RESUMO
Hsp90 has long been recognized as an attractive and crucial molecular target for cancer therapy. Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported as a natural inhibitor of Hsp90. Here, we present the structure-activity relationship of Garcinia xanthones analogues as Hsp90 inhibitors and identify that compound 25, with a simplified skeleton, had an improved inhibitory effect toward Hsp90. Compound 25 inhibited the ATPase activity of Hsp90 with an IC50 value of 3.68±0.18µM. It also exhibited potent antiproliferative activities in some solid tumor cells. In SK-BR-3 cells with high Hsp90 expression, compound 25 induced the degradation of Hsp90 client proteins including Akt and Erk1/2 without causing the heat shock response. Additionally, compound 25 inhibited angiogenesis in HUVEC cells through Hsp90 regulation of the HIF-1α pathway. These results demonstrate that compound 25 as an Hsp90 inhibitor with a new structure could be further studied for the development of tumor therapy.
Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Garcinia/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Xantonas/química , Xantonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Relação Estrutura-AtividadeRESUMO
High-performance liquid chromatography (HPLC) plays an important role in biotechnology, and a majority of chromatographic separations use gradient elution. While gradient generators can be built in different formats, binary pumps or quaternary pumps are most frequently used for gradient generator constructions. We have recently developed a high-pressure electroosmotic pump (EOP); the pump can be manufactured at a cost of a few hundred dollars. However, it is challenging to use this pump to deliver a gradient eluent directly. In this study, we first improve the monolith preparation by applying a pressure to the monomer solution during polymerization. We assemble a binary EOP gradient generator and discuss the relationship between the gradient profile and voltage applied to the EOP. We demonstrate the feasibility of the binary EOP gradient generator for generating a smooth and reproducible nanoflow gradient. After integration of the gradient generator into a miniaturized HPLC system, we use the HPLC system for separating peptide mixtures from trypsin-digested proteins. The performance comparison between the above miniaturized HPLC system and an Agilent 1200 HPLC system exhibits comparable efficiencies, resolutions, and peak capacities.
RESUMO
Here, we construct an open-channel on-chip electroosmotic pump capable of generating pressures up to â¼170 bar and flow rates up to â¼500 nL/min, adequate for high performance liquid chromatographic (HPLC) separations. A great feature of this pump is that a number of its basic pump units can be connected in series to enhance its pumping power; the output pressure is directly proportional to the number of pump units connected. This additive nature is excellent and useful, and no other pumps can work in this fashion. We demonstrate the feasibility of using this pump to perform nanoflow HPLC separations; tryptic digests of bovine serum albumin (BSA), transferrin factor (TF), and human immunoglobulins (IgG) are utilized as exemplary samples. We also compare the performance of our electroosmotic (EO)-driven HPLC with Agilent 1200 HPLC; comparable efficiencies, resolutions, and peak capacities are obtained. Since the pump is based on electroosmosis, it has no moving parts. The common material and process also allow this pump to be integrated with other microfabricated functional components. Development of this high-pressure on-chip pump will have a profound impact on the advancement of lab-on-a-chip devices.
Assuntos
Eletro-Osmose/métodos , Dispositivos Lab-On-A-Chip , Nanotecnologia/métodos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Eletro-Osmose/instrumentação , Humanos , Imunoglobulina G/análise , Nanotecnologia/instrumentação , Soroalbumina Bovina/análiseRESUMO
Beta-1,3-glucuronosyltransferase (B3GAT3), overexpressed in hepatocellular carcinoma (HCC) and negatively correlated to prognosis, is a promising target for cancer therapy. Currently, no studies have reported small molecule inhibitors of B3GAT3. In this study, we designed and synthesized a series of small-molecule inhibitors of B3GAT3 through virtual screening and structure optimization. The lead compound TMLB-C16 exhibited potent B3GAT3 inhibitory activity (KD = 3.962 µM) by effectively suppressing proliferation and migration, and inducing cell cycle arrest and apoptosis in MHCC-97H (IC50= 6.53 ± 0.18 µM) and HCCLM3 (IC50= 6.22 ± 0.23 µM) cells. Furthermore, compound TMLB-C16 demonstrated favorable pharmacokinetic properties with a relatively high bioavailability of 68.37%. It significantly inhibited tumor growth in both MHCC-97H and HCCLM3 xenograft tumor models without causing obvious toxicity. These results indicate that compound TMLB-C16 is an effective small molecule inhibitor of B3GAT3, providing a basis for the future development of B3GAT3-targeted drugs.
