A method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes

Abstract

The present invention discloses a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes, belonging to the technical field of determination of active ingredients in Lentinula edodes. The method includes: detecting the extract of Lentinula edodes by ultra-high performance liquid chromatography-tandem mass spectrometry: the chromatographic column is an HILIC or C18 chromatographic column; mobile phase A and mobile phase B are an aqueous solution containing formic acid and a methanol or acetonitrile solution containing formic acid, respectively; the mass spectrometry conditions include: an electrospray ionization source; 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. This method has the characteristics of high sensitivity, high selectivity and high accuracy, and is applicable to the quantitative detection of ergothioneine, eritadenine, ergosterol peroxide and ergosterol compounds in Lentinula edodes.

Description

Technical Field

[0001] The present invention relates to the technical field of determination of active ingredients in Lentinula edodes, and more particularly to a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes. Background Art

[0002] Lentinula edodes is a widely cultivated edible and medicinal fungus, containing rich nutritional components and bioactive compounds. In recent years, with the in-depth research on functional foods, ergothioneine, eritadenine, ergosterol and ergosterol peroxide, as important active ingredients of Lentinula edodes, have received increasing attention.

[0003] Among them, ergothioneine is a natural amino acid derivative derived from fungi and cyanobacteria, with powerful antioxidant functions, and has been proven in various studies to be able to resist oxidative stress and protect cells from damage. Eritadenine is an alkaloid, first extracted from Lentinula edodes, and Lentinula edodes is also considered to be the main source of eritadenine. Eritadenine can reduce cholesterol synthesis by inhibiting S-adenosyl-L-homocysteine hydrolase (SAH hydrolase), which helps to reduce the cholesterol level in serum. Ergosterol is a sterol compound with a relatively high content in Lentinula edodes, which is a precursor of vitamin D2, widely present in the fungal cell membrane, and has antioxidant, anti-tumor and immunomodulatory effects. Ergosterol peroxide has attracted extensive 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 eritadenine in Lentinula edodes.

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

[0006] The purpose of the present invention is to provide a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes to solve or improve the above technical problems.

[0007] The present invention can be implemented as follows:

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

[0009] Detecting the Lentinula edodes extract by ultra-high performance liquid chromatography-tandem mass spectrometry method under the following conditions:

[0010] The chromatographic conditions include: using an HILIC or C18 chromatographic column; the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is an aqueous solution containing formic acid, and mobile phase B is a methanol or acetonitrile solution containing formic acid; the elution time and the volume content of mobile phase B within the elution time are as follows: 0 min to 0.2 min, 80%; 0.2 min to 1 min, 80% to 90%; 1 min to 4 min, 90% to 100%; 4 min to 8.5 min, 100%; 8.5 min to 8.6 min, 100% to 80%; 8.6 min to 11 min, 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 3500 V to 3700 V; the sheath gas is 25 psi to 35 psi; the auxiliary gas is 5 psi to 15 psi; the tail gas is 0 psi.

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

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

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

[0015] In an alternative embodiment, the volume percentages of formic acid contained in mobile phase A and mobile phase B are respectively 0.2%.

[0016] In an alternative 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 alternative 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 alternative embodiment, the injection volume of the extract is 5 μL, and / or the temperature of the column oven is 35 °C.

[0020] In an alternative 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 exhaust gas is 0 psi.

[0021] In an alternative embodiment, the preparation of the Lentinula edodes extract includes: cutting the fresh Lentinula edodes sample to be tested, soaking it in liquid nitrogen, and homogenizing it, then adding an extraction solvent for extraction to obtain an extract; or, grinding the dry Lentinula edodes sample to be tested, sieving it, and then adding an extraction solvent for extraction to obtain an extract.

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

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

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

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

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

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

[0028] In an alternative embodiment, ultrasonic extraction is used for extraction.

