Method and kit for rapidly identifying russula vinosa

Abstract

The invention relates to a method and a kit for rapidly identifying russula vinosa, and belongs to the technical field of detection methods. A fresh mushroom sample is prepared into a mushroom extracting solution, and the mushroom extracting solution is subjected to vortex, drying and centrifugation to obtain supernate; adding an N-methyl phenylethylamine solution, a triphenylphosphine solution and a 2, 2 '-dithiobipyridine solution into the supernate, and carrying out derivatization reaction to obtain a primary derivatization sample solution; a magnesium monoperoxyphthalate hexahydrate solution is added for a derivatization reaction, and then a NaHCO3 solution is added for a secondary derivatization reaction; and performing LC-MS / MS analysis on the primary derivatization sample solution and the secondary derivatization sample solution to identify whether a derivatization product of cyclopropyl-2-alkene carboxylic acid exists or not. The method is short in required time, mild in condition and good in specificity.

Description

Technical Field

[0001] The invention relates to a method and a kit for rapidly identifying Russula subspecies, belonging to the technical field of detection methods. Background Art

[0002] Currently, identification methods for Russula subsparsely glabrous are primarily morphological and molecular. Morphological identification relies on morphological characteristics, observing the shape and color of the fruiting body's cap, stipe, and gills. This method relies heavily on subjective judgment, carries significant uncertainty, and requires extensive experience and expertise, making accurate identification difficult for the general public, especially after mushrooms have been processed for consumption. Only a few molecular methods have been reported for laboratory identification of Russula subsparsely glabrous. For example, PCR and loop-mediated isothermal amplification (LAMP) have been used for molecular identification of Russula subsparsely glabrous. Molecular techniques are widely used in species identification research due to their specificity, accuracy, and sensitivity. Furthermore, DNA, as the genetic material of life, is highly stable and can be detected in various tissues and developmental stages of an organism. However, these methods often require specialized personnel, are time-consuming, and costly, making them unsuitable for initial screening of large numbers of samples. Therefore, there is an urgent need to develop a rapid, accurate, and specific LC-MS / MS method for the identification of Russula subsparsely glabrous. Summary of the Invention

[0003] In light of this, the present invention aims to provide a method and kit for the rapid identification of Russula subsparsely edging. By analyzing the molecular structural characteristics of cycloprop-2-enecarboxylic acid, the primary toxic factor in Russula subsparsely edging, and targeting its active functional groups (carboxyl group and carbon-carbon double bond), a derivatization strategy was developed. Through a two-step derivatization reaction, accurate identification of Russula subsparsely edging was achieved. This method is simple to operate, requires minimal time, and requires minimal operator intervention. It exhibits high specificity and accuracy, and can be applied to the rapid identification of large numbers of samples.

[0004] To achieve the above objectives, the technical solutions of the present invention are as follows.

[0005] A method for rapidly identifying Russula subsparsely gilled, comprising the following steps:

[0006] S1: Weigh fresh mushroom samples and prepare mushroom extract;

[0007] S2: adding acetonitrile to the mushroom extract, vortexing to mix, adding excess anhydrous sodium sulfate to dry, and centrifuging to obtain the supernatant;

[0008] S3: taking the supernatant and adding N-methylphenylethylamine solution, triphenylphosphine solution and 2,2'-disulfide dipyridine solution to carry out a derivatization reaction to obtain a primary derivatization sample solution;

[0009] S4: Take a portion of the primary derivatization sample solution and add magnesium monoperoxyphthalate hexahydrate solution to carry out a derivatization reaction, then add NaHCO3 solution and vortex; then add ethyl acetate for extraction, vortex, centrifuge, and collect the upper layer; concentrate to dryness in a centrifugal concentrator, and reconstitute with acetonitrile aqueous solution to obtain a secondary derivatization sample solution;

[0010] S5: The remaining primary derivatization sample solution and the secondary derivatization sample solution are diluted with acetonitrile-water solution, respectively, and subjected to LC-MS / MS analysis to identify whether there is a derivatization product of cycloprop-2-enecarboxylic acid. If so, the mushroom sample is determined to be Russula subspecies.

