A method for detecting NMN in cosmetics

Abstract

The present invention belongs to the field of cosmetics testing, and specifically relates to a quantitative analysis and detection method for trace amounts of NMN in cosmetics. The quantitative analysis and detection method includes a kit for detecting NMN in cosmetics, the kit comprising a QuEChERS cleanup column based on multi-walled carbon nanotube adsorption, a 50% methanol aqueous solution, a 0.1% formic acid aqueous solution, and methanol; the QuEChERS cleanup column comprises multi-walled carbon nanotubes, PSA, and C18, and the method steps specifically include sample cleanup, concentration, and high-performance liquid chromatography detection.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics testing, and specifically relates to a method for detecting NMN in cosmetics. Background Art

[0002] NMN, or β-nicotinamide mononucleotide, is a biologically active nucleotide that is naturally formed by the reaction of a phosphate group and a nucleoside-containing ribose with nicotinamide. In animal cells, NMN is a cellular energy source and a metabolite of nicotinamide adenine dinucleotide (NAD+) biosynthesis, playing a vital role in cell proliferation and function. As we age, NAD levels in the body decrease. + The decrease in levels leads to a decrease in DNA repair ability, driving the aging process. NMN is quickly absorbed in the body and can quickly increase NAD in cells. + Further research has found that NMN, as a cosmetic raw material, has anti-wrinkle, antioxidant, and whitening effects.

[0003] In January 2022, the National Medical Products Administration (NMPA) announced the registration approval of NMN as a cosmetic ingredient, allowing it to be used as a raw material in cosmetics. Based on the current registration information, the maximum concentration of NMN added to cosmetics by various companies is no more than 3%. However, due to the lack of an internationally standardized method for determining NMN content in cosmetics, it is impossible to accurately measure the NMN content in cosmetics, and therefore it is impossible to determine whether the NMN concentrations registered by various companies are the actual concentrations. Therefore, it is extremely necessary to develop a standardized method for the quantitative detection of NMN content in cosmetics to promote the high-quality and compliant development of these cosmetics.

[0004] NMN has a high polarity, is not very volatile, is easily soluble in water, and is difficult to dissolve in organic solvents. Currently, there are few reports on its quantitative analysis and detection, and the relevant reports that can be retrieved are mainly for the quantitative detection of NMN in the food field, and its detection methods mainly include the use of triple quadrupole liquid chromatography-mass spectrometry, nuclear magnetic resonance or high performance liquid chromatography for analysis. Due to the special physical and chemical properties of NMN, the current quantitative analysis and detection methods are mainly carried out using triple quadrupole liquid chromatography-mass spectrometry and nuclear magnetic resonance, but the cost of routine product testing is relatively high. On the other hand, there are currently no reports on the quantitative detection and analysis of NMN in the cosmetics field, and the detection of conventional highly polar substances in cosmetics mainly includes high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, etc., but the properties of cosmetics are diverse (including water-like, emulsion-like, ointment-like, etc.) and the ingredient matrix is ​​complex. Therefore, in order to accurately quantitatively detect the ingredients in cosmetics, it is often necessary to pre-treat the sample to be tested.

[0005] Patent application number CN202011584631.4, entitled "A Liquid Phase Detection Method for Bose-In in Cosmetics," discloses the use of HPLC-differential refractometer detection to detect the content of Bose-In in cosmetics. The patent pre-treats the cosmetic sample by dispersing it with an organic reagent, then extracting it with ultrapure water by vortexing. The supernatant is then filtered and analyzed by HPLC. Patent application number CN202111659754.4, entitled "A Method for Simultaneously Determining Multiple Pigments in Cosmetics," discloses the use of HPLC-DAD to detect the content of multiple pigments in cosmetics. The patent pre-treats the cosmetic sample by mixing the sample with multiple organic reagents for extraction, followed by centrifugal filtration, and finally HPLC analysis. Currently, the detection of specific substances in cosmetic samples primarily relies on a combination of pre-treatment and liquid chromatography separation; pre-treatment often involves extraction with an organic reagent, followed by centrifugal filtration. However, this commonly used sample pre-treatment method is more suitable for samples with relatively high levels of the substance to be detected.

