nicotinamide is 0.000065 mg/mL-0.065 mg/mL;
the standard curve of nicotinamide is y= 320.5360x-0.0177, the correlation coefficient R 2 ＝1.0000。
The linear range of the 5-phosphoric acid-D-ribose is 0.003 mg/mL-0.015 mg/mL.
The standard curve of the 5-phosphoric acid-D-ribose is y= 659.1430x-0.3987, and the correlation coefficient R 2 ＝0.9985。
As can be seen from the above, the correlation coefficient of each standard curve is not lower than 0.998, and the standard curves have good linear relationship.
The separation degree of nicotinamide is more than 28.
The separation degree of the 5-phosphoric acid-D-ribose is more than 2.7.
From the above, it can be seen that the degree of separation is high and the specificity is strong.
The NMN purity, nicotinamide content and impurity number in the plurality of samples are consistent, and the 5-phosphoric acid-D-ribose is not detected, which indicates that the detection method of the invention has good repeatability,
in conclusion, the HPLC-UV-CAD combined detection method is adopted, so that the method has the advantages of strong specificity, high sensitivity, good linear correlation, high repeatability and good solution stability, and the pretreatment process of the sample is simple and quick, can accurately detect the purity of NMN in the NMN sample, and the content of nicotinamide and 5-phosphoric acid-D-ribose, and is beneficial to the control of product quality.
The invention provides an HPLC separation method of beta-nicotinamide mononucleotide and degradation impurities thereof, which comprises the following steps: a) Dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution;
dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution; b) Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector; the chromatographic parameters are: the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, and the mobile phase is eluted at equal degree; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate. According to the invention, HPLC-UV-CAD is used for analysis and detection, acetonitrile is used for post-column compensation, CAD background interference is reduced, sensitivity of detecting 5-phosphoric acid-D-ribose is improved, and NMN purity, nicotinamide and 5-phosphoric acid-D-ribose content can be accurately detected.
To further illustrate the present invention, the following describes in detail, with reference to examples, an HPLC separation method of β -nicotinamide mononucleotide from its degradation impurities.
Example 1
The embodiment provides a detection method for detecting purity of NMN and main degradation impurities of nicotinamide and 5-phosphoric acid-D-ribose in NMN synthesized by an enzyme catalysis method by taking nicotinamide and D-ribose as raw materials, which comprises the following steps:
(1) instrument chromatographic conditions: the instrument is UltiMate 3000 double ternary liquid chromatograph (DAD diode array detector and Corona Veo electric fog detector), the mobile phase A is acetonitrile, the mobile phase B is 100mmol/L ammonium formate aqueous solution (0.2% formic acid); elution ratio mobile phase A mobile phase B was 65:35 (acetonitrile post column compensation, compensation flow rate 1.3 ml/min); the flow rate is 1.0ml/min; the column was Ultimate HILIC Amide (length 250mm, inner diameter 4.6mm, particle size 5 μm); the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, the atomization temperature is 50 ℃, and the carrier gas pressure is 35psi; the sample injection volume is 15 mu L; the column temperature is 25 ℃; the temperature of the sample tray was controlled at 15 ℃.
(2) Solvent: the 65% acetonitrile aqueous solution was filtered through a 0.45 μm organic phase filter, and the subsequent filtrate was collected and examined.
(3) Sample solution preparation: 25mg of NMN sample is precisely weighed, placed in a 10mL volumetric flask, added with 65% acetonitrile aqueous solution for dissolution, diluted to volume to scale, shaken uniformly and filtered by a 0.45 mu m organic phase filter membrane, and the subsequent filtrate is taken for detection.
(4) Preparing a 5-phosphoric acid-D-ribose reference substance solution: precisely weighing 20mg of 5-phosphoric acid-D-ribose, placing in a 100mL volumetric flask, adding 65% acetonitrile aqueous solution for dissolution and volume fixing to a scale, shaking uniformly, precisely weighing the solution in the 1 mL-100 mL volumetric flask, adding 65% acetonitrile aqueous solution for dilution and volume fixing to the scale, shaking uniformly, filtering with a 0.45 μm organic phase filter membrane, and detecting the subsequent filtrate.
(5) Preparing NMN reference substance solution: accurately weighing 25mg of NMN reference substance, placing in a 10mL volumetric flask, adding 65% acetonitrile water solution for dissolution, fixing volume to scale, shaking, filtering with 0.45 μm organic phase filter membrane, and collecting filtrate.
