CN111610268B - Method for separating mannitol, adenosine and ergosterol in sample to be detected and application - Google Patents
Method for separating mannitol, adenosine and ergosterol in sample to be detected and application Download PDFInfo
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Abstract
The invention provides a method for separating mannitol, adenosine and ergosterol in a sample to be detected. The method comprises the following steps: sequentially eluting a sample to be detected in a solid phase extraction column by using a first eluent, a second eluent, a third eluent and a fourth eluent, and respectively collecting an elution product of the first eluent, an elution product of the second eluent and an elution product of the fourth eluent so as to sequentially obtain eluents containing mannitol, adenosine and ergosterol, wherein the first eluent, the second eluent, the third eluent and the fourth eluent are respectively and independently methanol water solution, ethanol water solution, acetonitrile water solution or any combination of the three. The method can realize the simultaneous purification of the adenosine and the ergosterol in the sample to be detected, and the purity of the obtained adenosine and ergosterol is greatly improved.
Description
Technical Field
The invention relates to the field of analysis and detection, in particular to a method for separating mannitol, adenosine and ergosterol in a sample to be detected and application thereof, and more particularly relates to a method for separating mannitol, adenosine and ergosterol in a sample to be detected and a method for detecting the content of mannitol, adenosine and ergosterol in a sample to be detected.
Background
Ergosterol and mannitol are important pharmaceutical and chemical raw materials and are widely applied in food, medicine and chemical industries. Mannitol is a pharmaceutically good diuretic, lowering intracranial pressure, intraocular pressure and treating renal drugs, dehydrators, sugar substitutes, also as excipients for tablets and diluents for solids and liquids. Adenosine is recognized as an important modulator of neurotransmission and is involved in many physiological functions, such as regulating sleep, anxiety, cognition, and memory. The edible fungi contain abundant ergosterol and mannitol, and adenosine is also used as a quality evaluation index of the edible fungi such as cordyceps sinensis.
The existing technology for simultaneously measuring mannitol, adenosine and ergosterol in edible fungi adopts the methods of ultrasonic, hot reflux or pressurized fluid extraction and the like to respectively extract, and then respectively uses liquid phase to carry out analysis or content measurement [ Journal of Chromatography A,2004,1036(2): 239-; 2016(6) 66-67,70 parts of edible fungi; journal of Food and Drug Analysis,2005,13(4): 338-; chinese Medicine,2010,5(1): 4-4; the Chinese journal of experimental prescriptions, 2013,19(6), 161-163; shanghai agricultural science, 2014,30(2):60-64] has the defects of long time consumption for extraction and analysis, precision and high cost of required instruments and the like. At present, the reports of simultaneously separating and preparing ergosterol and adenosine from one medicinal material are less, and the traditional silica gel column chromatography (Chinese traditional medicine journal, 1989,14(10): 32-33; 2915-2917 in China Chinese medicine journal, 2008,33 (24); chinese medicinal materials 2009(2) 226-228; food Science and Human Wellness,2018,7(4): 282-286; chinese patent CN 107556358], the method is time-consuming and labor-consuming, the sample loss is large, and the environment is easily polluted.
Therefore, methods for simultaneously determining mannitol, adenosine and ergosterol in edible fungi still need to be developed and improved.
Disclosure of Invention
In order to solve the defects of long extraction and analysis time in the prior art for simultaneously measuring mannitol, adenosine and ergosterol in edible fungi and the defects of time and labor waste and sample loss in the prior art for simultaneously separating and preparing ergosterol and adenosine from one medicinal material, the invention establishes a method for synchronously preparing and quickly and quantitatively detecting mannitol, adenosine and ergosterol in edible fungi.
In a first aspect of the invention, the invention provides a method for separating mannitol, adenosine and ergosterol from a sample to be tested. According to an embodiment of the invention, the method comprises: sequentially eluting a sample to be detected in a solid phase extraction column by using a first eluent, a second eluent, a third eluent and a fourth eluent, and respectively collecting an elution product of the first eluent, an elution product of the second eluent and an elution product of the fourth eluent so as to sequentially obtain eluents containing mannitol, adenosine and ergosterol; wherein the first eluent, the second eluent, the third eluent and the fourth eluent are respectively methanol water solution, ethanol water solution, acetonitrile water solution or any combination of the three. The elution product of the first eluent comprises mannitol, the elution product of the second eluent mainly comprises adenosine, and the elution product of the fourth eluent mainly comprises ergosterol. The inventor finds out through experiments that the method can realize the simultaneous and complete separation of mannitol, adenosine and ergosterol in a sample to be detected, realize the simultaneous purification of the adenosine and the ergosterol, completely remove impurities with the polarity greater than that of the adenosine and impurities with the polarity less than that of the adenosine greater than that of the ergosterol, and greatly improve the purity of the obtained adenosine and ergosterol.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to the embodiment of the invention, the sample to be detected is edible fungi. The method according to the embodiment of the invention is particularly suitable for the separation of mannitol, adenosine and ergosterol and the purification of adenosine and ergosterol in edible fungi.