[0006] The patent with application number CN202111382244.7 and invention name “A method for determining the content of NMN in dietary supplement products” discloses the use of HPLC method to detect NMN in dietary supplements. The sample pretreatment method in this patent is to mix the sample with an organic reagent, perform ultrasonic extraction, and then centrifuge. However, this method of sample pretreatment using organic reagent extraction combined with centrifugal filtration is not suitable for the detection of NMN in cosmetics. Because the content of NMN in cosmetics is extremely low, about 1 to 20 mg / kg, but the content of NMN in dietary supplements can reach 10,000 mg / kg, the use of organic solvent extraction combined with centrifugal filtration can easily lead to the direct removal of trace amounts of NMN in the sample, or the inability to be effectively extracted from the oil sample, or the destruction of the NMN molecules, which in turn leads to subsequent detection failures or inaccurate content detection.

[0007] Based on this, it is necessary to propose a new method for the quantitative detection of trace amounts of NMN in cosmetics. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a method for quantitative detection of trace NMN in cosmetics, and the specific technical solution is as follows.

[0009] A quantitative analysis and detection method for trace NMN in cosmetics, comprising a kit for detecting NMN in cosmetics, the kit comprising a QuEChERS cleanup column based on multi-walled carbon nanotube adsorption, a 50% methanol aqueous solution, a 0.1% formic acid aqueous solution, and methanol; the QuEChERS cleanup column comprises multi-walled carbon nanotubes, PSA, and C18; the specific steps of the quantitative analysis and detection method are as follows:

[0010] 1) Sample purification: Weigh an appropriate amount of the cosmetic sample to be tested, add a 50% methanol aqueous solution, and sonicate for 10-20 minutes to fully dissolve the sample. The mass ratio of the sample to the added 50% methanol aqueous solution is 1:10. Repeat the ultrasonic extraction once to obtain a sample extract. The sample extract is purified once through a QuEChERS cleanup column based on multi-walled carbon nanotube adsorption at a flow rate of 1-3 drops / second to obtain a sample purified solution. The sample purified solution is centrifuged twice, and the supernatant is collected.

[0011] 2) Concentration: The supernatant obtained in step 1) was evaporated in a vacuum rotary evaporator at 50-60° C. until nearly dry (appearing as a fuzzy substance). Heating was then stopped, and the supernatant was extracted with a 50% methanol-water solution. The solution was then filtered through a 0.45 μm aqueous filter to collect the supernatant.

[0012] 3) HPLC detection: the supernatant obtained in step 2) was isocratically eluted using 0.1% formic acid aqueous solution as mobile phase A and methanol solution as mobile phase B.

[0013] Furthermore, the sample extract in step 1) has a column flow rate of 1 drop / second, 2 drops / second, or 3 drops / second.

[0014] Furthermore, the sample purification solution in step 1) is centrifuged at a speed of 8000-12000 rpm for 10-20 min.

[0015] Preferably, the sample purification solution in step 1) is centrifuged at a speed of 10,000 rpm for 15 minutes.

[0016] Furthermore, the elution condition in step 3) is 15% A-85% B isocratic elution for 0-12 min.

[0017] Furthermore, the chromatographic column of the high performance liquid chromatography method is a HILIC chromatographic column, the injection volume is 10 μL, and the flow rate is 0.8 mL / min.

[0018] Furthermore, the column temperature of the high performance liquid chromatography is 35°C.

[0019] Furthermore, the detector of the high performance liquid chromatography is a DAD detector, and the detection wavelength is 235 nm.

[0020] Furthermore, the cosmetic sample includes facial cream, facial milk and / or facial mask essence.

[0021] Furthermore, the cosmetic sample is weighed with an accuracy of 0.001 g.

[0022] Beneficial technical effects

[0023] 1) The present invention provides a method for quantitative analysis and detection of trace NMN in cosmetics. Since the content of NMN in cosmetics is extremely low, usually only 1 to 20 mg / kg, and the types of cosmetic samples are diverse and the matrix is ​​complex, the present invention has introduced a breakthrough QuEChERS cleanup column for purification treatment before sample detection. Although QuEChERS cleanup columns are currently widely used in the pretreatment of chemical drug and antibacterial drug residue detection in various foods, there are no reports on their use in pretreatment of cosmetic sample detection. The experiments of the present invention have shown that QuEChERS cleanup columns can effectively remove most of the interferences in the cosmetic matrix (for example: lipid substances in facial milk and cream) without destroying the NMN structure, and have the advantages of simple operation, low instrument and equipment cost, and less environmental pollution.

