Method for detecting reduced nicotinamide mononucleotide and application thereof

Abstract

The application belongs to the technical field of quality detection, and particularly relates to a detection method of reduced nicotinamide mononucleotide (NMNH) and application thereof. The detection method of reduced nicotinamide mononucleotide provided by the application establishes a specific detection method based on high performance liquid chromatography technology, clearly controls the quality of a product containing reduced nicotinamide mononucleotide, fills a vacancy in the technical field of quality standard of reduced nicotinamide mononucleotide product, and can be used as a quality control detection method of reduced nicotinamide mononucleotide product and a sample in a production process.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology, specifically relating to a method for detecting reduced nicotinamide mononucleotide (NMNH) and its application. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are essential metabolites for a variety of electron exchange-dependent biochemical reactions in which they act as metabolic cofactors. This fine-tuning, altered by increased enzyme activity or decreased biosynthesis through NAD+ consumption, is a common feature of the natural aging process. In recent years, this relationship between declining NAD+ levels and disease has drawn attention to NAD+ supplementation strategies as a potential therapeutic approach. In this regard, supplementing NAD+ precursors to activate NAD+ rescue pathways and increase NAD+ levels has proven particularly effective in improving or preventing disease in many animal models of metabolic disorders, neurodegeneration, and aging, as well as in humans.

[0003] According to relevant literature, reduced nicotinamide mononucleotide (NMNH) is a novel NAD+ supplement precursor whose anti-aging properties surpass those of NMN (nicotinamide mononucleotide) and NR (nicotinamide-ribose chloride). Studies have found that NMNH not only significantly increases NAD+ levels in cells but also accelerates the rate of NAD+ concentration increase, effectively acting as an NAD+ enhancer. Compared to NMN, NMNH increases cellular NAD+ levels both in vitro and in vivo, particularly in various human tissues, with the greatest impact on the liver and kidneys. Simultaneously, NMNH significantly increases NAD+ content in the brain, calf muscles, brown adipose tissue, and heart—something NMN cannot achieve. Furthermore, NMNH supplementation can reduce cellular damage in hypoxia / reoxygenation injury and enhance cellular repair capabilities by targeting NAD+ regeneration, strengthening mitochondrial activity, and nucleotide metabolism. It can also increase intracellular NADH accumulation, inhibit cellular glycolysis and the tricarboxylic acid cycle (TCA cycle), and suppress cell growth, while NMN only has limited inhibitory effects on cellular glycolysis. Therefore, NMNH, as an innovative frontier and rising star in the anti-aging product family, has received widespread attention and application.

[0004] However, there are currently no publicly available reports on quality standards and testing methods for NMNH in the industry. The field looks forward to developing a method that can characterize and detect the quality status and effective content of NMNH, which would be of positive significance for the development and promotion of NMNH in the field of anti-aging. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting reduced nicotinamide mononucleotide (NMNH), wherein the detection method utilizes high performance liquid chromatography to characterize the related substances and content, providing data support for the quality control of NMNH-related products;

[0006] The second technical problem to be solved by the present invention is to improve the application of the detection method for the above-mentioned reduced nicotinamide mononucleotide (NMNH) in the field of NMNH quality characterization and quality monitoring.

[0007] To solve the above-mentioned technical problems, the detection method of reduced nicotinamide mononucleotide of the present invention includes the step of performing high performance liquid chromatography detection on the test sample solution;

[0008] In the high performance liquid chromatography detection step, the chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, using an 8-47 mM potassium dihydrogen phosphate aqueous solution containing 0.1% triethylamine as mobile phase A, and using methanol solvent as mobile phase B, and performing gradient elution according to the procedure in Table 1 below.

[0009] Table 1 Elution Procedure

[0010] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 8 95 5 10 90 10 15 90 10 15.01 98 2 20 98 2

[0011] Specifically, in the method for detecting reduced nicotinamide mononucleotide, the chromatographic conditions in the high-performance liquid chromatography (HPLC) detection step further include:

[0012] Column temperature 28-32℃; and / or;

[0013] The flow rate is 0.65-0.75 ml / min; and / or;

[0014] The detection wavelength is 338-342nm; and / or.

