Novel Cell Membrane Capillary Chromatography for Screening Active Compounds from Natural Products

Cell membrane chromatography (CMC) is a useful method for the simultaneous isolation and identification of active compounds from natural products. However, it suffers from high cell membrane consumption and is time-consuming to operate. In this study, CMC was performed for the first time with a silica capillary, termed cell membrane capillary chromatography (CMCC). Pancreatic islet cell membranes from a mouse were immobilized onto the capillary inner wall functionalized with aldehyde groups. Scanning electron microscopy observation of the prepared column showed that the cell membrane was evenly coated onto the capillary inner wall. Three model analytes with the pharmacological property of hypoglycemic activity including glibenclamide, glipizide and berberine were tested. They were all retained by the prepared column. Furthermore, the retention factors of the analytes in CMCC correlated well with their pharmacological action. The analytical procedure including washing (to obtain a flat baseline), injection and separation was accomplished within 10 min. The CMCC column was also used for screening active compounds from a natural plant (Coptis chinensis). The hypoglycemia activity of active components such as berberine was verified using the method. The results indicated that CMCC is a viable alternative method for screening active compounds from natural products.     

Novel Cell Membrane Capillary Chromatography for Screening Active Compounds from Natural Products

Cell membrane chromatography (CMC) is a useful method for the simultaneous isolation and identification of active compounds from natural products. However, it suffers from high cell membrane consumption and is time-consuming to operate. In this study, CMC was performed for the first time with a silica capillary, termed cell membrane capillary chromatography (CMCC). Pancreatic islet cell membranes from a mouse were immobilized onto the capillary inner wall functionalized with aldehyde groups. Scanning electron microscopy observation of the prepared column showed that the cell membrane was evenly coated onto the capillary inner wall. Three model analytes with the pharmacological property of hypoglycemic activity including glibenclamide, glipizide and berberine were tested. They were all retained by the prepared column. Furthermore, the retention factors of the analytes in CMCC correlated well with their pharmacological action. The analytical procedure including washing (to obtain a flat baseline), injection and separation was accomplished within 10 min. The CMCC column was also used for screening active compounds from a natural plant (Coptis chinensis). The hypoglycemia activity of active components such as berberine was verified using the method. The results indicated that CMCC is a viable alternative method for screening active compounds from natural products.

     

Enhanced stability designs of cell membrane chromatography for screening drug leads

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.     

On-line concentration of neomycin and screening aminoglycosides in milk by short capillary column and tandem mass spectrometry

An MS-MS method was established for the trace analysis of neomycin and screening aminoglycoside antibiotics (such as amikacin, gentamicin, kanamycin, and tobramycin) in a milk sample. The extraction and purification are based on ion-pair SPE technology on a short fused-silica capillary RP C18 column. The capillary SPE column provided the stationary phase to retain aminoglycoside antibiotics and MS-MS compatible organic acid heptafluorobutyric acid (HFBA) was used as protein precipitation and ion-pair reagent. Aminoglycosides were extracted in this short column and directly eluted to MS-MS without evaporating to dryness and reconstituted with MS-MS compatible solvent after SPE. The LOQ was 0.1 microg/mL and the calibration curve was linear up to 6.4 microg/mL. A small amount of milk product, 10 microL, is sufficient for the analysis and application of this method as the trace analysis of neomycin in the biological matrix proved simple and workable.     

Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure. The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaCl solution. Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e., the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product from the unreacted substrate. The measured peak area of the product then represented the enzyme activity. For enzyme inhibitor screening, the mixture solution of the substrate and the inhibitor was injected and assayed the reduction of the enzyme activity. The immobilized enzyme could withstand 100 consecutive assays by only losing 10% activity. The reproducibility in terms of time-to-time, day-to-day, and batch-to-batch was measured with RSD% less than 4.7%. Furthermore, the screening system was validated by a known inhibitor. Finally, screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.     

Dopamine-polyethyleneimine co-deposition of a capillary for α-glucosidase immobilization and its application in enzyme inhibitor screening

An online method based on CE was established to screen α-glucosidase inhibitors from traditional Tibetan medicine extracts. First, the inner wall at the inlet of capillary column was simply and effectively functionalized by dopamine-polyethyleneimine co-deposition method, which combines the adhesion property of dopamine and easy cationization of polyethyleneimine. Then α-glucosidase was rapidly immobilized on the inner wall of the capillary column by electrostatic adsorption. The inter- and intraday repeatability of the peak area of the enzymatic reaction product (p-nitrophenol) in a capillary was evaluated, and RSD% (n = 3) was 0.94% and 1.09%, respectively. Good batch-to-batch reproducibility of the peak area between different capillaries (RSD = 2.1%, n = 5) shows that the preparation method has good reproducibility. The Michaelis–Menten constant of the immobilized α-glucosidase was measured to be 1.18 mM, and the capillary column enzyme reactor retained 85.9% of initial activity after 30 cycles. Finally, it was applied to the screening of enzyme inhibitors in 20 traditional Tibetan medicine extracts. Sixteen medicines with inhibitory activity were screened out, and Rheum australe had the strongest inhibitory effect with an inhibitory rate of 83.3 ± 0.4%. These results showed that this method is effective to find potential enzyme inhibitors.     

