Comparative Liquid Chromatographic Studies for the Determination of Bioactive Peptides

Reliable and sensitive chromatographic methods have been developed and applied to determine derivatized di- and tripeptides: aspartame, carnosine, glutathione, alanyl-glutamine, and γ-glutamyl-cysteine. Photodiode-array detection was used for dansyl-chloride derivatives, while fluorescent detection was carried out for orthophthaldialdehyde derivatized peptides to obtain higher sensitivity. The sensitivity and detection limits were determined and compared in order to select the most efficient analytical method. Transition metal complexes [zinc(II), cadmium(II), silver(I), and copper(II)] with cysteine containing peptides were also determined with ultraviolet-visible detection. The structural identification was carried out by the analysis of the mass-spectra recorded with an atmospheric pressure chemical ionization method in separate chromatographic experiments.     

Changes in the fluorescence spectrum of individual single-wall carbon nanotubes induced by light-assisted oxidation with hydroperoxide

Through fluorescence-spectrum measurements, we investigated the effects of light-assisted oxidation with H2O2 (LAOx) on single-wall carbon nanotubes (SWNTs) that were individually dispersed in an aqueous solution of surfactant. The intensities of the fluorescence spectra were decreased remarkably by the LAOx when the light's wavelength was 400-500 nm and a little when 600-700 nm. The spectrum intensity did not recover even when the pH was restored to an original value of 6.5. The spectra changed little when the LAOx wavelength was 500-600 nm or the light was not irradiated. In addition, the effect of LAOx on SWNTs was related to the diameters of SWNTs. We inferred that these phenomena reflected that H2O2 was dissociated by absorbing the fluorescence light emitted from optically excited SWNTs, which, in turn, accelerated the burning out of SWNTs.     

Some applications of near-ultraviolet laser-induced fluorescence detection in nanomolar- and subnanomolar-range high-performance liquid chromatography or micro-high-performance liquid chromatography

In this work we present some applications of near-UV laser-induced fluorescence (LIF) with micro-HPLC (microHPLC) and HPLC. To test the sensitivity of the detection, we used pyrene and aflatoxins, because both of these molecules exhibit native fluorescence. Then we studied catecholamines derivatized with 1,2-diphenylethylenediamine. The results show that we were able to reach better sensitivity levels than previously described in LIF studies. For catecholamines, a 50-fold increase in sensitivity compared to conventional fluorescence was obtained. These results indicate that LIF detection associated with HPLC or microHPLC can be used to detect very low concentrations of substances that can be excited in the near-UV range after labeling at nanomolar concentrations.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

Postcolumn derivatization of amino acids using reaction flow chromatography columns with fluorescence detection: A fast new approach to selective derivatization techniques

Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yielded R²?≥?0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously.     

Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.     

Miniaturized thermal lens and fluorescence detection system for microchemical chips

We have developed a miniaturized two-way detection system using thermal lens and fluorescence spectroscopies for microchip chemistry. The system was composed of laser diode (LD) modules, fiber-based optics combined with a gradient index lens, and miniaturized detection units for thermal lens and fluorescence signals. The detection limits in the thermal lens and fluorescence spectroscopies were 6.3 x 10(-9)M for Ni(II) phthalocyanine tetrasulfonic acid and 3.0 x 10(-9)M for cy5, respectively. The performance of the system with the miniaturized thermal lens was equivalent to that of a conventional thermal lens microscope. The fluorescence sensitivity was comparable to sensitivities offered by conventional miniaturized systems.     

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed.In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.     

A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography/tandem mass spectrometry

Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.     

Peptide maps at picomolar levels obtained by reversed-phase high-performance liquid chromatography and pre-column derivatization with phenyl isothiocyanate. Microsequencing of Phenylthiocarbamyl Peptides

A new reversed-phase high-performance liquid chromatography approach to the production of analytical peptide maps by pre-column derivatization using phenylisothiocyanate is described. Tryptic peptide digests were derivatized with phenyl isothiocyanate to form the phenylthiocarbamyl peptides followed by reversed-phase high-performance liquid chromatographic analysis. The phenylthiocarbamyl peptides were separated by reversed-phase high-performance liquid chromatography with the conventional gradient elution system of water-acetonitrile containing trifluoroacetic acid. The sensitivity of detection of these peptide derivatives was within the range 5-10 pmol with a constant baseline at 254-260 nm. The isolated phenylthiocarbamyl peptides can be subjected to automatic Edman degradation. The effectiveness of this method was exemplified by microsequencing of phenylthiocarbamyl peptides isolated from tryptic digests of three different proteins: alpha-lactalbumin, beta-lactoglobulin and a lambda light-chain immunoglobulin.     

Single-walled carbon nanotube as an effective quencher

Over the past few years, single-walled carbon nanotubes (SWNTs) have been the focus of intense research motivated by their unique physical and chemical properties. This review specifically summarizes recent progress in the development of fluorescence biosensors that integrate the quenching property of SWNTs and the recognition property of functional nucleic acids. SWNTs are substantially different from organic quenchers, showing superior quenching efficiency for a variety of fluorophores, with low background and high signal-to-noise ratio, as well as other advantages derived from the nanomaterial itself. As the second key component of biosensors, functional nucleic acids can bind to either their complementary DNA or a target molecule with the ability to recognize a broad range of targets from metal ions to organic molecules, proteins, and even live cells. By taking advantage of the strengths and properties of both SWNTs and nucleic acid based aptamers, a series of fluorescence biosensors have been designed and fabricated for the detection of a broad range of analytes with high selectivity and sensitivity.     

