CN112415103B - Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC - Google Patents
Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC Download PDFInfo
- Publication number
- CN112415103B CN112415103B CN202011146591.5A CN202011146591A CN112415103B CN 112415103 B CN112415103 B CN 112415103B CN 202011146591 A CN202011146591 A CN 202011146591A CN 112415103 B CN112415103 B CN 112415103B
- Authority
- CN
- China
- Prior art keywords
- sample
- furocoumarin
- mekc
- mcds
- fesi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC, which comprises the following steps: mixing a sample to be detected with an adsorbent, crushing to obtain mixed powder, eluting the mixed powder by using a solid phase extraction column, collecting effluent liquid, and volatilizing a solvent to obtain an extract; respectively pretreating the capillary and the extract, carrying out electrophoretic analysis based on FESI-MCDS-MEKC to obtain the peak area of furocoumarin in the extract, and substituting the peak area into a corresponding standard curve to obtain the furocoumarin content in the sample to be detected. The method has the advantages of simple operation, short analysis time, high separation efficiency, accuracy, reliability, good reproducibility, and enrichment multiple up to 302 times, and can accurately determine the contents of Notopterygii rhizoma alcohol, isoimperatorin and imperatorin in a sample to be tested.
Description
Technical Field
The invention relates to the technical field of analysis and detection, and particularly relates to a method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC.
Background
Furocoumarin is a lactone compound containing a benzo alpha-pyrone structure in plant, animal and microorganism metabolites, has various biological activities such as pain relieving, inflammation diminishing, microorganism resisting, convulsion resisting, cancer resisting and the like, is widely applied in the pharmaceutical industry, but the phototoxicity shown by the furocoumarin is also widely concerned by students.
Trace amounts of furocoumarin under uv irradiation may cause skin burns, and high doses of furocoumarin have shown toxicity to different tissues in mice even in the absence of uv irradiation. Therefore, furocoumarin is listed as a forbidden substance in the chinese cosmetic industry regulations. However, in many so-called natural whitening products, the furocoumarin is still reported to be contained, which has great safety hazards for consumers. Therefore, developing a rapid, low-cost, green assay is particularly important for monitoring trace amounts of furocoumarin in cosmetics.
Imperatorin, isoimperatorin, notopterygium alcohol and the like are typical furocoumarins. In particular imperatorin and isoimperatorin, as main ingredients in angelica dahurica, are often likely to be added into natural whitening products. Currently, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and other methods are commonly used for determination of coumarins, but these methods are expensive in materials and often require a large amount of organic reagents and cumbersome procedures.
The capillary electrophoresis Chromatography (CE) method has the advantages of high resolution, low solvent consumption, simple operation and the like, but has low detection sensitivity on neutral substances. To overcome this drawback, one method is to adopt the on-line enrichment technique of CE, and the other method is to adopt an effective sample pretreatment means (off-line enrichment).
Neutral species are not charged in the electrolyte and cannot be separated in the conventional Capillary Zone Electrophoresis (CZE) mode, which is a problem in capillary electrophoresis separation. Micellar electrokinetic capillary chromatography (MEKC) is another mode of CE that accomplishes the separation and determination of neutral analytes by adding micelles to background Buffer (BGE). However, the traditional Chinese medicine components are complex and have low content, and the CE quantitative analysis of neutral substances such as coumarins, saponins, terpenoids and the like is still greatly limited. In order to improve the detection sensitivity of neutral substances, on-line enrichment methods based on MEKC are gradually developed, such as purging technology (Sweeping), field amplification sample introduction-reverse micelle migration technology (FESI-RMM), micellar cyclodextrin accumulation technology (MCDS), and the like.
FESI is a method of injecting a low conductivity sample solution into a capillary by its own electrophoretic mobility under electrokinetic sample injection, thereby increasing the sample injection amount to achieve enrichment. It was reported to be used in combination with RMM to enrich for neutral species.
