AU2020100965A4 - Method For Preparation Of Solid Phase Extraction Column Of Sophocarpine Molecularly Imprinted Polymer - Google Patents

Abstract

The present invention discloses a method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer, including precipitation polymerization method for preparation of dual-template imprinted polymer. Dissolve the template (OSC, 0.04 mmol) in 5 mL of chloroform, refrigerate it at 4 °C for 30 min, add cross-linking agent (ethylene glycol dimethacrylate, 1 mmol), initiator (ABN, 0.15 mmol) and ACN (25 mL) and degas the solution with N2 for 15 min, then place the solution in 60 °C water bath for 24 h, at 150 rpm. After polymerization, screen the polymer particles in ACN, and retain the solid polymer particles with 22 m nylon membrane. In order to remove the template, extract the polymer particles by Soxhlet extraction until OSC can't be detected by HPLC-MS/MS. Clean the residues in d-MIPs with ACN and water. d-NIPs are prepared in the same way without template. Drawings 15.OkV 16.0mm x50.0k 11/5/2017 21:02 1.00un FIG. 1 60 -EXI3OSC - [-----sc 0) E 5o 0) 40 o 30 E C 20 0 FG - 101 0 (, MeOH ACN Water loading solvent FIG.2 1/2

Description

Drawings

15.OkV 16.0mm x50.0k 11/5/2017 21:02 1.00un

FIG. 1

60 -EXI3OSC 0) - [-----sc E 5o 0) 40

o 30 E C 20 0 FG - 101 0 (,

MeOH ACN Water loading solvent

FIG.2

1/2

Descriptions

Method for Preparation of Solid Phase Extraction Column of

Sophocarpine Molecularly Imprinted Polymer

The present invention relates to the technical field of traditional Chinese medicine, in particular to the preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer and the extraction and separation technology of sophocarpidine compounds from S. moorcroftiana.

Background Technology S. moorcroftiana is a kind of shrub endemic to the Qinghai-Tibet Plateau, mainly distributed in the middle and upper reaches of the Yarlung Zangbo River. With good environmental adaptability, it is used for wind prevention and sand fixation and water and soil erosion. Its branches and leaves are usually used as fodder for cattle and sheep, and the seeds are used as the main medicine for the treatment of vomiting, jaundice, diphtheria, dysentery and indigestion. The main compounds include sophocarpine, Oxysophocarpine, matrine, oxymatrine and flavonoids. Sophocarpine alkaloid has many pharmacological effects, such as reducing scar tissue proliferation, inhibiting cancer cell proliferation, resisting cachexia and protecting myocardial injury, etc. Sophocarpine and Oxysophocarpine often coexist in S. moorcroftiana. Therefore, it is of great value to establish an effective extraction method for the effective separation and determination of Sophocarpine alkaloid. It is reported that the matrine active substances from sophora flavescens, Sophora alopecuroide and subprostrate sophora are mainly extracted by conventional solid phase extraction (CE), capillary electrophoresis (CE) and pH-zone-refining countercurrent chromatography (CCC), etc. However, low selectivity and the use of a large amount of solvent are major disadvantages of conventional solid phase extraction, which seriously affects the extraction, separation and enrichment of compounds in medicinal plants from complex substrates. CCC has been used to separate and purify a large number of active ingredients from traditional Chinese medicine and other natural drugs, but this method uses too many harmful solvents, seriously polluting the environment and increasing extraction costs. Therefore, the development of a simple, rapid,

Descriptions

environmentally friendly and highly specific modern separation method is of great

significance for the extraction, purification and enrichment of matrine, oxymatrine

and sophocarpine in S. moorcroftiana.