[0011] Furthermore, in step S1, a fresh mushroom sample is weighed, homogenized with water, extracted by shaking, and centrifuged to obtain a supernatant. Water is then added, vortexed to mix, and centrifuged to obtain a supernatant. The two supernatants are combined and concentrated by centrifugation to obtain a mushroom extract.

[0012] Furthermore, in step S2, the volume ratio of the mushroom extract to acetonitrile is 100:1000-5000.

[0013] Furthermore, in step S3, the solvent of the N-methylphenethylamine solution is acetonitrile, and the concentration is 0.01-50 mg / mL;

[0014] The solvent of triphenylphosphine solution is acetonitrile, and the concentration is 0.01-50 mg / mL;

[0015] The solvent of the 2,2'-disulfide dipyridine solution is acetonitrile, and the concentration is 0.01-50 mg / mL.

[0016] Furthermore, in step S3, the volume ratio of the supernatant, N-methylphenethylamine solution, triphenylphosphine solution and 2,2'-disulfide dipyridine solution is 100:10-100:10-100:10-100.

[0017] Furthermore, in step S3, the derivatization reaction temperature is 30-50° C., and the reaction time is 5-60 min.

[0018] Furthermore, in step S4, the solvent of the magnesium monoperoxyphthalate hexahydrate solution is water, and the concentration is 50-173 mg / mL;

[0019] The solvent of the NaHCO3 solution is water, and the concentration is 40-84 mg / mL.

[0020] Furthermore, in step S4, the volume ratio of the primary derivatization sample solution, the magnesium monoperoxyphthalate hexahydrate solution and the NaHCO3 solution is 100:50-200:100.

[0021] Furthermore, in step S4, the derivatization reaction temperature is 30-40° C., and the reaction time is 5-30 min.

[0022] Furthermore, in step S5, the chromatographic conditions for LC-MS / MS analysis are as follows:

[0023] Chromatographic column: Phenomenex Luna Omega C18 column, 50×2.1 mm, 1.6 μm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; column temperature: 40°C; injection volume: 5 μL; mobile phase gradient:

[0024]

[0025]

[0026] Furthermore, in step S5, the mass spectrometry conditions for LC-MS / MS analysis are as follows:

[0027] Positive ion mode, scanning mode was full scan-auto-trigger secondary mass spectrometry scan (Full MS / dd-MS2), spray voltage was 3.2 kV, ion transmission temperature was 320 ° C, nebulization temperature was 320 ° C, sheath gas flow rate was 35 arb, auxiliary gas flow rate was 10 arb, primary resolution was 35000 FWHM, secondary mass spectrometry resolution was 17500 FWHM, scanning range was m / z 50-1000 Da, normalized collision energy (NCE) was 20, 35 and 45 eV.

[0028] A kit for rapidly identifying Russula subspecies, comprising an N-methylphenylethylamine solution, a triphenylphosphine solution, a 2,2'-disulfide dipyridine solution and a magnesium monoperoxyphthalate hexahydrate solution.

[0029] Beneficial effects

[0030] Cycloprop-2-enecarboxylic acid is the main toxic factor in Russula subsparsely covered, and its detection is of great significance in preventing poisoning of Russula subsparsely covered. The present invention analyzes the molecular structure characteristics of cycloprop-2-enecarboxylic acid and constructs a two-step derivatization strategy for the possible active functional groups (carboxyl group, carbon-carbon double bond) of cycloprop-2-enecarboxylic acid. In the absence of a cycloprop-2-enecarboxylic acid standard, cycloprop-2-enecarboxylic acid can be accurately identified using a high-resolution mass spectrometer. The derivatization method can not only increase the characteristic diagnostic ions and help identify cycloprop-2-enecarboxylic acid, but also greatly improve the sensitivity of mass spectrometry detection, effectively solving the detection challenge caused by the low response of this type of compound in the mass spectrum. The identification method provided by the present invention improves the detection sensitivity, making it possible to detect cycloprop-2-enecarboxylic acid under a wider range of experimental conditions.

