CN112557556A - Method for detecting glucoside alkaloid in potatoes - Google Patents
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Abstract
The invention relates to the field of chemical analysis, and particularly discloses a method for detecting glucoside alkaloid in potatoes, which comprises the following steps: (1) taking a potato sample, and drying and crushing the potato sample to obtain a potato analysis sample; (2) mixing a potato analysis sample with a solvent, ultrasonically extracting glucoside alkaloid in the potato analysis sample, filtering after extraction, collecting filtrate, and evaporating the filtrate to dryness to obtain a glucoside alkaloid crude extract; (3) preparing a glucoside alkaloid crude extract solution from the glucoside alkaloid crude extract obtained in the step (2), extracting glucoside alkaloid in the glucoside alkaloid crude extract solution by using a three-phase electric film extraction technology by taking the glucoside alkaloid crude extract solution as a donor phase, and detecting the concentration of the glucoside alkaloid in a receiving phase in the three-phase electric film extraction after the extraction is finished. The method has outstanding sample purification capacity, can reduce the interference of other impurities in the potatoes on the detection of the glucoside alkaloid, and greatly improves the detection accuracy and precision of the glucoside alkaloid.
Description
Technical Field
The invention relates to the field of chemical analysis, in particular to a method for detecting glucoside alkaloid in potatoes.
Background
The potato is one of the main food crops in China, has rich nutrition and medicinal value, and is widely applied to the food and medicine industries. Potato plants and tubers also contain a highly toxic glycoside alkaloid, which is present in very low levels in fresh, mature potatoes, but 50 times as much in sprouted and greened potatoes, and is highly likely to cause toxicity and even death in humans. Therefore, the development of a new method for efficiently separating and accurately detecting glucoside alkaloids in different parts of potato tubers has important significance for improving the quality safety of potatoes and processed products thereof.
The potato sample has the characteristics of complex matrix, serious interference and the like, the sample needs to be pretreated before the analysis and detection of the glucoside alkaloid so as to eliminate the interference of the matrix in the sample, and the pretreatment method for selecting the sample with strong purification capacity and high selectivity is particularly important. Domestic and foreign researches show that the traditional extraction method of glucoside alkaloids in potatoes adopts a liquid-liquid extraction method using water or acid water as an extraction solvent, and comprises a single solvent method, a double solvent method and a multi-solvent mixing method, so that the method has the advantage of low cost, but the extraction process is complex, the time consumption is long, and the extraction rate is low. In order to improve the extraction efficiency and reduce the energy consumption, researchers propose an ultrasonic extraction method and supercritical CO2New techniques such as extraction and microwave extraction, although this is trueThe methods greatly shorten the pretreatment time of the sample, but starch, sugar, fat and the like in the sample are difficult to completely remove, and great interference is generated on the detection result.
The electromembrane extraction technology is a novel sample pretreatment technology proposed by Pedersen-Bjergaard et al in 2006, and the separation principle is that under the action of an electric field, charged analytes move to an electrode with opposite electrical property to the charged analytes and pass through a supporting liquid membrane to enter receiving liquid. In the EME extraction process, the mass transfer process of an analyte mainly takes electromigration as a main process, so that the extraction time is greatly shortened; meanwhile, due to the selectivity of the supported liquid film, the EME has stronger purification capacity, so that the EME is rapidly developed in the field of sample pretreatment.
Disclosure of Invention
In view of the problems and deficiencies of the prior art, it is an object of the present invention to provide a method for detecting glycoside alkaloids in potatoes.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
a method for detecting glucoside alkaloids in potato comprises the following steps:
(1) taking a potato sample, and drying and crushing the potato sample to obtain a potato analysis sample;
(2) mixing a potato analysis sample with a solvent, ultrasonically extracting glucoside alkaloid in the potato analysis sample, filtering after extraction, collecting filtrate, and evaporating the filtrate to dryness to obtain a glucoside alkaloid crude extract;
(3) preparing a glucoside alkaloid crude extract solution from the glucoside alkaloid crude extract obtained in the step (2), extracting glucoside alkaloid in the glucoside alkaloid crude extract solution by using a three-phase electric film extraction technology by taking the glucoside alkaloid crude extract solution as a donor phase, and detecting the concentration of the glucoside alkaloid in a receiving phase in the three-phase electric film extraction after the extraction is finished.
According to the method, preferably, the membrane used in the three-phase electromembrane extraction technology in step (3) is a porous polypropylene fiber membrane, an extraction solvent is loaded on the porous polypropylene fiber membrane, and the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether.
According to the above method, preferably, the volume percentage of the bis (2-ethylhexyl) phosphate in the extraction solvent mixed solution is 5% to 30%.
According to the above method, preferably, the porous polypropylene fiber membrane has a pore size of 0.2 μm and a thickness of 100 μm to 200. mu.m.