Recently, it has been reported that molecularly imprinted polymers can be effectively extracted from sophocarpine and Oxysophocarpine. Related studies have found that when matrine molecule is used as a single template, only matrine and sophocarpine are extracted, while the molecularly imprinted polymer synthesized with Oxysophocarpine as a single template can only extract oxymatrine and oxysophocarpine from radix sophorae. Therefore, considering the high coexistence of these active substances, the adsorbent can only be quantitatively analyzed for many times, which is time-consuming and laborious, and severely limits the rapid and synchronous extraction, separation and determination of sophocarpine-alkaloids. It is of great significance to prepare a kind of solid phase adsorbent for simultaneous detection of matrine alkaloid molecules in the extraction of several active compounds such as S. moorcroftiana, radix sophorae flavescentis and subprostrate sophora. The dual-template molecular imprinting technique optimizes the cavity size of adsorbents because it can be more accurately simulated by a large and a small imprinted molecule, rather than just a compound with a molecular structure. So dual-template molecularly imprinted polymers can overcome these difficulties by extracting more similar pharmacological active substances from interested plants, such as matrine alkaloids, flavones, etc., which are of great research value.

Invention Summary The raw materials include sophocarpine, Oxysophocarpine and quercetin, S. moorcroftiana roots, stems, leaves and fruit, Ethylene Glycol Dimethacrylate (EGDMA) as cross-linking agent, methacrylic acid (MAA) as functional monomer, azobisisobutyronitrile as initiator, acetic acid and formic acid, acetonitrile (ACN), methanol (MeOH) and chloroform;

The dual-template molecularly imprinted polymer is prepared by precipitation polymerization method. Dissolve the template (OSC, 0.04 mmol) in 5 mL of chloroform, refrigerate it, add cross-linking agent (ethylene glycol dimethacrylate, 1 mmol), initiator (ABN, 0.15 mmol) and ACN (25 mL) and degas the solution, then place the solution in 60 °C water bath for 24 h, at 150 rpm. After polymerization, screen the polymer particles in ACN, and retain the solid polymer particles with 22 m nylon membrane. In order to remove the template, extract the polymer particles by Soxhlet extraction until OSC can't be detected by HPLC-MS/MS. Clean the residues in d-MIPs with ACN and water. d-NIPs are prepared in the same way without template;

Preparation of dual-template molecularly imprinted polymer solid phase extraction column: place a sieve plate on one end of a 5 mL polyethylene solid phase extraction

Descriptions

column with the coarse surface facing the polymer. Take 10 mg d-MIPs, activate them with 5 mL acetone, and fill them into the column. Finally, place another sieve plate on top of the d-MIPs, then wash and activate them thoroughly with water and ACN prior to extraction and purification;

Dual-template molecularly imprinted polymer solid phase extraction: accurately weigh Ig of roots, stems, leaves and seeds samples of S. moorcroftiana each, naturally dry for 2 weeks, and add 50 mL of methanol-water (1: 1, v / v) to each sample. The supernatant is obtained after ultrasonic treatment for 20 min and centrifugation for 5000 revolutions for 5 min. After repeated extraction by ultrasonic treatment and centrifugation for three times, the extract is almost evaporated to dry in the nitrogen stream. Then the samples are reduced to a dry state in the nitrogen stream. Finally, before d-MISPE, redissolve the residue in 1mL acetonitrile, and add the above extract to the prepared MISPE. Under optimum conditions, filter the solution with 0.22 m nylon membrane to obtain a dual-template molecularly imprinted solid phase extraction column.

Description of Drawings In order to more clearly explain the embodiments of this invention or the technical proposals in existing technology, the figures to be used in the embodiments will be briefly introduced below. Obviously, the figures in the following description are only some embodiments of this invention. Ordinary technicians in this field can also obtain other figures according to these figures without making creative effort.

FIG.1 is Scanning electron microscope images of MIPs.

FIG. 2 is Effect of Different Loading Solvents on MIPs

FIG. 3 is Recovery (%) of MT, OMT, SC in the washing solvent after rinsing with n-hexane, carbon tetrachloride (CCl4), dichloromethane (CH2C12), acetonitrile (ACN), and methanol (MeOH).

