Biomedical subjects

H Thomas Karnes

Publications and source records attributed to H Thomas Karnes.

13 recordsLinked to original sources

PubMed2006

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography.

The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-microm x 25-mm column packed with 1-microm silica beads was chosen for use with a 500-microL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R2=0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.

Animals↗

PubMed2006

Characterization of a potential medium for 'biorelevant' in vitro release testing of a naltrexone implant, employing a validated stability-indicating HPLC method.

A variety of factors have been recognized that influence media optimization for drug release studies of implant dosage forms. Of primary importance is selection of a medium that physiologically mimics the milieu at the site of administration (a condition termed 'biorelevance'). We describe in this paper, the characterization of Hanks' balanced salts solution, with necessary modification, for application as a 'biorelevant' medium for in vitro release studies of a biodegradable, subcutaneous implant of naltrexone. A detailed investigation of changes in pH, osmolality and ultraviolet (UV) spectrum as a function of time and temperature was conducted. Variation in the parameters evaluated was found to be within acceptable limits. Validation of a simple and selective, high performance liquid chromatography (HPLC) assay method for naltrexone was carried out to evaluate stability. The calibration curves were linear from 0.16 to 20.00 microg ml(-1). Imprecision and inaccuracy were less than 2% and no interference was observed from degradation peaks. Stability studies of naltrexone indicated the media should be replaced every 7-8 days for real-time testing. This was applied to an investigation of in vitro drug release. The method has been proven to be suitable for investigation of naltrexone released from the implant.

Buffers↗

PubMed2006

Profiling in vitro drug release from subcutaneous implants: a review of current status and potential implications on drug product development.

This review presents current methods and strategies for studying the release characteristics of drugs from subcutaneous implant dosage forms. Implants are dosage forms that are subcutaneously placed with the aid of surgery or a hypodermic needle, and are designed to release drugs over a prolonged period of time. In most cases, the objective of a release test is to identify sufficiently discriminatory procedures that in turn would provide data to set meaningful specifications. Additional information obtained from successful in vitro-in vivo correlations (IVIVC) and accelerated drug release tests are extremely useful during drug product development. Although several workers have employed different methods to monitor drug release from these dosage forms, the use of the compendial Apparatus 4 (flow-through) device has been recommended in a publication on FIP/AAPS Guidelines for drug release testing of modified release dosage forms. However, most of method development with this device has focused on oral immediate or controlled release dosage forms and little published information is available on implants. Two recent reports on workshops provide useful information on methods to evaluate drug release from controlled-release parenterals such as implants, including IVIVC and accelerated release testing. Details on such studies, however, are generally not found in the literature; possibly because of the high proprietary value of methodologies for establishing release specifications of implant dosage forms. This article reviews the current status of methodologies used in the investigation of drug release from subcutaneous implants with an emphasis on mechanistic, product development and regulatory perspectives.

Drug Design↗

PubMed2005

Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds.

When biomolecules such as proteins, lipids, and DNA are subjected to oxidative attack by free radicals or other reactive species, a number of measurable biomarkers may be produced. The study of oxidative DNA damage is valuable in research concerning cancer and aging. The current review includes methodology involving various separation science techniques for the analysis of DNA oxidation biomarkers, mainly 8-hydroxy-2'-deoxyguanosine. This review will present recent analytical developments with respect to sample preparation and instrumental considerations, noting key outcomes and biological relevance where appropriate.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

Bioanalytical method validation for macromolecules in support of pharmacokinetic studies.

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.

Calibration↗

PubMed2005

Determination of aflatoxin B1 in sidestream cigarette smoke by immunoaffinity column extraction coupled with liquid chromatography/mass spectrometry.

