Synthesis and characterization of solid 2-methoxycinnamylidenepyruvic acid

The 2-methoxycinnamylidenepyruvic acid (2-MeO-HCP) was synthesized and characterized for nuclear magnetic resonance (¹H and 13C NMR), mass spectrometry (MS), Infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The application of DSC for purity determination is well documented in literature and is used in the analysis of pure organic compounds. The molecular geometry and vibrational frequencies of 2-MeO-HCP have been calculated.

A novel indicator reaction for the catalytic determination of V(V) at ppb levels by the kinetic spectrophotometric method

A novel sensitive and relatively selective kinetic method is presented for the determination of V(V), based on its catalytic effect on the oxidation reaction of Ponceau Xylydine by potassium bromate in presence of 5-sulfosalicylic acid (SSA) as activator. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of Ponceau Xylydine at 640 nm between 0.5 to 7 min (the fixed time method) in H3PO4 medium at 25ºC. The effect of various parameters such as concentrations of H3PO4, SSA, bromate and Ponceau Xylydine, temperature and ionic strength on the rate of net reaction were studied. The method is free from most interferences, especially from large amounts of V(IV). The decrease in absorbance is proportional to the concentration of V(V) over the entire concentration range tested (1-15 ng mL−1) with a detection limit of 0.46 ng mL-1 (according to statistical 3Sblank/k criterion) and a coefficient of variation (CV) of 1.8% (for ten replicate measurement at 95% confidence level). The proposed method suffers few interferences such as Cr(VI) and Hg(II) ions. The method was successfully applied to the determination of V(V) in tap water, drinking water, bottled mineral water samples and a certified standard reference material such as SRM-1640 with satisfactory results. The vanadium contents of water samples were also determined by FAAS for a comparison. The recovery of spiked vanadium(V) was found to be quantitative and the reproducibility was satisfactory. It was observed that the results of the SRM 1640 were in good agreement with the certified value.

A pH optode based on thymol blue: application to determination of CO2 using flow injection analysis system

An optode based on thymol blue (TB), an acid-based indicator, has been constructed and evaluated as a detector in FIA system for CO2 determination. The dye was chemically immobilised on the surface of a bifurcated glass optical fibre bundle, using silanisation in organic media. In FIA system, hydrogen carbonate or carbonate samples are injected in a buffer carrier solution, and then are mixed with phosphoric acid solution to generate CO2, which diffuses through a PTFE membrane, in order to be collected in an acceptor carrier fluid, pumped towards to detection cell, in which the optode was adapted. The proposed system presents two linear response ranges, from 1.0 x 10-3 to 1.0 x 10-2 mol l-1, and from 2.0 x 10-2 to 0.10 mol l-1. The sampling frequency was 11 sample h-1, with good repeatability (R.S.D < 4 %, n = 10). In flow conditions the optode lifetime was 170 h. The system was applied in the analysis of commercial mineral water and the results obtained in the hydrogen carbonate determination did not differ significantly from those obtained by potentiometry, at a confidence level of 95 %.

New reagents for the spectrophotometric determination of ranitidine hydrochloride

A new spectrophotometric method is proposed for the assay of ranitidine hydrochloride (RNH) in bulk drug and in its dosage forms using ceric ammonium sulphate (CAS) and two dyes, malachite (MAG) green and crystal violet (CV) as reagents. The method involves the addition of a known excess of ceric ammonium sulphate to ranitidine hydrochloride in acid medium, followed by the determination of unreacted CAS by reacting with a fixed amount of malachite green or crystal violet and measuring the absorbance at 615 or 582 nm respectively against the reagent blank. The Beer's law is obeyed in the concentration range of 0.4-8.0 µg/ ml of ranitidine hydrochloride (RNH) for RNH-MAG system and 0.2-1.6µg/ml of ranitidine hydrochloride for RNH-CV system. The molar Absorptivity, Sandell's sensitivity for each system were calculated. The method has been successfully applied to the determination of ranitidine hydrochloride in pure and dosage forms.

A simple spectrophotometric method for the determination of captopril in pharmaceutical preparations using ammonium molybdate

A simple, rapid and sensitive spectrophotometric method for the determination of captopril (CPT) in pharmaceutical formulations is proposed. This method is based on the reduction reaction of ammonium molybdate, in the presence of sulphuric acid, for the group thiol of CPT, producing a green compound (λ max 407 nm). Beer's law is obeyed in a concentration range of 4.60 x 10-4 - 1.84 x 10-3 mol l-1 of CPT with an excellent correlation coefficient (r = 0.9995). The limit of detection and limit of quantification were 7.31 x 10-6 e 2.43 x 10-5 mol l-1 of CPT, respectively. The proposed method was successfully applied to the determination of CPT in commercial brands of pharmaceuticals. No interferences were observed from the common excipients in the formulations. The results obtained by the proposed method were favorably compared with those given by the official reported method at 95 % confidence level.

Potentiometric and conductimetric studies of chemical equilibria for pyridoxine hydrochloride in aqueous solutions: simple experimental determination of pKa values and analytical applications to pharmaceutical analysis

The combination of two low-cost classical procedures based on titrimetric techniques is presented for the determination of pyridoxine hydrochloride in pharmaceuticals samples. Initially some experiments were carried out aiming to determine both pKa1 and pKa2 values, being those compared to values of literature and theoretical procedures. Commercial samples containing pyridoxine hydrochloride were electrochemically analysed by exploiting their acid-base and precipitation reactions. Potentiometric titrations accomplished the reaction between the ionizable hydrogens present in pyridoxine hydrochloride, being NaOH used as titrant; while the conductimetric method was based on the chemical precipitation between the chloride of pyridoxine hydrochloride molecule and Ag+ ions from de silver nitrate, changing the conductivity of the solution. Both methods were applied to the same commercial samples leading to concordant results when compared by statistical tests (95 and 98% confidence levels). Recoveries ranging from 99.0 to 108.1% were observed, showing no significant interference on the results.