FIG. 4 is Recovery of OSC, SC in eluant after elution with acetic acid-methanol (3: 7, v / v), acetic acid-methanol (2: 8, v / v), and acetic acid-methanol (1: 9, v / v)

Detailed description of the preferred embodiments 2.2.1 Materials

Prepare sophocarpine, oxysophocarpine and quercetin (purity > 99%), roots, stems, leaves and fruit of S. moorcroftiana, and use ethylene glycol dimethacrylate (EGDMA) as crosslinking agent and methacrylic acid (MAA) as functional monomer, and azobisisobutyronitrile as initiator. Distill MAA twice before use to remove the polymerization inhibitor, acetic acid and formic acid, acetonitrile (ACN), methanol (MeOH), and chloroform. Use the microporous Milli-Q water purification system

Descriptions

(Milli Pore, Bedford, USA) to prepare ultrapure water. All other chemicals are of analytical grade. By dissolving appropriate amount of the stock solution (100 mg L-1) in ACN, the stock solution is kept in dark at 4 °C for no more than 3 months. The solution is diluted according to different requirements of subsequent experiments.

2.2.2 Instruments and operating parameters

Hitachi S-4800 (Hitachi Co., Ltd., Tokyo, Japan) is imaged by scanning electron microscopy (SEM) using a 2000-CMB US PE ultraviolet and visible spectrophotometer with a wavelength of 210 nm under high vacuum conditions, characterizing the shapes and sizes of various polymers. FT-IR spectra are recorded by Philips PU9800 infrared spectrometer, and the adsorption performance of d-MIP is determined by HPLC-MS/MS Technology. The device is a triple quadrupole mass spectrometer (Applied Biosystems, AB SCIEX, Foster City, CA, USA) connected in tandem to a multistage monitoring mode by high performance liquid chromatography (Agilent Technologies, Palo Alto, USA). Analytical column is C18 column (Aguilent Eclipse, XDB: 1mm x 100mm Id: 3.5tm; Agilent Technologies, separate at column temperature of 25 °C, with MeOH and 0.5% (v / v) formic acid as mobile phase at a flow rate of 0.5 mL min-1. In order to ensure the test results, all samples are tested three times, and the applicability of this method is verified by testing the standard samples of MT and OMT with the same concentration.

The mass spectrometer operates at an ion source temperature of 500 °C, a spray voltage of 5000 V, an atomization pressure of 172.38 x 103 Pa, an auxiliary gas and a curtain gas pressure of 310.28 x 103 Pa and 275.80 x 103 Pa, respectively, with a blocking voltage and a collision voltage of 15 V and 6V, mother ions m / z of 263.3 (Oxysophocarpine), 247.2 (sophocarpine) and 270.3 (quercetin), and daughter ions of 205.1 and 136.2 (OSC), 96.2 and 136.2 (SC), 210.2 and 87.4, quantitative ions m / z of 205.1 (OSC), 96.2 (SC) and 210.2 (quercetin).

2.2.3 Preparation of dual-template molecularly imprinted polymers

The dual-template molecularly imprinted polymer is prepared by precipitation polymerization method. Dissolve the template (OSC, 0.04 mmol) in 5 mL of chloroform, refrigerate it at 4 °C for 30min, add cross-linking agent (ethylene glycol dimethacrylate, 1 mmol), initiator (ABN, 0.15 mmol) and ACN (25 mL) and degas the solution with N2 for 15 min, then place the solution in 60 °C water bath for 24 h, at 150 rpm. After polymerization, screen the polymer particles in ACN, and retain the solid polymer particles with 22 m nylon membrane. In order to remove the template, extract the polymer particles by Soxhlet extraction until OSC can't be detected by HPLC-MS/MS. Clean the residues in d-MIPs with ACN and water. d-NIPs are prepared in the same way without template.

