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H T Karnes

Publications and source records attributed to H T Karnes.

At least 37 records · Page 2Linked to original sources
PubMed1997

Single pump column switching technique employing a flow gradient and wavelength programmed fluorescence for simultaneous monitoring of serotonin, fluoxetine and norfluoxetine in rat brain microdialysate.

A single pump column switching technique with multidimensional chromatography, flow gradient and wavelength programmed fluorescence detection was developed for simultaneous quantitation of serotonin, fluoxetine and norfluoxetine in rat brain microdialysate. The column switching was configured such that position I of the switching valve employed column I (50 mm length) and column II (250 mm length) in series. This configuration resulted in optimal resolution of serotonin from interfering neurochemicals from rat brain. After elution of serotonin at 13.2 min the valve was switched to position II in which the flow of the mobile phase was directed through column I only. Flow gradient programming was then used to ramp the flow rate from 0.1 to 0.4 ml min-1 which resulted in optimal elution of fluoxetine and norfluoxetine. Strategic optimization of the single mobile phase enabled use of a single pump and detector making the analytical system simple and cost effective. Wavelength programmed fluorescence enabled sensitive detection of the analytes despite the difference in their fluorescence spectrum. The limit of detection for serotonin, norfluoxetine and fluoxetine were 10, 612 and 523 fmol, respectively. Rat brain microdialysate samples demonstrated selectivity for serotonin, fluoxetine and norfluoxetine. The method demonstrates application to the study of site specific neuropharmacokinetics and neuropharmacodynamics of fluoxetine in vivo.

Animals↗
PubMed1997

Evaluation of the neuropharmacodynamics of paroxetine in vivo utilizing microdialysis.

The effect of paroxetine, a selective serotonin reuptake inhibitor used in the treatment of depression, on extracellular serotonin levels was evaluated in freely moving conscious rats. Microdialysis, a powerful in vivo technique to monitor the extracellular levels of neurotransmitters, was used to monitor the baseline changes in the levels of serotonin in rat brain anterior lateral striatum post paroxetine administration, which is a measure of the neuropharmacodynamic effect of the drug. Microdialysis sampling was performed for 210 min prior to and for 240 min after intraperitoneal administration of paroxetine (10 mg/kg). Paroxetine caused a statistically significant increase in the extracellular levels of serotonin in the anterior lateral striatum sampled by microdialysis. The present study demonstrates the utility of microdialysis for studying the in vivo neuropharmacodynamics of paroxetine in conscious rats.

Animals↗
PubMed1996

Analysis of gamma-(cholesteryloxy)butyric acid in biologic samples by derivatization with 5-(bromomethyl)fluorescein followed by high-performance liquid chromatography with laser-induced fluorescence detection.

This report describes the first application of 5-(bromo-methyl)fluorescein (5-BMF) for the quantitation of a pharmaceutically relevant carboxyl-containing analyte in a biological matrix. An analytical method for quantitation of gamma-(cholesteryloxy)butyric acid (CBA), a relatively new antitumor agent, in different tissues of Sprague-Dawley rats was developed. 5-BMF was employed to form a stable and spectrally well-characterized conjugate of CBA. The derivatization yield was maximized by optimizing several reaction variables. The conjugate was separated by high-performance liquid chromatography and quantitated by a laboratory-constructed argon ion laser fluorometer. The detection limits for CBA were 4.6 x 10(-9) and 6.34 x 10(-11) M by conventional and laser-induced fluorescence (LIF), respectively. A derivatization limit of detection of 1.85 x 10(-9) M was achieved by LIF for the conjugate The analytical method was useful for quantitation of CBA in various tissues in the picogram per milliliter range.

Animals↗
PubMed1996

An oxazine reagent for derivatization of carboxylic acid analytes suitable for liquid chromatographic detection using visible diode laser-induced fluorescence.