[0015] Specifically, in the method for detecting reduced nicotinamide mononucleotide, the high-performance liquid chromatography (HPLC) detection step uses a YMC-PACK ODS-AQ column or an equivalent column.

[0016] Specifically, the method for detecting reduced nicotinamide mononucleotide includes the following steps for preparing the test solution: precisely adding a diluent to the test sample, mixing, filtering, and collecting the filtrate.

[0017] Specifically, the method for detecting reduced nicotinamide mononucleotide further includes the step of preparing a reference solution, which specifically includes: accurately adding the reference standard NMNH to a diluent and mixing, filtering and collecting the filtrate to obtain the solution.

[0018] Specifically, in the method for detecting reduced nicotinamide mononucleotide, the diluent includes purified water.

[0019] Specifically, the method for detecting reduced nicotinamide mononucleotide further includes a step of fitting a linear equation for NMNH;

[0020] The linear equation for NMNH is Y = 12524X + 286.23, R = 0.9999.

[0021] The present invention also discloses the application of the detection method of reduced nicotinamide mononucleotide in the field of reduced nicotinamide mononucleotide quality control.

[0022] The present invention also discloses a detection model for reduced nicotinamide mononucleotide, wherein the linear equation of the model is Y = 12524X + 286.23, R = 0.9999; where X is the sample concentration and Y is the peak area.

[0023] The present invention also discloses the application of the detection model of the reduced nicotinamide mononucleotide in the field of reduced nicotinamide mononucleotide quality control.

[0024] The detection method for reduced nicotinamide mononucleotide (NMN) described in this invention establishes a specific detection method based on high-performance liquid chromatography (HPLC), clarifies the quality control of products containing NMN, fills a gap in the technical field of quality standards for NMN products, and can be used as a quality control detection method for NMN products and samples during the production process.

[0025] The detection method for reduced nicotinamide mononucleotide described in this invention has the advantages of simple sample preparation and rapid and efficient quality analysis. It is easy to operate and highly efficient in detection, solving the problem of quality control of reduced nicotinamide mononucleotide components in the production process. At the same time, it fills the gap in research on quality control and can be used as a quality control method for reduced nicotinamide mononucleotide related products. Attached Figure Description

[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0027] Figure 1 This is the result of the attribute overlay in Example 2;

[0028] Figure 2 The linear fitting results for NMNH in Example 2;

[0029] Figure 3 The results are the durability test results under the condition of adding 7 mmol of potassium dihydrogen phosphate to mobile phase A in Example 3. Detailed Implementation

[0030] In the following embodiments of the present invention, the standards involved in methodological verification and durability testing are shown in Table 2 below.

[0031] Table 2 Validation Standards

[0032]

[0033]

[0034] Example 1

[0035] Preparation of reference solution: Accurately weigh 100 mg of NMNH reference standard and place it in a 100 ml volumetric flask. Add purified water to dissolve and dilute to the mark, and shake well to obtain the reference solution.

[0036] Test solution: Accurately weigh 100 mg of NMNH test sample and place it in a 100 ml volumetric flask. Add purified water to dissolve and dilute to the mark, then shake well to obtain the test solution.

[0037] Blank solution (diluent): purified water.

[0038] High performance liquid chromatography (HPLC) was used to determine the content of NMNH and related substances. The specific chromatographic parameters are as follows:

[0039] Chromatographic column: YMC-PACK ODS-AQ, 5μm 4.6*250mm;

[0040] Mobile phase A: 10 mM potassium dihydrogen phosphate aqueous solution containing 0.1% triethylamine;

[0041] Mobile phase B: methanol solvent;

[0042] Washing procedure: as specified in Table 1 above;

[0043] Detection wavelength: 340nm;

[0044] Flow rate: 0.7 ml / min;

[0045] Injection volume: 10 μL;

[0046] Column temperature: 30℃.

