Method for detecting ergothioneine in gel-based food products

Abstract

The application discloses a detection method of ergothioneine in gel matrix food. The application adopts a liquid chromatography tandem mass spectrometry method to detect ergothioneine in gel matrix food, wherein the detection method is obtained by setting the composition of the mobile phase and the elution program of the mobile phase of the liquid chromatography detection, and parameters such as mass spectrometry characteristic ion pairs and ion spray temperature are jointly constructed, so that accurate qualitative and quantitative detection of ergothioneine in gel matrix food is realized, a more comprehensive and effective quality control and evaluation method is provided for gel matrix food containing ergothioneine, the blank of the liquid chromatography tandem mass spectrometry technology in the detection of ergothioneine in a complex food matrix is filled, and solid technical support is provided for quality control and standard establishment of related gel matrix products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, in particular to a detection method of ergothioneine in gel matrix food and application thereof. BACKGROUND

[0002] Ergothioneine (EGT) is a rare natural amino acid with multiple biological activities, including anti-inflammatory, cell protection, inhibition of UV damage, maintenance of DNA synthesis and repair, promotion of normal cell growth and immune regulation, and potential value in improving brain function, enhancing cognitive ability, relieving depression, and activating immune response. Ergothioneine cannot be synthesized by the human body itself and must rely on exogenous intake. The main natural sources include various mushrooms, cereal grains, and some animal tissues. At present, with the deepening of people's understanding of its health benefits, ergothioneine has been widely used in dietary supplements and functional foods, especially in gel matrix type dietary supplements and functional foods. By adding ergothioneine to the gel matrix, it is not only convenient for consumers to eat, but also the gel macromolecular structure can wrap ergothioneine, reducing its contact with the external environment and ensuring its functional activity. Therefore, making ergothioneine into gel matrix type dietary supplements and functional foods is in line with consumer demand. However, in related gel matrix type dietary supplements and functional foods, the content of ergothioneine is usually low, and the gel matrix composition is more complex. The traditional liquid chromatography method is often insufficient in sensitivity and anti-interference ability, and the accuracy of qualitative and quantitative analysis is poor, which cannot realize the quality control and evaluation of related dietary supplements and functional foods. SUMMARY

[0003] Therefore, it is necessary to provide a method for accurately detecting ergothioneine in gel matrix food.

[0004] In a first aspect, the present application provides a method for detecting ergothioneine in gel matrix food, which adopts a liquid chromatography tandem mass spectrometry method to detect ergothioneine in gel matrix food.

[0005] The conditions of liquid chromatography include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A includes a formic acid aqueous solution, and the mobile phase B includes methanol; the elution program of the mobile phase adopts gradient elution, and the program of gradient elution is as follows:

[0006] 0 min-4 min, the volume percentage of the mobile phase B changes from 15% to 30%;

[0007] 4 min-5 min, the volume percentage of the mobile phase B changes from 30% to 15%;

[0008] 5 min-7 min, the volume percentage of the mobile phase B maintained at 15%;

[0009] The conditions of the mass spectrum include: the mass of the parent ion is 230 Da, the mass of the daughter ion is 127 Da, the scanning mode is MRM, the temperature of the electrospray ion source is 80-150°C.

[0010] In some embodiments, the chromatographic column is a C18 chromatographic column, and the model of the chromatographic column is ACQUITY UPLC BEH C18.

[0011] In some embodiments, the column temperature of the chromatographic column is 30-35°C; and / or, the flow rate of the mobile phase is 0.2-0.5 mL / min; and / or, the injection volume is 1-20 μL; and / or, the volume fraction of the formic acid in the mobile phase A is 0.05-0.1%.

[0012] In some embodiments, the conditions of the mass spectrum further include: the desolvation gas temperature is 500°C, the desolvation gas flow rate is 800 L / Hr; and / or, the capillary voltage is 3.0 kV, the cone voltage is 30 V, the entrance voltage is 2 V; and / or, the collision energy is 35 eV; and / or, the collision cell exit voltage is 0.5 V; and / or, the pressure of the gas curtain is 40 psi, the pressure of the dry gas is 35 psi, the pressure of the nebulizer gas is 70 psi, and the gas of the gas curtain, the dry gas and the nebulizer gas includes nitrogen; and / or, the gas pressure of the collision cell is 95 psi, and the gas includes nitrogen.

[0013] In some embodiments, the gel matrix food includes a gel candy, and the gel candy includes a gel candy containing a filling; and the ingredients of the filling include astaxanthin.

[0014] In some embodiments, the detection method further includes the steps of preparing a standard solution and detecting the standard solution by using the liquid chromatography tandem mass spectrometry method, wherein the preparation method of the standard solution includes: mixing the ergothioneine standard with a first solvent to prepare the standard solution.

[0015] In some embodiments, the first solvent includes water; and / or, the concentration of the standard solution is 5-500 ng / mL.

[0016] In some embodiments, the detection method further includes the steps of preparing a sample solution and detecting the sample solution by using the liquid chromatography tandem mass spectrometry method, wherein the preparation method of the sample solution includes: mixing the gel matrix food with a second solvent, ultrasonic treatment, filtration, taking the filtrate and adding a third solvent to prepare the sample solution.

[0017] In some embodiments, the second solvent and the third solvent each independently comprise one or more of an alcohol solvent and water; and / or, the mass-volume ratio of the gel matrix food to the second solvent is 1 g: (70-90) mL.

