Detection method for simultaneously detecting ergothioneine, alpha-ketoglutaric acid and vitamin C

Abstract

The invention discloses a method for detecting ergothioneine, alpha-ketoglutaric acid and vitamin C at the same time. The detection method for simultaneously detecting ergothioneine, alpha-ketoglutaric acid and vitamin C comprises the following steps: uniformly mixing a sample to be detected with water, and performing ultrasonic treatment to prepare a solution to be detected; and performing high performance liquid chromatography detection on the solution to be detected. The detection method provided by the invention is simple and convenient to operate, can be used for synchronously and quantitatively detecting the ergothioneine, the alpha-ketoglutaric acid and the vitamin C quickly, efficiently and accurately, and can be used for simultaneously determining the contents of the ergothioneine, the alpha-ketoglutaric acid and the vitamin C in a to-be-detected sample.

Description

Technical Field

[0001] This application belongs to the field of chemical analysis technology and relates to a method for the simultaneous detection of ergothioneine, α-ketoglutarate and vitamin C. Background Technology

[0002] Ergothioneine (EGT) is a potent antioxidant that effectively neutralizes various reactive oxygen species (ROS), protecting cells from oxidative damage, enhancing skin barrier function, and brightening skin tone. It specifically distributes within mitochondria and the cell nucleus, protecting them from UV radiation damage, thereby inhibiting collagen degradation, delaying photoaging, and exerting anti-wrinkle and anti-aging effects. Alpha-ketoglutarate (AKG) is a key molecule in cellular energy metabolism and collagen synthesis, providing skin cells with the energy needed for metabolism and repair, helping to improve skin elasticity, firm the skin, reduce wrinkles, and promote damage repair. Vitamin C (Ascorbic Acid, VC) is a multifunctional antioxidant and cofactor that can directly scavenge free radicals, protect cell structure, and regenerate oxidized vitamin E, thereby enhancing the overall antioxidant defense system. Meanwhile, as an essential cofactor for collagen hydroxylase, vitamin C plays a key role in collagen synthesis and stabilization. It can also inhibit tyrosinase activity, reduce melanin production, and achieve comprehensive effects of whitening, fading spots, anti-oxidation, and anti-aging.

[0003] Because ergothioneine, α-ketoglutarate, and vitamin C have significant synergistic effects in anti-oxidation and beauty, and can jointly alleviate core skin aging problems such as oxidative stress, collagen loss, and insufficient cellular energy, they are increasingly being used in compound formulation products to achieve holistic beauty and health benefits from the inside out. Examples include DNG AKG Time Capsules (containing EGT and AKG), Brieofood AKG Ergothioneine Capsules (containing EGT and AKG), CELFULL Bright & Young (containing EGT and VC), Wholly You Ergothioneine Plus (containing EGT, Ca-AKG, and VC), QUICKSILVER Liposome-L-Ergothioneine (containing EGT and VC), Swisse Ultra Vitalizing Serum (containing EGT and VC), SMNutrition AKG Supplement (containing AKG and VC), and biovoz Ergothioneine Capsules.

[0004] Simultaneous and accurate analysis of ergothioneine, α-ketoglutarate, and vitamin C is of great significance for improving product quality control, supporting efficacy verification, and optimizing production processes, and can effectively avoid the problems of low efficiency and high error risk of separate detection. However, due to the significant differences in the physicochemical properties, large differences in stability, difficulty in balancing pretreatment conditions, and similar chromatographic retention behaviors of the three, establishing a unified method that can accurately quantify all three simultaneously still faces considerable challenges, and no method for simultaneous detection of the three has been reported to date. Summary of the Invention

[0005] Therefore, it is necessary to provide a method for the simultaneous detection of ergothioneine, α-ketoglutarate, and vitamin C.