[0047] The above-described procedures were used to detect NMNH-containing test solutions by high-performance liquid chromatography.

[0048] Example 2 Methodological Investigation

[0049] This embodiment relates to the high-performance liquid chromatography method for the determination of NMNH content developed above. The method validation items include: system usability, specificity, precision, linearity, range, accuracy, and robustness.

[0050] 1. System adaptability verification

[0051] Solution preparation: Accurately weigh 100 mg of the reference standard, place it in a 100 ml volumetric flask, dissolve and dilute it to the mark with purified water to prepare the reference standard solution, and inject it into 6 injections consecutively.

[0052] Blank solution: purified water.

[0053] In this embodiment, the system adaptability was verified according to the scheme described in Embodiment 1, and the results are shown in Table 3 below.

[0054] Table 3 System Applicability Results

[0055] No. 1 2 3 4 5 6 average value RSD Peak area 13312.2 13279.2 13267.0 13264.5 13250.3 13243.6 13269.5 0.18％ Retention time 12.173 12.176 12.178 12.191 12.208 12.187 12.191 0.12％ Theoretical number of plates 18161 18358 17924 18132 17985 17870 18072 1.00％ Resolution 6.21 6.22 6.22 6.20 6.24 6.19 6.21 0.28％

[0056] As can be seen, the retention time RSD% of the main peak is 0.12%, the peak area RSD% is 0.18%, and the separation degree between the main peak and the adjacent impurity peak is greater than 1.5, which meets the requirements and indicates that the method has good system applicability.

[0057] 2. Specificity test

[0058] Preparation of high-temperature forced degradation solution: Take an appropriate amount of NMNH test sample, place it in an oven at 105℃ for 24 hours, take it out, accurately weigh 100mg of the test sample, place it in a 100ml volumetric flask, dissolve and dilute it to the mark with purified water to prepare the sample solution.

[0059] Blank solution: purified water.

[0060] In this embodiment, the verification was performed according to the scheme described in Embodiment 1, and the specific overlay image results are shown in the appendix. Figure 1 The separation results are shown in Table 4 below.

[0061] Table 4. Separation results of NMNH and impurities

[0062] name Retention time (min) Resolution Impurities before the main peak 11.277 1.93 NMNH 11.737 1.67 Impurities after the main peak 12.570 2.06

[0063] It can be seen that the blank solution has no interference at the NMNH chromatographic peak. In the forced degradation solution, the resolution between adjacent impurities and the NMNH chromatographic peak is 1.67 and 2.06, respectively, both greater than 1.5, indicating that the method has good specificity.

[0064] 3. Accuracy Test

[0065] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions.

[0066] Preparation of 50% accuracy solution: Accurately weigh 50 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare three parallel solutions.

[0067] Preparation of 100% accuracy solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare three parallel solutions.

[0068] Preparation of 150% accuracy solution: Accurately weigh 150 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare three parallel solutions.

[0069] In this embodiment, the verification was carried out according to the scheme described in Example 1, and the accuracy solution results are shown in Table 5 below.

[0070] Table 5. Accuracy Results of NMNH

[0071]

[0072]

[0073] As can be seen, the recovery rate and the average recovery rate are between 98.07% and 99.55%, and the RSD% of the nine samples is 0.65%, which is less than 2.0%, meeting the requirements and indicating that the method has good accuracy.

[0074] 4. Precision testing

[0075] Repeatability: Experimenter 1 accurately weighs 100 mg of the test sample, places it in a 100 ml volumetric flask, dissolves it in purified water, and dilutes it to the mark. Prepare 6 parallel samples.

[0076] Intermediate precision: Experimenter 2 accurately weighs 100 mg of the test sample, places it in a 100 ml volumetric flask, dissolves and dilutes it to the mark with purified water. Prepare 6 parallel samples.

[0077] In this embodiment, the verification was performed according to the scheme described in Example 1. The repeatability and intermediate detection precision results are shown in Tables 6-8 below.