化妆品中挥发性有机溶剂的通用检测方法

以化妆品配方中常见及禁用的36种有机溶剂为研究模板,建立了化妆品中挥发性有机溶剂残留评价初筛知识库、确证知识库和定量方法。初筛知识库包括双柱保留指数知识库和NIST质谱库。双柱保留指数知识库以保留指数为定性指标,选择极性的VF-1301ms和非极性的DB-5ms两根色谱柱,用顶空气相色谱-质谱法考察了36种有机溶剂在两种色谱分离系统中的保留特性。利用NIST MS search 2.0作为检索工具,同时建立了36种挥发性有机溶剂的顶空气相色谱-质谱定量方法。样品经60 ℃、30 min静态顶空后以连接了VF-1301ms石英毛细管色谱柱的气相色谱-质谱仪检测,外标法定量。方法检出限为0.01~3.3 μg/g,加标回收率为60.77%~126.60%。该方法从通用性的角度,为化妆品中挥发性有机溶剂残留的筛查、鉴别和定量提供方法,部分解决了测定化妆品中挥发性有机溶剂时需要针对不同检测目标建立不同方法以及潜在溶剂存在备选筛查的问题。     

氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究

基于石墨烯优良的物化性能,利用层层组装法将氧化石墨烯修饰于石英毛细管内壁,制备了氧化石墨烯基质的毛细管电色谱,通过电渗流、拉曼光谱等对其进行表征。在此基础上,基于离子键合法将胰蛋白酶固定于毛细管电色谱柱头,制备胰蛋白酶微反应器。两者结合构成毛细管电色谱胰蛋白酶微反应器。实验结果显示,氧化石墨烯作为基质既可提高样品的分离效率,还能促进胰蛋白酶的催化性能。氧化石墨烯修饰的毛细管电色谱对N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸毛细管的3.70提升至4.71,而其固定化酶活性(米氏常数K_m=1.10 mmol/L,最大反应速率V_(max)=0.32 mmol·L~(-1)·s~(-1))也明显优于裸毛细管(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1))。利用所制备的微反应器从10种中药材中筛选胰蛋白酶抑制剂活性成分的药材,结果发现三七和大黄中均存在胰蛋白酶抑制剂活性成分。     

In-tube solid-phase microextraction-capillary liquid chromatography as a solution for the screening analysis of organophosphorus pesticides in untreated environmental water samples

This paper describes a method for the selective screening of organophosphorus pesticides in water. In-tube solid-phase microextraction (SPME) in an open capillary column coupled to capillary liquid chromatography (LC) with UV detection has been used to effect preconcentration, separation and detection of the analytes in the same assembly. For in-tube SPME two capillary columns of the same length and different internal diameters and coating thicknesses have been tested and compared, a 30 cm x 0.25 mm I.D., 0.25 micro m thickness coating column, and a 30 cm x 0.1 mm I.D., 0.1 micro m of coating thickness column. In both columns the coating was 95% dimethylpolysiloxane (PDMS)-5% diphenylpolysiloxane. The proposed methodology provided limits of detections (LODs) for the tested organophosphorus pesticides in the 0.1-10 micro g/L range, whereas the direct injection of the samples onto the capillary LC system provided LODs in the 50-1000 micro g/L range. The sensitivity of the proposed in-tube SPME-capillary LC method is adequate to monitorize the analyte levels in drinking water. Several triazines, polycyclic aromatic hydrocarbons (PAHs), nonylphenol, organochloride pesticides or polybrominated diphenyl ethers (PBDEs) have been evaluated as possible interferents. The reliability of the described method is demonstrated by analysing different real water samples.     