[0006] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, comprising the following steps:

[0007] The sample to be tested is mixed with water and subjected to ultrasonic treatment to prepare the test solution;

[0008] The test solution is subjected to high-performance liquid chromatography (HPLC) detection, and the HPLC detection conditions include:

[0009] A mobile phase A is a sodium hydrogen phosphate aqueous solution, and a mobile phase B is acetonitrile, wherein the total volume percentage of mobile phase A and mobile phase B is 100%.

[0010] The elution process includes:

[0011] From 0 to 6 minutes, the volume percentage of the mobile phase B changed from 0% to 20%.

[0012] Over 6 to 8 minutes, the volume percentage of mobile phase B changed from 20% to 0%.

[0013] For 8 to 12 minutes, maintain the volume percentage of the mobile phase B at 0%.

[0014] In some embodiments, the provided method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C satisfies one or more of the following conditions:

[0015] (1) The ultrasonic treatment time is 2 min to 15 min;

[0016] (2) The power of ultrasonic treatment is 80W~200W;

[0017] (3) The frequency of ultrasonic treatment is 40kHz~60kHz.

[0018] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C involves ultrasonic treatment at a temperature of 25 °C or less.

[0019] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: the detection wavelength for ergothioneine is 220 nm to 280 nm, the detection wavelength for α-ketoglutarate is 200 nm to 220 nm, and the detection wavelength for vitamin C is 250 nm to 280 nm.

[0020] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: the concentration of disodium hydrogen phosphate in the mobile phase A is 10 mM to 50 mM.

[0021] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: a flow rate of 0.8 mL / min to 1.0 mL / min, and / or an injection volume of 5 μL to 20 μL, and / or a column temperature of 25°C to 35°C.

[0022] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: the chromatographic column packing material being C18 alkylsilane-bonded silica gel.

[0023] In some embodiments, the provided method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C satisfies one or both of the following conditions:

[0024] (1) The sample to be tested is a dietary supplement and / or functional food;

[0025] (2) The sample to be tested is a powder.

[0026] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, the method further comprising the following steps:

[0027] Take the standard of ergothioneine, the standard of α-ketoglutarate, and the standard of vitamin C to prepare mixed standard solutions of different concentrations;

[0028] The mixed standard solutions of different concentrations were subjected to high performance liquid chromatography (HPLC) for detection, and a standard curve was constructed based on the peak area in the detection results and the concentration of the mixed standard solutions.

[0029] Substitute the detection results of the high performance liquid chromatography of the test solution into the standard curve to calculate the contents of ergothioneine, α-ketoglutarate and vitamin C in the test solution.

[0030] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided for use in the quality control of dietary supplements and / or functional foods.

[0031] The aforementioned detection method is simple to operate and can rapidly, efficiently, and accurately perform simultaneous quantitative detection of ergothioneine, α-ketoglutarate, and vitamin C. It can be used to simultaneously determine the content of these three substances in a sample. This method solves the problem of difficulty in simultaneous detection due to the significant differences in their physicochemical properties, meeting the urgent needs of quality control for compound formulation products, significantly improving detection efficiency, reducing detection costs and time, and is suitable for rapid screening and routine analysis of large batches of samples. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0033] Figure 1 This is a full-wavelength scan of ergothionein;

[0034] Figure 2 This is a full-wavelength scan of α-ketoglutarate;

[0035] Figure 3 This is a full-wavelength scan of vitamin C;

[0036] Figure 4 The chromatogram is for ergothioneine standard.

[0037] Figure 5 The chromatogram is for α-ketoglutaric acid standard.

[0038] Figure 6 The chromatogram of vitamin C standard is shown below.

[0039] Figure 7 The image shows the HPLC chromatogram of the standard, where pink represents α-ketoglutarate; black represents vitamin C; and blue represents ergothioneine.

[0040] Figure 8 This is a graph showing the effect of ultrasound time on the extraction of compounds from capsule powder;

[0041] Figure 9 This is a diagram of the detection results in Comparative Example 1;

[0042] Figure 10 The image shows the chromatograms of the three compounds detected in the product in Example 2. Detailed Implementation

[0043] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0044] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0045] 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 pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0046] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0047] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0048] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0049] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0050] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0051] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0052] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0053] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0054] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0055] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0056] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0057] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0058] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0059] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0060] In this application, the concentration unit "mM" in the context refers to "mmol / L".