[0078] Table 6. Repeatability test data of Experimenter 1

[0079]

[0080] Table 7. Intermediate precision results for Experimenter 2

[0081]

[0082] Table 8 Intermediate Precision Results

[0083]

[0084]

[0085] As can be seen, the RSD of the 6 content results under Experimenter 1's operation was 0.51%, and the RSD of the 12 content results was 0.78%, which met the requirements, indicating that the method has good repeatability and intermediate precision.

[0086] 5. Linearity and range testing

[0087] Linear stock solution: Weigh 1 g of NMNH reference standard, place it in a 100 ml volumetric flask, add purified water to dissolve and dilute to the mark.

[0088] Linearity test solution 1: Accurately transfer 0.5 ml of the linearity stock solution into a 10 ml volumetric flask and dilute to the mark with purified water.

[0089] Linearity test solution 2: Accurately transfer 0.8 ml of the linearity stock solution into a 10 ml volumetric flask and dilute to the mark with purified water.

[0090] Linearity test solution 3: Accurately transfer 1.0 ml of linearity stock solution into a 10 ml volumetric flask and dilute to the mark with purified water.

[0091] Linearity test solution 4: Accurately transfer 1.2 ml of linearity stock solution into a 10 ml volumetric flask and dilute to the mark with purified water.

[0092] Linearity test solution 5: Accurately transfer 1.5 ml of linearity stock solution into a 10 ml volumetric flask and dilute to the mark with purified water.

[0093] Take 1-5 of the above linear test solutions and perform the procedures described in Example 1. Record the chromatograms and perform linear regression calculations with the standard concentration (mg / ml) as the abscissa and the main peak area as the ordinate. Obtain the linear regression equation and correlation coefficient. The results are shown in Table 9 below, and the fitted linear graph is attached. Figure 2 .

[0094] Table 9. Linearity test results

[0095]

[0096]

[0097] As can be seen, the detection method described in this invention exhibits a linear relationship between peak area and solution concentration within the range of 0.5014 mg / ml to 1.5041 mg / ml: y = 12524x + 286.23, R = 0.9999. This meets the requirements, indicating that the method has good linearity.

[0098] Example 3 Durability Test

[0099] This embodiment examines the durability of the method described in Embodiment 1.

[0100] 1. Durability - Diluent at different pH values ​​- pH 4.0

[0101] Preparation of diluent: Take an appropriate amount of purified water and adjust the pH of the diluent to 4.0 using phosphoric acid solution.

[0102] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with diluent. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0103] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with diluent. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, respectively.

[0104] In this embodiment, the results of the investigation of the diluent pH 4.0 are shown in Table 10 below.

[0105] Table 10 Durability Test Results - Diluent pH 4.0

[0106]

[0107]

[0108] As can be seen, in this embodiment, with the diluent selected at pH 4.0, the peak area RSD of reference standard-1 for 6 consecutive injections was 2.41%, and the recovery rate of reference standard-2 for 2 consecutive injections was 94.27%. However, the test result of the test sample, compared with the content under normal conditions (reproducible results of experimenter 1), showed a recovery rate of 97.05%, indicating that neither the system suitability nor the sample recovery rate met the requirements.

[0109] 2. Durability - Diluent at different pH values ​​- pH 8.0

[0110] Preparation of diluent: Take an appropriate amount of purified water and adjust the pH of the diluent to 8.0 with triethylamine solution.

[0111] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with diluent. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0112] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with diluent. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject them multiple times for testing.

[0113] In this embodiment, the results of the diluent pH 8.0 test are shown in Table 11 below.

[0114] Table 11 Durability Test Results - Diluent pH 8.0

[0115]

[0116] As can be seen, in this embodiment, with the diluent selected at pH 8.0, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.19%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.73%. Compared with the test results of the test sample under normal conditions (reproducible results of experimenter 1), the recovery rate was 101.42%. The system suitability and sample recovery rate both meet the requirements.

[0117] 3. Durability - Investigation of different chromatographic columns

[0118] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0119] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0120] In this embodiment, a SHIMADZU chromatographic column was used for testing.

[0121] In this embodiment, the results of different chromatographic columns are shown in Table 12 below.