According to an embodiment of the present invention, the edible fungi is optionally cordyceps sinensis, fermented cordyceps sinensis powder, bacillus bovis, straw mushroom, morchella esculenta or fermented cordyceps sinensis powder.
According to an embodiment of the present invention, the first eluent is a 7% -10% methanol solution or a 2% -3% ethanol solution, the second eluent is a 11% -35% methanol solution or a 4% -20% ethanol solution, the third eluent is a 36% -97% methanol solution or a 21% -87% ethanol solution, and the fourth eluent is a 98% -100% methanol solution or an 88% -100% ethanol solution.
According to a specific embodiment of the present invention, the first eluent is a 7% to 10% methanol solution, the second eluent is a 11% to 35% methanol solution, the third eluent is a 36% to 97% methanol solution, and the fourth eluent is a 98% to 100% methanol solution.
According to a specific embodiment of the present invention, the first eluent is a 2% to 3% ethanol solution, the second eluent is a 4% to 20% ethanol solution, the third eluent is a 21% to 87% ethanol solution, and the fourth eluent is an 88% to 100% ethanol solution.
According to a specific embodiment of the present invention, the first eluent is 9% methanol solution, the second eluent is 30% methanol solution, the third eluent is 96% methanol solution, and the fourth eluent is 100% methanol solution.
The inventor finds that the first eluent is a 7-10% methanol solution or a 2-3% ethanol solution, which can not only further improve the purity of adenosine in the elution product of the second eluent, but also reduce the loss of adenosine; the second eluent is 11-35% methanol solution or 4-20% ethanol solution, which can further improve the purity and extraction rate of adenosine; the third eluent is 36-97% methanol solution or 21-87% ethanol solution, which can not only further improve the purity of ergosterol in the elution product of the fourth eluent, but also reduce the loss of ergosterol. Thereby effectively improving the purity of the adenosine and ergosterol obtained by separation.
According to an embodiment of the present invention, a sample to be tested is sequentially eluted in a solid phase extraction column with at least 6 column volumes of the first eluent, at least 3 column volumes of the second eluent, at least 6 column volumes of the third eluent, and at least 6 column volumes of the fourth eluent. The inventors have found that the purity of the adenosine and ergosterol obtained is further increased at the above column volumes for the first eluent, the second eluent, the third eluent and the fourth eluent.
It should be noted that the "column volume" as used herein refers to the volume of solvent remaining in the filler. The algorithm for column volume is many. According to a specific embodiment of the present invention, the algorithm for the column volume of the present invention is: weighing 1.0-1.5g of filler, adding a proper volume of methanol for suspension (volume A), pouring the suspension into an empty solid-phase extraction column provided with a sieve plate, knocking the outer wall of the column by a heavy object to enable the filler to be settled until the filler does not settle any more, placing a graduated centrifuge tube below the solid-phase extraction column, then opening a piston of the solid-phase extraction column to enable methanol to flow out, closing the piston when the methanol reaches the filler liquid level, and enabling the volume of the centrifuge tube to be B. A-B is the volume of one column. The 1.0g of the packing of the present invention had a column volume of 1.5 mL. If A is added in a larger amount, the amount of B evolved will increase accordingly, B will vary with A, and (A-B) is a fixed value, i.e.the solvent remaining in the column is constant and only influenced by the amount of packing.
According to the embodiment of the invention, the sample to be tested is provided in the form of edible fungus-diatomite mixed powder.
According to an embodiment of the invention, the dispersant is diatomaceous earth, florisil, silica gel or basic alumina.
According to the embodiment of the invention, the mass ratio of the edible fungi and the dispersing agent in the edible fungi-dispersing agent mixed powder is as follows: 1:(5-20).
According to the specific embodiment of the invention, the dispersing agent is diatomite, and the sample to be tested is provided in the form of edible fungus-diatomite mixed powder.
According to the embodiment of the invention, the mass ratio of the edible fungi to the diatomite in the edible fungi-diatomite mixed powder is 1 (5-20).
According to the embodiment of the invention, the edible fungus-diatomite mixed powder is obtained by mixing and grinding the edible fungus and the diatomite for 1-3 min. Further improving the grinding efficiency and improving the dispersion uniformity of the edible fungi.
According to an embodiment of the present invention, the solid phase extraction column is loaded with ODS packing.
According to the embodiment of the invention, the mass ratio of the ODS filler to the sample to be tested is as follows: (1.0-1.5): (0.12-0.4).
According to the embodiment of the invention, the mass ratio of the ODS filler to the edible mushroom-dispersant mixed powder is as follows: (1.0-1.5): (0.12-0.4).
According to an embodiment of the present invention, the ODS filler is previously activated by soaking in methanol. Thereby opening groups in the ODS filler to remove interferents, and further improving the purity of the target substance.