Comparison of methods for determination of nutrient digestibility of a dry kibble diet for ocelots

In this experiment, methods of total fecal collection (TFC) and internal markers (acid-insoluble ash - AIA, crude fiber - CF, and acid-detergent fiber - ADF) were compared for determination of the coefficients of apparent digestibility (CAD) for dry matter (DM), crude protein (CP), ether extract (EE), nitrogen-free extracts (NFE), and gross energy (GE) of commercial feline dry kibble for ocelots (Leopardus pardalis). Six adult animals, weighing 12.45±1.37 kg, gradually received experimental kibble in their usual diet until the beginning of the experiment and were submitted to an adaptation period ten days prior to the collection period. CAD obtained by TFC, AIA, CF, and ADF were, respectively, 73.7, 76.83, 62.01, and 46.03% for dry matter; 81.9, 84.8, 75.8, and 63.8% for crude protein; 85, 86.7, 78.5, and 69.1% for ether extract; 78.52, 79.55, 69.11, and 53.04% for nitrogen-free extracts; and 80.5, 82.2, 71.4, and 58.4% for gross energy. The AIA method showed to be efficient in determining coefficients of apparent digestibility and may contribute to investigations on the digestibility of diets for wild felines. In comparison to the items of ocelot's usual diet, the kibble used in this paper provided an adequate nutritional supply with reduced daily costs per animal.

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients

Aluminum (Al3+) overload is frequently associated with lipid peroxidation and neurological disorders. Aluminum accumulation is also reported to be related to renal impairment, anemia and other clinical complications in hemodialysis patients. The aim of the present study was to determine the degree of lipid peroxidation, platelet aggregation and serum aluminum in patients receiving regular hemodialytic treatment. The level of plasma lipid peroxidation was evaluated on the basis of thiobarbituric acid reactive substances (TBARS). Mean platelet peroxidation in patients undergoing hemodialysis was significantly higher than in normal controls (2.7 ± 0.03 vs 1.8 ± 0.06 nmol/l, P<0.05). Platelet aggregation and serum aluminum levels were determined by a turbidimetric method and atomic absorption spectrophotometry, respectively. Serum aluminum was significantly higher in patients than in normal controls (44.5 ± 29 vs 10.8 ± 2.5 µg/l, P<0.05). Human blood platelets were stimulated with collagen (2.2 µg/ml), adenosine diphosphate (6 µM) and epinephrine (6 µM) and showed reduced function with the three agonists utilized. No correlation between aluminum levels and platelet aggregation or between aluminum and peroxidation was observed in hemodialyzed patients.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR ß mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 µM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of ß-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR ß, as well as RXR a, ß and g expression was low in both HC11 and HC11ras cells. In addition, RAR ß mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

ENZYMATIC DETERMINATION OF STARCH IN DOCE DE LEITE USING DIALYSIS

The importance of starch for the food industry makes it necessary to develop new, fast, economic and accurate methodologies for its quantification. In the present paper starch hydrolysis using commercial enzymes of industrial grade are studied aiming to develop an easy and cheap analysis, available to a greater number of industries and technicians. The proposed method is simple, divided in a first step where soluble sugars are eliminated from the samples by using dialysis, followed by starch hydrolysis of the retained fraction with a thermoresistent bacterial alfa-amylase (Termamyl 120L®) and an amyloglucosidase (AMG 300L®). The hydrolysis conditions were those suggested by the enzyme producer. After the hydrolysis step the material was dialysed again for the extraction of glucose that was quantified by the glucose-oxidase colorimetric reactant. The results allowed the construction of calibration equations for starch determination on the analyzed samples. These samples were produced on a laboratory scale and native and acid-modified corn starches were added in known concentrations. By considering the final dilutions employed for glucose determination on the samples, it was possible to confirm that they were identical to that of the glucose-oxidase reactant calibration.

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.

Comparison of methodologies for moisture determination on dried bee pollen samples

Bee pollen moisture value is one of the quality parameters for this product. Some countries such as Argentina, Brazil, Bulgaria, Poland and Switzerland have bee pollen regulations on quality parameters, but these are not clear regarding which method should be used for moisture determination. The aim of this paper was to compare six methods of moisture determination in dried bee pollen samples. The methods were: conventional oven at 100 °C, vacuum oven at 70 °C, desiccator with sulfuric acid, drying out process with infrared light at 85 °C, lyophilization and Karl Fisher's method. Based on the results, the best methods for moisture determination of bee pollen were the drying process with infrared and the lyophilization, since these have shown lower moisture values.

Determination of structural and mechanical properties, diffractometry, and thermal analysis of chitosan and hydroxypropylmethylcellulose (HPMC) films plasticized with sorbitol

In this work, the structural, mechanical, diffractometric, and thermal parameters of chitosan-hydroxypropylmethylcellulose (HPMC) films plasticized with sorbitol were studied. Solutions of HPMC (2% w/v) in water and chitosan (2% w/v) in 2% acetic acid solution were prepared. The concentration of sorbitol used was 10% (w/w) to both polymers. This solutions were mixed at different proportions (100/0; 70/30; 50/50; 30/70, and 0/100) of chitosan and HPMC, respectively, and 20 mL was cast in Petri dishes for further analysis of dried films. The miscibility of polymers was assessed by X-ray diffraction, scanning electronic microscopy (SEM), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA). The results obtained indicate that the films are not fully miscible at a dry state despite the weak hydrogen bonding between the polymer functional groups.