[0061] Ergothioneine, α-ketoglutarate, and vitamin C have very different properties. Although they are easy to separate, they are difficult to detect simultaneously. Establishing a unified, efficient, and accurate single analytical method faces a huge challenge: (1) Pretreatment extraction conditions must be met to extract the three components from the sample simultaneously, efficiently, and without loss. However, their chemical stability, especially their tolerance to the environment, varies greatly: vitamin C is sensitive to light, heat, and oxygen and is easily degraded in neutral or alkaline solutions, while EGT and AKG are relatively stable; (2) vitamin C and ergothioneine have poor retention in reverse chromatographic columns, with similar elution times, making them prone to co-elution and overlapping chromatographic peaks, which affects accurate quantification; (3) the current detection methods for AKG all use fluorescent reagent derivatization, which is complicated and the fluorescence is easily quenched, requiring immediate testing after sample preparation; (4) EGT has no retention in ordinary chromatographic columns and has an early elution time of less than 3 minutes. Most literature uses hydrophilic interaction liquid chromatography columns (HILIC columns) to extend the retention time.

[0062] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, comprising the following steps: mixing the sample to be tested with water, subjecting it to ultrasonic treatment to prepare a test solution; and performing high-performance liquid chromatography (HPLC) on the test solution, wherein the HPLC conditions include:

[0063] A mobile phase A is a sodium hydrogen phosphate aqueous solution, and a mobile phase B is acetonitrile, wherein the total volume percentage of mobile phase A and mobile phase B is 100%.

[0064] The elution process includes:

[0065] From 0 to 6 minutes, the volume percentage of the mobile phase B changed from 0% to 20%.

[0066] Over 6 to 8 minutes, the volume percentage of mobile phase B changed from 20% to 0%.

[0067] For 8 to 12 minutes, maintain the volume percentage of the mobile phase B at 0%.

[0068] The provided detection method uses disodium hydrogen phosphate aqueous solution as mobile phase A, acetonitrile as mobile phase B, and the above-mentioned elution procedure. It uses a common reversed-phase C18 column for separation, which not only prolongs the peak time of ergothioneine, but also achieves the separation of ergothioneine and vitamin C.

[0069] The provided detection method is simple to operate, does not require derivatization, and can quickly, efficiently, and accurately perform simultaneous quantitative detection of ergothioneine, α-ketoglutarate, and vitamin C. It can be used to simultaneously determine the content of ergothioneine, α-ketoglutarate, and vitamin C in the sample to be tested.

[0070] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C provides an ultrasonic treatment time of 2 min to 15 min, for example, 2 min, 5 min, 10 min, 15 min, etc., or a range consisting of any two of the aforementioned values.

[0071] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C provides an ultrasonic treatment power of 80W to 200W, for example, 80W, 100W, 120W, 150W, 200W, etc., or any range of the aforementioned two values.

[0072] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C uses an ultrasonic treatment frequency of 40 kHz to 60 kHz, for example, 40 kHz, 50 kHz, 60 kHz, or any combination of the aforementioned values.

[0073] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C involves ultrasonic treatment at a temperature of 25°C or less, or in some embodiments, ultrasonic treatment at a temperature of 0°C to 25°C.

[0074] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided. The high-performance liquid chromatography (HPLC) detection conditions include: a detection wavelength of 220 nm to 280 nm for ergothioneine, a detection wavelength of 200 nm to 220 nm for α-ketoglutarate, and a detection wavelength of 250 nm to 280 nm for vitamin C. For example, the detection wavelength of ergothioneine can be 220 nm, 240 nm, 245 nm, 250 nm, 257 nm, 260 nm, 265 nm, 280 nm, etc., or any range of any two of the aforementioned values; the detection wavelength of α-ketoglutarate can be 200 nm, 210 nm, 220 nm, or any range of any two of the aforementioned values; and the detection wavelength of vitamin C can be 250 nm, 260 nm, 266 nm, 270 nm, 280 nm, etc., or any range of any two of the aforementioned values.