MCDS-MEKC is a novel on-line enrichment technology proposed by Quirino, and has the advantage of no need of complex sample pretreatment process. The method comprises the steps of filling a background buffer solution containing micelles in a capillary, then respectively feeding cyclodextrin and a micelle solution containing a sample, and reversing the effective mobility of an analyte to be detected through the combination action of the micelles and the cyclodextrin so as to stack the analyte at the boundary of a sample zone and a cyclodextrin zone. The method is also an effective neutral substance on-line enrichment technology. In order to further improve the sensitivity of CE detection on neutral substances, field amplification sample introduction and micelle cyclodextrin accumulation are combined under a reverse migration micelle electrokinetic chromatography mode, separation and enrichment conditions are optimized, and a method for determining the neutral substances, which is simple, convenient, good in reproducibility and environment-friendly, is expected to be established.
However, the enrichment factor of these methods is mostly only several times to several tens times, and the sensitivity thereof still needs to be improved.
Disclosure of Invention
The invention provides a method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC, which is characterized in that a molecular sieve is used as an adsorbent, and matrix solid phase dispersion extraction (MSPD) and field amplification sample accumulation (FESI) -micelle cyclodextrin accumulation reverse migration micelles (MCDS-MEKC) are combined to be used for determining trace furocoumarin compounds in a complex matrix, so that the detection sensitivity of capillary electrophoresis chromatography is further improved, and notopterygium alcohol, isoimperatorin or imperatorin with similar properties can be separated.
A method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC comprises the following steps:
mixing a sample to be detected with an adsorbent, crushing to obtain mixed powder, eluting the mixed powder by using a solid phase extraction column, collecting effluent liquid, and volatilizing a solvent to obtain an extract;
respectively preprocessing the capillary and the extract, performing electrophoretic analysis based on FESI-MCDS-MEKC to obtain the peak area of furocoumarin in the extract, and substituting the peak area into a corresponding standard curve to obtain the furocoumarin content in the sample to be detected.
The adsorbent is a molecular sieve; preferably molecular sieve KIT-6, KIT-6 is used as adsorbent, and the extraction effect of isoimperatorin and imperatorin is the best.
The invention discloses a method for on-line determination of furocoumarin content, which is a method for analyzing and determining trace photosensitive furocoumarin components in cosmetics by a combined technology of matrix solid phase dispersion extraction (MSPD) combined with field amplification sample accumulation (FESI) -micelle cyclodextrin accumulation reverse migration micelle (MCDS-MEKC). Combining a matrix solid phase dispersion extraction technology with a field amplification sample accumulation combined micelle cyclodextrin accumulation reverse migration micelle technology, using a molecular sieve as an adsorbent in MSPD to be adsorbed and combined with target molecules of a sample to be detected, separating the target molecules from a complex matrix, eluting with a proper solvent to obtain eluent containing the target molecules, and then separating and detecting the eluent by adopting a FESI-MCDS-MECK electrophoresis enrichment mode.
Matrix solid phase dispersion extraction (MSPD) can selectively extract target analytes while destroying the structure of a solid or semisolid material, thereby not only reducing the loss of samples, but also avoiding the problems of large amount of organic solvents used in liquid-liquid extraction, the phenomenon of 'emulsification' and low extraction speed.
The furocoumarin is notopterygium alcohol, isoimperatorin or imperatorin. Notopterygium incisum alcohol, isoimperatorin or imperatorin have similar structures and similar physical properties, and the conventional separation method has low separation degree.
The mass ratio of the sample to be detected to the adsorbent is 1: 1.5 to 2.5. When the dosage of the adsorbent is too small, the adsorption is incomplete; when the amount of the adsorbent is too much, the sample to be detected is difficult to elute.
The grinding is carried out through uniform-speed grinding, the time for uniform-speed grinding is 140-160 s, the sample and the adsorbent reach adsorption balance, the grinding time is increased, the acting force of the sample and the adsorbent is too strong, and the extraction efficiency is reduced.
The eluent for elution is methanol, ethanol, acetonitrile, acetone or ethyl acetate; the extraction efficiency of methanol is best, and the eluent for elution is preferably methanol.