According to the above method, preferably, the crude glycoside alkaloid extract solution in step (3) is prepared using an acidic aqueous solution as a solvent, wherein the acidic aqueous solution is any one of 10mmol/L hydrochloric acid, 20mmol/L formic acid, and 10mmol/L phosphate buffer solution having a pH of 4.
According to the method, preferably, the receiving phase adopted in the three-phase electromembrane extraction technology in the step (3) is any one of a 10mmol/L hydrochloric acid solution, a 20mmol/L formic acid solution, a 20mmol/L acetic acid solution and a 10mmol/L trifluoroacetic acid solution.
According to the above method, preferably, the voltage for applying the electric field between the donor phase and the receiving phase in the three-phase electromembrane extraction technology in step (3) is 40V-150V.
According to the above method, preferably, the solvent in step (2) is a methanol solution containing 5% acetic acid, and the temperature of ultrasonic extraction is 30-40 ℃.
According to the above method, preferably, the extraction time in the step (3) is 5min to 30 min.
According to the above method, preferably, the glycoside alkaloid is alpha-solanine and/or alpha-kaline.
The invention adopts three-phase electric membrane extraction technology to extract glucoside alkaloid in glucoside alkaloid crude extract solution, wherein one operation method comprises the following steps: a) sealing one end of the hollow fiber membrane, and loading an extraction solvent on the hollow fiber membrane; b) injecting a receiving phase into the inner cavity of the hollow fiber membrane treated in the step a); c) immersing the closed end of the hollow fiber membrane treated in the step b) in a donor phase (glucoside alkaloid crude extract solution), inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, connecting the working electrode with a power supply anode, connecting the counter electrode with a power supply cathode, applying voltage to the working electrode and the counter electrode to form an electric field, and extracting glucoside alkaloid in the donor phase under the action of the electric field.
The invention adopts another operation method for extracting glucoside alkaloid from a glucoside alkaloid crude extract solution by adopting a three-phase electric membrane extraction technology, which comprises the following steps: a) selecting a tubular receiving phase container with two open ends, sealing and fixing the porous polypropylene fiber membrane at one end of the tubular receiving phase container at high temperature of an electric iron, and loading an extraction solvent on the porous polypropylene fiber membrane; b) injecting a receiving phase into the inner cavity of the receiving compatilizer; c) immersing the lower end of the receiving phase container treated in the step b) in a donor phase (glucoside alkaloid crude extract solution), inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, connecting the working electrode with a power supply anode, connecting the counter electrode with a power supply cathode, applying voltage to the working electrode and the counter electrode to form an electric field, and extracting glucoside alkaloid in the donor phase under the action of the electric field.
Compared with the prior art, the invention has the following beneficial effects:
(1) before the glucoside alkaloid in the potatoes is detected, the three-phase electric film microextraction technology is adopted to extract the glucoside alkaloid in the potatoes, the mass transfer process of the glucoside alkaloid in the extraction process mainly takes electromigration as a main part, and the extraction time is greatly shortened; in addition, separation, purification and enrichment of glucoside alkaloid in the potatoes can be realized during extraction through the selectivity of the membrane and the extraction solvent loaded on the membrane in the extraction process, so that the interference of other impurities in the potatoes on the detection of the glucoside alkaloid is greatly reduced, and the detection accuracy and precision of the glucoside alkaloid are improved.
(2) The invention adopts acidic aqueous solution as solvent to prepare donor phase solution, namely glucoside alkaloid crude extract solution, and the acidic solvent can ensure that glucoside alkaloid exists in a form of single charged ions, thereby being beneficial to the extraction and separation of the glucoside alkaloid under the action of electric field migration.
(3) When the glucoside alkaloid is extracted, 10mmol/L hydrochloric acid solution, 20mmol/L formic acid solution, 20mmol/L acetic acid solution or 10mmol/L trifluoroacetic acid solution is used as a receiving phase, so that the acidic solvent can enable the glucoside alkaloid to exist in a form of single charged ions on the basis that the electric field migration effect is the main mass transfer driving force in the electric membrane extraction process, and the glucoside alkaloid is favorably extracted into a receptor solution under the electric field migration effect.
(4) According to the invention, the extraction solvent is loaded on the porous polypropylene fiber membrane, so that the consumption of the extraction solvent is low, and the problems of high organic solvent consumption and harm to the environment in the traditional extraction technology are avoided; moreover, the liquid phase extraction process and the back extraction process are carried out simultaneously, so that the extraction efficiency is effectively improved.
(5) The detection method is suitable for detecting the glucoside alkaloid in different samples, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of a three-phase electromembrane extraction apparatus in example 1 of the present invention;
FIG. 2 is a standard working curve for the glycoside alkaloid of example 6 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Extraction condition optimization of (I) three-phase electric membrane extraction glucoside alkaloid
Example 1: extraction solvent optimization experiment
In order to investigate the influence of different extraction solvents on the separation and enrichment effects of glucoside alkaloids, the invention takes a mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline as a donor phase to carry out three-phase electromembrane extraction, calculates the recovery rates of the alpha-solanine and the alpha-kaline by detecting the concentrations of the alpha-solanine and the alpha-kaline in a receiving phase after the extraction is finished, and screens the extraction solvents according to the recovery rates. For details of the experiment, see the experiments of examples 1-1 to 1-6 below.
