Nanofluid of magnetic-activated charcoal and hydrophobic deep eutectic solvent: Application in dispersive magnetic solid-phase extraction for the determination and preconcentration of warfarin in biological samples by high-performance liquid chromatography.

In this study, for the first time, nanofluid of magnetic-activated charcoal and hydrophobic deep eutectic solvent (AC@Fe3 O4 -DES) based dispersive magnetic solid-phase extraction was successfully applied for the determination and preconcentration of warfarin in plasma and urine samples. The hydrophobic DES was prepared by mixing tetramethylammonium chloride (as hydrogen bond acceptor) and thymol (as hydrogen bond donor) and acted simultaneously as both carrier and stabilizer for magnetic nanoparticles. In this method, the nanofluid as a new extraction solvent was rapidly injected into the aqueous sample, which led to improvement of the mass transfer of the analytes into the sorbent and reduction of the extraction time. In the screening step, the fractional factorial design was applied for selecting some important parameters which significantly affected the extraction procedure. The effective parameters were then optimized by Box-Behnken design. Under the optimal conditions, the limits of detection were in the range of 0.3-1.6 ng/ml. A good linear range was observed in the range of 1.0-500.0 ng/ml for water and 5.0-500.0 ng/ml for urine and plasma. The intra- and inter-day relative standard deviations were 2.7-3.2 and 1.9-4.5% for five replications, respectively. Based on the results, the proposed method was successfully applied for the determination of warfarin in biological samples, using high-performance liquid chromatography.

Enantioseparation of warfarin derivatives on molecularly imprinted polymers for (S)- and (R)-chlorowarfarin.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4'-chlorowarfarin (CWF), (S)-CWF and (R)-CWF (MIPWF, MIPCWF, MIP(S)-CWF and MIP(R)-CWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and a crosslinker, respectively, by multi-step swelling and polymerization. The molar ratio of a template molecule, 4-VPY to EDMA was 6:18:25 or 4:18:25. The retention and molecular recognition properties of MIPWF and MIPCWF were evaluated using a mixture of sodium phosphate buffer or ammonium formate and acetonitrile in reversed-phase LC. WF and CWF on these MIPs gave the maximal retentions at mobile phase pH 7, and those retentions were decreased with an increase of acetonitrile content. The retention and imprinting factors were in the order of WF < CWF < 4'-bromowarfarin (BWF) on MIPWF and MIPCWF in neutral mobile phases. On the other hands, in acidic mobile phases the retention factors were in the same order with those in neutral mobile phases, while the imprinting factors of WF and CWF were higher on the respective MIPs. These results suggest that ionic or hydrogen bonding interactions, hydrophobic interactions and π-π interactions could work for the retention and molecular recognition of WF, CWF and BWF on these MIPs in a reversed-phase mode. Furthermore, MIP(S)-CWF and MIP(R)-CWF could separate WF, CWF and BWF enantiomers in acidic mobile phases.

Hydrophobic borneol-based natural deep eutectic solvents as a green extraction media for air-assisted liquid-liquid micro-extraction of warfarin in biological samples.

In the present study, a new generation of water-immiscible natural deep eutectic solvents (DESs) was synthesized using borneol as a hydrogen-bonding acceptor and decanoic acid, oleic acid, and thymol as a hydrogen-bonding donor in different molar ratios. These green hydrophobic solvents which are chemically stable in aqueous solutions were used as extraction solvents for isolation and pre-concentration of warfarin in biological samples. In this method, fine droplets of DESs were dispersed into the sample solution by using the air-assisted liquid-liquid micro-extraction method to accelerate the cloudy emulsion system formation and increase the mass transfer of the analyte to the DES-rich phase. The borneol based deep eutectic solvent is a worthy generation of the extraction solvents in the ALLME method due to low-cost and less toxicity. A Plackett-Burman design was utilized for screening the experimental parameters. The effective parameters were then optimized by Box-Behnken design (BBD). Optimized extraction conditions were pH of sample solution of 3.9, number of aspiration/dispersion cycles of 15, the volume of DES of 60 µL, and rate and time of centrifuge of 6000 rpm and 10 min, respectively. Under the optimized conditions, the developed NADES-ALLME method exhibited a wide linear range of 5-500 µg L - 1 for plasma and urine samples with satisfactory recoveries above 88.80%. Limit of detections (LODs) and Limit of quantifications (LOQs) of warfarin were in the ranges of 0.5-2.7 and 1.65-8.91, respectively. The enrichment factors were obtained in the range of 148-164 and precisions were lower than 5.87%. Finally, the proposed method was successfully employed for the analysis of warfarin in human urine and plasma samples.