[0031] The kit and identification method of the present invention are suitable for identifying Russula subspecies in samples of mushrooms and processed products, providing technical support for emergency response to foodborne mushroom poisoning. The detection method requires a short time, uses mild conditions, has good specificity, and is simple to operate, making it easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the mass spectrometry result of the primary derivatization product of cycloprop-2-enecarboxylic acid in Example 1.

[0033] Figure 2 This is the mass spectrometry result of the secondary derivatization product of cycloprop-2-enecarboxylic acid in Example 1.

[0034] Figure 3 This is the mass spectrometric response result of the primary derivatization product of cycloprop-2-enecarboxylic acid in Example 2.

[0035] Figure 4 This is the mass spectrometric response result of the primary derivatization product of cycloprop-2-enecarboxylic acid in Example 3.

[0036] Figure 5 This is the mass spectrometric response result of the primary derivatization product of cycloprop-2-enecarboxylic acid in Example 4.

[0037] Figure 6 This is the mass spectrometry response result of the secondary derivatization product of cycloprop-2-enecarboxylic acid in Example 5. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below with reference to specific embodiments.

[0039] All materials, reagents, and instruments in the following examples, unless otherwise specified, can be purchased from commercial channels.

[0040] Prepare the following reagents: N-methylphenethylamine solution (N-methylphenethylamine dissolved in acetonitrile); triphenylphosphine solution (triphenylphosphine dissolved in acetonitrile); 2,2'-disulfide pyridine solution (2,2'-disulfide pyridine dissolved in acetonitrile); magnesium monoperoxyphthalate hexahydrate solution (the dissolving reagent for magnesium monoperoxyphthalate hexahydrate is water).

[0041] Instruments used: Dionex UltiMate 3000 liquid chromatograph and QExactive mass spectrometer (Thermo Fisher Scientific, USA); 3-18KS low-temperature centrifuge (Sigma, USA); Advantage A10 Milli-Q purified water system (Millipore, USA).

[0042] Example 1

[0043] (1) Sample pretreatment

[0044] Weigh a fresh mushroom sample, add ultrapure water, homogenize, vortex, and centrifuge to obtain the supernatant. Repeat the above steps, combine the two extraction supernatants, and concentrate to 1 g / mL by centrifugation. Take 20 μL of the 1 g / mL mushroom extract and add 100 μL of acetonitrile, vortex to mix, add excess anhydrous sodium sulfate to dry, and centrifuge to obtain the supernatant.

[0045] (2) Derivatization reaction

[0046] Take 20 μL of supernatant, add 100 μL of acetonitrile, and 10 μL each of derivatization reagents N-methylphenylethylamine solution, triphenylphosphine solution, and 2,2'-disulfide dipyridine solution (concentrations are 0.5 mg / mL, 1 mg / mL, and 1 mg / mL, respectively), vortex mix, and react at 40°C for 15 min to obtain the primary derivatization sample solution.

[0047] Take 20 μL of the primary derivatization sample solution, add 40 μL of 500 mM monoperoxyphthalic acid solution and react at 30°C for 5 minutes to obtain a secondary derivatization sample solution.

[0048] (3) LC-MS / MS detection

[0049] The obtained primary derivatization sample solution and secondary derivatization sample solution were diluted with 60% acetonitrile water (v:v) to obtain test sample solutions 1 and 2. The test sample solutions were transferred to injection vials for LC-MS / MS analysis to identify the primary derivatization product and secondary derivatization product of cycloprop-2-enecarboxylic acid.