[0075] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: the concentration of disodium hydrogen phosphate in mobile phase A is 10 mM to 50 mM, for example, the concentration of disodium hydrogen phosphate in mobile phase A is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, etc., or it can be a range composed of any two of the aforementioned values.

[0076] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: a flow rate of 0.8 mL / min to 1.0 mL / min, and / or an injection volume of 5 μL to 20 μL, and / or a column temperature of 25°C to 35°C.

[0077] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the high-performance liquid chromatography (HPLC) detection conditions include: the chromatographic column packing material being C18 alkylsilane-bonded silica gel.

[0078] In some implementations, high-performance liquid chromatography (HPLC) detection employs a diode array detector (DAD) or a UV-Vis detector capable of multi-wavelength detection.

[0079] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the sample to be tested is a dietary supplement and / or functional food. For example, in some embodiments, the sample to be tested includes one or more of DNG AKG Time Capsules, Brieofood AKG Ergothioneine Capsules, CELFULL Bright & Young, Wholly You Ergothioneine Plus, QUICKSILVER Liposome-L-Ergothioneine, Swisse Ultra Vitalizing Serum, SMNutrition AKG Supplement, and Biovoz Ergothioneine Capsules.

[0080] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, wherein the sample to be tested is a powder.

[0081] In some embodiments, a method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided, the method further comprising the following steps:

[0082] Take the standard of ergothioneine, the standard of α-ketoglutarate, and the standard of vitamin C to prepare mixed standard solutions of different concentrations;

[0083] The mixed standard solutions of different concentrations were subjected to high performance liquid chromatography (HPLC) for detection, and a standard curve was constructed based on the peak area in the detection results and the concentration of the mixed standard solutions.

[0084] Substitute the detection results of the high performance liquid chromatography of the test solution into the standard curve to calculate the contents of ergothioneine, α-ketoglutarate and vitamin C in the test solution.

[0085] In some embodiments, the method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C is provided for use in the quality control of dietary supplements and / or functional foods.

[0086] The provided detection method can be applied to the quality control of dietary supplements and functional foods, as well as to formulation research and product development. For example, in some embodiments, the provided detection method can be applied to the quality control of beauty dietary supplements, and in other embodiments, it can be applied to the quality control of capsule-type beauty dietary supplements.

[0087] The following are specific embodiments. They are intended to provide further detailed explanation of this application to help those skilled in the art and researchers to better understand it. The technical conditions and other details do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims of this application.

[0088] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0089] The reagents and instruments used in the examples were sourced as follows: Disodium hydrogen phosphate: analytical grade, purchased from Aladdin Shanghai; Acetonitrile: chromatographic grade, purchased from Aladdin Shanghai; Ergothioneine (CAS: 497-30-3, purity ≥98%) and α-ketoglutarate (CAS: 328-50-7, purity ≥98%): analytical grade, purchased from Aladdin Shanghai; Vitamin C (CAS: 50-81-7, purity ≥99%): analytical grade, purchased from Yuanye Shanghai; Filter membrane (0.45μm) and filter (0.22μm) were purchased from Jinteng Experimental Equipment Co., Ltd. The high-performance liquid chromatograph was purchased from Shimadzu, Japan. The core components of the system were a high-pressure pump (LC-20AT), a diode array detector (SPD-M20A), an autosampler (SIL-20AC), and a column oven (CTO-20AC).

[0090] In the examples, "capsule powder" refers to the contents of the capsules.

[0091] Example 1

[0092] This embodiment provides a method for the simultaneous detection of ergothioneine, α-ketoglutarate, and vitamin C. The capsule powder was purchased from Biovoz ergothioneine capsules.