The volume of the eluent is 400-600 mu L per 25mg of sample to be detected, the analyte is sufficiently eluted at the moment, and the elution volume is increased continuously to dilute the analyte, so that the extraction efficiency is reduced.
The capillary is pretreated, and a new capillary column is washed and activated by NaOH (1mol/L and 20min), NaOH (0.1mol/L and 10min), pure water (10min) and buffer (5min) in sequence under the pressure of 50mbar before use.
The pretreatment of the extract comprises the following steps: the extract was redissolved with 20mmol/L sodium dodecyl sulfate to obtain an extract solution with a sample matrix of sodium dodecyl sulfate.
The conditions of the electrophoretic analysis are as follows:
using 80-120 mmol/L sodium dodecyl sulfate, 5-15 mmol/L sodium dihydrogen phosphate, 40-60 mmol/L phosphoric acid and 10% -40% methanol as buffer solution; firstly adding cyclodextrin solution at 40-60 mbar pressure, then adding pure water at 40-60 mbar pressure, and finally adding the extract solution of which the sample matrix is sodium dodecyl sulfate at-5 to-10 kV voltage, wherein the separation voltage is-15 to-25 kV, the temperature is 20-30 ℃, and the detection wavelength is 254 nm.
The content of methanol in the buffer solution is 28-32%, peaks of three analytes can be completely separated, and the response value is highest.
The sample injection time of the cyclodextrin solution is 45-225 s; the sample introduction time of the sample solution is 160-200 s.
The sensitivity and the separation degree are comprehensively considered, and the sample injection time of the cyclodextrin solution is preferably 110-130 s; the sample introduction time of the sample solution is preferably 140-160 s.
The sample introduction time of the pure water is 1-3 s.
The cyclodextrin is any one of isopropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, alpha-cyclodextrin or beta-cyclodextrin.
The standard curve establishing method comprises the following steps: and respectively carrying out the electrophoretic analysis on the standard solutions of the notopterygium alcohol, the isoimperatorin and the imperatorin with the concentrations of 1.0-12.5 mu g/mL, and respectively drawing standard curves of the notopterygium alcohol, the isoimperatorin and the imperatorin by taking the peak area in an electrophoretic spectrogram as a vertical coordinate and the concentrations as a horizontal coordinate.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a method for extracting coumarin by using a molecular sieve as an adsorbent and combining a matrix solid-phase dispersion extraction technology. Compared with the conventional extraction method, the method is simple, time-saving and low in cost, and the consumption of the organic solvent is low, so that the pollution to the environment and the damage to the health of a human body are reduced. In addition, the molecular sieve has the excellent performances of large specific surface area, ordered pore size distribution, good hydrothermal stability and the like, and the extraction efficiency is also greatly improved.
2. The method uses a capillary electrophoresis online enrichment technology (field amplification sample accumulation combined micelle cyclodextrin accumulation reverse migration micelle technology) for analyzing coumarin, has the advantages of simple and convenient operation, short analysis time, high separation efficiency, accuracy and reliability, good reproducibility, and enrichment times up to 302 times, is more suitable for the analysis of traditional Chinese medicine components of complex matrixes than a high performance liquid chromatography, better meets the requirement of green chemistry, and can accurately determine the contents of notopterygium alcohol, isoimperatorin and imperatorin in a sample to be detected.
Drawings
FIG. 1 shows structural formulas of Notopterygii rhizoma alcohol, isoimperatorin and imperatorin.
Fig. 2 is an electrophoretic image of an actual sample, in which 2 represents isoimperatorin and 3 represents imperatorin.
FIG. 3 is a graph showing the effect of the type of adsorbent.
FIG. 4 effect of sample to adsorbent ratio.
FIG. 5 is a graph showing the effect of milling time.
FIG. 6 influence of eluent species.
Fig. 7 effect of eluent volume.
FIG. 8 shows the effect of organic solvents in the buffer, 1 for notopterygium alcohol, 2 for isoimperatorin and 3 for imperatorin.
