Hollow-fiber liquid-phase microextraction of amphetamine-type stimulants in human hair samples

A fast method was optimized and validated in order to quantify amphetamine-type stimulants (amphetamine, AMP; methamphetamine, MAMP; fenproporex, FPX; 3,4-methylenedioxymethamphetamine, MDMA; and 3,4-methylenedioxyamphetamine, MDA) in human hair samples. The method was based in an initial procedure of decontamination of hair samples (50 mg) with dichloromethane, followed by alkaline hydrolysis and extraction of the amphetamines using hollow-fiber liquid-phase micro extraction (HF-LPME) in the three-phase mode. Gas chromatography-mass spectrometry (GC-MS) was used for identification and quantification of the analytes. The LoQs obtained for all amphetamines (around 0.05 ng/mg) were below the cut-off value (0.2 ng/mg) established by the Society of Hair Testing (SoHT). The method showed to be simple and precise. The intra-day and inter-day precisions were within 10.6% and 11.4%, respectively, with the use of only two deuteratecl internal standards (AMP-d5 and MDMA-d5). By using the weighted least squares linear regression (1/x(2)), the accuracy of the method was satisfied in the lower concentration levels (accuracy values better than 87%). Hair samples collected from six volunteers who reported regular use of amphetamines were submitted to the developed method. Drug detection was observed in all samples of the volunteers. (c) 2012 Elsevier B.V. All rights reserved.

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DEVELOPMENT AND VALIDATION OF AN ANALYTICAL METHOD FOR QUANTITATION OF THE DRUG BEVACIZUMAB BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY. In this study, an analytical method was developed and validated for quantitation of the drug bevacizumab (Avastin (R)) by high performance liquid chromatography (HPLC). The HPLC column was a BioSuite 250 (R) HR SEC, 300 x 7.8 mm x 5 mu m (Waters, USA). The mobile phase consisted of phosphate buffered saline (PBS). The results revealed that the method was specific, precise. accurate, robust and linear (r(2) = 0.998) from 5 to 75 mu g mL(-1). Therefore, this method can be used in drug release studies or in quality control ampoules of the drug.

Flow-injection spectrophotometric determination of captopril in pharmaceutical formulations using a new solid-phase reactor containing AgSCN immobilized in a polyurethane resin

A simple flow-injection analysis procedure was developed for determining captopril in pharmaceutical formulations employing a novel solid-phase reactor containing silver thiocyanate immobilized in a castor oil derivative polyurethane resin. The method was based on silver mercaptide formation between the captopril and Ag(I) in the solid-phase reactor. During such a reaction, the SCN- anion was released and reacted with Fe3+, which generated the FeSCN2+ complex that was continuously monitored at 480 nm. The analytical curve was linear in the captopril concentration range from 3.0 x 10(-4) mol L-1 to 1.1 x 10(-3) mol L-1 with a detection limit of 8.0 x 10(-5) mol L-1. Recoveries between 97.5% and 103% and a relative standard deviation of 2% for a solution containing 6.0 x 10(-4) mol L-1 captopril (n = 12) were obtained. The sample throughput was 40 h(-1) and the results obtained for captopril in pharmaceutical formulations using this procedure and those obtained using a pharmacopoeia procedure were in agreement at a 95% confidence level.

Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography for fatty acid profiling of cell wall phospholipids

The combination of solid-phase microextraction (SPME) with comprehensive two-dimensional gas chromatography is evaluated here for fatty acid (FA) profiling of the glycerophospholipid fraction from human buccal mucosal cells. A base-catalyzed derivatization reaction selective for polar lipids such as glycerophospholipid was adopted. SPME is compared to a miniaturized liquidliquid extraction procedure for the isolation of FA methyl esters produced in the derivatization step. The limits of detection and limits of quantitation were calculated for each sample preparation method. Because of its lower values of limits of detection and quantitation, SPME was adopted. The extracted analytes were separated, detected, and quantified by comprehensive two-dimensional gas chromatography with flame ionization detection (FID). The combination of SPME and comprehensive two-dimensional gas chromatography with FID, using a selective derivatization reaction in the preliminary steps, proved to be a simple and fast procedure for FA profiling, and was successfully applied to the analysis of adult human buccal mucosal cells.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Automated analysis of lidocaine and its metabolite in plasma by in-tube solid-phase microextraction coupled with LC-UV for pharmacokinetic study

