Enzymatic Detection of Urinary Steroids on Thin-Layer Chromatograms

Abstract

Enzymatic detection of urinary steroids on thin-layer chromatograms (TLC) is described. Steroids in urine such as neutral 3α-hydroxysteroids, 3β-hydroxysteroids, acidic 3α-hydroxysteroids and 17β-hydroxysteroids are detected on thin-layer plate using enzyme reactions.

Combination of chromatographic techniques and enzyme reactions give more specific analysis of the compounds in a body fluid. Some clinical application by the use of these enzymatic methods is also described and these enzymatic detection methods are shown to be useful for the diagnosis of some disease of abnormal steroid metabolism.     

Piezoelectric Detection of Ricin and Affinity-Purified Goat Anti-ricin Antibody

Abstract

Detection of ricin, in a piezoelectric quartz crystal microbalance format, can be accomplished with the use of capture antibody techniques. These techniques allow for the specific attachment of ricin to immobilized capture antibodies on the quartz crystal transducer area. A reversed format can also be used to detect antibody in solution. In this case, the antigen is immobilized and antibody attaches specifically, thus increasing the overall mass and decreasing the resonant frequency. In this report we describe detection of both ricin and anti-ricin antibody using immunological piezoelectric quartz crystal microbalance techniques.

     

Pre-Column Switching Techniques for the Determination of Drugs and Metabolites in Body Fluids in Research and Routine Analysis

Abstract

The use of a column switching system for direct injection of samples and of a sample clean-up on reversed phase pre-columns is described. The pre-columns were filled with spherical C-18 silica gel of particle size 30 μm.

Two applications are reported on: (1) the direct injection of serum samples for the simultaneous analysis of nine antiepileptic drugs and metabolites and (2) the determination of phenytoin and of carbamazepine in serum ultra-filtrates.

The purge liquid for the sample clean-up was diluted phosphoric acid, and the eluent mixture for the chromatographic separation was water/acetonitrile. The analytical column (length 12.5 cm) was filled with C-18 silica gel of particle size 5 μm. A gradient elution was chosen for the first application, while the second application was carried out using isocratic chromatographic conditions.     

Analysis of Malodorous Sulfur Gases and Volatile Organometalloid Compounds in Landfill Gas Emissions Using Capillary Gas Chromatography with Programmed Temperature Vaporization Injection and Atomic Emission Detection

Volatile compounds containing Group V and Group VI elements in landfill gases are of concern as a source of toxic pollutants and unpleasant odors. Conventional analytical techniques for these compounds e.g. ICP-MS, ICP-AES are complicated, expensive and time consuming. The use of a simple programmed temperature vaporization injection (PTV) technique coupled to gas chromatography with atomic emission detection (GC-AED) has been successfully demonstrated to identify compounds containing arsenic, antimony, and sulfur in landfill gas. With an adapted PTV injection system (using a combination of a ten-port and a six-port Valco valves), problems associated with AED discharge tube damage due to high carrier gas flow rate during sample loading can be overcome. The gas samples generated from both a laboratory biowaste digester and a domestic landfill site were characterized using these techniques. Large sample gas volumes were adsorbed onto a cooled sorbent trap containing Porapak Q, followed by rapid liberation onto a porous layer open tubular column (PLOT) using programmed thermal desorption. Arsenic and antimony were also detected in the landfill leachate collected from the same landfill site using hydride generation-atomic absorption spectrometry (Hy-AAS). The efficiency of different traps has also been compared.     

Amperometric Immunosensor for the Detection of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Water

ABSTRACT

An amperometric immunosensor for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in water has been developed using sequential injection analysis techniques. The system is based on a rapid competitive enzyme immunoassay employing an alkaline phosphatase-labeled monoclonal antibody directed against the herbicide and an immunoreactor with 2,4-D immobilized via bovine serum albumin either to Eupergit in a column or directly to the surface of a glass capillary. The detection limit of the immunosensor at 0.1 μg 2,4-D/l without enrichment of the analyte makes automatic measurements of 2,4-D in drinking and ground water feasible.     

Direct Determination of Glucose in Blood Serum Using Trinder's Reaction

Abstract

A very simple flow injection analysis system for direct determination of glucose in blood serum based on Trinder's reaction is described. The sera samples (15 μl) can be injected directly to the system without the deproteinization or the use of a dialyzer.

Calibration curves are linear in the range 50–400 mg/dl. The sampling frequency is 60 samples per hour. Results obtained by the proposed procedure are compared with those acquired at a local hospital using their routine glucose procedure also based on Trinder's reaction. It is shown that a better mix between sample and reagents is achieved using the single bead string reactor (SBSR).     