2.2.4 Performance evaluation of single template molecularly imprinted polymers

D e s cr i p t i o n s

In order to evaluate the adsorption performance of dual-template molecularly imprinted polymer, static and dynamic adsorption experiments are carried out, which are determined at different time (0.5, 1, 3, 6, 8, 10, 16, 24 hour) with the dual-template standard solution of the same concentration (1ml, 25 mg L-1). Adsorption experiments are conducted at the same time (24 h) with different concentrations of standard solutions (0.5, 1, 2, 3, 5, 7,9,10, and 12 mg L-1). These results are used to evaluate the binding capacity of d-MIPs and d-NIPs to active molecules. The isothermal adsorption equilibrium curves are established using OSC and SC as model compounds, and 5 mg of d-MIPs and d-NIPs samples are weighed respectively, dispersed in the above series of standard solutions, and shaken by mechanical table at 25 °C for 10 min (10000 rpm). Balance the adsorbent, dry the supernatant in nitrogen stream, wash with acetonitrile three times, dissolve in ACN, and determine the concentration of active molecule by HPLC-MS / MS.

In the equilibrium adsorption experiment, according to Equation (1), the adsorption capacity (Qe, mg g-1) per mg of the polymer is calculated according to the change between the initial concentration CO of the standard solution and the concentration Ce after adsorption equilibrium. V(mL) is the volume of the standard solution, and m(mg) is the mass of d-MIPs. The Scatchard Equation (2) is used to determine the dissociation constant (Kd) of the polymer and the maximum binding capacity (Qmax) of the polymer.

m= (CO - C()V M

Q Qmax

C, Kd (2)

2.2.5 Optional experiments

As its molecular structure is similar to those of SC and OSC, the selectivity of D-MIP is studied by using OSC. A mixed standard solution (0.5 mgL-1) containing the same amount of MT, OMT and SC is prepared with 1mL acetonitrile as solvent. Add d-MIP or d-NIP (5 mg) to 1 mL of tube, shake it at 25 0 C for 24 h, then centrifuge it (5000 rpm, 10 min). Keep the supernatant, and analyze it by HPLC-MS / MS. Each sample is determined three times. The equations below are three classical models for studying the selectivity of molecularly imprinted polymers. The distribution coefficient (KD) is calculated by using the concentrations of CO and Ce before and after d-MIP equilibrium adsorption, and the distribution coefficient (KD) before and after d-nip adsorption is calculated by using Eq (3). This mainly reflects the adsorption capacity of d-MIPs on templates and structural analogs (SC). The larger the KD value, the stronger the adsorption capacity. The selectivity coefficient of d-MIP for SC and OSC

Descriptions

of competing species (quercetin) can be obtained from the binding curve by Eq (4), in which S represents the competing species and K represents the selectivity coefficient, indicating the selectivity of d-MIP to the template; the relative selectivity coefficient ( K ' ) indicates the template adsorption selectivity of d-MIPs relative to d-NIPs, which mainly reflects the difference between d-MIPs and d-NIPs in molecular recognition and selectivity. It reflects the advantages and disadvantages of imprinting effect. In addition, the greater the value of , the better the imprint

effect.

C -o C KD C e (3)

Kd(tomplato) Kd(s) (4)

K =K D-MIP K D-NIP (5)

2.2.6 Drawing of calibration curve

First prepare 1 mg mL-1 of mixed standard mother liquor of sophocarpine alkaloid, then dilute to 0, 5, 10, 20, 50, 100, 200 ng / mL- respectively, and use the established LC-MS / MS method for analysis and a calibration curve is drawn.

2.2.7 Preparation of dual-template molecularly imprinted solid phase extraction column

Place a sieve plate on one end of a 5 mL polyethylene solid phase extraction column with the coarse surface facing the polymer. Take 10 mg d-MIPs, activate them with 5 mL acetone, and fill them into the column. Finally, place another sieve plate on top of the d-MIPs, then wash and activate them thoroughly with water, acetic acid-methanol (20:80, v/v) and ACN prior to extraction and purification.

