Laser-induced dispersed fluorescence detection of polycyclic aromatic compounds in soil extracts separated by capillary electrochromatography.

Polycyclic aromatic hydrocarbons (PAHs) and nitrogen containing aromatic compounds (NCACs) are characterized in soil extracts and laboratory standards by capillary electrochromatography (CEC) with laser-induced dispersed fluorescence (LIDF) detection using a liquid-nitrogen cooled charge-coupled device detector. The LIDF detection technique provides information on compound identity and, when coupled with the high separation efficiencies of the CEC technique, proves useful in the analysis of complex mixtures. Differences in fluorescence spectra also provide a means of identifying co-eluting compounds by using deconvolution algorithms. Detection limits range from 0.5 to 96x10(-10) M for selected PAHs and 0.9-3.7x10(-10) M for selected NCACs. Soil extracts are also injected onto the CEC column to evaluate chromatographic method performance with respect to complex samples and the ability to withstand exposure to environmental samples.

Separation of related opiate compounds using capillary electrochromatography.

Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m.

Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence.

Limited by the lack of a sensitive, universal detector, many capillary-based liquid-phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid-phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti-biotin-IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser-induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC-biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC-biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).

Imaging of Pressure- and Electrokinetically Driven Flows through Open Capillaries.

A new tool for imaging both scalar transport and velocity fields in liquid flows through microscale structures is described. The technique employs an ultraviolet laser pulse to write a pattern into the flow by uncaging a fluorescent dye. This is followed, at selected time delays, by flood illumination with a pulse of visible light which excites the uncaged dye. The resulting fluorescence image is collected onto a sensitive CCD camera. The instrument is designed as an oil immersion microscope to minimize beam steering effects. The caged fluorescent dye is seeded in trace quantities throughout the active fluid, thus images with high contrast and minimal distortion due to any molecular diffusion history can be obtained at any point within the microchannel by selectively activating the dye in the immediate region of interest. We report images of pressure- and electrokinetically driven steady flow within round cross section capillaries having micrometer scale inner diameters. We also demonstrate the ability to recover the velocity profile from a time sequence of these scalar images by direct inversion of the conserved scalar advection-convection equation.

Upper respiratory tract disease and mycoplasmosis in desert tortoises from Nevada.

A population of desert tortoises (Gopherus agassizii) at Yucca Mountain (Nevada, USA) was monitored during four sampling periods using enzyme-linked immunosorbent assays (ELISA) to determine the percentage of individuals that had been exposed to Mycoplasma agassizii, a causative agent of upper respiratory tract disease. Respiratory tract disease has been considered a significant factor in the decline of desert tortoise populations in the Mojave Desert (USA). Few differences between sexes in ELISA values or percentages testing positive were noted. From 15 to 23% of samples per period tested positive for exposure to the mycoplasma. However, we noted few clinical signs of upper respiratory tract disease. This is in contrast to an earlier study which reported a similar proportion of seropositive tortoises as well as a high percentage of tortoises with clinical signs. However, our results are consistent with that study's conclusion that seropositivity for M. agassizii was a poor predictor of the likelihood to exhibit clinical signs of upper respiratory tract disease. Earlier reported epizootics of mycoplasma-associated respiratory disease occurred mainly during times of drought. Our samples were collected during a period of average to above-average rainfall, suggesting that manifestation of clinical signs of the disease may depend upon the physiological condition of tortoises which, in turn, is related to environmental conditions.

Automated capillary electrochromatography: reliability and reproducibility studies.

The routine application of capillary electrochromatography (CEC) is demonstrated by incorporating 75 microns I.D. capillaries packed with 3 microns octadecylsilica (ODS) particles into a commercial CZE instrument. A mixture of several neutral compounds is separated into its components with an average efficiency up to 181 000 plates/m in less than 8 min. Hundreds of consecutive runs are performed over a period of weeks from which it is concluded that the reproducibility of the capacity factors is better than 2% and that CEC separations can be achieved in a reliable and routine manner.

Gradient elution in capillary electrochromatography.

