Detection method of beta-nicotinamide nucleotide and related substances thereof

Abstract

The invention discloses a method for detecting beta-nicotinamide nucleotide (NMN), beta-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in cosmetics. The method comprises the following steps: (1) preparing a standard sample; (2) drawing a standard curve; (3) preparing a sample to be detected; (4) extracting the to-be-detected sample prepared in the step (3) with water, and (5) filtering an extracting solution obtained in the step (4), then detecting in high performance liquid chromatography, and calculating the contents of beta-nicotinamide nucleotide (NMN), beta-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in the to-be-detected sample according to a formula.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics detection, and more specifically relates to a method for detecting the contents of β-nicotinamide mononucleotide, β-nicotinamide adenine dinucleotide, nicotinamide, and nicotinic acid in cosmetics. Background Art

[0002] β-Nicotinamide mononucleotide (NMN) is an active nucleoside monophosphate naturally present in the body and belongs to a derivative of the vitamin B family. NMN is an intermediate in the biosynthesis of β-nicotinamide adenine dinucleotide (NAD). NAD is a derivative of vitamin PP (nicotinic acid (NA) and nicotinamide (NAM)) and is a crucial metabolic redox coenzyme in eukaryotes, playing an important role in various biological processes of the body such as cell death, aging, gene expression, neuroinflammation, and DNA repair. The consumption and reduction of NAD lead to the downregulation of mitochondrial function, which will cause aging. NMN was first successfully filed for the record as a new cosmetic raw material in China in January 2022. The record information shows that the purpose of using this raw material is skin protectant, moisturizer, and antioxidant, and it can be used in various cosmetics except lip products, oral hygiene products, and spray products. The successful record of β-nicotinamide mononucleotide means that NMN raw materials can be used as cosmetic ingredients in Chinese cosmetics.

[0003] Since NMN is relatively unstable, if the formulation is improper or the storage is improper, it may be inactivated before reaching consumers. Therefore, it is particularly important for cosmetic companies to effectively monitor the contents of NMN and its related substances. However, so far, there has been no relevant report on the detection method for the contents of NMN and its related substances in cosmetics.

[0004] Therefore, a detection method for accurately and reliably, quickly and conveniently, and highly practical determination of β-nicotinamide mononucleotide and its related substances in cosmetics is needed. Summary of the Invention

[0005] On the one hand, the present invention provides a method for detecting β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA), and nicotinamide (NAM) in cosmetics, comprising the following steps:

[0006] (1) Prepare a standard sample containing β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA), and nicotinamide (NAM);

[0007] (2) The standard prepared in step (1) is detected by high performance liquid chromatography, and a standard curve is plotted with the peak area as the ordinate and the concentration as the abscissa;

[0008] (3) Prepare a test sample containing β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM);

[0009] (4) Extract the test sample prepared in step (3),

[0010] (5) Filter the extract obtained in step (4), then place it in high performance liquid chromatography for detection, and calculate the contents of β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in the test sample according to the following formula:

[0011]

[0012] In the formula:

[0013] X represents the content of the analyte in the cosmetic, in weight percentage (%);

[0014] C represents the concentration of the analyte in the test sample obtained from the standard curve, in μg / mL;

[0015] V represents the total volume of the test sample, in mL;

[0016] M represents the sample mass, in g.

[0017] In a preferred embodiment, water is used for extraction in step (4).

[0018] In a preferred embodiment, the concentrations of NMN, NAD, NA and NAM in the standard sample prepared in step (1) are 16 μg / mL, 80 μg / mL, 160 μg / mL, 400 μg / mL, 800 μg / mL.

[0019] In a preferred embodiment, the detection wavelength of high performance liquid chromatography is 250 - 300 nm, preferably 260 nm.

[0020] In a preferred embodiment, the volume fraction of the mobile phase used in high performance liquid chromatography varies in the range of "methanol + water = 5 + 95" to "methanol + water = 10 + 90", preferably "methanol + water = 8 + 92".

[0021] In a preferred embodiment, the chromatographic column used in high performance liquid chromatography is a C18 column.

[0022] In a preferred embodiment, the column temperature of the chromatographic column in high performance liquid chromatography is 30 °C.