Aflatoxins produced by food-borne molds are known carcinogenic toxins. Aflatoxin B1 (AFB1) is reported as the most toxic of this class of mycotoxins. We have coupled immunoaffinity column extraction with LC/MS to produce a sensitive and selective approach for the study of AFB1. As AFB1 can be potentially found in tobacco it is of interest to establish whether AFB1 can be transferred from a cigarette fortified with AFB1, to the sidestream smoke. Previous studies have found that AFB1 does not transfer to the mainstream smoke. Since sidestream smoke may contain higher concentrations of some smoke components, a method was developed to analyze the sidestream smoke produced from machine-smoked cigarettes. Sidestream smoke condensates collected on Cambridge filter pads were extracted with isopropanol, then further purified using immunoaffinity extraction columns. The extracts were then analyzed by LC/MS and LC/MS/MS. An instrumental limit of detection (LOD) was established at 3.75 pg injected on column, with the limit of quantitation (LOQ) equal to 11.25 pg on column for both LC/MS and LC/MS/MS. The instrument was found to be linear from 11.25 pg to 150 pg (r > 0.995.) Precision ranged from 4.2% to 8.4% at the LOQ, while accuracy ranged from 0.53% to 1.33%. The immunoaffinity extraction method LOD was determined to be 100 pg fortified onto the Cambridge filter. The LOQ was 350 pg. The average recovery of the AFB1 from the Cambridge pad was 82.9% over the range of 100-1000 pg fortified onto the pad. AFB1 was not detected in unfortified cigarettes. A transfer experiment, fortifying cigarettes at 1 microg/cigarette determined that AFB1 was transferred only slightly from the burning cigarette to the sidestream smoke. The mean percent transfer was 0.087%.

Aflatoxin B1↗

PubMed2005

Evaluation of volatile ion-pair reagents for the liquid chromatography-mass spectrometry analysis of polar compounds and its application to the determination of methadone in human plasma.

A liquid chromatography method using volatile ion-pairing reagents and tandem mass spectrometry was developed to obviate observed matrix effect for ionizable polar compounds. The present study investigated the addition of volatile ion-pair reagents to the reconstitution solution instead of the mobile phase to enhance the efficiency of chromatographic separation and minimize the sensitivity loss due to the formation of ion-pairs. The volatile ion-pair reagents used were perfluorinated carboxylic acids with n-alkyl chains: heptafluorobutanoic acid (HFBA), nonafluoropentanoic acid (NFPA), tridecafluoroheptanoic acid (TDFHA) and pentadecafluorooctanoic acid (PDFOA). The model analytes evaluated were N-methylnicotinamide (MNA) chloride, N-methyl 2-pyridone 5-carboxamide (2PY) and phenylephrine. The effects of alkyl chain length and the concentrations of the ion-pair reagents on the retention of analytes were studied, as well as the effect of pH on the retention of phenylephrine. The volatile ion-pair reagents in the reconstitution solution showed significant effect on the retention of the ionizable polar compounds, and the sensitivity of detection was improved for plasma samples through decreasing the matrix effect. This methodology was successfully applied to establish a quantitative assay for the polar drug substance methadone in human plasma with a concentration range from 0.1 to 50 ng/mL. Ion-pair reagents not only shifted the retention time but also reduced the carry-over peak for methadone.

Analgesics, Opioid↗

PubMed2005

Use of capillary electrophoresis in drug quality assessment of synthetic porcine secretin.

The purity profile for porcine secretin attributable to contamination by equilibrium products such as aspartoyl(3) secretin has been shown to be dependent on the pH of the analytical system. Capillary zone electrophoresis (CZE) methods have been developed for the efficient separation of synthetic porcine secretin, its equilibrium products and other impurities in aqueous solutions at both acidic and alkaline pH. These conditions are more representative of those used for the reconstitution and administration of porcine secretin, and good results cannot be achieved using HPLC due to poor peak shape above pH 5.8. The influence of various CZE operational parameters was systematically examined. The methods were validated for accuracy, precision, linearity, LOD and LOQ. A comparative evaluation of the stability of test solutions was determined using CZE and HPLC over a range of pH values. HPLC and CZE methods produced similar results at low pH.

Animals↗

PubMed2005

A molecular model to explain paclitaxel and docetaxel sensitivity changes through adduct formation with primary amines in electrospray ionization mass spectrometry.

