Method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and lentinine in lentinus edodes

Abstract

The invention discloses a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and lentinine in lentinus edodes, and belongs to the technical field of measurement of active ingredients of the lentinus edodes. The method comprises the following steps: detecting a shiitake mushroom extracting solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry mode, wherein a chromatographic column is an HILIC or C18 chromatographic column; the mobile phase A and the mobile phase B are an aqueous solution containing formic acid and a methanol or acetonitrile solution containing formic acid respectively; the mass spectrum conditions comprise an electrospray ionization source; a positive ion multiple reaction monitoring mode; the ion spraying temperature is 315-325 DEG C; the temperature of the ion transmission tube is 320-330 DEG C; and the ion spraying voltage is 3500 V to 3700 V. The method has the characteristics of high sensitivity, high selectivity and high accuracy, and is suitable for quantitative detection of ergothioneine, lentinine, ergosterol peroxide and ergosterol compounds in shiitake mushrooms.

Description

Technical Field

[0001] The invention relates to the technical field of determination of active ingredients of shiitake mushrooms, and in particular to a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and shiitake purine in shiitake mushrooms. Background Art

[0002] Shiitake mushrooms are a widely cultivated edible and medicinal fungus that is rich in nutrients and bioactive compounds. In recent years, with the deepening of research on functional foods, ergothioneine, shiitake purine, ergosterol and ergosterol peroxide, as important active ingredients of shiitake mushrooms, have received increasing attention.

[0003] Among them, ergothioneine is a natural amino acid derivative derived from fungi and cyanobacteria. It has a powerful antioxidant function and has been shown in many studies to resist oxidative stress and protect cells from damage. Shiitake purine is an alkaloid that was first extracted from shiitake mushrooms, which are also considered to be the main source of shiitake purine. Shiitake purine can reduce the synthesis of cholesterol by inhibiting S-adenosyl-L-homocysteine ​​hydrolase (SAH hydrolase), which helps to lower serum cholesterol levels. Ergosterol is a sterol compound with a high content in shiitake mushrooms. It is a precursor of vitamin D2 and is widely present in fungal cell membranes. It has antioxidant, anti-tumor and immunomodulatory effects. Ergosterol peroxide has attracted widespread attention in anti-cancer, anti-inflammatory and antibacterial research due to its unique structure and pharmacological activity.

[0004] However, at present, there is no accurate and feasible simultaneous detection method for effectively detecting the contents of ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms, so as to solve or improve the above technical problems.

[0007] The present invention can be implemented like this:

[0008] In a first aspect, the present invention provides a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms, comprising the following steps:

[0009] The shiitake mushroom extract was tested by ultra-high performance liquid chromatography-tandem mass spectrometry under the following conditions:

[0010] The chromatographic conditions include: using a HILIC or C18 chromatographic column; the mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing formic acid, and the mobile phase B is a methanol or acetonitrile solution containing formic acid; the elution time and the volume content of the mobile phase B during the elution time are as follows: 0min to 0.2min, 80%; 0.2min to 1min, 80% to 90%; 1min to 4min, 90% to 100%; 4min to 8.5min, 100%; 8.5min to 8.6min, 100% to 80%; 8.6min to 11min, 80%;

[0011] The mass spectrometry conditions include: the ion source type is an electrospray ionization source; the scanning mode is a positive ion multiple reaction monitoring mode; the ion spray temperature is 315°C to 325°C; the ion transfer tube temperature is 320°C to 330°C; the ion spray voltage is 3500V to 3700V; the sheath gas is 25psi to 35psi; the auxiliary gas is 5psi to 15psi; and the tail gas is 0psi.

[0012] In an alternative embodiment, the chromatographic column is an ACQUITY UPLC BEH HILIC column or an ACQUITY BEH C18 column.

[0013] In an alternative embodiment, ACQUITY BEH C18 was used as the chromatographic column, and the column specifications were 2.1 mm × 100 mm, 1.7 μm.

[0014] In an optional embodiment, the volume percentage of formic acid contained in the mobile phase A and the mobile phase B respectively is 0.1% to 0.3%.

[0015] In an optional embodiment, the volume percentage of formic acid contained in the mobile phase A and the mobile phase B is 0.2%.

[0016] In an optional embodiment, the flow rate of the mobile phase is 0.2 mL / min to 0.4 mL / min.

[0017] In an alternative embodiment, the flow rate of the mobile phase is 0.3 mL / min.

