Kenneth R. Wehmeyer

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma.

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.

Fluorogenic assay for β-glucuronidase using microchip-based capillary electrophoresis

Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with beta-glucuronidase of the conjugated glucuronide, fluorescein mono-beta-D-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess beta-glucuronidase activity. Michaelis-Menten enzyme kinetics analysis yielded the Km value. The results were compared with those from experiments done by conventional CE. The Km value for beta-glucuronidase with FMG is being reported for the first time as 18 microM. The inhibition of beta-glucuronidase by the competitive inhibitor D-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant beta-glucuronidase and FMG concentrations. A dose-response plot was acquired and the IC50 value for SL was determined to be 3 microM.

Determination of dextromethorphan and dextrorphan in human plasma by liquid chromatography/tandem mass spectrometry

Rapid, sensitive and selective methods were developed for the determination of dextromethorphan and its major metabolite, dextrorphan, in human plasma using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope internal standards were prepared for analysis by a liquid-liquid back-extraction procedure. Dextromethorphan and dextrorphan were chromatographed on a short reversed-phase column, using separate isocratic mobile phase conditions optimized to elute each compound in approximately 1.1 min. For both analytes, calibration curves were obtained over four orders of magnitude and the limit of quantitation was 5 pg ml-1 using a 1 ml plasma sample volume. The accuracy across the entire range of spiked DEX and DOR concentrations was, in general, within 10% of the spiked value. The precision was generally better than 6% for replicate sample preparations at levels of 50 pg ml-1 or higher and typically better than 12% at levels below 50 pg ml-1. The method was applied for the evaluation of the pharmacokinetic profiles of dextromethorphan and dextrorphan in a human volunteer following peroral administration of a commercially available cough formulation.

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography-tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography-tandem mass spectrometry-negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25-100 microl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-microl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100+/-10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 microg/kg) produced an approximately 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.

Liquid Chromatography with Electrochemical Detection of Phenol and NADH for Enzyme Immunoassay

Abstract Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.

Protein-aptamer binding studies using microchip affinity capillary electrophoresis

The use of traditional CE to detect weak binding complexes is problematic due to the fast‐off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare‐silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin‐binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin–aptamer complex from the free aptamer was achieved in less than 10 s on a single‐cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three‐fold S/N.

Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

Isolation and quantification of fluoroacetate in rat tissues, following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor.

Peptidyl fluoromethyl ketones (PFMK) are irreversible inhibitors of cathepsin B, a cysteine proteinase thought to be involved in the degradation of cartilage. It has been speculated that PFMK inhibitors may metabolize in rodents to form fluoroacetate (FAC), an extremely toxic poison. A highly selective and sensitive separation and detection scheme was developed to measure trace levels of FAC in rat tissues following PFMK dosing. The procedure consisted of extracting FAC from tissue and spiking the extract with [18O]2-fluoroacetate (18O-FAC) as an internal standard. FAC and 18O-FAC were further isolated from matrix components using ion-exchange, solid-phase extraction. The pentafluorobenzyl esters of FAC and 18O-FAC were formed to facilitate the chromatographic separation. Two-dimensional gas chromatography coupled with selected-ion-monitoring detection provided the final measurement. The assay had a limit of detection of 2 ng FAC per g tissue, and was capable of accurately quantitating as little as 10 ng FAC per g tissue with a S/N ratio of 40:1. Linearity was established over two orders of magnitude, from 2-500 ng ml-1, with 5 microliters injected on-column. The method was used to demonstrate that FAC was formed in rats following dosing with Z-Phe-Ala-CH2-F, a PFMK cathepsin enzyme inhibitor.

Evaluation of a benchtop ion trap gas chromatographic-tandem mass spectrometric instrument for the analysis of a model drug, tebufelone, in plasma using a stable-isotope internal standard

The performance of a benchtop GC-ion trap MS-MS instrument, the Varian Saturn 4D, was evaluated for the analysis of a model drug, tebufelone, in plasma. The sample preparation scheme was designed to provide a highly complex extract with matrix-derived interferences eluting near and at the retention time of tebufelone and its stable-isotope-labeled analog. The performance of the ion trap in the selected-reaction-monitoring mode was evaluated and also compared with results obtained on a benchtop GC-MS linear quadrupole instrument operated in the selected-ion-monitoring mode. The ion trap, operated in the selected-reaction-monitoring mode, was found to provide a higher degree of selectivity for the analysis of tebufelone. The increased selectivity obtained on the ion trap operated in the selected-reaction-monitoring mode resulted in superior accuracy and precision, as well as a lower limit of quantitation relative to that obtained by the GC-MS analysis. A linear standard curve was obtained over three orders of magnitude and the limit of quantitation for tebufelone in plasma was 100 pg/ml using the GC-ion trap MS-MS instrument.

Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE–LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts

Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE-LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE-LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE-LIF approach. The advantages of MCE-LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.