[0018] In some embodiments, the alcohol solvent comprises one or more of methanol and ethanol; and / or, the ultrasonic treatment time is 10 min-30 min; and / or, the ultrasonic treatment power is 100 W-200 W, and the ultrasonic treatment frequency is 30 kHz-50 kHz; and / or, the filter membrane pore size used for filtration is 0.22 μm.

[0019] Compared with the conventional technology, the technical scheme of the present application has the beneficial effects including:

[0020] The present application adopts a liquid chromatography tandem mass spectrometry detection method to detect ergothioneine in gel matrix food, and by using a mobile phase system formed by an aqueous solution containing formic acid and methanol for gradient elution, and by using mass spectrometry characteristic ion pairs and ion spray temperature parameters to jointly construct a detection method, accurate qualitative and quantitative detection of ergothioneine in gel matrix food is realized, and a more comprehensive and effective quality control and evaluation method for gel matrix food containing ergothioneine is provided. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A total ion current chromatogram of an ergothioneine standard sample solution is shown.

[0022] Figure 2 Ergothioneine characteristic peak areas of sample solution prepared by different extraction solvents in Example 1 are shown.

[0023] Figure 3 Total ion current chromatograms of ergothioneine in samples detected by different mobile phases in Example 2 are shown, where A) is the mobile phase 1, B) is the mobile phase 2, and C) is the mobile phase 3.

[0024] Figure 4 Total ion current chromatograms of samples detected by different pretreatments in Example 3 are shown.

[0025] Figure 5 Total ion current chromatograms of samples detected by different flow rates in Example 4 are shown.

[0026] Figure 6 Ergothioneine characteristic peak areas of samples detected by different ultrasonic treatment times in Example 5 are shown.

[0027] Figure 7 A standard curve graph for linear correlation investigation of the detection method of the present application is shown. DETAILED DESCRIPTION

[0028] For the purposes of this application, reference will be made to the accompanying drawings in which the preferred embodiments of the present application are illustrated. It should be understood that the present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It will be apparent that those skilled in the art will be able to make modifications to the embodiments disclosed herein without departing from the scope of the present application.

[0029] 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 application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0030] As used herein, the terms "optionally", "optional" or "optional" mean to choose from either of the two alternatives "yes" or "no". If there are multiple "options" in a technical solution, and there is no special description, and there is no contradictory relationship or mutual restriction, each "option" is independent of each other. The term "and / or" used herein includes any and all combinations of one or more related listed items. The terms "a plurality of", "a plurality of", and the like, as used herein, refer to a number greater than 2 or equal to 2, for example, "one or more" means one, two or more. The open technical features or technical solutions described herein with the terms "contain", "include", "comprise" and the like, if there is no other description, can be regarded as providing both the closed features or solutions composed of the listed members and the open features or solutions including additional members outside the listed members.

[0031] In this application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for description purposes and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0032] If there is no special description, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0033] Ergothioneine cannot be synthesized by the human body itself and must rely on exogenous intake. Its main natural sources include various types of mushrooms (such as Pleurotus citrinopileatus, i.e. Pleurotus eryngii), cereal grains and certain animal tissues (such as liver and red blood cells). Therefore, traditional detection techniques are mainly used for detecting natural substances containing ergothioneine, for example, using liquid chromatography to detect Pleurotus citrinopileatus, a high-ergothioneine-enriched species.

[0034] As people's understanding of the health benefits of ergothioneine deepens, ergothioneine has been widely used in dietary supplements and functional foods. In order to control and evaluate the quality of related products, it is necessary to detect ergothioneine in dietary supplements and functional foods. However, the content of ergothioneine in such products is relatively low, and the sensitivity and anti-interference ability of liquid chromatography are often insufficient, making it impossible to achieve accurate qualitative and quantitative detection. Especially for products with complex matrix components, such as gel matrix foods, the detection technology is difficult, and currently there is no technology that can accurately detect ergothioneine in gel matrix foods containing ergothioneine.

[0035] The gel matrix in food usually includes macromolecular or high molecular substances, which can be a gel formed by natural high molecular substances, synthetic or semi-synthetic high molecular substances. As non-limiting examples, natural high molecular substances include one or more of animal gelatin, pectin, algal gum, agar, starch and konjac gum. Synthetic or semi-synthetic high molecular substances include sodium polyacrylate, etc. Based on the gel matrix, it is extremely difficult to detect related small molecule active substances (such as ergothioneine) in gel matrix foods using traditional liquid chromatography LC or high performance liquid chromatography HPLC. The main reasons include: (1) The gel matrix of macromolecules is a high molecular polymer. In chromatographic analysis, on the one hand, there is no specific characteristic peak, and the characteristic peak is very wide, causing the signal of the small molecule active substance to be covered and producing superimposed interference signals, resulting in the inability to detect; on the other hand, the gel matrix may be degraded into small molecular peptides, polysaccharide fragments or small particles during the detection process, which compete with the target small molecule substance for retention on the chromatographic column, resulting in the chromatographic peak of the target substance being widened, deformed or even completely covered by the matrix peak, making it impossible to accurately qualify by retention time. (2) Based on the molecular characteristics of the gel matrix, the gel matrix molecular structure contains a large number of hydroxyl groups, which can form hydrogen bonds and other interactions with small molecules, and even form complexes, affecting the chromatographic behavior of small molecule active substances, and the repeatability is poor, making it impossible to form a stable and standard food quality control and evaluation method.