[0023] In a preferred embodiment, the filtration in step (5) is carried out using a 0.22 μm filter membrane. Description of the Drawings

[0024] Figure 1 Shows the ultraviolet absorption spectra of β-nicotinamide nucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM).

[0025] Figure 2 Shows the high performance liquid chromatogram of the mixed standard working solution of β-nicotinamide nucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM), where 1: NMN; 2: NAD; 3: nicotinic acid (NA); 4: NAM.

[0026] Figures 3a - 3d Respectively show the standard curves of β-nicotinamide nucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM). Detailed Description of the Invention

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0028] The present invention relates to a method for detecting cosmetics, specifically to a method for detecting the contents of β-nicotinamide nucleotide, β-nicotinamide adenine dinucleotide, nicotinamide and nicotinic acid in cosmetics. The detection method of the present invention at least includes the following steps: a. providing a detection instrument combination; b. setting detection parameter conditions; c. preparing a standard solution and drawing a standard curve; d. pre-treating the sample to be tested and reading on the standard curve; e. calculating the results. The detection method of the present invention has the characteristics of an appropriate linear range, simple pre-treatment, reliable sample separation, accurate quantification, good recovery rate, etc.

[0029] The technical problem to be solved by the present invention is to select a suitable extraction solvent and chromatographic separation conditions to detect the contents of β-nicotinamide nucleotide, β-nicotinamide adenine dinucleotide, nicotinic acid and nicotinamide in cosmetics, meeting the advantages of simple steps, reliable method, high accuracy, low detection limit, etc.

[0030] The present invention discloses a method for detecting β-nicotinamide nucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in cosmetics, including the following steps:

[0031] (1) Provide detection instruments:

[0032] High-performance liquid chromatograph Waters Acquity Arc + diode array detector Waters 2998 combination, or equivalent model.

[0033] (2) Set detection conditions:

[0034] Chromatographic column: Agilent SB-C18 chromatographic column (250mm×4.6mm, 5μm, Agilent);

[0035] Mobile phase: Mobile phase A: methanol, Mobile phase B: water (999ml 0.05mol / L sodium dihydrogen phosphate + 1g sodium heptanesulfonate, pH 3.0); A:B = 8:92; Flow rate: 1.0 mL / min;

[0036] Injection volume: 5 μL; Column temperature: 30 °C; Detection wavelength: 260 nm.

[0037] (3) Plot the standard curve:

[0038] Accurately weigh appropriate amounts of NMN, NAD, NA, and NAM reference substances, dissolve them in water, make up the volume, shake well, and prepare a mixed standard stock solution. Store it refrigerated in a 4 °C refrigerator. Take the above mixed standard stock solution and gradually dilute it with the mobile phase to obtain mixed standard working solutions of 16 μg / mL, 80 μg / mL, 160 μg / mL, 400 μg / mL, and 800 μg / mL. Inject and detect according to the above detection conditions (2), plot the standard curve with the peak area as the ordinate and the concentration as the abscissa, and calculate the linear regression equation of the standard curve.

[0039] (4) Sample treatment and detection:

[0040] Take 0.5 - 1 g (accurate to 0.1 mg) of the cosmetic in a 50 mL stoppered volumetric flask, add 20 mL of water, vortex for 1 min to disperse, and ultrasonically extract for 15 min. Make up the volume with the mobile phase, shake well, take a part of the liquid and filter it through a 0.22 μm filter membrane to obtain the test solution. Inject and detect according to the above detection conditions (2), and quantify the sample on the standard curve. The response value of the analyte in the sample solution should be within the linear range of the standard curve.

[0041] (5) Calculation formula, quantitative analysis by external standard method, the calculation formula for the content of the analyte in the sample is:

[0042]

[0043] Where:

[0044] X——Mass fraction of the analyte in the sample, %;

[0045] C—the concentration of the analyte in the test solution obtained from the standard curve, in micrograms per milliliter (μg / mL);

[0046] V—the total volume of the diluted sample, in milliliters (mL);

[0047] M—the mass of the sample, in grams (g);

[0048] The calculation result is retained to three significant figures.

[0049] (6) Selecting a detector wavelength of 260 nm is an optimized technical solution of this method.