Nanoparticle targeting at cells

Gold nanoparticles have been used for analytical and biomedical purposes for many years. In fact, the labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. We report in this study the derivatization of tiopronin-protected nanoparticles with ethylenediamine and poly(ethylene glycol) bis(3-aminopropyl) terminated and their functionalization with the GRGDSP peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line to determine the biocompatibility, and the cell-particle interactions, using fluorescence and scanning electron microscopies. The results indicate that tiopronin gold nanoparticles aggregate due to culture medium proteins, whereas the tiopronin gold nanoparticles derivatized with ethylenediamine induce endocytosis, and the same nanoparticles derivatized with poly(ethylene glycol) derivative promote particle-cell adhesion.     

Performance of an ultraviolet light-emitting diode-induced fluorescence detector in capillary electrophoresis

The performance of a fluorescence detector in capillary electrophoresis (CE) using a light-emitting diode (LED) as excitation source is reported. An ultraviolet LED pulsed at a repetition rate of 500 Hz, combined with a time-discrimination and averaging acquisition system, was used. Limits of detection of 3 and 18 fmoles (at a signal-to-noise ratio equal to 3) were achieved for fluorescamine-derivatized bradykinin and lysine, respectively. This system exhibited a linear response for a concentration range between 54 and 417 microM for derivatized lysine, and between 1.81 and 23.58 microM for derivatized bradykinin. This detection system showed to be very convenient for routine analytical applications.     

Electrochemical detection of the 3,5-dinitrobenzoyl derivative of digoxin by high-performance liquid chromatography

Electrochemical detection of 3,5-dinitrobenzoyl derivatives of digoxin and its metabolites following high-performance liquid chromatography is reported. Partial resolution of derivatized digoxin and dihydrodigoxin was obtained using a Spherisorb ODS II analytical column. Both single- and dual-electrode detection were investigated and a maximum sensitivity equivalent to 0.39 ng of digoxin was found with the dual-electrode method. This system has the necessary sensitivity and selectivity for development into a therapeutic monitoring assay method.     

Determination of individual bile acids in serum by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4-bromomethyl-7-methoxycoumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep-Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18-crown-6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm. Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free-bile acids. Recoveries of individual bile acids varied from 83-96% for free and glycine conjugates and 72-83% for taurine conjugates. Coefficients of variation were in the range of 5.1-12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods.     

Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products

Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.     

Determination of Underivatized Polyamines: A Review of Analytical Methods and Applications

Putrescine, cadaverine, spermidine, and spermine are ubiquitous polyamines that are essentially found in diverse organisms (e.g., bacteria, fungi), animals, and higher plants. Analysis of these analytes is traditionally performed either by chromatographic or electrophoretic techniques. The majority of assays employ liquid chromatography with fluorimetric detection either with pre-column or post-column derivatization. However, the derivatization procedures have several disadvantages to the whole analytical process. This article describes the analytical method developments in the determination of underivatized polyamines. This includes flow injection analysis, high performance liquid chromatography (using conductivity, condensation nucleation light scattering, chemiluminescence, indirect fluorescence, and mass spectrometric detection) and electromigration methods. The reported systems are essentially based on the work developed by the authors since 1995. A comparison of the methods highlighting their main advantages and disadvantages and sensitivity was also provided. The most promising system seems to be the liquid chromatography-tandem mass spectrometric due to its high sensitivity and specificity.     

Time-resolved high-performance liquid chromatography fluorescence detector using a nanosecond pulsed light source for detecting lanthanide-chelated compounds

We have constructed a time-resolved fluorescence detection (TRFD) system for the analysis of amino compounds with high-performance liquid chromatography (HPLC) using lanthanide ion chelates. In order to carry out time-resolved measurements, we have employed a nanosecond pulsed xenon-arc lamp as an excitation light source. Amino compounds derivatized by isothiocyanobenzyl-EDTA (IEDTA) with the lanthanide chelate are mixed with an enhancer solution in a post-column manner and detected by TRFD. Taking advantage of a property of the long fluorescence lifetime of the lanthanide chelates, high selectivity against background fluorescence was achieved. In order to demonstrate the usefulness of TRFD, fundamental performance tests were carried out. Details of the system are also described.     

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for rapid and sensitive analysis of two new bioactive reagents using dynamic covalent coating

A simple, rapid, selective, and sensitive micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection (LIF) method was developed, using hexamethyldisilazane (HMDS) as dynamic covalent coating (DCC), for the analysis of two new bioactive agents N-n-hexyl-N'-(sodium p-aminobenzenesulfonate) thiourea (HXPT) and N-n-undecyl-N'-(sodium p-aminobenzenesulfonate) thiourea (UPT) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. MEKC methods both not using DCC and using DCC were investigated. In a series of optimization steps, DCC and a running buffer of 20 mM Na2B4O7 + 16 mM SDS + 8% acetonitrile were applied for determination of the derivatives. Linear relationships for HXPT and UPT were obtained in the range of 5 to 100 microM (correlation coefficient: 0.9986 for HXPT, 0.9978 for UPT), and the detection limits for HXPT and UPT were 16.5 and 39.0 ng mL(-1). The sensitivity was improved over that of fluorescence spectroscopy methods. The method was applied to the analysis of the two reagents in lab water waste with recoveries in the range of 95.6-107.5%.