Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed.     

Coupling HPLC to on-line, post-column (bio)chemical assays for high-resolution screening of bioactive compounds from complex mixtures

It is challenging to screen and identify bioactive compounds from complex mixtures. We review a recently developed technique that couples high-performance liquid chromatography (HPLC) to on-line, post-column (bio)chemical assays and parallel chemical analysis to screen and identify bioactive compounds from complex mixtures without the need for cumbersome purification and subsequent screening. In this system, HPLC separates complex mixtures and a post-column (bio)chemical assay determines the activity of the individual compounds present in the mixtures. Parallel chemical-detection methods (e.g., diode-array detection, mass spectrometry and nuclear magnetic resonance) identify and quantify the active compounds simultaneously. We focus on relatively widely used on-line, post-column assays for antioxidant screening and less widely used hyphenated systems involving assays based on enzymes and receptors. These strategies have proved to be very useful for rapid profiling and identification of individual active components in mixtures to provide a powerful method for natural product-based drug discovery.     

Antioxidant activity assays on-line with liquid chromatography

Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS(+) have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.     

Screening of radical scavengers in Scutellaria baicalensis using HPLC with diode array and chemiluminescence detection

Scutellaria baicalensis Georgi is a well-known medical plant widely used as a famous traditional Chinese medicine. It has been reported that S. baicalensis can protect against oxidative stress and possess anti-inflammatory effect. In the present paper, an HPLC-diode array-chemiluminescence detection method for on-line detection was successfully developed to screen antioxidants in complex S. baicalensis extracts. Using the proposed approach, eight compounds in the S. baicalensis extracts were found to possess a potential antioxidant activity. Furthermore, the effects of purified compounds on protecting RAW 264.7 cells from hydrogen peroxide injury were also investigated in vitro to confirm the established method, which were consistent with the results of HPLC-diode array-chemiluminescence detection method. These results demonstrated that this method was a useful technique for rapidly screening and identifying bioactive components from complex herbal medicines.     

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 μM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.     

Coupling of capillary electrophoresis with reaction detection for the on-line evaluation of radical scavenging activity of analytes

The main task of this work was to create a rapid, simple and less material and time consuming method involving capillary electrophoretic separation in order to separate analytes and evaluate antioxidant activity within a single analytical run. Several interfaces were developed and used to couple CE to the reaction detector. The method developed enables simultaneous electrophoretic separation and evaluation of antioxidant activity of each separated compound in the mixture. The analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) as synthetic radical reagent. The on-line capillary electrophoresis-reaction (antioxidant activity) detection method can be used for a rapid evaluation of individual antioxidants in complex mixtures, particularly extracts of natural products. Possibility to evaluate radical scavenging activity of extracts of natural products components is demonstrated on an example of aqueous propolis extract. Four phenolic acids where separated and their radical scavenging activity was on-line evaluated.     

On-line screening methods for antioxidants scavenging superoxide anion radical and hydrogen peroxide by liquid chromatography with indirect chemiluminescence detection

The identification of radical species is possible by the electron spin resonance technique. However, the antioxidants in complex matrices such as biological and food samples are difficult to determine. Hence, we developed novel screening systems for antioxidants, which are mainly eliminating superoxide anion radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), by HPLC with luminol-based chemiluminescence (CL) detection. When the sample contains antioxidants, inhibited peaks corresponding to each antioxidant are observed on the chromatogram. The antioxidant activities of catechins and flavones were determined with flow injection analysis by the proposed indirect CL. The scavenging activity for H(2)O(2) and O(2)(-) were different from each catechin and flavone. Furthermore, the potential was dependent upon the number and the position of OH functional group in the structure. Some applications such as the screening of antioxidants in tea products were also investigated. In spite of many peaks appeared on the chromatogram at UV detection, only the peaks corresponding to the compounds having elimination effect to O(2)(-) and/or H(2)O(2) were detected as inhibited peaks. Consequently, the proposed HPLC-CL seems to provide new screening systems for antioxidants possessing inhibition activity of O(2)(-) and H(2)O(2).     

Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2'-diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.     

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC₅₀ values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.     

Biomimetic modeling of oxidative drug metabolism

The prediction of drug metabolism is an important task in drug development. Besides well-established in vitro and in vivo methods using biological matrices, several biomimetic models have been developed. This review summarizes three different nonenzymatic strategies, including metalloporphyrins as surrogates of the active centre of cytochrome P450, Fenton’s reagent, and the electrochemical oxidation of drug compounds. Although none of the systems can simulate the whole range of cytochrome P450-catalyzed reactions adequately, the biomimetic models show some advantages over standard in vitro methods. For example, metalloporpyhrin catalysts allow the synthesis of certain metabolites in sufficient amounts and with sufficient purities to permit characterization and further pharmacological and toxicological tests. The electrochemical generation of metabolites coupled on-line to liquid chromatography/mass spectrometry is a promising tool for studying reactive metabolites and can be applied in automated high-throughput screening approaches. In this paper, detailed comparisons with cytochrome P450 catalysis are drawn, advantages and disadvantages of the respective methods are revealed, and possible applications are discussed.     