[0018] In an optional embodiment, the injection volume of the extract is 4 μL to 6 μL, and / or the temperature of the column oven is 34° C. to 36° C.

[0019] In an optional embodiment, the injection volume of the extract is 5 μL, and / or the temperature of the column oven is 35° C.

[0020] In an optional embodiment, the ion spray temperature is 320° C.; the ion transfer tube temperature is 325° C.; the ion spray voltage is 3600 V; the sheath gas is 30 psi; the auxiliary gas is 10 psi; and the tail gas is 0 psi.

[0021] In an optional embodiment, the preparation of the shiitake mushroom extract includes: cutting the fresh shiitake mushroom sample to be tested into pieces, soaking it in liquid nitrogen, homogenizing it, and then adding an extraction solvent to extract it to obtain an extract; or, grinding and sieving the dried shiitake mushroom sample to be tested, and then adding an extraction solvent to extract it to obtain an extract.

[0022] In an optional embodiment, the extraction solvent used for extraction includes at least one of methanol, acetonitrile, isopropanol and water.

[0023] In an optional embodiment, the extraction solvent used for extraction is a methanol-water solution with a methanol volume of not less than 80%.

[0024] In an optional embodiment, the extraction solvent is a methanol-water solution with a volume of 85% to 95% methanol;

[0025] In an alternative embodiment, the extraction solvent is a methanol-water solution with a volume percentage of 90% methanol.

[0026] In an optional embodiment, during the extraction process, the solid-liquid ratio of the extraction solvent to the shiitake mushroom sample to be tested is 10 mL:1 g to 120 mL:1 g.

[0027] In an optional embodiment, the solid-liquid ratio is 120mL:1g.

[0028] In an optional embodiment, the extraction is carried out by ultrasonic extraction.

[0029] In an optional embodiment, the ultrasonic extraction temperature is room temperature.

[0030] In an optional embodiment, the ultrasonic extraction time is 10 min to 160 min.

[0031] In an optional embodiment, the ultrasonic extraction time is 30 min to 60 min.

[0032] In an optional embodiment, the ultrasonic extraction time is 60 min.

[0033] The beneficial effects of the present invention include:

[0034] The embodiment of the present invention provides a simultaneous detection and analysis method for ergothioneine, ergosterol, ergosterol peroxide and shiitake purine in shiitake mushrooms, which adopts an ultra-high performance liquid chromatography-tandem mass spectrometry method to detect the four active compounds in shiitake mushrooms, and optimizes the pre-treatment conditions of shiitake mushrooms and the instrument parameters of ultra-high performance liquid chromatography-tandem mass spectrometry, so that the target compounds can be efficiently extracted and accurately detected with high selectivity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 is the UHPLC-MS / MS ion chromatogram corresponding to Example 1;

[0037] Figure 2 is the UHPLC-MS / MS ion chromatogram corresponding to Example 2;

[0038] Figure 3 is the UHPLC-MS / MS ion chromatogram corresponding to Example 3;

[0039] Figure 4 is the UHPLC-MS / MS ion chromatogram corresponding to Example 4;

[0040] Figure 5 is the UHPLC-MS / MS ion chromatogram corresponding to Example 5;

[0041] Figure 6 is the UHPLC-MS / MS ion chromatogram corresponding to Example 6;

[0042] Figure 7 is the UHPLC-MS / MS ion chromatogram corresponding to Example 7;

[0043] Figure 8 is the UHPLC-MS / MS ion chromatogram corresponding to Example 8;

[0044] Fig. 9 It is the UHPLC-MS / MS ion chromatogram corresponding to the mixed standard solution of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in Test Example 1;

[0045] Fig.10The results of the analysis of the contents of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms with different extraction solvents in Experimental Example 2;

[0046] Fig.11 The results of the analysis of the contents of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms with different ratios of methanol and water in Experimental Example 2;

[0047] Fig.12 The results of the analysis of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms at different solid-liquid ratios in Experimental Example 3;

[0048] Fig.13 The results of the analysis of the contents of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms at different ultrasonic extraction temperatures in Experimental Example 4;

[0049] Fig.14 These are the analysis results of the contents of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms at different ultrasonic extraction times in Experimental Example 5. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0051] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided by the present invention is described below.

[0052] The invention provides a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and shiitake purine in shiitake mushrooms. Ultra high performance liquid chromatography-tandem mass spectrometry is used to detect ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms.