[0036] The present application aims to provide a detection method capable of accurately qualitatively and quantitatively detecting ergothioneine in gel matrix foods, and to provide a quality control and evaluation method for related gel matrix foods.

[0037] In a first aspect, the application provides a method for detecting ergothioneine in a gel matrix food, which uses a liquid chromatography tandem mass spectrometry method to detect ergothioneine in a gel matrix food.

[0038] The conditions of the liquid chromatography include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A includes aqueous formic acid, and the mobile phase B includes methanol; the elution program of the mobile phase uses gradient elution, and the gradient elution program is as follows:

[0039] 0 min-4 min, the volume percentage of the mobile phase B changes from 15% to 30%;

[0040] 4 min-5 min, the volume percentage of the mobile phase B changes from 30% to 15%;

[0041] 5 min-7 min, the volume percentage of the mobile phase B maintains 15%;

[0042] The conditions of the mass spectrometry include: the mass of the parent ion is 230 Da, the mass of the daughter ion is 127 Da, the scanning mode is MRM, and the temperature of the electrospray ion source is 80℃-150℃.

[0043] The application detects ergothioneine in a gel matrix food by a liquid chromatography tandem mass spectrometry method, uses aqueous formic acid-methanol as a mobile phase system, optimizes the elution program of the mobile phase system, and realizes rapid and efficient detection of ergothioneine in a gel matrix food. At the same time, by controlling the characteristic ion pair and ion temperature and other parameters in the mass spectrometry, the technical problems of ion suppression and co-elution interference can be effectively solved. Compared with the anti-interference ability and sensitivity of the traditional liquid chromatography, the accuracy of the detection method is significantly improved.

[0044] In some embodiments, the liquid chromatography uses a high-performance liquid chromatograph, Waters, ACQUITY UPLC I-Class PLUS system.

[0045] In some embodiments, the mass spectrometry uses AB Sciex Triple Quad 5500+.

[0046] In some embodiments, the chromatographic column uses a C18 chromatographic column. As a non-limiting example, the chromatographic column uses ACQUITY UPLC BEH C18, 1.7 μm, 2.1×50 mm.

[0047] In some embodiments, the column temperature of the chromatographic column is 30℃-35℃, including but not limited to 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, or a range formed by any two of the foregoing and a value in the range.

[0048] In some embodiments, the flow rate of the mobile phase is 0.2 mL / min - 0.5 mL / min, including but not limited to 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, or a range formed by any two of the foregoing, and values therein.

[0049] In some embodiments, the injection volume is 1 μL - 20 μL, including but not limited to 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, or a range formed by any two of the foregoing, and values therein. Further, the injection volume is 8 μL - 10 μL.

[0050] In some embodiments, the mass fraction of formic acid in the mobile phase A is 0.05% - 0.1%, including but not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or a range formed by any two of the foregoing, and values therein.

[0051] In some embodiments, the conditions of the mass spectrometer further include: the desolvation gas temperature is 500 °C, the desolvation gas flow rate is 800 L / Hr; and / or, the capillary voltage is 3.0 kV, the cone voltage is 30 V, the entrance voltage (EP) is 2 V; and / or, the collision energy (CE) is 35 eV; and / or, the collision cell exit voltage (CXP) is 0.5 V; and / or, the curtain gas pressure is 40 psi, the dry gas pressure is 35 psi, the nebulizer gas pressure is 70 psi, the curtain gas, the dry gas, and the nebulizer gas include nitrogen; and / or, the collision cell gas pressure is 95 psi, the gas includes nitrogen.

[0052] In some embodiments, the gel matrix food includes an ergothioneine-containing gel candy, and the gel matrix in the gel candy generally includes natural high molecular substances, such as animal gelatin, plant polysaccharides (carrageenan, agar), starch, and the like. The gel matrix is generally a substance with physical elasticity, and the gel candy includes, illustratively, an ergothioneine-containing gel jelly. Further, the ergothioneine-containing gel candy includes a gel candy with a filling, and based on the qualitative and quantitative difficulties of the active substance brought by the gel matrix, the qualitative and quantitative difficulties of the small molecule active substance (ergothioneine) in the gel candy with a filling, such as the ergothioneine-containing gel jelly, are greater, and the main reason is that the filling ingredients in the gel candy are complex, further increasing the difficulty of chromatographic separation. As a non-limiting example, the gel candy with a filling includes an astaxanthin-filled gel candy, and in the present application, astaxanthin affects the chromatographic behavior of ergothioneine, and the main reasons include:

[0053] (1) Severe ion suppression effect

[0054] Matrix effect: During extraction, a large amount of impurities such as lipids and pigments in astaxanthin sandwich will enter the mass spectrometry ion source together with ergothioneine.

[0055] Ion suppression: These impurities will compete with ergothioneine for charge and energy, resulting in a significant decrease in the ionization efficiency of ergothioneine, i.e., "ion suppression" occurs. Its direct manifestation is that the signal intensity of ergothioneine is severely depressed or even completely disappears, making it impossible to be effectively detected.

[0056] (2) Contamination and interference with the mass spectrometry system

[0057] Contamination of ion source: Liposoluble substances such as astaxanthin are prone to adhere to the key components of the mass spectrometry ion source, forming stubborn dirt. This will cause the performance of the instrument to decrease, the baseline noise to increase, and the sensitivity to be unstable, making it impossible to achieve stable detection of trace ergothioneine.