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for determination of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in human plasma has been developed, validated, and further applied to pharmacokinetic study in pregnant women with gestational diabetes mellitus (GDM) subjected to epidural anesthesia. Important factors in the optimization of in-tube SPME performance are discussed, including the draw/eject sample volume, draw/eject cycle number, draw/eject flow rate, sample pH, and influence of plasma proteins. The limits of quantification of the in-tube SPME/LC method were 50 ng/mL for both metabolite and lidocaine. The interday and intraday precision had coefficients of variation lower than 8%, and accuracy ranged from 95 to 117%. The response of the in-tube SPME/LC method for analytes was linear over a dynamic range from 50 to 5000 ng/mL, with correlation coefficients higher than 0.9976. The developed in-tube SPME/LC method was successfully used to analyze lidocaine and its metabolite in plasma samples from pregnant women with GDM subjected to epidural anesthesia for pharmacokinetic study.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)(2)SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6HSO7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents. (C) 2011 Elsevier Inc. All rights reserved.

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective beta-blocker activity but both enantiomers present similar activity on a1-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T (R) (Teicoplanin) column. Protonated ions [M + H]+ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml-1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC08 205.52 vs. 82.61 (ng h) ml-1), with an enantiomer ratio of 2.48. Chirality, 2012. (C) 2012 Wiley Periodicals, Inc.

Solid phase microextraction and LC-MS/MS for the determination of paliperidone after stereoselective fungal biotransformation of risperidone

The present work describes for the first time the use of SPME coupled to LC-MS/MS employing the polar organic mode in a stereoselective fungal biotransformation study to investigate the fungi ability to biotransform the drug risperidone into its chiral and active metabolite 9-hydroxyrisperidone (9-RispOH). The chromatographic separation was performed on a Chiralcel OJ-H column using methanol:ethanol (50:50, v/v) plus 0.2% triethylamine as the mobile phase at a flow rate of 0.8 mL min(-1). The SPME process was performed using a C18 fiber, 30 min of extraction time and 5 min of desorption time in the mobile phase. The method was completely validated and all parameters were in agreement with the literature recommendations. The Cunninghamella echinulata fungus was able to biotransform risperidone into the active metabolite, (+)-9-RispOH, resulting in 100% of enantiomeric excess. The Cunninghamella elegans fungus was also able to stereoselectively biotransform risperidone into (+)- and (-)-9-RispOH enantiomers at different rates. (C) 2012 Elsevier B.V. All rights reserved.

Enzymatic Solid-Phase Reactor Based on Silica Organofunctionalized with p-Phenylenediamine for Electrochemical Detection of Phenolic Compounds

A new biomaterial, based on silica organofunctionalized with p-phenylenediamine (p-PDA) and the enzyme peroxidase, was used in the development of an enzymatic solid-phase reactor. The analytical techniques used in the characterization showed that the organic ligand was incorporated into the silica matrix. Thus, the silica modified with p-PDA allowed the incorporation of peroxidase by the electrostatic interaction between the carboxylic groups present in the enzyme molecules and the amino groups attached to the silica. The enzymatic solid-phase reactor was used for chemical oxidation of phenols in 1, 4-benzoquinone that was then detected by chronoamperometry. The system allowed the analysis of hydroquinone with a detection limit of 83.6 nmol L-1. Thus, the new material has potential in the determination of phenolic compounds river water samples.

Magnetic Properties of Liquid-Phase Sintered CoFe2O4 for Application in Magnetoelastic and Magnetoelectric Transducers

Cobalt ferrite is a ferrimagnetic magnetostrictive ceramic that has potential application in magnetoelastic and magnetoelectric transducers. In this work, CoFe2O4 was obtained using a conventional ceramic method and Bi2O3 was used as additive in order to obtain liquid-phase sintered samples. Bi2O3 was added to the ferrite in amounts ranging from 0.25 mol% to 0.45 mol% and samples were sintered at 900 degrees C and 950 degrees C. It was observed the presence of Bi-containing particles in the microstructure of the sintered samples and the magnetostriction results indicated microstructural anisotropy. It was verified that it is possible to get dense cobalt ferrites, liquid-phase sintered, with relative densities higher than 90% and with magnetostriction values very close to samples sintered without additives.