[0053] When performing liquid chromatography, the chromatographic column used may be a HILIC chromatographic column or a C18 chromatographic column. In some typical embodiments, the chromatographic column used may be an ACQUITY UPLC BEH HILIC chromatographic column or an ACQUITY BEHC18 chromatographic column. The ACQUITY UPLC BEH HILIC chromatographic column can be purchased from Waters, and has a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; ACQUITY The BEH C18 column can be purchased from Waters, and has a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm. In some more typical embodiments, the chromatographic column used is ACQUITY BEH C18 column, which has better peak shape and higher resolution than ACQUITY UPLC BEH HILIC column.

[0054] Before detection, the instrument conditions can be optimized. The present invention first creates a scanning method, determines the standard of ergothioneine, shiitake purine, ergosterol, and ergosterol peroxide, clarifies the precursor ion, and then determines the daughter ion and optimizes the collision energy, and finally completes the MRM method establishment. In order to obtain a suitable separation effect for the target compound, the liquid chromatography parameters are further optimized.

[0055] The retention times of ergothioneine, shiitake purine, ergosterol, ergosterol peroxide and in shiitake mushrooms as well as the MRM qualitative and quantitative ion pairs and collision energy information are shown in Table 1.

[0056] Table 1 Chromatographic and mass spectrometric information of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms

[0057]

[0058] The mobile phase includes mobile phase A and mobile phase B. The mobile phase A is an aqueous solution containing formic acid, and the mobile phase B is a methanol or acetonitrile solution containing formic acid, and preferably the mobile phase B is methanol containing formic acid.

[0059] In some embodiments, the volume percentage of formic acid contained in mobile phase A and mobile phase B can be 0.1% to 0.3%, such as 0.1%, 0.15%, 0.2%, 0.25% or 0.3%, etc., or other values ​​within the range of 0.1% to 0.3%. In some preferred embodiments, the volume percentage of formic acid contained in mobile phase A and mobile phase B is 0.2%.

[0060] By using an acetonitrile-water system as the mobile phase for comparison, ergothioneine, ergosterol, ergosterol peroxide and shiitake purine required to be detected in the present invention can produce peaks in the acetonitrile-water system and the methanol-water system, but show a higher response in the methanol-water system, so the present invention uses the methanol-water system as the mobile phase to obtain a more accurate detection effect.

[0061] Generally, in chromatographic detection, acid or salt can be added to the mobile phase to increase the sensitivity of the analyte and improve the shape of the chromatographic peak. The present invention uses four different water-methanol systems as comparisons, one is to add 0.2% (v / v) formic acid to the water-methanol system, the second is to add 5mmol / L of ammonium formate to the water-methanol system, the third is to add 0.2% (v / v) formic acid and 5mmol / L of ammonium formate to the water-methanol system, and the fourth is not to add acid and salt to the water-methanol system. By comparison, adding 0.2% (v / v) formic acid can more effectively improve the detection sensitivity and more effectively improve the peak shape.

[0062] In some embodiments, the flow rate of the mobile phase can be 0.2mL / min to 0.4mL / min, such as 0.2mL / min, 0.25mL / min, 0.3mL / min, 0.35mL / min or 0.4mL / min, etc., or other values ​​within the range of 0.2mL / min to 0.4mL / min. In some preferred embodiments, the flow rate of the mobile phase is 0.3mL / min.

[0063] In some embodiments, the injection volume of the extract can be 4 μL to 6 μL, such as 4 μL, 4.5 μL, 5 μL, 5.5 μL or 6 μL, etc., or other values ​​within the range of 4 μL to 6 μL. In some preferred embodiments, the injection volume of the extract is 5 μL.

[0064] In some embodiments, the column oven temperature can be 34° C. to 36° C., such as 34° C., 34.5° C., 35° C., 35.5° C. or 36° C., or other values ​​within the range of 34° C. to 36° C. In some preferred embodiments, the column oven temperature is 35° C.

[0065] In the present invention, the elution time and the volume content of mobile phase B during the elution time are as follows: 0min-0.2min, 80%; 0.2min-1min, 80%-90%; 1min-4min, 90%-100%; 4min-8.5min, 100%; 8.5min-8.6min, 100%-80%; 8.6min-11min, 80%.