Example 1-1:
separation and enrichment of glycoside alkaloid:
(1) preparing a mixed standard solution of 1mg/L alpha-solanine and 1mg/L alpha-kaline: taking standard products of alpha-solanine and alpha-kaline, taking 10mmol/L phosphate buffer solution with pH 4 as a solvent to prepare mixed standard product solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, taking the mixed standard product solution as a donor phase, and carrying out subsequent three-phase electro-membrane extraction.
(2) Construction and extraction of a three-phase electric membrane extraction device:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 5: 95;
c) injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is 20mmol/L formic acid aqueous solution; a sample bottle is taken, and 1000 mu L of donor phase is added, wherein the donor phase is the mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure schematic diagram of the three-phase electric membrane extraction device is shown in figure 1.
(3) Separation and enrichment:
and (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 120V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 20 min.
Examples 1 to 2:
the contents of example 1-2 are substantially the same as those of example 1-1, except that:
the extraction solvent adopted in the step (2) is a mixed solution of di (2-ethylhexyl) phosphate (DEHP) and 2-nitrobenzene octyl ether, and the volume ratio of the di (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 10: 90.
Examples 1 to 3:
examples 1 to 3 are substantially the same as examples 1 to 1 except that:
the extraction solvent adopted in the step (2) is a mixed solution of di (2-ethylhexyl) phosphate (DEHP) and 2-nitrobenzene octyl ether, and the volume ratio of the di (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 15: 85.
Examples 1 to 4:
examples 1 to 4 are substantially the same as examples 1 to 1 except that:
the extraction solvent adopted in the step (2) is a mixed solution of di (2-ethylhexyl) phosphate (DEHP) and 2-nitrobenzene octyl ether, and the volume ratio of the di (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80.
Examples 1 to 5:
examples 1 to 5 are substantially the same as examples 1 to 1 except that:
the extraction solvent adopted in the step (2) is a mixed solution of bis (2-ethylhexyl) phosphate (DEHP) and 2-nitrophenyloctyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrophenyloctyl ether in the mixed solution is 25: 75.
Examples 1 to 6:
examples 1 to 6 are substantially the same as examples 1 to 1 except that:
the extraction solvent adopted in the step (2) is a mixed solution of di (2-ethylhexyl) phosphate (DEHP) and 2-nitrobenzene octyl ether, and the volume ratio of the di (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 30: 70.
And (3) measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase after the extraction of the embodiment 1-1 to the embodiment 1-6 is finished by adopting a high performance liquid chromatography mass spectrometer, and calculating the recovery rates of the alpha-solanine and the alpha-kaline. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Agilent Eclipse Plus C18 column with a specification of 3.0X 100mm and a particle size of 1.8. mu.m. The mobile phase is 30% water-70% methanol, the flow rate is 0.4mL/min, the sample injection amount is 2.0 muL, the mass spectrometry adopts an ion trap as a mass analyzer, the detection is carried out in a positive ion mode, and the quantification is carried out in an MRM mode, wherein the parent ion of alpha-solanine is 869, the daughter ion thereof is 398.5 and 98, the parent ion of alpha-kaline is 853, and the daughter ion thereof is 398.5 and 98. Specific results are shown in table 1.
TABLE 1 Effect of extraction solvent on the separation and enrichment of glycoside alkaloids
| Examples | Extraction solvent | Recovery ratio of alpha-solanine (%) | Recovery of alpha-Kasolanine (%) |
| Examples 1 to 1 | DEHP: 2-Nitrobenzene octyl ether 5:95 | 31 | 31 |
| Examples 1 to 2 | DEHP: 2-Nitrobenzene octyl ether 10:90 | 37 | 35 |
| Examples 1 to 3 | DEHP: 2-Nitrophenyl octyl ether 15:85 | 40 | 50 |
| Examples 1 to 4 | DEHP: 2-Nitrophenyl octyl ether ═ 20:80 | 78 | 83 |
| Examples 1 to 5 | DEHP: 2-Nitrophenyl octyl ether 25:75 | 53 | 50 |
| Examples 1 to 6 | DEHP: 2-Nitrobenzene octyl ether 30:70 | 43 | 45 |
As can be seen from table 1, the recovery rates of α -solanine and α -kaline increased with the increase of the volume percentage of DEHP in the mixed extraction solvent and then decreased, and when the volume ratio of DEHP to 2-nitrophenyloctyl ether in the extractant was 20:80, the recovery rates of α -solanine and α -kaline reached the highest, respectively 78% and 83%. In subsequent experiments, a mixed solvent of DEHP and 2-nitrobenzene octyl ether with the volume ratio of 20:80 is used as a preferred extractant.