Studies of drug interactions with alpha1-acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography.

A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α1-acid glycoprotein (AGP). Affinity microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72nmol AGP when using a 20mm×2.1mm i.d. microcolumn. These microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13-76%); however, disopyramide gave a 21-25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.

On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) µg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

Fast separation of warfarin and 7-hydroxywarfarin enantiomers by cyclodextrin-assisted capillary electrophoresis.

The enantioseparation of warfarin and its main metabolite has been achieved using several cyclodextrin types and buffers at different pH, including conditions that have not been attempted so far. Methyl-ß-cyclodextrin, highly sulfated-ß-cyclodextrin and highly sulfated-γ-cyclodextrin were the most efficient chiral selectors. The pH range, within which particular cyclodextrins support chiral separation, has been approximately determined for the first time. By shortening the effective capillary length to 10 cm, the time of analysis has been vastly reduced <2 min. Hence, baseline separations of warfarin and 7-hydroxywarfarin enantiomers have been achieved in times unreported for those species until now. The established conditions are promising for the further development of new highly selective and fast methods involving warfarin, its derivatives, as well as the same cyclodextrin types.

Comprehensive characterization of rodenticides in wastewater by liquid chromatography-tandem mass spectrometry.

Rodenticides are used as pest control to eradicate rodents and have emerged as new environmental contaminants due to their widespread use in domestic and urban infrastructures. In this study, we have developed and validated an analytical methodology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of 13 anticoagulant rodenticides in wastewater. In a first step, ionization conditions were tested in electrospray mode, and positive ionization gave the highest sensitivity. Fragmentation patterns were determined and two selected reaction monitoring (SRM) transitions were selected for each compound. Using a Zorbax Eclipse XDB-C18 column and specific SRM transitions, 13 compounds were resolved. The LC-MS/MS method provided good linearity, sensitivity, intra- and inter-day precision, and good identification capabilities for these compounds in wastewaters. Thereafter miniaturized liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were optimized. Oasis HLB and Strata WA SPE cartridges with methanol/dichloromethane as eluting solvents provided good recoveries and limits of detection ranged between 0.34 and 20 ng L(-1), whereas LLE failed to recover some compounds. Finally, the performance of both LLE and SPE methods was evaluated by analyzing rodenticides in a set of wastewaters. Warfarin was the only detected compound at nanogram per liter level, and good agreement was observed between LLE and SPE.

Chromatographic separation and assignment of absolute configuration of hydroxywarfarin isomers.

The absolute configuration of several hydroxywarfarin isomers was assigned using a comparison of elution order on chiral stationary phases, optical rotation, and circular dichroism (CD) spectra, with confirmation of assignments made by comparison between experimental and calculated CD spectra and selective synthesis of hydroxywarfarin isomers from enantiopure warfarin using human liver microsomes.

[On the history of vitamin K, dicoumarol and warfarin]. / Pionerer bag vitamin K, dikumarol og warfarin.

