CN115112805A - Detection method of nicotinamide content and application thereof - Google Patents

Detection method of nicotinamide content and application thereof Download PDF

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CN115112805A
CN115112805A CN202210911339.1A CN202210911339A CN115112805A CN 115112805 A CN115112805 A CN 115112805A CN 202210911339 A CN202210911339 A CN 202210911339A CN 115112805 A CN115112805 A CN 115112805A
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nicotinamide
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付春香
李守军
刘拓
张晓鹏
刘爱玲
高明涛
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Ringpu Hi Tech Tianjin Biotechnology Co ltd
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Abstract

The invention discloses a detection method of nicotinamide content and application thereof, wherein the detection method adopts High Performance Liquid Chromatography (HPLC) to detect, and comprises the steps of mixing a mobile phase A, a mobile phase B and a mobile phase C in proportion, and carrying out isocratic elution; wherein the mobile phase A is 0.1-0.2% sodium heptanesulfonate water solution (pH is 1.9-2.5), the mobile phase B is methanol, and the mobile phase C is isopropanol. The method of the invention can separate the main component, namely the nicotinamide, in the test sample and carry out content measurement. The method can effectively detect the nicotinamide within the concentration range of 1 mu g/mL to 20 mu g/mL, has accurate and reliable result, can greatly improve the monitoring efficiency of the nicotinamide, and has strong practicability and popularization value.

Description

Detection method of nicotinamide content and application thereof
Technical Field
The invention belongs to the technical field of preparation analysis, and particularly relates to a method for detecting nicotinamide content and application thereof.
Background
At present, the determination methods for nicotinamide content include the following methods:
the method comprises the following steps: in the detection of the feed additive nicotinamide in GB7301-2017, glacial acetic acid is used as a solvent, a potentiometric titration method is adopted, and amino in a sample is titrated by perchloric acid standard titration liquid as a principle to carry out detection.
The second method comprises the following steps: the measurement of nicotinamide in the Chinese veterinary pharmacopoeia also employs a perchloric acid titration reaction method, which is substantially the same as that in the first method.
The third method comprises the following steps: niacinamide was measured by HPLC method: HPLC chromatographic conditions: the mobile phase is potassium dihydrogen phosphate solution (0.01moL/L) -water-methanol-acetonitrile (1: 79: 15: 5), the pH value is adjusted to 8.0 +/-0.2 by ammonia water, the flow rate is 0.8mL per minute, the column temperature is 35 ℃, the detection wavelength is 262nm, and the sample injection amount is 10 uL.
Sample detection: precisely weighing about 10mg of the sample, placing in a 100mL measuring flask, adding appropriate amount of water, shaking to dissolve, diluting with water to scale, shaking to obtain sample solution (10 μ g/mL), and precisely weighing 10 μ L. Injecting into a liquid chromatograph, and recording a chromatogram; and taking a proper amount of nicotinamide control substance. Dissolving in water, diluting to obtain control solution containing nicotinamide 100 μ g/mL, and determining by the same method. And calculating the content of nicotinamide by peak area according to an external standard method. (Nynes 352100 test for Ningde City of Fujian province) in the pharmaceutical composition of Yangxi pharmaceutical 2009, volume 21, phase 10, determination of nicotinamide content by high performance liquid chromatography)
The defects of the first method and the second method are as follows: the perchloric acid titration requirement is strict, the reproducibility is poor, and the requirement on the personnel ability is high.
The third method has the following disadvantages: firstly, the use range of the chromatographic column should be selected to be wide range of the chromatographic column with pH more than 8.0, the cost is high, secondly, the absorption peak with the wavelength of 262nm is selected to be closer to the general 254nm wavelength, the interference is high, the data accuracy is low, and finally, the components of the mobile phase are excessive, the cost is high and the preparation is time-consuming.
Therefore, there is a need in the art to develop a method for detecting nicotinamide content, so as to overcome the shortcomings of the above three methods.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for detecting nicotinamide content and application thereof.
The technical scheme of the invention is as follows:
the invention provides a method for detecting the content of nicotinamide, which comprises the following steps: detecting by HPLC method, wherein the chromatographic condition is that a mobile phase A, a mobile phase B and a mobile phase C are mixed in proportion and isocratic elution is carried out; wherein the mixing proportion of the mobile phase A, the mobile phase B and the mobile phase C is (91-93): 7-4): 2-3, the mobile phase A is 0.1-0.2% sodium heptanesulfonate aqueous solution, and the pH is 1.9-2.5; the mobile phase B is methanol; the mobile phase C is isopropanol.