Example 2: screening experiment of Donor phase solvent
In order to investigate the influence of different donor phase solvents on the separation and enrichment effects of glucoside alkaloids, the invention adopts different solvents to prepare a mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, uses the mixed standard solution as a donor phase, carries out three-phase electromembrane extraction, calculates the recovery rates of the alpha-solanine and the alpha-kaline by detecting the concentrations of the alpha-solanine and the alpha-kaline in a receiving phase after the extraction is finished, and screens the donor phase solvents according to the recovery rates. For details of the experiment, see the experiment of example 2-1 to example 2-3 below.
Example 2-1:
separation and enrichment of glycoside alkaloid:
(1) preparing a mixed standard solution of 1mg/L alpha-solanine and 1mg/L alpha-kaline: taking standard substances of alpha-solanine and alpha-kaline, taking 10mmol/L hydrochloric acid as a solvent to prepare a mixed standard substance solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, taking the mixed standard substance solution as a donor phase, and carrying out three-phase electro-membrane extraction.
(2) Construction and extraction of a three-phase electric membrane extraction device:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80;
c) injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is 20mmol/L formic acid aqueous solution; a sample bottle is taken, and 1000 mu L of donor phase is added, wherein the donor phase is the mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure schematic diagram of the three-phase electric membrane extraction device is shown in figure 1.
(3) Separation and enrichment:
and (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 120V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 20 min.
Example 2-2:
the contents of example 2-2 are substantially the same as those of example 2-1 except that:
in the step (1), 20mmol/L formic acid is used as a sample phase solvent to prepare a mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-solanine.
Examples 2 to 3:
the contents of example 2-3 are substantially the same as example 2-1 except that:
in the step (1), a mixed standard solution containing 1mg/L of alpha-solanine and 1mg/L of alpha-kaline is prepared by using a phosphate buffer solution with the pH value of 710 mmol/L as a solvent.
And (3) measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase after the extraction of the embodiment 2-1 to the embodiment 2-3 by adopting a high performance liquid chromatography mass spectrometer, and calculating the recovery rates of the alpha-solanine and the alpha-kaline. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Agilent Eclipse Plus C18 column with a specification of 3.0X 100mm and a particle size of 1.8. mu.m. The mobile phase is 30% water-70% methanol, the flow rate is 0.4mL/min, the sample injection amount is 2.0 muL, the mass spectrometry adopts an ion trap as a mass analyzer, the detection is carried out in a positive ion mode, and the quantification is carried out in an MRM mode, wherein the parent ion of alpha-solanine is 869, the daughter ion is 398.5 and 98, the parent ion of alpha-kaline is 853, and the daughter ion is 398.5 and 98. Specific results are shown in table 2.
TABLE 2 Effect of Donor phase solvent on glycoside alkaloid separation and enrichment
As can be seen from table 2, when the sample phase solvent is a phosphate buffer solution with a pH of 410 mmol/L, the extraction recovery rates of α -solanine and α -kaline are the highest, which is probably because the polarities of α -solanine (logP ═ 0.43) and α -kaline (logP ═ 1.12) are relatively large, and the addition of the phosphate buffer solution to the sample phase can form an ion pair with α -solanine and α -kaline, thereby increasing the partition coefficient in the extraction solvent, and promoting the electromembrane extraction of α -solanine and α -kaline. Therefore, the donor phase solvent is preferably phosphate buffer at pH 410 mmol/L.
Example 3: screening experiments for phase-accepting Agents
In order to investigate the influence of separation and enrichment effects of different receiving phases on glucoside alkaloids, the method takes different solutions as receiving phases to perform three-phase electro-membrane extraction, calculates the recovery rates of alpha-solanine and alpha-kaline by detecting the concentrations of the alpha-solanine and the alpha-kaline in the receiving phases after the extraction is finished, and screens a donor phase according to the recovery rates. For details of the experiment, see the experiment of example 3-1 to example 3-3 below.
Example 3-1:
separation and enrichment of glycoside alkaloid:
(1) preparing a mixed standard solution of 1mg/L alpha-solanine and 1mg/L alpha-kaline: taking standard products of alpha-solanine and alpha-kaline, taking 10mmol/L phosphate buffer solution with pH 4 as a solvent to prepare mixed standard product solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, taking the mixed standard product solution as a donor phase, and carrying out three-phase electro-membrane extraction.
(2) Construction and extraction of a three-phase electric membrane extraction device:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80;
c) injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is 10mmol/L hydrochloric acid aqueous solution; a sample bottle is taken, and 1000 mu L of donor phase is added, wherein the donor phase is the mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure schematic diagram of the three-phase electric membrane extraction device is shown in figure 1.
(3) Separation and enrichment:
and (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 120V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 20 min.