2.2.8 Optimization of solid phase extraction procedure

The solvent is optimally loaded, rinsed and eluted by a solid phase extraction column. After each step, the washing solvent is collected and dried in 25 °C nitrogen, the residues are redissolved in 1 mL CAN. The resulting solution is filtered through a nylon membrane with a pore diameter of 22 m, and analyzed by HPLC-MS / MS. Each test is repeated three times. The loaded solvents under investigation are MeOH,

Descriptions

ACN and water, standard solutions including MT and OMT (1 mL, 10 mg L-1) loaded onto d-MISPE solid phase extraction column containing d-MIP, cleaning solvents (2 mL) involved in the determination of N-hexane, carbon tetrachloride (CCl4), methylene chloride (CH2C12), ACN, MeOH and water, elution solvent of acetic acid-methanol mixture (3: 7, 2: 8, 1: 10, v / v) in different proportions.

2.2.9 Application of dual-template molecularly imprinted solid phase extraction combined with LC-MS / MS technique in practical samples(2 mL)

Accurately weigh Ig of roots, stems, leaves and seeds samples of S. moorcroftiana each, naturally dry for 2 weeks, and add 50 mL of methanol-water (1: 1, v / v) to each sample. The supernatant is obtained after ultrasonic treatment for 20 min and centrifugation for 5000 revolutions for 5 min. After repeated extraction by ultrasonic treatment and centrifugation for three times, the extract is almost evaporated to dry in the nitrogen stream. Then the samples are reduced to a dry state in the nitrogen stream. Finally, before d-MISPE, redissolve the residue in 1mL acetonitrile, and add the above extract to the prepared MISPE. Under optimum conditions, filter the solution with 0.22 m nylon membrane, and test it by LC-MS/MS.

2.3 Results and discussion

2.3.1 Synthesis of single template molecularly imprinted polymers and interaction of dual-template molecules with functional monomers

Dickert et al. reported an effective method for the preparation of molecularly imprinted materials with excellent specific binding ability. A single template molecularly imprinted polymer is prepared using Oxysophocarpine as a template molecule. Functional monomers are selected according to UV-VIS spectrum and crosslinking agent and initiator are selected according to previous reports. Electrostatic interactions, hydrogen bonding, it-n interaction and hydrophobic interaction may be one of the reasons for the formation of binding sites in MIPs. The selectivity and specificity of the binding sites may be caused by these binding sites. Since both MT and OMT are Lewis alkali, acidic MAA is used as functional monomer. In addition, it is reported that when OSC is used as a monodisperse template agent, MAA is selected as a functional monomer, showing good adsorption capacity and recognition performance to target molecules.

UV-Vis results study the interaction with d-MIPs when MAA and two templates (0.01 mmol) (also in ACN) are added to CAN. The carbonyl group on OSC and hydroxyl group on MAA may be involved in the interaction, electrostatic interactions can occur between templates and MAA, and lone pair electrons on the carbonyl group of methacrylic acid may initiate interactions with functional monomers with primary or tertiary amine nitrogen of the OSC. The absorption peak of the compound formed by the reaction of the template molecule with the functional monomer is not observed

Descriptions

until the red shift occurs when the molar ratio of the two templates to MAA is 1:3, which may mean that the functional monomer MAA interacts with the template molecule at the appropriate concentration. In the subsequent experiments, we select the ratio of template to functional monomer as 1: 3. Since EGDMA is the most widely used crosslinking agent and has been reported in sophocarpine molecularly imprinted polymers, EGDMA is used in this study.

