Flávia Ferreira de Carvalho Marques

Is it possible to screen for milk or whey protein adulteration with melamine, urea and ammonium sulphate, combining Kjeldahl and classical spectrophotometric methods?

The Kjeldahl method and four classic spectrophotometric methods (Biuret, Lowry, Bradford and Markwell) were applied to evaluate the protein content of samples of UHT whole milk deliberately adulterated with melamine, ammonium sulphate or urea, which can be used to defraud milk protein and whey contents. Compared with the Kjeldahl method, the response of the spectrophotometric methods was unaffected by the addition of the nitrogen compounds to milk or whey. The methods of Bradford and Markwell were most robust and did not exhibit interference subject to composition. However, the simultaneous interpretation of results obtained using these methods with those obtained using the Kjeldahl method indicated the addition of nitrogen-rich compounds to milk and/or whey. Therefore, this work suggests a combination of results of Kjeldahl and spectrophotometric methods should be used to screen for milk adulteration by these compounds.

Characterization of newfound natural luminescent properties of melamine, and development and validation of a method of high performance liquid chromatography with fluorescence detection for its determination in kitchen plastic ware

Experimental studies of the natural photoluminescence of melamine in aqueous solutions showed that its fluorescence intensity (at 250/365 nm) was appropriated for analytical purposes. The exploitation of such melamine property provided the basis of development of a new, simple, precise and accurate method based on high performance liquid chromatography with fluorescence detection (HPLC-Fluo) to determine melamine in kitchen plastic ware following aqueous extraction using a microwave oven. Optimization of analytical parameters such as solvent composition, pH and extraction conditions led to limits of detection and quantification of melamine of 0.0081 and 0.027 μg mL(-1), respectively, with a linear range up to 10 μg mL(-1). Sample extracts fortified with melamine at three concentration levels produced an average recovery of 98±6%, which was in agreement with the results achieved with a reference HPLC-UV method. Different samples of kitchen plastic ware analyzed by the developed and optimized method showed melamine concentrations in the aqueous extract up to 17 µg mL(-1), which corresponded to 86.0 mg kg(-1) in these utensils. The results obtained indicate that the use of kitchen plastic ware made of melamine can contaminate food with this compound after heating in a microwave oven.

Capillary zone electrophoresis method for the direct determination of amino acids in recombinant human erythropoietin preparations used for the treatment of anemia.

A method based on CZE for the determination of glutamic acid, glycine, and alanine in a biopharmaceutical formulation containing recombinant human erythropoietin was developed. The separation was achieved within less than 5 min, using a fused‐silica capillary column (55 cm × 50 μm id) and 30 mmol/L phosphate buffer at pH 11.5, containing 0.6 mmol/L CTAB and 10% v/v methanol, as BGE solution. Applied potential of –25 kV, temperature of 15°C and hydrodynamic injection time of 15 s, at 50 mbar, were employed. The detection of the analytes was carried out without any derivatization reaction, at 220 nm using an UV‐DAD detector. Linear ranges from 50 to 2500 mg/L and quantification limits of 40, 39, and 37 mg/L were obtained for glutamic acid, glycine, and alanine, respectively. Sample preparation required only a dilution step. Considering peak area and migration time values, the method presented good repeatability (RSD <1.7%; n = 9) and intermediate precision (RSD <1.0%; n = 6). Recovery evaluation using a commercial sample led to values between 97.5 ± 5.2% and 101.5 ± 4.6%, demonstrating the feasibility of the method, which was successfully applied in the quantification of the amino acids of interest in biopharmaceutical samples.

Laser induced fluorescence and photochemical derivatization for trace determination of camptothecin.

Laser-excited fluorescence was used for the selective determination of camptothecin in samples containing anti-cancer camptothecin-analogs (irinotecan and topotecan). The selectivity of the method was based on the UV photochemical derivatization in basic solution which increased the analyte fluorescence (337/450 nm) and eliminated fluorescence from the two campthotecin-analogs. The influence of UV exposure time and sodium hydroxide concentration was studied using an experimental design. Limit of detection was 4 × 10(-10) mol L(-1) with linear fluorescence response up to 1 × 10(-6)mol L(-1). Average recoveries of camptothecin (added to the samples to simulate a contamination) were 92 ± 4 and 94 ± 6% (n=3) respectively in irinotecan and topotecan based pharmaceuticals.

Determination of ascorbic acid in the retina during chicken embryo development using high performance liquid chromatography and UV detection

The retina is a specialized tissue of the central nervous system (CNS) and it is the only part of the CNS that can be visualized non-invasively. During vertebrate development, the retina originates together with the optic nerve as outgrowths of the developing brain, and in this respect, the avian retina is a very convenient model for neurochemical studies of the CNS. In this study, a HPLC-UV method was developed and validated for the determination of ascorbic acid (AA) in the chicken embryo retina. AA has an important role in the retina because of its antioxidant properties. The developed method showed very good figures of merit (recovery = 91 ± 2%; repeatability and intermediate precision better than 1.67% and 2.53% and a limit of quantification of 0.03 mg L−1). Retinas of two embryo ages (12 days and 18 days) showed AA concentrations of 0.0107 ± 0.0010 and 0.0055 ± 0.0005 μg of AA per μg of protein, respectively, and the statistical comparison of results confirmed the decrease of the AA level. These results seem to correlate well with oxidative stress protection, but this fact is still under investigation. As far as we are aware, this is the first study that demonstrates the HPLC-UV determination of ascorbic acid in the chicken embryo retina and its variation along embryo development.