The Determination of Non-Oxidizable Species Using Electrochemical Detection in Ion Chromatography

Abstract

The use of an electrochemical detector for the ion chromatographic detection of non-oxidizable anions such as F^?, PO₄ ^?3, NO₃ ^?, and SO₄ ^?2 is described. The electrochemical detector is placed in line after a fiber suppressor and responds to eluent pH changes as the dissociated acids pass through the detector. The intensity of the signal is dependent on the applied potential at the cell with 0.3V being an optimum. Minimum detection limits with a 0.10 mL sample injection volume are below 0.5 ppm for F^?, Cl^?, PO₄ ^?3, NO₃ ^?, and SO₄ ^?2. No adverse effects on the silver working electrode have been observed.     

Bioanalysis of Naproxen by High Performance Reversed Phase Liquid Chromatography with Photometric and Fluorimetric Detection

Abstract

Methods for the quantitative determination of NAPROXEN and its main metabolite in plasma and urine are described. The separation is based on reversed phase liquid chromatography with LiChrosorb RP 8 (5 μm) as the support and methanol/phosphate buffer pH 7 as mobile phase, in some cases with addition of tetrabutyl ammonium ion as ion-pairing agent to improve the chromatographic selectivity. With UV-detector and a simple filter fluorometer an extraction-evaporation procedure is used for both plasma and urine determinations, while the high selectivity and sensitivity of a sophisticated fluorescence detector permits the direct injection of diluted samples on to the column. Use of an internal standard improves the within-run precision (s_rel%), which for plasma determinations of NAPROXEN are - with UV-detection, 0.2 – 1.7% (range 10 – 40 μg/ml), with filter fluorometer, 2.4 – 5.9% (range 12 – 58 μg/ml), and with fluorescence detector, 0.8 – 4.1% (range 5 – 20 μg/ml).     

Rapid Determination of Aldehydes in Air Analyses

Abstract

A simple analytical method for aldehyde determinations in ambient air is described. The method involved trapping the aldehydes as their DNPH derivatives in acetonitrile solution and then direct injection and analysis of the resulting solution on a reversed phase HPLC column with simultaneous detection at 254 and 360 nm. Several comparison studies are described.     

Multi-Element Analysis of Landfill Gas by ICP Emission and Mass Spectrometry

Methodology for the determination of trace elements in landfill gas by inductively coupled plasma emission and mass spectrometry is described. The approach is based on direct injection of landfill gas into the ICP using a simple gas sampling manifold. Chlorine and sulphur were quantified in landfill gas by ICP-OES while Sn, Mg, Cu and Zn were measured by ICP-MS. Due to the complex chemical composition of landfill gas, measurements in both emission and mass spectrometry were compromised by spectral interferences. The possibility for multi-element analysis of landfill gas samples based on the use of aqueous standard solutions was demonstrated.     

Simultaneous Flow Injection Analysis of Paracetamol and Ascorbic Acid with Multiple Pulse Amperometric Detection

The present work reports a simple and quick strategy for simultaneous determination of paracetamol (PC) and ascorbic acid (AA) in pharmaceutical formulations using flow injection method with multiple pulse amperometric detection. The method allows the resolution of the mixture without chemical pretreatment of the sample or electrode modification or the use of chemometric techniques for data analysis. The compounds are detected by applying four sequential pulses (waveform) in function of time to a three‐electrode amperometric system that uses a wall‐jet cell with gold as working electrode. AA is direct detected at +0.40 V and PC is indirectly detected at 0.0 V by the reduction (desorption) of the oxidation product (N‐acetyl‐p‐benzoquinoneimine) electrochemically generated at +0.65 V. The fourth potential pulse (?0.05 V) is applied for the complete regeneration (cleaning) of the gold electrode surface. The linear response range was optimized between 5 and 24 mg L^?1 for AA and 50 and 240 mg L^?1 for PC. The difference between the two responses ranges (10‐fold) present correlation with the concentration of these compounds in two different pharmaceutical formulations available in the Brazilian market. The analytical frequency was calculated in 60 injections per hour. The use of the proposed methodology for PC quantification in the presence of higher AA concentrations was also carried out. Using the standard addition method, it was possible to detect PC in trace levels (LD=0.2 mg L^?1) in the presence of 880‐fold more of AA (176 mg L^?1).     

Gaseous Nitrogen Dioxide for Sustainable Oxidative Deprotection of Trimethylsilyl Ethers

Abstract

In this study, trialkylsilyl ethers of indispensable protected alcohols are oxidatively deprotected in neat form with gaseous nitrogen dioxide (NO₂). Quantitative yields of aldehydes or ketones are obtained without the necessity of chromatography. The byproducts, nitrogen monoxide, anhydrous nitric acid, and hexamethyldisiloxane, can be quantitatively separated by evaporation and distillation in closed systems for recycling or further use. The direct new method supersedes the previous techniques that produce dangerous wastes and require chromatographic workup, while the atmospheric gas NO₂ and its gaseous reduction products are easily kept in closed systems until further use.     

