Eduardo Costa Figueiredo

Analysis of fluoxetine and norfluoxetine in human plasma by liquid-phase microextraction and injection port derivatization GC-MS.

A two-phase liquid phase microextraction using a hollow fiber combined with injection port derivatization and gas chromatographic analysis was developed for extracting and detecting fluoxetine (FLU) and norfluoxetine (NOR) in human plasma. Simultaneous extraction in a multiple tube shaker was used and, afterward, the organic phase was simply injected together with the derivatizing agent n-methyl-bis(trifluoroacetamide) (MBTFA). Factors influencing injection port derivatization, and several extraction parameters were optimized. Under optimal conditions the proposed method provided linearity between 10 and 500ngmL(-1) (R(2)=0.9973) for FLU, and between 15 and 500ngmL(-1) (R(2)=0.9972) for NOR. Intra-assay precision (RSD) between 4.8 and 13.1% and inter-assay between 5.4 and 14.2% were obtained, with detection and quantification limits of 3 and 10ngmL(-1), and of 5 and 15ngmL(-1) for FLU and NOR, respectively, using selected ion monitoring mode. Selectivity, short term stability and extraction efficiency were also evaluated. This method was simple, cheap, and environmentally friendly, yielding significant reduction of solvents and derivatizing agent consumption. The method was successfully applied to the analysis of samples from 5 patients under fluoxetine treatment.

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry.

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb(2+) ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb(2+) adsorption capacity of 34.5mg g(-1). High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb(2+) adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1µg L(-1), an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb(2+) spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum.

Restricted access carbon nanotubes for direct extraction of cadmium from human serum samples followed by atomic absorption spectrometry analysis

In this paper, we propose a new sorbent that is able to extract metal ions directly from untreated biological fluids, simultaneously excluding all proteins from these samples. The sorbent was obtained through the modification of carbon nanotubes (CNTs) with an external bovine serum albumin (BSA) layer, resulting in restricted access carbon nanotubes (RACNTs). The BSA layer was fixed through the interconnection between the amine groups of the BSA using glutaraldehyde as cross-linker. When a protein sample is percolated through a cartridge containing RACNTs and the sample pH is higher than the isoelectric point of the proteins, both proteins from the sample and the BSA layer are negatively ionized. Thus, an electrostatic repulsion prevents the interaction between the proteins from the sample on the RACNTs surface. At the same time, metal ions are adsorbed in the CNTs (core) after their passage through the chains of proteins. The Cd(2+) ion was selected for a proof-of-principle case to test the suitability of the RACNTs due to its toxicological relevance. RACNTs were able to extract Cd(2+) and exclude almost 100% of the proteins from the human serum samples in an online solid-phase extraction system coupled with thermospray flame furnace atomic absorption spectrometry. The limits of detection and quantification were 0.24 and 0.80 μg L(-1), respectively. The sampling frequency was 8.6h(-1), and the intra- and inter-day precisions at the 0.80, 15.0, and 30.0 μg L(-1) Cd(2+) levels were all lower than 10.1% (RSD). The recoveries obtained for human blood serum samples fortified with Cd(2+) ranged from 85.0% to 112.0%. The method was successfully applied to analyze Cd(2+) directly from six human blood serum samples without any pretreatment, and the observed concentrations ranged from

Molecularly imprinted pipette-tip solid phase extraction for selective determination of fluoroquinolones in human urine using HPLC-DAD.

A simple method using HPLC-DAD was developed for the determination of fluoroquinolones in human urine including ciprofloxacin (CIPRO), enrofloxacino (ENRO), marbofloxacino (MARBO) and norfloxacin (NOR). In addition, it was studied the extraction of fluoroquinolones in human urine samples using pipette tip-based molecularly imprinted polymers solid phase extraction (PT-MIPs-SPE). With the goal of finding the best procedure for extraction of four fluoroquinolones in human urine, several parameters that are likely to affect the efficiency of extraction during sample preparation, including the washing solvent, type and volume of eluent, amount of material, the volume of the sample, pH and the ionic strength were systematically optimized. Chromatographic separations of fluoroquinolones were hit within 10min using a Synergi(®) C18 (250×4.6mm, 4μm) column and mobile phase consisting of water (10mM of phosphoric acid, the pH adjusted at 3.29 with triethylamine) : acetonitrile (85.7: 14.3, v/v) at a flow rate of 1.5mLmin(-1). Detection was performed at 290nm. The average extraction recoveries/standard deviation relative to ENRO, CIPRO, NOR and MARBO were 96.40±5.51%, 42.47±4.81%, 41.82±7.99% and 87.49±4.70, respectively. The method was liner from 39 to 1260ngmL(-1) for each fluoroquinolone with correlation coefficient of 0.9904, 0.9910, 0.9914 and 0.9919, to ENRO, CIPRO, NOR and MARBO, respectively. The assays of within-day and between-day precision and accuracy for all analytes were studied at three concentration levels and were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of CIPRO to a healthy volunteer.