[0066] The total running time for the simultaneous determination of four non-volatile compounds by the above method was 11 min, and the peak elution order was ergothioneine, lentinan, ergosterol peroxide, and ergosterol. In the present invention, the mass spectrometry conditions include: the ion source type is an electrospray ionization source; the scanning mode is a positive ion multiple reaction monitoring mode; the ion spray temperature is 315°C to 325°C (such as 315°C, 318°C, 320°C, 322°C or 325°C, etc.); the ion transfer tube temperature is 320°C to 330°C (such as 320°C, 322°C, 325°C, 328°C or 330°C, etc.); the ion spray voltage is 3500V to 3700V (such as 3500V, 3550V, 3600V, 3650V or 3700V, etc.); the sheath gas is 25psi to 35psi (such as 25psi, 28psi, 30psi, 32psi or 35psi, etc.); the auxiliary gas is 5psi to 15psi (such as 5psi, 8psi, 10psi, 12psi or 15psi, etc.); and the tail gas is 0psi.

[0067] In some preferred embodiments, the ion spray temperature is 320° C.; the ion transfer tube temperature is 325° C.; the ion spray voltage is 3600 V; the sheath gas is 30 psi; the auxiliary gas is 10 psi; and the tail gas is 0 psi.

[0068] In order to evaluate the feasibility of the analytical method of ergothioneine, shiitake purine, ergosterol and ergosterol peroxide in shiitake mushrooms in the present invention, the present invention is evaluated by detection limit (LOD), quantification limit (LOQ), linear curve, accuracy, matrix effect and precision. LOD and LOQ are compound concentrations of 3 times and 10 times the signal-to-noise ratio (S / N), respectively; the drawing of the standard curve is set with at least 7 series of concentrations; the accuracy is evaluated by the spiked recovery rate, and in the evaluation process of the recovery rate, the spiked levels of L (low), M (medium) and H (high) are designed, and the calculation formula is: recovery rate = (detection amount - original amount) / added amount × 100%. The matrix effect is evaluated by the slope method, and the calculation formula is: matrix effect = matrix standard curve slope / solvent standard curve slope. When the matrix effect is 0.8-1.2, it means that the matrix effect has little effect on the method, and solvent standards can be used for quantification; when the matrix effect is lower than 0.8 or higher than 1.2, it means that the matrix effect has an inhibitory or enhancing effect on the method, and matrix standards need to be used for quantification. The precision of the method was evaluated based on repeatability (intra-day precision) and reproducibility (inter-day precision). The intra-day precision was evaluated by performing six replicates of ergothioneine, shiitake purine, ergosterol, and ergosterol peroxide mixed standards at different time points on the same day, and the inter-day precision was evaluated by performing six replicates for three consecutive days. The results were expressed as relative standard deviations (RSD).

[0069] In the present invention, the preparation of the shiitake mushroom extract comprises: cutting the fresh shiitake mushroom sample to be tested into pieces, soaking it in liquid nitrogen, homogenizing it, and then adding an extraction solvent to extract it to obtain an extract; or, grinding and sieving the dried shiitake mushroom sample to be tested (such as passing through a 60-mesh sieve), and then adding an extraction solvent to extract it to obtain an extract.

[0070] In some embodiments, the extraction solvent used for extraction includes at least one of methanol, acetonitrile, isopropanol and water. In some preferred embodiments, the extraction solvent used for extraction is a methanol-water solution with a methanol volume of not less than 80%. For example, the volume of methanol in the methanol-water solution can be 80%, 85%, 90%, 95% or 100%, etc., or other values ​​within the range of not less than 80%. In some preferred embodiments, the extraction solvent is a methanol-water solution with a methanol volume of 85% to 95%. In some more preferred embodiments, the extraction solvent is a methanol-water solution with a methanol volume of 90%.

[0071] In some embodiments, during the extraction process, the solid-liquid ratio of the extraction solvent to the shiitake mushroom sample to be tested is 10 mL: 1 g to 120 mL: 1 g, such as 10 mL: 1 g, 20 mL: 1 g, 40 mL: 1 g, 60 mL: 1 g, 80 mL: 1 g, 100 mL: 1 g or 120 mL: 1 g, etc., and can also be other values ​​within the range of mL: 1 g to 120 mL: 1 g. In some preferred embodiments, the solid-liquid ratio of the extraction solvent to the shiitake mushroom sample to be tested is 120 mL: 1 g.

[0072] In some embodiments, the extraction is performed by ultrasonic extraction, and the ultrasonic extraction temperature is room temperature.

[0073] In some embodiments, the ultrasonic extraction time can be 10 min to 160 min, such as 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, 140 min or 160 min, etc., or other values ​​within the range of 10 min to 160 min. In some preferred embodiments, the ultrasonic extraction time is 30 min to 60 min. In some more preferred embodiments, the ultrasonic extraction time is 60 min.