FIG. 9 Effect of the injection time of Cyclodextrin (standard concentration 1. mu.g/mL), 1 represents Notopterygium incisum alcohol, 2 represents isoimperatorin, and 3 represents imperatorin.
FIG. 10 influence of sample injection time (standard concentration of 1. mu.g/mL), 1 for Notopterygii rhizoma alcohol, 2 for isoimperatorin, 3 for imperatorin; a is an electrophoretogram under different sample injection time, and B is the peak area of each analyte under different sample injection time.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Reagents or materials used in embodiments of the invention are commercially available:
the solid phase extraction column is a 1mL column tube made of PP and purchased from Shanghai' an spectral laboratory science and technology Co.
The size of the small column sieve plate is 1mL, the material is PE, and the small column sieve plate is purchased from Shanghai' an spectral experiment science and technology company Limited.
Molecular sieve KIT-6, preferably available from Nanjing Ginko nanotechnology GmbH.
Molecular sieve SBA-15, preferably available from Nanjing Ginko nanotechnology GmbH.
Florida, preferably from Shanghai' an spectral laboratory science and technology, Inc.
Al 2 O 3 Preferably from Shanghai' an spectral laboratory science and technology, Inc.
Silica gel mSiO 2 ·nH 2 O, preferably from Shanghai's spectral laboratory science and technology, Inc.
Example 1
Mixing 25mg of radix Angelicae Dahuricae powder with 50mg of molecular sieve KIT-6, grinding for 150s to obtain mixed powder, eluting the mixed powder with solid phase extraction column with 500 μ L methanol as eluent, and volatilizing methanol in rapid solvent volatilizer to obtain the extract.
Pretreating the capillary, and washing and activating the new capillary column with NaOH (1mol/L,20min), NaOH (0.1mol/L,10min), pure water (10min) and buffer solution (5min) in sequence under 50mbar pressure before use;
the extract is re-dissolved by 20mmol/L sodium dodecyl sulfate to obtain an extract solution with a sample matrix of sodium dodecyl sulfate.
And then carrying out electrophoretic analysis based on the FESI-MCDS-MEKC to obtain the peak area of the furocoumarin in the extracting solution (extract), substituting the peak area into a corresponding standard curve to obtain the furocoumarin content in the sample to be detected, wherein the establishment of the standard curve is embodiment 4.
The electrophoresis conditions are as follows: buffer solution: 100mmol/L SDS,10mmol/L sodium dihydrogen phosphate, 50mmol/L phosphoric acid and 30% methanol; cyclodextrin solution: 50mmol/L hydroxypropyl-beta-cyclodextrin, and 100mmol/L phosphoric acid as solvent; sample matrix: 20mmol/L SDS; sample injection procedure: sequentially adding cyclodextrin solution 120s and pure water 2s under 50mbar pressure; sample solution was injected for 150s at-8 kv. And (3) detecting the temperature: 25 ℃; detecting voltage: -20 kv; detection wavelength: 254 nm.
The structural formulas of Notopterygii rhizoma alcohol, isoimperatorin and imperatorin are shown in figure 1.
The electrophoresis spectrogram of radix Angelicae Dahuricae under the preferred condition of MSPD-FESI-MCDS-MEKC is shown in FIG. 2, and the result shows that 3 effective components can be completely separated, and the feasibility of the extraction method and detection conditions is determined. Wherein Notopterygii rhizoma alcohol is not detected, 2 represents isoimperatorin, and 3 represents imperatorin.
Example 2 MSPD Condition optimization
(1) Influence of adsorbent species
Referring to the method in example 1, radix angelicae is taken as an actual sample, and the influence of the molecular sieve on the MSPD enrichment effect is examined.
The influence of two molecular sieves KIT-6 and SPA-16 and other traditional adsorbents of florisil silica, alumina and silica gel on the extraction efficiency of MSPD is examined, and the result is shown in figure 3. The results show that KIT-6 has the best extraction effect of two target analytes when used as an adsorbent. This may be due to its 3D steric structure increasing the specific surface area, the sample being able to contact the adsorbent more fully. Therefore, KIT-6 was chosen as the adsorbent.