[0093] 1. Preparation of standard solutions

[0094] Accurately weigh appropriate amounts of ergothioneine, α-ketoglutarate, and vitamin C standards, place them in 10 mL volumetric flasks, dilute to the mark with 0.1% (w / v) metaphosphoric acid aqueous solution, and shake well to prepare a series of mixed standard solutions: ergothioneine standard solution concentrations of 32, 64, 128, 256, and 512 μg / mL; α-ketoglutarate standard solution concentrations of 43, 86, 172, 344, and 688 μg / mL; and vitamin C standard solution concentrations of 75, 150, 300, 600, and 1200 μg / mL. All standard solutions should be prepared fresh before use.

[0095] 2. HPLC chromatographic conditions

[0096] The high-performance liquid chromatograph (HPLC) was a Shimadzu LC-20AT (Japan); the column was an InterSustain AQ-C18 250×4.6mm, 5μm; the column temperature was 30℃; mobile phase A was 50mM disodium hydrogen phosphate solution, and mobile phase B was acetonitrile; the flow rate was 1.0mL / min, and the injection volume was 10μL. The elution gradient was 0-6min, 0-20%B, 6-8min, 20-0%B, 8-12min, 0%. The standard solution was scanned at all wavelengths, and the results are as follows: Figures 1-3 As shown.

[0097] Based on the full-wavelength scan results of the standard samples, the optimal ultraviolet absorption wavelengths were selected as follows: ergothionein 257nm, α-ketoglutarate 210nm to eliminate interference, and vitamin C 266nm.

[0098] The HPLC chromatographic conditions were determined through experiments as follows: The HPLC system was purchased from Shimadzu, Japan. The core components of the system were a high-pressure pump (LC-20AT), a diode array detector (SPD-M20A), an autosampler (SIL-20AC), and a column oven (CTO-20AC). The chromatographic column was an InterSustain AQ-C18 250×4.6mm, 5μm; the column temperature was 30℃; mobile phase A was 50mM disodium hydrogen phosphate solution, and mobile phase B was acetonitrile; the flow rate was 1.0mL / min, and the injection volume was 10μL. The elution gradient was 0-6min, 0-20%B, 6-8min, 20-0%B, 8-12min, 0%. The detection wavelengths were ergothionein 257nm, α-ketoglutarate 210nm, and vitamin C 266nm.

[0099] The standard solution was analyzed under the determined HPLC chromatographic conditions, and the results are as follows: Figures 4-7 As shown. From Figures 4-7 As can be seen, the peak time of EGT is 5.410 min, the peak time of AKG is 2.396 min, and the peak time of VC is 2.757 min.

[0100] 3. Calculation and Expression of Test Results for the Samples to be Tested

[0101] The formula for calculating the content of active substances in the sample to be tested is as follows:

[0102] ;

[0103] In the formula:

[0104] X - Percentage of active substances in the sample to be tested (g / 100g);

[0105] C - Calculate the concentration (μg / mL) of the active substance in the sample from the standard curve;

[0106] V - The final volume of the sample to be tested (mL);

[0107] F - Dilution factor;

[0108] M - Sample volume (mg) to be tested.

[0109] 4. Create a standard curve

[0110] The prepared series of standard working solutions were injected and analyzed under the liquid chromatography conditions described above. A series of standard curves were plotted with the concentration of each compound on the x-axis and the peak area (mAU) response value on the y-axis, and the linear regression equation of the standard curves was obtained.

[0111] Limit of detection (LOQ): the concentration at a signal-to-noise ratio of 3:1; Limit of quantitation (LOD): the concentration at a signal-to-noise ratio of 10:1.

[0112] Results: The linearity was good, meeting the requirements of the chromatographic methodology.

[0113] Table 1. Linear equations, correlation coefficients, and detection limits of the analytes.