[0074] In the present invention, the shiitake mushroom extract can be firstly subjected to solid-liquid separation, and then the liquid can be collected for detection.

[0075] The solid-liquid separation may be performed by centrifugation. For example, the centrifugation may be performed at 3°C ​​to 5°C and 7000 rpm to 9000 rpm for 5 min to 10 min.

[0076] In some embodiments, the pretreated shiitake mushroom sample can be processed and tested in the following manner: accurately weigh 0.250 g (± 0.001 g) of the sample into a 50 mL centrifuge tube, add 30 mL of methanol-water solution, vortex for 2 min using a parallel vortexer, ultrasonicate for 40 min at room temperature, centrifuge at 4°C, 8000 rpm, 10 min, extract the supernatant, filter through a 0.20 μm filter membrane into a sampling vial for testing.

[0077] The features and performance of the present invention are further described in detail below in conjunction with the examples. The data were analyzed using Xcalibur software from Thermo Fisher Scientific, including qualitative and quantitative analysis.

[0078] Example 1

[0079] The present embodiment provides a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms, which comprises the following steps:

[0080] S1: Preparation of standard solution

[0081] Due to the different properties of different compounds, methanol was selected as the solvent to prepare the standard stock solution of ergosterol peroxide, ethanol was selected as the solvent to prepare the standard stock solution of ergosterol, and water was used as the solvent to prepare the standard stock solutions of shiitake purine and ergothioneine.

[0082] Weigh the required standard accurately on an analytical balance with an accuracy of 0.01 mg, transfer the standard to a 10 mL volumetric flask, dissolve the standard with the corresponding solvent and dilute to the mark on the volumetric flask, and store the resulting stock solution in a -80°C refrigerator. Based on the above standard stock solution, prepare a mixed standard intermediate solution with a concentration of 5 mg / L, and also store it in a -80°C refrigerator for subsequent use.

[0083] The mixed solution of ergothioneine, lentinan, ergosterol and ergosterol peroxide was prepared with methanol into mixed standard working solutions with mass concentrations of 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L and 5000 μg / L; each standard solution was prepared before use.

[0084] S2: Preprocessing.

[0085] About 500 g of shiitake mushroom samples were selected. Fresh shiitake mushrooms were treated with liquid nitrogen and then ground using a grinder. Dried shiitake mushrooms were directly ground using a grinder and passed through a 60-mesh sieve. The sieved samples were placed at -80°C for testing.

[0086] S3: Extraction.

[0087] Accurately weigh 0.250 g (± 0.001 g) of the mushroom powder obtained after pretreatment into a 50 mL centrifuge tube, add 30 mL of methanol aqueous solution (methanol volume percentage is 90%) at a solid-liquid ratio of 120 mL: 1 g, vortex with a parallel vortex instrument for 2 min, ultrasonicate at room temperature for 60 min, centrifuge at 4 ° C, 8000 rpm for 10 min, extract the supernatant, filter through a 0.20 μm filter membrane into a sampling vial, prepare the sample to be tested, and set aside.

[0088] S4: Liquid chromatography-triple quadrupole / linear ion trap mass spectrometer (TSQ Endura) was used for UPLC-QQQ-MS detection and analysis.

[0089] Chromatographic conditions include: chromatographic column is ACQUITY BEH C18 column (Waters Company, USA) (2.1mm×100mm, 1.7μm); column oven temperature was 35°C; mobile phase A was aqueous solution, and mobile phase B was pure methanol solution; flow rate was 0.3mL / min; elution time and volume content of mobile phase B during elution time were as follows: 0min~0.2min, 80%; 0.2min~1min, 80%~90%; 1min~4min, 90%~100%; 4min~8.5min, 100%; 8.5min~8.6min, 100%~80%; 8.6min~11min, 80%; injection volume was 5μL.

[0090] The TSQ triple quadrupole mass spectrometry conditions included: the ion source type was electrospray ionization (ESI) source; the scan mode was positive ion multiple reaction monitoring (MRM) mode; the ion spray temperature was 320°C; the ion transfer tube temperature was 325°C; the ion spray voltage was 3600V; the sheath gas was 30psi; and the auxiliary gas was 10psi. UHPLC-MS / MS ion flow chromatography Figure 1 shown.

[0091] Example 2

[0092] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0093] The chromatographic conditions include: the chromatographic column is an ACQUITY UPLC BEH HILIC column (Waters, USA) (2.1 mm×100 mm, 1.7 μm).