Example 3-2:
the contents of example 3-2 are substantially the same as those of example 3-1 except that:
the receiving phase in the step (2) is a 20mmol/L acetic acid solution.
Examples 3 to 3:
the contents of example 3-3 are substantially the same as those of example 3-1 except that:
the receiving phase in the step (2) is a 10mmol/L trifluoroacetic acid solution.
And (3) measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase after the extraction of the embodiment 3-1 to the embodiment 3-3 by adopting a high performance liquid chromatography mass spectrometer, and calculating the recovery rates of the alpha-solanine and the alpha-kaline. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Agilent Eclipse Plus C18 column with a specification of 3.0X 100mm and a particle size of 1.8. mu.m. The mobile phase is 30% water-70% methanol, the flow rate is 0.4mL/min, the sample injection amount is 2.0 muL, the mass spectrometry adopts an ion trap as a mass analyzer, the detection is carried out in a positive ion mode, and the quantification is carried out in an MRM mode, wherein the parent ion of alpha-solanine is 869, the daughter ion is 398.5 and 98, the parent ion of alpha-kaline is 853, and the daughter ion is 398.5 and 98. Specific results are shown in table 3.
Table 3 receives the effect of phase solvent on the separation and enrichment of glycoside alkaloids
| Examples | Receiving phase | Recovery ratio of alpha-solanine (%) | Recovery of alpha-Kasolanine (%) |
| Example 3-1 | 10mmol/L hydrochloric acid | 25 | 25 |
| Examples 3 to 2 | 20mmol/L acetic acid solution | 27 | 26 |
| Examples 3 to 3 | 10mmol/L trifluoroacetic acid solution | 13 | 16 |
| Examples 1 to 4 | 20mmol/L formic acid solution | 78 | 83 |
As is clear from Table 3, the highest recovery rates of α -solanine and α -kaline were obtained when a 20mmol/L formic acid solution was used as the receiving phase solution. The main reasons are: during the process of electro-membrane extraction of alpha-solanine and alpha-kaline, the pH of a receiving phase solution can gradually rise, but analytes can be back-extracted, so that the extraction rate is reduced. The 20mmol/L formic acid has certain buffering capacity and can stabilize the pH of a receiving phase and the current of a system in the extraction process; however, when a 20mmol/L acetic acid solution is used as the receiving phase solution, the acetic acid solution is easily leaked into the extraction solvent at the beginning of extraction, and the composition of the extraction solvent is changed, so that the extraction of alpha-solanine and alpha-kaline is inhibited. When 10mmol/L trifluoroacetic acid solution is the receiving phase solution, the current is larger when the alpha-solanine and the alpha-kaline are extracted by the electric film, the system is unstable, and the phenomenon of double electric layers is possibly generated, thereby inhibiting the extraction of the alpha-solanine and the alpha-kaline. Therefore, the phase solvent is preferably 20mmol/L formic acid solution.
Example 4: screening experiment of extraction Voltage
In order to discuss the influence of different extraction voltages on the separation and enrichment effects of glucoside alkaloids, the method carries out three-phase electric membrane extraction under different voltages, calculates the recovery rates of alpha-solanine and alpha-kaline by detecting the concentrations of the alpha-solanine and the alpha-kaline in a receiving phase after the extraction is finished, and screens the extraction voltages according to the recovery rates. For details of the experiment, see the experiment of example 4-1 to example 4-5 below.
Example 4-1:
separation and enrichment of glycoside alkaloid:
(1) preparing a mixed standard solution of 1mg/L alpha-solanine and 1mg/L alpha-kaline: taking standard products of alpha-solanine and alpha-kaline, taking 10mmol/L phosphate buffer solution with pH 4 as a solvent to prepare mixed standard product solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, taking the mixed standard product solution as a donor phase, and carrying out three-phase electro-membrane extraction.
(2) Construction and extraction of a three-phase electric membrane extraction device:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80;
c) injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is 20mmol/L formic acid aqueous solution; a sample bottle is taken, and 1000 mu L of donor phase is added, wherein the donor phase is the mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure schematic diagram of the three-phase electric membrane extraction device is shown in figure 1.
(3) Separation and enrichment:
and (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 40V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 20 min.
Example 4-2:
the contents of example 4-2 are substantially the same as those of example 4-1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 60V.
Examples 4 to 3:
the contents of example 4-3 are substantially the same as those of example 4-1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 80V.
Examples 4 to 4:
the contents of example 4-4 are substantially the same as those of example 4-1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 100V.
Examples 4 to 5:
examples 4 to 5 are substantially the same as example 4 to 1 except that:
and (4) the voltage of the power supply of the electrophoresis apparatus in the step (3) is 150V.
And (3) measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase after the extraction of the embodiment 4-1 to the embodiment 4-5 is finished by adopting a high performance liquid chromatography mass spectrometer, and calculating the recovery rates of the alpha-solanine and the alpha-kaline. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Agilent Eclipse Plus C18 column with a specification of 3.0X 100mm and a particle size of 1.8. mu.m. The mobile phase is 30% water-70% methanol, the flow rate is 0.4mL/min, the sample injection amount is 2.0 muL, the mass spectrometry adopts an ion trap as a mass analyzer, the detection is carried out in a positive ion mode, and the quantification is carried out in an MRM mode, wherein the parent ion of alpha-solanine is 869, the daughter ion is 398.5 and 98, the parent ion of alpha-kaline is 853, and the daughter ion is 398.5 and 98. Specific results are shown in table 4.
TABLE 4 Effect of Voltage on separation and enrichment of glycoside alkaloid
| Examples | Voltage (V) | Recovery ratio of alpha-solanine (%) | Recovery of alpha-Kasolanine (%) |
| Example 4-1 | 40 | 37 | 40 |
| Example 4 to 2 | 60 | 45 | 47 |
| Examples 4 to 3 | 80 | 59 | 62 |
| Examples 4 to 4 | 100 | 69 | 74 |
| Examples 1 to 4 | 120 | 78 | 83 |
| Examples 4 to 5 | 150 | 36 | 40 |
As shown in table 4, as the extraction voltage is increased from 40V to 120V, the recovery rate of α -solanine is increased from 37% to 78%, the recovery rate of α -kalopane is increased from 40% to 83%, the extraction voltage is further increased to 150V, and the recovery rates of α -solanine and α -kalopane are respectively decreased to 36% and 40%. The main reason is that electromigration is a mass transfer process of electromembrane extraction, and increasing extraction voltage in a certain range can promote the extraction of alpha-solanine and alpha-kaline, but too high extraction voltage can cause electrolysis, so that the system current is increased, the system is unstable, and the extraction of alpha-solanine and alpha-kaline can be inhibited. Therefore, the extraction voltage is preferably 120V.
Example 5: screening experiment for extraction time
In order to discuss the influence of different extraction times on the separation and enrichment effects of glucoside alkaloids, the invention extracts the glucoside alkaloids by a three-phase electric film at different times, calculates the recovery rates of alpha-solanine and alpha-kaline by detecting the concentrations of the alpha-solanine and the alpha-kaline in a receiving phase after the extraction is finished, and screens extraction voltage according to the recovery rates. For details of the experiment, see the experiment of example 5-1 to example 5-5 below.
Example 5-1:
separation and enrichment of glycoside alkaloid:
(1) preparing a mixed standard solution of 1mg/L alpha-solanine and 1mg/L alpha-kaline: taking standard products of alpha-solanine and alpha-kaline, taking 10mmol/L phosphate buffer solution with pH 4 as a solvent to prepare mixed standard product solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline, taking the mixed standard product solution as a donor phase, and carrying out three-phase electro-membrane extraction.
(2) Construction and extraction of a three-phase electric membrane extraction device:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80;
c) injecting 200 mu L of receiving phase into the receiving compatilizer; the receiving phase is 20mmol/L formic acid aqueous solution; a sample bottle is taken, and 1000 mu L of donor phase is added, wherein the donor phase is the mixed standard solution containing 1mg/L alpha-solanine and 1mg/L alpha-kaline prepared in the step (1); inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving phase compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of the electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. The structure schematic diagram of the three-phase electric membrane extraction device is shown in figure 1.
(3) Separation and enrichment:
and (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 120V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 5 min.
Example 5-2:
the contents of example 5-2 are substantially the same as those of example 5-1 except that:
the extraction time in the step (3) is 10 min.
Examples 5 to 3:
the contents of example 5-3 are substantially the same as those of example 5-1 except that:
the extraction time in the step (3) is 15 min.
Examples 5 to 4:
the contents of example 5-4 are substantially the same as example 5-1 except that:
the extraction time in the step (3) is 25 min.
Examples 5 to 5:
the contents of example 5-5 are substantially the same as example 5-1 except that:
the extraction time in the step (3) is 30 min.
And (3) measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase after the extraction of the embodiment 5-1 to the embodiment 5-5 is finished by adopting a high performance liquid chromatography mass spectrometer, and calculating the recovery rates of the alpha-solanine and the alpha-kaline. The detection conditions of the high performance liquid chromatography mass spectrometer are as follows: the column was an Agilent Eclipse Plus C18 column with a specification of 3.0X 100mm and a particle size of 1.8. mu.m. The mobile phase is 30% water-70% methanol, the flow rate is 0.4mL/min, the sample injection amount is 2.0 muL, the mass spectrometry adopts an ion trap as a mass analyzer, the detection is carried out in a positive ion mode, and the quantification is carried out in an MRM mode, wherein the parent ion of alpha-solanine is 869, the daughter ion is 398.5 and 98, the parent ion of alpha-kaline is 853, and the daughter ion is 398.5 and 98. Specific results are shown in table 5.
TABLE 5 Effect of Voltage on separation and enrichment of glycoside alkaloid
| Examples | Extraction time (min) | Recovery ratio of alpha-solanine (%) | Recovery of alpha-Kasolanine (%) |
| Example 5-1 | 5 | 25 | 26 |
| Examples 5 and 2 | 10 | 37 | 48 |
| Examples 5 to 3 | 15 | 54 | 60 |
| Examples 1 to 4 | 20 | 78 | 83 |
| Examples 5 to 4 | 25 | 57 | 59 |
| Examples 5 to 5 | 30 | 56 | 64 |
As can be seen from table 5, as the extraction time increases, the recovery rate of α -solanine and α -kaline increases first and then decreases, and when the extraction time is 20min, the recovery rates of α -solanine and α -kaline reach the highest values, respectively 78% and 83%, and the extraction time continues to be prolonged, but the recovery rates of α -solanine and α -kaline decrease instead, because the extraction time continues to be prolonged after the extraction system reaches equilibrium, which in turn causes the α -solanine and α -kaline to be back-extracted to the membrane phase or the sample phase, so that the recovery rates decrease. Therefore, 20min was chosen as the optimal extraction time.
(II) separation, enrichment and detection of glucoside alkaloid in potato
The glucoside alkaloid in the potato is separated, enriched and detected under the optimal extraction condition obtained by optimizing the embodiment (I).
Example 6:
a method for detecting glucoside alkaloids in potato comprises the following steps:
(1) preparation of potato samples:
washing fresh potato purchased in farmer market in clear water for 3-5 times until the surface of the potato has no impurities, soil and the like, and exposing the potato to sunlight until the potato skin turns green or germinates. Peeling off the buds from the matrix after the buds turn green, simultaneously peeling the green potato peels, cutting the green potato peels into strips with uniform sizes, respectively placing the peels and the buds in a drying box, drying to constant weight, then placing in a crusher, crushing and sieving by a 40-mesh sieve to obtain potato analysis samples, and placing the potato analysis samples in a polyethylene plastic bag for storage at normal temperature for later use.
(2) Extracting a crude extract of the patatin alkaloid:
weighing 5.0g potato sample, adding 20mL methanol solution containing 5% acetic acid, performing ultrasonic treatment at 35 deg.C for 20min, vacuum filtering, extracting for 2-3 times, mixing the two filtrates, and adding into 50mL volumetric flask to desired volume. Transferring 20mL of extracting solution from the raw material, and evaporating the extracting solution under reduced pressure to obtain a crude glycoside alkaloid extract.
(3) Three-phase electromembrane extraction:
dissolving the crude glucoside alkaloid extract obtained in the step (2) by using a phosphate buffer solution with the pH value of 410 mmol/L to obtain a crude glucoside alkaloid extract solution, and performing three-phase electromembrane extraction by using the solution as a donor phase and using a 20mmol/L formic acid solution as a receiving phase. The specific operation of the three-phase electromembrane extraction is as follows:
a) selecting a 1mL pipette suction head, fixing a porous polypropylene fiber membrane with the aperture of 0.2 mu m and the thickness of 200 mu m at the wide-mouth end of the pipette suction head at the high temperature of an electric soldering iron, and cutting off part of the length of the other end of the suction head to obtain a receiving phase container.
b) Coating 10 mu L of extraction solvent on the surface of the porous polypropylene fiber membrane, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrobenzene octyl ether, and the volume ratio of the bis (2-ethylhexyl) phosphate to the 2-nitrobenzene octyl ether in the mixed solution is 20: 80;
c) injecting 200 mu L of receiving phase into a receiving compatilizer, adding 1000 mu L of donor phase solution into a sample bottle, inserting a working electrode into the donor phase, inserting a counter electrode into the receiving phase, then placing the receiving compatilizer into the sample bottle, enabling the porous polypropylene fiber membrane to be just contacted with the liquid level of the donor phase solution of the sample bottle, connecting the working electrode with the positive electrode of an electrophoresis apparatus power supply, and connecting the counter electrode with the negative electrode of the electrophoresis apparatus power supply. And (3) placing the sample bottle on a constant-temperature blending instrument, starting a power supply of the electrophoresis instrument and the blending instrument, and extracting the glucoside alkaloid under the driving of an external voltage and the auxiliary action of oscillation. The voltage of the power supply of the electrophoresis apparatus is 120V, the rotating speed of the constant-temperature blending apparatus is 1000rpm, and the extraction time is 20 min.
(4) And after extraction is finished, taking out the receiving phase solution in the receiving phase container, and measuring the concentrations of the alpha-solanine and the alpha-kaline in the receiving phase by adopting a high performance liquid chromatography mass spectrometer.
When the concentrations of the alpha-solanine and the alpha-kaline in a receiving phase are measured by adopting a high performance liquid chromatography mass spectrometer, a standard working curve of the alpha-solanine and the alpha-kaline needs to be firstly made.
The standard working curve of the alpha-solanine and the alpha-kaline is prepared by the following steps:
cutting a fresh potato sample into strips with uniform size, drying the strips in a drying oven to constant weight, then crushing the strips in a crusher and sieving the strips with a 40-mesh sieve to obtain a blank matrix sample. Weighing 5.0g of blank matrix sample, adding 20mL of methanol solution containing 5% acetic acid, performing ultrasonic treatment at 35 deg.C for 20min, vacuum filtering, extracting for 2-3 times, mixing the two filtrates, and placing in a 50mL volumetric flask to constant volume. Removing 20mL of the extract therefrom, and evaporating under reduced pressure to obtain a blank matrix sample solution. A series of glucoside alkaloid standard substances (alpha-solanine and alpha-kaline) with different concentrations are respectively added into the blank matrix sample solution to prepare a glucoside alkaloid blank matrix labeling working solution with the concentration gradient of 20 mu g/L-1000 mu g/L. Extracting the glycoside alkaloid blank matrix standard solution according to the three-phase electric membrane extraction operation in the step (3), measuring chromatogram peak areas of alpha-solanine and alpha-kaline in a receiving phase by using a high performance liquid chromatography mass spectrometer after extraction, drawing working curves of the alpha-solanine and the alpha-kaline (as shown in figure 2), and obtaining related performance parameters of the detection method provided by the invention, wherein the related performance parameters comprise a linear range, a linear correlation coefficient, a detection limit and a quantitative limit, and are shown in a table 6.
Table 6 relevant parameters for the glycoside alkaloid blank matrix spiked solution detection method.
| Analyte | Linear Range (μ g/L) | Linear correlation coefficient R2 | Detection limit | Limit of quantification |
| Alpha-solanine | 5-1000 | 0.9993 | 1.5 | 5.2 |
| Alpha-kaline | 5-1000 | 0.9992 | 1.2 | 4.1 |
Through detection, the concentrations of the alpha-solanine and the alpha-kaline in the green potato peel are respectively 32.36mg/kg and 79.76mg/kg, and the concentrations of the alpha-solanine and the alpha-kaline in the green potato tuber are respectively 637.70mg/kg and 784.90 mg/kg.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting glucoside alkaloids in potatoes is characterized by comprising the following steps:
(1) taking a potato sample, and drying and crushing the potato sample to obtain a potato analysis sample;
(2) mixing a potato analysis sample with a solvent, ultrasonically extracting glucoside alkaloid in the potato analysis sample, filtering after extraction, collecting filtrate, and evaporating the filtrate to dryness to obtain a glucoside alkaloid crude extract;
(3) preparing a glucoside alkaloid crude extract solution from the glucoside alkaloid crude extract obtained in the step (2), extracting glucoside alkaloid in the glucoside alkaloid crude extract solution by using a three-phase electric film extraction technology by taking the glucoside alkaloid crude extract solution as a donor phase, and detecting the concentration of the glucoside alkaloid in a receiving phase in the three-phase electric film extraction after the extraction is finished.
2. The method according to claim 1, wherein the membrane used in the three-phase electromembrane extraction technology in step (3) is a porous polypropylene fiber membrane, and an extraction solvent is loaded on the porous polypropylene fiber membrane, and the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether.
3. The method according to claim 2, wherein the extraction solvent is a mixed solution of bis (2-ethylhexyl) phosphate and 2-nitrophenyloctyl ether, and the volume percentage of the bis (2-ethylhexyl) phosphate in the mixed solution is 5-30%.
4. The method according to claim 2, wherein the porous polypropylene fiber membrane has a pore size of 0.2 μm and a thickness of 100 μm to 200 μm.
5. The method according to claim 1, wherein the crude glycoside alkaloid extract solution in step (3) is prepared by using an acidic aqueous solution as a solvent, wherein the acidic aqueous solution is any one of 10mmol/L hydrochloric acid, 20mmol/L formic acid and 10mmol/L phosphate buffer solution with pH = 4.
6. The method of claim 5, wherein the receiving phase used in the three-phase electromembrane extraction technique in step (3) is any one of a 10mmol/L hydrochloric acid solution, a 20mmol/L formic acid solution, a 20mmol/L acetic acid solution and a 10mmol/L trifluoroacetic acid solution.
7. The method according to claim 1, wherein the voltage for applying the electric field between the donor phase and the receiving phase in the three-phase electromembrane extraction technique in step (3) is 40-150V.
8. The method according to claim 1, wherein the solvent in step (2) is a methanol solution containing 5% acetic acid, and the temperature of the ultrasonic extraction is 30-40 ℃.
9. The method according to claim 1, wherein the extraction time in step (3) is 5min to 30 min.
10. The method of claim 1, wherein the glycoside alkaloid is alpha-solanine and/or alpha-kaline.
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