[0114]

[0115] 5. Preparation of sample solution

[0116] Accurately weigh the capsule powder and place it in a 50 mL volumetric flask. Discard the outer shell and add 0.1% metaphosphoric acid aqueous solution. Vortex for 1 min to dissolve the capsule powder. Sonicate for 2 min, 5 min, 10 min, and 15 min respectively. During sonication, the temperature may rise. Add ice packs to prevent degradation due to excessive temperature. Draw up the supernatant with a syringe, filter it through a 0.22 μm filter, dilute it appropriately, and put it into a liquid chromatography vial. Perform high performance liquid chromatography detection under the following chromatographic conditions and calculate the contents of ergothioneine, α-ketoglutarate, and vitamin C in the samples treated with different sonication times.

[0117] HPLC chromatographic conditions: The high-performance liquid chromatograph (HPLC) was purchased from Shimadzu, Japan. The core components of the system were a high-pressure pump (LC-20AT), a diode array detector (SPD-M20A), an autosampler (SIL-20AC), and a column oven (CTO-20AC). The chromatographic column was an InterSustain AQ-C18 250×4.6 mm, 5 μm; the column temperature was 30℃; mobile phase A was 50 mM disodium hydrogen phosphate solution, and mobile phase B was acetonitrile; the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The elution gradient was 0–6 min, 0–20% B, 6–8 min, 20–0% B, 8–12 min, 0%. The detection wavelengths were ergothionein 257 nm, α-ketoglutarate 210 nm, and vitamin C 266 nm.

[0118] Test results as follows Figure 8 As shown, from Figure 8 It can be seen that the content of ergothioneine, α-ketoglutarate and vitamin C was highest when the ultrasound time was 5 minutes.

[0119] The sample processing method is as follows: accurately weigh the capsule powder, put it into a 50mL volumetric flask, discard the outer shell, add 0.1% metaphosphoric acid aqueous solution, vortex for 1min to dissolve, sonicate for 5min, use a syringe to draw up the supernatant, filter it through a 0.22μm filter, dilute it appropriately, put it into a liquid chromatography vial, and analyze it on the instrument.

[0120] 6. Methodological Validation

[0121] (1) Repeatability experiment

[0122] Under the same instrument conditions, prepare a 200 μg / mL solution of the compound standard, repeat the process 6 times, and calculate the relative standard deviation based on the chromatographic peak area.

[0123] The results are shown in Table 2. The RSD of ergothioneine was 1.83%; the RSD of α-ketoglutarate was 2.35%; and the RSD of vitamin C was 1.04%. The relative standard deviations were less than 3%, which met the methodological requirements.

[0124] (2) Precision test

[0125] Accurately weigh 6 portions of capsule powder samples, extract compounds according to the pretreatment procedure, dilute to the appropriate concentration, load the samples for testing, and store in a refrigerator at 4 degrees Celsius in the dark (to prevent degradation). Measure the samples for three consecutive days, calculate the relative standard deviation based on the chromatographic peak area, and evaluate the intra-day and inter-day precision.

[0126] The results are shown in Table 2. The intra-day and inter-day precision of the three analytes were all less than 5%, which met the methodological requirements.

[0127] Table 2. Methodological validation of the analytes (repeatability, intra-day and inter-day precision)

[0128]

[0129] (3) Stability test

[0130] Accurately weigh six portions of the capsule powder sample, extract the compound according to the pretreatment procedure, dilute to an appropriate concentration, and analyze the sample using an chromatographic instrument. Analyze every four hours for a total of three measurements. Calculate the relative standard deviation based on the peak area.

[0131] The results are shown in Table 2. The RSD of stability is less than 5%, which meets the methodological requirements.

[0132] (4) Spike recovery experiment

[0133] Accurately weigh 6 portions of capsule powder samples, and add low, medium and high concentrations of standards. Extract the compounds according to the pretreatment procedure, dilute to the appropriate concentration, load the samples for detection, calculate the spiked recovery rate and relative standard deviation based on the peak area, and examine the accuracy of the method.

[0134] The calculation formula is as follows:

[0135] ;

[0136] In the formula:

[0137] C1 - Calculate the ergothioneine concentration (μg / mL) of the spiked sample from the standard curve.

