Quantification of beta-lactam antibiotics in veterinary drugs: amoxicillin and ampicillin determination by high performance liquid chromatography

This work focused on the development and validation of an RP-HPLC-UV method for quantification of beta-lactam antibiotics in three pharmaceutical samples. Active principles analyzed were amoxicillin and ampicillin, in 3 veterinary drugs. Mobile phase comprised 5 mmol L-1 phosphoric acid solution at pH 2.00, acetonitrile with gradient elution mode and detection wavelength at 220 nm. The method was validated according to the Brazilian National Health Surveillance regulation, where linear range and linearity, selectivity, precision, accuracy and ruggedness were evaluated. Inter day precision and accuracy for pharmaceutical samples 1, 2 and 3 were: 1.43 and 1.43%; 4.71 and 3.74%; 2.72 and 1.72%, respectively, while regression coefficients for analytical curves exceeded 0.99. The method had acceptable merit figure values, indicating reliable quantification. Analyzed samples had active principle concentrations varying from -12 to +21% compared to manufacturer label claims, rendering the medicine unsafe for administration to animals.

Bioanalytical method for the quantification of the monastrol derivative anticancer candidate lasom 65 in pre-clinical pharmacokinetic investigations

A simple HPLC/UV method was developed for the determination of the anticancer candidate LaSOM 65 in rat plasma. Samples were cleaned by protein precipitation with acetonitrile (recovery > 95%), after which they were subjected to chromatography under the isocratic elution of an acetonitrile:water (45:55, ν/ν) solution with detection at 303 nm. The method was linear (r² > 0.98) over the concentration range (0.05-2 µg mL-1) with intra- and inter-day precision ranging from 9.6% to 13.6% and 4.3% to 5.4%, respectively. The accuracy of the method ranged from 85% to 113.6%, and it showed sufficient sensitivity to determine pharmacokinetic parameters of LaSOM 65 after intravenous administration to Wistar rats.

HPLC-DAD based method for the quantification of flavonoids in the hydroethanolic extract of Tonina fluviatilis Aubl. (Eriocaulaceae) and their radical scavenging activity

This article describes the isolation and identification of flavonoids in the hydroethanolic extract of the aerial parts from Tonina fluviatilis and evaluation of their antiradical activity. A method based on HPLC-DAD was developed and validated for detecting and quantifying flavonoids in hydroethanolic extracts. The flavonoids identified and quantified in the extract were 6,7-dimethoxyquercetin-3-O-β-D-glucopyranoside (1), 6-hydroxy-7-methoxyquercetin-3-O-β-D-glucopyranoside (2), and 6-methoxyquercetin-3-O-β-D-glucopyranoside (3). The developed method presented good validation parameters, showing that the results obtained are consistent and can be used in ensuring the quantification of these constituents in the extracts. Compounds 2 and 3 showed strong antiradical activity when compared with the positive controls (quercetin and gallic acid).

Optimization of chromatographic conditions and comparison of extraction efficiencies of four different methods for determination and quantification of pesticide content in bovine milk by UFLC-MS/MS

This paper describes the optimization of a multiresidue chromatographic analysis for the identification and quantification of 20 pesticides in bovine milk, including three carbamates, a carbamate oxime, six organophosphates, two strobilurins, a pyrethroid, an oxazolidinedione, an aryloxyphenoxypropionate acid/ester, a neonicotinoid, a dicarboximide, and three triazoles. The influences of different chromatographic columns and gradients were evaluated. Furthermore, four different extraction methods were evaluated; each utilized both different solvents, including ethyl acetate, methanol, and acetonitrile, and different workup steps. The best results were obtained by a modified QuEChERS method that lacked a workup step, and that included freezing the sample for 2 hours at -20 ºC. The results were satisfactory, yielding coefficients of variation of less than 20%, with the exception of the 50 µg L-1 sample of famoxadone, and recoveries between 70 and 120%, with the exception of acephate and bifenthrin; however, both analytes exhibited coefficients of variation of less than 20%.

THE APPLICATION OF AUTOMATED CORRELATION OPTIMIZED WARPING TO THE QUALITY EVALUATION OF Radix Puerariae thomsonii: CORRECTING RETENTION TIME SHIFT IN THE CHROMATOGRAPHIC FINGERPRINTS

The application of automated correlation optimized warping (ACOW) to the correction of retention time shift in the chromatographic fingerprints of Radix Puerariae thomsonii (RPT) was investigated. Twenty-seven samples were extracted from 9 batches of RPT products. The fingerprints of the 27 samples were established by the HPLC method. Because there is a retention time shift in the established fingerprints, the quality of these samples cannot be correctly evaluated by using similarity estimation and principal component analysis (PCA). Thus, the ACOW method was used to align these fingerprints. In the ACOW procedure, the warping parameters, which have a significant influence on the alignment result, were optimized by an automated algorithm. After correcting the retention time shift, the quality of these RPT samples was correctly evaluated by similarity estimation and PCA. It is demonstrated that ACOW is a practical method for aligning the chromatographic fingerprints of RPT. The combination of ACOW, similarity estimation, and PCA is shown to be a promising method for evaluating the quality of Traditional Chinese Medicine.

DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OF Ilex paraguariensis BY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

Potentiometric quantification and speciation of oxygenated groups in humic substances using BEST7 software

In this study the BEST7 software was employed to quantify different classes of functional groups and to model the proton titration behavior of humic substances. To illustrate the process, the Suwannee River fulvic acid of the IHSS (International Humic Substances Society) was used. Five categories - two classes of phenolic groups (phenol and cathecol), two classes of carboxylic groups (benzoic and phtalic) and the combination between them (salicylic) - of oxygenated groups were considered as being responsible for the potentiometric behavior of the sample and were quantitatively determined. The most and the least abundant groups were cathecol (3.300 ± 0.010 mmol g-1) and phenol (1.225 ± 0.070 mmol g-1), respectively. The estimated equilibrium constants were also determined and were in good agreement with the literature values for phenol and cathecol groups and for benzoic, phtalic and salicylic acids. Distribution diagrams of the species were generated with the software SPE and SPEPLOT.

Automated, adaptive methods for forest inventory

Forest inventories are used to estimate forest characteristics and the condition of forest for many different applications: operational tree logging for forest industry, forest health state estimation, carbon balance estimation, land-cover and land use analysis in order to avoid forest degradation etc. Recent inventory methods are strongly based on remote sensing data combined with field sample measurements, which are used to define estimates covering the whole area of interest. Remote sensing data from satellites, aerial photographs or aerial laser scannings are used, depending on the scale of inventory. To be applicable in operational use, forest inventory methods need to be easily adjusted to local conditions of the study area at hand. All the data handling and parameter tuning should be objective and automated as much as possible. The methods also need to be robust when applied to different forest types. Since there generally are no extensive direct physical models connecting the remote sensing data from different sources to the forest parameters that are estimated, mathematical estimation models are of "black-box" type, connecting the independent auxiliary data to dependent response data with linear or nonlinear arbitrary models. To avoid redundant complexity and over-fitting of the model, which is based on up to hundreds of possibly collinear variables extracted from the auxiliary data, variable selection is needed. To connect the auxiliary data to the inventory parameters that are estimated, field work must be performed. In larger study areas with dense forests, field work is expensive, and should therefore be minimized. To get cost-efficient inventories, field work could partly be replaced with information from formerly measured sites, databases. The work in this thesis is devoted to the development of automated, adaptive computation methods for aerial forest inventory. The mathematical model parameter definition steps are automated, and the cost-efficiency is improved by setting up a procedure that utilizes databases in the estimation of new area characteristics.

Damage quantification and reaction of bean genotypes (Phaseolus vulgaris L.) to Meloidogyne incognita race 3 and M. javanica

The damage and the resistance levels of cultivars and accessions of common beans rescued in the South and mountain regions of Espírito Santo State, Brazil, to M. incognita race 3 and M. javanica parasitism were evaluated under a greenhouse. Four rescued bean genotypes ("FORT-10", "FORT-13", "FORT-16" and "FORT-19") and 2 commercial cultivars: "Pérola", and "Aporé", were tested. The cultivar "Rico-23" was included as standard of susceptibility to nematodes and non-inoculated plants constituted the control. Thus, the experiment was carried out in a completely randomized design in 3 (treatments considering nematodes) x 7 (genotypes and bean cultivars) factorial arrangement, with 7 replicates. Data were measured at 50 days after plant inoculation. For damage quantification, the following variables were evaluated: plant height (PHE), number of nodes (NNO), number of trifoliate leaves (NRT), fresh matter weight (FWE) and dry matter weight (DWE) of shoots, root weight (RWE), number of root nodules (NRO) and final population (FPO) of nematodes per root system. There were no significant differences between the effects caused by M. incognita and M. javanica, but both species showed inferior values of PHE, NNO, NRT, RWE, FWE and DWE compared to controls. Concerning the levels of resistance of bean plants to M. incognita, the genotypes "FORT-10", "FORT-13", "Aporé" and "FORT-16" behaved as moderately resistant, the cultivars "Rico 23" and "Pérola" low resistant, and the genotype "FORT-19" as highly susceptible. When parasitized by M. javanica, the beans "FORT-19", "Rico-23", "FORT-16" and "FORT-13" were low resistant, "Pérola" and "Aporé" susceptible and "FORT-10" highly susceptible.