According to the embodiment of the invention, the ODS filler after activation treatment is subjected to elution and replacement in advance with the first eluent. Further, the target substance in the sample to be detected is prevented from being directly eluted, so that the yield of the target substance is prevented from being reduced.
According to an embodiment of the present invention, the sample to be tested is an edible fungus, and the method for separating mannitol, adenosine and ergosterol from the edible fungus comprises:
(1) grinding edible fungus powder and diatomite according to the mass ratio of 1 (5-20) and uniformly mixing to obtain edible fungus-diatomite mixed powder;
(2) according to the mass ratio (0.12-0.4) of the edible fungus-diatomite mixed powder to the ODS filler: (1.0-1.5), loading the edible fungus-diatomite mixed powder in the step (1) into a solid phase extraction column filled with ODS filler, sequentially eluting with a first eluent, a second eluent, a third eluent and a fourth eluent, and respectively collecting an elution product of the first eluent, an elution product of the second eluent and an elution product of the fourth eluent so as to sequentially obtain eluents containing mannitol, adenosine and ergosterol; wherein the first eluent is a 7-10% methanol solution or a 2-3% ethanol solution, the second eluent is a 11-35% methanol solution or a 4-20% ethanol solution, the third eluent is a 36-97% methanol solution or a 21-87% ethanol solution, and the fourth eluent is a 98-100% methanol solution or an 88-100% ethanol solution.
According to an embodiment of the present invention, the present invention provides a method for separating mannitol, adenosine and ergosterol from edible fungi, comprising:
(1) grinding edible fungus powder and diatomite according to the mass ratio of 1 (5-20) and uniformly mixing to obtain edible fungus-diatomite mixed powder;
(2) according to the mass ratio (0.12-0.4) of the edible fungus-diatomite mixed powder to the ODS filler: (1.0-1.5), loading the edible fungus-diatomite mixed powder in the step (1) into a solid phase extraction column filled with ODS filler, sequentially eluting with a first eluent, a second eluent, a third eluent and a fourth eluent, and respectively collecting an elution product of the first eluent, an elution product of the second eluent and an elution product of the fourth eluent so as to sequentially obtain eluents containing mannitol, adenosine and ergosterol; the first eluent is a 9% methanol solution, the second eluent is a 30% methanol solution, the third eluent is a 96% methanol solution, and the fourth eluent is a 100% methanol solution.
According to an embodiment of the present invention, the present invention provides a method for separating mannitol, adenosine and ergosterol from edible fungi, comprising:
(1) grinding edible fungus powder and diatomite according to the mass ratio of 1 (5-20) and uniformly mixing to obtain edible fungus-diatomite mixed powder;
(2) according to the mass ratio (0.12-0.4) of the edible fungus-diatomite mixed powder to the ODS filler: (1.0-1.5), loading edible fungus-diatomite mixed powder (0.12-0.4) g in the step (1) into a solid phase extraction column filled with ODS filler, eluting with 9mL of 9% methanol solution, 5mL of 30% methanol solution, 9mL of 96% methanol solution and 9mL of 100% methanol solution in sequence, and collecting the elution product of the 9% methanol solution, the elution product of the 30% methanol solution and the elution product of the 100% methanol solution respectively to obtain an eluent containing mannitol, adenosine and ergosterol.
In a second aspect of the invention, the invention provides a method for detecting the contents of mannitol, adenosine and ergosterol in a sample to be detected. According to an embodiment of the invention, the method comprises: separating the sample to be tested by the method so as to obtain eluent containing mannitol, adenosine and ergosterol in sequence; respectively measuring the eluates containing mannitol, adenosine and ergosterol by high performance liquid chromatography to obtain chromatograms of mannitol, adenosine and ergosterol; and determining the content of the mannitol, the adenosine and the ergosterol in the sample to be tested based on the obtained chromatogram of the mannitol, the adenosine and the ergosterol. According to the method provided by the embodiment of the invention, the contents of mannitol, adenosine and ergosterol in the sample to be detected, especially the edible fungi, can be synchronously prepared and quickly detected.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to the embodiment of the present invention, the eluate containing mannitol is measured by high performance liquid chromatography under the following chromatographic conditions: a chromatographic column: shodex Asahipak NH 2P-504E, 4.6 × 250mm,5 μm; an ELSD detector; the drift tube temperature is 40 ℃; carrier gas N2The flow rate is 1.6 mL/min; the sample injection amount is 10 mu L; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the mobile phase is as follows: an aqueous solution of acetonitrile, wherein the volume of acetonitrile and water is 82.5: 17.5.
according to still another embodiment of the present invention, the mannitol control is detected by high performance liquid chromatography under the chromatographic conditions for detecting the eluent containing mannitol, so as to obtain a chromatogram of the mannitol control.