[0138] C2 - Calculate the ergothioneine concentration (μg / mL) of the unspiked sample from the standard curve;

[0139] C3-ergothioneine spiking concentration (μg / mL).

[0140] The results are shown in Table 3. The spiked recoveries were 97-104%, and the RSDs were all less than 5%, which met the methodological requirements.

[0141] Table 3. Methodological Validation of Analytes (Spiked Recovery)

[0142]

[0143] Example 2

[0144] The test capsules were purchased from Biovoz, product name Biovoz Ergothioneine Capsules, and the content was determined according to the following method. Each sample was tested in triplicate, and the data are presented as mean ± standard deviation.

[0145] 1. Sample pretreatment

[0146] Accurately weigh the contents of the capsules (approximately equivalent to one capsule) and place them in a 50 mL volumetric flask. Add 0.1% metaphosphoric acid aqueous solution and vortex for 1 min to fully disperse and dissolve the contents. Then, perform ultrasonic extraction for 5 min, using an ice-water bath to control the temperature to no more than 25°C during the ultrasonic process. Filter the extract through a 0.22 μm microporous membrane and use the filtrate as the test solution.

[0147] 2. Chromatographic analysis

[0148] The HPLC chromatographic conditions were as follows: The high-performance liquid chromatograph was purchased from Shimadzu, Japan. The core components of the system were a high-pressure pump (LC-20AT), a diode array detector (SPD-M20A), an autosampler (SIL-20AC), and a column oven (CTO-20AC). The chromatographic column was an InterSustain AQ-C18 250×4.6mm, 5μm; the column temperature was 30℃; mobile phase A was 50mM disodium hydrogen phosphate solution, and mobile phase B was acetonitrile; the flow rate was 1.0mL / min, and the injection volume was 10μL. The elution gradient was 0-6min, 0-20%B, 6-8min, 20-0%B, 8-12min, 0%. The detection wavelengths were ergothionein 257nm, α-ketoglutarate 210nm, and vitamin C 266nm.

[0149] Perform high-performance liquid chromatography (HPLC) analysis on the test solution. Record the retention time and peak area of ​​each target compound.

[0150] 3. Content Calculation: The peak areas of ergothioneine, α-ketoglutarate, and vitamin C were substituted into the corresponding linear regression equations obtained in "4. Preparation of Standard Curve" of Example 1 (see Table 1) to calculate the concentration of each compound in the test solution. Then, based on the sampling amount, final volume, and dilution factor, the content of each active substance in each capsule (mg / capsule) was calculated.

[0151] 4. Test Results

[0152] Chromatograms of the three active substances are shown below Figure 10 As shown, the chromatographic analysis results demonstrate that the established analytical method possesses good detection efficiency and separation selectivity. At three UV detection wavelengths of 266 nm, 210 nm, and 257 nm, the target components VC, AKG, and EGT exhibited clear and symmetrical chromatographic peaks at 2.758 min, 2.394 min, and 5.385 min, respectively, with retention times consistent with the corresponding standards. This proves that each component exhibits characteristic absorption in the UV region, enabling stable and sensitive qualitative and quantitative analysis. Furthermore, the chromatograms show baseline separation between each target peak and adjacent impurity peaks, with no significant overlap or interference. Particularly at 210 nm, the peaks are sharp and without tailing, indicating that the chromatographic conditions are well-optimized and the system achieves good separation. The effective differentiation of target components and impurities under multi-wavelength detection further confirms the method's good specificity and anti-interference ability in complex matrices.

[0153] After data processing, the test results were as follows: ergothioneine 25.4 mg / capsule, α-ketoglutarate 10.5 mg / capsule, and vitamin C 75.2 mg / capsule.

[0154] The test results show that the detection method provided by the present invention can accurately determine the content of ergothioneine, α-ketoglutarate and vitamin C in capsule contents samples, and can achieve accurate quantification, proving its good universality and practicality.