[0050] (7) Taking three times the noise signal as the method detection limit and ten times the noise signal as the quantification limit, the detection limits of NMN, NAD, NA, and NAM components are 0.3 μg / mL, and the quantification limit is 1 μg / mL.

[0051] In a specific embodiment, the detection instrument used in the detection method of the present invention is a combination of a high-performance liquid chromatograph Waters Acquity Arc and a diode array detector Waters 2998. Of course, other equivalent models of high-performance liquid chromatographs can also be used.

[0052] In the detection method of the present invention, the chromatographic column used is an Agilent SB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm, Agilent). In the detection method of the present invention, the mobile phase is A: methanol, B: 0.05 mol / L sodium dihydrogen phosphate aqueous solution (containing 0.1% sodium heptanesulfonate, adjusted to pH 3.0 with phosphoric acid); A:B = 8:92. In the detection method of the present invention, the flow rate of the mobile phase is 1.0 mL / minute. In a specific embodiment, the injection volume is 5 μL. In a specific embodiment, the column temperature of the high-performance liquid chromatograph is 30 °C. In a specific embodiment, the detection wavelength is 260 nm.

[0053] Selecting a detector wavelength of 260 nm is an optimized technical solution of this method. Actually, according to the ultraviolet spectrophotometric scanning spectrum, considering the situation that other components in individual samples may co-elute with β-nicotinamide nucleotide and its related substances, wavelengths between 250 nm and 300 nm are all optional detection wavelengths.

[0054] Mobile phase condition: methanol + water = 8 + 92 is an optimized solution of this method. Actually, considering the detection time cost and the situation that other components in the sample co-elute with β-nicotinamide nucleotide and its related substances, mobile phase conditions: "methanol + water = 5 + 95" to "methanol + water = 10 + 90" are all optional mobile phase conditions.

[0055] In a preferred embodiment, if the method detection limit is three times the noise signal, the detection limits of NMN, NAD, NA, and NAM components are 0.3 μg / mL; if the noise ten times the signal is used as the quantitation limit, the quantitation limit is 1 μg / mL.

[0056] In the detection method of the present invention, standard curves of NMN, NAD, NA, and NAM need to be plotted. In a specific embodiment, the process of plotting the standard curve is as follows: Take the mixed standard stock solution, add methanol, and use 0.05 mol / L sodium dihydrogen phosphate aqueous solution (containing 0.1% sodium heptanesulfonate, adjusted to pH 3.0 with phosphoric acid) to make the volume ratio of methanol to the aqueous phase in the solution 8:92, and prepare a series of mixed standard working solutions with concentrations of 16 μg / mL, 80 μg / mL, 160 μg / mL, 400 μg / mL, and 800 μg / mL. Inject the samples for detection according to the above instrument detection conditions, and plot the standard curve with the peak area as the ordinate and the concentration as the abscissa.

[0057] In the detection method of the present invention, the sample needs to be pretreated. In a specific embodiment, water extraction is used for the sample to be tested. For example, in a specific embodiment, the sample pretreatment process is as follows: Take 1 g of the cosmetic in a 50 mL stoppered colorimetric tube, add 20 mL of water, vortex for 1 min to disperse, ultrasonically extract for 15 min, and make up the volume with the mobile phase.

[0058] In the detection method of the present invention, before high performance liquid chromatography detection, the pretreated sample needs to be filtered. In a specific embodiment, a 0.22 μm filter membrane is used for filtration.

[0059] In the detection method of the present invention, external standard method is used for quantification, and the calculation formula is as follows:

[0060]

[0061] In the formula:

[0062] X—the mass fraction of the analyte in the sample, %;

[0063] C—the concentration of the analyte in the test solution obtained from the standard curve, in micrograms per milliliter (μg / mL);

[0064] V—the total volume of the diluted sample, in milliliters (mL);

[0065] M—the mass of the sample, in grams (g).

[0066] The present invention will be further described below in conjunction with specific examples. However, it should be understood that these examples are only used to illustrate the present invention and do not constitute a limitation of the scope of the present invention. The test methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0067] 1. Reagents

[0068] Reference substances: β-nicotinamide nucleotide (purity ≥ 99%, McLean), β-nicotinamide adenine dinucleotide (purity ≥ 98%, McLean), nicotinic acid (purity ≥ 99%, Aladdin), nicotinamide (purity ≥ 99%, McLean).