The objective of this study was to adopt a molecular modeling approach to understand changes in signal intensity due to adduct formation with short-chain alkylamines for two anticancer agents, paclitaxel and docetaxel, during electrospray mass spectrometric analysis. We describe a simple and intuitive modeling procedure using a comparison of hydropathic interaction (HINT) scores to explain differences in responses of amine adducts formed with the two analytes. The responses of paclitaxel and docetaxel were generally enhanced considerably (up to approximately 500% in some instances) on adding the amines. However, for the docetaxel adduct formed with added decylamine in the mobile phase, the response dropped by 32%. A mechanistic understanding for this behavior is proposed, and binding scores calculated from corresponding molecular models were found to be consistent with the trend obtained from mass spectrometric data.

Amines↗

PubMed2004

Evaluation of deuterium isotope effects in normal-phase LC-MS-MS separations using a molecular modeling approach.

Molecular modeling of stationary phases presents a unique challenge because there is little available experimentally derived structural information. Verified interaction mechanisms at a molecular level with analytes are also rare. Molecular mechanics calculations using the Tripos force field were carried out to qualitatively and quantitatively assess stationary phase interactions. Binding energy values of -15.40, 15.28, -12.53, and -12.34 kcal/mol, respectively, are obtained for olanzapine (OLZ), OLZ-D3, des-methyl olanzapine (DES), and DES-D8 that corresponded to the retention behavior of the four compounds observed using liquid chromatography-mass spectrometry (MS)-MS. The model explains, semiquantitatively, the deuterium isotope effect in the normal-phase chromatographic separation of these compounds.

Journal Article↗

PubMed2001

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe.

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 microg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were -12.58 and -0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 microg/ml. 3-OHQ formation complied with Michaelis-Menten kinetics and the mean values+/-S.D. of Km and Vmax were 36.98+/-4.62 microg/ml and 321.39+/-3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.

Animals↗

PubMed1985

Simultaneous determination of codeine and ibuprofen in plasma by high-performance liquid chromatography.

A procedure is described for the simultaneous determination of codeine and ibuprofen in human plasma following the administration of the two substances in a proposed combination dosage form. The two substances were extracted separately from plasma and then determined together by high-performance liquid chromatography (HPLC) using a fluorescence detector. The codeine was first extracted from alkalinized plasma with hexane-dichloromethane (2:1, v/v) and then washed with sodium hydroxide solution. The ibuprofen was then extracted with hexane from the plasma acidified with sulphuric acid. The organic layers were collected, evaporated to dryness and the reconstituted residue was subjected to HPLC. The detection limit for codeine was 8 microg 1(-1) and for ibuprofen 1 mg 1(-1).

Journal Article↗

PubMedDate not supplied

Heterogeneous post-column immunoreaction detection using magnetized beads and a laboratory-constructed electromagnetic separator.

The nature of immune reactors allows development of quantitative analytical methods that are highly selective and can often be used directly with complex biological matrixes such as blood, plasma or urine. A major limitation of immunoassay is that antibodies are sometimes unable to discriminate structurally similar species such as drug metabolites and synthetic analogs. The problem associated with the lack of discrimination can be circumvented by coupling immunoassay with liquid chromatography post-column. The most commonly used separation method in post-column immunoreaction detection is the affinity column. Affinity columns may create undesired effects such as a compromise of the chromatographic separation efficiency, the requirement for an antibody with fast reaction kinetics and the need for flushing the column. This paper reports a post-column immunoreaction detection system coupled with a laboratory-constructed on-line magnetic separation flow chamber that is designed to overcome these problems. The system uses disposable magnetic beads as a solid-phase support for separation that can be easily removed from the system. The model analytes chosen for this study were digoxin and its metabolites due to the commercial availability of monoclonal antibodies for these compounds. Digoxin was separated using a chromatographic method prior to being interfaced through a liquid handler system to the immunoreactor. Compatibility of the HPLC mobile phase was determined to be acceptable with a mixing ratio of 1:3 between the LC fraction and immunoreagent solution. The dynamic range of the calibration curve in digoxin-spiked phosphate buffer was found to be 0.25-12 ng/ml and a quadratic fit was found to provide the best fit to the data with a correlation coefficient of 0.9974. The residual error for all standards was less than 15%. The percentage RSDs for the two controls, 2 and 10 ng/ml, were 6.88 and 4.82% (n = 6) and the percentage errors were 7.07 and -6.89% (n = 6), respectively.

Chromatography, High Pressure Liquid↗