Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.

Nonaqueous capillary zone electrophoresis of synthetic organic polypeptides

Poly(Nε-trifluoroacetyl-l-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanol/acetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of ∼50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

Development of advanced capillary electrophoresis techniques with UV and mass spectrometry detection for forensic, pharmaceutical and environmental applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. ^ Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. ^ Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. ^ It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. ^ Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.^

Development of Advanced Capillary Electrophoresis Techniques with UV and Mass Spectrometry Detection for Forensic, Pharmaceutical and Environmental Applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

The Applicability Of Ordered Mesoporous Sba-15 And Its Hydrophobic Glutaraldehyde-bridge Derivative To Improve Ibuprofen-loading In Releasing System.

The mesoporous SBA-15 silica with uniform hexagonal pore, narrow pore size distribution and tuneable pore diameter was organofunctionalized with glutaraldehyde-bridged silylating agent. The precursor and its derivative silicas were ibuprofen-loaded for controlled delivery in simulated biological fluids. The synthesized silicas were characterized by elemental analysis, infrared spectroscopy, (13)C and (29)Si solid state NMR spectroscopy, nitrogen adsorption, X-ray diffractometry, thermogravimetry and scanning electron microscopy. Surface functionalization with amine containing bridged hydrophobic structure resulted in significantly decreased surface area from 802.4 to 63.0 m(2) g(-1) and pore diameter 8.0-6.0 nm, which ultimately increased the drug-loading capacity from 18.0% up to 28.3% and a very slow release rate of ibuprofen over the period of 72.5h. The in vitro drug release demonstrated that SBA-15 presented the fastest release from 25% to 27% and SBA-15GA gave near 10% of drug release in all fluids during 72.5 h. The Korsmeyer-Peppas model better fits the release data with the Fickian diffusion mechanism and zero order kinetics for synthesized mesoporous silicas. Both pore sizes and hydrophobicity influenced the rate of the release process, indicating that the chemically modified silica can be suggested to design formulation of slow and constant release over a defined period, to avoid repeated administration.

Fases estacionárias monolíticas para separações cromatográficas

Monolithic stationary phases represent a new generation of chromatographic separation media. These phases consist of a continuous separation bed prepared by in situ polymerization or consolidation inside the column tubing. In recent years, their simple preparation procedure, unique properties and excellent performance have attracted quite remarkable attention in liquid chromatography and capillary electrochromatography. This review summarizes the preparation, characterization and applications of monolithic stationary phases. The analytical potential of these columns is demonstrated with separations involving various families of compounds in different separation modes.

Determination of Vecuronium Bromide in Pharmaceuticals: Development, Validation and Comparative Study of HPLC and CZE Analytical Methods

Vecuronium bromide is a neuromuscular blocking agent used for anesthesia to induce skeletal muscle relaxation. HPLC and CZE analytical methods were developed and validated for the quantitative determination of vecuronium bromide. The HPLC method was achieved on an amino column (Luna 150 x 4.6 mm, 5 mu m) using UV detection at 205 nm. The mobile phase was composed of acetonitrile:water containing 25.0 mmol L(-1) of sodium phosphate monobasic (50:50 v/v), pH 4.6 and flow rate of 1.0 mL min(-1). The CZE method was achieved on an uncoated fused-silica capillary (40.0 cm total length, 31.5 cm effective length and 50 mu m i.d.) using indirect UV detection at 230 nm. The electrolyte comprised 1.0 mmol L(-1) of quinine sulfate dihydrate at pH 3.3 and 8.0% of acetonitrile. The results were used to compare both techniques. No significant differences were observed (p > 0.05).

Stereoselective determination of midodrine and desglymidodrine in culture medium: Application to a biotransformation study employing endophytic fungi

A CE method was developed and validated for the stereoselective determination of midodrine and desglymidodrine in Czapek culture medium to be applied to a stereoselective biotransformation study employing endophytic fungi. The electrophoretic analyses were performed using an uncoated fused-silica capillary and 70 mmol/L sodium acetate buffer solution (pH 5.0) containing 30 mmol/L heptakis (2, 3, 6-tri-O-methyl)-beta-CD as running electrolyte. The applied voltage and temperature used were 15 kV and 15 C, respectively. The UV detector was set at 200 nm. The sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.1-12 mu g/mL for each enantiomer of midodrine and desglymidodrine (r >= 0.9975). Within-day and between-day precision and accuracy evaluated by RSDs and relative errors, respectively, were lower than 15% for all analytes. The method proved to be robust by a fractional factorial design evaluation. The validated method was used to assess the midodrine biotransformation to desglymidodrine by the fungus Phomopsis sp. (TD2), which biotransformed 1.1% of (-)-midodrine to (-)-desglymidodrine and 6.1% of (+)-midodrine to (+)-desglymidodrine.

Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4%w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 mu g/mL for each 4-OH-Prop enantiomer and 0.10-10.0 mu g/mL for each Prop enantiomer (r >= 0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)4-OH-Prop in 72 h of incubation.

Box-Behnken design for the optimization of an enantioselective method for the simultaneous analysis of propranolol and 4-hydroxypropranolol by CE

An experimental design optimization (Box-Behnken design, BBD) was used to develop a CE method for the simultaneous resolution of propranolol (Prop) and 4-hydroxypropranolol enantiomers and acetaminophen (internal standard). The method was optimized using an uncoated fused silica capillary, carboxymethyl-beta-cyclodextrin (CM-beta-CD) as chiral selector and triethylamine/phosphoric acid buffer in alkaline conditions. A BBD for four factors was selected to observe the effects of buffer electrolyte concentration, pH, CM-beta-CD concentration and voltage on separation responses. Each factor was studied at three levels: high, central and low, and three center points were added. The buffer electrolyte concentration ranged from 25 to 75 mM, the pH ranged from 8 to 9, the CM-beta-CD concentration ranged from 3.5 to 4.5%w/v, and the applied run voltage ranged from 14 to 20 W. The responses evaluated were resolution and migration time for the last peak. The obtained responses were processed by Minitab (R) to evaluate the significance of the effects and to find the optimum analysis conditions. The best results were obtained using 4%w/v CM-beta-CD in 25 mM triethylamine/H(3)PO(4) buffer at pH 9 as running electrolyte and 17 kV of voltage. Resolution values of 1.98 and 1.95 were obtained for Prop and 4-hydroxypropranolol enantiomers, respectively. The total analysis time was around of 15 min. The BBD showed to be an adequate design for the development of a CE method, resulting in a rapid and efficient optimization of the pH and concentration of the buffer, cyclodextrin concentration and applied voltage.

Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils

Copaifera species (Leguminoseae) are popularly known as ""copaiba"" or ""copaiva"". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of beta-caryophyllene, alpha-copaene, and alpha-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100 mu L) were dissolved in 20 mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0 mM) in a 25 mL glass flask. A 1 mu L aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenylmethylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74 mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091 mM. beta-Caryophyllene, alpha-copaene, and alpha-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies. (C) 2010 Elsevier B.V. All rights reserved.

Nanocomposite Polymer Electrolyte Membranes: Methanol Crossover and Conductivity

Commercial Nafion® 117 membranes were successfully modified by in-situ reactions (sol-gel of TEOS and/or polymerization of aniline) within Nafion structures. Water-methanol permeability and proton conductivity were investigated in order to determine the potential performance of these membranes for DMFC systems. Silica-polyaniline modification resulted in 84% methanol crossover reduction, from 2.45x10^-5 cm2.s^-1 for conventional Nafion membranes to 3.71x10^-6 cm2.s^-1 for the modified silica-polyaniline composite membrane at 75 degrees C. In addition, conductivity was not hindered, as the polyaniline-Nafion membrane increased from 12.2 to 15 mS.cm^-1 as compared to Nafion, while a reduction of 11% was observed for silica-polyaniline-Nafion composite membrane. The results in this work strongly suggest the potential of polyaniline nanocomposites to enhance the performance of DMFCs.

Spontaneous Eyeblink Activity

Spontaneous blinking is essential for maintaining a healthy ocular surface and clarity of vision. The spontaneous blink rate (SBR) is believed to reflect a complex interaction between peripheral influences mediated by the eye surface and the central dopaminergic activity. The SBR is thus extremely variable and dependent on a variety of psychological and medical conditions. Many different methods have been employed to measure the SBR and the upper eyelid kinematics during a blink movement. Each has its own merits and drawbacks, and the choice of a specific method should be tailored to the specific needs of the investigation. Although the sequence of muscle events that leads to a blink has been fully described, knowledge about the neural control of spontaneous blinking activity is not complete. The tear film is dynamically modified between blinks, and abnormalities of the blink rate have an obvious influence on the ocular surface.