(2) Effect of sample to adsorbent ratio
Referring to the method of example 1, radix angelicae dahuricae is used as an actual sample, and the influence of the ratio of the sample to the adsorbent on the factors of the MSPD enrichment effect is examined.
The sample to adsorbent ratio is one of the important factors affecting the extraction efficiency. The results of the extraction of isoimperatorin and imperatorin at sample to adsorbent ratios of 1:1, 1:2, 1:3 and 2:1, respectively, were examined at 25mg, and are shown in figure 4. It can be seen from the figure that the peak areas of the two analytes are highest when the amount ratio of the sample to the adsorbent is 1: 2. This is probably because when the sample to adsorbent ratio was 1:1 and 2:1, the adsorbent was used in too little amount and adsorption was incomplete; when the ratio is 1:3, the amount of the adsorbent is too large, resulting in difficulty in elution of the analyte. Thus, the experiment chose a sample to adsorbent dosage ratio of 1: 2.
(3) Influence of grinding time
Referring to the method of example 1, radix angelicae dahuricae was used as an actual sample, and the influence of grinding time on the MSPD enrichment effect was examined.
One of the basic operations of the MSPD is to mix and grind a sample and an adsorbent to destroy the tissue structure of the sample, so that the sample is completely dispersed on the surface of the adsorbent, the contact surface between the sample and an eluent is increased, and the extraction efficiency is improved. Therefore, the present study examined the effect of milling times of 90, 120, 150 and 180s, respectively, on extraction efficiency, and the results are shown in fig. 5. As can be seen, the milling time increased from 90s to 180s, the peak areas of both analytes increased and then decreased, and the peak area of the analyte reached the maximum at a milling time of 150 s. This shows that when the grinding time is 150s, the sample and the adsorbent have reached adsorption equilibrium, and then the grinding time is increased, which in turn causes too strong force between the sample and the adsorbent, and the extraction efficiency is decreased. In summary, 150s was selected as the final grinding time.
(4) Influence of eluent type
Referring to the method of example 1, radix angelicae dahuricae was used as an actual sample to examine the influence of the eluent on the MSPD enrichment effect.
Suitable eluents should meet the requirement of dissolving the target analyte but leaving the matrix impurities in the filler. In this experiment, peak areas of target analytes when eluents are methanol, ethanol, acetonitrile, acetone and ethyl acetate respectively are investigated, and the result is shown in fig. 6. It is seen from the figure that the peak areas of the two analytes are the largest and the extraction efficiency is the best when methanol is used as the eluent. This is probably due to isoimperatorin and imperatorin having a lactone structure, with a small polarity, closest to methanol polarity. According to the principle of similarity and compatibility, the extraction efficiency of methanol is the best. Therefore, methanol was chosen as eluent for the experiment and used in the next optimization of conditions.
(5) Effect of eluent volume
Referring to the method of example 1, the effect of the volume of eluent on the factor of MSPD enrichment effect was examined by using radix angelicae as an actual sample.
This experiment investigated the efficiency of extraction of target analytes by MSPD at methanol volumes of 250, 500, 750, and 1000 μ L, respectively, and the results are shown in fig. 7. The results show that the peak areas of both analytes increase when the elution volume is increased from 250. mu.L to 500. mu.L; the peak area decreased as the elution volume continued to increase from 500. mu.L to 1000. mu.L. This indicates that 500. mu.L of methanol is sufficient to elute the analyte, and increasing the elution volume further dilutes the analyte, reducing the extraction efficiency. Therefore, 500 μ L was chosen as the final elution volume for the experiment.
Example 3 FESI-MCDS-MEKC Condition optimization
(1) Influence of organic solvent in buffer
Referring to the method in example 1, the concentration of the standard substance is 1 μ g/mL, and dahurian angelica root is used as an actual sample to examine the influence of the organic solvent in the buffer solution on the factors of the MSPD enrichment effect.