[0058] Interference with mass analysis: A large amount of impurities will produce complex background noise, making it difficult for even high-resolution mass spectrometry to accurately identify and quantify the characteristic signal of ergothioneine, resulting in a too low signal-to-noise ratio and failure of qualitative and quantitative analysis.

[0059] (3) Challenge of chromatographic separation

[0060] Even though mass spectrometry itself has high specificity, the premise is that liquid chromatography (LC) can provide effective separation. Astaxanthin and its degradation products may form strong retention "ghost peaks" on the chromatographic column or co-elute with ergothioneine. This not only exacerbates the above-mentioned ion suppression, but also makes it difficult to accurately integrate the chromatographic peak of ergothioneine during data analysis.

[0061] In summary, the presence of astaxanthin oil sandwich, through the introduction of a large amount of complex matrix components, mainly triggers the "ion suppression effect", which is the most deadly in LC-MS analysis, and is accompanied by a serious risk of instrument contamination. The two factors work together to fundamentally destroy the sensitivity, stability, and accuracy necessary for LC-MS detection of ergothioneine. Therefore, physically separating the astaxanthin sandwich from the gel matrix to be tested is a necessary and key pretreatment step to ensure the successful establishment of the LC-MS analysis method.

[0062] In some embodiments, the detection method further comprises a step of preparing a standard solution and detecting the standard solution by using the liquid chromatography tandem mass spectrometry method, wherein the preparation method of the standard solution comprises: mixing ergothioneine standard with a first solvent to prepare a standard solution.

[0063] In some embodiments, the first solvent comprises water.

[0064] In some embodiments, the concentration of the standard solution is 5 ng / mL-500 ng / mL, including but not limited to 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 220 ng / mL, 250 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL or a range formed by any two of the foregoing and numerical values within the range.

[0065] In some embodiments, the detection method further comprises a step of preparing a test sample solution and detecting the test sample solution by using the liquid chromatography tandem mass spectrometry method, wherein the preparation method of the test sample solution comprises: mixing the gel matrix food with a second solvent, ultrasonic treatment, filtration, taking the filtrate to add a third solvent, and preparing a test sample solution.

[0066] In some embodiments, the second solvent and the third solvent each independently comprise one or more of an alcohol solvent and water. Further, the second solvent and the third solvent comprise water. Among them, the second solvent uses water as an extraction solvent, which can improve the dissolution and enrichment of ergothioneine in the gel matrix, and improve the accuracy of the detection result.

[0067] In some embodiments, the mass-volume ratio of the gel matrix food to the second solvent is 1 g: (70-90) mL. It can be understood that the gel matrix food is the gel matrix part in the food.

[0068] In some embodiments, the alcohol solvent comprises one or more of methanol and ethanol.

[0069] In some embodiments, the ultrasonic treatment time is 10 min-30 min, including but not limited to 10 min, 15 min, 20 min, 25 min, 30 min or a range formed by any two of the foregoing and numerical values within the range. Ultrasonic treatment can improve the dissolution of ergothioneine in the matrix, and the dissolution of ergothioneine can be maximized within the ultrasonic time range.

[0070] In some embodiments, the ultrasonic treatment power is 100 W-200 W, including but not limited to 100 W, 120 W, 150 W, 180 W, 200 W or a range formed by any two of the foregoing and numerical values within the range.

[0071] In some embodiments, the ultrasonic treatment frequency is 30 kHz-50 kHz, including but not limited to 30 kHz, 35 kHz, 40 kHz, 45 kHz, 50 kHz or a range formed by any two of the foregoing and numerical values within the range.

[0072] In some embodiments, the filter membrane used in the filtration has a pore size of 0.22 μm. As a non-limiting example, the filter membrane used in the filtration is a 0.22 μm nylon membrane.

[0073] In some embodiments, the preparation of the test sample solution further comprises pre-treating the gel matrix food, including: taking the gel matrix part of the gel matrix food and crushing it.

[0074] In some embodiments, the gel matrix food comprises a sandwich gel food, and the pre-treatment of the sandwich gel food comprises: removing the sandwich part of the sandwich gel food, and cleaning and crushing the remaining gel matrix part.

[0075] In some embodiments, the detection limit of ergothioneine in the gel matrix food detected by the detection method is ≥ 20.2 pg / mL.

[0076] In some embodiments, the quantification limit of ergothioneine in the gel matrix food detected by the detection method is ≥ 67.3 pg / mL.

[0077] The present application uses the method of liquid chromatography-mass spectrometry (HPLC-MS / MS) to realize rapid and accurate detection of ergothioneine in gel matrix food. The detection method has low detection limit and quantification limit, and is suitable for the detection of ergothioneine in complex matrix products. It provides an effective quality control and evaluation method for related products, and ensures the quality of food.

[0078] It should be noted that the experimental methods in the following embodiments of the present application without specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. The various common chemical reagents used in the embodiments are commercially available products or can be prepared by known means by those skilled in the art.

[0079] Example 1

[0080] The present embodiment provides a method for detecting ergothioneine in gel candy, which is as follows:

[0081] The sample to be detected is a sandwich gel candy containing astaxanthin.

[0082] Preparation of the test sample solution: remove the sandwich (astaxanthin oil) in the gel candy, wipe it clean with an oil-absorbing paper, weigh the gel shell at 285 mg, then cut it into pieces, and then use the extraction solvents shown in Table 1 to make up to 25 mL, ultrasonic (180 W, 40 kHz) for 30 min, filter with a 0.22 μm nylon membrane, dilute 100 times with water, filter with a 0.22 μm nylon membrane, and prepare the test sample solution.