Preferably, the mixing ratio of the mobile phase A, the mobile phase B and the mobile phase C is 91:7: 2.
Preferably, the mobile phase a is a 0.1% aqueous solution of sodium heptanesulfonate at pH 2.1.
The mobile phase A is sodium heptanesulfonate water solution, and the preparation method comprises the steps of mixing the sodium heptanesulfonate and water according to the mass-volume ratio of (10-20) to 1000, shaking uniformly, and adjusting the pH value of the water solution to 1.9-2.5 by perchloric acid.
Preferably, the preparation method of the mobile phase A comprises the following steps: mixing sodium heptanesulfonate and water according to the mass-volume ratio of 10:1000, shaking up, and adjusting the pH value of the aqueous solution to 2.1 by perchloric acid.
The detection conditions of HPLC are as follows: octadecylsilane chemically bonded silica is used as a filler of a chromatographic column, the specification of the chromatographic column is 4.6mm multiplied by 150mm, the flow rate is 0.8-1.2mL/min, the detection wavelength is 250-280nm, the column temperature is 25-35 ℃, and the sample injection amount is 20 mu L.
Preferably, the flow rate is 1.0mL/min, the detection wavelength is 267nm, and the column temperature is 30 ℃.
The detection steps of the nicotinamide content are as follows:
step one, preparing an extracting solution, namely adding 50mL of acetonitrile and 10mL of glacial acetic acid into 800mL of water, mixing, diluting the water to 1000mL, and uniformly mixing to obtain the product;
accurately weighing about 0.1g to 100mL of a test sample in a volumetric flask, dissolving with the extracting solution and fixing the volume, precisely weighing 5mL to 100mL of the volumetric flask, diluting with the extracting solution and fixing the volume to prepare a solution with the final concentration of 5 mu g/mL;
step three, preparing a nicotinamide control solution, dissolving and diluting the extract to prepare the nicotinamide control solution containing 5.0 mu g of nicotinamide in each 1 ml;
step four, setting HPLC chromatographic conditions: mixing the mobile phase A, the mobile phase B and the mobile phase C in proportion, and performing isocratic elution; wherein the mixing proportion of the mobile phase A, the mobile phase B and the mobile phase C is (91-93): (7-4): (2-3); the mobile phase A is 0.1-0.2% sodium heptanesulfonate water solution, and the pH value is 1.9-2.5; the mobile phase B is methanol; the mobile phase C is isopropanol; the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and the specification is 4.6mm multiplied by 150 mm; the flow rate is 0.8-1.2 mL/min; the detection wavelength is 250-280 nm; the column temperature is 25-35 ℃; the sample injection amount is 20 mu L;
step five, precisely measuring a sample solution, injecting the sample solution into a liquid chromatograph, and recording a chromatogram map 1; and injecting the nicotinamide reference substance solution into a liquid chromatograph, recording a chromatogram 2, and calculating the content of nicotinamide in the test sample by peak area according to an external standard method.
Preferably, the assay comprises the steps of:
step one, preparing an extracting solution: adding 50mL of acetonitrile and 10mL of glacial acetic acid into 800mL of water, uniformly mixing, adding water to a constant volume of 1000mL, and uniformly mixing to obtain the product;
step two, preparing a test solution: weighing 0.1g of a test sample, diluting the test sample by using an extracting solution, wherein the mass of the test sample and the extracting solution is as follows: the volume ratio is 0.1 g: 100mL, continuously taking a proper amount of the solution, diluting the solution by 200 times with the extracting solution, and shaking up to obtain a test solution of 5.0 mu g/mL;
step three, preparing a reference substance solution: taking the nicotinamide standard substance, adding the extracting solution to prepare a solution containing 5.0 mug of nicotinamide in each 1mL, namely a control solution of 5.0 mug/mL;
step four, setting HPLC detection conditions: octadecylsilane chemically bonded silica is used as a filler of a chromatographic column, the specification of the chromatographic column is 4.6mm multiplied by 150mm, the flow rate of a mixed mobile phase is 1.0mL/min, the detection wavelength is 267nm, the column temperature of the chromatographic column is 30 ℃, an HPLC mixed mobile phase adopts an isocratic elution mode, and the mixed mobile phase consists of a mobile phase A, a mobile phase B and a mobile phase C, wherein the mobile phase A is a 0.1% sodium heptanesulfonate (pH 2.1) aqueous solution, the mobile phase B is methanol, the mobile phase C is isopropanol, and the mixing ratio of the mobile phase A, the mobile phase B and the mobile phase C is 91:7: 2;
and step five, precisely measuring 20 mu L of the test sample solution, injecting the test sample solution into a liquid chromatograph, recording a chromatogram 1, after the chromatogram is finished, injecting 20 mu L of the reference sample solution into the liquid chromatograph, recording a chromatogram 2, and calculating the content of the nicotinamide in the test sample by peak area according to an external standard method.