[0050] The chromatographic conditions are as follows:

[0051] Chromatographic column: Phenomenex Luna Omega C18 column (50×2.1mm, 1.6μm)

[0052] Mobile phase A: 0.1% formic acid aqueous solution;

[0053] Mobile phase B: acetonitrile;

[0054] Column temperature: 40°C;

[0055] Injection volume: 5 μL;

[0056] The mobile phase gradient was as follows:

[0057]

[0058]

[0059] Mass spectrometry conditions are as follows:

[0060] Positive ion mode, scanning mode was full scan-auto-trigger secondary mass spectrometry scan (Full MS / dd-MS2), spray voltage was 3.2 kV, ion transmission temperature was 320 ° C, nebulization temperature was 320 ° C, sheath gas flow rate was 35 arb, auxiliary gas flow rate was 10 arb, primary resolution was 35000 FWHM, secondary mass spectrometry resolution was 17500 FWHM, scanning range was m / z 50-1000 Da, normalized collision energy (NCE) was 20, 40 and 60 eV.

[0061] Based on the structural formulas of the primary and secondary derivatization products of cycloprop-2-enecarboxylic acid, combined with the retention time of the extracted ion chromatogram obtained by LC-MS / MS detection, and the parent ion and product ion of the secondary mass spectrum, the presence of cycloprop-2-enecarboxylic acid in the sample was identified.

[0062] The reaction equation is:

[0063]

[0064] The parent ion [M+H]+ of the primary derivatization product of cycloprop-2-enecarboxylic acid is m / z 202.12264, and the characteristic fragments are m / z 70.06513, 91.05423, 98.06004, 105.06988, 134.09643 and 174.12773; Figure 1 shown.

[0065] The parent ion [M+H]+ of the secondary derivatization product of cycloprop-2-enecarboxylic acid is m / z 218.11756, and the characteristic fragments are m / z 55.0186, 79.0549, 83.0132, 105.0704, 136.11243 and 218.1176; Figure 2 shown.

[0066] Example 2

[0067] In this example, the ratio of N-methylphenethylamine (MPEA), triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS) remains unchanged, the concentration ratio is 1:2:2, and the volume ratio is 1:1:1; the concentration of N-methylphenethylamine is changed to 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL, respectively, and the other conditions are the same as in Example 1; the mass spectrometry response results of the primary derivatization product are shown in FIG. Figure 3 shown.

[0068] Example 3

[0069] This example investigates the initial derivatization at three reaction temperatures (30°C, 40°C, and 50°C) and at different reaction times (2 min, 5 min, 10 min, 15 min, 20 min, 30 min, and 60 min), with the remaining conditions being the same as in Example 1. The results are shown in FIG. Figure 4 shown.

[0070] Example 4

[0071] This example investigates the effect of different water contents in the supernatant of step (1) on the derivatization reaction, with the remaining conditions being the same as in Example 1; the results are shown in FIG. Figure 5 shown.

[0072] Example 5

[0073] This example investigates the secondary derivatization at four reaction temperatures (30°C, 40°C, 50°C, and 60°C) and at different reaction times (2 min, 5 min, 10 min, 30 min, and 60 min), with the remaining conditions being the same as in Example 1. The results are shown in FIG. Figure 6 shown.

[0074] Example 6

[0075] In this example, samples of Russula subsparsely glabrous, Russula sparsely glabrous, Russula japonica, and Amanita mushrooms were tested using the method of Example 1. The results showed that cycloprop-2-enecarboxylic acid was detected only in the freshly collected Russula subsparsely glabrous sample and not in the other samples. Given the instability of cycloprop-2-enecarboxylic acid, it is speculated that it may degrade during long-term storage.

[0076] In summary, the invention includes but is not limited to the above embodiments. Any equivalent replacement or partial improvement made under the spirit and principle of the present invention shall be deemed to be within the scope of protection of the present invention.