[0094] UHPLC-MS / MS ion current chromatography Figure 2 shown.

[0095] Example 3

[0096] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0097] Mobile phase A was aqueous solution, and mobile phase B was pure acetonitrile solution.

[0098] UHPLC-MS / MS ion current chromatography Figure 3 shown.

[0099] Example 4

[0100] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0101] Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was a methanol solution containing 0.1% formic acid.

[0102] UHPLC-MS / MS ion current chromatography Figure 4 shown.

[0103] Example 5

[0104] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0105] Mobile phase A was an aqueous solution containing 0.2% formic acid, and mobile phase B was a methanol solution containing 0.2% formic acid.

[0106] UHPLC-MS / MS ion current chromatography Figure 5 shown.

[0107] Example 6

[0108] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0109] Mobile phase A was an aqueous solution containing 0.3% formic acid, and mobile phase B was a methanol solution containing 0.3% formic acid.

[0110] UHPLC-MS / MS ion current chromatography Figure 6 shown.

[0111] Example 7

[0112] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0113] Mobile phase A was an aqueous solution containing 5 mmol / L of ammonium formate, and mobile phase B was a methanol solution containing 5 mmol / L of ammonium formate.

[0114] UHPLC-MS / MS ion current chromatography Figure 7 shown.

[0115] Example 8

[0116] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms provided in this embodiment is different from that in embodiment 1 only in that:

[0117] Mobile phase A was an aqueous solution containing 0.2% formic acid and 5 mmol / L ammonium formate, and mobile phase B was a methanol solution containing 0.2% formic acid and 5 mmol / L ammonium formate.

[0118] UHPLC-MS / MS ion current chromatography Figure 8 shown.

[0119] Test Example 1

[0120] according to Figures 1 to 8 The peak shape was finally determined, and the mobile phase A was an aqueous solution containing 0.2% formic acid, and the mobile phase B was a methanol solution containing 0.2% formic acid as the optimal conditions. Taking the 100μg / L mixed standard solution as an example, the corresponding UHPLC-MS / MS total ion chromatogram is shown in the figure. Fig. 9 shown.

[0121] Fig. 9 Four peaks of compounds appeared in the results, namely Peak 2 corresponding to ergothioneine, Peak 1 corresponding to shiitake purine, Peak 4 corresponding to ergosterol, and Peak 3 corresponding to ergosterol peroxide.

[0122] Test Example 2

[0123] Taking Example 1 as an example, the extraction solvents therein were replaced to compare the effects of different extraction solvents on the extraction results of shiitake mushrooms. Specifically, the five different extraction solvents were methanol, acetonitrile, ethanol, isopropanol and water. The results are shown in FIG. Fig.10 shown.

[0124] Depend on Fig.10 It can be seen that water has a better extraction effect on ergothioneine, shiitake purine, ergosterol, and ergosterol peroxide, and methanol has a better solubility for ergothioneine and shiitake purine, but among these five extraction solvents, methanol has the worst extraction effect on ergosterol and ergosterol peroxide. Acetonitrile has a better extraction effect on ergosterol and ergosterol peroxide, but the worst extraction effect on ergothioneine and shiitake purine.

[0125] Considering the extraction of the target compound, methanol and water were selected as the extraction agents. Different ratios of methanol and water (pure water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, 85% methanol aqueous solution, 90% methanol aqueous solution, 95% methanol aqueous solution, pure methanol) were further selected for comparison. The results are as follows: Fig.11 shown.

[0126] Depend on Fig.11 It can be seen that 90% methanol aqueous solution has the best extraction effect on ergothioneine, lentinan, and ergosterol peroxide, and has a better extraction effect on ergosterol. Therefore, 90% methanol aqueous solution was finally selected as the optimal extraction solvent.

[0127] Test Example 3

[0128] Taking Example 1 as an example, the solid-liquid ratio was changed to compare the effects of different solid-liquid ratios on the extraction effect of shiitake mushrooms. Specifically, the solid-liquid ratio (in g:mL) was 1:20, 1:40, 1:60, 1:80, 1:120, 1:160 and 1:180. The results are as follows: Fig.12 shown.