The method has the best effect in detecting the nicotinamide within the concentration range of 1-20 mu g/mL, the concentration of the nicotinamide and the peak area obtained by HPLC (high performance liquid chromatography) detection have good linear relation within the concentration range of 1-20 mu g/mL, and the linear equation is that y is 36.347x +6.3405, R is 2 0.9997, wherein x is the concentration of nicotinamide,. mu.g/mL, and y is the peak area by HPLC, mAV × s.
Preferably, the detection method can be used for detecting the content of nicotinamide in the feed additive.
Determination of mobile phase according to the invention: sodium alkyl sulfonates such as sodium dodecyl sulfate, sodium octane sulfonate and sodium heptane sulfonate are ion pair reagents used for HPLC detection of alkaline compounds, the longer the carbon chain of alkyl in the ion pair reagent, the weaker the polarity, the poorer the separation effect, i.e. the sodium alkyl sulfonate has different separation conditions for different substances, the separation selectivity is increased, the nicotinamide belongs to amide compounds and has alkalescence, and according to the length sequence of the carbon chain of alkyl in the sodium alkyl sulfonate, the mobile phase A in the application selects sodium heptane sulfonate which is superior to other ion pair reagents such as sodium dodecyl sulfate and sodium octane sulfonate, so that the detection wavelength of the nicotinamide can be prolonged to 267nm and is far away from the general wavelength of 254 nm.
The polarity of acetonitrile is higher than that of methanol, and one aspect of methanol is that the acetonitrile can completely dissolve more types of samples, the peak shape of an object to be detected is well improved, the selectivity of methanol is different from that of acetonitrile, the elution time and retention time are also well improved, so that the mobile phase B of the application selects methanol.
The invention has the beneficial effects that:
the method is established based on an HPLC method, the HPLC method is adopted to replace a detection method for nicotinamide content in Chinese veterinary pharmacopoeia and GB7301-2017, a mobile phase which is simpler than the existing method is determined through tests, meanwhile, the mobile phase of the method enables the detection wavelength of nicotinamide to be changed from 262nm to 267nm, the general wavelength is away from 254nm, interference is reduced, data accuracy is improved, and the purpose of separating and determining the content of main component nicotinamide in a test sample is achieved.
The method improves the separation of nicotinamide and nicotinic acid, has simple operation and high accuracy, can well guide the clinical detection of the nicotinamide content, and can greatly improve the detection efficiency of the nicotinamide.
Compared with the existing detection method of nicotinamide content, the method provided by the invention can achieve the detection purpose by adopting the universal chromatographic column and the ultraviolet detector, is simple and convenient to operate and higher in accuracy, greatly reduces the cost in the aspect of material consumption, and can be realized by using a high performance liquid chromatograph equipped with the ultraviolet detector.
