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Biomedical subjects

M J Sepaniak

Publications and source records attributed to M J Sepaniak.

30 records · Page 2Linked to original sources

Pharmaceutical analysis using micellar electrokinetic capillary chromatography.

The potential utility in pharmaceutical analysis of a capillary electrokinetic separation technique that employs a micellar "pseudo-stationary phase" is discussed and illustrated. Chromatograms of separations of vitamin metabolites and derivatized amino acids are presented to illustrate the high efficiency of the technique and the ability to simultaneously separate the charged and neutral components of pharmaceutical samples. The analytical characteristics of the technique and the importance of optimizing experimental parameters, such as surfactant concentration and capillary column diameter, are discussed and demonstrated with the aid of chromatograms.

Chromatography↗

Analysis of B6 vitamers by micellar electrokinetic capillary chromatography with laser-excited fluorescence detection.

Micellar electrokinetic capillary chromatography (MECC) involves the application of a high voltage (10 to 40 kV) across a capillary column (25 to 75 microns i.d.) that is filled with a solution containing micelles. The mobile phase in this work consists of sodium dodecyl sulfate in an aqueous phosphate/borate buffer system. Pyridoxine (vitamin B6) and five of its metabolites are separated, with efficiencies as high as 60,000 theoretical plates/meter. Pyridoxic acid, a metabolite of B6, is separated and quantitated in human urine using laser-excited fluorescence detection. Limits of detection are less than a picogram injected.

Chromatography, Liquid↗

Dynamically modified capillary columns for liquid chromatographic separations of biological compounds.

Dynamically modified open tubular capillary columns were evaluated for separations of biological and clinical interest. Small diameter, dynamically modified columns were readily prepared by simple addition of cetyltrimethylammonium salts to the mobile phase. Separations similar to those obtained on conventional reversed-phase columns were observed for neutral solutes, and ion-pair reversed-phase separations were observed for charged solutes. Borosilicate and standard flint glasses were evaluated with respect to common etching procedures. A potential clinical application is presented in the separation of antitumor drugs.

Antineoplastic Agents↗

The clinical use of laser-excited fluorometry.

Many areas of spectroscopy have benefited from the use of laser radiation sources. I present a discussion of the basic properties of the laser and how these properties can be advantageous when laser excitation is used in fluorometry. Although the laser has not yet been accepted as a routine instrument in the clinical laboratory, its unique properties have rendered it useful in several analytical methodologies that are based on fluorometry and used in the clinical or biological fields. Accordingly, I briefly review the practical aspects of some clinical applications of laser-excited fluorometry.

Biological Transport↗

Liquid chromatography in open tubes.

Liquid chromatography in open tubes is evaluated for applicability to clinical analysis. The principal advantages of this technique include high separation power, reduced solvent consumption, and small sample volume requirements. Technology for the production of columns as small as 8 micron (i.d.) is described. Preliminary results are encouraging and, with further development in column and detector technology, the technique should find wide applicability in clinical analysis.

Chromatography, Liquid↗

Optical fiber fluoroprobes in clinical analysis.

We used quartz optical fibers and laser excitation in developing single-fiber fluoroprobes, to be used to measure molecular fluorescence in minute volumes of body fluids. We illustrate and discuss the analytical capabilities of these fluoroprobes. Limits of detection for the anti-tumor drug doxorubicin are about 10(-7) mol/L, by either conventional fluorescence or sequentially excited fluoroscence modes of detection. We also report the results of preliminary in vivo measurements of doxorubicin in the interstitial fluids of human tumors, heterotransplanted in immune-deficient laboratory mice.

Animals↗

Determination of adriamycin and daunorubicin in urine by high-performance liquid chromatography with laser fluorometric detection.

A separation and detection scheme is presented for the determination of the antitumor drugs adriamycin and daunorubicin in human urine. Separation is accomplished by reversed-phase high-performance liquid chromatography and the drugs are detected down to the low picogram level by laser excited fluorescence using a unique fiber optic based flow-cell. Excellent detector selectivity and linearity are reported, and some of the factors influencing the performance of the detector are discussed. Possible extension of the procedure to other biologically important determinations are mentioned.

Chromatography, High Pressure Liquid↗

Development of a new capillary electrophoresis-based fibre optic sensor.

A new fluorescence-based fibre optic sensor is described which combines the sensitivity offered by laser-induced fluorescence with the selectivity offered by capillary electrophoresis (CE). A single optical fibre directly probes the terminus of a 5-8 cm separation capillary. The linear geometry associated with this sensor necessitates a 'single reservoir' design, thus presenting major challenges to overcome in comparison to the conventional two-reservoir configuration common to a typical laboratory setup. Some of the challenges confronted by the design features presented in this work include the reduction of gravity-driven hydrostatic flow, the ejection of electrolytic gases evolved at the detection-side electrode and the establishment a suitable compromise between detectability and separation performance. The success of such design features demonstrates the feasibility of a CE-based sensor which offers several amenities particularly useful for in situ sensing. Such attributes include selectivity, diminutive size, flexibility, reusability, high sensitivity, speed, and remote control. Detailed descriptions of sensor fabrication are included, including two variations on a general design concept. In addition, the single-fibre optical detection system is described. Separation characteristics of the new CE-based sensor are presented, highlighted by an observed separation efficiency of up to 8000 theoretical plates (for a 5 cm capillary). The separation of a three-component mixture of the laser dyes, Rhodamine 6G, fluorescein isothyocyanate and sodium fluorescein, is demonstrated.

Biosensing Techniques↗

Enhancing chemi-mechanical transduction in microcantilever chemical sensing by surface modification.

The use of chemically selective thin-film coatings has been shown to enhance both the chemical selectivity and sensitivity of microcantilever (MC) chemical sensors. As an analyte absorbs into the coating, the coating can swell or contract causing an in-plane stress at the associated MC surface. However, much of the stress upon absorption of an analyte may be lost through slippage of the chemical coatings on the MC surface, or through relaxation of the coating in a manner that minimizes stress to the cantilever. Structural modification of MC chemical sensors can improve the stress transduction between the chemical coating and the MC. Surfaces of silicon MC were modified with focused ion beam milling. Sub-micron channels were milled across the width of the MC. Responses of the nanostructured, coated MCs to 2,3-dihydroxynaphthalene and a series of volatile organic compounds (VOCs) were compared to smooth, coated MCs. The analytical figures of merit for the nanostructured, coated MCs in the sensing of VOCs were found to be better than the unstructured MCs. A comparison is made with a previously reported method of creating disordered nanostructured MC surfaces.

Journal Article↗

Examination of cyanine intercalation dyes for rapid and sensitive detection of DNA fragments by capillary electrophoresis.

Recently, a series of cyanine dyes has been developed that are reported to offer superior fluorescence upon intercalation relative to ethidium bromide (EB). The dimeric cyanine dyes, such as POPO-3 (Molecular Probes, Eugene, OR, U.S.A.), are reported to offer the highest fluorescence enhancement of the cyanine dyes. Although POPO-3 does offer an improvement in sensitivity, achieving reproducible, high-efficiency results by employing pre-column, on-column, and postcolumn labeling strategies proved to be problematic. Evaluation of the labeling rate of these dimeric dyes indicates that perhaps the most limiting factor to using this dye for rapid CE analysis of DNA fragments is the apparent slow rate at which the dye fully intercalates into the DNA structure. Use of the monomeric analog PO-PRO-3 (Molecular Probes) resulted in much better separations and low limits of defection when employed for on-column intercalation. However, we find that the kinetics are too slow to employ this dye in a sheath flow cell for postcolumn derivatization.

Benzoxazoles↗