Assuntos
Acetamidas , Antineoplásicos , Carcinoma Hepatocelular , Proliferação de Células , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/síntese química , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Acetamidas/química , Acetamidas/farmacologia , Acetamidas/síntese química , Acetamidas/uso terapêutico , Camundongos , Relação Estrutura-Atividade , Apoptose/efeitos dos fármacos , Camundongos Nus , Descoberta de Drogas , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto , Simulação de Acoplamento Molecular , Masculino , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/síntese químicaRESUMO
Targeting tumor stemness is an innovative approach to cancer treatment. Zinc Finger Protein 207 (ZNF207) is a promising target for weakening the stemness of glioma cells. Here, a series of novel N-(anthracen-9-ylmethyl) benzamide derivatives against ZNF207 were rationally designed and synthesized. The inhibitory activity was evaluated, and their structure-activity relationships were summarized. Among them, C16 exhibited the most potent inhibitory activity, as evidenced by its IC50 values ranging from 0.5-2.5 µM for inhibiting sphere formation and 0.5-15 µM for cytotoxicity. Furthermore, we found that C16 could hinder tumorigenesis and migration and promote apoptosis in vitro. These effects were attributed to the downregulation of stem-related genes. The in vivo evaluation demonstrated that C16 exhibited efficient permeability across the blood-brain barrier and potent efficacy in both subcutaneous and orthotopic glioma tumor models. Hence, C16 may serve as a potential lead compound targeting ZNF207 and has promising therapeutic potential for glioma.
Assuntos
Antineoplásicos , Glioma , Humanos , Glioma/tratamento farmacológico , Glioma/patologia , Relação Estrutura-Atividade , Apoptose , Benzamidas/farmacologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proliferação de Células , Proteínas Associadas aos MicrotúbulosRESUMO
In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.
Assuntos
Fracionamento Químico/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/análise , Dodecilsulfato de Sódio/análise , Focalização Isoelétrica/métodos , Mioglobina/análise , Dodecilsulfato de Sódio/químicaRESUMO
The pressure output of a pump cannot be increased simply by connecting several of them in series. This barrier is eliminated with the micropump developed in this work. The pump is actually an assembly of a number of fundamental pump units connected in series. The maximum pressure output of this pump assembly is directly proportional to the number of serially connected pump units. Theoretically, one can always enhance the pressure output by adding more pump units in the assembly, but in reality the upper pressure is constrained by the microtees or microunions joining the pump components. With commercially available microtees and microunions, pressures of more than 1200 bar have been achieved. We have recently experimented using open capillaries to build this pump, but many capillaries have to be utilized in parallel to produce an adequate flow to drive HPLC separations. In this paper, we synthesize polymer monoliths inside 75 µm i.d. capillaries, use these monoliths to assemble miniaturized pumps, characterize the performance of these pumps, and employ these pumps for HPLC separations of intact proteins. By tuning the experimental parameters for monolith preparations, we obtain both negatively and positively charged submicrometer capillary channels conveniently. Each monolith in a 75 µm i.d. capillary is equivalent to several thousands of open capillaries.
RESUMO
Glycidyl methacrylate-bonded ß-cyclodextrin (GMA-ß-CD) is synthesized as a new chiral monomer by direct chemical bonding with GMA using a fast and simple alternative procedure. Next, rigid and homogenous monolithic columns were prepared by polymerization of GMA-ß-CD monomer with ethylene dimethacrylate (EDMA), in the presence of commonly used porogens and a charged achiral monomer to form a versatile chiral monolith. This is the first report in which a preparation procedure for a methacrylate-bonded CD is introduced for chiral separations in CEC. The degree of substitution of GMA-ß-CD monomer and mobile-phase parameters were optimized to achieve the highest enantioselectivity and plate number. To evaluate the GMA-ß-CD monolithic column, different classes of chiral compounds were screened. Under the optimized ß-CD monolith phase and the optimum mobile-phase conditions, 30 neutral and basic chiral compounds and two acidic compounds could be separated. The high chemical and mechanical stability, homogenous microflow and no loss of material at the interface allows for the first time the feasibility of applying this polymer-based monolithic column for CEC coupled to ESI-MS. Compared with CEC-UV, CEC-ESI-MS showed higher sensitivity and lower resolution. However, resolution greater than 1.0 can still be obtained for majority of the select tested compound in CEC-ESI-MS with at least three out of seven compound providing Rs≥1.5. The results reinforce the potential of GMA-ß-CD monolithic columns for chiral separations with high sensitivity in CEC-ESI-MS. Finally, using hexobarbital as the model chiral analyte, the monolithic column demonstrated excellent stability and reproducibility of retention time and enantioselectivity.