Ultraviolet Photolysis and Ortho-Phthalaldehyde-Mercaptoethanol Derivatization of Pharmaceuticals for Enhanced Fluorescence Detection in HPLC

Abstract

Several classes of nitrogenous pharmaceutical were examined for fluorescence after ultraviolet (UV) radiation induced photolysis followed by reaction with o-phthalaldehyde-2-mercaptoethanol (OPA-MERC), and after UV photolysis alone. Photolyses were examined in water, mixtures of methanol/water (1:1), and acetonitrile/water (1:1). Acetone was assessed as a photosensitizer to enhance photolysis and fluorescence response. Flow injection analysis and high-performance liquid chromatographic techniques were used for several pharmaceuticals. The analytes were subjected to UV photolysis and reaction with OPA-MERC reagent for generation of fluorophores that responded to fluorescence detection. During photolysis, solvent type as well as the presence of photosensitizers seem to play a significant role in the formation of primary amines and fluorophores. Photochemical transformation products of some of the pharmaceutical chemicals are proposed. Analytical figures of merit were determined for some analytes. This fluorescence detection approach is applicable for a number of pharmaceuticals at nanogram level.     

Detection of Mycoplasma Contamination in Lymphoblastoid Cultures by a Simple,HPLC Method

Abstract

A simple method for detecting mycoplasma contamination of lymphoid cell cultures is discussed. This method is based on high pressure liquid chromatographic (HPLC) detection of citrulline by reversed-phase separation of its ortho-phthaldialdehyde (OPA) derivative. Formation of citrulline via conversion of arginine using the enzyme arginine deiminase present in most of the commonly encountered strains of mycoplasma is measured by this sensitive technique. The use of HPLC techniques offers an extremely sensitive indication of mycoplasma contamination in cells having large nuclei which make detection by staining with Hoechst dyes difficult to interpret.     

Flow Injection Analysis of Famotidine with Spectrophotometric Detection

Abstract

A flow injection determination of famotidine has been described. The method is based on the reaction of the drug with cupric acetate to form a blue coloured complex which shows absorption maxima at 314 nm and 630 nm. For an injection volume of 100 μl calibration graphs were rectilinear from 10- 50 μg. ml^?1 and 50–500 μg.ml^?1 of drug at the two wavelengths respectively. Samples could be analysed at rates upto 60 per hour with a relative standard deviation less than 1.4%. The method was evaluated by analysis of the pure drug and commercial formulations. The results compare well with those obtained by official methods.     

New Analytical Techniques for the Detection of BSE and other TSEs

Abstract

This review discusses new methods for detecting BSE and other TSE prions. A number of different analytical techniques have been applied in the past few years. New immunoassays (as well as new antibodies), an improved Western blot, CE-based methods, and a Raman-based method are some of the analytical techniques reviewed. In addition, methods to increase the concentration of prions and some cautionary notes are presented in this review.     

Determination of Phenols In Natural Waters with A Flow-Analysis Method and Chemiluminescence Detection

ABSTRACT

A simple flow-manifold with time based sample introduction system was elaborated and used to determine phenols in natural waters. Use of computer controlled solenoid valves and direct chemiluminescence detection enables very low reagent consumption. A quantity of less than 1.5 μmol of potassium permanganate and 600μmol of sulphuric acid per determination is used. The use of on-line preconcentration column filled with XAD-4 resin enables determination with the detection limit of 5 ng-ml^?. The sample throughput is 12-h^? when the column is used and 60-h^? without a column.     

Comparison of Fluorescence Immunochromatographic Assay Strip and Gold Colloidal Immunochromatographic Assay Strip for Detection of Microcystin

Abstract

Microcystins are a family of cyclic polypeptides produced by different species of cyanobacteria (blue green algae), which can form blooms in lakes and water reservoirs. However, it is difficult to detect microcystins directly in the water since the concentration of the toxins in water is usually too low. It is necessary to develop a simple and quick method to detect microcystins. In this paper, different detection characteristics of fluorescence immunochromatography and gold colloidal immunochromatography for analysis of cyanobacterial toxins were studied. These two immunochromatography assays are easy to perform, rapid, sensitive, and their quantitative range is within detectable microcystin concentrations in water samples. The fluorescence immunochromatographic system has the unique advantages of low detection limit, and satisfactory accurate results are obtained. The gold colloidal immunochromatographic system has the strong advantage of direct detection of microcystins at the test site without having to bring the samples back to the laboratory. Therefore, these two techniques supplement each other.     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.