Molecularly imprinted solid phase extraction of urinary diethyl thiophosphate and diethyl dithiophosphate and their analysis by gas chromatography–mass spectrometry

An analytical method involving molecularly imprinted solid phase extraction (MISPE) and gas chromatography-mass spectrometry (GC-MS) was developed for the analysis of organophosphates metabolites (diethyl thiophosphate--DETP and diethyl dithiophosphate - DEDTP) in human urine samples. A DETP molecularly imprinted polymer (MIP) was synthesized using 4-vinylpiridine as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The conditioning step of the MISPE was conducted by running 3 mL of acetonitrile, 3 mL of 0.1 mol L⁻¹ dibasic phosphate buffer at pH 11 and 2 mL of water through the molecularly imprinted polymer (MIP) cartridge. The extraction step was executed using 1.0 mL of a urine sample, with the pH previously adjusted to 3.0. Finally, the analytes were eluted with 3 mL of acetonitrile and derivatized with 3% 2,3,4,5,6-pentafluorobenzyl bromide solution at room temperature for 1h. The sample was analyzed by GC-MS in the SIM (selected ion monitoring) mode. Analytical calibration curves for DETP and DEDTP were constructed using a pool of urine samples and six levels of concentration. The method was found to be linear from 10 to 500 μg L⁻¹ (r>0.99) with limits of quantification of 10 μg L⁻¹ for both analytes. The within-day and between-day precisions were evaluated (as %RSD) and all the results were <15% for both analytes. The method was accurate (relative error<±15%), with good robustness.

On-line restricted access molecularly imprinted solid phase extraction of ivermectin in meat samples followed by HPLC-UV analysis.

A new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) was synthesized, characterized and used for direct analysis of ivermectin from bovine meat samples, in a two-dimensional liquid chromatography system with UV detection. Ivermectin, 4-vynilpiridine and ethylene glycol dimethacrylate were employed as template, functional monomer and cross-linker, respectively. A BSA layer was cross-linked around the polymer, resulting in a biocompatible chemical barrier able to eliminate about 100% of protein from the samples. Ivermectin was extracted from the minced meat samples through a solvent extraction using methanol:water (70:30, v:v), and the extracts were directly injected into the two-dimensional liquid chromatography system, without any other treatment. Samples, fortified with ivermectin from 50 to 500 μg kg(-1), were used to build the analytical calibration curve (r=0.996). The limit of quantification was 50 μg kg(-1). Precision and accuracy presented variation coefficients, as well as relative errors lower than 17.0% and within -18.5% and 22.0%, respectively.

Development of pipette tip-based on molecularly imprinted polymer micro-solid phase extraction for selective enantioselective determination of (−)-(2S,4R) and (+)-(2R,4S) ketoconazole in human urine samples prior to HPLC-DAD

In this work a simple and selective sample preparation technique employing pipette tip-based on molecularly imprinted polymer micro-solid phase extraction (PT-MIP-μ-SPE) coupled to HPLC/DAD was developed for the determination of the cis-enantiomers of ketoconazole (KTZ) in human urine samples. Several parameters affecting the proposed PT-MIP-μ-SPE technique were investigated, namely the washing solvent, type and volume of the eluent, amount of material (molecularly imprinted polymer, MIP), sample volume, pH and salt addition. The recoveries were around 100% using 5 μg mL−1 for the (−)-(2S,4R) and (+)-(2R,4S) KTZ enantiomers. Optimum chromatographic conditions for determination of the (−)-(2S,4R) and (+)-(2R,4S)-KTZ enantiomers were found using the chiral stationary phase polysaccharide derived of amylose tris(3,5-dimethylphenylcarbamate). The mobile phase consisted of ethanol:water (95:5, v/v) at a flow rate of 1 mL min−1 and the detection was performed at 250 nm. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, robustness and stability were assessed and they were within the recommended guidelines. The method showed to be linear over the concentration range from 6.25 to 650.00 ng mL−1 for each enantiomer, with the correlation coefficients of 0.9962 and 0.9952 for (+)-(2R,4S)-KTZ and (−)-(2S,4R)-KTZ, respectively. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. The results for the application of the method in preliminary urinary excretion showed that there are not significant differences in the analyzed concentrations of KTZ cis-enantiomers in urine samples.