(6) Preparing nicotinamide reference substance solution: accurately weighing nicotinamide reference substance 25mg, placing in a 25mL volumetric flask, adding 65% acetonitrile aqueous solution for dissolution, fixing the volume to a scale, shaking uniformly, filtering with a 0.45 μm organic phase filter membrane, and taking the subsequent filtrate for detection.
(7) And (3) measuring: and (3) detecting the solution to be detected in (2), (3) and (4) by adopting the chromatographic conditions in (1) to obtain a chromatogram. In the UV chromatograms (see fig. 1, 2, 3), after subtraction of the blank solvent peak, NMN purity and nicotinamide content (relative retention time 0.32, correction factor 0.53) were calculated as corrected peak area normalization. In the CAD chromatogram (see FIG. 4, FIG. 5 and FIG. 6), the content of 5-phosphate-D-ribose is calculated by an external standard method.
The results are shown in Table 1
TABLE 1
Example 1 shows that NMN products measured by the method of the invention have high purity and low impurity content.
Example 2
A detection method for destroying purity of NMN in a sample and main degradation impurities in NMN solution at high temperature, alkali and illumination comprises the following steps:
(1) instrument chromatographic conditions
Same as in example 1
(2) Sample preparation
Alkali damage samples: 50mg of NMN sample is taken and placed in a 20mL volumetric flask, after a proper amount of 65% acetonitrile aqueous solution is added for dissolution, 0.8mL of ammonia water is added, the mixture is uniformly shaken, the mixture is placed at 15 ℃ for 10 hours, then the mixture is neutralized to be neutral by formic acid, the 65% acetonitrile aqueous solution is fixed to volume to scale, after the mixture is uniformly shaken, the mixture is filtered by a 0.45 mu m organic phase filter membrane, and the subsequent filtrate is detected (sample to be detected).
High Wen Huai sample: 25mg of NMN sample is taken and placed in a 10mL volumetric flask, and an appropriate amount of 65% acetonitrile aqueous solution is added for dissolution, and shaking is carried out. The flask was placed in an oven (105 ℃ C.) for 10 minutes, taken out, cooled to room temperature, fixed to volume with 65% acetonitrile aqueous solution to scale, shaken well, filtered with a 0.45 μm organic phase filter membrane, and the subsequent filtrate was taken for detection (sample to be tested).
Light destroys the sample: 25mg of NMN sample is taken and placed in a 10mL transparent volumetric flask, 65% acetonitrile aqueous solution is added for dissolution, the volume is fixed to the scale, and shaking is carried out uniformly. After 10 days of standing under 5000lux condition, the mixture is taken out, filtered by a 0.45 mu m organic phase filter membrane, and the subsequent filtrate is taken for detection (sample to be detected).
(3) And (3) measuring: and (3) detecting the solution to be detected by adopting the chromatographic condition in the step (1) to obtain a chromatogram. In the UV chromatogram (see fig. 7, 8 and 9), after the blank solvent peak is subtracted, the NMN purity and nicotinamide content (relative retention time is 0.32, correction factor is 0.53) are calculated according to the correction peak area normalization method, the NMN purities in the high-temperature destroyed sample, the alkali destroyed sample and the light destroyed sample are 94.87%, 98.07%, 99.2% respectively, and the nicotinamide content is 5.13%, 1.93% and 0.80% respectively. In the CAD chromatogram (see FIG. 10, FIG. 11 and FIG. 12), the content of 5-phosphate-D-ribose is calculated by an external standard method, and the contents of 5-phosphate-D-ribose in the high temperature damage sample, the alkali damage sample and the light damage sample are respectively 9.67%, 3.63% and 1.52%.
Example 2 shows that the invention is adopted to effectively determine the purity of NMN solution under high temperature, alkali and illumination damage and the content of nicotinamide and 5-phosphoric acid-D-ribose which are main degradation impurities, and the result shows that NMN is unstable to high temperature and alkali and is easy to degrade.
Example 3
The embodiment provides a method for detecting purity and main degradation impurities of NMN raw materials synthesized by enzyme catalysis after 18 months of standing at 40 ℃,75% RH and 25 ℃ and 18 months of standing at 60% RH, which comprises the following steps:
(1) instrument chromatographic conditions
Same as in example 1
(2) Sample preparation
Sample 1: accurately weighing 25mg of NMN raw material after being placed for 18 months at 40 ℃ and 75% RH, placing the NMN raw material into a 10mL volumetric flask, adding 65% acetonitrile aqueous solution for dissolution, diluting to a fixed volume to a scale, shaking uniformly, filtering by using a 0.45 mu m organic phase filter membrane, and taking a subsequent filtrate for detection.