SEM photographs show that the surface of the d-MIPs spherical particles is relatively rough (FIG.1), and the average particle size of the d-MIPs is 0.25 0.03 m. On the contrary, instead of forming spherical polymers, rigid polymers are formed for d-NIPs. Compared to the d-MIPs, d-NIPs have deep pores, which may be detrimental to the adsorption and desorption of template molecules. After the adsorption of template molecules, d-NIPs become harder and the pore size increases, which may lead to their lack of imprinting selectivity and recognition. In contrast to this, when the template molecules are adsorbed, d-MIPs became denser and the pores become smaller, indicating that the interaction between the template and d-MIP occurs. The d-MIPs loaded and unloaded are analyzed directly by FTIR. The strong peak observed at 1737cm-1 after unloading is caused by the tensile vibration of -C =0 (FIG. 1). Comparing the two types of d-MIPs, it is found that the template binds to d-MIPs, which is a displacement from 1737 cm-i to 1733 cm-1, indicating that free C = 0 in the template may be related to the hydrogen bond formed between MAA. The other main absorption peaks of IR spectra are: Methyl tensile vibration 2973 cm-1, -C = C stretching vibration 1638 cm-1, -C-0-C-absorption peak moving from 1151cm-i to 1159 cm-1, and the top of C-N at peak positions of 1262 and 1267 cm- respectively stretching. All of these changes indicate that on MT and OMT imprinted polymers, this may occur by intermolecular hydrogen bonding or a pair of electrons on the carbonyl group, primary or tertiary amines on the template interacting with hydroxyl hydrogen on the functional monomer.

2.3.2 Evaluation of adsorption

By evaluating the adsorption capacity of d-MIPs and d-NIPs over different time periods, the adsorption capacity of d-NIPs increases rapidly in the 5th hour and then reaches the equilibrium rapidly, and the adsorption capacity of d-MIPs increases until gradually reaching saturation after 10 h. These results show that d-NIPs do not have specific adsorption sites or appropriate pores. For d-MIPs, the molecular recognition site on the surface is the first place to bind to the template, and when the surface center is fully occupied, the deeper place starts to bind to the template. With the saturation of these centers, the adsorption rate gradually decreases, so the adsorption time should be kept within 10 hours. Combined with isotherm analysis, the adsorption capacity of d-MIPs increases with the increase of OSC and SC concentration, and the adsorption capacity of d-NIPs on OSC and SC is very low. Under the same conditions, the adsorption capacity of d-MIPs on OSC and SC is high, and the saturated adsorption concentration reaches 10 mg 1-i.Therefore, the adsorption selectivity of

Descriptions

d-MIPs to the target is higher than that of d-NIPs.

2.3.4 Optimization of dual-template molecularly imprinted column program

The process of extraction, separation, purification and enrichment on MISPE column focuses on the loading, washing and elution steps of d-MISPE column. In this study, the optimum loading, washing and elution conditions of the two sophocarpine alkaloids are evaluated. The loaded solvent increases the amount of OSC and SC adsorbed on MIPs. The washing solvent can effectively remove other impurities without removing OSC and SC. The eluant can effectively remove OSC and SC in d-MISPE column. In the case of polar stationary phase, the loaded solvent must have a weak or equivalent polarity to the stationary phase. When the polarity of the loaded solvent is too strong, the OSC is not easy to be retained, and the adsorption capacity of d-MIPs decreases. The results showed that when ACN is used as the solvent, OSC adsorbs d-MIPs more strongly than other solvents, and quercetin could not be adsorbed, which may be because the minimal expansion of d-MIPs in water and methanol would not be affected (FIG. 2). The recovery rates of OSC and SC (%) are calculated by leaching SPE column with different solvents. CCl4 removes the most impurities compared to MT, OMT and SC in the solvents tested (FIG. 3). So CCl4 is selected as the washing solvent and acetic acid-methanol (2: 8, v / v) as eluant (FIG. 4). Therefore, ACN, CCl4 and acetic acid-methanol (2:8, v/v) are selected in the MISPE protocol.

