Indole-3-acetic Acid Levels of Plant Tissue as Determined by a New High Performance Liquid Chromatographic Method

A method for the analysis of indole-3-acetic acid (IAA) in plant extracts has been developed based on high performance liquid chromatography separation of IAA on a microparticulate strong anion exchange column followed by quantitation with two selective detectors: an electrochemical, carbon paste amperometric detector and/or a fluorescence detector. The detection limit for IAA is less than 1 nanogram with the fluorescence detector and less than 50 picograms with the electrochemical detector.

The IAA levels are reported for various tissues of wheat, pinto beans, soybeans, cotton, and corn.

     

Narrow-bore liquid chromatography-tandem mass spectrometry with simultaneous radioactivity monitoring for partially characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor

Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C₁₈, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the ¹⁴C trace. A ¹⁴C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the ¹⁴C profile) were characterized by mass spectrometry.     

Narrow-bore liquid chromatography-tandem mass spectrometry with simultaneous radioactivity monitoring for partially characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg,a potent thrombin inhibitor

Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C₁₈, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the ¹⁴C trace. A ¹⁴C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the ¹⁴C profile) were characterized by mass spectrometry.     

Narrow-bore liquid chromatography-tandem mass spectrometry with simultaneous radioactivity monitoring for partially characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of -MePhe-Pro-Arg, a potent thrombin inhibitor

Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C₁₈, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the ¹⁴C trace. A ¹⁴C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of -MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the ¹⁴C profile) were characterized by mass spectrometry.     

Separation of oxidatively damaged DNA nucleobases and nucleosides on packed and monolith C18 columns by HPLC-UV-EC

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40min to just under 4min, was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV-vis and EC detector, which is especially useful for the analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2'-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal.     

High-throughput characterization and quality control of small-molecule combinatorial libraries

To fully realize the potential of combinatorial synthesis and high-throughput screening for increasing the efficiency of the drug discovery and development process, issues related to compound purity must be addressed. Impurities, often present after synthesis, can lead to ambiguous screening results and inhibit the development of quality structure-activity relationships. The demand for high-throughput analytical characterization of combinatorial libraries has prompted the development of more rapid methods to keep pace with compound production. Recent progress has focused upon the development of parallel separation methods, multiplexed detector interfaces, and synergistic combinations of different detectors possessing complementary selectivities.     

Computational modelling of motion detectors: responses to two-frame displays

Schemes for motion detection fall into two classes. Reichardt correlators compare spatial luminance patterns at two locations at different times; gradient detectors compare spatial and temporal luminance gradients. Both are candidate operators for biological and machine vision systems. A large body of perceptual data exists, defining the properties of motion detectors used by human observers, which can form a basis for determining which class of detector is appropriate for the human visual system. Plausible versions of each detector were implemented, and their responses to a variety of two-frame stimuli were computed. Results indicated that both detectors can predict most of the data, but on balance gradient detectors offer the best working hypothesis for motion detection by human observers. This conclusion is necessarily limited to the type of stimuli used, and may require modification in the light of responses to continuously moving stimuli.     

Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.     

Sensitive electrochemical detection method for alpha-acids, beta-acids and xanthohumol in hops (Humulus lupulus L.)

A new HPLC method with coulometric detection for the quantification of xanthohumol, alpha-acids and beta-acids in hops was developed. The separation of compounds was accomplished with a C18 column and isocratic elution with methanol: 50 mM potassium phosphate: ortho-phosphoric acid=80:20:0.25 (v/v/v). The method was validated and UV and electrochemical detectors (ECD) were compared. The HPLC method with ECD was precise, accurate and very sensitive for detection of xanthohumol and alpha- and beta-acids. The detection limits of analytes were at least 8.8 to 24 times lower with ECD than those of the UV detector. The ECD method was successfully applied for quantification of studied compounds in hop pellets. The concentrations of all compounds obtained with ECD and UV were found to be equivalent. This is the first study demonstrating a very sensitive and validated method for the quantification of xanthohumol, alpha- or beta-acids in hop samples with the use of the electrochemical detector.     

Development of the YAP-PEM scanner for breast cancer imaging

A prototype for positron emission mammography is under development at the Department of Physics of Pisa University. The device will be composed of two opposing detectors (parallel plane geometry). The active part of each detector head consists of a matrix of 900 YAP: Ce pixel scintillators, with a 2×2 mm² pitch and a 30 mm thickness. The read out is performed by an array of nine metal channel dynode PSPMTS (mod. R8520-00-C12) from Hamamatsu. In the previous version of the head, the PSPMTS were independently read out. For the clinical implementation of the prototype we have designed a simplified circuitry for the readout of the nine tubes based on a multiplexed resistive divider, reducing the number of channels from 36 to 4. A simulation study for an optimised amplifier has been carried out. The housing for each of the two yap-pem detectors has been fully engineered and is in the assembly stage.     

Instrumentation for advanced microseparations in pharmaceutical analysis and proteomics

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

High-performance liquid chromatography of biologically important pyrimidine derivatives with ultraviolet—voltammetric—polarographic detection

High-performance liquid chromatographic separation of a number of biologically important pyrimidine derivatives was studied in the reversed-phase system. Good results were obtained using a C₁₈ alkyl-bonded silica column and an aqueous citrate—phosphate mobile phase of pH 3.5. All eluted components are detected with the UV absorbance detector at 254 nm, whereas the voltammetric detector with a polymeric carbon-paste electrode detects only derivatives containing oxidizable functional groups (amino, mercapto) and the polarographic detector with a mercury electrode only those with reducible groups (nitro, aza). The signal of the electrochemical detectors is proportional to the number of electroactive groups in the solute molecule. The use of two or three detectors in series thus improves the resolution of complex mixtures and facilitates identification.     

High-throughput comprehensive analysis of D- and L-amino acids using ultra-high performance liquid chromatography with a circular dichroism (CD) detector and its application to food samples

A rapid and comprehensive analytical method for D- and L-enantiomers of proteinogenic amino acids was developed using ultra-high performance liquid chromatography (UHPLC) equipped with a circular dichroism (CD) detector. Pre-column derivatization reagents were examined for enhanced sensitivity and selectivity for UV and CD detection: 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected. The method, using a CD detector, does not require separation of optical isomers on a column to calculate the enantio ratio (%D) using the g-factor value and produces a simple chromatogram in comparison to other reported methods. Using this advantage, combined with UHPLC technology, analysis time for the derivatized proteinogenic amino acids was within 5.5 min. The UV detection limit was 4.9-23 pmol/injection and the CD detection limit was 11-64 pmol/injection. The method was applied to the analysis of D- and L-amino acids in food samples. D-Ala, D-Asp, D-Glu and D-Ser were detected at high concentrations in some Japanese black vinegars, fermented milks and yogurts. The results were identical to the results determined by the OPA method. We suggest the UHPLC-CD method would be useful in screening the D-amino acid content of foods and in helping to clarify the importance and reason for the presence of D-amino acids in foods.     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. METHODS: Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.     

Automated methods for multiplexed pathogen detection

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.     

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.     

Assay of the enantiomers of ibutilide and artilide using solid-phase extraction,derivatization, and achiral-chiral column-switching high-performance liquid chromatography

Ibutilide fumarate and artilide fumarate, new drugs for the treatment of cardiac arrhythmias, each contain a stereogenic center bearing a secondary alcohol group. Reversed-phase achiral-chiral column-switching HPLC separations of the enantiomers of each compound were developed and validated for quantitation in plasma and other biofluids. The key component of the method was derivatization with 1-naphthyl isocyanate, which enhanced the sensitivity of fluorescence detection and enabled the enantiomers to be separated on a Pirkle column (covalent 3,5-dinitrobenzoyl-d-phenylglycine stationary phase). The lower limit of quantitation of ibutilide fumarate was typically 0.1 ng/ml or less of each enantiomer in 1 ml of plasma. Two of the special features of the column-switching system included operation with two samples in the system at one time, which reduced analysis time to 16 min/sample for ibutilide and 19 min/sample for artilide, and a relay-operated switching of detector outputs, which allowed achiral and chiral column chromatographic data to be gathered from two detectors into a single data acquisition channel.     

A flow immunoassay for studies of human exposure and toxicity in biological samples

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.     

Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).