[0122] Table 12 Robustness - Results of tests on different chromatographic columns

[0123]

[0124] As can be seen, in the investigation of different chromatographic columns, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.27%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.35%; while the test sample showed a recovery rate of 101.05% compared with the content under normal conditions (reproducible results of experimenter 1). The system suitability and sample recovery rate both meet the requirements.

[0125] 4. Durability - Mobile Phase A System (Adjust the pH of Mobile Phase A to 7.0 using ammonia)

[0126] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0127] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0128] The pH of mobile phase A was adjusted to 7.0 using ammonia water, and other conditions were the same as in Example 1. The results are shown in Table 13 below.

[0129] Table 13. Durability Results - Ammonia Adjustment of Mobile Phase A pH to 7.0

[0130]

[0131] As can be seen, in this embodiment, the pH of mobile phase A was adjusted to 7.0 using ammonia. The peak area RSD of reference standard-1 for 6 consecutive injections was 0.09%, and the recovery rate of reference standard-2 for 2 consecutive injections was 100.24%. The test result of the test sample showed a recovery rate of 101.62% compared with the content under normal conditions (reproducible results of experimenter 1). The system suitability and sample recovery rate both meet the requirements.

[0132] 5. Durability Test - Mobile Phase A System (Adjust the pH of mobile phase A to 7.0 using potassium hydroxide solution)

[0133] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0134] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0135] The pH of mobile phase A was adjusted to 7.0 using potassium hydroxide solution, and other conditions were the same as in Example 1. The results are shown in Table 14 below.

[0136] Table 14. Durability Results - Adjustment of Mobile Phase A pH to 7.0 with Potassium Hydroxide Solution

[0137]

[0138] As can be seen, in this embodiment, the pH of mobile phase A was adjusted to 7.0 using potassium hydroxide solution. The peak area RSD of reference standard-1 for 6 consecutive injections was 0.45%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.67%. Compared with the content of the test sample under normal conditions (reproducible results of experimenter 1), the recovery rate was 101.95%. The system suitability and sample recovery rate both meet the requirements.

[0139] 6. Durability Test - Different concentrations of triethylamine (0.05%) in mobile phase A

[0140] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0141] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0142] 0.05% triethylamine was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 15 below.

[0143] Table 15. Robustness Results - Triethylamine in Mobile Phase A (0.05%)

[0144]

[0145] It can be seen that when 0.05% triethylamine is added to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections is 0.24%, and the recovery rate of reference standard-2 for 2 consecutive injections is 99.52%; while the test result of the test sample, compared with the content under normal conditions (reproducible results of experimenter 1), has a recovery rate of 101.13%. The system suitability and sample recovery rate both meet the requirements.

[0146] 7. Robustness - Different triethylamine concentrations (0.15%) in mobile phase A

[0147] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0148] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0149] 0.15% triethylamine was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 16 below.

[0150] Table 16. Robustness Results - Triethylamine in Mobile Phase A (0.15%)

[0151]

[0152] It can be seen that when 0.05% triethylamine is added to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections is 2.39%, and the recovery rate of reference standard-2 for 2 consecutive injections is 101.06%; while the recovery rate of the test sample is 101.08% compared with the content under normal conditions (reproducible results of experimenter 1), and the system suitability does not meet the requirements.

[0153] 8. Robustness - Different buffer salts (10 mmol sodium dihydrogen phosphate) in mobile phase A

[0154] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0155] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0156] 10 mmol of sodium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 17 below.

[0157] Table 17 Robustness – Results of different buffer salts (10 mmol sodium dihydrogen phosphate) in mobile phase A

[0158]

[0159]

[0160] As can be seen, with the addition of 10 mmol of sodium dihydrogen phosphate to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.07%, and the recovery rate of reference standard-2 for 2 consecutive injections was 101.76%; while the recovery rate of the test sample was 100.76% compared with the content under normal conditions (reproducible results of experimenter 1). Both the system suitability and the recovery rate of the test sample meet the requirements.