Efficient molecularly imprinted polymer as a pipette-tip solid-phase sorbent for determination of carvedilol enantiomers in human urine

In this work, an efficient pipette tip based on molecularly imprinted polymers solid-phase extraction (PT-MIP-SPE) method was developed for carvedilol (CAR) analysis. This compound is available in clinical practice as a racemic mixture, in which (-)-(S)-CAR is a β- and α1-adrenergic antagonist, while (+)-(R)-CAR only acts as an α1-adrenergic antagonist. Enantioseparation of CAR presented satisfactory retention times (5.85 and 14.84min), acceptable theoretical plates (N=2048 and 2018) and good resolution (Rs=9.27). The separation was performed using a Chiralpak® IA column (100mm×4.6mm, 3μm), a mixture of methanol:ethanol:water (64:15:21, v/v/v) plus 0.3% diethylamine as mobile phase, temperature of 35°C and flow rate of 1.5mLmin-1. After density functional theory calculations based on prepolymerization complexes, the best protocol for the MIP synthesis was chosen. Then, some parameters that affect the PT-MIP-SPE technique were investigated. After optimization, the best conditions were 300μL of water as washing solvent, 500μL of acetonitrile:acetic acid (7:3, v/v) as eluting solvent, 20mg of MIP, 500μL of urine sample (pH 12.5) and no addition of NaCl. Recoveries±relative standard deviation (RSD%) for (+)-(R)-CAR and (-)-(S)-CAR were 101.9±4.8% and 104.6±2.1%, respectively. The method was linear over the concentration range from 20 to 1280ngmL-1 for each enantiomer, with correlation coefficients larger than 0.99 for both enantiomers. The method was applied successfully in a preliminary study of urinary excretion after administration of CAR racemate to a healthy volunteer.

In vitro evaluation of transdermal nicotine delivery systems commercially available in Brazil

The aim of this study was to develop and validate a method for evaluating the release and skin permeation from transdermal nicotine patches using the vertical diffusion cell (VDC). The VDC is an experimental apparatus employed in research, development, and the pharmaceutical field because it can simulate conditions closest to those established in clinical trials. Two transdermal nicotine delivery systems marketed in Brazil to release 14 mg over 24 hours were evaluated. Release studies were carried out using a regenerated cellulose dialysis membrane and permeation studies were carried out using excised porcine ear skin. The results indicated that nicotine release from both evaluated patches follows Higuchis release kinetics, while skin permeation studies indicated zero-order release kinetics. Nicotine release rates were different between both evaluated patches, but drug permeation rates were not significantly different. According to validation studies, the method was appropriate for evaluating in vitro performance of nicotine patches. The proposed method can be applied to in vitro comparative studies between different commercial nicotine patches and may be used as an auxiliary tool in the design of new transdermal nicotine delivery systems.

Solid-phase extraction of triazole fungicides from water samples using disks impregnated with carbon nanotubes followed by GC-MS analysis

ABSTRACT Triazole fungicides are pesticides widely employed in the cultivation of fruits, vegetables and grains. However, their ability to change the steroid hormone biosynthesis may result in endocrine complications for mammals, as well as changes in cholesterol and triglyceride levels and hepatotoxicity. The analysis of the triazole fungicides in superficial waters is important in order to monitor the risk for the biota. However, the use of efficient extraction procedures has been necessary in order to concentrate these pesticides before the analysis. In-disk solid-phase extraction (SPE) can be highlighted as a potential pre-concentration technique, mainly because the possibility to extract the analytes from a large sample volume, increasing the method detectability. Carbon nanotubes (CNTs) have been often used as solid extraction phase due to their high sorption capacity, surface area and internal volume, as well as mechanical, chemical and thermal stability. In this paper, we proposed the preparation of a new SPE disk impregnated with CNTs for the extraction of triazole fungicides from environmental water samples. The disks were obtained by acid corrosion of a cellulose membrane followed by its impregnation with CNTs. The developed method was validated for the analysis of triadimenol, tebuconazole and epoxiconazole, according to international validation protocols. The limits of quantification obtained for triadimenol, tebuconazole and epoxiconazole were 0.1, 0.1 and 0.05 µg L−1, respectively. The linearity ranged from 0.05 to 10.00 µg L−1 for epoxiconazole and from 0.1 to 10.00 µg L−1 for triadimenol and tebuconazole, with correlation coefficients higher than 0.999 for all of them. The precisions, expressed as relative standard deviation, were lower than 12%. The accuracies were within −12.07% to 17.7% (expressed as relative error).