2.3.5 Verification of the method

In order to verify the accuracy and precision of the LC-MS /MS method, 7 concentrations in the range of 10 ~ 200OgL-1 are used to analyze the standards in the samples of OSC and SC S. moorcroftiana for 3 times. The results show that three analytes are obtained under the improved MISPE condition. The method has a good linear relationship (R>0.99), the SNR of the three alkaloids is 3, and the LOD value is between 0.15 gL-1 and 0.39 gL-1 (Tables 1-3). Combining dMISPE and HPLC-MS /

MS, determine their content after 10 times dilution. The contents of OSC and SC in S. moorcroftiana are 1554.32 g g-1, 3604.67 -1 and 21.54 g g-1. To verify the accuracy of MISPE-LC-MS / MS, add three analytes to the diluted solution extracted from the active compound. Evaluate the accuracy of MISPE-LC-MS / MS method. The average recovery rates of OSC and SC are 96.51%-98.42%, 80.34%-92.48%, 73.25%-76.02%, respectively, with intra-day precision ranging from 2.15% to 6.82%(n=3)(Tables 1-4). This method is effective for the quantitative determination of OSC and SC in actual samples.

Linear range Calibration curve R kg-1)(n=5) (p g kg-1)

Descriptions

S. moorcroftian OSC 50-1000 y=225x+0.0087 0.9981 15.42 a SC 50-1000 y=578x+0.0013 0.9984 11.53

Table 2-3 Linear range, linear regression equation, correlation coefficient (R) and detection limit of the determination of MT, OSC and SC by HPLC-MS / MS

Fortified Intra-day Real content le Recovery prciio Compounds g -)level (%(=) precision Copud 8g1 ( P g kg-1) (%)(n5) (RSD, %)(n=5) 50 80.34 2.61 OSC 3604.67 100 91.43 2.15 200 92.48 2.37 50 76.02 4.03 SC 21.54 100 73.25 6.82 200 74.52 4.76

Table 2-4 Real Contents of OSC and SC in S. Moorcroftiana Seeds and Determination of Recovery by d-MISPE-HPLC-MS / MS

2.3.6 Sample test

The method is applied to the actual samples to further verify its practicability. With S. Moorcroftiana as the actual samples, add 50 mL methanol-water (1: 1) to the samples, evaporate the extract to dry. Dissolve it in acetonitrile (1.0 mL) and then purify it according to the optimized MISPE program. MISPE is used to reduce the matrix interference and improve the extraction efficiency, so that the average contents of OMT and MT in the seeds reach the maximum. The method of MISPE-LC-MS / MS is used to detect S. Moorcroftiana. The detection values are 3793.88 and 1828.25 g mL-1, respectively. However, the average maximum content of SC in matrine roots is 35.13 pg mL-. These results not only prove that d-MISPE column has a good ability to extract and enrich OSC and SC from different tissue samples, but also indicate that S. Moorcroftiana growing in the marshland of Qinghai-Tibet plateau will be a good source of OSC and SC.

C la i m s

1. A method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer has the following characteristics, including the following steps:

The raw materials include sophocarpine, Oxysophocarpine and quercetin, S. moorcroftiana roots, stems, leaves and fruit, Ethylene Glycol Dimethacrylate (EGDMA) as cross-linking agent, methacrylic acid (MAA) as functional monomer, azobisisobutyronitrile as initiator, acetic acid and formic acid, acetonitrile (ACN), methanol (MeOH) and chloroform;

The dual-template molecularly imprinted polymer is prepared by precipitation polymerization method. Dissolve the template (OSC, 0.04 mmol) in 5 mL of chloroform, refrigerate it, add cross-linking agent (ethylene glycol dimethacrylate, 1 mmol), initiator (ABN, 0.15 mmol) and ACN (25 mL) and degas the solution, then place the solution in 60 °C water bath for 24 h, at 150 rpm. After polymerization, screen the polymer particles in ACN, and retain the solid polymer particles with 22 m nylon membrane. In order to remove the template, extract the polymer particles by Soxhlet extraction until OSC can't be detected by HPLC-MS/MS. Clean the residues in d-MIPs with ACN and water. d-NIPs are prepared in the same way without template;