[0161] 9. Robustness - Different buffer salts (7 mmol potassium dihydrogen phosphate) in mobile phase A

[0162] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0163] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0164] Add 7 mmol of potassium dihydrogen phosphate to mobile phase A, and use the same chromatographic conditions as in Example 1. See the attached chromatogram for the detection results. Figure 3 .

[0165] As can be seen, the retention time of the principal components drifts significantly under this condition, failing to meet the requirements, and therefore no results were calculated.

[0166] 10. Robustness - Different buffer salts (8 mmol potassium dihydrogen phosphate) in mobile phase A

[0167] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0168] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0169] 8 mmol of potassium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 18 below.

[0170] Table 18. Robustness – Results of different buffer salts (8 mmol potassium dihydrogen phosphate) in mobile phase A

[0171]

[0172] The results showed that when 8 mmol of potassium dihydrogen phosphate was added to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.09%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.41%. The recovery rate of the test sample was 101.67% compared with the content under normal conditions (reproducible results of experimenter 1). Both the system suitability and the recovery rate of the test sample met the requirements.

[0173] 11. Robustness - Different buffer salts (47 mmol potassium dihydrogen phosphate) in mobile phase A

[0174] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0175] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0176] 47 mmol of potassium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 19 below.

[0177] Table 19. Robustness – Results of different buffer salts (47 mmol potassium dihydrogen phosphate) in mobile phase A

[0178]

[0179] As can be seen, with the addition of 47 mmol of potassium dihydrogen phosphate to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.13%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.82%; while the test result of the test sample, compared with the content under normal conditions (reproducible results of experimenter 1), showed a recovery rate of 101.73%. Both the system suitability and the recovery rate of the test sample met the requirements.

[0180] 12. Robustness - Different buffer salts (48 mmol potassium dihydrogen phosphate) in mobile phase A

[0181] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0182] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0183] 48 mmol of potassium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The main component peak in the sample showed a splitting trend, and the results were not calculated.

[0184] 13. Robustness - Different buffer salts (50 mmol potassium dihydrogen phosphate) in mobile phase A

[0185] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0186] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0187] 50 mmol of potassium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 20 below.

[0188] Table 20. Robustness – Results of different concentrations of buffer salt (50 mmol potassium dihydrogen phosphate) in mobile phase A

[0189]

[0190] It can be seen that when 50 mmol of potassium dihydrogen phosphate is added to mobile phase A, the peak area RSD of reference standard-1 for 6 consecutive injections is 0.51%, and the recovery rate of reference standard-2 for 2 consecutive injections is 98.28%. However, the recovery rate of the test sample is 101.43% compared with the content under normal conditions (reproducible results of experimenter 1). The system suitability and the recovery rate of the test sample both meet the requirements, but there is a leading peak before the main peak, which does not meet the requirements.

[0191] 14. Robustness - Different buffer salts (100 mmol potassium dihydrogen phosphate) in mobile phase A

[0192] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0193] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0194] 100 mmol of potassium dihydrogen phosphate was added to mobile phase A, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 21 below.

[0195] Table 21. Robustness – Results of buffer salts (100 mmol potassium dihydrogen phosphate) at different concentrations in mobile phase A.

[0196]

[0197] It can be seen that when 100 mmol of potassium dihydrogen phosphate is added to mobile phase A, the peak area RSD of reference standard-1 in 6 consecutive injections is 0.21%, and the recovery rate of reference standard-2 in 2 consecutive injections is 99.19%. However, the recovery rate of the test sample is 101.58% compared with the content under normal conditions (reproducible results of experimenter 1). The system suitability and the recovery rate of the test sample both meet the requirements, but there is a leading peak before the main peak, which does not meet the requirements.

[0198] 15. Durability - Changing the mobile phase ratio

[0199] Preparation of high-temperature forced degradation solution: Take an appropriate amount of NMNH test sample, place it in an oven at 105℃ for 24 hours, take it out, accurately weigh 100mg of the test sample, place it in a 100ml volumetric flask, dissolve and dilute it to the mark with purified water to prepare the sample solution.