Organic solvents in the Buffer (BGE) can affect the magnitude of the electroosmotic flow and thus indirectly the separation process. The experiment examined the peak appearance of three analytes at 0%, 10%, 20% and 30% methanol content, and the results are shown in fig. 8. As can be seen, the peaks of the three analytes cannot be separated when no methanol is added to the buffer. The peaks of notopterygium alcohol and isoimperatorin are not completely separated when the methanol content is 10% and 20%. Only when the methanol content was 30%, the peaks of the three analytes could be completely separated and the response value was the highest. Thus, 30% methanol was chosen as the organic additive in the buffer for the experiment.
(2) Effect of Cyclodextrin injection time
Referring to the method of example 1, the concentration of the standard substance was 1. mu.g/mL, and the effect of the cyclodextrin injection time on the MSPD enrichment effect was examined using Angelica dahurica as an actual sample.
In the experiment, the peak appearance conditions of three analytes are examined when the sample injection time is fixed to 150s and the sample injection time of the cyclodextrin is respectively 90s, 120s, 150s and 180 s. As a result, as shown in FIG. 9, the response became high when the cyclodextrin injection time was increased from 90s to 120 s. This is probably due to the fact that the increased amount of cyclodextrin allows for a more facile release of the analyte and a better enrichment. However, with the further increase of the injection time of the cyclodextrin, the peak height is not obviously increased, but the separation degree of the notopterygium alcohol and the isoimperatorin peak is reduced. Conditions for feeding 120s of cyclodextrin solution and 150s of sample solution (feeding time ratio of 4:5) were selected for the experiment in consideration of sensitivity and resolution.
(3) Influence of sample introduction time of sample solution
Referring to the method of example 1, the concentration of the standard substance is 1 μ g/mL, and radix angelicae dahuricae is used as an actual sample to examine the influence of the sample injection time of the sample solution on the MSPD enrichment effect.
Increasing the sample injection time is the most direct method for improving the enrichment effect, but too long sample injection time can cause peak broadening and poor resolution. Therefore, in this experiment, under the condition of the optimal injection ratio (cyclodextrin: sample: 4:5), the peak emergence conditions of the analytes when the injection time of cyclodextrin is 72, 96, 120 and 180s and the injection time of sample is 90, 120, 150 and 180s are examined, and the result is shown in fig. 10, wherein, a graph a and a graph B are respectively the electrophoretogram and the peak area of each analyte at different injection times. The results show that the peak areas of the three analytes increase when the injection time is increased from 90s to 150 s. When the injection time is 150s, the response value of the three analytes is the highest, and each peak is completely separated. In conclusion, 150s was determined as the optimal sample injection time.
Example 4 methodology examination
Method linearity, detection limit, reproducibility and enrichment factor
A proper amount of 1mg/mL mixed reference solution is taken, mixed standard solutions with the concentrations of the notopterygium alcohol, the isoimperatorin and the imperatorin being 0.1, 1.0, 3.0, 5.0, 7.5 and 10.0 mu g/mL respectively are accurately prepared, and the mixed standard solutions are parallelly measured for three times under the condition of the example 1. Taking the peak areas of the three coumarin compounds as the ordinate and the concentration as the abscissa to make a standard curve. The results show that the linear relationship of the three target analytes is good between 0.1 and 10 mu g/mL. And continuously injecting the mixed standard solution of 1 mu g/mL for 6 times within one day and continuously injecting the mixed standard solution for 3 times per day for 3 days respectively to evaluate the intra-day precision and the inter-day precision, wherein the peak areas RSD obtained by the result are less than 3.31 percent, and the reproducibility of the method is proved. The results are shown in Table 1.
TABLE 1
The enrichment factor calculation formula of the MSPD-FESI-MCDS-MEKC method is as follows:
conventional sample introduction conditions are as follows: the buffer solution is 100mmol/L SDS, 50mmol/L H 3 PO 4 And 30% methanol at 50mbar for 5s into a 50. mu.g/mL cocktail of isoimperatorin and imperatorin (solvent is buffer). The enrichment times of the method for isoimperatorin and imperatorin are calculated to be 302 and 283 times respectively, and the detection sensitivity of CE to the two coumarin compounds is effectively improved.