This study reports the use of Nile Blue, an oxazine dye, as a derivatization reagent that fluoresces in the far-red spectral region and is suitable for derivatization with carboxylic-acid-containing analytes. Model carboxylic acid analytes such as benzoic acid, acetic acid, phenylacetic acid and hexanoic acid have been reacted as acid chlorides to form Nile Blue derivatives. The synthesis product of the Nile Blue benzoic acid derivative was confirmed using electrospray-mass spectrometry, infrared spectrometry, 1H and 13C nuclear magnetic resonance, reversed phase liquid chromatography (RP-HPLC), normal phase-thin layer chromatography, and spectral characterization. The synthesized Nile Blue derivatives, separated from reaction by-products with RP-HPLC, all demonstrated an approximately 10-fold drop in molar absorptivity and relative quantum yield. In addition, a 40 nm increase in Stokes shift was observed. A portion of the fluorescence was regained through post-column ionization of the Nile Blue benzoic acid derivative at pH 12. A RP-HPLC limit of detection of 88.25 fmol on column has been reported with conventional fluorescence detection-post-column ionization of the Nile Blue benzoic acid derivative. A limit of detection of 1.99 fmol on column (3.98 x 10(-11) M) has been demonstrated for the Nile Blue benzoic acid derivative with the use of a laboratory-constructed visible diode laser fluorescence detector.

Carboxylic Acids↗
PubMed1995

5-Bromomethyl fluorescein (5-BMF) for derivatization of carboxyl containing analytes for use with laser-induced fluorescence detection.

PURPOSE: 5-Bromomethyl fluorescein (5-BMF) was evaluated in this work as a pre-column, off-line derivatizing reagent for analytes containing a free carboxyl group. The reagent possessed high molar absorptivity and quantum yield and it's excitation maximum matched the intense 488.0 nm emission line of an argon ion laser. The thermal and photo-stability of the reagent were also suitable for our work. METHODS: A mixture of 5-BMF, palmitic acid, 18-crown-6 and potassium carbonate was reacted at 76 degrees C for one hour. The reaction yield was maximized by a sequential single-factor optimization of the reaction variables. Spectral characterizations were accomplished using conventional instrumentation and detection limits were evaluated using a laboratory constructed laser-induced fluorescence (LIF) detector which was optimized for concentration detection. RESULTS: Two apparent conjugates of palmitic acid were formed under the reaction conditions employed. The conjugate chosen for quantitative purposes was stable at room temperature over 24 hours and possessed high molar absorptivity (128,832 M-1cm-1) and quantum yield (0.82) under basic conditions. The excitation and emission maxima of the conjugate were 500 nm and 523 nm respectively which maintains suitability for argon-ion laser excitation. An argon-ion LIF detector suitable for use with conventional liquid chromatography was constructed. A detection limit of 7.56 x 10(-10)M of palmitic acid was achieved at a signal to noise ratio of three, using the strong 488.0 nm laser emission line and a commercially available flow cell component. This corresponds to 38 femtomoles of palmitic acid on-column. CONCLUSION: The concentration detection limit was superior to literature reports for detection of fatty acids. The mass detection limit provided approximately an order of magnitude improvement over conventional fluorescence. The reagent is potentially useful for analysis of carboxyl containing analytes at low concentrations.

Carboxylic Acids↗
PubMed1995

Chemical compatibility of inotropic and vasoactive agents delivered via a multiple line infusion system.

OBJECTIVES: To determine the chemical compatibility of three different triple drug admixtures diluted with either 5% dextrose in water or 0.9% NaCl solution when administered via a multiple line infusion system (Omni-Flow 4000, Abbott Laboratories, Abbott Park, IL). The triple drug admixtures were: a) dobutamine, dopamine, and norepinephrine; b) nitroglycerin, sodium nitroprusside, and dobutamine; and c) nitroglycerin, dopamine, and dobutamine. DESIGN: Two phase in vitro compatibility study. SETTING: Pharmaceutical laboratory. SUBJECTS: None. INTERVENTIONS: Phase I assessed chemical stability when the triple drug admixture was placed in a single container. In phase II, individual drug components of the admixtures were infused via the multiple line infusion system. Samples were collected at time 0, 1 hr, 2 hrs, 4 hrs, 12 hrs, and 24 hrs. All samples were frozen and stored at -70 degrees C until assayed. MEASUREMENTS AND MAIN RESULTS: Samples were assayed using stability-indicating high performance liquid chromatography. The triple drug admixtures were considered to be chemically stable if there was < or = 10% loss of stated potency over 24 hrs. In phase I, chemical stability was observed for all triple drug admixtures at 24 hrs. In phase II, dobutamine, dopamine, norepinephrine, and sodium nitroprusside showed chemical stability at 24 hrs. Nitroglycerin showed a two-fold increase in concentration at 24 hrs compared with the initial concentration through the test infusion system; however, this amount was still one third lower than originally anticipated. CONCLUSIONS: All triple drug admixtures were chemically stable when placed in single containers. Dobutamine, norepinephrine, and sodium nitroprusside showed chemical stability when delivered via a multiple line infusion system. The reduced recovery of nitroglycerin from the test infusion system may result from adsorption of the nitroglycerin to the polyvinyl chloride plastic cassette and tubing during infusion.

