Development and Validation of Stability-Indicating HPLC Methods for Quantitative Determination of Pravastatin, Fluvastatin, Atorvastatin, and Rosuvastatin in Pharmaceuticals

High-performance liquid-chromatographic (HPLC) methods were validated for determination of pravastatin sodium (PS), fluvastatin sodium (FVS), atorvastatin calcium (ATC), and rosuvastatin calcium (RC) in pharmaceuticals. Two stability-indicating HPLC methods were developed with a small change (10%) in the composition of the organic modifier in the mobile phase. The HPLC method for each statin was validated using isocratic elution. An RP-18 column was used with mobile phases consisting of methanol-water (60:40, v/v, for PS and RC and 70:30, v/v, for FVS and ATC). The pH of each mobile phase was adjusted to 3.0 with orthophosphoric acid, and the flow rate was 1.0mL/min. Calibration plots showed correlation coefficients (r)0.999, which were calculated by the least square method. The detection limit (DL) and quantitation limit (QL) were 1.22 and 3.08 mu g/mL for PS, 2.02 and 6.12 mu g/mL for FVS, 0.44 and 1.34 mu g/mL for ATC, and 1.55 and 4.70 mu g/mL for RC. Intraday and interday relative standard deviations (RSDs) were 2.0%. The methods were applied successfully for quantitative determination of statins in pharmaceuticals.

Development and validation of high performance liquid chromatographic and UV-derivative spectrophotometric methods for the determination of sotalol hydrochloride in tablets

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.

LC-MS-MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi

The purpose of this study was the development and validation of an LC-MS-MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 x 4.6 mm, 5 mu m particle size), column temperature 8 degrees C, and the mobile phase hexane-isopropanol-trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min(-1). Post-column infusion with 10 mmol L(-1) ammonium acetate in methanol at a flow rate of 0.3 mL min(-1) was performed to enhance MS detection (positive electrospray ionization). Liquid-liquid extraction was used for sample preparation with hexane-ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1-20 mu g mL(-1) for IBP, 0.05-7.5 mu g mL(-1) for each 2-OH-IBP enantiomer, and 0.025-5.0 mu g mL(-1) for each COOH-IBP stereoisomer (r >= 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at -20 degrees C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and p-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22 degrees C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Effect of modifiers for As, Cu and Pb determinations in sugar-cane spirits by GF AAS

Palladium plus magnesium nitrates with and without Ir, Ru and W were evaluated for the simultaneous determination of As, Cu and Pb in cachaca by graphite furnace atomic absorption spectrometry. For 20 mu L sample, 5 mu L Pd(NO(3))(2) and 3 mu L Mg(NO(3))(2) dispensed together onto the Ir-coated platform of the THGA, analytical curves in the 0-30.0 mu g L(-1) As, 0-1.50 mg L(-1) Cu and 0-60.0 mu g L(-1) Pb were built up and typical linear correlation coefficients were always better than 0.999. The limit of detection was 1.30 mu g L(-1) As, 140 mu g L(-1) Cu and 0.90 mu g L(-1) Pb. As, Cu and Pb contents in 10 cachaca samples agreed with those obtained by ICP-MS. Recoveries of spiked samples varied from 96% to 106% (As), 97% to 112% (Cu) and 92% to 108% (Pb). The relative standard deviation (n = 12) was typically 2.7%, 3.3% and 1.9%. (C) 2008 Elsevier Ltd. All rights reserved.

A valuation of patients` willingness-to-pay for insulin delivery in diabetes

Objectives: The aim of this study was to determine the insulin-delivery system and the attributes of insulin therapy that best meet patients` preferences, and to estimate patients` willingness-to-pay (WTP) for them. Methods: This was a cross-sectional discrete choice experiment (DCE) study involving 378 Canadian patients with type 1 or type 2 diabetes. Patients were asked to choose between two hypothetical insulin treatment options made up of different combinations of the attribute levels. Regression coefficients derived using conditional logit models were used to calculate patients` WTP. Stratification of the sample was performed to evaluate WTP by predefined subgroups. Results: A total of 274 patients successfully completed the survey. Overall, patients were willing to pay the most for better blood glucose control followed by weight gain. Surprisingly, route of insulin administration was the least important attribute overall. Segmented models indicated that insulin naive diabetics were willing to pay significantly more for both oral and inhaled short-acting insulin compared with insulin users. Surprisingly, type 1 diabetics were willing to pay $C11.53 for subcutaneous short-acting insulin, while type 2 diabetics were willing to pay $C47.23 to avoid subcutaneous short-acting insulin (p < .05). These findings support the hypothesis of a psychological barrier to initiating insulin therapy, but once that this barrier has been overcome, they accommodate and accept injectable therapy as a treatment option. Conclusions: By understanding and addressing patients` preferences for insulin therapy, diabetes educators can use this information to find an optimal treatment approach for each individual patient, which may ultimately lead to improved control, through improved compliance, and better diabetes outcomes.