Pesticide residue analysis in foodstuffs applying capillary gas chromatography with mass spectrometric detection. State-of-the-art use of modified DFG-multimethod S19 and automated data evaluation

This paper focuses on recent developments in the author's laboratory and reports on the "ultimate" analysis scheme which has evolved over the last 20 years in our laboratory. This demonstrates the feasibility of screening analyses for pesticide residue identification, mainly by full scan GC-MS, down to the 0.01 ppm concentration level in plant foodstuffs. It is based on a miniaturized DFG S19 extraction applying acetone for extraction followed by liquid-liquid extraction with ethyl acetate-cyclohexane followed by gel permeation chromatography. The final chromatographic determination is carried out with a battery of three parallel operating gas chromatographic systems using effluent splitting to electron-capture and nitrogen-phosphorus detection, one with a SE-54 the other with a OV-17 capillary column and the third one with a SE-54 capillary column and mass selective detection for identification and quantitation. The method is established for monitoring more than 400 pesticides amenable to gas chromatography. These pesticide residues are identified in screening analyses by means of the dedicated mass spectral library PEST.L containing reference mass spectra and retention times of more than 400 active ingredients and also metabolites applying the macro program AuPest (Automated residue analysis on Pesticides) for automated evaluation which runs with Windows based HP ChemStation software. The two gas chromatographic systems with effluent splitting to electron-capture and nitrogen-phosphorus detection are used to check the results obtained with the automated GC-MS screening and also to detect those few pesticides which exhibit better response to electron-capture and nitrogen-phosphorus detection than to mass spectrometry in full scan.     

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.     

Rapid determination of aliphatic amines in water samples by pressure-assisted monolithic octadecylsilica capillary electrochromatography-mass spectrometry

A pressure-assisted capillary chromatography-mass spectrometry method based on the use of a monolithic octadecylsilica (ODS) capillary is proposed for the determination of aliphatic amines. A 25 mM citric acid buffer containing 10% methanol is used as running electrolyte. Separation is achieved by simultaneously applying a capillary electrophoresis (CE) voltage of 13 kV and an overimposed pressure of 8 bar. The use of pressure is required to ensure stable electrospray conditions. Analysis times are reduced by using a capillary column consisting of a 30 cm long monolithic silica capillary column bound with ODS and a fused-silica capillary column also 30 cm long. The proposed method was successfully applied to the determination of low-molecular-weight aliphatic amines in tap and river water. The analysis of real samples requires cleanup and preconcentration, which can be performed automatically by inserting a minicolumn in the replenishment system of the commercial instrument.     

Automated capillary gas chromatographic analysis of pesticide residues in food

A method for multiresidue pesticide analysis in food is described. After a conventional clean-up, gas chromatographic analysis is performed in a gas chromatograph equipped with two fused-silica capillary columns coated with methylsilicone SP 2100 and methylphenylsilicone OV-17. The effluent from each column is split to electron-capture and nitrogen-phosphorus detectors, which are connected to a dual channel integrator. Therefore, from each gas chromatographic run parallel records of signals from the two detectors are obtained. Calibration of the system is carried out for the SP 2100 column with three test mixtures covering all pesticides. Additionally, four internal standards are included, two responding to the electron-capture detector and the other two to the nitrogen-phosphorus detector. Automated analysis is performed with test mixtures and food samples on the SP 2100 column overnight as a screening procedure. After selection of positive samples a confirmatory test and quantitation are carried out manually applying appropriate test mixtures according to the results of the screening runs.     

Development of nano Bio LC columns for the search of acetylcholinesterase molecular targets

A novel acetylcholinesterase Nano liquid chromatography capillary column (75 μm i.d. × 50 mm length) was developed for the fast screening of acetylcholinesterase inhibitors and the evaluation of their molecular recognition mechanism. Biotinylated acetylcholinesterase was immobilized on a streptavidin Nano liquid chromatography capillary column. Because of the very strong streptavidin-biotin interaction, the acetylcholinesterase immobilization step performed by frontal analysis is very fast (required less than 10 min), and the amount of immobilized acetylcholinesterase was in the microgram range (1 μg). The yellow anion obtained from the enzymatic reaction detected at 412 nm was achieved within 60 s, and the immobilized acetylcholinesterase retained 96% of the initial activity beyond 90 days. This column was successfully applied for the discrimination of weak affinity ligands to acetylcholinesterase from nonbinders, which is the heart of fragment-based drug design. This column was used for the determination of the IC₅₀ values of a series of inhibitor molecules. In addition, it was demonstrated that competitive experiments could be performed with our miniaturized system to confirm the existence and binding pocket of a ligand to acetylcholinesterase contained in a methanol plant extract. The results revealed that our acetylcholinesterase Nano liquid chromatography capillary column developed in this work represents a useful tool for the rapid screening of inhibitor candidates and evaluation of the action mechanism.     