A Phase I clinical trial of lodenafil carbonate, a new phosphodiesterase Type 5 (PDE5) inhibitor, in healthy male volunteers

Lodenafil carbonate is a new phosphodiesterase Type 5 (PDE5) inhibitor used in treatment of erectile dysfunction. Objective: The present study was conducted to evaluate the safety, tolerability, and pharmacokinetics of lodenafil carbonate after administering ascending (1 - 100 mg) single oral doses to healthy male volunteers (n = 33). Methods: The study was an open-label, dose-escalation, Phase I clinical trial involving the administration of single oral doses of lodenafil carbonate. Lodenafil carbonate was administered sequentially, escalating in single doses of 1 mg - 100 mg with a washout period of at least 1 week between each dose. The progression to the next dose was allowed after clinical and laboratory exams, Ambulatory Monitoring of Arterial Pressure (AMAP) without relevant clinical modifications and adverse events without clinical relevancy. Blood samples were collected at pre-dose, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 14, 16, 20 and 24 h post-dosing. Plasma samples for measurement of lodenafil carbonate and lodenafil were analyzed by liquid chromatography coupled to tandem mass spectrometry. Results: No serious adverse events were observed, and none of the subjects discontinued the study due to intolerance. The AMAP measurements, clinical and laboratory exams and ECG revealed no significant changes even at higher doses. Lodenafil carbonate was not detected in any samples, indicating that it acts as a prodrug. The mean lodenafil pharmacokinetic parameters for t(max) and t(1/2) were 1.6 (+/- 0.4) h and 3.3 (+/- 1.1) h, respectively. This study demonstrated that lodenafil carbonate was well tolerated and showed a good safety profile in healthy male volunteers.

AN EFFICIENT HPLC-UV METHOD FOR THE QUANTITATIVE DETERMINATION OF CEFADROXIL IN HUMAN PLASMA AND ITS APPLICATION IN PHARMACOKINETIC STUDIES

Cefadroxil is a semi-synthetic first-generation oral cephalosporin used in the treatment of mild to moderate infections of the respiratory and urinary tracts, skin and soft tissue infections. In this work a simple, rapid, economic and sensitive HPLC-UV method is described for the quantitative determination of cefadroxil in human plasma samples using lamivudine as internal standard. Sample pre-treatment was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed with a mobile phase consisting of a mixture of sodium dihydrogen phosphate monohydrate solution, methanol and acetonitrile in the ratio of 90:8:2 (v/v/v) at a flow rate of 1.0mL/min. The proposed method is linear between 0.4 to 40.0 mu g/mL and its average recovery is 102.21% for cefadroxil and 97.94% for lamivudine. The method is simple, sensitive, reproducible, less time consuming for determination of cefadroxil in human plasma. The method can therefore be recommended for pharmacokinetics studies, including bioavailability and bioequivalence studies.

An environmentally friendly analytical procedure for nickel determination by atomic and molecular spectrometry after cloud point extraction in different samples

Cloud point extraction (CPE) was employed for separation and preconcentration prior to the determination of nickel by graphite furnace atomic absorption spectrometry (GFAAS), flame atomic absorption spectrometry (FAAS) or UV-Vis spectrophotometry. Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) was used for the first time as a complexing agent in CPE. The nickel complex was extracted from the aqueous phase using the Triton X-114 surfactant. Under optimized conditions, limits of detection obtained with GFAAS, FAAS and UV-Vis spectrophotometry were 0.14, 0.76 and 1.5 mu g L-1, respectively. The extraction was quantitative and the enrichment factor was estimated to be 27. The method was applied to natural waters, hemodialysis concentrates, urine and honey samples. Accuracy was evaluated by analysis of the NIST 1643e Water standard reference material.