According to the present invention, the adenosine-containing eluate is measured by high performance liquid chromatography under the following conditions: a chromatographic column: poroshell120SB-C18,4.6 × 30mm,2.7 μm; a DAD detector; monitoring at a wavelength of 260 nm; the sample injection amount is 2 mu L; the column temperature was 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is as follows: an aqueous methanol solution, wherein the volume ratio of methanol to water is 20: 80.
according to still another embodiment of the present invention, the adenosine control is detected by high performance liquid chromatography in advance under the above-mentioned chromatographic conditions for detecting an eluent containing adenosine, so as to obtain a chromatogram of the adenosine control.
According to an embodiment of the present invention, the ergosterol-containing eluate is measured by high performance liquid chromatography under the following chromatographic conditions: a chromatographic column: poroshell120SB-C18,4.6 × 30mm,2.7 μm; DAD detector, 283nm wavelength monitoring; the sample injection amount is 2 mu L; the column temperature was 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is as follows: an aqueous ethanol solution, wherein the volume ratio of ethanol to water in the aqueous ethanol solution is 94: 6.
according to still another embodiment of the present invention, the ergosterol control is detected by high performance liquid chromatography under the above-mentioned chromatographic conditions for detecting an ergosterol-containing eluate in advance, so as to obtain a chromatogram of the ergosterol control.
According to the embodiment of the invention, the obtained chromatogram of the mannitol, the adenosine and the ergosterol in the sample to be detected, the chromatogram of the mannitol reference substance, the chromatogram of the adenosine reference substance and the chromatogram of the ergosterol reference substance are used for determining the contents of the mannitol, the adenosine and the ergosterol in the sample to be detected by adopting an external standard method.
In conclusion, the method for synchronously preparing and rapidly and quantitatively detecting mannitol, adenosine and ergosterol in the sample to be detected, which is established by the invention, has the following main advantages:
(1) the content determination of mannitol, adenosine and ergosterol and the purification of adenosine and ergosterol in the edible fungi can be realized simultaneously;
(2) the adenosine and ergosterol are purified by one step by using matrix solid-phase dispersion extraction operation, the purification time is short, the operation is simple, the sample loss is less, and the compound purity is high;
(3) the chromatographic method has short analysis time, and adenosine and ergosterol peak within 1 min.
(4) The invention can provide a method for rapid content determination for quality control of various edible fungi and provides reference for quality evaluation of the edible fungi.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a liquid phase diagram of methanol elution 1-7 groups a (A), b (B) according to example 1 of the present invention;
FIG. 2 is a liquid phase diagram of groups b of 3, 8-12 of methanol elutions according to example 1 of the present invention;
FIG. 3 is a liquid phase diagram of methanol elution groups 1-7, c (A), d (B), according to example 1 of the present invention;
FIG. 4 is a liquid phase diagram of ethanol elution 1-4 groups a (A), b (B) according to example 1 of the present invention;
FIG. 5 is a liquid phase diagram of groups b of ethanol elution 8-15 according to example 1 of the present invention;
FIG. 6 is a liquid phase diagram of ethanol elution groups 1-7, c (A), d (B), according to example 1 of the present invention;
FIG. 7 is a liquid phase diagram of the 1 st to 6 th column volumes (V1-V6) of a 9% methanol elution section according to example 2 of the present invention;
FIG. 8 is a liquid phase diagram of column volumes 1-3 (V1-V3) of a 30% methanol elution fraction according to example 2 of the present invention;
FIG. 9 is a liquid phase diagram of column volumes 1-6 (V1-V6) of a 96% methanol elution fraction according to example 2 of the present invention;
FIG. 10 is a liquid phase diagram of column volumes 1-6 (V1-V6) of a 100% methanol elution fraction according to example 2 of the present invention;
FIG. 11 is a liquid chromatogram of each eluate fraction of Cordyceps sinensis according to example 3 of the present invention;
FIG. 12 is a liquid chromatogram of each elution fraction of fermented Cordyceps sinensis bacterial powder according to example 3 of the present invention;
FIG. 13 is a liquid phase chromatogram of each eluted fraction of Boletus edulis according to example 3 of the present invention;
FIG. 14 is a liquid phase diagram of various elution sections of volvariella volvacea according to example 3 of the present invention;
FIG. 15 is a liquid phase diagram of each eluted section of Morchella esculenta according to example 3 of the present invention; and