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

Quantification of incubation, latent and infection periods of Phakopsora pachyrhizi in soybean, according to chronological time and degree-days

ABSTRACT In experiments conducted in a growth chamber, the chronological time and the accumulated degree-days were determined for the duration of incubation, latent and infectious periods of Phakopsora pachyrhizi cultivars BRSGO 7560 and BRS 246 RR. Detached soybean leaflets were placed in gerbox-type acrylic boxes and inoculated with 20 x 103 uredospores/mL. The study was conducted at 12-h photoperiod and temperatures of 10ºC, 15ºC, 22ºC, 25ºC and 30°C for 30 days. Lesions and uredia/cm2were evaluated and the number of uredia per lesion was quantified after the beginning of sporulation. The sporulation potential was also quantified for cultivars BRSGO 7560 and BRS 246 RR. The steps of the infection process can be quantified based on both the chronological time and the accumulated heat. The cultivar BRSGO 7560 produced 4,012.8 spores/cm2 and BRS 246 RR, 7,348.4 uredospores/cm2. The largest number of uredia was produced at 25ºC in both cultivars; however, BRS 246 RR presented 372.7 uredia/cm2 and BRSGO 7560, 231.6 uredia/cm2. At 10ºC and 30°C, leaf infection did not occur in both cultivars.

Automated Software Testing in Machine Automation

The problem of software (SW) defaults is becoming more and more topical because of increasing amount of the SW and its complication. The majority of these defaults are founded during the test part that consumes about 40-50% of the development efforts. Test automation allows reducing the cost of this process and increasing testing effectiveness. In the middle of 1980 the first tools for automated testing appeared and the automated process was implemented in different kinds of SW testing. In short time, it became obviously, automated testing can cause many problems such as increasing product cost, decreasing reliability and even project fail. This thesis describes automated testing process, its concept, lists main problems, and gives an algorithm for automated test tools selection. Also this work presents an overview of the main automated test tools for embedded systems.

Benefits of automated acclimatization during the pre-milking phase of lactating girolando cows

The aim of this paper was to evaluate the automated acclimatization effects during pre-milking of cows on thermal conditioning, physiology, milk production and cost-benefit of the automated adiabatic evaporative cooling system (AECS). The treatments 20; 30; 40 min and control consisted of exposure time of pre-milking cows to the automated AECS. Sixteen cows were used with an average daily milk yield of 19 kg, distributed in a 4 x 4 Latin square design. The Tukey's test (P<0.05) was used to compare the means. The environmental variables, dry bulb temperature (DBT, ºC) and relative humidity (RH, %), were recorded every minute, which allowed the determination of the system efficiency through the Temperature and Humidity Index (THI). The respiratory rate (RR), rectal temperature (RT) and temperature of the coat (TC) were measured before and after the acclimatization. The 40 min treatment kept the environmental variables and the comfort indexes within recommended limits. The physiological variables (RR, RT and TC) were lower in the 40 min treatment and reflected positively on milk production, which increased 3.66% compared to the control treatment. The system was profitable, having a 43 days return on investment and a monthly revenue increase of R$ 1,992.67.

Development of an automated bench top electronic penetrometer

The aim of this study was to develop a an automated bench top electronic penetrometer (ABEP) that allows performing tests with high rate of data acquisition (up to 19,600 Hz) and with variation of the displacement velocity and of the base area of cone penetration. The mechanical components of the ABEP are: a supporting structure, stepper motor, velocity reducer, double nut ball screw and six penetration probes. The electronic components of ABEP are: a "driver" to control rotation and displacement, power supply, three load cells, two software programs for running and storing data, and a data acquisition module. This penetrometer presented in compact size, portable and in 32 validation tests it proved easy to operate, and showed high resolution, high velocity in reliability in data collection. During the validation tests the equipment met the objectives, because the test results showed that the ABEP could use different sizes of cones, allowed work at different velocities, showed for velocity and displacement, were only 1.3% and 0.7%, respectively, at the highest velocity (30 mm s-1) and 1% and 0.9%, respectively for the lowest velocity (0.1 mm s-1).

Effect of working pressure at different spray nozzles on drift quantification in wind tunnel

Each year, there is an increase in pesticide consumption and in its importance of use in the large-scale agricultural production, being fundamental the knowledge of application technology to the activity success. The objective of the present study was to evaluate the influence of working pressure on the drift generated by different spray nozzles, assessed in wind tunnel. The treatments were composed of two spray nozzles AXI 110015 and AXI 11002 with pressure levels of 276 and 414 kPa. The spray solution was composed by water and NaCl at 10%. The applications were conducted at wind speed of 2.0 m s-1, being the drift collected at 5.0; 10.0 and 15.0 m away from the spray boom and at heights of 0.2; 0.4; 0.6; 0.8 e 1.0 m from the tunnel floor. To both spray nozzles, the greatest drift was collected at the smallest distance to the spray-boom and at the lowest height. The AXI 11002 nozzle gave a smaller drift relative to the AXI 110015 nozzle for the two tested pressures and for all the collection points. Regardless of the nozzle, a rise in the working pressure increases the spray drift percentage at all distances in the wind tunnel.