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

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

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

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

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

[0034] The simultaneous detection and analysis method for ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes provided by the embodiments of the present invention uses an ultra-high performance liquid chromatography-tandem mass spectrometry method to detect four active compounds in Lentinula edodes. By optimizing the pretreatment conditions of Lentinula edodes and the instrument parameters of ultra-high performance liquid chromatography-tandem mass spectrometry, the target compounds can be efficiently extracted and accurately detected with high selectivity and high sensitivity. Description of the Drawings

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

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

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

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

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

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

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

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

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

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

[0045] Figure 10Analysis results of the contents of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes with different extraction solvents in Test Example 2;

[0046] Figure 11 Analysis results of the contents of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes with different ratios of methanol to water in Test Example 2;

[0047] Figure 12 Analysis results of the contents of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes with different solid-to-liquid ratios in Test Example 3;

[0048] Figure 13 Analysis results of the contents of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes with different ultrasonic extraction temperatures in Test Example 4;

[0049] Figure 14 Analysis results of the contents of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes with different ultrasonic extraction times in Test Example 5. Detailed implementation manners

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide, and eritadenine in Lentinula edodes provided by the present invention will be described below.

[0052] The present invention provides a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide, and eritadenine in Lentinula edodes, and ultra-high performance liquid chromatography-tandem mass spectrometry is used to detect ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes.

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

[0054] Before detection, the instrument conditions can be optimized first. In the present invention, a scanning method is first created to measure the standards of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide, and the precursor ions are identified. Secondly, the product ions are determined and the collision energy is optimized. Finally, the MRM method is established. In order to obtain a suitable separation effect for the target compounds, the liquid chromatography parameters are further optimized.

[0055] The retention times of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide 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, eritadenine, ergosterol, and ergosterol peroxide in shiitake mushrooms

[0057]

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

[0059] In some embodiments, the volume percentages of formic acid contained in mobile phase A and mobile phase B can be 0.1% - 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% - 0.3%. In some preferred embodiments, the volume percentages of formic acid contained in mobile phase A and mobile phase B are both 0.2%.

[0060] By using the acetonitrile - water system as the mobile phase for comparison, ergothioneine, ergosterol, ergosterol peroxide, and eritadenine to be detected in the present invention can all elute peaks in both the acetonitrile - water system and the methanol - water system. However, they show a higher response in the methanol - water system. Therefore, the methanol - water system is used as the mobile phase in the present invention to obtain a more accurate detection effect.

[0061] Generally, in chromatographic detection, the sensitivity of the analyte can be increased and the chromatographic peak shape can be improved by adding an acid or a salt to the mobile phase. In this invention, four different water - methanol systems are used for comparison. One is adding 0.2% (v / v) formic acid to the water - methanol system, the second is adding 5 mmol / L ammonium formate to the water - methanol system, the third is adding 0.2% (v / v) formic acid and 5 mmol / L ammonium formate to the water - methanol system, and the fourth is adding no acid or salt to the water - methanol system. Through 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 0.2 mL / min to 0.4 mL / min, such as 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min or 0.4 mL / min, etc., or other values within the range of 0.2 mL / min to 0.4 mL / min. In some preferred embodiments, the flow rate of the mobile phase is 0.3 mL / 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 temperature of the column oven can be 34°C to 36°C, such as 34°C, 34.5°C, 35°C, 35.5°C or 36°C, etc., or other values within the range of 34°C to 36°C. In some preferred embodiments, the temperature of the column oven is 35°C.

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

[0066] The total running time for simultaneously determining four non-volatile compounds by the above method is 11 minutes, and the elution order is ergothioneine, eritadenine, ergosterol peroxide, and ergosterol in sequence. 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 25 psi to 35 psi (such as 25 psi, 28 psi, 30 psi, 32 psi, or 35 psi, etc.); the auxiliary gas is 5 psi to 15 psi (such as 5 psi, 8 psi, 10 psi, 12 psi, or 15 psi, etc.); the exhaust gas is 0 psi.

[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 3600V; the sheath gas is 30 psi; the auxiliary gas is 10 psi; the exhaust gas is 0 psi.

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

[0069] In the present invention, the preparation of the Lentinus edodes extract includes: cutting the fresh Lentinus edodes samples to be tested, soaking them in liquid nitrogen, and homogenizing them, then adding an extraction solvent for extraction to obtain an extract; or, grinding the dry Lentinus edodes samples to be tested and sieving them (such as sieving through a 60-mesh sieve), then adding an extraction solvent for extraction 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 more preferred embodiments, the extraction solvent is a methanol-water solution with a methanol volume of 85% - 95%. In some even 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 Lentinus edodes samples 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., or other values within the range of mL:1 g to 120 mL:1 g. In some more preferred embodiments, the solid-liquid ratio of the extraction solvent to the Lentinus edodes samples to be tested is 120 mL:1 g.