Hierarchical determinants of capital structure

We analyze the influence of time-, firm-, industry- and country-level determinants of capital structure. First, we apply hierarchical linear modeling in order to assess the relative importance of those levels. We find that time and firm levels explain 78% of firm leverage. Second, we include random intercepts and random coefficients in order to analyze the direct and indirect influences of firm/industry/country characteristics on firm leverage. We document several important indirect influences of variables at industry and country-levels on firm determinants of leverage, as well as several structural differences in the financial behavior between firms of developed and emerging countries. (C) 2010 Elsevier B.V. All rights reserved.

Neighborhood and Efficiency in Manufacturing in Brazilian Regions: A Spatial Markov Chain Analysis

This paper analyzes the geography of regional competitiveness in manufacturing in Brazil. The authors estimate stochastic frontiers to calculate regional efficiency of representative firms in 137 regions in the period 2000-2006, in four sectors defined by technological intensity. The efficiency results are analyzed using Markov Spatial Transition Matrices to provide insights into the transition of regions between efficiency levels, considering their local spatial context. The results indicate that geography plays an important role in manufacturing competitiveness. In particular, regions with more competitive neighbors are more likely to improve their relative efficiency (pull effect) over time, and regions with less competitive neighbors are more likely to lose relative efficiency (drag effect). The authors find that the pull effect is stronger than the drag effect.

Heterogeneity, saving-investment dynamics and capital mobility in Latin America

The purpose of this paper is to analyze the dynamics of national saving-investment relationship in order to determine the degree of capital mobility in 12 Latin American countries. The analytically relevant correlation is the short-term one, defined as that between changes in saving and investment. Of special interest is the speed at which variables return to the long run equilibrium relationship, which is interpreted as being negatively related to the degree of capital mobility. The long run correlation, in turn, captures the coefficient implied by the solvency constraint. We find that heterogeneity and cross-section dependence completely change the estimation of the long run coefficient. Besides we obtain a more precise short run coefficient estimate compared to the existent estimates in the literature. There is evidence of an intermediate degree of capital mobility, and the coefficients are extremely stable over time.

Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high-performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Separation was obtained using a reverse phase C-18 column with UV detection (210 nm). The mobile phase consisted of water: acetonitrile (78:22, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.08-40.0 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125-40.0 mu g mL(-1) for phenytoin, The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.0, 4.0 and 20.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125 mu g mL(-1) for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. Based on figures of merit results, the SBSE/HPLC-UV proved adequate for antiepileptic drugs analyses from therapeutic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (c) 2008 Elsevier B.V. All rights reserved.

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirrazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50 ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500 ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants. (c) 2007 Elsevier B.V. All rights reserved.

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C(8) column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125-50.0 mu g mL(-1). The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 mu g mL(-1). Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (C) 2009 Elsevier B.V. All rights reserved.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

The nymph of Tortopus harrisi Traver (Ephemeroptera: Polymitarcyidae)

Polymitarcyidae is a family of burrowing mayflies (Ephemeroptera: Ephemeroidea) distributed throughout the world but with highest diversity in the Neotropics. Tortopus Needham & Murphy, with a Panamerican distribution, is known from twelve species described in the adult stage. Nymphs are only known for three species: T. puella (Pictet), T. obscuripennis Dominguez and T. sarae Dominguez, and present a rather homogeneous morphology (Molineri 2008). They were firstly described for T. puella by Scott et al. (1959) and later Molineri (2008) described the other two. Both studies reported that these species burrow U-shaped tunnels in clay banks of rivers and streams, thus preventing them from being sampled in most limnological studies (that use surbers, drags, or drift nets). The aim of the present contribution is to describe and illustrate the previously unknown nymph of Tortopus harrisi Traver that shows important anatomical differences with the other nymphs known in the genus. This morphological differentiation suggests a different habitat use by these nymphs, sampled with drag and surber samplers in sandy substrate. New locality records are given for T. harrisi in Brazil. The nymphs are preserved in alcohol, mouthparts, legs and genital rudiments were mounted in microscope slides with Canada Balsam. Drawings were made with a camera lucida attached to a stereo microscope. The material is deposited in CUIC (Cornell University Insect Collection, Ithaca, NY), IML (Instituto Miguel Lillo, Tucuman) and in MZSP (Museu de Zoologia da Universidade de Sao Paulo, Sao Paulo). Catalogs and bibliography were consulted at Ephemeroptera Galactica (Hubbard 2009).