Sample 2: accurately weighing 25mg of NMN raw material after being placed for 18 months at 25 ℃ and 60% RH, placing the NMN raw material into a 10mL volumetric flask, adding 65% acetonitrile aqueous solution for dissolution, diluting to a fixed volume to a scale, shaking uniformly, filtering by using a 0.45 mu m organic phase filter membrane, and taking a subsequent filtrate for detection.
(3) Sample detection
And (3) detecting the sample to be detected by adopting the chromatographic condition in the step (1) to obtain a chromatogram. In the UV chromatogram (see fig. 13, 14), after the blank solvent peak was subtracted, the NMN purity and nicotinamide content (relative retention time 0.32, correction factor 0.53), NMN purity 87.69% in sample 1, nicotinamide content 6.07%, NMN purity 99.93% in sample 2, nicotinamide content 0.05% were calculated by corrected peak area normalization. In the CAD chromatogram (see FIG. 15, FIG. 16), the 5-phosphate-D-ribose content was calculated by the external standard method, the 5-phosphate-D-ribose content in sample 1 was 11.44%, and the 5-phosphate-D-ribose content in sample 2 was undetectable.
Example 3 shows that the invention is adopted to effectively determine the purity of the NMN raw material after being stored at different temperatures and different humidity conditions and the content of nicotinamide and 5-phosphoric acid-D-ribose which are main degradation impurities, and the result shows that the NMN raw material is unstable to store at 40 ℃ and 75% RH and is degraded.
The method for detecting NMN liquid phase provided by the invention is examined by the following experiments, and the results are as follows:
(1) investigation of specificity
The mixed solution of NMN, nicotinamide and 5-phosphoric acid-D-ribose and the diluent are prepared, and the analysis is carried out by the detection method provided in the example 1, so that the result shows that the diluent has no interference on NMN and the peak positions of various impurities, and the separation degree of three compounds is more than 2.7, so that the detection method has strong specificity. The peak time and the degree of separation of each peak are shown in Table 1 (map see FIG. 17 and FIG. 18)
TABLE 2
(3) Sensitivity investigation
Adopting a progressive dilution method, analyzing NMN, nicotinamide and 5-phosphoric acid-D-ribose solutions with different concentrations by using the detection method provided in the example 1, and measuring the lowest quantitative detection concentration of NMN, nicotinamide and 5-phosphoric acid-D-ribose under the condition that the signal to noise ratio is not less than 10; the lowest detection concentrations of NMN, nicotinamide and 5-phosphate-D-ribose were measured with a signal to noise ratio of not less than 3. The specific results are shown in Table 3, and the results show that the detection method of the invention has high sensitivity.
TABLE 3 Table 3
(3) Linear investigation
Linear NMN solution: accurately weighing 50mg of NMN reference substance, placing into a 20mL volumetric flask, adding 65% acetonitrile water solution for dissolution, diluting to volume, and shaking. Gradually diluting to prepare linear solution with NMN concentration ranging from 0.0005mg/mL to 2.5 mg/mL.
Linear nicotinamide solution: accurately weighing nicotinamide reference substance 13mg, placing in a 20mL volumetric flask, adding 65% acetonitrile aqueous solution for dissolution, diluting to volume, and shaking. Gradually diluting to prepare linear solution with nicotinamide concentration ranging from 0.000065mg/mL to 0.065 mg/mL.
Linear solution of 5-phosphate-D-ribose: 13mg of 5-phosphoric acid-D-ribose reference substance is precisely weighed, placed in a 20mL volumetric flask, added with 65% acetonitrile aqueous solution for dissolution, diluted to volume to scale, and shaken well. Gradually diluting to prepare a linear solution with the concentration of 5-phosphoric acid-D-ribose ranging from 0.003mg/mL to 0.015mg/mL.
Analysis was performed by the detection method provided in example 1, and a standard curve was drawn with the concentration as the abscissa and the peak area as the ordinate, see fig. 19, 20, and 21, where the nmn standard curve is y= 182.2557x-0.0929, and the correlation coefficient R 2 =1.0000; the standard curve of nicotinamide is y= 320.5360x-0.0177, the correlation coefficient R 2 =1.0000; the standard curve of the 5-phosphoric acid-D-ribose is y= 659.1430x-0.3987, and the correlation coefficient R 2 =0.9985; from this, the correlation coefficient of each standard curve is not lower than 0.998, and the standard curves have good linear relationship.