[0083] Table 1: Composition of test sample extraction solvent

[0084]

[0085] Preparation of standard solution: an appropriate amount of ergothioneine standard was accurately weighed into a 10 mL volumetric flask, diluted to the mark with water, shaken well, and prepared into a standard solution.

[0086] Detection method: the test sample solution and the standard solution were detected by HPLC-MS / MS.

[0087] Liquid chromatography system and parameters: high performance liquid chromatograph (Waters, ACQUITY UPLC I-Class PLUS system), chromatographic column ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm, column temperature 35℃, mobile phase A 0.1% (v / v) formic acid in water, mobile phase B methanol, gradient elution (gradient elution program see Table 2), flow rate 0.4 mL / min, injection volume 10 μL.

[0088] Table 2: Gradient elution program

[0089]

[0090] Mass spectrometry system and parameters: mass spectrometer AB Sciex Triple Quad 5500+, scan mode MRM, parent ion 230.0 Da, daughter ion 127.0 Da, ion spray temperature 320℃, electrospray ion source temperature 150℃, desolvation gas temperature 500℃, desolvation gas flow rate 800 L / Hr, capillary voltage 3.0 kV, cone voltage 30 V, entrance voltage (EP) 2 V, collision energy (CE) 35 eV, collision cell exit voltage (CXP) 0.5 V, curtain gas (nitrogen) pressure 40 psi, drying gas (nitrogen) pressure 35 psi, nebulizer gas (nitrogen) pressure 70 psi, collision cell gas (nitrogen) pressure 95 psi.

[0091] The results of the detection according to the above detection method are as follows:

[0092] Appendix Figure 1The total ion flow chromatogram of the ergothioneine standard solution. The test sample solutions prepared using water, methanol: water = 1:1 (v / v) and methanol as the extraction solvent were subjected to chromatographic analysis, and the characteristic peak area of ergothioneine in each test sample solution was counted. The results are shown in Table 1. The chromatographic peak area of the test sample solution prepared using water as the extraction solvent was 272000, the chromatographic peak area of the test sample solution prepared using methanol: water = 1:1 (v / v) as the extraction solvent was 232000, and the chromatographic peak area of the test sample solution prepared using methanol as the extraction solvent was 199000. Comparison shows that the ergothioneine chromatographic peak area detected by HPLC-MS / MS in the gel candy prepared using water as the extraction solvent is the largest, indicating that water as the extraction solvent can better dissolve ergothioneine in the gel candy, and the quantification is more accurate.

[0093] Example 2

[0094] The present embodiment provides a method for detecting ergothioneine in gel candy, which is as follows:

[0095] Test sample: astaxanthin sandwich gel candy.

[0096] Preparation of test sample solution: Remove the filling (astaxanthin oil) in the gel candy, wipe it clean with oil-absorbing paper, weigh the gel shell at 285 mg, then cut it into pieces, extract with water to a constant volume of 25 mL, ultrasonic (180 W, 40 kHz) for 30 min, filter with a 0.22 μm nylon membrane, dilute 100 times with water, filter with a 0.22 μm nylon membrane, and prepare the test sample solution.

[0097] Preparation of standard solution: accurately weigh an appropriate amount of ergothioneine standard, place it in a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare the standard solution.

[0098] Detection method: HPLC-MS / MS method was used for detection, the test sample solution and the standard solution were loaded onto HPLC / HPLC-MS / MS for measurement, and elution was performed using the mobile phase system shown in Table 3, the elution program was the same as Table 2, and the rest of the liquid chromatography system and parameters, mass spectrometry system and parameters were the same as Example 1.

[0099] Table 3: Mobile phase composition system

[0100]

[0101] The results of the detection according to the above detection method are shown in the accompanying Figure 3As shown in the figure, adding a certain concentration of formic acid can provide pH value, as shown in the figure A), B) and C) comparison, ergothioneine chromatographic peak in A) and B) split peak, and A) compared to B) split more obvious, and C) no chromatographic peak splitting and good shape, can be seen, with the increase of formic acid concentration, the elution effect of ergothioneine is better and better, the chromatographic peak shape is further optimized, and the response value of the chromatographic peak is higher and higher. When the concentration of 0.1% (v / v) formic acid is reached, the ergothioneine in the test sample can be accurately quantified.

[0102] Example 3

[0103] The present embodiment provides a method for detecting ergothioneine in gel candies, which is specifically as follows:

[0104] The sample to be detected is astaxanthin sandwich gel candy.

[0105] Preparation of test sample solution: weigh the whole gel candy (containing 0.5 g astaxanthin oil and 0.285 g gel matrix) 0.785 g, then cut it into pieces, use water as the extraction solvent to make up to 25 mL, ultrasonic (180 W, 40 kHz) for 30 min, filter with 0.22 μm nylon membrane, dilute 100 times with water, filter with 0.22 μm nylon membrane, and prepare the test sample solution.

[0106] Preparation of standard solution: accurately weigh a proper amount of ergothioneine standard, put it into a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare the standard solution.

[0107] Detection method: use HPLC-MS / MS method for detection, and load the test sample solution and the standard solution into HPLC / HPLC-MS / MS for measurement. The liquid chromatography system and parameters and the mass spectrometry system and parameters are the same as those in Example 1.

