Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC
阅读说明：本技术 一种基于mspd提取联合fesi-mcds-mekc在线测定呋喃香豆素含量的方法 (Method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC ) 是由 楚楚 刘彩婧 连琳敏 杨斐 童胜强 颜继忠 于 2020-10-23 设计创作，主要内容包括：本发明公开了一种基于MSPD提取联合FESI-MCDS-MEKC在线测定呋喃香豆素含量的方法,包括如下步骤：将待测样品与吸附剂混合,粉碎后获得混合粉末,将混合粉末用固相萃取柱进行洗脱,收集流出液后挥干溶剂得到提取物；分别对毛细管和提取物进行预处理,再基于FESI-MCDS-MEKC进行电泳分析得到提取物中呋喃香豆素的峰面积,将峰面积代入到相应的标准曲线中得到待测样品中呋喃香豆素含量。该方法操作简便、分析时间短、分离效率高而且准确可靠,重现性好,富集倍数达到302倍,可准确测定待测样品中羌活醇、异欧前胡素和欧前胡素的含量。(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 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 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.)
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.
2. The method for online determination of furocoumarin content based on MSPD extraction in combination with FESI-MCDS-MEKC as claimed in claim 1, wherein the furocoumarin is notopterygium alcohol, isoimperatorin or imperatorin.
3. 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 adsorbent is molecular sieve.
4. The method for online determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 1, wherein the mass ratio of the sample to be tested to the adsorbent is 1: 1.5 to 2.5.
5. The method for online determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 1, wherein the pulverization is constant speed grinding pulverization, and the time of the constant speed grinding pulverization is 140-160 s.
6. 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.
7. The method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 6, wherein the volume of the eluent is 400 μ L-600 μ L per 25mg of sample to be tested.
8. The method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in any one of claims 1 to 7, wherein 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; 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.
9. The method for on-line determination of furocoumarin content based on MSPD extraction combined with FESI-MCDS-MEKC as claimed in claim 8, 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.
10. 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 8, wherein the cyclodextrin is any one of isopropyl- β -cyclodextrin, methyl- β -cyclodextrin, α -cyclodextrin or β -cyclodextrin.
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 detected, 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 the like are commonly used as coumarin measurement methods, but these methods are expensive in materials and often require a large amount of organic reagents and complicated operation steps.
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 approach is to use an on-line enrichment technique for CE, and the other is to use an efficient sample pre-treatment (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 that the analyte is stacked 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 tested 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; 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.
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 considered comprehensively, and the sample introduction 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 incisum alcohol, 2 for isoimperatorin, 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.
Florilite is preferably available from Shanghai' an spectral laboratory science and technology, Inc.
Al2O3Preferably from Shanghai' an spectral laboratory science and technology, Inc.
Silica gel mSiO2·nH2O, preferably from Shanghai' an 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 introduction procedure: sequentially adding cyclodextrin solution 120s and pure water 2s under 50mbar pressure; sample solution was injected for 150s at-8 kv. 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 of example 1, the effect of the molecular sieve on the MSPD enrichment effect is examined by taking radix angelicae as an actual sample.
The influence of two molecular sieves KIT-6 and SPA-16 and other traditional adsorbents 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 to examine the influence of grinding time on the factors of the MSPD enrichment effect.
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 similar phase dissolution, 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. Thus, 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 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 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 solution injection time
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 the separation of each peak is complete. 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 (3) 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 in-day precision and the in-day precision, wherein the RSD of the peak area obtained by the result is less than 3.31 percent, which shows that the method has good reproducibility. 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 H3PO4And 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.