[0069] Methanol (chromatographic grade, Merck), sodium dihydrogen phosphate (analytical grade, Sinopharm), phosphoric acid (analytical grade, Sinopharm), ammonium heptane sulfonate (analytical grade, Aladdin), deionized water (homemade from a pure water machine).

[0070] The cosmetic samples include GF Multi-Effect Defense Toner, QICHU Baby Soothing Solution, Double Sisters Shanghai Fragrance Body Lotion, MAXAM Almond Oil Moisturizing Cream, and Herborist New Seven Whites Whitening and Brightening White Mud Mask, all provided by Shanghai Jahwa.

[0071] 2. Instruments and Equipment

[0072] Waters ARC high performance liquid chromatograph with diode array detector (Waters); Cary 100 UV-visible spectrophotometer (Agilent); METTLER TOLEDO multi-parameter tester S470–K (METTLER TOLEDO); KQ-800DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XS205 analytical balance (METTLER TOLEDO).

[0073] Agilent SB-C18 chromatographic column (250 mm×4.6 mm, 5 μm, Agilent); Millex-GP filter membrane (0.22 μm, Merck).

[0074] Example 1

[0075] 1. Preparation of standard stock solution

[0076] Accurately weigh an appropriate amount of NMN, NAD, NA and NAM reference substances into a 10 mL volumetric flask, dissolve with water and make up to volume, shake well, and prepare a mixed standard stock solution with a concentration of 8 mg / mL for each component. Store in a refrigerator at 4°C.

[0077] 2. Preparation of reference solution

[0078] Take the mixed standard stock solution, add an appropriate amount of methanol, and use 0.05 mol / L sodium dihydrogen phosphate aqueous solution (containing 0.1% sodium heptanesulfonate, adjusted to pH 3.0 with phosphoric acid) to make the volume ratio of methanol to the aqueous phase in the solution 8:92, and prepare a series of mixed standard working solutions with concentrations of 16 μg / mL, 80 μg / mL, 160 μg / mL, 400 μg / mL, and 800 μg / mL.

[0079] 3. HPLC Detection

[0080] Use high performance liquid chromatography (HPLC) to detect the above series of reference substance solutions with different concentrations respectively, obtain the linear relationship between the chromatographic peak areas and contents of 4 components, draw the corresponding standard working curves, and obtain the regression equations and correlation coefficients. The specific results are shown in Table 1.

[0081] Table 1: Linear Equations of Standard Curves

[0082]

[0083] Meanwhile, use high performance liquid chromatography (HPLC) to detect the mixed standard working solution of 4 components, and obtain the high performance liquid chromatograms of 4 components. The specific results are shown in Figures 3a to 3d .

[0084] Among them, the high performance liquid chromatography includes the following detection conditions:

[0085] The detector is a photodiode array detector (PDA); the chromatographic column is an Agilent SB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm), and the column temperature is 30°C. Mobile phase A is methanol, and mobile phase B is 0.05 mol / L sodium dihydrogen phosphate aqueous solution (containing 0.1% sodium heptanesulfonate, adjusted to pH 3.0 with phosphoric acid), A:B = 8:92. Isocratic elution is adopted, the flow rate is 1.0 mL / min. The injection volume is 5 μL. The detection wavelength is 260 nm.

[0086] Example 2

[0087] Take the mixed standard working solution of 4 components with a concentration of 160 μg / mL. According to the HPLC conditions in Step 3 of Example 1, accurately pipette 5 μL of the same reference substance standard solution into the liquid chromatograph, inject samples repeatedly for 6 times, and measure the peak areas of each component. The specific results of precision are shown in Table 2. The results show that the RSDs of the 4 components are 0.061% for β-nicotinamide ribonucleotide, 0.18% for β-nicotinamide adenine dinucleotide, 0.47% for nicotinic acid, and 0.29% for nicotinamide. The RSD of the chromatographic peak areas is less than 0.5%, indicating that the precision of the instrument is good.