Claims

1. A method for rapid identification of Russula subsparsely covered, characterized by: The method steps include: S1: Weigh fresh mushroom samples and prepare mushroom extract; S2: adding acetonitrile to the mushroom extract, vortexing to mix, adding excess anhydrous sodium sulfate to dry, and centrifuging to obtain the supernatant; S3: taking the supernatant and adding N-methylphenylethylamine solution, triphenylphosphine solution and 2,2'-disulfide dipyridine solution to carry out a derivatization reaction to obtain a primary derivatization sample solution; S4: Take a portion of the primary derivatization sample solution and add magnesium monoperoxyphthalate hexahydrate solution to carry out a derivatization reaction, then add NaHCO3 solution and vortex; then add ethyl acetate for extraction, vortex, centrifuge, and collect the upper layer; concentrate to dryness in a centrifugal concentrator, and reconstitute with acetonitrile aqueous solution to obtain a secondary derivatization sample solution; S5: The remaining primary derivatization sample solution and the secondary derivatization sample solution are diluted with acetonitrile-water solution, respectively, and subjected to LC-MS / MS analysis to identify whether there is a derivatization product of cycloprop-2-enecarboxylic acid. If so, the mushroom sample is determined to be Russula subspecies.

2. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S1, a fresh mushroom sample is weighed, homogenized with water, extracted by shaking, and centrifuged to obtain a supernatant. Water is then added, vortexed to mix, and centrifuged to obtain a supernatant. The two supernatants are combined and concentrated by centrifugation to obtain a mushroom extract.

3. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S2, the volume ratio of the mushroom extract to acetonitrile is 100:1000-5000.

4. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S3, the solvent of the N-methylphenethylamine solution is acetonitrile, and the concentration is 0.01-50 mg / mL; The solvent of triphenylphosphine solution is acetonitrile, and the concentration is 0.01-50 mg / mL; The solvent of the 2,2'-dithiodipyridine solution is acetonitrile, and the concentration is 0.01-50 mg / mL; And / or, the volume ratio of the supernatant, N-methylphenethylamine solution, triphenylphosphine solution and 2,2'-disulfide dipyridine solution is 100:10-100:10-100:10-100.

5. A method for rapid identification of Russula subsparsely according to claim 1 or 4, characterized in that: In step S3, the derivatization reaction temperature is 30-50° C., and the reaction time is 5-60 min.

6. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S4, the solvent of the magnesium monoperoxyphthalate hexahydrate solution is water, and the concentration is 50-173 mg / mL; The solvent of NaHCO3 solution is water, and the concentration is 40-84 mg / mL; And / or, the volume ratio of the primary derivatization sample solution, the magnesium monoperoxyphthalate hexahydrate solution and the NaHCO3 solution is 100:50-200:

100.

7. A method for rapid identification of Russula subsparsely described in claim 1 or 6, characterized in that: In step S4, the derivatization reaction temperature is 30-40° C., and the reaction time is 5-30 min.

8. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S5, the chromatographic conditions for LC-MS / MS analysis are as follows: Chromatographic column: Phenomenex Luna Omega C18 column, 50×2.1 mm, 1.6 μm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile; Column temperature: 40°C; injection volume: 5 μL; mobile phase gradient: 。 9. The method for rapid identification of Russula subsparsely according to claim 1, wherein: In step S5, the mass spectrometry conditions for LC-MS / MS analysis are as follows: Positive ion mode, scanning mode was full scan-auto-trigger secondary mass spectrometry scan (Full MS / dd-MS2), spray voltage was 3.2 kV, ion transmission temperature was 320 ° C, nebulization temperature was 320 ° C, sheath gas flow rate was 35 arb, auxiliary gas flow rate was 10 arb, primary resolution was 35000 FWHM, secondary mass spectrometry resolution was 17500 FWHM, scanning range was m / z 50-1000 Da, normalized collision energy (NCE) was 20, 35 and 45 eV.

10. A kit for rapid identification of Russula subspecies, characterized by: The method comprises the N-methylphenethylamine solution, triphenylphosphine solution, 2,2'-disulfide dipyridine solution and magnesium monoperoxyphthalate hexahydrate solution used in the method according to any one of claims 1 to 9.

Images