[0024] 2) Since NMN is present in very low levels in cosmetics and is encapsulated in lipid samples, it is difficult to effectively extract NMN from cosmetics using traditional organic reagent extraction methods, and thus it cannot be effectively detected using HPLC methods. The present invention uses a QuEChERS cleanup column to remove most of the interfering substances in the cosmetic matrix to obtain NMN. Supplemented with a concentration step, the purified NMN is enriched for subsequent detection. The detection and analysis method of the present invention can detect NMN added at a content of less than 20 mg / kg.

[0025] 3) The method of the present invention has fast analysis speed, high sensitivity, and accurate qualitative and quantitative analysis, which is of great significance for promoting the quality improvement and effective supervision of NMN cosmetic products at home and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0027] Figure 1 This is the chromatogram of facial milk directly tested after extraction with organic solvent;

[0028] FIG2 is a chromatogram of the analysis and detection of cosmetic samples treated by the demulsification method (a: face cream, b: face lotion);

[0029] Figure 3 is the chromatogram of NMN and cosmetic samples analyzed by different chromatographic columns (a: HILIC column analysis of NMN standard, b: C 18 Column analysis and detection of NMN standards, c: HILIC column analysis and detection of cosmetic samples; d: C 18 Column analysis of cosmetic samples);

[0030] Figure 4 is a chromatogram of the analysis and detection of cosmetic samples under different chromatographic conditions (a: mobile phase AB = 80%-20%; b: mobile phase AB = 50%-50%; c: mobile phase AB = 15%-85%; d: flow rate 0.8 ml / L). DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0032] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0033] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0034] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0035] Materials and instruments

[0036] NMN standard: β-nicotinamide mononucleotide (C 11 H 15 N2O8P); CAS: 1094-61-7, purity ≥98%, Shanghai Yuanye Biotechnology Co., Ltd. Methanol (CH4O): chromatographically pure, Merck, Germany. Formic acid (CH2O2): chromatographically pure, Sinopharm Chemical Reagent Co., Ltd. Purification column: KNORTH Clean-up LPAS cleanup column, model: ZC69032, KNORTHCo. Ltd. Nylon 66 aqueous filter membrane: 13 mm, 0.45 μm, Jinteng Experimental Equipment Co., Ltd.

[0037] iChrom5100 analytical domestic liquid chromatograph, Dalian Yilite Analytical Instrument Co., Ltd. Venusil HILIC column (4.6 mm × 250 mm, 5 μm, Tianjin Bona Aijieer Technology Co., Ltd.; Thmorgan-VM200 vortex mixer, Thmorgan Biotechnology Co., Ltd.; HITACHI-CF15RXII centrifuge, Hitachi Ltd.; Sartorius analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; ELGA water purifier, Beijing Chengyi Hengyi Technology Co., Ltd.; LMDTC-15F ultrasonic cleaner, Beijing Green Cotton Technology Co., Ltd.; MPE series high-throughput vacuum parallel concentrator, Ruike Group Co., Ltd.

[0038] Solution preparation

[0039] Mobile phase: Mobile phase A consisted of 0.1% formic acid in water, prepared using a laboratory water purifier with a conductivity of 18.2 MΩ. The solution was filtered through an aqueous filter membrane and ultrasonically degassed before use. Mobile phase B consisted of methanol (chromatographic grade) that was filtered through an organic filter membrane and ultrasonically degassed before use.

[0040] Standard stock solution: Accurately weigh 10 mg of NMN standard and place it in a 10 mL volumetric flask. Dissolve it in 50% methanol and water and dilute to 10 mL to prepare a standard stock solution with a concentration of 1000 μg / mL. Protect from light and store in a refrigerator at 4°C until used.