The history of the discovery and development of vitamin K and its antagonists, the oral anticoagulants dicoumarol and warfarin, are fascinating, triumphant landmarks in the annals of medicine. Vitamin K was found by Carl Peter Henrik Dam and Fritz Schønheyder from the University of Copenhagen. The discovery was initiated by Dam, by a lucky choice of chicks in the dissertation of sterol metabolism, since the vitamin is not formed by intestinal bacteria in these animals. In these experiments the lack of an unknown factor in the synthetic diet caused internal bleeding similar to that found in scurvy, but the bleeding was not reversed by vitamin C and it could not be explained by the lack of classical vitamins. In 1935 the unknown antihaemorrhagic factor was named vitamin K and a few months later the phenomenon was also observed by H.J. Almquist and E.L.R. Stokstad in Berkeley. The activity of the factor was determined by bioassay in different extracts of green vegetables and alfalfa by Dam and Schønheyder. Vitamin K was isolated in 1939 by Dam and Paul Karrer in Zurich and the structure was determined by Edward Adelbert Doisy. Dam and Doisy were awarded the Nobel Prize in 1943. A dramatic story starts the discovery of dicoumarol. In the 1920s cattle in Canada began dying of internal bleeding with no obvious precipitating cause. Frank W. Schofield, a veterinary pathologist in Alberta, found that the mysterious disease was connected to the consumption of spoiled sweet clover hay and noted a prolonged clotting time. Ten years after a farmer traveled in a blizzard with his dead cow and a milk can of the unclotted blood to the University of Wisconsin. Only the door to the biochemical department of Karl Paul Link was open. This event started the isolation of the anticoagulant agent dicou- marol which was formed by microbial induced oxidation of coumarin in the mouldy sweet clover hay. More than hundred dicoumarol-like anticoagulants were synthesized by Link and his co-workers. A potent hemorrhagic agent named warfarin was first used as an effective rat poison. However, warfarin became the drug of choice and the break- through in the treatment of thromboembolic diseases. Today new oral anticoagulants are competing with warfarin.

Chromatographic resolution of closely related species: separation of warfarin and hydroxylated isomers.

Recent developments in the field of organic synthesis are leading to increasingly complex mixtures of closely related species (positional isomers, regioisomers, diastereomers, etc.) that often prove challenging for chromatographic analysis and separation. In this study we investigate the separation of a representative mixture of warfarin and 5 different monohydroxylation isomers to assess whether conventional techniques are suitable for addressing this separation challenge, or whether 'next generation' separation tools such as multidimensional chromatography may be required. In this example, modifications of results obtained from conventional achiral and chiral chromatography method development screening platforms afford rapid separation of all components for both achiral and chiral analysis, with supercritical fluid chromatography showing the best performance in both cases (1.8min for separation of six components by achiral SFC and 8.0min for separation of twelve components by chiral SFC). While other more complex mixtures may require additional tools, these results suggest that new applications of existing separation platforms may be useful for creating the chromatographic methods required to support this new area of synthetic chemistry.

Development of a chiral micellar electrokinetic chromatography-tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites: application to the analysis of patients serum samples.

The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE-MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-L,L-leucylvalinate (poly-L,L-SULV) as a chiral pseudophase in MEKC-MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45 min. This is in comparison to 100 min required for separation of the same mixture with packed column CEC-MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC-MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000 ng/mL for R- and S-warfarin, 5 to 1000 ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000 ng/mL for R and S-4'-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC-MS/MS were found to be at levels of 2 and 5 ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC-MS/MS method to accurately quantitate with high sensitivity.

[Vitamin K antagonists: from discovery to pharmacogenetics]. / Les antagonistes de la vitamine K : de leur découverte à la pharmacogénétique.

Vitamin K antagonists (VKA) are used for 60 years in the treatment and prevention of thromboembolic disease. VKA were first used as rodenticides. There was a growing use of VKA in humans after President Eisenhower received them after a heart attack in 1955. However, the use of VKA is still challenging because they are characterized by a narrow therapeutic index and a great inter-individual variability in the dose response to the drug. This variability can partly be explained by demographic, clinical and therapeutic factors, but also by genetic variations. The main enzyme responsible for VKA metabolism is the hepatic cytochrome P450 2C9 (CYP2C9). Vitamin K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle and was identified as the pharmacological target of VKA. Genetic variations affecting both CYP2C9 and VKORC1 are associated with a significant decrease in the VKA dose requirements and an increased risk of bleeding. Genotyping both CYP2C9 and VKORC1 before the initiation of VKA allows to identify a subgroup of patients with an early response to VKA therapy, that expose them to overdosage and a higher bleeding risk. More recently, a polymorphism in the gene encoding CYP4F2 has been identified and may partly explain the variability in warfarin maintenance dose by altering the metabolism of vitamin K. In addition, rare mutations have been found in VKORC1 that could explain very high VKA dose requirements and pharmacodynamic resistance.

Low-density solvent-based dispersive liquid-liquid microextraction followed by high performance liquid chromatography for determination of warfarin in human plasma.

