The respiratory mechanics of the yacare caiman (Caiman yacare).

The structure and function of crocodilian lungs are unique compared with those of other reptiles. We examined the extent to which this and the semi-aquatic lifestyle of crocodilians affect their respiratory mechanics. We measured changes in intratracheal pressure in adult and juvenile caiman (Caiman yacare) during static and dynamic lung volume changes. The respiratory mechanics of juvenile caiman were additionally measured while the animals were floating in water and submerged at 30, 60 and 90 deg to the water's surface. The static compliance of the juvenile pulmonary system (2.89±0.22 ml cmH2O-1 100 g-1) was greater than that of adults (1.2±0.41 ml cmH2O-1 100 g-1), suggesting that the system stiffens as the body wall becomes more muscular and keratinized in adults. For both age groups, the lungs were much more compliant than the body wall, offering little resistance to air flow (15.35 and 4.25 ml cmH2O-1 100 g-1 for lungs, versus 3.39 and 1.67 ml cmH2O-1 100 g-1 for body wall, in juveniles and adults, respectively). Whole-system dynamic mechanics decreased with increasing ventilation frequency (fR), but was unaffected by changes in tidal volume (VT). The vast majority of the work of breathing was required to overcome elastic forces; however, work to overcome resistive forces increased proportionally with fR Work of breathing was higher in juvenile caiman submerged in water at 90 deg because of an increase in work to overcome both elastic and flow resistive forces. The lowest power of breathing was found to occur at high fR and low VT for any given minute ventilation (V̇E) in caiman of all ages.

Dealing with Hydrogen Bonding on the Conformational Preference of 1,3-Aminopropanols: Experimental and Molecular Dynamics Approaches.

This study expands the knowledge on the conformational preference of 1,3-amino alcohols in the gas phase and in solution. By employing Fourier transform infrared spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT) calculations, quantum theory of atoms in molecules (QTAIM), natural bond orbital (NBO) analysis, and molecular dynamics (MD), the compounds 3-aminopropan-1-ol (1), 3-methylaminopropan-1-ol (2), and 3-dimethylaminopropan-1-ol (3) are evaluated. The results show that the most stable conformation of each compound in the gas phase and in nonpolar solvents exhibited an O-H···N intramolecular hydrogen bond (IHB). Based on the experimental and theoretical OH-stretching frequencies, the IHB becomes stronger from 1 to 3. In addition, from the experimental NMR J-couplings, the IHB conformers are predominant in nonbasic solvents, representing 70-80% of the conformational equilibrium, while in basic solvents, such conformers only represent 10%. DFT calculations and QTAIM analysis in the gas phase support the occurrence of IHBs in these compounds. The MD simulation indicates that the non-hydrogen-bonded conformers are the lowest energy conformations in the solution because of molecular interactions with the solvent, while they are absent in the implicit solvation model based on density. NBO analysis suggests that methyl groups attached on the nitrogen atom affect the charge transfer energy involved in the IHB. This effect occurs mostly because of a decrease in the s-character of the LPN orbital along with weakening of the charge transfer from LPN to σ*OH, which is caused by an increase in the C-C-N bond angle.

Trends of intramolecular hydrogen bonding in substituted alcohols: a deeper investigation.

Intramolecular hydrogen bonding (IAHB) is one of the most important intramolecular interactions and a critical element in adopted molecular arrangements. Moreover, slight substitution in a molecule can affect its strength to a great extent. It is well established that alkyl groups play a positive role in IAHB strength. However, the effects that drive it are specific to each system. To investigate the influence of IAHB and its strength dependency on different acyclic compounds, the conformational preferences of propane-1,3-diol, 3-methoxypropan-1-ol, 3-ethoxypropan-1-ol, 3-isopropoxypropan-1-ol, 3-(tert-butoxy)propan-1-ol, butane-1,3-diol, 3-methoxybutan-1-ol, 3-methylbutane-1-diol, and 3-methoxy-3-methylbutan-1-ol were evaluated experimentally using infrared spectroscopy theoretically supported by topological and natural bond orbital analyses. The most stable conformation of each compound exhibited IAHB and these conformers are more populated in the equilibrium for all studied compounds. Experimental infrared and topological data suggest that the strength of IAHB increases for each methyl group addition. NBO analyses indicate that methyl groups in different positions related to an OH moiety affect the charge transfer energy involved in intramolecular hydrogen bonding. This occurs mostly due to an increase in the spn-hybridized lone pair (LP1O) contribution to the charge transfer , which is a result of changes in s-character and orbital energy caused by geometrical rearrangements, rehybridization, and/or electronic effects.