In analogy to pressure-driven gradient techniques in high-performance liquid chromatography, a system has been developed for delivering electroosmotically driven solvent gradients for capillary electrochromatography (CEC). Dynamic gradients with submicroliter per minute flow rates are generated by merging two electroosmotic flows that are regulated by computer-controlled voltages. These flows are delivered by two fused-silica capillary arms attached to a T-connector, where they mix and then flow into a capillary column that has been electrokinetically packed with 3-µm reversed-phase particles. The inlet of one capillary arm is placed in a solution reservoir containing one mobile phase, and the inlet of the other is placed in a second reservoir containing a second mobile phase. Two independent computer-controlled, programmable, high-voltage power supplies (0-50 kV) [Formula: see text] one providing an increasing ramp and the other providing a decreasing ramp [Formula: see text] are used to apply variable high-voltage potentials to the mobile phase reservoirs to regulate the electroosmotic flow in each arm. The ratio of the electroosmotic flow rates between the two arms is changed with time according to the computer-controlled voltages to deliver the required gradient profile to the separation column. Experiments were performed to confirm the composition of the mobile phase during a gradient run and to determine the change of the composition in response to the programmed voltage profile. To demonstrate the performance of electroosmotically driven gradient elution in CEC, a mixture of 16 polycyclic aromatic hydrocarbons was separated in less than 90 min. This gradient technique is expected to be well-suited for generating not only solvent gradients in CEC but also other types of gradients, such as pH and ionic strength gradients, in capillary electrokinetic separations and analyses.

Multiplex spectroscopy: determining the transition moments and absolute concentrations of molecular species.

A procedure is described that uses two spectroscopic techniques, absorption and infrared degenerate four-wave mixing, in tandem (multiplex) to measure the transition dipole moments and absolute concentrations of molecular species in situ. The method is demonstrated by the measurement of the relative transition moments and concentrations of two dissimilar sample gas components, hydrogen chloride and nitrogen dioxide, but is applicable to a wide variety of molecules and, thus, can provide new information for transient molecular species. Further, difficulties in obtaining quantitative information through techniques such as laser-induced fluorescence, coherent anti-Stokes Raman scattering, and degenerate four-wave mixing spectroscopies can be overcome when a multiplex approach is used.

Induction of cytochromes P-450 2B and 3A in mice following the dietary administration of the novel cognitive enhancer linopirdine.

The effects of the novel cognitive enhancer linopirdine [3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one] on hepatic cytochromes P-450 (CYP) and linopirdine metabolism were determined in female mice fed 0, 10, 50, and 250 mg/kg/day of linopirdine in the diet for 4, 7, 14, and 28 days. Linopirdine induced CYP maximally by day 4 only at the highest dose, as demonstrated by significant (p < 0.05) increases in total spectral CYP and liver weight. SDS-PAGE revealed induced 52 kDa microsomal protein(s), identified as CYP2B and 3A by immunoblotting. Linopirdine also increased the rates of reactions selectively catalyzed by CYP2B and 3A (pentoxyresorufin O-dealkylation, benzphetamine N-demethylation, erythromycin N-demethylation, and testosterone 2 beta-, 6 beta-, 16 beta-hydroxylation), 1.7- to 3.0-fold vs. control, similar to increases produced by the prototypical CYP2B and 3A inducers phenobarbital and dexamethasone. No increase in microsomal CYP1A or 2E levels was demonstrated by immunoblotting or selective substrate assays. CYP induction increased the metabolism of linopirdine. The area under the plasma concentration-time curve of linopirdine after a 250 mg/kg/day dose decreased 11-fold from day 1-28, and microsomes from a parallel 250 mg/kg/day group metabolized linopirdine 1.9-fold faster than control (p < 0.05). This autoinduction was due primarily to the induced CYP3A, because antibodies recognizing CYP3A inhibited the microsomal metabolism of linopirdine by 85%, whereas antibodies to CYP2B were not inhibitory. In summary, the dietary consumption of 250 mg/kg/day of linopirdine by female mice coinduced CYP2B and 3A maximally by day 4, and resulted in an increased rate of metabolism of linopirdine, predominantly due to CYP3A.

Determination of amantadine in human plasma by capillary gas chromatography using electron-capture detection following derivatization with pentafluorobenzoyl chloride.

A specific, sensitive, and accurate capillary gas chromatographic method for the quantitation of amantadine in human plasma is described. Amantadine and the internal standard, rimantadine were extracted from plasma under alkaline conditions into toluene. Both compounds were derivatized with pentafluorobenzoyl chloride. The derivatives were separated on a HP-1 capillary column at 180 degrees C and detected using a 63Ni electron-capture detector. The minimum quantifiable limit of the assay is 2.3 ng ml-1 of amantadine base using 1 ml of plasma. The method was used to evaluate the bioequivalence of two different formulations of amantadine hydrochloride.

Detection of trace molecular species using degenerate four-wave mixing.

Spectroscopies that make use of laser light have provided an important tool to modern researchers for the nonintrusive analysis of chemical systems. The strengths and limitations of these spectroscopic techniques often determine the viability of scientific investigations. The unique properties of degenerate four-wave mixing, a nonlinear optical technique, have recently been found to provide powerful capabilities for a wide range of applications.