[0041] Standard working solution: Using 50% methanol aqueous solution as the solvent, dilute the NMN standard stock solution to a series of standard working solutions of 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, and 5 μg / mL. This solution must be prepared before use.

[0042] Extraction solution: Prepare the sample extraction solution with a volume ratio of methanol to water of 1:1.

[0043] Cosmetic samples: Seven cosmetic samples containing NMN were tested. The cosmetics included facial masks, essences, creams, and lotions.

[0044] Example 1

[0045] 1.1 Sample extraction

[0046] Accurately weigh 1.0 g (accurate to 0.001 g) of the cosmetic sample into a 15 mL stoppered centrifuge tube and record the mass. Add 10 mL of extraction solution and sonicate for 10-20 minutes to fully dissolve the cosmetic sample. Sonicate again for 10-20 minutes to obtain the sample extract.

[0047] Because facial lotions and creams are lipid-based, they are not conducive to the enrichment and concentration of trace substances in the sample. Therefore, the matrix must be purified to remove most interfering substances. In some cases, facial mask essences are a mixture of water and oil, so pretreatment is also required for these samples.

[0048] 1.2 Sample purification

[0049] The extract was purified by passing it through a purification column (KNORTH Clean-up LPAS purification column, model: ZC69032) at a flow rate of 1-3 drops / second (1 drop / second, 2 drops / second, or 3 drops / second). The purified sample after passing through the column was centrifuged at 10,000 rpm for 15 minutes. The supernatant was taken and centrifuged again at 10,000 rpm for 15 minutes to obtain the centrifuged purified sample.

[0050] 1.3 Concentration

[0051] The purified sample solution after centrifugation was placed in a heart-shaped bottle and placed in a vacuum rotary evaporator. Concentrate to near dryness at 55°C, dissolve in 1 mL of sample extraction solution, aspirate with a syringe, and filter through a 0.45 μm aqueous filter before HPLC analysis. Because the amount of NMN added to cosmetics is very low, the purified solution must be concentrated after purification. The purpose of concentration is to enrich the NMN to be detected for subsequent HPLC analysis.

[0052] Example 2

[0053] Selection and optimization of extraction solution

[0054] This embodiment selected four extraction solutions: (1) 100% water; (2) methanol: water = 20:80; (3) methanol: water = 50:50; (4) methanol: water = 85:15. The same samples of facial cream, facial milk, and facial mask essence (hereinafter referred to as facial mask) were taken, and 10 μg / mL NMN standard was added to each of them. The sample extraction method of Example 1 was adopted, and the above four extraction solutions were used for extraction, and the spiked recovery rate was measured. The results are shown in Table 1 below. As can be seen from the table, when 50% methanol aqueous solution was used as the extraction solution, the sample recovery rate can be controlled between 98.25% and 107.57%, and the results have better stability. Therefore, the present invention selects 50% methanol aqueous solution for sample extraction.

[0055] Table 1 Effect of different solvents on NMN extraction efficiency

[0056]

[0057] Conclusion: The recovery rates of facial masks, creams and milks were between 80% and 120% when extracted with 50% methanol aqueous solution, so 50% methanol aqueous solution was selected as the extraction solution.

[0058] Example 3

[0059] Selection and optimization of pretreatment methods

[0060] Since facial lotion and cream samples contain a lot of esters, which are not conducive to sample filtration and will also mask the detection effect of low-abundance analytes in the sample, it is necessary to first degrease the sample. This example investigates different purification methods for facial lotion and cream samples.

[0061] Method 1: Organic reagent extraction: Accurately weigh 1.0 g of cosmetic sample (accurate to 0.001 g) and add 10 mL of 50% methanol-water solution for extraction. Ultrasonicate for 10-20 minutes to fully dissolve the cosmetic sample. Ultrasonicate again for 10-20 minutes to obtain the sample extract. HPLC analysis is then performed directly.

[0062] Conclusion: Since the content of NMN in cosmetics is very low and contained in lipid samples, direct extraction with 50% methanol solution cannot effectively extract NMN in lipid microsomes, so it cannot be detected in the final chromatogram. Figure 1 . Figure 1 There is only a matrix peak on the surface, but no NMN peak.