Preparation of dual-template molecularly imprinted polymer solid phase extraction column: place a sieve plate on one end of a 5 mL polyethylene solid phase extraction column with the coarse surface facing the polymer. Take 10 mg d-MIPs, activate them with 5 mL acetone, and fill them into the column. Finally, place another sieve plate on top of the d-MIPs, then wash and activate them thoroughly with water and ACN prior to extraction and purification;

Dual-template molecularly imprinted polymer solid phase extraction: accurately weigh Ig of roots, stems, leaves and seeds samples of S. moorcroftiana each, naturally dry for 2 weeks, and add 50 mL of methanol-water (1: 1, v / v) to each sample. The supernatant is obtained after ultrasonic treatment for 20 min and centrifugation for 5000 revolutions for 5 min. After repeated extraction by ultrasonic treatment and centrifugation for three times, the extract is almost evaporated to dry in the nitrogen stream. Then the samples are reduced to a dry state in the nitrogen stream. Finally, before d-MISPE, redissolve the residue in 1mL acetonitrile, and add the above extract to the prepared MISPE. Under optimum conditions, filter the solution with 0.22 m nylon membrane to obtain a dual-template molecularly imprinted solid phase extraction column;

2. The method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer according to claim 1 has the following characteristic: the purity of sophocarpine, Oxysophocarpine and quercetin in the raw materials is greater than 99%;

C la i m s

3. The method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer according to claim 1 has the following characteristics: distill MAA twice before use to remove the polymerization inhibitor. Use the microporous Milli-Q water purification system (Milli Pore, Bedford, USA) to prepare ultrapure water. All other chemicals are of analytical grade. By dissolving appropriate amount of the stock solution (100 mg L-1) in ACN, the stock solution is kept in dark at 4 °C for no more than 3 months. The solution is diluted according to different requirements of subsequent experiments;

4. The method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer according to claim 1 has the following characteristics: in the preparation of the dual-template molecularly imprinted polymer, dissolved the template (OSC, 0.04mmol) in 5mL chloroform and refrigerate it at 4C for 30 min. Degas it with N2 for 15min;

5. The method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer according to claim 1 has the following characteristics: in the preparation of dual-template molecularly imprinted solid phase extraction column, the ratio of water to acetic acid-methanol is 20:80, v/v;

6. The method for preparation of solid phase extraction column of sophocarpine molecularly imprinted polymer according to claim 1 has the following characteristics: the performance evaluation method of the solid phase extraction column of the sophocarpine molecularly imprinted polymer: in order to evaluate the adsorption performance of dual-template molecularly imprinted polymer, static and dynamic adsorption experiments are carried out, which are determined at different time (0.5, 1, 3, 6, 8, 10, 16, 24 hours) with the dual-template standard solution of the same concentration (1ml, 25 mg L-1). Adsorption experiments are conducted at the same time (24 h) with different concentrations of standard solutions (0.5, 1, 2, 3, 5, 7,9,10, and 12 mg L-1). These results are used to evaluate the binding capacity of d-MIPs and d-NIPs to active molecules. The isothermal adsorption equilibrium curves are established using OSC and SC as model compounds, and 5 mg of d-MIPs and d-NIPs samples are weighed respectively, dispersed in the above series of standard solutions, and shaken by mechanical table at 25 °C for 10 min (10000 rpm). Balance the adsorbent, dry the supernatant in nitrogen stream, wash with acetonitrile three times, dissolve in ACN, and determine the concentration of active molecule by HPLC-MS/ MS;

In the equilibrium adsorption experiment, according to Equation (1), the adsorption capacity (Qe, mgg-1) per mg of the polymer is calculated according to the change between the initial concentration CO of the standard solution and the concentration Ce after adsorption equilibrium. V(mL) is the volume of the standard solution, and m(mg) is the mass of d-MIPs. The Scatchard Equation (2) is used to determine the

Claims (1)

1. Claims

   dissociation constant (KD) of the polymer and the maximum binding capacity (Qmax) of the polymer.

   Q = (CO - C,)v/m (1

   _ _ Qmax - 0

   C Kd (2)