FIG. 16 is a liquid phase diagram of each elution section of fermented Cordyceps sinensis bacterial powder according to example 3 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following described embodiments are exemplary and are intended to be illustrative of the invention and are not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the first aspect, the invention provides a method for separating mannitol, adenosine and ergosterol in edible fungi by using a matrix solid-phase dispersion extraction technology, and provides a method for purifying adenosine and ergosterol in edible fungi by using the matrix solid-phase dispersion extraction technology. According to a particular embodiment of the invention, the method comprises the following steps:
weighing an appropriate amount of dry edible fungus powder, putting into a mortar, adding an appropriate amount of diatomite according to a mass ratio of 1:5-1:20(g/g), fully grinding and uniformly mixing (1-3min) to obtain edible fungus-diatomite mixed powder, and weighing 0.12-0.4g of the powder for later use. Weighing a proper amount of ODS filler (1.0-1.5g), soaking in pure methanol overnight for activation, filling the ODS filler into a small solid-phase extraction column (with the inner diameter of 16mm, the column height of 80mm and 12mL with a sieve plate), settling, compacting, leveling the filler surface, carefully adding filter paper with the size of the cross section of the column above the filler, eluting and replacing the ODS column with about 30mL of initial elution solution (7-10% methanol solution (or 2-3% ethanol solution)), putting the eluent on the filler surface, carefully pouring prepared sample-diatomite mixed powder into the filler surface to level the sample powder, and sequentially eluting with organic solvents with similar polarities as the eluent, specifically 9mL of 7-10% methanol solution (or 2-3% ethanol solution), 5mL of 11-35% methanol solution (or 4-20% ethanol solution), 9mL of 36% -97% methanol solution (or 21% -87% ethanol solution) and 9mL of 98% -100% methanol (88% -100% ethanol solution), and collecting the eluent according to polarity. Concentrating under reduced pressure 11-35% methanol eluate (or 4-20% ethanol eluate) and 98-100% methanol eluate (or 88-100% ethanol eluate), recovering, and lyophilizing to obtain adenosine and ergosterol reference substances. Additionally, the 7% -10% methanol eluate (or 2% -3% ethanol eluate) contains mannitol.
In a second aspect, the invention provides a method for quantitative analysis of mannitol, adenosine and ergosterol in edible fungi by high performance liquid chromatography. According to a particular embodiment of the invention, the method comprises the following steps:
taking the 7% -10% methanol eluent, the 11% -35% methanol eluent and the 98% -100% methanol eluent obtained in the first aspect, respectively metering to 10mL, 5mL and 10mL, and filtering with a 0.2 μm organic filter membrane to obtain a mannitol test solution, an adenosine test solution and an ergosterol test solution.
The detection chromatographic conditions of 7-10% methanol eluent (or 2-3% ethanol eluent) are as follows: chromatography columns (Shodex Asahipak NH 2P-504E, 4.6 x 250mm,5 μm); ELSD Detector, drift tube temperature 40 deg.C, carrier gas (N)2) The flow rate is 1.6 mL/min; the sample volume is 10 mu L; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; mobile phase V (acetonitrile): v (water) ═ 82.5: 17.5; and (4) calculating the content by an external standard method according to the peak area of the mannitol reference substance solution.
The detection chromatographic conditions of 11-35% methanol eluent (or 4-20% ethanol eluent) are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (methanol): v (water) ═ 20: 80; and (4) calculating the content by using an external standard point method according to the peak area of the adenosine reference substance solution.
The detection chromatographic conditions of 98-100% methanol eluent (or 88-100% ethanol eluent) are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 283nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (ethanol): v (water) ═ 94: 6; and (4) calculating the content by using an external standard point method according to the peak area of the ergosterol reference substance solution.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
1. Laboratory apparatus
Agilent high performance liquid chromatograph model 1260 (Agilent technologies, Inc., USA); evaporative light scattering detector type Sedex90 (sedre corporation, france); chromatography columns Shodex Asahipak NH 2P-504E (4.6 mm. times.250 mm,5 μm), Poroshell120SB-C18 (4.6. times.30 mm,2.7 μm); one in ten thousand electronic balance model XS204 (mettler-toledo instruments ltd, switzerland); a one-tenth-ten-thousandth electronic balance model XS205 (mertler-toledo instruments ltd, switzerland); ultrasonic cleaning apparatus SBL-22 DT (Ningbo Xinzhi Biotechnology GmbH).
2. Medicinal materials
Cordyceps is from Hubei, fermented Cordyceps powder (Cs-4) is from Jiangxi province, fermented Cordyceps powder (Cs-C-Q80) is from Zhejiang province, straw mushroom is from Guangdong province, Boletus is from Yunnan province, and Morchella esculenta is from Hebei province.
3. Reagent
Chromatographic methanol (stoichiometrically chemical limited), chromatographic acetonitrile (stoichiometrically chemical limited), chromatographic ethanol (semer fly), and the remaining reagents are analytically pure; the ultrapure water was self-made in the laboratory (Milli-Q). Control mannitol (purity 99.1%), adenosine (purity 99.7%), and ergosterol (purity 96.2%) were purchased from the institute of food and drug testing, China.
