Monolithic silica liquid chromatography columns for the determination of cyclooxygenase II inhibitors in human plasma.

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib (I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6 x 50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.

Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection.

Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.

Filtration capture immunoassay for bacteria: optimization and potential for urinalysis.

A novel assay utilizing immuno-labeling, filtration, and electrochemistry for the rapid detection of bacteria has been optimized for the detection of Escherichia coli O157:H7. Bacteria were specifically labeled with alkaline phosphatase conjugated polyclonal antibodies and captured on a polycarbonate track-etched membrane filter (0.2 microm pore size). The filter was then placed directly against a glassy carbon electrode, incubated with enzyme substrate, and the product detected by square wave voltammetry. The high speed and capture efficiency of membrane filtration and inherent sensitivity of electrochemical detection produced a 25-min assay with a detection limit of 5 x 10(3) E. coli O157:H7 per ml using a filtration volume of 100 microl (i.e. 500 cells filtered). The labeling, filtration, and electrochemical steps were optimized, and the assay performance using electrochemical and colorimetric detection methods was compared. The assay was used to detect E. coli O157:H7 that was spiked into filter-sterilized urine at clinically relevant concentrations.

Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7.

A new approach for rapid assay of bacteria in liquid samples is described. Cells were labeled by incubation with an enzyme-antibody conjugate and captured by filtration of the sample/conjugate mixture through a 0.2 microm filter. The enzyme-labeled cells were detected by placing the filter on the surface of an electrode, incubating with enzyme substrate, and measuring the current produced by oxidation of the electroactive enzyme product. Assay time was 25 min and a detection limit of approximately 5000 cells/ml was obtained for E. coli O157:H7. Background current due to non-specific binding of conjugate to the filter was the primary factor controlling the detection limit, and fewer than 50 cells could be detected when very small sample volumes (10 microl) were used to minimize background current.

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids.

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and alpha-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R(F) value as the hygromycin B standard.

Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella.

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.

Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium.

There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.