Chromatography, High Pressure Liquid↗
PubMed1994

Stability of indocyanine green in human serum stored at -20 and -70 degrees C.

The stability of indocyanine green (ICG) in human serum after storage at -20 and -70 degrees C for an extended time was studied. Serum samples were fortified with ICG at 6.5, 2.0 and 0.3 microgram ml-1, aliquoted, and frozen at -20 or -70 degrees C. The analytic methodology used an internal standard and separation on a reverse phase liquid chromatographic column with UV detection at 240 nm. Samples were prepared for analysis by protein precipitation with acetonitrile. The assay was stability indicating as shown by the complete loss of the ICG peak upon storage at -20 degrees C for 8 weeks. ICG was not stable, as determine by a t test, for 1 week in human serum when stored at -20 degrees C. Storage at -70 degrees C improved stability but the ICG concentration began to fall steadily after 4 weeks of storage.

Blood↗
PubMed1993

HPLC analysis of salinomycin in human plasma using pre-column oxidation and automated heart cut column switching.

Salinomycin is a polyether antibiotic used to promote growth in cattle and poultry. Workers may be exposed to salinomycin through handling of animal feeds that contain the drug and it is necessary to monitor plasma samples from these workers for salinomycin to ensure safety. A method for analysis of salinomycin in plasma samples was therefore developed. Salinomycin and the internal standard narasin are extracted into iso-octane then subjected to silica gel solid-phase extraction in which the sample is washed with methylene chloride-methanol (98.5:15) then eluted with a 90:10 proportion of the same mixture. Both salinomycin and narasin are oxidized with pyridinium dichromate to form a chromophore absorbing at 225 nm. The concentrated product was injected onto a C18 pre-column and heart cut from 1.85 to 3.65 min onto a C18 analytical column. The method was shown to be selective for salinomycin and narasin in six blank plasma samples. The method was linear over a range of 15-300 ng ml-1 with a detection limit of approximately 5 ng ml-1. The mean absolute recovery was found to be 93.4 and 97.9% for salinomycin and narasin, respectively. The method was accurate to within 5% at all concentrations studied. Within-run and between-run precision were both less than 8% RSD at all concentrations studied and the method was suitable for the purpose of monitoring plasma from exposed agricultural workers.

Anti-Bacterial Agents↗
PubMed1993

Solid-state diode laser-induced fluorescence detection in high-performance liquid chromatography.

The need to measure ultralow levels of pharmaceuticals in biological matrices at femtogram and attogram levels presents a significant challenge to bioanalysts. Liquid chromatography has proven to be a versatile and valuable tool for separating analytes from complex biological matrices and fluorescence detection provides both the sensitivity and the selectivity required to measure femtogram and attogram levels of analytes. Solid-state diode lasers have been used primarily in printers, compact disc recorders, and bar code scanners but have recently been adapted for use as light sources for fluorescence detection. Excellent spectral features in the visible and near-infrared regions of the spectrum, together with low cost and ruggedness, make diode lasers an attractive alternative for use as light sources in analytical measurement. Biological matrices demonstrate minimal background signals in the far-red regions of the spectrum which diode lasers emit and diode lasers are among the most stable light sources available. These facts along with expected developments in labeling systems make the potential for use of diode lasers in LC-detection quite promising. This paper reviews characteristics of diode lasers, the properties of potential visible and near-infrared fluorescent probes, instrumental aspects of diode laser fluorometers, and future trends that can be expected in this exciting field of bioanalytical research.

Chromatography, High Pressure Liquid↗
PubMed1993

Precision, accuracy, and data acceptance criteria in biopharmaceutical analysis.