Example 1 elution of impurities and elution of target Compound eluent and determination of eluent concentration
The preparation of adenosine and ergosterol based on matrix solid phase dispersion extraction separation comprises the following steps:
(1) separate preparation
Weighing 0.1g of fermented cordyceps sinensis powder, adding 2.0g of diatomite into a mortar, fully grinding and uniformly mixing (1min) to obtain fermented cordyceps sinensis powder-diatomite (1:20) mixed powder, weighing 0.4g of the powder, putting the powder into a solid phase extraction column (12mL solid phase extraction column with a sieve plate and 1g of ODS filler), and respectively setting 12 test groups (1-12) for groping and eluting pre-adenosine impurity (containing mannitol), adenosine, impurity between adenosine and ergosterol and methanol (ethanol) concentration of ergosterol. Impurities and target compounds are eluted by respectively using 9mL of a solution, 5mL of b solution, 9mL of c solution and 9mL of d solution which are sequentially arranged according to the table 1 and the table 2, so that the optimal eluent concentration is determined, and the eluent is collected according to the elution sequence in the experimental process.
Table 1: gradient of elution concentration of solid phase extraction methanol
Table 2: gradient of elution concentration of solid phase extraction ethanol
(2) HPLC detection
And (3) respectively carrying out HPLC detection on each section of eluent obtained in the step (1).
The chromatographic conditions of the eluent detection of the groups a and b sections 1-7 in the tables 1 and 2 are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (methanol): v (water) ═ 20: 80 (pre adenosine elution impurity conditions);
the chromatographic conditions of the eluent detection of the groups 8-12 in the table 1 and the groups 8-15 in the table 2 are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the elution procedure is 0-2min, 20% methanol, 2-7min, 20% -100% methanol, 7-8min, 100% methanol, 8-10min, 100% -20% methanol (conditions for adenosine elution);
the chromatographic conditions of the eluent detection of the groups c and d sections 1-7 in the tables 1 and 2 are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 283 wavelength monitoring; the sample volume is 2 mu L; the column temperature is 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (ethanol): v (water) ═ 94: 6 (elution of impurities between adenosine and ergosterol and conditions for elution of ergosterol);
the results are shown in FIGS. 1-6, respectively. As can be seen from the figure, the a section mainly contains impurities with polarity greater than that of adenosine, the b section mainly contains adenosine, the c section mainly contains impurities with polarity less than that of adenosine and greater than that of ergosterol, and the d section mainly contains ergosterol. As can be seen from FIG. 1 or FIG. 4, the section a is eluted by using 7% -10% methanol solution or 2% -3% ethanol solution, which can ensure the purity of adenosine in the eluate of the section b without loss of adenosine; as can be seen from fig. 1 and 2 or fig. 4 and 5, the section b adopts 11% -35% methanol solution or 4% -20% ethanol solution for elution, so as to ensure the purity and maximum extraction rate of adenosine; as can be seen from the combination of FIGS. 2 and 3 or FIGS. 5 and 6, the ergosterol in the eluent of the section d can be ensured without loss of the ergosterol by eluting the section c with 36% -97% methanol solution or 21% -87% ethanol solution. And in the range of the elution conditions, carrying out HPLC detection on the adenosine and ergosterol elution sections according to the liquid phase conditions, and calculating the purity of the two compounds by using a peak area normalization method, wherein the result shows that the HPLC purity of the adenosine reaches more than 80%, and the HPLC purity of the ergosterol reaches more than 98%.
EXAMPLE 2 optimization of different elution volumes
The preparation of adenosine and ergosterol based on matrix solid phase dispersion extraction separation comprises the following steps:
(1) separation of
Weighing 0.1g of fermented cordyceps sinensis powder, adding 0.5g of diatomite into a mortar, fully grinding and uniformly mixing (1min) to obtain edible fungus-diatomite (1:5) mixed powder, weighing 0.12g of the powder, putting the powder on a solid phase extraction column (12mL of solid phase extraction column with a sieve plate and 1g of ODS filler), sequentially eluting with 9% methanol solution, 30% methanol solution, 96% methanol solution and 100% methanol solution, and collecting one tube according to polarity and each column volume (1.5 mL).
(2) HPLC detection
And (3) respectively carrying out HPLC detection on the eluents of the sections and the tubes obtained in the step (1).
The detection chromatographic conditions of 9% and 30% methanol eluent are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (methanol): v (water) ═ 20: 80;
the chromatographic conditions of 96% and 100% methanol eluent detection are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 283 wavelength monitoring; the sample volume is 2 mu L; the column temperature is 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (ethanol): v (water) ═ 94: 6;
the results are shown in FIGS. 7-10, respectively. As can be seen, 6 column volumes of 9% methanol solution can completely remove impurities with a polarity greater than adenosine, 3 column volumes of 30% methanol solution can completely extract adenosine, 6 column volumes of 96% methanol solution can completely remove impurities with a polarity less than adenosine greater than ergosterol, and 6 column volumes of 100% methanol can completely extract ergosterol. The volume of eluent is the minimum eluent volume that maximizes the purity of adenosine and ergosterol.