[0108] The results of the detection according to the above detection method are shown in the following table: Figure 4 As shown in the figure, the pretreatment of astaxanthin sandwich gel candy does not remove the sandwich, and the whole astaxanthin sandwich gel candy is cut into pieces and extracted with the extraction solvent. There is a lot of matrix interference, and ergothioneine is not detected, and there is no chromatographic peak. Compared with the chromatogram shown in Figure 3 As shown in the chromatogram, for the gel candy containing sandwich, the sandwich needs to be removed to avoid interference.

[0109] Example 4

[0110] The present embodiment provides a method for detecting ergothioneine in gel candies, which is specifically as follows:

[0111] The sample to be detected is astaxanthin sandwich gel candy.

[0112] Preparation of test sample solution: remove the filling (astaxanthin oil) in the gel candy, wipe it clean with oil-absorbing paper, weigh the gel shell 285 mg, then cut it into pieces, use water as the extraction solvent to make up to 25 mL, ultrasonic (180 W, 40 kHz) for 30 min, filter with 0.22 μm nylon membrane, dilute 100 μL filtrate with water by 100 times, filter with 0.22 μm nylon membrane, and prepare the test sample solution.

[0113] Preparation of standard sample solution: accurately weigh a certain amount of ergothioneine standard sample, put it into a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare the standard sample solution.

[0114] Detection method: use HPLC-MS / MS method for detection, and load the test sample solution and the standard sample solution into HPLC / HPLC-MS / MS for measurement, the flow rate is 0.6 min / mL, and the rest of the liquid chromatography system and parameters and the mass spectrometry system and parameters are the same as those in Example 1.

[0115] The results detected by the above detection method are shown in FIG. 1, and it can be seen from the figure that in the chromatogram detected by the flow rate of 0.6 min / mL, the chromatographic peak of ergothioneine does not appear split, but compared with FIG. 2, the chromatographic peak is obviously detected in advance, and there is obvious tailing, resulting in poor quantitative accuracy. Figure 5 Figure 2 The results detected by the above detection method are shown in FIG. 1, and it can be seen from the figure that in the chromatogram detected by the flow rate of 0.6 min / mL, the chromatographic peak of ergothioneine does not appear split, but compared with FIG. 2, the chromatographic peak is obviously detected in advance, and there is obvious tailing, resulting in poor quantitative accuracy.

[0116] Example 5

[0117] The present embodiment provides a detection method of ergothioneine in gel candy, which is as follows:

[0118] Detection sample: astaxanthin filling gel candy.

[0119] Preparation of test sample solution: remove the filling (astaxanthin oil) in the gel candy, wipe it clean with oil-absorbing paper, weigh the gel shell 285 mg, then cut it into pieces, use water as the extraction solvent to make up to 25 mL, ultrasonic (180 W, 40 kHz) for different times (as shown in Table 4), filter with 0.22 μm nylon membrane, dilute 100 μL filtrate with water by 100 times, filter with 0.22 μm nylon membrane, and prepare the test sample solution.

[0120] Preparation of standard sample solution: accurately weigh a certain amount of ergothioneine standard sample, put it into a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare the standard sample solution.

[0121] ​Detection method: the method of liquid chromatography tandem mass spectrometry HPLC-MS / MS is used for detection, and the test sample solution and the standard solution are respectively loaded on HPLC / HPLC-MS / MS for measurement, and the liquid chromatography system and parameters and the mass spectrometry system and parameters are the same as those in example 1.

[0122] The chromatographic peak area of ergothioneine in the sample is counted, and the results are shown in table 4 and the attached Figure 6

[0123] Table 4: relationship between ultrasonic time and peak area

[0124]

[0125] The results of table 4 and the attached Figure 6 It is shown that with the increase of ultrasonic time, the chromatographic peak area increases, indicating that the dissolution of ergothioneine in the sample is more, and when the ultrasonic time is more than 20 min, the chromatographic peak area remains at a certain level, therefore, considering comprehensively, the ultrasonic time of 20 min has better detection effect, and saves cost, realizes faster quantification. The gel shell is cut into a size of a piece of cereal, and since the gel matrix needs a certain time to absorb water, the dissolution is not carried out in the first 5 min, and the piece is clearly visible, and gradually absorbs and swells and dissolves in 5 min-10 min, the gel matrix dissolves in 10 min-30 min, and the ultrasonic dissolves and uniformly mixes, and gradually extracts ergothioneine.

[0126] In summary, the preferred detection method of ergothioneine in gel candies is obtained through a large number of experimental verification, which is specifically:

[0127] Preparation of test sample solution: remove the filling (astaxanthin oil) in the gel candy, wipe it clean with oil-absorbing paper, weigh the gel shell 285 mg, then cut it into pieces, and respectively use the extraction solvent shown in table 1 to constant volume to 25 mL, ultrasonic (180W, 40 kHz) for 20 min, filter with 0.22 μm nylon membrane, take 100 μL filtrate, dilute 100 times with water, filter with 0.22 μm nylon membrane, and prepare the test sample solution.

[0128] Preparation of standard solution: accurately weigh a certain amount of ergothioneine standard, place it in a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare the standard solution.