[0088] Table 2: Precision Data

[0089]

[0090] Example 3

[0091] Select 5 kinds of cosmetics with different matrices, namely, aqueous solution (GUFU Multi-Effect Defense Toner) without β-nicotinamide nucleotide and its related substances, liquid (QICHU Baby Soothing Lotion), lotion (SHUANGMEI Night Shanghai Perfumed Body Lotion), cream (MAXAM Almond Oil Nourishing Cream), and smearable mask (HERBORIST New Seven White Whitening & Brightening White Clay Mask).

[0092] Take 3 portions of each kind of cosmetic, with each portion being 1 g (accurate to 0.1 mg). Place them in 10-mL stoppered colorimetric tubes, and respectively add 0.75 mL, 0.25 mL, and 0.025 mL of the mixed standard stock solution in Example 1. Add 2 mL of water, vortex for 1 min to disperse, make up the volume with the mobile phase, and perform ultrasonic extraction for 15 min. Take a part of the liquid and filter it through a 0.22-μm filter membrane to obtain the spiked recovery samples. The concentrations of each component are 600 μg / mL, 200 μg / mL, and 20 μg / mL respectively.

[0093] Measure the peak areas according to the HPLC conditions in Step 3 of Example 1, perform quantification on the standard curve, calculate the recovery rate and RSD. The specific results are shown in Table 3. The recovery rate ranges from 92.6% to 106.6%. The results show that the recovery rates of β-nicotinamide nucleotide and its related substances are good, with high accuracy, and can meet the test requirements.

[0094] Table 3: Spiked Recovery Results

[0095]

[0096]

[0097] The above embodiments are only for illustrating the present invention and are not intended to limit the patent of the present invention. It should be noted that for those of ordinary skill in the art, various changes and modifications can be made without departing from the scope of the present invention's concept, and these all belong to the protection scope of the present invention; therefore, all equivalent changes and modifications made to the scope of the claims of the present invention shall fall within the coverage scope of the claims of the present invention.

Claims

1. A method for detecting β-nicotinamide ribonucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in cosmetics, comprising the following steps: (1) Prepare standard samples containing β-nicotinamide ribonucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM); (2) Detect the standard prepared in step (1) by high performance liquid chromatography, and draw a standard curve with the peak area as the ordinate and the concentration as the abscissa; (3) Prepare a sample to be detected containing β-nicotinamide ribonucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM); (4) Extract the sample to be detected prepared in step (3); (5) Filter the extract obtained in step (4), then detect it by high performance liquid chromatography, and calculate the contents of β-nicotinamide ribonucleotide (NMN), β-nicotinamide adenine dinucleotide (NAD), nicotinic acid (NA) and nicotinamide (NAM) in the sample to be detected according to the following formula: In the formula: X represents the content of the analyte in the cosmetics, in weight percentage (%); C represents the concentration of the analyte in the sample to be detected obtained from the standard curve, in μg / mL; V represents the total volume of the sample to be detected, in mL; M represents the sample mass, in g.

2. The detection method according to claim 1, wherein Water is used for extraction in step (4).

3. The detection method according to claim 1, characterized in that, The concentrations of NMN, NAD, NA and NAM in the standard sample prepared in step (1) are 16 μg / mL, 80 μg / mL, 160 μg / mL, 400 μg / mL, 800 μg / mL.

4. The detection method according to claim 1, wherein The detection wavelength of high performance liquid chromatography is 250 - 300 nm.

5. The detection method according to claim 4, wherein The detection wavelength of high performance liquid chromatography is 260 nm.

6. The detection method according to claim 1, characterized in that The volume fraction of the mobile phase used in high performance liquid chromatography varies within the range of "methanol + water = 5 + 95" to "methanol + water = 10 + 90".

7. The detection method according to claim 6, characterized in that, The volume fraction of the mobile phase used in high performance liquid chromatography is "methanol + water = 8 + 92".

8. The detection method according to claim 1, wherein The chromatographic column used in high performance liquid chromatography is a C18 column.

9. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column in high performance liquid chromatography is 30 °C.

10. The detection method according to claim 1, wherein A 0.22 μm filter membrane is used for filtration in step (5).

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