Assuntos
Eletrocromatografia Capilar/métodos , Ciclodextrinas/química , Compostos de Epóxi/química , Metacrilatos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetonitrilas/química , Eletrocromatografia Capilar/instrumentação , Concentração de Íons de Hidrogênio , Sensibilidade e Especificidade , Estereoisomerismo , TemperaturaRESUMO
A surfactant bound poly (11-acrylaminoundecanoic acid-ethylene dimethacrylate) monolithic column was simply prepared by in situ co-polymerization of 11-acrylaminoundecanoic acid and ethylene dimethacrylate with 1-propanol, 1,4-butanediol and water as porogens in 100 microm id fused-silica capillary in one step. This column was used in CEC-atmospheric pressure photoionization (APPI)-MS system for separation and detection of N-methylcarbamates pesticides. Numerous parameters are optimized for CEC-APPI-MS. After evaluation of the mobile phase composition, sheath liquid composition and the monolithic capillary outlet position, a fractional factorial design was selected as a screening procedure to identify factors of ionization source parameters, such as sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizing gas pressure, vaporizer temperature and capillary voltage, which significantly influence APPI-MS sensitivity. A face-centered central composite design was further utilized to optimize the most significant parameters and predict the best sensitivity. Under optimized conditions, S/Ns around 78 were achieved for an injection of 100 ng/mL of each pesticide. Finally, this CEC-APPI-MS method was successfully applied to the analysis of nine N-methylcarbamates in spiked apple juice sample after solid phase extraction with recoveries in the range of 65-109%.
Assuntos
Carbamatos/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Praguicidas/química , Acetatos/química , Acetonitrilas/química , Acrilamidas/química , Aminoácidos/química , Análise de Variância , Bebidas/análise , Modelos Lineares , Metacrilatos/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase SólidaRESUMO
A novel anionic surfactant bound monolithic stationary phase based on 11-acrylaminoundecanoic acid is designed for CEC. The monolith possessing bonded undecanoyl groups (hydrophobic sites) and carboxyl groups (weak cationic ion-exchange sites) were evaluated as a mixed-mode stationary phase in CEC for the separation of neutral and polar solutes. Using a multivariate D-optimal design the composition of the polymerization mixture was modeled and optimized with five alkylbenzenes and seven alkyl phenyl ketones as test solutes. The D-optimal design indicates a strong dependence of electrochromatographic parameters on the concentration of 11-acrylaminoundecanoic acid monomer and porogen (water) in the polymerization mixture. A difference of 6, 8 and 13% RSD between the predicted and the experimental values in terms of efficiency, resolution and retention time, respectively, indeed confirmed that the proposed approach is practical. The physical (i.e. morphology, porosity and permeability) and chromatographic properties of the monolithic columns were thoroughly investigated. With the optimized monolithic column, high efficiency separation of N-methylcarbamates pesticides and positional isomers was successfully achieved. It appears that this type of mixed-mode monolith (containing both chargeable and hydrophobic sites) may have a great potential as a new generation of CEC stationary phase.
Assuntos
Acrilamidas/química , Aminoácidos/química , Eletrocromatografia Capilar/instrumentação , Tensoativos/química , Acrilamidas/síntese química , Aminoácidos/síntese química , Derivados de Benzeno/isolamento & purificação , Eletrocromatografia Capilar/métodos , Cetonas/isolamento & purificaçãoAssuntos
DNA/isolamento & purificação , Eletroforese Capilar/métodos , Técnicas Analíticas Microfluídicas/métodos , DNA/genética , Eletro-Osmose , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Escherichia coli/genética , Técnicas Analíticas Microfluídicas/instrumentação , Plasmídeos , SoluçõesRESUMO
We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Proteínas/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia por Troca Iônica/instrumentação , Cromatografia de Fase Reversa/instrumentação , Misturas Complexas/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , HumanosRESUMO
Polymer monolithic columns with I.D. between 100 and 320 microm were prepared by in-situ polymerization of styrene and divinylbenzene in fused silica capillaries. The effects of monolithic column I.D. on the separation of proteins in reversed-phase capillary-liquid chromatography under gradient elution were systemically studied. The loading capacity was positively proportional to the volume of the stationary phase. It was found that the smaller diameter columns showed better performance for protein separation. The minimum plate height decreases from 34.99 microm (320 microm I.D. column) to 5.39 microm (100 microm I.D. column) for a retained protein. After studying the three parameters of the Van Deemter equation, it was interpreted that the smaller diameter can provide less flow resistance and the better performance may also be improved by the increasing of the effective diffusion. This conclusion was also supported by the data of separation permeability and breakthrough curves.