[0063] Method 2: Demulsification: Demulsification, also known as demulsification, refers to the complete destruction of the emulsion into two immiscible phases. Take the same cream and lotion, add 10 μg / mL NMN standard and 0.01g demulsifier (sodium chloride) to each of them for demulsification. After demulsification, the sample forms several liposome particles, and NMN may be wrapped inside the liposome particles. Then use the sample extraction method of Example 1 and measure the spike recovery rate.

[0064] Conclusion: The results are shown in Figure 2. Figure 2a This is the sample analysis diagram after the cream is demulsified. Figure 2b The chromatogram in Figure 2 is a messy one, obscuring and destroying the structure of the target substance, NMN.

[0065] Method 3: QuEChERS cleanup column: Take the same facial cream and facial lotion, add 10 μg / mL NMN standard to each, use the sample extraction method of Example 1, and measure the spike recovery rate. The results are shown in the table below (using the m-PFC cleanup column as a control).

[0066] Table 2 Effect of different purification rates on sample purification effect

[0067]

[0068] Note: This batch of facial mask essence does not contain oil, so it was not tested. This example aims to verify the effect of different pre-treatment methods on samples containing oily substances.

[0069] The results show that NMN cannot be detected by direct detection after extraction with an organic solvent. This is because NMN is wrapped in the lipid particles of the lipid sample and the organic reagent cannot be effectively extracted. Although the demulsification method can disperse the lipid sample into several lipid particles to a certain extent, which is conducive to the detection of NMN wrapped therein. However, the chromatogram obtained after the sample extract after demulsification is messy and has the effect of covering up and destroying the structure of the target substance NMN, and demulsification will also cause damage to the chromatographic column. Therefore, the traditional organic reagent extraction and demulsification methods are not suitable for the analysis and detection of NMN in cosmetics.

[0070] Example 4

[0071] Selection and optimization of liquid chromatography conditions

[0072] According to the literature, the reverse phase C 18Chromatographic column is used to separate NMN, but due to the high polarity of NMN, there is almost no retention, the peak time is too early, and it is easy to be confused with the sample matrix peak. Therefore, the present invention considers using HILIC chromatographic column with better separation effect for polar samples to separate the samples, and C 18 Comparison of the separation performance between the column and the HILIC column. Figure 3a 、 3b The following are the results of HILIC and C-HPLC analysis of NMN standards. 18 From the chromatogram of the column, we can see that the retention times of NMN are 6.93min and 2.58min respectively. 18 The chromatographic column was used to separate three matrix samples. The results are as follows Figure 3c 、 3d As shown. It can be seen that although C 18 The retention time of the column is earlier than that of the HILIC column, but C 18 The separation effect of the chromatographic column is far inferior to that of the HILIC column. 18 When the matrix sample is analyzed using a chromatographic column, NMN cannot be distinguished from the cosmetic matrix. Therefore, the present invention uses a HILIC column for better separation effect.

[0073] In order to separate the NMN to be tested from other components of the three matrices, namely, mask extract, cream, and milk, as much as possible, the mobile phase gradient program was further optimized. 50% methanol-water extract was used as the background solution (extraction solution) of the mask essence, cream, and milk, and a mixed standard was added thereto to prepare a test solution with a concentration of 10 μg / mL. The samples were chromatographically separated using an isocratic elution program. (1) Mobile phase A: mobile phase B = 15:85, (2) Mobile phase A: mobile phase B = 50:50, and (3) Mobile phase A: mobile phase B = 80:20 were selected. These three elution modes were compared, and the results are shown in Figure 4.

[0074] The results in Figure 4 show that for the three matrix samples, when the organic phase ratio was 20% ( Figure 4a ), the retention time of NMN is 3.0min, which makes it impossible to effectively separate NMN from the matrix. When the organic phase ratio is 50% isocratic elution ( Figure 4b ), the retention time of NMN is 3.8min, and NMN and the matrix cannot be effectively separated, which affects the efficiency of the analysis method and is accompanied by peak broadening. When the organic phase ratio is 85% isocratic elution ( Figure 4c), the separation effect is the best, the peak time of NMN is short, and the peak shape basically satisfies the Gaussian distribution, which can achieve the effect of separating the impurity peak in the matrix from the chromatographic peak of the analyte, reducing the possibility of interference from the impurity peak. However, under this isocratic elution condition, although the separation degree of the mask essence is good, the separation degree of the cream and the facial milk needs to be improved. Therefore, the flow rate was changed from the conventional 1mL / min to 0.8mL / min ( Figure 4d ), demonstrating that the resolution was improved by reducing the flow rate.