[0129] ​Chromatographic system and parameters: high performance liquid chromatograph (Waters, ACQUITY UPLC I-Class PLUS system), the chromatographic column uses ACQUITY UPLC BEH C18, 1.7 μm, 2.1×50 mm, the chromatographic column temperature is 35 DEG C, the flow rate is 0.4 mL / min, the injection amount is 10 μL, the mobile phase A is 0.1% (v / v) formic acid aqueous solution, the mobile phase B is methanol, gradient elution procedure is as follows: 0 min-4 min, the volume percentage of the mobile phase B changes from 15% to 30%; 4 min-5 min, the volume percentage of the mobile phase B changes from 30% to 15%; 5 min-7 min, the volume percentage of the mobile phase B maintains 15%.

[0130] Mass spectrometry system and parameters: mass spectrometer AB Sciex Triple Quad 5500+, the scanning mode is MRM, the parent ion is 230.0 Da, the daughter ion is 127.0 Da, the ion spray temperature is 320 DEG C, the electrospray ion source temperature is 150 DEG C, the desolvation gas temperature is 500 DEG C, the desolvation gas flow rate is 800 L / Hr, the capillary voltage is 3.0 kV, the cone hole voltage is 30 V, the inlet voltage (EP) is 2 V, the collision energy (CE) is 35 eV, the collision cell outlet voltage (CXP) is 0.5 V, the pressure of curtain gas (nitrogen) is 40 psi, the pressure of drying gas (nitrogen) is 35 psi, the pressure of atomization gas (nitrogen) is 70 psi, the pressure of collision cell gas (nitrogen) is 95 psi.

[0131] In order to further verify the limit of quantification, specificity, precision, recovery rate and stability of the preferred detection method, the relevant methodology experiments are carried out, as follows:

[0132] Test Example 1: Investigation of linear correlation

[0133] This test example investigates the quantitative accuracy of the preferred detection method for ergothioneine in gel candy by constructing a standard curve, and a series of ergothioneine standard solutions are prepared: 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL.

[0134] The content of ergothioneine in astaxanthin sandwich gel candy is calculated as shown in formula (I):

[0135] (I)

[0136] In the formula: X- percentage of ergothioneine in the sample to be tested (g / 100 g); C- concentration of ergothioneine in the sample to be tested calculated from the standard curve (ng / mL); V- constant volume of the sample to be tested (mL); F- dilution multiple; M- sample amount of the sample to be tested (g).

[0137] The standard curve of ergothioneine marker concentration and chromatographic peak area was constructed (see Figure 1). Figure 7 The regression equation is: y = 14853x + 662237, R 2 = 0.999, which meets the linear requirement, the detection limit LOD = 20.2 pg / mL, and the quantification limit LOQ = 67.3 pg / mL. It can be seen that the detection method provided in the application can detect very low content of ergothioneine in gel candies, which is accurate in quantification and has a significant advantage compared with traditional techniques.

[0138] Test Example 2: Specificity Investigation

[0139] The specificity of the above detection method was verified by diluting the sample by different multiples, and the peak areas of different dilution multiples were compared.

[0140] Preparation of test sample solution: The filling (astaxanthin oil) in the gel candy was removed, and the gel shell was wiped clean with an oil-absorbing paper, weighed 285 mg, then cut into pieces, and then water was used as the extraction solvent to make up to 25 mL, ultrasonic (180 W, 40 kHz) for 20 min, filtered with a 0.22 μm nylon membrane, 100 μL of the filtrate was diluted with water by 100 times, filtered with a 0.22 μm nylon membrane, and prepared into a test sample solution.

[0141] The test sample solution was diluted by 1000 times and 2000 times, respectively, and the sample was measured on the machine, and the matrix factor (Matrix Factor, MF) was calculated according to the peak area, and the calculation formula is shown in formula (II):

[0142] (II)

[0143] In the formula: MF- matrix factor (0.8-1.2, considered stable); A F1 - chromatographic peak area of the sample to be tested diluted by 1000 times; A F2 - chromatographic peak area of the sample to be tested diluted by 2000 times; F1- multiple of dilution by 1000 times, F2- multiple of dilution by 2000 times;

[0144] According to the above investigation method, the matrix factor MF of astaxanthin filling gel candy is 0.92, which is within a reasonable range, and there is no influence of other interfering substances, and the specificity of the detection method is good.

[0145] Test Example 3: Intra-day and Inter-day Precision Investigation

[0146] Prepare ergothioneine standard solution with low, medium and high concentrations, respectively, namely quality control sample (QC), LQC = 30 ng / mL, MQC = 80 ng / mL, HQC = 160 ng / mL. Three parallel samples are prepared for each concentration. Repeat the three concentrations within a day for three days. Calculate the relative standard deviation (RSD%) of the data of the first day to obtain the intra-day precision, and calculate the data of the three consecutive days to obtain the inter-day precision (RSD%), and calculate the accuracy (RE%), the calculation formula is shown in formula (III):

[0147] (III)

[0148] In the formula: RE-accuracy (%); C1-ergothioneine concentration of the quality control sample calculated from the standard curve (ng / mL); C2-ergothioneine quality control sample concentration (ng / mL).

[0149] The results obtained according to the above investigation method are shown in Table 5:

[0150] Table 5: Intra-day and inter-day precision investigation results

[0151]

[0152] As shown in Table 5, the accuracy of the intra-day and inter-day precision investigation of the detection method provided by the present application is 98%-105.5%, and the RSD is less than 6%, which meets the detection requirements.