[0200] The ratio of the mobile phase for 8 min was changed to mobile phase A: mobile phase B = 97:3, while other parameters remained unchanged. The results are shown in Table 22 below. The separation degree between the main peak and the adjacent impurities was 2.58 and 4.86, which met the requirements.

[0201] Table 22 Durability - Results of Changing Mobile Phase Ratio over 8 Minutes

[0202] name Retention time (min) Resolution Impurities before the main peak 11.860 1.12 NMNH 12.738 2.58 Impurities after the main peak 14.822 4.86

[0203] As can be seen, by changing the mobile phase ratio for 15 min to mobile phase A: mobile phase B = 95: 5, while keeping other parameters unchanged, the results are shown in Table 23 below. The separation degree between the main peak and the adjacent impurities is 1.59 and 2.14, which meets the requirements.

[0204] Table 23 Durability - Results of Changing Mobile Phase Proportion for 15 Minutes

[0205] name Retention time (min) Resolution Impurities before the main peak 11.309 2.39 NMNH 11.753 1.59 Impurities after the main peak 12.570 2.14

[0206] 16. Durability - Flow rate 0.65 ml / min

[0207] Reference solution: Accurately weigh 100 mg of reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0208] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0209] The flow rate was controlled at 0.65 ml / min, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 24 below.

[0210] Table 24 Durability - Flow Rate 0.65 ml / min

[0211]

[0212]

[0213] As can be seen, when the flow rate was adjusted to 0.65 ml / min, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.31%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.51%; while the test result of the test sample, compared with the content under normal conditions (reproducible result of experimenter 1), showed a recovery rate of 101.26%, and both the system applicability and the recovery rate of the test sample met the requirements.

[0214] 17. Durability - Flow rate 0.75 ml / min

[0215] Reference solution: Accurately weigh 100 mg of reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0216] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0217] The flow rate was controlled at 0.75 ml / min, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 25 below.

[0218] Table 25 Durability - Flow Rate 0.75 ml / min

[0219]

[0220]

[0221] Conclusion: When the flow rate was adjusted to 0.75 ml / min, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.24%, and the recovery rate of reference standard-2 for 2 consecutive injections was 100.19%. Compared with the content under normal conditions (reproducible results of experimenter 1), the recovery rate of the test sample was 101.55%. Both the system suitability and the recovery rate of the test sample met the requirements.

[0222] 18. Durability - Column temperature 28℃

[0223] Reference solution: Accurately weigh 100 mg of reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0224] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0225] The column temperature was controlled at 28°C, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 26 below.

[0226] Table 26 Durability - Column Temperature 28°C

[0227]

[0228] As can be seen, when the column temperature was adjusted to 28℃, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.21%, and the recovery rate of reference standard-2 for 2 consecutive injections was 99.48%; while the test result of the test sample, compared with the content under normal conditions (reproducible result of experimenter 1), showed a recovery rate of 101.06%. The system suitability and the recovery rate of the test sample both met the requirements.

[0229] 19. Durability - Column temperature 32℃

[0230] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0231] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0232] The column temperature was controlled at 32°C, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 27 below.

[0233] Table 27 Durability - Column Temperature 32℃

[0234]

[0235] As can be seen, when the column temperature was adjusted to 32℃, the peak area RSD of reference standard-1 for 6 consecutive injections was 0.31%, and the recovery rate of reference standard-2 for 2 consecutive injections was 100.38%; while the test result of the test sample, compared with the content under normal conditions (reproducible result of experimenter 1), showed a recovery rate of 100.13%. The system suitability and the recovery rate of the test sample both met the requirements.

[0236] 20. Durability - Wavelength 338nm

[0237] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0238] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0239] The wavelength was controlled at 338 nm in the chromatographic conditions, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 28 below.

[0240] Table 28 Durability - Wavelength 338nm

[0241]

[0242] As can be seen, when the wavelength is adjusted to 338 nm, the peak area RSD of reference standard-1 for 6 consecutive needle tests is 0.19%, and the recovery rate of reference standard-2 for 2 consecutive needle tests is 99.75%; while the recovery rate of the test sample is 100.18% compared with the content under normal conditions (reproducer 1's repeatability results). The system suitability and the recovery rate of the test sample both meet the requirements.