(4) Repeatability investigation
25mg of NMN sample is precisely weighed, placed in a 10mL volumetric flask, added with 65% acetonitrile aqueous solution for dissolution, diluted to volume to scale, shaken uniformly, filtered by a 0.45 mu m organic phase filter membrane, detected by a subsequent filtrate, prepared into six sample solutions in parallel, analyzed by the detection method provided in example 1, the results show that the NMN purity, nicotinamide content and impurity number in 6 samples are consistent, and the 5-phosphoric acid-D-ribose is not detected, thus showing that the detection method has good repeatability, and the specific results are shown in Table 4.
TABLE 4 Table 4
(5) Solution stability investigation
25mg of NMN sample is precisely weighed, placed in a 10mL volumetric flask, added with 65% acetonitrile aqueous solution for dissolution, diluted to volume to scale, uniformly shaken and filtered by a 0.45 mu m organic phase filter membrane, and after the subsequent filtrate is stored for 2 hours, 4 hours, 6 hours, 8 hours and 10 hours in a 15 ℃ sample injection tray, the detection method provided in example 1 is adopted to detect, and the result shows that the NMN purity of the NMN solution is 99.91% from 99.97% after the NMN solution is placed for 10 hours at 15 ℃, the nicotinamide content is increased to 0.07% from 0.01%, the absolute amount of impurity increase is controlled within 0.1%, the impurity number is consistent, and the NMN solution is stable when placed for 10 hours at 15 ℃. The specific results are shown in Table 5
TABLE 5
Comparative example 1
Comparative example 1 is different from example 1 in that a reversed-phase C18 column resistant to pure water of one polarity is usedHPLC analysis was performed on NMN, 5-phosphate-D-ribose, nicotinamide reference solutions with concentrations of 1mg/ml, 0.5mg/ml, and mobile phase of 100mM ammonium formate, respectively, with Perfect T3 (5 μm 4.6X 250 mM): acetonitrile=90: 10, the final HPLC chromatogram thereof is shown in fig. 22, and fig. 22 is a C18 column detection spectrum of comparative example 1; as shown in FIG. 22, NMN, 5-phospho-D-ribose and nicotinamide have early peak times and NMN coincides with the peak of 5-phospho-D-ribose.
Comparative example 2
Comparative example 2 differs from the detection method in example 1 in that HPLC analysis was performed on NMN, 5-phospho-D-ribose, nicotinamide reference solution with polymer matrix amino column Shodex Asahipak NH2P-50 4e (5 μm 4.6 x 250 mm) at concentrations of 1mg/ml, 0.5mg/ml, respectively, and the final HPLC chromatogram obtained, as shown in fig. 23, fig. 23 is comparative example 2 amino column detection profile; as can be seen from fig. 23, the NMN, the 5-phosphoric acid-D-ribose and the nicotinamide have early peak-out time, chromatographic peaks of the NMN, the 5-phosphoric acid-D-ribose and the nicotinamide are basically overlapped, and the detection background noise is large and the detection sensitivity is low due to the column loss of the amino column.
Comparative example 3
Comparative example 3 differs from the detection method in example 1 in that acetonitrile is used as the mobile phase: water=65: carrying out HPLC analysis on NMN, 5-phosphoric acid-D-ribose and nicotinamide reference substance solutions, wherein the concentrations of the NMN, 5-phosphoric acid-D-ribose and nicotinamide reference substance solutions are respectively 1mg/ml, 1mg/ml and 0.5mg/ml, and the final HPLC chromatograms are shown in FIG. 24, and FIG. 24 is a different mobile phase diagram of comparative example 3; as can be seen from FIG. 24, NMN, 5-phosphate-D-ribose and nicotinamide have early peak times, the chromatographic peaks of nicotinamide and NMN coincide, and NMN and 5-phosphate-D-ribose have asymmetric peak shapes, with asymmetric factors of 0.58 and 0.75, respectively.
Comparative example 4
Comparative example 4 differs from the detection method in example 1 in that acetonitrile was used as mobile phase A, 100mmol/L of ammonium formate aqueous solution (0.2% formic acid) as mobile phase B, and that mobile phase A was eluted at a ratio of 75:25 (acetonitrile post column compensation, compensation flow rate 1.3 ml/min) to carry out HPLC analysis on NMN, 5-phosphoric acid-D-ribose and nicotinamide reference substance solutions, wherein the concentrations of the NMN, 5-phosphoric acid-D-ribose and nicotinamide reference substance solutions are respectively 1mg/ml, 1mg/ml and 0.5mg/ml, and the final obtained HPLC chromatograms are shown in FIG. 25, and FIG. 25 is different elution gradient maps of comparative example 4; as can be seen from FIG. 25, NMN and nicotinamide peak form trailing, the trailing factors are 2.37 and 2.41,5-phosphoric acid-D-ribose peak form wide and fat, the trailing is a straight triangle peak, and the trailing factor is 6.11.