Functionalized graphene quantum dots loaded with free radicals combined with liquid chromatography and tandem mass spectrometry to screen radical scavenging natural antioxidants from Licorice and Scutellariae

A novel screening method was developed for the detection and identification of radical scavenging natural antioxidants based on a free radical reaction combined with liquid chromatography with tandem mass spectrometry. Functionalized graphene quantum dots were prepared for loading free radicals in the complex screening system. The detection was performed with and without a preliminary exposure of the samples to specific free radicals on the functionalized graphene quantum dots, which can facilitate charge transfer between free radicals and antioxidants. The difference in chromatographic peak areas was used to identify potential antioxidants. This is a novel approach to simultaneously evaluate the antioxidant power of a component versus a free radical, and to identify it in a vegetal matrix. The structures of the antioxidants in the samples were identified using tandem mass spectrometry and comparison with standards. Fourteen compounds were found to possess potential antioxidant activity, and their free radical scavenging capacities were investigated. The order of scavenging capacity of 14 compounds was compared according to their free radical scavenging rate. 4′,5,6,7‐Tetrahydroxyflavone (radical scavenging rate: 0.05253 mL mg^?1 s^?1) showed the strongest capability for scavenging free radicals.     

Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures

In this study, an end-point-based fluorescence assay for soluble epoxide hydrolase (sEH) was transformed into an on-line continuous-flow format. The on-line biochemical detection system (BCD) was coupled on-line to liquid chromatography (LC) to allow mixture analysis. The on-line BCD was based on a flow system wherein sEH activity was detected by competition of analytes with the substrate hydrolysis. The reaction product was measured by fluorescence detection. In parallel to the BCD data, UV and MS data were obtained through post-column splitting of the LC effluent. The buffer system and reagent concentrations were optimized resulting in a stable on-line BCD with a good assay window and good sensitivity (S/N > 60). The potency of known sEH inhibitors (sEHis) obtained by LC–BCD correlates well with published values. The LC–BCD system was applied to test how oxidative microsomal metabolism affects the potency of three sEHis. After incubation with pig liver microsomes, several metabolites of sEHis were characterized by MS, while their individual potencies were measured by BCD. For all compounds tested, active metabolites were observed. The developed method allows for the first time the detection of sEHis in mixtures providing new opportunities in the development of drug candidates.     

Hyphenated HSCCC–DPPH· for Rapid Preparative Isolation and Screening of Antioxidants from Selaginella moellendorffii

A method for rapid preparative isolation and screening of antioxidants has been developed by combining preparative HSCCC with on-line radical scavenging detection by use of DPPH· (1,1-diphenyl-2-picrylhydrazyl) as model radical. The HSCCC effluent was split into two parts. The major part was collected and the rest was introduced, through a switch valve, directly into a reaction coil for on-line evaluation of antioxidant activity. Each of the antioxidants separated by HSCCC reacted postcolumn with the DPPH· solution in methanol and was observed as a negative peak at 517 nm of size corresponding to its antioxidant activity. The on-line method was successfully used for preparative isolation and screening of antioxidant components from an ethyl acetate extract of the traditional Chinese medicine Selaginella moellendorffii. A total of 114.7 mg amentoflavone, 12.4 mg hinokiflavone, 7.2 mg podocarpusflavone A, and 1.1 mg ginkgetin, identified as the main radical scavengers, were obtained with purity of 99.4, 98.6, 98.9, and 99.2%, respectively, as determined by LC, from 1.0 g ethyl acetate extract. Their chemical structures were identified by MS and by UV and NMR spectroscopy.     

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.     

In planta biocatalysis screen of P450s identifies 8-methoxypsoralen as a substrate for the CYP82C subfamily, yielding original chemical structures

An in vivo plant screen that allows for the analysis of exogenously applied substrates against transgenic Arabidopsis lines overexpressing individual cytochrome P450s has been developed. By deploying this screen with a subset of 91 P450s, we have identified an original substrate for members of the CYP82C subfamily. The therapeutic compound 8-methoxypsoralen was hydroxylated by plants overexpressing CYP82C2 or CYP82C4, forming 5-hydroxy-8-methoxypsoralen. Additionally, plants further modified this product to create a glycosylated compound, likely the compound 5-O-beta-D-glucopyranosyl-8-methoxypsoralen. The discovery of adducts of therapeutic compounds demonstrates the potential of this biocatalysis screening approach to create compounds that may be of pharmacological value. Additionally, this platform provides a means to expand the general knowledge base of P450 enzyme/substrate combinations and may provide valuable tools for a vast array of biocatalytic and bioremediation processes.     

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.     

Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations

On-line method, which combines HPLC distribution and post-column reaction, was designed for the search of individual antioxidants. Optimization of the assay was performed evaluating optimal ABTS(+) radical cation concentration in the reactor, reaction time, impact of flow rate, reaction coil length. HPLC-ABTS assay validation in this work was performed by assessing reference antioxidant negative peak areas in radical scavenging chromatogram. Sample free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). Optimized and validated method was applied in detection of compounds possessing free radical scavenging ability in complex mixtures. Antioxidant compounds were studied in perilla (Perilla frutescens (L.) Britton var. crispa f. viridis) herbal raw material and its preparations. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometer, using a reference mass for determining accurate masses. Radical scavenging characteristics of rosmarinic acid, which is the dominant phenolic compound in medicinal herbal raw material of perilla and its preparations, were confirmed by the calculated TEAC values. Compounds responsible for antioxidant effect in herbal raw materials and herbal preparations were identified, evaluated and compared.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.     

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.     

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.