Screening of aflatoxins in feed samples using a flow system coupled to capillary electrophoresis

A method is proposed which presents a new approach to the joint use of capillary electrophoresis (CE) commercial equipment and a flow system. This flow system allows the total determination of several compounds by using a fluorimetric screening system. The individual determination for each analyte is performed by the CE proposed method. The screening procedure uses simple equipment and operations and provides a yes/no binary response that occasionally requires confirmation. A fast, simple, and reliable method has been developed in order to determine the most frequent mycotoxins in feed samples using micellar electrokinetic capillary chromatography (MECC). An extraction step followed by a purification step was carried out on the samples in order to remove interference substances before analysis. A C18 column was chosen to concentrate the mycotoxins, and the analytes were eluted from C18 using methanol. The MECC method allows the separation of six mycotoxins within 50 min with a reproducibility as RSD between 7.45 and 13.06%, and a limit of detection (LOD) between 0.02 and 0.06 mg l(-1) for all the mycotoxins. These LODs were clearly below legal limits (0.05 mg l(-1)).     

Semipreparative-scale gas-chromatographic separation of filbertone enantiomers

The enantioselectivity of heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl-beta-CD) toward racemic filbertone (E-5-methyl-hep-2-en-4-one) was studied by performing the chiral separation on a capillary column, a thick-film wide-bore column and a semipreparative column. The semipreparative enantioseparation of filbertone was achieved at 80 degrees C by using a packed column providing (R)- and (S)-enantiomers of filbertone with ee 90 and 96%, respectively. The isolated enantiomers (approximately 250 microg each, ee = 90-96%) may be used for studies on the relationship of chirality and biological activity by olfactory screening and toxicological studies.     

Use of dual-column fused-silica capillary gas chromatography in combination with detector response factors for analytical toxicology

Retention and detector response factor data have been given for 188 compounds on the DB1 capillary column using a dual nitrogen--phosphorus and flame ionization detection system. Factors affecting the detector response factor parameter in a dual-capillary column system have been discussed showing its advantage in drug screening.     

Through-a-chip partial filling affinity capillary electrophoresis for estimating binding constants of ligands to receptors

In this paper, we describe the development of a microfluidic/capillary electrophoresis (CE) technique employing partial filling affinity capillary electrophoresis (PFACE) to estimate binding constants of ligands to receptors using as model systems carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Using multilayer soft lithography (MSL), a microfluidic device (MD) consisting of fluid and control channels is fabricated and fitted with an external capillary column. Multiple flow channels allows for manipulation of a zone of ligand and sample containing receptor and non-interacting standards into the MD and subsequently into the capillary column. Upon electrophoresis the sample components migrate into the zone of ligand where equilibrium is established. Changes in migration time of the receptor are used in the analysis to obtain a value for the binding interaction. The manipulation of small volumes of solution on the MD minimizes the need of time-consuming pipetting steps.     

Development and thermodynamic evaluation of novel lipid raft stationary phase chromatography for screening potential antitumor agents

Novel lipid raft stationary phase chromatography (LRSC), with lipid rafts that contain abundant tropomyosin‐related tyrosine kinase A receptors immobilized on the stationary phase, was developed for a high‐throughput screening of potentially active antitumor agents. Lestaurtinib was used as a model compound to determine the operational parameters of the LRSC. Of all the factors considered, the particle size of column packing, the column temperature and the flow rate were of immense importance in determining the performance of the established LRSC system. In order to profoundly comprehend the binding interaction between the model drug and the receptors on the column, thermodynamic studies were employed. The results revealed that the interaction was spontaneous and exothermic, a typical enthalpy‐driven process. Additionally, the primary forces were hydrogen bonding and van der Waals forces. In evaluating the applicability of the method, active extracts from Albizziae Cortex were screened out using the LRSC system under the optimized conditions. The bioactive components were successfully confirmed by the MTT assay. In conclusion, it could be said that the LRSC is a good model for screening potential antitumor agents because of its viability, rapid response and scalable features. Copyright © 2014 John Wiley & Sons, Ltd.     

Radiochemical detection for packed capillary liquid chromatography-mass spectrometry

For the identification of trace level organic molecules, such as drug or pesticide metabolites, there is need of a practical method to do packed capillary liquid chromatography-mass spectrometry (LC-MS) with radiochemical detection. This problem has been successfully solved by use of a post column flow-splitter, with coaxially transported makeup flow that increases the split flow rate to a flow compatible with commercially available radiochemical flow cells. To test the device, 14C-labeled azoxystrobin, a commercial fungicide, was analyzed by liquid chromatography-radiochemical activity monitor-mass spectrometry (LC-RAM-MS) using a 0.32 mm i.d. packed capillary column. Azoxystrobin could be detected at 500 pCi with good signal/noise. The method is general and can be used with capillary LC columns of smaller diameters. Column efficiency of about 20,000 theoretical plates/m was achieved using either radiochemical or mass spectrometric data, thus demonstrating the lack of band broadening using the described method for radiochemical detection. The simple hardware described allows the routine use of packed capillary LC with radiochemical detection.