Extraction and determination of warfarin, a widely used anticoagulant drug, in human plasma were performed using a new generation of dispersive liquid-liquid microextraction (DLLME) and high performance liquid chromatography (HPLC). The extraction procedure is based on extraction solvents lighter than water and performing of extraction in a specially designed extraction cell. Some important parameters, including kind and volume of extraction and disperser solvents, pH of the sample solution, salt concentration in the sample solution and extraction time were investigated and optimized. Under the optimized conditions (150 µL 1-octanol as extraction solvent, 150 µL methanol as disperser solvent, pH(sample)=2.3, extraction time of 2 min, without salt addition), limit of detection (LOD) of 5 ng mL⁻¹ and extraction recovery of 91.0% were obtained. The calibration curve was linear within the range of 15-3000 ng mL⁻¹ with the square of correlation coefficient (R²) of 0.998. Repeatability and reproducibility of method based on five replicate extraction and determination were 2.8% and 6.5%, respectively. The proposed method was applied successfully for the determination of warfarin in plasma sample from a patient under treatment with this drug, and was demonstrated to be sensitive, efficient, and convenient.

Analysis of R- and S-hydroxywarfarin glucuronidation catalyzed by human liver microsomes and recombinant UDP-glucuronosyltransferases.

Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by K(m), V(max), and K(i), comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved.

Three-phase hollow fiber liquid phase microextraction of warfarin from human plasma and its determination by high-performance liquid chromatography.

A simple, inexpensive and efficient sample preparation technique, three-phase hollow fiber liquid phase microextraction (HF-LPME), followed by high-performance liquid chromatography-ultra violet detection (HPLC-UV) was used for the analysis of warfarin in human plasma. Warfarin was extracted from 11.0 ml of aqueous solution with pH=2.3 (donor phase) into 1-octanol immobilized in the wall pores of a porous hollow fiber and then extracted into the acceptor phase with pH=11.0 located in the lumen of the hollow fiber. After the extraction, the acceptor phase was directly injected into the HPLC system for quantification. Different factors affecting the HF-LPME including nature of organic extraction solvent, pH of donor and acceptor phases, stirring rate, extraction time and salt addition to the sample solution (donor phase) were investigated and optimized. Under the optimized conditions (1-octanol as organic solvent, pH(donor)=2.3, pH(acceptor)=11.0, stirring speed of 1000 rpm, extraction time of 30 min, without addition of salt), limit of detection (LOD) of 5 ng ml(-1) and enrichment factor of 225 were obtained. The calibration curve was linear within the range of 15-550 ng ml(-1) with the square of correlation coefficient of 0.9984. The values of intra-day relative standard deviation (RSD) and inter-day RSD were 4.2% and 11.1%, respectively. The method was applied successfully for the analysis of warfarin in plasma sample from a patient under treatment with this drug and excellent sample clean-up was observed.

Enantioseparation of organic acids of pharmaceutical interest using eremomycin as a chiral selector.

Strong adsorption of eremomycin on the fused-silica capillary wall was used for separation of enantiomers by CE. The capillary with adsorbed chiral selector was shown to be easily prepared and has reproducible properties. The effect of the chiral selector concentration, pH and composition of the BGE, and applied voltage on enantioseparation of acidic compounds, such as profens and aromatic carboxylic acids, was investigated. Two native α-amino acids, aspartic acid and glutamic acid, were enantioseparated. Fourteen tested compounds (including amino acids) were baseline resolved. Good selectivity of separation (α>1.09) was achieved. The migration order of ibuprofen and ketoprofen enantiomers was determined. The procedures were proposed for the analysis of flurbiprofen and warfarin in pharmaceuticals. Linearity was achieved in the concentration range of 4.0×10(-5)-2.0×10(-3) M for flurbiprofen and 3.2×10(-6)-4.9×10(-6) M for warfarin. The detection limits were found to be about 1×10(-5) M for flurbiprofen and 1×10(-6) M for warfarin.

Removal of pharmaceuticals from water: using liquid-core microcapsules as a novel approach.