Drawings
FIG. 1 is an HPLC chromatogram of mobile phase ApH1.9;
FIG. 2 is an HPLC profile of mobile phase ApH2.1;
FIG. 3 is an HPLC chromatogram of mobile phase ApH2.5;
FIG. 4 is an HPLC plot at 25 ℃ column temperature;
FIG. 5 is an HPLC chromatogram at a column temperature of 30 ℃;
FIG. 6 is an HPLC chromatogram at a column temperature of 35 ℃;
FIG. 7 is an HPLC plot of flow rate 0.8 mL/min;
FIG. 8 is an HPLC plot of flow rate 1.0 mL/min;
FIG. 9 is an HPLC plot of flow rate 1.2 mL/min;
FIG. 10 is an HPLC plot of nicotinamide concentration of 1 μ g/mL;
FIG. 11 is an HPLC plot of nicotinamide concentration of 2 μ g/mL;
FIG. 12 is an HPLC plot of nicotinamide concentration of 5 μ g/mL;
FIG. 13 is an HPLC plot of nicotinamide concentration of 10 μ g/mL;
FIG. 14 is an HPLC plot of nicotinamide concentration of 20 μ g/mL;
FIG. 15 is an HPLC chromatogram of a blank solvent, i.e., an extract;
FIG. 16 is an HPLC profile of a control solution;
FIG. 17 is a graph showing the linear relationship between the concentration of nicotinamide and the peak area obtained by HPLC.
Detailed Description
The following examples are intended to illustrate preferred embodiments of the present invention, and it will be understood by those skilled in the art that the following descriptions should not be construed as limiting the scope of the present invention.
Example 1 detection method of nicotinamide content
And (3) testing the sample: nicotinamide.
Step one, preparing an extracting solution: adding 50mL of acetonitrile and 10mL of glacial acetic acid into 800mL of water, uniformly mixing, adding water to a constant volume of 1000mL, and uniformly mixing to obtain the product;
step two, preparing a test solution: weighing 0.1g of a test sample, diluting the test sample by using an extracting solution, wherein the mass of the test sample and the extracting solution is as follows: the volume ratio is 0.1 g: 100mL, continuously taking a proper amount of the solution, diluting the solution by 200 times with the extracting solution, and shaking up to obtain a test solution with the concentration of 5.0 mu g/mL;
step three, preparing a reference substance solution: collecting nicotinamide standard, adding the extractive solution to obtain solution containing nicotinamide 5.0 μ g per 1mL, i.e. 5.0 μ g/mL control solution;
step four, setting HPLC chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of a chromatographic column, the specification of the chromatographic column is 4.6mm multiplied by 150mm, the flow rate is 1.0mL/min, the detection wavelength is 267nm, the column temperature is 30 ℃, isocratic elution is adopted, and the mobile phase consists of a mobile phase A, a mobile phase B and a mobile phase C, wherein the mobile phase A is a 0.1% sodium heptanesulfonate (pH 2.1) aqueous solution, the mobile phase B is methanol, the mobile phase C is isopropanol, and the ratio of the mobile phase A to the mobile phase B to the mobile phase C is 91:7: 2;
and step five, precisely measuring 20 mu L of the test sample solution, injecting the test sample solution into a liquid chromatograph, recording a chromatogram 1, after the chromatogram is finished, injecting 20 mu L of the reference sample solution into the liquid chromatograph, recording a chromatogram 2, and calculating the content of the nicotinamide in the test sample by peak area according to an external standard method.
EXAMPLE 2 examination of the durability of the invention
Selecting C of 4.6mm x 150mm 18 The durability of the chromatographic column focuses on the influence of the same proportion of mobile phase and different pH, column temperature and flow rate on the determination method.
1. Effect of mobile phase pH on the Process of the invention
In order to examine the influence of the pH value of the mobile phase on the content detection method, three mobile phases with the pH values of 1.9, 2.1 and 2.5 of the mobile phase A are respectively prepared, the other conditions are the same, chromatograms are respectively recorded, and the separation degree, the peak width, the number of theoretical plates, the retention time and the content are compared, and the results are shown in figures 1-3 and table 1:
TABLE 1 Effect of different pH of mobile phase on Nicotinamide detection
Figure BDA0003774039860000051
Figure BDA0003774039860000061
It is shown from table 1 that, when the pH of the mobile phase reaches 1.9, the retention time of nicotinamide is significantly advanced, and when the pH reaches 1.9, the retention time of nicotinamide is shifted backward, it can be seen that, when the pH of the mobile phase is in the range of 1.9-2.5, the separation degree and the content are both in accordance with the regulations, and when the pH is 1.9, the separation degree is significantly reduced, which indicates that the pH of the mobile phase is in the range of 1.9-2.1, nicotinamide is relatively stable, and the retention time, the separation degree, the peak width and the content are all in accordance with the regulations, and the pH of the mobile phase is the best at 2.1.