[0153] Test Example 4: Recovery rate

[0154] Prepare several portions of astaxanthin sandwich gel candy solution, add ergothioneine solutions with three different concentrations, so that the final spiked amounts are 30 ng / mL, 80 ng / mL and 160 ng / mL, which are 30%, 50% and 80% of the standard curve range, respectively, and then determine the recovery rate of ergothioneine. Each sample is prepared in triplicate. The recovery rate is ±20%, and the RSD% is less than 7%, which meets the detection requirements. The calculation formula of the recovery rate is shown in formula (IV):

[0155] (IV)

[0156] In the formula: C1-ergothioneine concentration of the spiked sample calculated from the standard curve (ng / mL); C2-ergothioneine concentration of the unspiked sample calculated from the standard curve (ng / mL); C3-ergothioneine spiked concentration (ng / mL).

[0157] The results obtained according to the above investigation method are shown in Table 6:

[0158] Table 6: Results of the recovery rate test

[0159]

[0160] The results of Table 6 show that the recovery rate of the detection method provided by the present application is 103%-107%, and the RSD is less than 6.5%, which meets the detection requirements.

[0161] Test Example 5: Stability test

[0162] Prepare several portions of astaxanthin sandwich gel candy solution, and store them under different conditions (4℃, 25℃, repeated freezing and thawing) for 0 h, 12 h and 24 h, respectively. Measure the ergothioneine content and calculate the RSD%. Verify the stability of the detection method, and do 6 parallel tests for each storage condition. The results are shown in Table 7:

[0163] Table 7: Results of the stability test

[0164]

[0165] The results of Table 7 show that the stability test of the detection method provided by the present application has an RSD of less than 5%, which meets the detection requirements.

[0166] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0167] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, some modifications and improvements can be made without departing from the concept of the present application, and these are within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for the detection of ergothioneine in a gel-based food product, characterized in that, The ergothioneine in the gel matrix food is detected by using a liquid chromatography tandem mass spectrometry method; The liquid chromatography conditions include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is methanol; the elution program of the mobile phase adopts gradient elution, and the gradient elution program is as follows: 0 min-4 min, the volume percentage of the mobile phase B changes from 15% to 30%; 4 min-5 min, the volume percentage of the mobile phase B changes from 30% to 15%; 5 min-7 min, the volume percentage of the mobile phase B is maintained at 15%; The mass spectrometry conditions include: the mass of the parent ion is 230 Da, the mass of the daughter ion is 127 Da, the scanning mode is MRM, the electrospray ion source temperature is 80-150 DEG C, the collision energy is 35 eV, and the capillary voltage is 3.0 kV.

2. The method for detecting ergothioneine in a gel base food according to claim 1, characterized by, The chromatographic column is a C18 chromatographic column, and the model of the chromatographic column is ACQUITY UPLC BEH C18.

3. The method for detecting ergothioneine in a gel base food according to claim 1, characterized by, The column temperature of the chromatographic column is 30-35 DEG C; and / or, the flow rate of the mobile phase is 0.2-0.5 mL / min; and / or, the injection amount is 1-20 μL; and / or, the volume fraction of the formic acid in the mobile phase A is 0.05-0.1%.

4. The method for detecting ergothioneine in a gel base food according to claim 1, characterized by, The mass spectrometry conditions further include: the desolvation gas temperature is 500 DEG C, the desolvation gas flow rate is 800 L / Hr; and / or, the cone voltage is 30 V, the inlet voltage is 2 V; and / or, the collision cell exit voltage is 0.5 V; and / or, the pressure of the gas curtain is 40 psi, the pressure of the drying gas is 35 psi, and the pressure of the nebulizing gas is 70 psi, and the gas curtain, the drying gas and the nebulizing gas include nitrogen; and / or, the gas pressure of the collision cell is 95 psi, and the gas includes nitrogen.

5. The method for detecting ergothioneine in a gel substrate food according to any one of claims 1 to 4, characterized in that, The gel matrix food includes gel candies, and the gel candies include gel candies containing fillings; the ingredients of the fillings include astaxanthin.

6. The method for detecting ergothioneine in a gel substrate food according to any one of claims 1 to 4, characterized in that, The detection method further includes the steps of preparing a standard solution and detecting the standard solution by using the liquid chromatography tandem mass spectrometry method, and the preparation method of the standard solution includes: mixing the ergothioneine standard product with a first solvent to prepare the standard solution.

7. The method for detecting ergothioneine in a gel substrate food product according to claim 6, wherein The first solvent includes water; and / or, the concentration of the standard solution is 5-500 ng / mL.

8. The method according to any one of claims 1 to 4, wherein the ergothioneine is detected in the gel base food. The detection method further includes the steps of preparing a test solution and detecting the test solution by using the liquid chromatography tandem mass spectrometry method, and the preparation method of the test solution includes: mixing the gel matrix food with a second solvent, ultrasonic treatment, filtration, taking the filtrate, adding a third solvent, and preparing the test solution.

9. The method for detecting ergothioneine in a gel base food product according to claim 8, characterized in that, The second solvent and the third solvent each independently include one or more of an alcohol solvent and water; and / or, the mass-volume ratio of the gel matrix food to the second solvent is 1 g: (70-90) mL.

10. The method for detecting ergothioneine in a gel substrate food according to claim 9, characterized by, The alcohol solvent includes one or more of methanol and ethanol; and / or, the ultrasonic treatment time is 10 min-30 min; and / or, the ultrasonic treatment power is 100 W-200 W, and the ultrasonic treatment frequency is 30 kHz-50 kHz; and / or, the filter membrane pore size used in the filtering is 0.22 μm.

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