Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Development of genital plates in nymphs of Triatoma pallidipennis, Stål 1872, (Hemiptera: Reduviidae) and its application as sexing method

Searching for morphometric differences between sexes in immature forms, the development of genital plates in the exuviae of Triatoma pallidipennis Stål 1872nymphs was studied. Differences were found in the form and size of the 9th genital urosternite, it is larger and wider in males as compared to females. This difference is reported in several South American Triatoma species. From our results it is possible to sex early stages from microscopic observation of genital plates in whole insects.

Application of synthetic peptides in development of a serologic method for laboratory diagnosis of schistosomiasis mansoni

The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003) on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019). These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni.

Development of a shelling method to recover whole kernels of the Cutia nut (Couepia edulis)

The kernel of the cutia nut (castanha-de-cutia, Couepia edulis (Prance) Prance) of the western Amazon, which is consumed by the local population, has traditionally been extracted from the nut with a machete, a dangerous procedure that only produces kernels cut in half. A shelling off machine prototype, which produces whole kernels without serious risks to its operator, is described and tested. The machine makes a circular cut in the central part of the fruit shell, perpendicular to its main axis. Three ways of conditioning the fruits before cutting were compared: (1) control; (2) oven drying immediately prior to cutting; (3) oven drying, followed by a 24-hour interval before cutting. The time needed to extract and separate the kernel from the endocarp and testa was measured. Treatment 3 produced the highest output: 63 kernels per hour, the highest percentage of whole kernels (90%), and the best kernel taste. Kernel extraction with treatment 3 required 50% less time than treatment 1, while treatment 2 needed 38% less time than treatment 1. The proportion of kernels attached to the testa was 93%, 47%, and 8% for treatments 1, 2, and 3, respectively, and was the main reason for extraction time differences.

Development and validation of an alternative titration method for the determination of sulfate ion in indinavir sulfate

A simple and rapid precipitation titration method was developed and validated to determine sulfate ion content in indinavir sulfate raw material. 0.1 mol L-1 lead nitrate volumetric solution was used as titrant employing potentiometric endpoint determination using a lead-specific electrode. The United States Pharmacopoeia Forum indicates a potentiometric method for sulfate ion quantitation using 0.1 mol L-1 lead perchlorate as titrant. Both methods were validated concerning linearity, precision and accuracy, yielding good results. The sulfate ion content found by the two validated methods was compared by the statistical t-student test, indicating that there was no statistically significant difference between the methods.

Development and application of spectrophotometric method for the determination of cefaclor in pharmaceutical formulations

A simple, fast and sensitive spectrophotometric method for the determination of cefaclor in pharmaceutical raw and dosage forms based on reaction with ninhydrin is developed, optimized and validated. The purple color (Ruhemenn's purple) that resulted from the reaction was stabilized and measured at 560 nm. Beer's law is obeyed in the concentration range of 4-80 µg mL-1 with molar absorptivity of 1.42 × 10(5) L mole-1 cm-1. All variables including the reagent concentration, heating time, reaction temperature, color stability period, and cefaclor/ninhydrin ratio were studied in order to optimize the reaction conditions. No interference was observed from common pharmaceutical adjuvant. The developed method is easy to use, accurate and highly cost-effective for routine studies relative to HPLC and other techniques.

Development of dissolution method for benznidazole tablets

The aim of this work was the development of a dissolution method for benznidazole (BNZ) tablets. Three different types of dissolution media, two stirring speeds and apparatus 2 (paddle) were used. The accomplishment of the drug dissolution profiles was compared through the dissolution efficiency. The assay was performed by spectrophotometry at 324 nm. The better conditions were: sodium chloride\hydrochloride acid buffer pH 1.2 with stirring speed of 75 rpm, volume of 900 mL and paddle as apparatus. Ahead of the results it can be concluded that the method developed consists in an efficient alternative for assays of dissolution for benznidazole tablets.

Development of a flotation method for preconcentration-separation of trace amounts of some metal ions in plant tissues prior to their FAAS determinations

An efficient flotation method based on the combination of flame atomic absorption spectrometry (FAAS) and separation and preconcentration step for determination of Cr3+, Cu 2+, Co2+, Ni2+, Zn2+, Cd 2+, Fe3+ and Pb2+ ions in various real samples by the possibility of applying bis(2-hydroxyacetophenone)-1,4-butanediimine (BHABDI) as a new collector was studied. The influence of pH, amount of BHABDI as collector, sample matrix, type and amount of eluting agent, type and amount of surfactant as floating agent, ionic strength and air flow rates i.e. variables affecting the efficiency of the extraction system was evaluated. It is ascertained that metal ions such as iron can be separated simultaneously from matrix in the presence of 0.012 mM ligand, 0.025% (w/v) of CTAB to a test sample of 750 mL at pH 6.5. These ions can be eluted quantitatively with 6 mL of 1.0 mol L-1 HNO3 in methanol which lead to the enrichment factor of 125. The detection limits for analyte ions were in the range of 1.3-2.4 ng mL-1. The method has been successfully applied for determination of trace amounts of ions in various real samples.