[0155] Comparative Example 1

[0156] Chromatographic conditions: High-performance liquid chromatograph (HPLC) was a Shimadzu LC-20AT (Japan); column was an InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature was 30℃; mobile phase A was 0.1% formic acid-water solution, and mobile phase B was 0.1% formic acid-acetonitrile solution; flow rate was 1.0 mL / min, injection volume was 10 μL. Elution gradient was 0-6 min, 0-20%B, 6-8 min, 20-0%B, 8-12 min, 0%. Detection wavelengths were ergothionein 257 nm, α-ketoglutarate 210 nm, and vitamin C 266 nm. Standard solutions of the three compounds were prepared using the same method as described in "1. Preparation of Standard Solutions" in Example 1, and the standards were then analyzed.

[0157] Test results as follows Figure 9 As shown. From Figure 9 As can be seen, ergothioneine and vitamin C have early and close elution times, which can easily lead to poor separation of the test sample solution or the two peaks merging together, resulting in inaccurate test results. In addition, ketoglutarate did not elute, indicating poor elution effect.

[0158] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0159] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C, characterized in that, Includes the following steps: The sample to be tested is mixed with water and subjected to ultrasonic treatment to prepare the test solution; The test solution is subjected to high-performance liquid chromatography (HPLC) detection, and the HPLC detection conditions include: A mobile phase A is a sodium hydrogen phosphate aqueous solution, and a mobile phase B is acetonitrile, wherein the total volume percentage of mobile phase A and mobile phase B is 100%. The elution process includes: From 0 to 6 minutes, the volume percentage of the mobile phase B changed from 0% to 20%. Over 6 to 8 minutes, the volume percentage of mobile phase B changed from 20% to 0%. For 8 to 12 minutes, maintain the volume percentage of the mobile phase B at 0%.

2. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to claim 1, characterized in that, One or more of the following conditions must be met: (1) The ultrasonic treatment time is 2 min to 15 min; (2) The power of ultrasonic treatment is 80W~200W; (3) The frequency of ultrasonic treatment is 40kHz~60kHz.

3. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to claim 1, characterized in that, Ultrasonic treatment is performed at a temperature of 25 °C or less.

4. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection include: the detection wavelength of ergothionein is 220nm~280nm, the detection wavelength of α-ketoglutarate is 200nm~220nm, and the detection wavelength of vitamin C is 250nm~280nm.

5. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to any one of claims 1 to 4, characterized in that, The conditions for high performance liquid chromatography detection include: the concentration of disodium hydrogen phosphate in mobile phase A is 10 mM to 50 mM.

6. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to any one of claims 1 to 4, characterized in that, The conditions for high-performance liquid chromatography detection include: a flow rate of 0.8 mL / min to 1.0 mL / min, and / or an injection volume of 5 μL to 20 μL, and / or a column temperature of 25℃ to 35℃.

7. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to any one of claims 1 to 4, characterized in that, The conditions for the high-performance liquid chromatography detection include: the column packing material is C18 alkylsilane bonded silica gel.

8. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to any one of claims 1 to 4, characterized in that, One or both of the following conditions must be met: (1) The sample to be tested is a dietary supplement and / or functional food; (2) The sample to be tested is a powder.

9. The method for simultaneously detecting ergothioneine, α-ketoglutarate, and vitamin C according to any one of claims 1 to 4, characterized in that, The method further includes the following steps: Take the standard of ergothioneine, the standard of α-ketoglutarate, and the standard of vitamin C to prepare mixed standard solutions of different concentrations; The mixed standard solutions of different concentrations were subjected to high performance liquid chromatography (HPLC) for detection, and a standard curve was constructed based on the peak area in the detection results and the concentration of the mixed standard solutions. Substitute the detection results of the high performance liquid chromatography of the test solution into the standard curve to calculate the contents of ergothioneine, α-ketoglutarate and vitamin C in the test solution.

10. The application of the detection method for simultaneous detection of ergothioneine, α-ketoglutarate and vitamin C as described in any one of claims 1 to 9 in the quality control of dietary supplements and / or functional foods.

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