EXAMPLE 3 application of the method-separation and purification of adenosine and ergosterol from various edible fungi Using matrix solid phase Dispersion extraction
Separating and purifying adenosine and ergosterol based on matrix solid phase dispersion extraction, comprising the following steps:
(1) separation of
Weighing 0.1g of dry edible fungus powder (cordyceps sinensis, fermented cordyceps sinensis powder, bolete, straw mushroom, morchella or fermented cordyceps sinensis powder), adding 0.5g of diatomite into a mortar, fully grinding and uniformly mixing (1min) to obtain edible fungus-diatomite (1:5) mixed powder, weighing 0.12g of the powder, putting the powder into a solid phase extraction column (12mL of solid phase extraction column with a sieve plate and 1g of ODS filler), parallelly arranging 6 test groups (corresponding to 6 edible fungi), sequentially eluting with 9mL of 9% methanol solution, 5mL of 30% methanol solution, 9mL of 96% methanol solution and 9mL of 100% methanol solution, and collecting eluent according to polarity.
(2) HPLC detection
Respectively detecting each section of eluent obtained in the step (1) by HPLC;
the detection chromatographic conditions of 9% and 30% methanol eluent are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (methanol): v (water) ═ 20: 80;
the chromatographic conditions of 96% and 100% methanol eluent detection are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 283 wavelength monitoring; the sample volume is 2 mu L; the column temperature is 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (ethanol): v (water) ═ 94: 6;
the results are shown in FIGS. 11-16, respectively. HPLC detection is carried out on the adenosine and ergosterol elution sections purified from each edible fungus, and the purity of the two compounds is calculated by applying a peak area normalization method, and the result is shown in Table 3.
Table 3: adenosine and ergosterol purity from edible fungus
Example 4 determination of the content of mannitol, adenosine and ergosterol
The quantitative analysis of adenosine and ergosterol in edible fungi comprises the following specific steps:
(1) respectively weighing appropriate amount of mannitol, adenosine and ergosterol, precisely weighing, respectively adding ultrapure water, 90% methanol and pure methanol to dissolve to obtain standard solutions with concentrations of 995.96 μ g/mL, 397.60 μ g/mL and 398.60 μ g/mL, and placing in a 4 deg.C refrigerator for use.
(2) The 9% methanol eluate, 30% methanol eluate and 100% methanol eluate obtained in example 3(1) were respectively metered to 10mL, 5mL and 10mL, and filtered through a 0.2 μm organic membrane to obtain a sample solution.
The chromatographic conditions of 9% methanol eluent detection are as follows: chromatography columns (Shodex Asahipak NH 2P-504E, 4.6 x 250mm,5 μm); ELSD Detector, drift tube temperature 40 deg.C, carrier gas (N)2) The flow rate is 1.6 mL/min; the sample volume is 10 mu L; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; mobile phase V (acetonitrile): v (water) ═ 82.5: 17.5; and (4) calculating the content by an external standard method according to the peak area of the mannitol reference substance solution.
The chromatographic conditions of 30% methanol eluent detection are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 260nm wavelength monitoring; the sample volume is 2 mu L; the column temperature is 25 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (methanol): v (water) ═ 20: 80; and (4) calculating the content by using an external standard point method according to the peak area of the adenosine reference substance solution.
The chromatographic conditions for detecting 100% methanol eluent are as follows: chromatography columns (Poroshell120SB-C18,4.6 x 30mm,2.7 μm); DAD detector, 283 wavelength monitoring; the sample volume is 2 mu L; the column temperature is 35 ℃; the flow rate is 1.0 mL/min; the mobile phase is V (ethanol): v (water) ═ 94: 6; and (4) calculating the content by using an external standard point method according to the peak area of the ergosterol reference substance solution.
The method is stable within 12 hours (RSD is less than 2 percent), good in reproducibility (RSD is less than 2 percent) and has the test results shown in the table 4.
Table 4: the contents of mannitol, adenosine and ergosterol in each edible fungus
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (20)
1. A method for separating mannitol, adenosine and ergosterol from a sample to be tested, comprising:
sequentially eluting a sample to be detected in a solid phase extraction column by using a first eluent, a second eluent, a third eluent and a fourth eluent, and respectively collecting an elution product of the first eluent, an elution product of the second eluent and an elution product of the fourth eluent so as to sequentially obtain eluents containing mannitol, adenosine and ergosterol;
the sample to be detected is edible fungi, the first eluent is a 7% -10% methanol solution or a 2% -3% ethanol solution, the second eluent is a 11% -35% methanol solution or a 4% -20% ethanol solution, the third eluent is a 36% -97% methanol solution or a 21% -87% ethanol solution, and the fourth eluent is a 98% -100% methanol solution or an 88% -100% ethanol solution.
2. The method of claim 1, wherein the edible fungi is Cordyceps sinensis, fermented Cordyceps sinensis powder, Boletus bovis, Volvariella volvacea, Morchella esculenta, or fermented Cordyceps sinensis powder.