Selective Spectrofluorimetric Method and Uncertainty Calculation for the Determination of Camptothecin in the Presence of Irinotecan and Topotecan

A selective fluorimetric method was developed to quantify camptothecin (CPT) in irinotecan (CPT-11) and in topotecan (TPT) based anti-cancer drugs. Selectivity was achieved by experimental optimization and using synchronized scanning and second-order spectral derivatization. The limits of quantification for CPT were in the order of 1 × 10−7 mol L−1 with a linear response up to 1 × 10−5 mol L−1. The combined uncertainty associated with the CPT fluorescence measurement at a 5 × 10−7 mol L−1 level were 10.9% and 6% of the reference level, respectively, for TPT and CPT-11 based pharmaceutical formulations. The CPT recoveries of 95 ± 12% and 91 ± 5.6% (n = 3) were achieved, respectively, in samples containing CPT-11 and TPT.

An alternative derivatization reaction to the determination of doramectin in bovine milk using spectrofluorimetry.

The doramectin (DOR), which belongs to the avermectins group (AVM), has a high antiparasitic activity and so it has been widely used in food-producing animals. The DOR shows low fluorescence quantum efficiency and as a consequence, chemical derivatization reactions are necessary to produce derivatives with improved luminescent properties before its determination by fluorimetry. As the presence of this compound in food represents a risk to human health, an easy, clean and low cost derivatization reaction, which is alternative to those usually employed and that enables its spectrofluorimetric determination in milk samples, was developed. Ethanolic solutions of DOR, containing sodium hydroxide at a final concentration of 0.25 mol L(-1), after 60 min of heating at 50 °C, produced fluorescent signals 1000 times higher than the original ethanolic solution. Using these optimized conditions, a linear response range that extended from 50.00 to 1000 μg L(-1), with a value of (R(2)) equal to 0.9970, was obtained. Average recovery of DOR was 92.5±1.5% (n=3) in bovine milk fortified samples submitted to a liquid-liquid extraction at low temperature and pre concentration process, indicating the usefulness and effectiveness of the proposed method. The proposed spectrofluorimetric method is an alternative to high-performance liquid chromatography (HPLC) based methods, allowing rapid and simple detection of doramectin in milk samples.

Determination of the Camptothecin Derivatives CPT-11 and SN-38 in Urine and Saliva by Micellar Electrokinectic Chromatography

The anti-cancer synthetic drug irinotecan (CPT-11) and its active metabolite SN-38 have been determined by micellar electrokinetic capillary chromatography (MEKC). The detection of the analytes was made at 368 nm and their separation took less than 7 min using a borate buffer (pH 8.8 at 25 mmol L−1) solution containing sodium dodecyl sulfate (45 mmol L−1) and acetonitrile (13.5% v/v). On-line analyte concentration (normal stacking mode) and the use of a highly sensitive cell (Z shaped cell) improved detection limits (at the 10−8 mol L−1 level). Recovery in fortified human saliva was 108 ± 5%, in agreement with the result achieved with the reference HPLC method. For the analysis of urine from rats submitted to a single dose of CPT-11 and SN-38, camptothecin was used as internal standard enabling recoveries close to 100% when compared to the results achieved using HPLC.

Overview of the scientific production in the Pharmacy area in Brazil: profile and productivity of researchers granted with fellowships by the National Council for Scientific and Technological Development

In Brazil, the National Council for Scientific and Technological Development (CNPq) distributes productivity fellowships in research (RS) as a recognition to individuals with outstanding productivity levels in their areas. The aim of this study is to evaluate the scientific production of the Brazilian Pharmacy area, one division of the Health Sciences Great Area of CNPq, considering the profile and productivity levels of RS fellows. The results showed that most of the 156 active RS fellows in 2015 were female, with doctorate completed in the Southeast region (mainly in University of São Paulo) and with research activities developed in the South and Southeast regions. Most of their work was published in journals classified as B1 and B2 Qualis in Pharmacy by the Coordination for the Improvement of Higher Education Personnel (CAPES), with high prevalence of publications in local journals and/or specialized on medicinal plants. Besides, they featured much dependence on advising and productivity indexes related to the category and level of RS fellowship. The evolution of such data must be continually evaluated to determine the influence of CNPq productivity fellowships on performance and stratification of researchers in the Pharmacy area in Brazil.

Implementation of a Spectrofluorimetric Method to the Determination of 8-Methoxypsoralen in Capsules and Creams Used in Treatment of Psoriasis and Vitiligo: an Evaluation of the Quality of Compounding Pharmacies in Rio de Janeiro

8-Methoxypsoralen is a widely sold drug in Brazil as handled capsules and creams because it is only industrially available as soft gel capsules of 10 mg. In order to contribute to the quality control of these pharmaceutical forms, a spectrofluorimetric method was implemented and applied for the evaluation of the quality of compounding pharmacies in the state of Rio de Janeiro (Brazil). Solutions of 8-methoxypsoralen prepared in ethanol/water (30:70 v/v) produced fluorescence signals at excitation/emission wavelengths of 305/495 nm. The method presented good precision (relative standard deviation, RSD < 4.8%) and recoveries in the pharmaceutical samples from 88.5-108% indicated its feasibility. The results of assay (dosing) and uniformity of content suggested that two of the three evaluated pharmacies did not have appropriate techniques in the preparation of these formulations, which could potentially reduce the efficiency of dermatoses treatment. The implemented method is an alternative to those described in literature for pharmaceutical forms containing 8-methoxypsoralen.