[0129] Fig.12 The results show that the volume of 90% methanol aqueous solution has a significant effect on the extraction efficiency of thioneine, shiitake purine, ergosterol and ergosterol peroxide. With the increase of solvent volume, the solid-liquid ratio shows an upward trend between 1:10 (g / mL) and 1:120 (g / mL), and the total amount of thioneine, shiitake purine, ergosterol and ergosterol peroxide extracted is improved. However, when the amount of solvent is not enough to achieve sufficient transfer compared with the solid, various balances may occur, resulting in undeniable resistance to mass transfer. At the same time, the increase in the amount of 90% methanol aqueous solution will also cause some other components such as lipids in shiitake mushrooms to be extracted, thereby affecting the extraction effect of thioneine, shiitake purine, ergosterol and ergosterol peroxide. Therefore, when the proportion of 90% methanol aqueous solution increases from 120 to 180, the extraction effect of the compound does not change significantly, and even at 180, the extraction effect of the compound shows a downward trend. Therefore, the optimal solvent solid ratio of the present invention is set to 1:120 (g / mL).

[0130] Test Example 4

[0131] Taking Example 1 as an example, the ultrasonic extraction temperature was changed to compare the effects of different ultrasonic extraction temperatures on the extraction effect of shiitake mushrooms. Specifically, the ultrasonic extraction temperature was set to an ice water bath and a room temperature water bath. The results are as follows: Fig.13 As shown, Figure 5 (b) is an enlarged view of the ergosterol peroxide extraction result in (a).

[0132] Depend on Fig.13 The results showed that the extraction effects of the four compounds at room temperature were greater than those at an ice water bath, so a room temperature water bath was used as the ultrasonic condition.

[0133] Test Example 5

[0134] Taking Example 1 as an example, the ultrasonic extraction time was changed to compare the effect of different ultrasonic extraction times on the extraction effect of shiitake mushrooms. Specifically, the ultrasonic extraction time was set to 0min, 10min, 20min, 30min, 40min, 60min, 120min and 160min. The results are as follows: Fig.14 shown.

[0135] Depend on Fig.14 The results showed that under ultrasound for 60 minutes, ergothioneine, ergosterol, ergosterol peroxide and shiitake purine could achieve better extraction effects.

[0136] Test Example 6

[0137] Taking Example 1 as an example, in order to prove the feasibility of the method, the linearity, sensitivity, accuracy and precision of the method were evaluated, and the results are summarized in Table 2.

[0138] Among the common matrix substances, this test example uses the slope method to evaluate the matrix effect. The calculation formula of the slope method is: slope = matrix standard curve slope / solvent standard curve slope. When the slope is 0.8-1.2, it means that the matrix effect has little effect on the method, and the solvent standard sample can be used for quantification; when the slope is lower than 0.8 or higher than 1.2, it means that the matrix effect has an inhibitory or enhancing effect on the method, and the matrix standard needs to be used for quantification. The relevant results of the evaluation of the matrix effect of ergothioneine, shiitake purine, ergosterol, and ergosterol peroxide in shiitake mushrooms using the slope method are shown in Table 2.

[0139] The relative standard deviation (RSD) was used to evaluate the inter-day precision and intra-day precision (the precision of the method was evaluated based on the intra-day and inter-day repeatability of low, medium and high spiked samples). The seven-point external standard method was used for accurate quantitative determination. The peak areas at concentrations of 1μg / L, 5μg / L, 50μg / L, 100μg / L, 500μg / L, 1000μg / L and 2000μg / L were plotted as standard curves to obtain the corresponding linear equations. The LODs and LOQs of the four compounds were determined when the signal-to-noise ratio (S / N) reached 3 and 10, respectively.

[0140] The accuracy of the method was evaluated by recovery studies at three spike concentrations (low, medium and high). In the recovery evaluation process, three spike levels of recovery were designed, L (low), M (medium) and H (high), and the added compound content was approximately 50%, 100% and 200% of the original content of shiitake purine (compound A), ergothioneine (compound B), peroxyergosterol (compound C) and ergosterol (compound D) in shiitake mushrooms. The linearity, sensitivity, accuracy and precision of the method for the four compounds are shown in Table 2.

[0141] Table 2 Statistical table of linearity, sensitivity, accuracy and precision results of the method

[0142]

[0143] As can be seen from Table 2, ergothioneine, ergosterol, ergosterol peroxide and shiitake purine all showed good linear relationships, with correlation coefficients (r 2 ) is greater than 0.9968. LOD and LOQ are between 2μg / L and 50μg / L (on a dry weight basis), respectively, which can meet the detection requirements. At all spiking levels, the intra-day and inter-day precisions expressed as RSD are less than 20%. The matrix effects of all compounds are between 0.8 and 0.9, and the matrix effects are weak, so solvent standards can be used for direct quantification.