[0243] 21. Durability - Wavelength 342nm

[0244] Reference solution: Accurately weigh 100 mg of the reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Reference Standard-1 and Reference Standard-2, and perform multiple injection tests on each.

[0245] Preparation of test solution: Accurately weigh 100 mg of the test sample and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with purified water. Prepare two parallel solutions, labeled as Test Sample-1 and Test Sample-2, and inject the samples multiple times for testing.

[0246] The wavelength was controlled at 342 nm in the chromatographic conditions, and other chromatographic conditions were the same as in Example 1. The results are shown in Table 29 below.

[0247] Table 29 Durability - Wavelength 342nm

[0248]

[0249] As can be seen, when the wavelength is adjusted to 342 nm, the peak area RSD of reference standard-1 for 6 consecutive needle tests is 0.23%, and the recovery rate of reference standard-2 for 2 consecutive needle tests is 99.80%; while the recovery rate of the test sample is 100.28% compared with the content under normal conditions (reproducer 1's repeatability results). The system suitability and the recovery rate of the test sample both meet the requirements.

[0250] In summary, the detection method for reduced nicotinamide mononucleotide described in this invention establishes a specific detection method based on high performance liquid chromatography (HPLC), clarifies the quality control of products containing reduced nicotinamide mononucleotide, and can be used as a quality control detection method for reduced nicotinamide mononucleotide products and samples during the production process.

[0251] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting reduced nicotinamide mononucleotide, characterized in that, This includes the step of performing high-performance liquid chromatography (HPLC) detection on the test sample solution; In the high-performance liquid chromatography (HPLC) detection step, the chromatographic conditions include: using octadecylsilane-bonded silica gel as the stationary phase, using an 8-47 mM potassium dihydrogen phosphate aqueous solution containing 0.1% triethylamine as mobile phase A, and using methanol as mobile phase B, and performing gradient elution according to the following procedure: 。 2. The method for detecting reduced nicotinamide mononucleotide according to claim 1, characterized in that, The chromatographic conditions in the high-performance liquid chromatography detection step also include: Column temperature 28-32℃; and / or; The flow rate is 0.65-0.75 ml / min; and / or; The detection wavelength is 338-342nm.

3. The method for detecting reduced nicotinamide mononucleotide according to claim 1 or 2, characterized in that, In the high-performance liquid chromatography detection step, the chromatographic column includes YMC-PACK ODS-AQ or an equivalent column.

4. The method for detecting reduced nicotinamide mononucleotide according to any one of claims 1-3, characterized in that, The preparation method of the test sample solution includes: taking the test sample, accurately adding a diluent and mixing, filtering and collecting the filtrate to obtain the solution.

5. The method for detecting reduced nicotinamide mononucleotide according to any one of claims 1-4, characterized in that, The method also includes the step of preparing a reference solution, specifically including: taking reference NMNH, accurately adding it to a diluent and mixing, filtering and collecting the filtrate to obtain the solution.

6. The method for detecting reduced nicotinamide mononucleotide according to claim 4 or 5, characterized in that, The diluent includes purified water.

7. The method for detecting reduced nicotinamide mononucleotide according to any one of claims 1-6, characterized in that, The method further includes the step of fitting a linear equation for NMNH; The linear equation for NMNH is Y = 12524X + 286.23, R = 0.9999.

8. The application of the detection method for reduced nicotinamide mononucleotide according to any one of claims 1-7 in the field of quality control of reduced nicotinamide mononucleotide.

9. A detection model for reduced nicotinamide mononucleotide, characterized in that, The linear equation of the model is Y = 12524X + 286.23, R = 0.9999; where X is the sample concentration and Y is the peak area.

10. The application of the detection model of reduced nicotinamide mononucleotide as described in claim 9 in the field of reduced nicotinamide mononucleotide quality control.

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