EXAMPLE 5 determination of actual samples
When the content of the angelica dahurica sample (purchased from the pharmacy of Wulin, Hangzhou, Zhejiang) is determined by the method established in the above example 1, notopterygium alcohol is not detected, imperatorin is 1.12mg/g, and isoimperatorin is 0.47 mg/g.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. A method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC is characterized by comprising the following steps:
mixing a sample to be detected with an adsorbent, crushing to obtain mixed powder, eluting the mixed powder by using a solid phase extraction column, collecting effluent liquid, and volatilizing a solvent to obtain an extract;
respectively preprocessing the capillary and the extract, performing electrophoretic analysis based on FESI-MCDS-MEKC to obtain the peak area of furocoumarin in the extract, and substituting the peak area into a corresponding standard curve to obtain the furocoumarin content in the sample to be detected;
the furocoumarin is notopterygium alcohol, isoimperatorin or imperatorin;
the adsorbent is a molecular sieve;
the mass ratio of the sample to be detected to the adsorbent is 1: 1.5-2.5;
the crushing is constant-speed grinding crushing, and the time for constant-speed grinding crushing is 140-160 s;
the conditions of the electrophoretic analysis are as follows:
taking sodium dodecyl sulfate of 80-120 mmol/L, sodium dihydrogen phosphate of 5-15 mmol/L, phosphoric acid of 40-60 mmol/L and 30% methanol as buffer solution; adding cyclodextrin solution under the pressure of 40-60 mbar, adding pure water under the pressure of 40-60 mbar, and adding the extract solution of which the sample matrix is sodium dodecyl sulfate under the voltage of-5 to-10 kV, wherein the separation voltage is-15 to-25 kV, the temperature is 20-30 ℃, and the detection wavelength is 254 nm.
2. The method for the on-line determination of furocoumarin content based on the combination of MSPD extraction and FESI-MCDS-MEKC as claimed in claim 1, wherein the eluent for elution is methanol, ethanol, acetonitrile, acetone or ethyl acetate.
3. The method for online determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 2, wherein the volume of the eluent is 400-600 μ L per 25mg of sample to be tested.
4. The method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 1, wherein the sample injection time of the cyclodextrin solution is 45-225 s; the sample introduction time of the sample solution is 160-200 s.
5. The method for on-line determination of furocoumarin content based on MSPD extraction in combination with FESI-MCDS-MEKC as claimed in claim 1, wherein the cyclodextrin is any one of isopropyl- β -cyclodextrin, methyl- β -cyclodextrin, α -cyclodextrin or β -cyclodextrin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011146591.5A CN112415103B (en) | 2020-10-23 | 2020-10-23 | Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011146591.5A CN112415103B (en) | 2020-10-23 | 2020-10-23 | Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112415103A CN112415103A (en) | 2021-02-26 |
| CN112415103B true CN112415103B (en) | 2022-08-26 |
Family
ID=74841063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011146591.5A Active CN112415103B (en) | 2020-10-23 | 2020-10-23 | Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112415103B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113390942B (en) * | 2021-06-08 | 2022-08-30 | 浙江工业大学 | Method for determining content of amaryllidaceae alkaloid based on EKS-CZE combined with MSPD |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770839A (en) * | 2017-03-02 | 2017-05-31 | 浙江工业大学 | The extraction detection method of flavone compound in a kind of subprostrate sophora |
| CN111007160A (en) * | 2019-10-28 | 2020-04-14 | 浙江工业大学 | Method for detecting content of neutral substance based on FESI-MCDS-MEKC |
-
2020
- 2020-10-23 CN CN202011146591.5A patent/CN112415103B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770839A (en) * | 2017-03-02 | 2017-05-31 | 浙江工业大学 | The extraction detection method of flavone compound in a kind of subprostrate sophora |
| CN111007160A (en) * | 2019-10-28 | 2020-04-14 | 浙江工业大学 | Method for detecting content of neutral substance based on FESI-MCDS-MEKC |
Non-Patent Citations (2)
| Title |
|---|