2. Effect of column temperature on the Process of the invention
In order to examine the influence of the column temperature change on the invention, the column temperature is controlled by a column temperature box in the test, three conditions of 25 ℃, 30 ℃ and 35 ℃ are respectively set, chromatographic signals of samples at corresponding temperatures are respectively collected, and the separation degree, the theoretical plate number, the retention time and the content in chromatogram parameters at different temperatures are compared, and the results are shown in figures 4-6 and table 2:
TABLE 2 influence of column temperature on Nicotinamide detection
Column temperature (. degree. C.) Retention time (min) Number of theoretical plate Peak width Degree of separation Content (% of indicated amount)
25 7.045 12093 2.11 1.22 99
30 6.939 16840 1.91 1.83 99
35 7.065 14687 2.09 1.66 100
As can be seen from the data in table 2 above, as the column temperature increases, the retention of nicotinamide is different but not significant; at different temperatures, the number of plates, the separation degree and the content of the nicotinamide all meet the regulations; it can thus be seen that the column temperature has less influence on nicotinamide.
3. Effect of flow Rate on the invention
In order to examine the influence of the flow rate on the invention, the flow rate is controlled by a quaternary pump in the experiment, three conditions of 0.8mL/min, 1.0mL/min and 1.2mL/min are respectively set, sample chromatographic signals under corresponding flow rates are respectively collected, and the separation degree, the theoretical plate number, the retention time and the content in chromatogram parameters under different flow rates are compared, and the results are shown in FIGS. 7-9 and Table 3:
TABLE 3 Effect of flow Rate on Nicotinamide detection
Figure BDA0003774039860000062
As can be seen from the data in the above table 3, the retention of nicotinamide is more obvious in advance with the increase of the flow rate, and the number of the plates and the content of the separation degree of nicotinamide meet the regulations under different flow rates, so that the influence of the flow rate on the nicotinamide is less.
Example 3 Effect of blank solvent on assay method
The blank solvent (namely the extracting solution), the reference substance solution and the test substance solution which are required by the nicotinamide are respectively processed according to the detection method of the invention, and the machine is arranged. As can be seen from the graphs of fig. 15-16, the blank solvent had no effect on nicotinamide.
Example 4 Linear relationship experiment
Since the concentration of nicotinamide is considered to be 5.0 μ g/mL in the method of the present invention, in this example, the nicotinamide extract is prepared into solutions with concentrations of 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL and 20.0 μ g/mL, respectively, shaken, 20 μ L of the reference solution is precisely measured and injected into the liquid chromatograph, and a chromatogram is recorded, as shown in fig. 10-14, by taking the peak area as the ordinate and the concentration of nicotinamide in the test solution as the abscissa, a linear chromatogram is obtained, as shown in fig. 17 and table 4:
TABLE 4 Linear relationship test results
Figure BDA0003774039860000071
According to the values in the table, the regression equation y of nicotinamide concentration and peak area in the test solution is 36.347x +6.3405, and the linear relation coefficient R is obtained 2 0.9997, i.e. concentration of nicotinamide in the range of 1.0-20.0 μ g/mL, concentration of nicotinamide is in a good linear relationship with peak area.
Example 5 repeatability test
Preparing nicotinamide into a solution with the concentration of 5.0 mug/mL by using the extracting solution, shaking up and filtering to obtain a test solution, injecting 20 mu L of the test solution into a liquid chromatograph, continuously injecting the sample for 5 times in the same volume, and recording a chromatogram, wherein the result is shown in the following table 5:
TABLE 5 examination of the repeatability tests
Number of times 1 2 3 4 5 RSD(％)
Area of nicotinamide 196.3 200.5 195.9 199.2 198.2 1.0
The experimental result shows that the RSD of the peak area measurement value in the repeatability is less than 2.0 percent, which indicates that the method has applicability to the product.
Example 6 intermediate precision test
Different testers accurately weigh the test samples respectively, intermediate precision tests of the method are carried out by using the method of the invention, and different operators use different instruments and operate according to the method of the invention at different time, wherein the test condition 1 is that the tester A respectively tests three groups of test samples according to the chromatographic condition of the embodiment, and the test condition 2 is that the tester B respectively tests three groups of test samples according to the chromatographic condition of the embodiment, and the results are shown in Table 6:
TABLE 6 Nicotinamide intermediate precision test
Figure BDA0003774039860000081
From the table, the nicotinamide content in the sample is measured by different persons and on different dates, the relative standard deviation of the results of six measurements is less than 2.0%, and the intermediate precision of the experimental method meets the requirement.