Assuntos
Eletrocromatografia Capilar/instrumentação , Fracionamento Químico/instrumentação , Proteínas/química , Proteínas/isolamento & purificação , Eletrocromatografia Capilar/métodos , Fracionamento Químico/métodos , Cromatografia Líquida de Alta Pressão/métodos , Difusão , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Modelos Químicos , Poliestirenos/síntese química , Poliestirenos/química , Porosidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício/química , Solventes/química , Espectrofotometria/métodosRESUMO
Toward developing a micro HPLC cartridge, we have recently built a high-pressure electroosmotic pump (EOP). However, we do not recommend people to use this pump to deliver an organic solvent directly, because it often makes the pump rate unstable. We have experimented several approaches to address this issue, but none of them are satisfactory. Here, we develop an innovative approach to address this issue. We first create an abruption (a dead-volume) within a fluid conduit. We then utilize an EOP to withdraw, via a selection valve, a train of eluent solutions having decreasing eluting power into the fluid conduit. When these solutions are further aspirated through the dead-volume, these solutions are partially mixed, smoothening concentration transitions between two adjacent eluent solutions. As these solutions are pushed back, through the dead-volume again, a smooth gradient profile is formed. In this work, we characterize this scheme for gradient formation, and we incorporate this approach with a high-pressure EOP, a nanoliter injection valve, and a capillary column, yielding a micro HPLC system. We then couple this micro HPLC with an electrospray ionization - mass spectrometer for peptide and protein separations and identifications.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Mioglobina/análise , Peptídeos/análise , Animais , Eletro-Osmose/instrumentação , Desenho de Equipamento , Cavalos , Espectrometria de Massas/instrumentação , Nanotecnologia/instrumentaçãoRESUMO
We integrate a high-pressure electroosmotic pump (EOP), a nanoflow gradient generator, and a capillary column into a miniaturized liquid chromatographic system that can be directly coupled with a mass spectrometer for proteomic analysis. We have recently developed a low-cost high-pressure EOP capable of generating pressure of tens of thousands psi, ideal for uses in miniaturized HPLC. The pump worked smoothly when it was used for isocratic elutions. When it was used for gradient elutions, generating reproducible gradient profiles was challenging; because the pump rate fluctuated when the pump was used to pump high-content organic solvents. This presents an issue for separating proteins/peptides since high-content organic solvents are often utilized. In this work, we solve this problem by incorporating our high-pressure EOP with a nano-flow gradient generator so that the EOP needs only to pump an aqueous solution. With this combination, we develop a capillary-based nano-HPLC system capable of performing nano-flow gradient elution; the pump rate is stable, and the gradient profiles are reproducible and can be conveniently tuned. To demonstrate its utility, we couple it with either a UV absorbance detector or a mass spectrometer for peptide separations.
Assuntos
Cromatografia Líquida/instrumentação , Peptídeos/análise , Osmose , PressãoRESUMO
This paper has established a simple method to detect directly phoxim in water. In the light of two-dimensional correlation analysis, the band of wavenumber for near-infrared spectroscopy of the model is between 5364.8 and 7552.9 cm(-1), the rate of accuracy for partial least squares discriminant analysis to calibration set (n=149) is 100%, prediction set (n=75) is 93.3% and the overall rate of accuracy for all the samples is 97.8% under the limit of detection 1 µg ml(-1) owing to the spectra preprocessing by standard normal variate transformation and multiplicative scatter correction. It is made clear that this method (two-dimensional correlation analysis combined with partial least squares discriminant analysis) is effective to detect directly phoxim in water.