| Comparison of Matrix Solid-Phase Dispersion and Liquid–Solid Extraction Methods Followed by Solid-Phase Extraction in the Analysis of Selected Furanocoumarins from Pimpinella Roots by HPLC–DAD;A. ONISZCZUK等;《Acta Chromatographica》;20151231;第687–696页 * |
| MichałWoźniakiewicz等.Separation of 20 coumarin derivatives using the capillary electrophoresis method optimized by a series of Doehlert experimental designs.《Talanta》.2017, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112415103A (en) | 2021-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Simultaneous analysis of antioxidants and preservatives in cosmetics by supercritical fluid extraction combined with liquid chromatography–mass spectrometry | |
| Qi et al. | Rapid and sensitive determination of anthraquinones in Chinese herb using 1-butyl-3-methylimidazolium-based ionic liquid with β-cyclodextrin as modifier in capillary zone electrophoresis | |
| Tian et al. | Separation and determination of lignans from seeds of Schisandra species by micellar electrokinetic capillary chromatography using ionic liquid as modifier | |
| Mullett et al. | Bio-compatible in-tube solid-phase microextraction capillary for the direct extraction and high-performance liquid chromatographic determination of drugs in human serum | |
| Yang et al. | Determination of free isomeric oleanolic acid and ursolic acid in Pterocephalus hookeri by capillary zone electrophoresis | |
| Kvasničková et al. | Application of capillary electrochromatography using macroporous polyacrylamide columns for the analysis of lignans from seeds of Schisandra chinensis | |
| Sun et al. | Analysis of rhubarb anthraquinones and bianthrones by microemulsion electrokinetic chromatography | |
| Sun et al. | Determination of volatile organic acids in oriental tobacco by needle-based derivatization headspace liquid-phase microextraction coupled to gas chromatography/mass spectrometry | |
| Li et al. | Simultaneous determination of matrine, sophoridine and oxymatrine in Sophora flavescens Ait. by high performance liquid chromatography | |
| Peng et al. | Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography | |
| CN101893612A (en) | Method for determining content of astaxanthin in antarctic krill oil by chromatography | |
| Ma et al. | The in-capillary-2, 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)-sweeping micellar electrokinetic chromatography-Diode array detector method for screening and quantifying trace natural antioxidants from Schisandra chinensis | |
| Thabano et al. | Capillary electrophoresis of neurotransmitters using in-line solid-phase extraction and preconcentration using a methacrylate-based weak cation-exchange monolithic stationary phase and a pH step gradient | |
| Beasley et al. | Determination and characterization of gallotannin by high performance liquid chromatography | |
| Wang et al. | Separation and on-line concentration of saponins from Panax notoginseng by micellar electrokinetic chromatography | |
| Cao et al. | Sensitive determination of pyrrolizidine alkaloids in Tussilago farfara L. by field‐amplified, sample‐stacking, sweeping micellar electrokinetic chromatography | |
| Liu et al. | Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography | |
| CN112415103B (en) | Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC | |
| Liu et al. | A sensitive two-step stacking by coupling field-enhanced sample injection and micelle to cyclodextrin stacking for the determination of neutral analytes | |
| Zhu et al. | Comparison of two sample preconcentration strategies for the sensitivity enhancement of flavonoids found in Chinese herbal medicine in micellar electrokinetic chromatography with UV detection | |
| Šlampová et al. | Micro-electromembrane extraction through volatile free liquid membrane for the determination of β-lactam antibiotics in biological and environmental samples | |
| Wang et al. | Simultaneous determination of eleven preservatives in cosmetics by micellar electrokinetic chromatography | |
| CN106610409A (en) | Chitosan filled micro-matrix solid-phase dispersion method | |
| CN106770839A (en) | The extraction detection method of flavone compound in a kind of subprostrate sophora | |
| Zhai et al. | Separation and determination of aristolochic acids in herbal medicines by microemulsion electrokinetic chromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |