Specific postcolumn detection method for HPLC assay of homocysteine based on aggregation of fluorosurfactant-capped gold nanoparticles

Gold nanoparticles (GNPs) capped with nonionic fluorosurfactant molecules (Zonyl FSN) were synthesized, and with the colloidal solution as a probe reagent, a new postcolumn colorimetric detection method for HPLC assay of homocysteine (Hcy) has been developed. The FSN-capped GNPs exhibited excellent stability in aqueous solutions, even in the presence of high salt. The aggregation of the GNPs could be induced by either Hcy or cysteine, resulting in an absorption decrease of the colloidal solution at 525 nm and an absorption increase at longer wavelengths (600-700 nm); however, the GNPs did not respond to other amino acids and biomolecules such as glutathione, cysteinylglycine, and glucose. Under optimal conditions (i.e., high salt, neutral pH, and approximately 70 degrees C), the color change of the GNP solution could almost complete ( approximately 90%) within approximately 30 s upon the addition of Hcy. The high selectivity and very fast kinetics of the reaction make it a promising system for HPLC postcolumn detection. The new technique has been employed to determine total Hcy levels in human urine and plasma samples, and the results are satisfactory.     

Application of rhodamine 800 for reversed phase liquid chromatographic detection using visible diode laser-induced fluorescence

This study reports the application of rhodamine 800, a far-red dye, suitable for excitation using visible diode laser-induced fluorescence (VDLIF) detection. A reagent synthesized from rhodamine 800 was evaluated as a precolumn reagent for derivatization with amino-containing analytes. The derivative of this reagent with primary amine analytes showed a loss of fluorescence. Rhodamine 800 was then applied as a mobile phase additive in the indirect mode for quantitation of valproic acid in plasma using reversed phase HPLC in combination with VDLIF detection. A visible diode laser (output power 8.50 mW) temperature-tuned to oscillate at 674.70 nm was used as a light source for a laboratory constructed HPLC fluorescence detector. A liquid/liquid extraction procedure was applied to human blank plasma. The selectivity of this method was validated by demonstration of a lack of interfering peaks in extracts of plasma (n = 3 sources). A calibration curve for valproic acid between 40 and 200 μg/mL was shown to be linear (r = 0.9932). The recoveries of valproic acid at concentrations of 50 and 100 μg/mL were evaluated and determined to be 73 and 72%, respectively. The precision and accuracy (n = 5) of the assay was within 7.0% RSD and 8.0% difference from the spiked concentration, respectively. The limits of detection (S/N = 3) for extracted and unextracted valproic acid were 15.0 and 11.54 μg/mL, respectively. The theoretical (C(lim)) and practical (C(det)) limits of detection in the detector flow cell for unextracted valproic acid at a S/N = 1 were found to be within 15%.     

Fluorescence detection of flavonols in HPLC by postcolumn chelation with aluminum

Flavonols are dietary antioxidants which may prevent coronary heart disease. To be able to study absorption of flavonols in humans, we developed a postcolumn derivatization with aluminum for HPLC with fluorescence detection. Variables governing postcolumn chelation, such as water content, buffer, organic modifier of the eluent, concentration of Al(3+), presence of acetic acid in the postcolumn reagent, and temperature, were studied and optimized. Of the flavonoids, only flavonols that contain a free 3-hydroxyl and 4-keto oxygen binding site form fluorescent complexes with Al(3+). The method has a detection limit of 0.15 ng/mL for quercetin, 0.05 ng/mL for kaempferol, 0.45 ng/mL for myricetin, and 0.05 mg/mL for isorhamnetin, thus improving detectability of quercetin 300-fold as compared to that possible with UV detection. The reproducibility relative standard deviation of the method is 1.4%. This extremely sensitive method enables, for the first time, determination of flavonols in body fluids after consumption of a normal diet.     

Determination of Iodide and Iodate by Ion Chromatography with Postcolumn Reaction and UV/Visible Detection

Iodide and iodate can be determined by two new methods using anion-exchange chromatography with postcolumn reaction and UV/visible detection. Iodide is determined as IBr(2)(-) at 249 nm. Iodate is determined as I(3)(-) at 288 nm. The analyses can be run completely automatically and do not require any sample pretreatment. The detection limits for iodide and iodate are 0.1 μg/L. The methods have been successfully applied to determine iodide and iodate in several mineral waters and in drinking water as well as for the determination of iodide in table salt.     

Rugged gap reactor device for postcolumn fluorescence detection in capillary electrophoresis

In this paper, the construction and performance of a rugged device for postcolumn derivatization in capillary electrophoresis (CE) are described. The device was based on a gap design, and a gap with a very small distance (<3 μm, estimated under microscope) could be easily constructed without micromanipulation. Addition of derivatizing reagents into the reaction capillary was attributable to gravity flow. The concentration of derivatizing reagents can be controlled through manipulating the electroosmotic flow in the reaction capillary and the height of the liquid levels from the derivatizing reagents to the buffer reservoirs. The device has been applied in fluorescence detection of amino acids using a mixture of o-phthaldialdehyde/2-mercaptoethanol as derivatizing reagent. Theoretical plate numbers for 11 amino acids separated in a pH 9.5 borate buffer were obtained in the order of 40?000-250?000. The detection limit for glycine (S/N = 2) was found to be 6.7 × 10(-)(7) mol/L using a commercial HPLC fluorescence detector modified for CE. Free amino acids in a wine sample were also determined. Because the device is quite stable, we believe that it can be used routinely in analytical laboratories.     

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection.

A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique.     

Stable tris(2,2'-bipyridine)ruthenium(III) for chemiluminescence detection

Two exceedingly stable [Ru(bipy)(3)](3+) reagents were prepared by dissolving either [Ru(bipy)(3)](ClO(4))(2) in acetonitrile (containing 0.05 M HClO(4)) or [Ru(bipy)(3)]Cl(2)·6H(2)O in 95:5 glacial acetic acid-acetic anhydride (containing 0.05 M H(2)SO(4)) followed by oxidation with PbO(2). These conveniently prepared solutions provide highly reproducible chemiluminescence detection over long periods of analysis, avoiding the need for recalibration or preparation of fresh reagent solutions and without the complications associated with online chemical or electrochemical oxidations. The reagent prepared in acetonitrile produced much greater signal intensities with a range of analytes and was deemed most suitable for high-performance liquid chromatography (HPLC) with postcolumn chemiluminescence detection.     

Laser ablation construction of on-column reagent addition devices for capillary electrophoresis

A simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis. This technique uses laser ablation with the second harmonic of a Nd:YAG laser (532 nm) at 13.5 mJ/pulse and a repetition rate of 15 Hz to create these gaps. A capillary is glued to a microscope slide and positioned at the focal point of a cylindrical lens using the focused beam from a laser pointer as a reference. Gaps of 14.0 +/- 2.2 microm (n = 33) at the bore of the capillary are produced with a success rate of 94% by ablation with 400 pulses. This simple method of gap construction requires no micromanipulation under a microscope, hydrofluoric acid etching, or use of column fittings. These structures have been used for reagent addition for postcolumn derivatization with laser-induced fluorescence detection and have been tested for the separation of proteins and amino acids. Detection limits of 6 x 10(-7) and 1 x 10(-8) M have been obtained for glycine and tranferrin, respectively. Separation efficiencies obtained using these gap reactors range from 38,000 to 213,000 theoretical plates.     

Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles

Silica nanoparticles with a diameter of 100 nm were covalently modified at their surface by adjustable amounts of amine and carboxyl functional groups. Bioconjugation studies of two proteins, streptavidin and streptactin, with the functional nanoparticles resulted in optimum binding of the proteins to a long-chain carboxyl-terminated linker. The surface functionalization of the nanoparticles was monitored by a variety of independent methods, including zeta-potential measurements, dynamic light scattering (DLS), scanning electron microscopy (SEM), particle charge detection titrations (PCD) and elemental analysis. At the surface of the nanoparticles, a functional surface group density of 1.8 amino groups per nm2 was realized. The amine functions were quantitatively transferred to carboxyl groups coupled with a linker elongation. Streptavidin was immobilized by covalent binding to the carboxyl linkers and resulted in a protein density at the surface of the nanoparticles that was three times higher than the highest binding densities at nanoparticles published to date. The binding capacity of the streptavidin-covered nanoparticles for ligand biotin was quantified by titration with biotin-4-fluorescein to 2.5 biotin binding sites per 100 nm2.     

A chemiluminescence reaction between hydrogen peroxide and acetonitrile and its applications.

Here we report a new chemiluminescence reaction between basic aqueous H2O2 and acetonitrile. Its ultraweak chemiluminescence could be greatly enhanced by luminol, isoluminol-labeled streptavidin, and an Edman-type fluorescent reagent. Light emission was intense and long-lived, and this facilitated the initiation of the reaction and the measurement of the light emission. The present results permit us to propose a series of convenient, highly sensitive, and enzyme-free techniques for the detection and quantification of luminol, related conjugates, acetonitrile, and amino acids. Overall, this new chemiluminescence reaction will be quite promising for numerous applications in immunoassay, DNA hybridization, environmental monitoring. and postcolumn chromatographic detection.     

On-line detection of antioxidative activity in high-performance liquid chromatography eluates by chemiluminescence

Luminol chemiluminescence (CL) was employed for the on-line detection of radical scavengers in HPLC eluates. Optimization of CL reagents and instrumental setup resulted in a steady postcolumn luminol photochemical reaction in the presence of microperoxidase and hydrogen peroxide at pH 10. Quenching of the CL signal was utilized to detect radical scavenging activity of both natural and synthetic antioxidants at the nanogram level. The detection system can be used with isocratic or gradient elution. Several antioxidative compounds were detected in thyme and sage acetone extracts. Quantitative results can be obtained when antioxidants are analyzed at certain concentrations. The method is suitable for rapid screening of antioxidants in crude extracts.     

Chromatographic detection using tris(2,2'-bipyridyl)ruthenium(III) as a fluorogenic electron-transfer reagent

Tris(2,2'-bipyridyl)ruthenium can be excited to fluorescence by visible light (lambda abs 454 nm, lambda em 607 nm) when in the M(II) oxidation state, but not in the M(III) state. A novel chromatographic detection method using the non-fluorescent M(III) form of the complex as a postcolumn fluorogenic reagent is demonstrated. The M(III) form is a powerful oxidizing agent (E degree = 1.27 V vs NHE, 1.05 V vs Ag/AgCl). The M(III) reagent is generated on-line from the M(II) form of the complex by a highly efficient porous carbon electrode and then reacted briefly with chromatographic effluent; the M(II) created by electron transfer from oxidation-susceptible analytes is then detected by fluorescence. The fluorescence detector can be calibrated for number of electrons transferred by injection of either M(II) or an oxidative standard such as ferrocyanide. It is hoped that this redox-based detection scheme will provide an alternative to electrochemical detection. Among the advantages are freedom from surface fouling and the potential for extremely low detection limits. The scheme was applied to detection of the peptide dynorphin A and several of its fragments. Dynorphin A contains tyrosine at the N-terminus (position 1) and tryptophan in position 15; these amino acid residues are susceptible to oxidation and peptides containing them can be detected on that basis. Flow injection testing of the model compounds Tyr-Gly-Gly-Phe-Leu and Gly-Gly-Trp-Gly indicated that tyrosine transferred approximately 1 electron to the M(III) reagent and that tryptophan transferred approximately 4 electrons. Similar results were obtained from the chromatographic runs. Dynorphin A and six dynorphin A fragments containing the N-terminal tyrosine were detected easily at 100 nM concentration (14 pmol) using laser-induced fluorescence. As expected, one fragment that did not contain tryptophan or tyrosine was not detected. A mass detection limit of 80 fmol was estimated for the tyrosine-containing fragments.     

Chromatographic detection of nitroaromatic and nitramine compounds by electrochemical reduction combined with photoluminescence following electron transfer

The oxidizing agent tris(bipyridyl)ruthenium(III), or Ru-(bpy)(3)3+, is used as a postcolumn reagent for the detection of nitroaromatic and nitramine explosive compounds. After separation, the explosives are reduced electrochemically to oxidizable products such as hydroxlamines and nitrosamines, and these products react readily with Ru-(bpy)(3)3+ and Ru(bpy)(3)2+. The photoluminescence from the latter is used for detection. A porous carbon electrode was used for on-line analyte reduction following chromatography. Another porous carbon electrode was used to generate the nonluminescent Ru(bpy)(3)3+ from Ru(bpy)(3)3+ on-line at high efficiency. The two streams were combined, and the Ru(bpy)(3)2+ produced by oxidation of the reduced analytes was detected by laser illumination and light detection. Reductive hydrodynamic voltammograms of nitrobenzene, 2,4,6-trinitrotoluene, and hexahydro-1,3,5-trinitro-1,3,5-triazine indicated that a potential of - 1500 mV vs Ag/AgCl was sufficient to achieve a maximum signal from the reduced analytes. HPLC with a water/acetonitrile gradient on a C-18 reversed-phase column was then used to determine these three compounds plus the four additional examples, 1,3,5,7-tetrazocine, 2,4-dinitrotoluene; 2,6-dinitrotoluene, and 4-nitrotoluene. For both hydrodynamic voltammetry and HPLC detection, the photoluminescence following electron-transfer signal was calibrated using the one-electron standards ferrocene and ferrocenecarboxylic acid. Detection limits were in the low-nanomolar range for 20-microL injections of nonpreconcentrated nitro compounds.     

An on-line HPLC method for detection of radical scavenging compounds in complex mixtures

A rapid on-line method for screening of complex mixtures for radical scavenging components was developed using a methanolic solution of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) stable free radical. The HPLC-separated analytes react postcolumn with the DPPH solution, and the induced bleaching is detected as a negative peak by an absorbance detector at 517 nm. An optimized instrumental setup is presented. The method is suitable for both isocratic and gradient HPLC runs with mobile-phase compositions ranging from 10 to 90% organic solvent in water or buffer (pH 3-6). The method is simple, has a broad applicability, and uses common instruments, inexpensive and stable reagents, and a time-saving and nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The limits of detection were 0.33-94 microg/mL, depending on the compound tested.     

Multidimensional biochemical detection of microcystins in liquid chromatography

The coupling of antibody-, receptor-, or enzyme-based inhibition assays postcolumn to chromatographic systems provides biological detectors with extraordinary high sensitivity and specificity. Three monoclonal antibodies (MC10E7, AD4G2, M8H5) directed against microcystins and protein phosphatase 1 (PP1) were used as off-line detectors for the HPLC separation of microcystins and nodularin in comparison to UV detection. For HPLC/ELISA coupling using antibody MC10E7, a detection limit of 0.04 ng microcystin-LR was achieved. The provisional guideline value for microcystin-LR (1 microg/L, WHO) could be monitored without prior sample concentration, in contrast to UV detection. Quantification of microcystin-LR and two cross-reactants was demonstrated. Furthermore, cross-reactivity or enzyme inhibition of new microcystins, only available in small amounts, can be determined by this method. Using a cyanobacterial extract, HPLC/ELISA coupling was compared to HPLC/UV and electrospray ionization mass spectrometry (ESI-TOFMS).     

Selective HPLC Analysis of n-Alkyl Hydroperoxides up to C(18)H(38)O(2)

A series of n-alkyl hydroperoxides are separated by HPLC and detected by their postcolumn reaction with horseradish peroxidase and p-hydroxyphenylacetic acid (HPAA) to yield a fluorescent product; several secondary and tertiary hydroperoxides, some 1-hydroxyalkyl hydroperoxides, and a few branched hydroperoxides are also examined. n-Alkyl hydroperoxides up to at least C-18 react with the enzyme with only minimally reduced efficiency at greater alkyl chain length. The effects of the column, the eluent, and the pH of the sample reaching the detector are described. The detection limit with gradient elution ranges from 0.4 μmol L(-)(1) for n-hexyl hydroperoxide to 1 μmol L(-)(1) for n-octadecyl hydroperoxide.     

Optical detection of chloramphenicol using molecularly imprinted polymers

A practical optical sensing system for the determination of chloramphenicol (CAP), utilizing molecularly imprinted polymers (MIPs) and HPLC, has been developed. The method is based on competitive displacement of a chloramphenicol-methyl red (CAP-MR) dye conjugate from specific binding cavities in an imprinted polymer by the analyte. The best of these polymers was obtained using (diethylamino)ethyl methacrylate as functional monomer at a monomer:template ratio of 2:1. HPLC with a mobile phase containing CAP-MR was used as the detection system, and injection of CAP and, to a lesser degree, thiamphenicol resulted in proportional displacement of the conjugate, which was detected at 460 nm. The detection system showed a linear response over a range of 3-1000 μg/mL and effectively detected CAP extracted from serum. This system offers a tailor-made, selective, and rapid method for CAP detection, is able to discriminate between similar molecules, and is effective below and above the therapeutic range (10-20 μg/mL serum, potentially toxic above 25 μg/mL). This technique is quite general and should enable the use of MIPs in a wide variety of applications involving the detection of families of molecules which possess a distinct arrangement of functional groups.     

Screening metal binding selectivities of macrocycle mixtures by HPLC--ESI-MS and postcolumn reactions

A rapid method for the screening of metal binding selectivities of host compounds in mixtures is presented. This method involves the separation of mixtures of hosts by HPLC, followed by postcolumn complexation with one or more metals, then analysis by mass spectrometry. The intensities of the host-guest complexes in the mass spectra correlate with the binding selectivities of the hosts. The method was applied to a series of lariat ethers that were synthesized as ion-selective reagents for ion-selective electrodes. The compounds most selective for Na+ vs Li+ and K+ were identified. Additionally, a mixture of substituted calixarenes was screened for alkali-metal-binding selectivity. These compounds were determined to be selective for Cs+ over Rb+, K+, and Na+.     

Affinity detection of low molecular weight analytes

In this paper we report attempts to detect directly the binding of a low molecular weight substance to a protein binding site. An optical transducer based on reflectometric interference spectroscopy (RIFS) was used to detect the binding of biotin (244 g/mol) to a thin silica film surface coated with streptavidin. RIFS allows measurement of changes in the optical thickness of thin transparent films with high resolution. During immobilization of streptavidin, an increase in layer thickness of about 5 nm was detected. Subsequent incubation with biotin (4 μM) resulted in a thickness increase of about 70 pm. Repeated incubation with biotin gave no further increase in layer thickness. The lowest biotin concentration showing significant effects was 40 nM. Incubation with benzoic acid (40 μM) gave no thickness change. The setup allowed significant detection of thickness increases of 2 pm and above. Therefore, the thickness effects observed in the study could be unambiguously and clearly identified.