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

[0073] In some embodiments, the ultrasonic extraction time can be 10 min - 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 - 160 min. In some more preferred embodiments, the ultrasonic extraction time is 30 min - 60 min. In some even more preferred embodiments, the ultrasonic extraction time is 60 min.

[0074] In the present invention, the Lentinus edodes extract can be first subjected to solid-liquid separation, and then the liquid is collected for detection.

[0075] Among them, the solid-liquid separation can be carried out by centrifugation. Exemplarily, the centrifugation can be carried out at 3°C - 5°C and 7000 rpm - 9000 rpm for 5 min - 10 min.

[0076] In some embodiments, the pretreated shiitake mushroom samples can be processed and then detected as follows: Accurately weigh 0.250 g (±0.001 g) of the sample into a 50 mL centrifuge tube, add 30 mL of methanol-aqueous solution, vortex for 2 min using a parallel vortex mixer, ultrasonicate for 40 min at room temperature, centrifuge at 4 °C, 8000 rmp for 10 min, extract the supernatant, filter through a 0.20 μm filter membrane into a vial for detection.

[0077] The features and properties of the present invention will be further described in detail below in conjunction with the examples. The data was analyzed using Thermo Fisher Scientific's Xcalibur software, including qualitative and quantitative analysis.

[0078] Example 1

[0079] This example provides a method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide, and eritadenine in shiitake mushrooms, which includes the following steps:

[0080] S1: Preparation of standard solutions

[0081] Due to the different characteristics 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 eritadenine and ergothioneine.

[0082] Accurately weigh the required standard substances on an analytical balance with a precision of 0.01 mg, transfer the standard substances to a 10 mL volumetric flask, dissolve the standard substances using the corresponding solvent and make up to the calibration line of the volumetric flask. The resulting stock solutions are stored in a -80 °C refrigerator. Based on the above standard stock solutions, a mixed standard intermediate solution with a concentration of 5 mg / L is prepared and also stored in a -80 °C refrigerator for subsequent use.

[0083] A mixed standard working solution 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 was prepared by mixing ergothioneine, eritadenine, ergosterol, and ergosterol peroxide solutions with methanol; each standard solution was prepared immediately before use.

[0084] S2: Pretreatment.

[0085] Select approximately 500 g of shiitake mushroom samples. For fresh shiitake mushrooms, after liquid nitrogen treatment, use a grinder to grind them. For dried shiitake mushrooms, directly use a grinder to grind them, pass through a 60-mesh sieve, and place the sieved samples at -80 °C for further testing.

[0086] S3: Extraction.

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

[0088] S4: Perform UPLC-QQQ-MS detection and analysis using a liquid chromatography-triple quadrupole / linear ion trap mass spectrometer (TSQ Endura).

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

[0090] The TSQ triple quadrupole mass spectrometry conditions include: the ion source type is an electrospray ionization (ESI) source; the scanning mode is a positive ion multiple reaction monitoring (MRM) mode; 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. The UHPLC-MS / MS ion current chromatogram is as Figure 1 shown.

[0091] Example 2

[0092] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and lentinan in lentinula edodes provided in this example is only different from that in Example 1 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] The UHPLC-MS / MS ion current chromatogram is as Figure 2 shown.

[0095] Example 3

[0096] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinus edodes provided in this example is only different from that in Example 1 in that:

[0097] Mobile phase A is an aqueous solution, and mobile phase B is a pure acetonitrile solution.

[0098] The UHPLC-MS / MS ion current chromatogram is as Figure 3 shown.

[0099] Example 4

[0100] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinus edodes provided in this example is only different from that in Example 1 in that:

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

[0102] The UHPLC-MS / MS ion current chromatogram is as Figure 4 shown.

[0103] Example 5

[0104] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinus edodes provided in this example is only different from that in Example 1 in that:

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

[0106] The UHPLC-MS / MS ion current chromatogram is as Figure 5 shown.

[0107] Example 6

[0108] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinus edodes provided in this example is only different from that in Example 1 in that:

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

[0110] The UHPLC-MS / MS ion current chromatogram is as Figure 6 shown.

[0111] Example 7

[0112] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinus edodes provided in this example is only different from that in Example 1 in that:

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

[0114] The UHPLC-MS / MS ion chromatogram is as Figure 7 shown.