3. The method of claim 1,
the first eluent is a 9% methanol solution, the second eluent is a 30% methanol solution, the third eluent is a 96% methanol solution, and the fourth eluent is a 100% methanol solution.
4. The method according to claim 1, characterized in that the sample to be tested is eluted in the solid phase extraction column with at least 6 column volumes of the first eluent, at least 3 column volumes of the second eluent, at least 6 column volumes of the third eluent and at least 6 column volumes of the fourth eluent in sequence.
5. The method of claim 2, wherein the sample to be tested is provided in the form of an edible mushroom-dispersant mixed powder.
6. The method of claim 5, wherein the dispersant comprises at least one selected from the group consisting of diatomaceous earth, Florisil, silica gel, and basic alumina.
7. The method according to claim 5, wherein the edible fungi-dispersing agent mixed powder comprises the edible fungi and the dispersing agent in a mass ratio of 1 (5-20).
8. The method according to claim 2, wherein the sample to be tested is provided in the form of edible fungi-diatomite mixed powder.
9. The method according to claim 8, wherein the edible fungi-diatomaceous earth mixed powder is obtained by subjecting edible fungi and diatomaceous earth to a mixing and grinding treatment for 1-3 min.
10. The process of claim 5, wherein the solid phase extraction column is loaded with ODS packing.
11. The method according to claim 10, wherein the mass ratio of the ODS filler to the sample to be tested is: (1.0-1.5):(0.12-0.4).
12. The method according to claim 10, wherein the mass ratio of the ODS filler to the edible fungus-dispersant mixed powder is: (1.0-1.5):(0.12-0.4).
13. A method for detecting the contents of mannitol, adenosine and ergosterol in a sample to be detected is characterized by comprising the following steps:
separating a sample to be tested by using the method of any one of claims 1 to 12 so as to obtain an eluent containing mannitol, adenosine and ergosterol in sequence;
respectively measuring the eluates containing mannitol, adenosine and ergosterol by high performance liquid chromatography to obtain chromatograms of mannitol, adenosine and ergosterol; and
and determining the contents of the mannitol, the adenosine and the ergosterol in the sample to be tested based on the obtained chromatogram of the mannitol, the adenosine and the ergosterol.
14. The method according to claim 13, wherein the mannitol-containing eluate is measured by high performance liquid chromatography under the following chromatographic conditions:
a chromatographic column: shodex Asahipak NH 2P-504E, 4.6 × 250mm,5 μm;
an ELSD detector;
the drift tube temperature is 40 ℃;
carrier gas N2The flow rate is 1.6 mL/min;
the sample injection amount is 10 mu L;
the column temperature is 30 ℃;
the flow rate is 1.0 mL/min;
the mobile phase is as follows: an aqueous solution of acetonitrile, wherein the volume of acetonitrile and water is 82.5: 17.5.
15. the method according to claim 14, wherein the mannitol control is detected by high performance liquid chromatography under the chromatographic conditions in advance so as to obtain a chromatogram of the mannitol control.
16. The method according to claim 13, wherein the adenosine-containing eluate is measured by high performance liquid chromatography under the following conditions:
a chromatographic column: poroshell120SB-C18,4.6 × 30mm,2.7 μm;
a DAD detector;
monitoring at a wavelength of 260 nm;
the sample injection amount is 2 mu L;
the column temperature was 25 ℃;
the flow rate is 1.0 mL/min;
the mobile phase is as follows: an aqueous methanol solution, wherein the volume ratio of methanol to water is 20: 80.
17. the method according to claim 16, wherein the adenosine control is detected by high performance liquid chromatography under the chromatographic conditions in advance to obtain a chromatogram of the adenosine control.
18. The method of claim 13, wherein the ergosterol-containing eluate is assayed by high performance liquid chromatography under the following conditions:
a chromatographic column: poroshell120SB-C18,4.6 × 30mm,2.7 μm;
DAD detector, 283nm wavelength monitoring;
the sample injection amount is 2 mu L;
the column temperature was 35 ℃;
the flow rate is 1.0 mL/min;
the mobile phase is as follows: an aqueous ethanol solution, wherein the volume ratio of ethanol to water in the aqueous ethanol solution is 94: 6.
19. the method of claim 18, wherein the ergosterol control is detected by high performance liquid chromatography under the chromatographic conditions in advance to obtain a chromatogram of the ergosterol control.
20. The method according to any one of claims 13 to 19, characterized in that the obtained chromatogram of mannitol, adenosine and ergosterol in the sample to be tested, the chromatogram of the mannitol reference, the chromatogram of the adenosine reference and the chromatogram of the ergosterol reference are used for determining the contents of mannitol, adenosine and ergosterol in the sample to be tested by an external standard method.
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| CN115112792A (en) * | 2022-06-15 | 2022-09-27 | 山东新华制药股份有限公司 | Method for extracting ergosterol from saccharomyces cerevisiae and detecting ergosterol by HPLC |
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