[0075] From the above experiments, it can be seen that by adjusting the proportion and flow rate of the mobile phase, the peak shape can be effectively improved, and the impurity peaks in the matrix can be separated from the chromatographic peaks of the analytes, thereby reducing the possibility of interference from impurity peaks, making the operation simple and time-saving, and improving the efficiency of analysis and detection.

[0076] In summary, this example uses high performance liquid chromatography to detect the content of NMN in three types of cosmetics. The liquid chromatography conditions are as follows: chromatographic column: Venusil HILIC (4.6 mm × 250 mm, 5 μm, ), injection volume was 10 μL, flow rate was 0.8 mL / min, column temperature was 35°C, detection wavelength was 235 nm, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was methanol solution, and isocratic elution conditions were 15% A-85% B elution 0-12 min.

[0077] Example 5

[0078] This example provides an example of using the method of the present invention to quantitatively detect the content of NMN in cosmetics

[0079] The method of the present invention comprises a detection kit, which comprises a QuEChERS purification column based on multi-walled carbon nanotube adsorption, a 50% methanol aqueous solution, a 0.1% formic acid aqueous solution and methanol; the QuEChERS purification column comprises multi-walled carbon nanotubes, PSA and C18.

[0080] 5.1 Sample pretreatment

[0081] Accurately weigh 1.0 g of sample (accurate to 0.001 g) into a 15 mL stoppered centrifuge tube and record the mass (mi). Add 10 mL of sample extraction solution and sonicate for 20 minutes to fully dissolve the sample. Ultrasonicate again for 20 minutes. Purify the extract using a KNORTH Clean-up LPAS cleanup column (model: ZC69032). Centrifuge at 10,000 rpm for 15 minutes. Remove the supernatant and centrifuge again at 10,000 rpm for 15 minutes. Place the supernatant in a heart-shaped bottle and place it in a vacuum rotary evaporator at 55°C. Concentrate to near dryness, dissolve in 1 mL of sample extraction solution, aspirate with a syringe, filter through a 0.45 μm aqueous filter, and prepare for HPLC analysis.

[0082] 5.2 Liquid chromatography conditions

[0083] The chromatographic column was Venusil HILIC (4.6 mm × 250 mm, 5 μm, ), injection volume was 10 μL, flow rate was 0.8 mL / min, column temperature was 35°C, detection wavelength was 235 nm, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was methanol solution, isocratic elution conditions were 0-12 min, 15% A-85% B.

[0084] 5.3 Standard curve

[0085] The prepared 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, and 5 μg / mL standard working solutions were injected and analyzed according to the liquid chromatography conditions of "5.2". The concentration of NMN was used as the horizontal axis and the peak area (MAU) response value was used as the vertical axis. A series of standard curves were drawn and linear regression was performed to obtain the linear regression equation of the standard curve of NMN.

[0086] 5.4 Instrument precision test

[0087] Under the same instrument conditions, take a 100 μg / mL standard working solution and repeat the injection 6 times according to the chromatographic conditions of "5.2". Measure the peak area of ​​each detected component and substitute it into the linear regression equation of the standard curve to obtain the measured concentration value. Then calculate the relative standard deviation (RSD) of the measured concentration.

[0088] The measured concentration and RSD of NMN standard are shown in the following table.

[0089] Table 3 Precision test results (mg / kg)

[0090] Standards 1 2 3 4 5 6 RSD (%) NMN 41.3 42.5 41.7 41.5 43.7 42.0 2.7

[0091] 5.5 Stability test

[0092] Under the same instrumental conditions, accurately weigh 1.0g (accurate to 0.001g) of three matrices, namely, facial mask essence, facial cream, and facial milk, and use the pretreatment method of "5.1" to prepare extracts of the three matrices. On the same day, the samples were injected and analyzed using the chromatographic conditions of "5.2". The samples were measured once every 2 hours, for a total of 6 times. The peak areas of the detected components were measured and substituted into the linear regression equation of the standard curve to obtain the measured concentration values. The relative standard deviation (RSD) of the measured concentrations was then calculated.