Accuracy and precision are the most important criteria in the assessment of an analytical method, and monitoring quality control during sample analysis is essential to ensure the validity of reported results. Various approaches to testing accuracy, precision, and quality control were applied to 10 analytes from seven chromatographic bioanalytical methods. These methods include fixed interval bias and significance testing for accuracy; fixed interval percentage relative standard deviation (%RSD) and analysis of variance (ANOVA) approaches for precision; +/- 20% fixed range, 99% confidence interval, multiple rules, and range chart for individuals approaches for quality control acceptance criteria. Quality control approaches were also applied to the entire run and to a bracketed approach whereby results are considered valid only if bracketed by acceptable quality control. Accuracy and precision were assessed for six runs of each analyte at three concentrations established to represent the calibration range of the analytical method. Quality control acceptance criteria were evaluated using all data sets from each of the analytical methods collected during the course of running various numbers of real samples. The data suggest that the fixed interval bias criteria for accuracy was a more liberal method of accuracy assessment because three of the seven methods would have been rejected according to the significance testing criteria whereas all were acceptable by the fixed internal bias criteria. Precision can be effectively assessed for between- and within-run data by criteria set on unconfounded %RSD values or by separation of the sources of variation using an ANOVA approach applied to confounded data.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

High performance liquid chromatographic determination of theophylline metabolites in human liver microsomes.

A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3-methylxanthine (3-MX), 1-methylxanthine (1-MX) and 1,3-dimethyluric acid (1,3-DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3-MX, 20 ng/mL for 1-MX and 20 ng/mL for 1,3-DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15-250 ng/mL for 3-MX, 20-250 ng/mL for 1-MX and 250-4000 ng/mL for 1,3-DMU. The absolute recovery ranged from 63-84% for 3-MX, 65-79% for 1-MX and 77-89% for 1,3-DMU over the calibration curve range. Accuracy for all three metabolites was within +/- 10% and adequate selectivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within-run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metabolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotics.

Chromatography, High Pressure Liquid↗
PubMed1991

Calibration and validation of linearity in chromatographic biopharmaceutical analysis.

Calibration in chromatographic biopharmaceutical analysis is a major determinate of method performance and many methods have been proposed to evaluate an appropriate calibration model, to determine the linear range and to evaluate the goodness of fit. Ten chromatographic bioanalytical methods have been evaluated in this work by observation of concentration-response curves, linearity plots, calculation of concentration residuals, correlation coefficients and lack of fit analysis. These methods were applied to univariant linear regression, weighted regression, polynomial regression and power fit models in order to determine the most appropriate way to establish and evaluate calibration functions. It was found that weighted linear regression provided the most appropriate calibration function for eight of the 10 methods studied, whereas unweighted regression and the power fit model proved appropriate for one each of the other two methods. The choice of calibration function was best accomplished through observation of calculated concentration residuals. Linearity and sensitivity plots were of little value for assessment of linearity through the selected calibration range if conventional (+/- 5%) tolerance limits are employed. Validation of the calibration model can be accomplished by demonstrating the concentration residuals and the slope of the log concentration-log response plots are within reasonable tolerance limits or by lack of fit analysis. Correlation coefficients were demonstrated to be of little value for this purpose and the quadratic approach to linearity validation was in disagreement with other methods in four of the 10 methods evaluated.

Calibration↗
PubMed1991

Validation of bioanalytical methods.

Validation of bioanalytical methods used to generate data for pharmacokinetic and bioavailability studies is approached by a variety of techniques and is subject to many different methods of interpretation. This Review puts the various techniques into perspective and discusses pitfalls which may occur in interpretation of validation data. Recovery studies, standardization techniques, and selectivity/specificity are discussed with regard to the intrinsic value of various techniques that are used in validation. Models used for analytical calibration curves are explained in terms of their validity and limitations, along with a presentation of the most common ways to validate the model. Analytical sensitivity and detection limits are presented and discussed with regard to the usefulness of the various definitions. Appropriate means of testing precision and accuracy, the most important factors in assessing method quality, are presented. Stability and ruggedness testing are discussed along with a presentation of ways to assess data acceptability on a daily run basis.

Biopharmaceutics↗
PubMed1990

Bioanalysis of anti-ulcer agents.