[0144] Therefore, the detection method provided by the present invention shows good accuracy and precision, indicating that the method is reliable and suitable for the qualitative and quantitative detection of ergothioneine, shiitake purine, ergosterol peroxide, and ergosterol compounds in shiitake mushroom samples.

[0145] In summary, the present invention adopts Therom TSQ Endura ultra high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS / MS) to successfully establish a qualitative and quantitative method for simultaneously analyzing four kinds of non-volatile compounds (ergothioneine, shiitake purine, ergosterol peroxide, ergosterol) in shiitake samples. The method provides good sensitivity, good matrix effect, good method accuracy and precision, which shows that the method established is reliable, and is suitable for the qualitative and quantitative detection of ergothioneine, shiitake purine, ergosterol peroxide, and ergosterol compounds in shiitake samples.

[0146] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and purine in shiitake mushrooms, characterized in that, The following steps are involved: The shiitake mushroom extract was tested by ultra-high performance liquid chromatography-tandem mass spectrometry under the following conditions: The chromatographic conditions include: using a HILIC or C18 chromatographic column; the mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing formic acid, and the mobile phase B is a methanol or acetonitrile solution containing formic acid; the elution time and the volume content of the mobile phase B during the elution time are as follows: 0min to 0.2min, 80%; 0.2min to 1min, 80% to 90%; 1min to 4min, 90% to 100%; 4min to 8.5min, 100%; 8.5min to 8.6min, 100% to 80%; 8.6min to 11min, 80%; The mass spectrometry conditions include: the ion source type is an electrospray ionization source; the scanning mode is a positive ion multiple reaction monitoring mode; the ion spray temperature is 315°C to 325°C; the ion transfer tube temperature is 320°C to 330°C; the ion spray voltage is 3500V to 3700V; the sheath gas is 25psi to 35psi; the auxiliary gas is 5psi to 15psi; and the tail gas is 0psi.

2. The method according to claim 1, characterized in that: The chromatographic column is ACQUITY UPLC BEH HILIC column or BEH C18 column; Preferably, using BEH C18 was used as the chromatographic column, and the column specifications were 2.1 mm × 100 mm, 1.7 μm.

3. The method according to claim 1, characterized in that The volume percentage of formic acid contained in mobile phase A and mobile phase B is 0.1% to 0.3% respectively; Preferably, the volume percentage of formic acid contained in mobile phase A and mobile phase B is 0.2%.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.2 mL / min to 0.4 mL / min; Preferably, the flow rate of the mobile phase is 0.3 mL / min.

5. The method according to claim 1, characterized in that The injection volume of the extract is 4 μL to 6 μL, and / or the temperature of the column oven is 34° C. to 36° C.; Preferably, the injection volume of the extract is 5 μL; and / or the temperature of the column oven is 35° C.

6. The method according to claim 1, characterized in that The ion spray temperature was 320°C; the ion transfer tube temperature was 325°C; the ion spray voltage was 3600V; the sheath gas was 30psi; the auxiliary gas was 10psi; and the tail gas was 0psi.

7. The method according to any one of claims 1 to 6, characterized in that: The preparation of the shiitake mushroom extract comprises: cutting the fresh shiitake mushroom sample to be tested into pieces, soaking it in liquid nitrogen, homogenizing it, and then adding an extraction solvent to extract it to obtain an extract; or, grinding and sieving the dried shiitake mushroom sample to be tested, and then adding an extraction solvent to extract it to obtain an extract.

8. The method according to claim 7, characterized in that The extraction solvent used for the extraction includes at least one of methanol, acetonitrile, isopropanol and water; Preferably, the extraction solvent used for the extraction is a methanol-water solution with a methanol volume of not less than 80%; Preferably, the extraction solvent is a methanol-water solution with a volume of 85% to 95% methanol; More preferably, the extraction solvent is a methanol-water solution with a methanol volume ratio of 90%.

9. The method according to claim 8, characterized in that During the extraction process, the solid-liquid ratio of the extraction solvent to the tested shiitake mushroom sample is 10 mL:1 g to 120 mL:1 g; Preferably, the solid-liquid ratio is 120mL:1g.

10. The method according to claim 7, characterized in that The extraction was carried out by ultrasonic extraction; Preferably, the ultrasonic extraction temperature is room temperature; and / or, the ultrasonic extraction time is 10 min to 160 min; Preferably, the ultrasonic extraction time is 30 min to 60 min; More preferably, the ultrasonic extraction time is 60 min.

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