[0093] The measured concentrations and RSDs of NMN in the three cosmetics are shown in the table below.

[0094] Table 4 Stability test results (mg / kg)

[0095] Standards 1 2 3 4 5 6 RSD (%) Facial mask 12.0 11.8 12.1 11.8 12.0 12.0 5.3 cream 151.0 149.6 149.9 150.6 150.4 149.8 1.2 facial lotion 13.6 13.7 13.5 13.7 13.6 13.7 2.5

[0096] 5.6 Repeatability test

[0097] Under the same instrumental conditions, accurately weigh 1.0g (accurate to 0.001g) of 6 portions of each of the three matrices: facial mask essence, facial cream, and facial milk. Follow the method under "5.1" and inject the treated test solution into the chromatographic conditions of "5.2" for analysis. Since the target substance NMN was not detected in the facial milk, a standard working solution with a concentration of 10μg / mL was added to the facial milk extract during the repeatability test. The peak area of ​​NMN was measured and substituted into the standard curve equation to calculate the standard deviation (RSD) of the measured concentration.

[0098] The concentrations and RSDs of NMN in 6 groups of parallel samples of facial masks, creams, and milk were calculated and summarized, and the results are shown in the following table.

[0099] Table 5 Repeatability test results (mg / kg)

[0100] Standards 1 2 3 4 5 6 RSD (%) Facial mask 12.2 12.0 11.8 11.9 11.8 11.9 5.3 cream 151.1 150.5 150.3 150.2 150.1 150.9 0.3 facial lotion 13.7 13.6 13.4 13.7 13.7 13.6 2.5

[0101] 5.7 Limit of Quantitation and Limit of Detection

[0102] The prepared standard solutions were analyzed using the liquid chromatography conditions described in Section 5.2. The peak area of ​​NMN was measured and incorporated into the regression equation for the standard curve. The limit of detection (LOD) and limit of quantification (LOQ) of this method were calculated using 3 times the average signal-to-noise ratio of the target component in the lowest-level standard solution and 10 times the average signal-to-noise ratio.

[0103] The NMN standard solution was measured and the standard curve regression equation was drawn using the ichrom5100 workstation. The linear range, correlation coefficient, LOD and LOQ are shown in the following table.

[0104] Table 6 Linear range, LOD and LOQ of NMN

[0105]

[0106] The data in the table above indicate that the method of the present invention exhibits good linearity within the range of 5.0 to 500 μg / mL, with correlation coefficients r > 0.999. Calculated limits of detection (LOD, S / N = 3) for NMN are both 1.5 mg / kg and limits of quantification (LOQ, S / N = 10) are both 5.0 mg / kg.

[0107] 5.8 Spike recovery

[0108] Accurately weigh 1.0g (accurate to 0.01g) of 12 portions each of facial cream, facial milk, and facial mask essence, and number them 1-12. Portions 1-3 are used as background, portions 4-6 are spiked with a 5μg / mL test solution, portions 7-9 are spiked with a 10μg / mL test solution, and portions 10-12 are spiked with a 50μg / mL test solution. Follow the procedure under "5.1" and analyze the treated background test solution and spiked samples at low, medium, and high concentrations by HPLC. Measure the peak areas and calculate the recovery.

[0109] Three cosmetic samples spiked at low, medium and high levels of 5 mg / kg, 10 mg / kg and 50 mg / kg were assayed using the method of the present invention, and the recoveries and RSD% were calculated. The results are shown in the following table.

[0110] Table 7 Spiked recovery test results (mg / kg)

[0111]

[0112] From the data in the above table, it can be seen that the recovery rate of NMN in cosmetics with three different matrices is between 93.9% and 109.4% (the recovery rate is between 80% and 120%, indicating that the method has good accuracy), and the relative standard deviation RSD is ≤3.7%, indicating that the recovery rate of this method is good and the results are accurate and reliable.