Development of new dissolution test and HPLC-RP method for anti-parasitic ornidazole coated tablets

This work aimed the development and validation of a new dissolution test for ornidazole coated tablets. The dissolution conditions were determined after testing Sink conditions, dissolution medium, apparatus, stirring speed, 24 h stability and medium filtration influence. The best conditions were paddle at a stirring speed of 75 rpm and 900 mL of 0.1 M HCl. A new HPLC quantification method was developed and validated. The dissolution test and quantification method showed to be adequate for their purposes and could be applied for quality control of ornidazole coated tablets, since there is no official monograph.

Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC

A rapid, economical, reproducible, and simple direct spectrophotometric method was developed and validated for the assay of nitazoxanide in pharmaceutical formulations. Nitazoxanide concentration was estimated in water at 345 nm and pH 4.5. The method was suitable and validated for specificity, linearity, precision, and accuracy. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. The proposed method was successfully applied in the determination of nitazoxanide in coated tablets and in powders for oral suspension. This method was compared to a previously developed and validated method for liquid chromatography to the same drug. There was no significative difference between these methods for nitazoxanide quantitation.

Development and validation of UV spectrophotometric method for determination of levofloxacin in pharmaceutical dosage forms

The objective of this research was to develop and validate an alternative analytical method for quantitative determination of levofloxacin in tablets and injection preparations. The calibration curves were linear over a concentration range from 3.0 to 8.0 μg mL-1. The relative standard deviation was below 1.0% for both formulations and average recovery was 101.42 ± 0.45% and 100.34 ± 0.85% for tablets and injection formulations, respectively. The limit of detection and limit of quantitation were 0.08 and 0.25 μg mL-1, respectively. It was concluded that the developed method is suitable for the quality control of levofloxacin in pharmaceuticals formulations.

Development and validation of a LC-method for the determination of phenols in a pharmaceutical formulation containing extracts from Stryphnodendron adstringens

A sensitive RP-HPLC method with UV detection successfully measured phenol(s) in an ointment containing 3% Stryphnodendron adstringens extract. Chromatography used acetonitrile (0.05% trifluoroacetic acid):water (0.05% trifluoroacetic acid) (v/v), flow rate 0.8 mL min-1. Quantitation was accomplished by the external-standard method. Linearity for 2.00 to 16.00 μg mL-1 (gallic acid) and 1.14 to 18.24 μg mL-1 (gallocatechin) was established. Intra- and inter-day precision levels were under 5%. LOD and LOQ were 0.231 and 0.770 μg mL-1 (gallic acid) and 0.151 and 0.504 μg mL-1 (gallocatechin), respectively. Determination of phenols was unaffected by product excipients.

Development and validation of an UV spectrophotometric method for the determination of aliskiren in tablets

For determination of aliskiren in commercial samples, an analytical UV spectrophotometric method was developed and validate according to ICH guideline. The method was linear in the range between 40 and 100 μg mL-1 (r² = 0.9997, n = 7) and exhibited suitable specificity, accuracy, precision, and robustness. It is simple, it has low cost, and it has low use polluting reagents. Therefore, the proposed method was successfully applied for the assay and dissolution studies of aliskiren in tablet dosage forms, and the results were compared to a validated RP-LC method, showing non-significant difference (P > 0.05).

Development and validation of a method for the analysis of tetracyclines in chicken-muscle by liquid chromatography-electrospray-mass spectrometry in tandem (LC-ESI-MS/MS)

A LC-ESI-MS/MS method was developed and validated according to the European Union decision 2002/657/EC, for the determination of tetracyclines (TCs) in chicken-muscle since Europe is one of the main markets for Brazilian products. Linearity of r > 0.9979, limits of quantification in the range of 7.0-35.0 ng/g, average recoveries of 89.38 - 106.27%, within-day and between-day precision were adequate for all TCs. The decision limit and the detection capability were 93.00-106.46 ng/g and 95.84-114.38 ng/g, respectively. This method is suitable for application in surveillance programmes of residues of TCs in chicken-muscle samples.