Genetic guidelines for captive breeding and reintroductions of the endangered Black-fronted Piping Guan, Aburria jacutinga (galliformes, cracidae), an Atlantic Forest endemic.

The survival of a number of birds rely on captive breeding and reintroduction into the wild, but captive populations are often small and can be exposed to the negative effects of inbreeding and genetic drift. Then, managers are concerned not only with producing as much offspring as possible, but also with the retention of the maximum genetic variability within and between populations. The Black-fronted Piping Guan, Aburria jacutinga, is an endangered cracid endemic to the Atlantic Forest of southeastern South America. Because of its conservation status and functional importance, a captive breeding program started independently, mainly in three aviaries, in the decade of 1980. Although they have supplied animals for reintroductions, genetic variability aspects have never been considered. Here we addressed levels of genetic variability within and between these aviaries. Bayesian clustering analyses revealed two lineages. Inbreeding was not detected, although we found evidences for a recent bottleneck in one of the aviaries. Then, our main management recommendations are: i) reintroducing the species in areas where it has been extinct is more prudent than supplementing natural populations, as it could involve risks of disrupting local adaptive complexes; ii) as far as inbreeding can be avoided, the captive groups should be managed separately to minimize adaptation to captivity; iii) crossbreedings in pre-release generations could improve reintroduction success; and iv) a studbook should be implemented. As populations of Black-fronted Piping Guan from conservation units are progressively declining, these captive genetic repositories may gain importance in a near future. Zoo Biol. 35:313-318, 2016. © 2016 Wiley Periodicals, Inc.

Nanostructures of Prussian blue supported on activated biochar for the development of a glucose biosensor.

This work emphasizes the utilization of biochar, a renewable material, as an interesting platform for anchoring redox mediators and bioreceptors in the development of economic, environmentally friendly biosensors. In this context, Fe(III) ions were preconcentrated on highly functionalized activated biochar, allowing the stable synthesis of Prussian blue nanostructures with an average size of 58.3 nm. The determination of glucose was carried out by indirectly monitoring the hydrogen peroxide generated through the enzymatic reaction, followed by its subsequent redox reaction with reduced Prussian blue (also known as Prussian white) in a typical electrochemical-chemical mechanism. The EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-Hydroxysuccinimide) pair was employed for the stable covalent immobilization of the enzyme on biochar. The biosensor demonstrated good enzyme-substrate affinity, as evidenced by the Michaelis-Menten apparent kinetic constant (4.16 mmol L-1), and analytical performance with a wide linear dynamic response range (0.05-5.0 mmol L-1), low limits of detection (0.94 µmol L-1) and quantification (3.13 µmol L-1). Additionally, reliable repeatability, reproducibility, stability, and selectivity were obtained for the detection of glucose in both real and spiked human saliva and blood serum samples.

How to Improve Sustainability in Fused Filament Fabrication (3D Printing) Research?

This review aims to provide an overview of sustainable approaches that can be incorporated into well-known procedures for the development of materials, pre- and post-treatments, modifications, and applications of 3D-printed objects, especially for fused filament fabrication (FFF). Different examples of conductive and non-conductive bespoke filaments using renewable biopolymers, bioplasticizers, and recycled materials are presented and discussed. The main final characteristics of the polymeric materials achieved according to the feedstock, preparation, extrusion, and treatments are also covered. In addition to recycling and remanufacturing, this review also explores other alternative approaches that can be adopted to enhance the sustainability of methods, aiming to produce efficient and environmentally friendly 3D printed products. Adjusting printing parameters and miniaturizing systems are also highlighted in this regard. All these recommended strategies are employed to minimize environmental damage, while also enabling the production of high-quality, economical materials and 3D printed systems. These efforts align with the principles of Green Chemistry, Sustainable Development Goals (SDGs), 3Rs (Reduce, Reuse, Recycle), and Circular Economy concepts.