In reviewing the analytical methods for the analysis of anti-ulcer drugs we observed an increase in the utilization of solid-phase extraction techniques, though the traditional liquid-liquid methods are still predominant. Liquid chromatographic techniques are employed more than gas chromatographic methods which reflects the general trend in chromatographic analysis for analytes in the nanogram range. We foresee a continued increase in the use of solid-phase extraction methodology (automated or otherwise) due to the potential for dramatic decreases in extraction times, cost and significant enhancement of extraction efficiency. Because the therapeutic concentrations of these drugs tend to be in the low nanogram range in plasma and the current trend in drug development is toward more potent agents, we anticipate the application of more sensitive liquid chromatographic detection techniques such as electrochemical and chemiluminescence detection to overcome the limitations of currently used technology.

Anti-Ulcer Agents↗
PubMed1990

Solid-phase extraction and liquid chromatography of torsemide and metabolites from plasma and urine.

Torsemide is a new diuretic drug with a profile of action similar to that of furosemide. The high potency of torsemide results in low dose therapy and causes problems for the pharmacokinetic study of the drug due to low plasma levels. Described here are methods for the analysis of torsemide and two metabolites in plasma and urine using solid-phase extraction and liquid chromatography. The limits of quantitation are 10 ng/mL for plasma and 20 ng/mL for urine. The relative standard deviations for precision are less than 10% for most analytes at most concentrations in the calibration range. The recoveries from plasma were 94.3, 92.9, and 95.6%, and from urine were 77.5, 66.6, and 76.5% for torsemide and metabolites M1 and M5, respectively. The method was suitable for pharmacokinetic studies. Data from a normal volunteer are presented.

Chromatography, Liquid↗
PubMed1990

In vitro effect of fluoroquinolones on theophylline metabolism in human liver microsomes.

Some quinolone antibiotics cause increases in levels of theophylline in plasma that lead to serious adverse effects. We investigated the mechanism of this interaction by developing an in vitro system of human liver microsomes. Theophylline (1,3-dimethylxanthine) was incubated with human liver microsomes in the presence of enoxacin, ciprofloxacin, norfloxacin, or ofloxacin. Theophylline, its demethylated metabolites (3-methylxanthine and 1-methylxanthine), and its hydroxylated metabolite (1,3-dimethyluric acid) were measured by high-pressure liquid chromatography, and Km and Vmax values were estimated. Enoxacin and ciprofloxacin selectively blocked the two N demethylations; they significantly inhibited the hydroxylation only at high concentrations. Norfloxacin and ofloxacin caused little or no inhibition of the three metabolites at comparable concentrations. The extent of inhibition was reproducible in five different human livers. Inhibition enzyme kinetics revealed that enoxacin caused competitive and mixed competitive types of inhibition. The oxo metabolite of enoxacin caused little inhibition of theophylline metabolism and was much less potent than the parent compound. Nonspecific inhibition of cytochrome P-450 was ruled out since erythromycin N demethylation (cytochrome P-450 mediated) was unaffected in the presence of enoxacin. These in vitro data correlate with the clinical interaction described for these quinolones and theophylline. We conclude that some quinolones are potent and selective inhibitors of specific isozymes of human cytochrome P-450 that are responsible for theophylline metabolism. This in vitro system may be useful as a model to screen similar compounds for early identification of potential drug interactions.

Biotransformation↗
PubMed1990

Loss of carbamazepine suspension through nasogastric feeding tubes.

The apparent loss of carbamazepine suspension during administration through polyvinyl chloride nasogastric feeding tubes in vitro was studied. Twelve methods of administering carbamazepine suspension (100 mg/5 mL) were tested; the methods differed with respect to nasogastric tube size, presence and type of diluent, and type of flush solution. Undiluted or 50% diluted carbamazepine suspension 200 mg was drawn up in a syringe and forced through adult or pediatric nasogastric feeding tubes. The tubes were immediately flushed twice with 50 mL of sterile water, 0.9% sodium chloride solution, or 5% dextrose solution, by using the same syringe used to administer the suspension. Samples were collected and analyzed for carbamazepine concentration by high-performance liquid chromatography. Each administration method was tested six times, and the results were subjected to analysis of variance. Significant loss of carbamazepine was noted for four of the six methods in which undiluted suspension was administered. In these methods, adult and pediatric tubes were flushed with sterile water or 0.9% sodium chloride. No significant loss of drug occurred for any of the methods involving the use of diluent. Significant losses were associated with diluent and flush solution but not nasogastric tube size. Carbamazepine suspension should be mixed with an equal volume of diluent before being administered through nasogastric feeding tubes.

Carbamazepine↗