Assuntos
Compostos Organotiofosforados/análise , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Água/análise , Calibragem , Análise Discriminante , Análise dos Mínimos Quadrados , Modelos Moleculares , Compostos Organotiofosforados/químicaRESUMO
Numerous micropumps have been developed, but few of them can produce adequate flow rate and pressure for high-performance liquid chromatography (HPLC) applications. We have recently developed an innovative hybrid electroosmotic pump (EOP) to solve this problem. The basic unit of a hybrid pump consists of a +EOP (the pumping element is positively charged) and a -EOP (the pumping element is negatively charged). The outlet of the +EOP is then joined with the inlet of the -EOP, forming a basic pump unit, while the anode of a positive high voltage (HV) power supply is placed at the joint. The inlet and outlet of this pump unit are electrically grounded. With this configuration, we can stack many of such pump units in series to boost the pumping power. In this work, we describe in details how an open-capillary hybrid EOP is constructed and characterize this pump systematically. We also show that a hybrid EOP with ten serially stacked pump units can deliver a maximum pressure of 21.5 MPa (â¼3100 psi). We further demonstrate the feasibility of using this hybrid EOP to drive eluents for HPLC separations of proteins and peptides.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Eletro-Osmose/instrumentação , Acetonitrilas/química , Peptídeos/isolamento & purificação , Pressão , Proteínas/isolamento & purificação , Ácido Trifluoracético/químicaRESUMO
A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA-EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.
Assuntos
Acrilamidas/química , Aminoácidos/química , Cromatografia Líquida de Alta Pressão/instrumentação , Proteínas/isolamento & purificação , Tensoativos/química , Butileno Glicóis/química , Cromatografia Líquida de Alta Pressão/métodos , Mioglobina/isolamento & purificação , Reprodutibilidade dos TestesRESUMO
As a new wave of technology, polymethacrylate-based monolithic column was prepared and its application in the separation of three kinds of microcystins (MCs) in pressurized capillary electrochromatography with ultraviolet detection was studied. The key factors affecting the separation performance, such as monolithic column, pressure of the pump, component and concentration of mobile phase and the voltage, were investigated and optimized in detail. A baseline separation could be achieved in less than 6 min using a 5 mM borate buffer with a pH of 9.6 and 10% acetonitrile as the mobile phases in isocratic elution, under a voltage of +13 kV and a supplementary pressure of 7.5 MPa. The calibration curves were linear with correlation coefficient r>0.998 over a range of 0.10-25.00 mg/L. The LODs for the three MCs were in the range of 0.03-0.09 mg/L. This method was successfully applied to separate MCs from other compounds in spiked tap water after solid-phase extraction. The lower LODs for MC-LR, MC-YR and MC-RR were obtained to be 0.10, 0.13, 0.16 microg/L, respectively. These results make it clear that this proposed system is accurate and robust enough to be used as a fast separation tool for routine monitoring of MCs in real water samples.
Assuntos
Eletrocromatografia Capilar/instrumentação , Microcistinas/análise , Microcistinas/isolamento & purificação , Eletrocromatografia Capilar/métodos , Pressão , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Polymethacrylate-based monolithic column was prepared in 150 microm i. d. capillary column in situ using butylmethacrylate and ethylene dimethacrylate. The application of methacrylate monolithic column in capillary high performance liquid chromatography ( micro-HPLC) for separation of microcystins (MCs) with ultraviolet (UV) detection has been studied. The properties of the monolithic column could be easily tailored by altering the preparation conditions. For the good reproducibility in preparation of monolithic column, this method could be used for the analysis of samples in practical. The effects of composition of mobile phase, pH value, concentration of the buffer, and the flow rate of the mobile phase on the separation of microcystins were investigated. The optimum separation for microcystins, MC-LR, MC-YR, and MC-RR, was achieved with a gradient elution of mobile phase A (0. 01 mol/L phosphate buffer, pH 2. 5 ) and mobile phosphate B (acetonitrile). The standard microcystins could be baseline-separated within 9 min. The limits of detection (LODs) (S/N = 3) for three standard microcystins were in the range of 0. 80 - 1. 03 mg/L. The intra-day and inter-day precisions of the method were obtained with the values of relative standard deviation less than 1. 0% and 2. 0%, respectively. This method was successfully used to analyze bloom samples and laboratory-cultured samples of cyanobacteria after performing solid phase extraction (SPE) using C18 cartridges for preconcentration. The whole procedure provided low LODs for MCs, e. g. the LOD for MC-LR was found to be 420 ng/L. This family of microcystin is analyzed by VL-HPLC using methacrylate monolithic column for the first time. It is shown that this method is promising in the routine analysis of microcystins in water samples in practical.