Solid-state characterization and dissolution properties of Fluvastatin sodium salt hydrates.

The present study reports the solid-state properties of Fluvastatin sodium salt crystallized from different solvents for comparison with crystalline forms of the commercially available raw material and United States Pharmacopeia (USP) reference standard. Fluvastatin (FLV) samples were characterized by several techniques; such as X-ray powder diffractometry, differential scanning calorimetry, thermogravimetry, liquid and solid-state nuclear magnetic resonance spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy, and scanning electron microscopy. In addition, intrinsic dissolution rate (IDR) of samples was performed in order to study the influence of crystalline form and other factors on rate and extent of dissolution. Three different forms were found. The commercial raw material and Fluvastatin-Acetonitrile (ACN) were identified as "form I" hydrate, the USP reference standard as "form II" hydrate and an ethanol solvate which presented a mixture of phases. Form I, with water content of 4%, was identified as monohydrate.

What is the traditional method of resistance training: a systematic review.

INTRODUCTION: Many resistance studies state that they used the traditional method of resistance training in the intervention. However, there is a wide difference on the characteristics of the training protocols used even though they are labeled as "the traditional method." There is no clear definition and characteristics for the traditional method of resistance training. The aim of this study was to describe the most common definitions and references, and also the main characteristics of the training variables of the studies using the traditional training method for strengthening. EVIDENCE ACQUISITION: Searches were carried out in Pubmed, Embase, SPORTDiscus and Web of Science. We included randomized controlled trials that included a strengthening program using the "traditional method" and that evaluated hypertrophy and/or maximum strength in healthy individuals. EVIDENCE SYNTHESIS: The initial search resulted in 26,057 studies, but only 39 studies were eligible and included in this review. The common characteristics of the traditional training protocol were frequency of 3 sessions/week, 3 sets of 9 repetitions, with weight =75% 1RM. The movement time was 2±1 seconds for the concentric and for the eccentric phases. Resting time between sets was 2±1 minutes. The concepts used to define the method as traditional and the characteristics of the intervention protocols were different. The American College of Sports Medicine (ACSM) was the most cited reference. CONCLUSIONS: The "traditional method of resistance training" can be defined as: "Three (±1) sets of 9±6 repetitions of concentric and eccentric exercises using an external load of 75±20% of one maximum repetition, completed 3±1 times/week.

Determination of lumiracoxib by a validated stability-indicating MEKC method and identification of its degradation products by LC-ESI-MS studies.

A stability-indicating MEKC method was developed and validated for the analysis of lumiracoxib (LMC) in pharmaceutical formulations using nimesulide as the internal standard (IS). Optimal conditions for the separation of LMC and degradation products were investigated. The method employed 50 mM borate buffer and 50 mM anionic detergent SDS solution at pH 9.0. MEKC method was performed on a fused-silica capillary (50 µm id; effective length, 40 cm) maintained at 30°C. The applied voltage was 20 kV and photodiode array (PDA) detector was set at 208 nm. The method was validated in accordance with the International Conference on Harmonisation requirements. The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using PDA detection. The degradation products formed under stressed conditions were investigated by LC-ESI-MS and the two degraded products were identified. MEKC method was linear over the concentration range of 5-150 µg/mL (r(2) =0.9999) of LMC. The method was precise, accurate, with LOD and LOQ of 1.34 and 4.48 µg/mL, respectively. The robustness was proved by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of LMC in tablets to support the quality control.

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability-indicating MEKC method.

A stability-indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused-silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5-100 and 60-1200 µg/mL for HCTZ and ALI, respectively (r(2) >0.9997). The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.

High-performance column liquid chromatographic method for the simultaneous determination of buclizine, tryptophan, pyridoxine, and cyanocobalamin in tablets and oral suspension.

An HPLC method was developed and validated for the simultaneous determination of buclizine, tryptophan, pyridoxine, and cyanocobalamin in pharmaceutical formulations. The chromatographic separation was carried out on an RP-C18 column using a mobile phase gradient of methanol, 0.015 M phosphate buffer (pH 3.0), and 0.03 M phosphoric acid at a flow rate of 1.0 mL/min and UV detection at 230, 280, and 360 nm, respectively, for buclizine, tryptophan, pyridoxine, and cyanocobalamin. The method validation yielded good results with respect to linearity (r>0.999), specificity, precision, accuracy, and robustness. The RSD values for intraday and interday precision were below 1.82 and 0.63%, respectively, and recoveries ranged from 98.11 to 101.95%. The method was successfully applied for the QC analysis of buclizine, tryptophan, pyridoxine, and cyanocobalamin in tablets and oral suspension.