[0115] Example 8

[0116] The method for simultaneously detecting ergothioneine, ergosterol, ergosterol peroxide and eritadenine in Lentinula edodes provided in this example is only different from that in Example 1 in that:

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

[0118] The UHPLC-MS / MS ion chromatogram is as Figure 8 shown.

[0119] Test Example 1

[0120] According to Figures 1 to 8 the peak shape, it is finally determined that mobile phase A being an aqueous solution containing 0.2% formic acid and mobile phase B being a methanol solution of 0.2% formic acid are the optimal conditions. Taking the mixed standard solution of 100 μg / L as an example, its corresponding UHPLC-MS / MS total ion chromatogram is as Figure 9 shown.

[0121] Figure 9 Peaks of four compounds appeared in , namely peak 2 corresponding to ergothioneine, peak 1 corresponding to eritadenine, 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 solvent was replaced to compare the influence of different extraction solvents on the extraction results of Lentinula edodes. Specifically, 5 different extraction solvents were methanol, acetonitrile, ethanol, isopropanol and water, and the results are as Figure 10 shown.

[0124] From Figure 10 it can be seen that water has a better extraction effect on ergothioneine, eritadenine, ergosterol and ergosterol peroxide. Methanol has a better solubility for ergothioneine and eritadenine, but among these 5 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 has the worst extraction effect on ergothioneine and eritadenine.

[0125] Considering the extraction of the target compound, two extractants, methanol and water, were selected for extraction. Further, 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 selected for comparison. The results are as Figure 11 shown.

[0126] As Figure 11 can be seen, the 90% methanol aqueous solution has the best extraction effect on ergothioneine, eritadenine, and ergosterol peroxide, and a good extraction effect on ergosterol. Therefore, the 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 influence of different solid-liquid ratios on the extraction effect of Lentinus edodes. Specifically, the solid-liquid ratios (unit: g:mL) were 1:20, 1:40, 1:60, 1:80, 1:120, 1:160, and 1:180. The results are as Figure 12 shown.

[0129] Figure 12 The results show that the volume of the 90% methanol aqueous solution has a significant impact on the extraction efficiency of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide. As the volume of the solvent increases, the solid-liquid ratio shows an upward trend between 1:10 (g / mL) and 1:120 (g / mL), and the total extraction amount of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide is improved. However, when the amount of the solvent is not sufficient compared to the solid to achieve sufficient transfer, various equilibria may occur, resulting in an undeniable resistance to mass transfer. At the same time, the increase in the amount of the 90% methanol aqueous solution will also extract some other components such as lipids in Lentinus edodes, thus affecting the extraction effect of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide. Therefore, when the proportion of the 90% methanol aqueous solution increases from 120 to 180, the extraction effect of the compound does not change significantly. 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 influence of different ultrasonic extraction temperatures on the extraction effect of Lentinus edodes. Specifically, the ultrasonic extraction temperatures were set to ice-water bath and normal-temperature water bath. The results are as Figure 13 shown, Figure 5 where (b) in it is an enlarged view of the extraction result of ergosterol peroxide in (a).

[0132] The Figure 13 results show that the extraction effects of the four compounds at room temperature are greater than those in an ice-water bath. Therefore, a constant-temperature water bath is 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 effects of different ultrasonic extraction times on the extraction effect of Lentinula edodes. Specifically, the ultrasonic extraction times were set to 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, 120 min, and 160 min, and the results are as Figure 14 shown.

[0135] The Figure 14 results show that under ultrasonic treatment for 60 min, optimal extraction effects of ergothioneine, ergosterol, ergosterol peroxide, and eritadenine can be obtained.

[0136] Test Example 6

[0137] Taking Example 1 as an example, 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 common matrix substances, in this test example, the matrix effect was evaluated by the slope method. The calculation formula of the slope method is: slope = slope of the matrix standard curve / slope of the solvent standard curve. When the slope is between 0.8 and 1.2, it indicates that the matrix effect has little influence on the method, and solvent standard samples can be used for quantification; when the slope is lower than 0.8 or higher than 1.2, it indicates that the matrix effect has an inhibitory or enhancing effect on the method, and at this time, matrix standard samples need to be used for quantification. The relevant results of evaluating the matrix effect of ergothioneine, eritadenine, ergosterol, and ergosterol peroxide in Lentinula edodes by 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 according to 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 1 μg / L, 5 μg / L, 50 μg / L, 100 μg / L, 500 μg / L, 1000 μg / L, and 2000 μg / L were plotted to obtain the corresponding linear equation. When the signal-to-noise ratio (S / N) reached 3 and 10 respectively, the LODs and LOQs of the 4 compounds were determined.