In recent years ever-increasing amounts of pharmaceuticals are being detected in the aquatic environment and in some cases, they have even been discovered in drinking water. Their presence is attributed mainly to the inability of sewage treatment plants to adequately remove these compounds from the sewage influent. The aim of this study was to investigate the feasibility, kinetics and efficiency of using liquid-core microcapsules as a novel methodology, termed capsular perstraction, to remove seven pharmaceuticals commonly found in the environment, from water. The process involves the envelopment of pre-selected organic solvents within a porous hydrogel membrane to form liquid-core microcapsules, which can be used to extract a large range of compounds. Results indicate that this novel approach is capable of extracting the seven chosen compounds rapidly and with a variable efficiency. The simultaneous use of both dibutyl sebacate and oleic acid liquid-core microcapsules at a liquid volume ratio of only 4% (v/v) resulted in the following extractions within 50min of capsule addition to contaminated water: furosemide 15%; clofibric acid 19%; sulfamethoxazole 22%; carbamazepine 54%; warfarin 80%; metoprolol 90% and diclofenac 100%. The effects of different agitation rates, microcapsule size and membrane thickness on the rate of mass transfer of warfarin into the liquid-core (dibutyl sebacate) of microcapsules was also examined. Results showed that the main rate-limiting step to mass transfer was due to the stagnant organic film (microcapsule size) within the core of the microcapsules. A volumetric mass transfer coefficient of 2.28x10(-6)m/s was obtained for the smallest microcapsules, which was nearly 4-fold higher compared to the value (0.6x10(-6)m/s) obtained for the largest microcapsules used in this study. Even with this resistance liquid-core microcapsules are still capable of the rapid extraction of the tested compounds and may provide a platform for the safe disposal of the pharmaceuticals after removal.

New high-performance liquid chromatography method for the determination of (R)-warfarin and (S)-warfarin using chiral separation on a glycopeptide-based stationary phase.

Warfarin is a well-known anticoagulant agent that occurs in two enantiomers, (R)-(+)-warfarin and (S)-(-)-warfarin. A new liquid chromatography method for the determination of both enantiomers was developed, validated and applied in in vitro studies with the aim of evaluating the accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line. OptiMEM cell cultivation medium samples and cellular lysates were purified using Waters Oasis MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic V2 column at a flow rate of 1.2mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (lambda(excit)=320nm, lambda(emiss)=415nm). The limit of detection was found to be 0.121micromol/L of (S)-warfarin and 0.109micromol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100micromol/L. The precision ranged from 1.3% to 12.2% of the relative standard deviation, and the accuracy reached acceptable values from 95.5% to 108.4%. The new bioanalytical method confirmed the same accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line.

Evaluation of silica monoliths in affinity microcolumns for high-throughput analysis of drug-protein interactions.

Silica monoliths in affinity microcolumns were tested for the high-throughput analysis of drug-protein interactions. HSA was used as a model protein for this work, while carbamazepine and R-warfarin were used as model analytes. A comparison of HSA silica monoliths of various lengths indicated columns as short as 1 to 3 mm could be used to provide reproducible estimates of retention factors or plate heights. Benefits of using smaller columns for this work included the lower retention times and lower back pressures that could be obtained versus traditional HPLC affinity columns, as well as the smaller amount of protein that is required for column preparation. One disadvantage of decreasing column length was the lower precision that resulted in retention factor and plate height measurements. A comparison was also made between microcolumns containing silica particles versus silica monoliths. It was demonstrated with R-warfarin that supports could be used in HSA microcolumns for the determination of retention factors or plate heights. However, the higher efficiency of the silica monolith made this the preferred support for work at higher flow rates or when a larger number of plates are needed during the rapid analysis of drug-protein interactions.

Preparation and characterization of poly(l-phenylalanine) chiral stationary phases with varying peptide length.

Three new chiral stationary phases with different lengths of l-phenylalanine peptide were prepared by solid-phase synthesis with tert-butoxycarbonyl (Boc)-l-phenylalanine on silica. The effect of phenylalanine peptide length on enantioselectivity was studied. The best separation of R/S-warfarin was achieved by the chiral stationary phase with intermediate peptide length. These stationary phases were found to exist mainly in alpha-helical conformation by using FT-IR spectra. The end-capping reagents for the N-terminus of the peptide were also evaluated.