Comparison between constant and decreasing rest intervals: influence on maximal strength and hypertrophy.

Most resistance training programs use constant rest period lengths between sets and exercises, but some programs use decreasing rest period lengths as training progresses. The aim of this study was to compare the effect on strength and hypertrophy of 8 weeks of resistance training using constant rest intervals (CIs) and decreasing rest intervals (DIs) between sets and exercises. Twenty young men recreationally trained in strength training were randomly assigned to either a CI or DI training group. During the first 2 weeks of training, 3 sets of 10-12 repetition maximum (RM) with 2-minute rest intervals between sets and exercises were performed by both groups. During the next 6 weeks of training, the CI group trained using 2 minutes between sets and exercises (4 sets of 8-10RM), and the DI group trained with DIs (2 minutes decreasing to 30 seconds) as the 6 weeks of training progressed (4 sets of 8-10RM). Total training volume of the bench press and squat were significantly lower for the DI compared to the CI group (bench press 9.4%, squat 13.9%) and weekly training volume of these same exercises was lower in the DI group from weeks 6 to 8 of training. Strength (1RM) in the bench press and squat, knee extensor and flexor isokinetic measures of peak torque, and muscle cross-sectional area (CSA) using magnetic resonance imaging were assessed pretraining and posttraining. No significant differences (p < or = 0.05) were shown between the CI and DI training protocols for CSA (arm 13.8 vs. 14.5%, thigh 16.6 vs. 16.3%), 1RM (bench press 28 vs. 37%, squat 34 vs. 34%), and isokinetic peak torque. In conclusion, the results indicate that a training protocol with DI is just as effective as a CI protocol over short training periods (6 weeks) for increasing maximal strength and muscle CSA; thus, either type of program can be used over a short training period to cause strength and hypertrophy.

Additive-manufactured (3D-printed) electrochemical sensors: A critical review.

Additive manufacturing or three-dimensional (3D)-printing is an emerging technology that has been applied in the development of novel materials and devices for a wide range of applications, including Electrochemistry and Analytical Chemistry areas. This review article focuses on the contributions of 3D-printing technology to the development of electrochemical sensors and complete electrochemical sensing devices. Due to the recent contributions of 3D-printing within this scenario, the aim of this review is to present a guide for new users of 3D-printing technology considering the required features for improved electrochemical sensing using 3D-printed sensors. At the same time, this is a comprehensive review that includes most 3D-printed electrochemical sensors and devices already reported using selective laser melting (SLM) and fused deposition modeling (FDM) 3D-printers. The latter is the most affordable 3D-printing technique and for this reason has been more often applied for the fabrication of electrochemical sensors, also due to commercially-available conductive and non-conductive filaments. Special attention is given to critically discuss the need for the surface treatment of FDM 3D-printed platforms to improve their electrochemical performance. The insertion of biochemical and chemical catalysts on the 3D-printed surfaces are highlighted as well as novel strategies to fabricate filaments containing chemical modifiers within the polymeric matrix. Some examples of complete electrochemical sensing systems obtained by 3D-printing have successfully demonstrated the enormous potential to develop portable devices for on-site applications. The freedom of design enabled by 3D-printing opens many possibilities of forthcoming investigations in the area of analytical electrochemistry.

Peroxiredoxin I is overexpressed in oncocytic lesions of salivary glands.

BACKGROUND: Oncocytic lesions, particularly frequent in the salivary glands, are characterized by cells with an atypical accumulation of mitochondria. This accumulation has been recognized as a compensatory mechanism to intrinsic functional defects of these organelles, resulting in energy production impairment and increased generation of reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)). Peroxiredoxin I (Prx I) is a H(2)O(2) scavenging protein and the expression of its yeast homolog was reported to be influenced by mitochondrial function. METHODS: In this study, we evaluated Prx I expression in oncocytic lesions of salivary glands by immunohistochemistry. RESULTS: Our results showed that Prx I is overexpressed in oncocytes regardless of the salivary gland lesion where they appear. CONCLUSIONS: These results suggest that Prx I expression in oncocytes is related to its ability to decompose mitochondrial-derived H(2)O(2) and that it could provide to the cells a protective role in an environment that, by continuously producing potential DNA-damaging ROS, predisposes to genome instability and cellular transformation.