[0140] The accuracy of the method was evaluated by the recovery rate study at three spiking concentrations (low, medium, and high). During the evaluation of the recovery rate, three spiking levels of recovery rate, namely L (low), M (medium), and H (high), were designed, and the added compound contents were approximately 50%, 100%, and 200% of the original contents of ergothioneine (compound B), ergosterol peroxide (compound C), and ergosterol (compound D) in Lentinula edodes. The linearity, sensitivity, accuracy, and precision of the method for the four compounds are shown in Table 2.

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

[0142]

[0143] As can be seen from Table 2, ergothioneine, ergosterol, ergosterol peroxide, and eritadenine all showed good linear relationships, and the correlation coefficient (r 2 ) was greater than 0.9968. The LOD and LOQ were between 2 μg / L and 50 μg / L (dry weight), which could meet the detection requirements. At all spiking levels, the intra-day and inter-day precisions expressed as RSD were both less than 20%. The matrix effects of all compounds were between 0.8 and 0.9, and the matrix effects were weak. Therefore, solvent standards could be directly used for quantification.

[0144] Therefore, the detection method provided by the present invention showed good accuracy and precision, indicating that the method was reliable and suitable for the qualitative and quantitative detection of ergothioneine, eritadenine, ergosterol peroxide, and ergosterol compounds in Lentinula edodes samples.

[0145] In summary, the present invention successfully established a qualitative and quantitative method for simultaneously analyzing four non-volatile compounds (ergothioneine, eritadenine, ergosterol peroxide, and ergosterol) in Lentinula edodes samples by using a Thermo TSQ Endura ultra-high performance liquid chromatography-tandem mass spectrometer (UHPLC-MS / MS). The method provided good sensitivity, good matrix effects, good method accuracy and precision, indicating that the established method was reliable and suitable for the qualitative and quantitative detection of ergothioneine, eritadenine, ergosterol peroxide, and ergosterol compounds in Lentinula edodes samples.

[0146] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within 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~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%; 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~325°C; the ion transfer tube temperature is 320°C~330°C; the ion spray voltage is 3500V~3700V; the sheath gas is 25psi~35psi; the auxiliary gas is 5psi~15psi; the tail gas is 0psi; The volume percentage of formic acid contained in mobile phase A and mobile phase B is 0.1%~0.3% respectively; 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; The extraction solvent used for the extraction is a methanol-water solution with a methanol volume of not less than 80%.

2. The method according to claim 1, characterized in that The chromatographic column is ACQUITY UPLC BEH HILIC column or ACQUITY UPLC ® BEHC18 column.

3. The method according to claim 2, characterized in that ACQUITY UPLC ® BEHC18 was used as the chromatographic column, and the column specifications were 2.1 mm × 100 mm, 1.7 μm.

4. 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 0.2%.

5. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.2mL / min~0.4mL / min.

6. The method according to claim 5, characterized in that The flow rate of the mobile phase was 0.3 mL / min.

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

8. The method according to claim 7, characterized in that The injection volume of the extract is 5 μL; and / or the temperature of the column oven is 35° C.

9. 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.

10. The method according to claim 1, characterized in that The extraction solvent is a methanol-water solution with a methanol volume ratio of 85% to 95%.

11. The method according to claim 10, characterized in that The extraction solvent was a methanol-water solution with a volume ratio of 90%.

12. The method according to claim 1, 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.

13. The method according to claim 12, characterized in that The solid-liquid ratio is 120mL:1g.

14. The method according to claim 1, characterized in that The extraction was carried out by ultrasonic extraction.

15. The method according to claim 14, characterized in that The ultrasonic extraction temperature was room temperature; And / or, the ultrasonic extraction time is 10 min to 160 min.

16. The method according to claim 15, characterized in that The ultrasonic extraction time is 30min~60min.

17. The method according to claim 16, characterized in that The ultrasonic extraction time was 60 min.

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