Example 7 accuracy test
Precisely weighing auxiliary material components required by nicotinamide and 9 parts of blank solution, respectively preparing 3 preparations according to the addition rates of 80%, 100% and 120% of the nicotinamide, and detecting according to the detection method of the invention, wherein the results are shown in Table 7:
TABLE 7 Nicotinamide accuracy test
Figure BDA0003774039860000082
The result shows that the addition amount of the nicotinamide is within the range of 80-120%, the recovery rate is 98-102%, and the method is accurate and reliable.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Claims (10)

1. The detection method of nicotinamide content is characterized by adopting an HPLC method for detection, wherein the chromatographic conditions are as follows: mixing the mobile phase A, the mobile phase B and the mobile phase C in proportion, and performing isocratic elution; wherein the mixing proportion of the mobile phase A, the mobile phase B and the mobile phase C is (91-93): (7-4): (2-3); the mobile phase A is 0.1-0.2% sodium heptanesulfonate water solution, and the pH value is 1.9-2.5; the mobile phase B is methanol; the mobile phase C is isopropanol.
2. The method for detecting nicotinamide content of claim 1, wherein a mixing ratio of mobile phase A, mobile phase B and mobile phase C is 91:7: 2.
3. The method for detecting nicotinamide content of claim 1, wherein mobile phase A is 0.1% sodium heptanesulfonate water solution, and pH is 2.1.
4. The method for detecting nicotinamide content of claim 1, wherein the preparation method of the mobile phase A comprises the following steps: mixing sodium heptanesulfonate and water according to the mass-volume ratio of (10-20) to 1000, shaking uniformly, and adjusting the pH value of the aqueous solution to 1.9-2.5 by using perchloric acid.
5. The method for detecting nicotinamide content of claim 4, wherein the preparation method of the mobile phase A comprises the following steps: mixing sodium heptanesulfonate and water according to the mass-volume ratio of 10:1000, shaking up, and adjusting the pH value of the aqueous solution to 2.1 by perchloric acid.
6. The method for detecting nicotinamide content of claim 1, wherein chromatographic conditions of HPLC are as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and the specification is 4.6mm multiplied by 150 mm; the flow rate is 0.8-1.2 mL/min; the detection wavelength is 250-280 nm; the column temperature is 25-35 ℃; the amount of sample was 20. mu.L.
7. The method of claim 6, wherein the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 267 nm.
8. The method for detecting nicotinamide content of claim 6, characterized in that the detection steps are as follows:
step one, preparing an extracting solution, namely adding 50mL of acetonitrile and 10mL of glacial acetic acid into 800mL of water, mixing, diluting the water to 1000mL, and uniformly mixing to obtain the product;
step two, accurately weighing 0.1g to 100mL of a test sample in a volumetric flask, dissolving the test sample in an extracting solution and fixing the volume, precisely weighing 5mL to 100mL of the volumetric flask, diluting the test sample with the extracting solution and fixing the volume to prepare a solution with the final concentration of 5 mu g/mL;
step three, preparing a nicotinamide control solution, dissolving and diluting the extract to prepare the nicotinamide control solution containing 5.0 mu g of nicotinamide in each 1 ml;
setting HPLC chromatographic conditions;
step five, precisely measuring a sample solution to be tested, injecting the sample solution into a liquid chromatograph, and recording a chromatogram map 1; and injecting the nicotinamide reference substance solution into a liquid chromatograph, recording a chromatogram 2, and calculating the content of nicotinamide in the test sample by peak area according to an external standard method.
9. Use of a method of detecting nicotinamide content of any one of claims 1-8 to detect nicotinamide at a concentration range of 1-20 μ g/mL.
10. Use of a method of detecting the level of nicotinamide according to any one of claims 1-8 in a method of detecting the level of nicotinamide in a feed additive.
CN202210911339.1A 2022-07-30 2022-07-30 Detection method of nicotinamide content and application thereof Pending CN115112805A (en)

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