Solid-State Characterization and Compatibility Studies of Penciclovir, Lysine Hydrochloride, and Pharmaceutical Excipients.

The physical and chemical characterization of the solid-state properties of drugs and excipients is fundamental for planning new formulations and developing new strategies for the treatment of diseases. Techniques such as differential scanning calorimetry, thermogravimetry, X-ray powder diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy are among the most commonly used techniques for these purposes. Penciclovir and lysine are individually used to treat the herpes virus. As such, the development of a formulation containing both drugs may have therapeutic potential. Solid-state characterization showed that both penciclovir and lysine were crystalline materials with melting points at 278.27 °C and 260.91 °C, respectively. Compatibility studies of penciclovir and lysine indicated a possible interaction between these substances, as evidenced by a single melting point at 253.10 °C. The compatibility of several excipients, including ethylenediaminetetraacetic acid, cetostearyl alcohol, sodium lauryl sulphate, di-tert-butyl methyl phenol, liquid petrolatum, methylparaben, nonionic wax, paraffin, propylene glycol, and propylparaben, was evaluated in ternary (penciclovir-lysine-excipient) mixtures (1:1:1, w/w/w) to determine the optimal formulation. The developed formulation was stable under accelerated and ambient conditions, which demonstrated that the interaction between penciclovir and lysine was suitable for the development of a formulation containing both drugs.

Green method for glucose determination using microfluidic device with a non-enzymatic sensor based on nickel oxyhydroxide supported at activated biochar.

This paper reports the use of nickel ions supported at activated biochar carbon paste electrode (NiAB-CPME) coupled in a microfluidic thread-based electroanalytical device (µTED) for non-enzymatic glucose determination. Biochar was initially prepared from castor oil cake at 400 °C and activated by HNO3 refluxing. Activation process promoted an increase of functional groups, surface area and porosity in comparison to precursor biochar. Activated biochar (AB) has shown an excellent performance to spontaneous preconcentration of Ni(II) ions. In alkaline conditions a stable voltammetric profile associated to Ni(OH)2/NiOOH redox pair was verified and a significant catalytic effect was observed in presence of glucose which was used for its monitoring. Microfluidic device was assembled at a plastic platform printed using 3D printer being easy to construction using low cost materials. Non-enzymatic amperometric glucose sensor coupled in µTED showed a good repeatability of 3.84% for successive injections of glucose (n = 10), a constant flow rate of 1.11 µL s-1 and an analytical frequency of 61 injections per hour. A linear dynamic range (LDR) from 5.0 to 100.0 µmol L-1, limit of detection (LOD) of 0.137 µmol L-1 and limit of quantification (LOQ) 0.457 µmol L-1 glucose were obtained. The proposed device was applied to glucose determination in real biological samples of human saliva and blood serum. Finally, the method was considered a green analytical procedure with Eco-Scale score of 81.

Solid-State Characterization of Different Crystalline Forms of Sitagliptin.