[0113] In this example, 7 NMN-containing cosmetics (including facial lotions, creams, and facial mask essences) of different brands were analyzed and tested. Matrix samples No. 1-7 were, respectively, No. 1 and No. 2 were facial mask essences, No. 3, 4, 5, and 6 were facial creams, and No. 7 was facial lotion. The test results are shown in the table below.

[0114] Table 8 NMN content in different cosmetics (mg / kg) (n=3)

[0115]

[0116]

[0117] Nd:not detected

[0118] Analysis of actual samples showed that NMN was detected in Mask No. 1 at a concentration of 12.1 mg / kg, while Mask No. 2 was not detected. Creams No. 3 and 4 were also negative. Creams No. 5 and 6 contained NMN at concentrations of 1501.8 mg / kg and 17.6 mg / kg, respectively. Facial Lotion No. 7 contained NMN at 13.7 mg / kg. This suggests that the authenticity of cosmetics currently advertised as containing NMN remains to be determined. Therefore, the development of a quantitative detection method for NMN in cosmetics is urgent.

[0119] 5.9 Data Processing

[0120] The linear regression equation and correlation coefficient of the standard curve in this article were obtained by processing with the ichrom5100 workstation. Other data were processed and plotted using Origin8.5 and Excel2010 software. The experimental data were the average of three parallel experiments.

[0121] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A method for quantitative analysis and detection of β-nicotinamide mononucleotide in cosmetics, characterized in that: The quantitative analysis and detection method includes a kit for detecting β-nicotinamide mononucleotide in cosmetics, the kit comprising a KNORTH Clean-up LPAS cleanup column, a 50% methanol aqueous solution, a 0.1% formic acid aqueous solution, and methanol. The specific steps of the quantitative analysis and detection method are as follows: 1) Sample purification: Weigh an appropriate amount of the cosmetic sample to be tested, add a 50% methanol-water solution, and sonicate for 10-20 minutes to fully dissolve the sample. The mass ratio of the sample to the added 50% methanol-water solution is 1:

10. Repeat the ultrasonic extraction once to obtain a sample extract. Purify the sample extract once through a KNORTH Clean-up LPAS cleanup column at a flow rate of 1-3 drops / second to obtain a sample purified solution. Centrifuge the sample purified solution and collect the supernatant. 2) Concentration: The supernatant obtained in step 1) was placed in a vacuum rotary evaporator and evaporated at 50-60°C until nearly dry. After that, heating was stopped and the supernatant was dissolved in 50% methanol-water solution. The solution was then filtered through a 0.45 µm aqueous filter to collect the supernatant. 3) High Performance Liquid Chromatography Detection: The supernatant obtained in step 2) was isocratically eluted using a 0.1% formic acid aqueous solution as mobile phase A and a methanol solution as mobile phase B; the isocratic elution conditions were mobile phase A:mobile phase B = 15:85; the HPLC column was a HILIC column, and the detection wavelength was 235 nm.

2. The quantitative analysis and detection method according to claim 1, wherein The sample extract in step 1) flows through the column at a rate of 1 drop / second, 2 drops / second, or 3 drops / second.

3. The quantitative analysis and detection method according to claim 1, wherein The sample purification solution in step 1) is centrifuged at a speed of 8000-12000 rpm for 10-20 minutes.

4. The quantitative analysis and detection method according to claim 1, wherein The sample purification solution in step 1) is centrifuged at a speed of 10,000 rpm for 15 minutes.

5. The quantitative analysis and detection method according to claim 1, wherein The injection volume of the HPLC method was 10 μL, and the flow rate was 0.8 mL / min.

6. The quantitative analysis and detection method according to claim 1, wherein The column temperature of the HPLC method was 35°C.

7. The quantitative analysis and detection method according to claim 1, wherein The detector of the high performance liquid chromatography is a DAD detector.

8. The quantitative analysis and detection method according to claim 1, wherein The cosmetic samples are facial cream, facial milk, and facial mask essence.

9. The quantitative analysis and detection method according to claim 1, wherein The cosmetic sample is weighed with an accuracy of 0.001 g.

Images