Comparative example 5
Comparative example 5 was different from the detection method in example 1 in that the method described in example 1 in CN114487218A was used, the NMN, 5-phosphoric acid-D-ribose, nicotinamide reference solution was subjected to HPLC analysis using Waters XBridge BEH Amide (250 mm×4.6mm×5 μm), the NMN, 5-phosphoric acid-D-ribose, nicotinamide reference solution concentrations were 1mg/mL, 0.5mg/mL, the detection wavelength of HPLC analysis was 265nm, the column temperature was 25 ℃, the sample injection amount was 20 μl, the flow rate was 1.0mL/min, an ammonium acetate aqueous solution (pH was adjusted to 9.0 with ammonia water) of 0.03mol/L was used as a buffer solution, the ammonium acetate aqueous solution and acetonitrile were used together as a mobile phase, the ratio of the ammonium acetate aqueous solution and acetonitrile was as shown in the following table, and the primary gradient elution, the secondary gradient elution, and the tertiary gradient elution were sequentially performed; the composition of the mobile phase and the time of gradient elution of each gradient elution are shown in Table 6, the final HPLC chromatogram is shown in FIG. 26, and the retention time of nicotinamide is 3.900min, and the peak shape of nicotinamide is sharp and symmetrical as can be seen from FIG. 26; the retention time of the beta-nicotinamide mononucleotide is 37.575min, the peak shape is asymmetric, the severe tailing is presented, and the tailing factor is 3.41; the 5-phosphate-D-ribose showed no peak at 265 nm. The detection method of the patent CN114487218A has the problem that NMN peak trailing and 5-phosphoric acid-D-ribose can not be detected.
TABLE 6
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 20 | 80 |
| 5 | 20 | 80 |
| 55 | 40 | 60 |
| 100 | 20 | 80 |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for HPLC separation of β -nicotinamide mononucleotide from its degraded impurities, comprising:
a) Dissolving a sample to be tested by adopting 65% acetonitrile to obtain a sample solution;
dissolving beta-nicotinamide mononucleotide, nicotinamide and 5-phosphoric acid-D-ribose by using 65% acetonitrile respectively to obtain a reference substance solution;
b) Detecting the sample solution and the reference solution by adopting high performance liquid chromatography; the detection adopts the combination of an ultraviolet detector and a CAD detector;
the chromatographic parameters are:
the chromatographic column is Ultimate HILIC Amide column; the wavelength of the ultraviolet detector is 261nm; the acquisition frequency of the CAD detector is 10Hz, and the mobile phase is eluted at equal degree; mobile phase a is acetonitrile; mobile phase B was 100mmol/L aqueous ammonium formate.
2. The separation method according to claim 1, wherein the mobile phase a: the volume ratio of mobile phase B was 65:35.
3. The separation method according to claim 1, wherein the column has a length of 250mm, an inner diameter of 4.6mm and a particle size of 5 μm.
4. The separation method of claim 1, wherein the CAD detector atomization temperature is 50 ℃ and the carrier gas pressure is 35psi.
5. The separation method according to claim 1, wherein the sample volume is 15 μl; the flow rate was 1.0ml/min.
6. The separation method according to claim 1, wherein the column temperature of the chromatographic column is 25 ℃; the temperature of the sample tray was controlled at 15 ℃.
7. The separation method according to claim 1, wherein the acetonitrile is post-column-compensated at a flow rate of 1.3ml/min.
8. The method according to claim 1, wherein the nicotinamide has a degree of separation of 28 or more and 5-phosphate-D-ribose has a degree of separation of 2.7 or more.
9. The separation method according to claim 1, wherein the degradation impurities include nicotinamide and 5-phosphate-D-ribose;
the quantitative limit concentration of nicotinamide is 0.06465 mug/mL, and the detection limit concentration is 0.03232 mug/mL; 5-phosphate-D-ribose quantitative limit concentration is 3.012 mug/mL, and detection limit concentration is 1.506 mug/mL;
the quantitative limit concentration of the beta-nicotinamide mononucleotide is 0.1287, and the detection limit concentration is 0.06435.
10. The method of claim 1, wherein the linear range of β -nicotinamide mononucleotide is 0.0005mg/mL to 2.5mg/mL;
the linear range of nicotinamide is 0.000065 mg/mL-0.065 mg/mL;
the linear range of the 5-phosphoric acid-D-ribose is 0.003 mg/mL-0.015 mg/mL.
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