Sitagliptin is an inhibitor of the enzyme dipeptidyl peptidase-4, used for the treatment of type 2 diabetes mellitus. The crystal structure of active pharmaceutical solids determines their physical and chemical properties. The polymorphism, solvates and hydrates can influence the free energy, thermodynamic parameters, solubility, solid-state stability, processability and dissolution rate, besides directly affecting the bioavailability. Thus, the physicochemical characterization of an active pharmaceutical ingredient is required to guarantee the rational development of new dosage forms. In this context, we describe herein the solid-state characterization of three crystalline forms of sitagliptin: sitagliptin phosphate monohydrate, sitagliptin phosphate anhydrous and sitagliptin base form. The investigation was carried out using differential scanning calorimetry (DSC), thermogravimetry (TG)/derivative thermogravimetry (DTG), spectroscopic techniques, X-ray powder diffraction (XRPD) and morphological analysis by scanning electron microscopy. The thermal analysis revealed that during the dehydration of sitagliptin phosphate monohydrate (Tpeak = 134.43 °C, ΔH = -1.15 J g-1) there is a characteristic crystalline transition event, which alters the physicochemical parameters of the drug, such as the melting point and solubility. The crystalline behavior of sitagliptin base form differs from that of sitagliptin phosphate monohydrate and sitagliptin phosphate anhydrous, mainly with regard to the lower temperature of the fusion event. The melting point (Tpeak) values obtained were 120.29 °C for sitagliptin base form, 206.37 °C for sitagliptin phosphate monohydrate and 214.92 °C for sitagliptin phosphate anhydrous. In relation to the thermal stability, sitagliptin phosphate monohydrate and sitagliptin phosphate anhydrous showed a slight difference; however, both are more thermostable than the base molecule. Therefore, through this study it was possible to establish the most suitable crystalline form of sitagliptin for the development of a safe, effective and appropriate pharmaceutical dosage form.

Conformational equilibria of trans -3-X-cyclohexanols (X = Cl, Br, CH3 and OCH3). A low temperature NMR study and theoretical calculations.

The integration of 1H and 13C NMR spectra, at - 90 degrees C in CS2/CD2Cl2 (9:1), for the trans-3-chlorocyclohexanol (1), trans-3-bromocyclohexanol (2), and trans-3-methoxycyclohexanol (4) showed that the equatorial-axial (ea) conformer occurs as ca 63, 63, and 69% in the conformational equilibrium, respectively. This corresponds to the following DeltaG(ea-ae) values (from (1)H spectrum): - 0.32 +/- 0.01, - 0.32 +/- 0.04, - 0.48 +/- 0.05 kcal mol(-1); and to (from 13C spectrum): - 0.31 +/- 0.04, - 0.35 +/- 0.05, and - 0.44 +/- 0.01 kcal mol(-1), respectively, in very good agreement within both series. Thus, although bromine is bulkier than chlorine, the 1,3-diaxial steric effects are similar in these equilibria. However, the integration of (1)H NMR spectrum for the trans-3-methylcyclohexanol (3) gave 90% of the 3ae conformer in the equilibrium, at - 90 degrees C on CS2/CD2Cl2 (9:1), corresponding to a DeltaG(ea-ae) value of 1.31 +/- 0.02 kcal mol(-1). The values obtained through the additivity rule, with data from monosubstituted cyclohexanes (DeltaG(Ad) = DeltaG(X) + DeltaG(OH)), for compounds 1, 2, and 4 (-0.37 +/- 0.15, - 0.34 +/- 0.09, and - 0.46 +/- 0.04 kcal mol(-1), respectively) are in very good agreement with the experimental values, but it is significantly smaller for compound 3 (0.79 +/- 0.02 kcal mol(-1)). Theoretical calculations through different levels of theory (HF/6-311 + g**, B3LYP/6-311 + g**, MP2/6-31 + g**, and CBS-4M) showed that CBS-4M is the best method for the study of conformational equilibria for these systems, since it provides DeltaG(ea-ae) values similar to the experimental values.

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.

Validation of the normocythemic mice bioassay for the potency evaluation of recombinant human erythropoietin in pharmaceutical formulations.

The normocythemic mice bioassay was validated for the potency evaluation of the recombinant human erythropoietin (rhEPO) against the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The bioassays were performed in 8-week-old female BALB/c mice, which received multiple daily injections of standard or sample solutions (3 + 3), for 4 days. The blood sampling was performed 24 h after the last injection and the reticulocytes were counted by automated flow cytometry. Method validation investigated parameters such as linearity, precision, accuracy, specificity, and robustness, giving results within the acceptable range. The dose-response curve was linear in the concentration range of 1-64 international units (IU)/mL, and the value of the determination coefficient (r2) was 0.9708. The bioassay was applied for the potency evaluation of rhEPO pharmaceutical products containing alfa or beta forms, expressed in different cell lines, giving biological potencies within 82.79 and 119.70% of the stated potency. The precision index calculated by the weight for the independent assays was >247. The results demonstrated the validity of the bioassay for the potency assessment of pharmaceutical formulations contributing to ensure the therapeutic efficacy of the biological medicine.