Influence of food on the oral absorption and bioavailability of moricizine.

Moricizine, a unique Class I antiarrhythmic agent, was orally administered with and without a meal to 24 healthy male subjects to determine the effect of food on moricizine absorption and bioavailability. Relative to the fasting state, a standardized breakfast delayed the time to peak plasma moricizine concentration (1.2 vs. 0.9 hr; P less than .03) and lowered peak plasma moricizine concentration by 24% (0.55 vs. 0.72 microgram/mL; P less than .03). Bioavailability, as measured by area under the plasma moricizine concentration versus time curve, was not significantly altered by the meal.

Nasal administration of a cognition enhancer provides improved bioavailability but not enhanced brain delivery.

Compound 1 [3,3-bis(4-pyridylmethyl)-1-phenylindolin-2-one] is an experimental cognition-enhancing drug now being developed for cognitive disorders. Oral bioavailability of 1 in rats was less than 10% of the dose. Nasal dosing improved bioavailability to greater than 50%. Brain levels of total radioactivity were measured after iv and nasal doses of radiolabeled 1. The ratio of AUCbrain:AUCplasma was the same by both routes, so nasal dosing did not enhance brain delivery. This is in contrast to other reports of large molecular weight substances and metals gaining direct access to the brain through the nasal epithelium.

Measurement of OH rotational temperatures in a flame using degenerate four-wave mixing.

Temperatures have been measured in a laminar premixed propane-air diffusion flame using degenerate four-wave mixing (DFWM) of the OH radical. The spectra were recorded simultaneously with laser-induced fluorescence through the (0, 0) band of the OH A(2)Sigma-X(2)II transition. Individual rotational lines are more clearly resolved in the DFWM spectrum than in the laser-induced fluorescence spectrum, although both are power broadened at laser intensities of 1-2 MW/cm(2) at 307 nm. Rotational temperatures have been determined from the DFWM spectra and are in close agreement with temperatures measured with coherent anti-Stokes Raman spectroscopy of nitrogen.

Two-dimensional imaging of OH in flames by degenerate four-wave mixing.

Degenerate four-wave mixing is used to produce two-dimensional images of OH distributions in atmosphericpressure flames. The phase-conjugated images from single laser pulses exhibit excellent signal-to-noise ratios and illustrate that degenerate four-wave mixing has outstanding potential as a multidimensional diagnostic for combustion environments.

Use of a charge-coupled device camera for broadband coherent anti-Stokes Raman scattering measurements.

The use of an unintensified charge-coupled device (CCD) camera for the acquisition of broadband CARS signals is demonstrated. The CCD camera offers significant advantages compared to intensified, linear photodiode array (PDA) detectors that are generally used for broadband CARS measurements. These advantages include higher spectral resolution and improved instrument function, larger dynamic range, and a 2-D format.

High-performance liquid chromatographic method for the determination of tiflamizole in plasma.

A sensitive, specific, high-performance liquid chromatographic procedure was developed for the measurement of plasma tiflamizole levels. Acidic plasma samples were extracted with three volumes of ether. The ether extracts were combined and evaporated to dryness. The residue was dissolved in acetonitrile, washed with hexane, and the acetonitrile was evaporated to dryness. The residue was dissolved in 0.5 mL of mobile phase consisting of acetonitrile and 0.007 M pH 3 sodium phosphate buffer (70:30, v/v) and then chromatographed on a octadecylsilane bonded microparticulate silica column. The assay is specific, precise, accurate, and can measure 10 ng of tiflamizole in 5 mL of plasma. The method was applied to human pharmacokinetic studies.

Tiflamizole elimination from plasma in rheumatoid arthritis.

Tiflamizole is a fluorinated diarylamidazole sulfone nonsteroidal anti-inflammatory drug not metabolized or excreted in urine. Its mean (+/- SD) elimination t 1/2 from plasma was 21.6 +/- 9 days (range 11.8 to 49.5 days) in 17 subjects with rheumatoid arthritis, and appeared to be first order in most of them. Plasma elimination t 1/2 was loosely related (r = -0.67) to stool frequency in eight subjects for whom stool frequency data were available. In one, cholestyramine decreased t 1/2 to 4.1 days. In two patients, synovial fluid total tiflamizole concentrations were approximately one-third of simultaneous plasma concentrations, but elimination t 1/2s from synovial fluid were of the same order as those from plasma. Even with infrequent dosing, the longer t 1/2 may help sustain the anti-inflammatory effects of this drug.