Isolation of mitraphylline from Uncaria tomentosa (Willd. ex Schult.) DC. barks and development of spectrophotometric method for total alkaloids determination in Cat's Claw samples.

INTRODUCTION: The usual quality control for Uncaria tomentosa (Willd. ex Schult.) DC. barks requires highly specific analytical standards and methods based on high-performance liquid chromatography (HPLC), which impacts the costs of the analytical process and the final products. OBJECTIVE: To obtain an analytical reference standard of mitraphylline by isolation from U. tomentosa barks and develop a spectrophotometric method for determination of total alkaloids in samples of U. tomentosa. METHODOLOGY: An alkaloid-enriched extract was obtained by acid-base partition and mitraphylline was selectively precipitated using an 80:20 v/v toluene/hexane solution. The compound was characterised by HPLC-UV/DAD (diode-array detector), mass spectrometry, UV-visible, infrared (IR) and 1 H- and 13 C-nuclear magnetic resonance (NMR) spectroscopy. Sample preparation for the spectrophotometric method consisted of an extraction with boiling methanol (3 × 10 mL, 15 min), followed by a strong cation exchange solid phase extraction (SCX-SPE) clean-up. RESULTS: Mitraphylline with a purity of 98% was isolated in 0.05% m/m yield. All characterisation results were in agreement with previous published data. The spectrophotometric method showed linear range between 0.40 and 20 µg/mL; limits of detection and quantification of 0.15 and 0.49 µg/mg, respectively; dispersion of results lower than 5% for repeatability and intermediate precision; statistically proven accuracy by comparison with reference values obtained by Soxhlet and an HPLC-UV/DAD method; and robustness in relation to sample mass extracted and extraction time. CONCLUSION: The methods developed to obtain mitraphylline analytical standard from U. tomentosa barks and to determine total alkaloids by spectrophotometry provided a cheaper and faster quality control alternative for U. tomentosa samples.

Simultaneous determination of alpha-, beta- and gamma-hydroxybutyric acids in micro-pulverized human hair by GC-MS: Method development, validation and application.

An analytical method was developed and validated for the simultaneous determination of alpha, beta and gamma-hydroxybutyric acids (AHB, BHB and GHB, respectively) in human hair. Hair samples (10 mg) were pulverized and submitted to extraction using methanol (1 mL) for 10 min. The extract was centrifuged, filtered, and evaporated to dryness at 40 °C under a gentle N2 flow, dissolved in ethyl acetate (0.050 mL) and derivatized using 0.050 mL of BSTFA containing 1% TMCS, at °C for 40 min. The derivatives were determined by GC/MS. Aliquots of natural hair ("blank") samples after water extraction (3 steps) were pulverized, spiked with AHB, BHB, GHB or d6-GHB and used for method validation. Figures of merit showed the method's feasibility. The LOQ values were 1 ng mg-1 for BHB and AHB and 0.6 ng mg-1 for GHB, and mean recoveries were 54%, 69% and 75% for AHB, BHB and GHB respectively. Precision and bias were better than 15% and 20% respectively. Calibration curves (LOQ to 20 ng mg-1 for AHB and BHB and LOQ to 16 ng mg-1 for GHB) obtained in 5 days were pooled for statistical analysis, but the data were heteroscedastic, as demonstrated by the White test. Therefore, weighted linear regression and data transformations were applied and the best results were obtained using the Box-Cox transformation [c' = (cλ-1)/λ] for GHB and log transformation for AHB and BHB. Authentic hair samples were collected from the posterior head vertex of 23 volunteers (16 females and 7 males). Four of them declared having type-2 diabetes (T2D). Only female volunteers reported hair treatments such as dyeing, flat ironing, straightening and/or bleaching. Dyeing appeared not to affect GHB levels in hair, but no data were found in the literature about other cosmetic treatments. GHB levels varied from < LOQ to 1.5 ng mg-1. BHB levels varied from < LOQ to 3.4 ng mg-1 and from < LOQ to 1.8 ng mg-1 in non-diabetic volunteers with or without family history of T2D, respectively. However, in diabetics, BHB levels varied from 3.4 to 5.2 ng mg-1. The levels of AHB varied from < LOQ to 1.2 ng mg-1 in non-diabetic individuals, and from 1.1 to 6.2 ng mg-1 in diabetics. Our results showed that AHB levels in hair have potential to as a biomarker for T2D, but this needs to be further studied with larger groups of volunteers. To our knowledge, this is the first published method for the determination of AHB and BHB in human hair.

Development and validation of a method for simultaneous determination of trace levels of five macrocyclic lactones in cheese by HPLC-fluorescence after solid-liquid extraction with low temperature partitioning.

A highly sensitive analytical method was developed and validated, following international guidelines, for the determination of the residues of five macrocyclic lactones (MLs) (abamectin, doramectin, eprinomectin, ivermectin and moxidectin) in cheese. The extracts were concentrated by rotary-evaporation and derivatized; no clean-up was necessary. Despite matrix complexity, no significant matrix-effect was verified, and standards were prepared in solvents. Linear working ranges varied from 0.25 to 5.0 µg L-1. Excellent limits of quantification (0.58-0.87 µg kg-1), mean recoveries (91-103%), and repeatability and intermediate precision (<5.8%) were obtained. Twenty-two samples of bovine and non-bovine cheeses were analyzed. Twenty-one samples showed residues of at least one ML (between 0.59 and 15.3 µg kg-1), but moxidectin was never detected; a sample of mozzarella was free of MLs. To the best of our knowledge, this is the first method describing the simultaneous evaluation of these MLs in cheese using HPLC and fluorescence detection.

Polycyclic aromatic hydrocarbons in biochar amended soils: Long-term experiments in Brazilian tropical areas.

The interest in charcoal for agricultural use (biochar) has sharply increased in recent years. However, biochar can contain groups of compounds such as polycyclic aromatic hydrocarbons (PAHs) that are considered persistent pollutants, and are formed concomitantly with biochar during its production by pyrolysis. Soil samples were collected in three experimental areas at different intervals (1, 3, 5 or 6 years) after the application of 16 Mg ha-1 of biochar. The total concentrations of PAHs; benzo[a]pyrene; and the estimated total cancer risk in biochar treated plots were larger than found in the control ones, but they decreased over time, equaling the control values after three years in the high C content soil (11.2 g C kg-1 soil), or after six years in the low C content soil (6.8 g C kg-1 soil). Nevertheless, the sum of PAH concentrations found in the biochar amended plots, in the range of 15.80-39.40 ng g-1, were around two orders of magnitude below the limits of prevention established by Brazilian legislation (8100 ng g-1) and some European regulations for soils (3000 ng g-1) and also lower than the observed in previous studies about biochar amended soils. Our results indicate that, under the conditions evaluated, the application of biochar to soil in the studied proportion (16 Mg ha-1, every six years) is safe concerning soil contamination by PAHs.

Repeated subcutaneous administrations of krokodil causes skin necrosis and internal organs toxicity in Wistar rats: putative human implications.

OBJECTIVE: "Krokodil" is the street name for an impure homemade drug mixture used as a cheap substitute for heroin, containing desomorphine as the main opioid. Abscesses, gangrene, thrombophlebitis, limb ulceration and amputations, jaw osteonecrosis, skin discoloration, ulcers, skin infections, and bleeding are some of the typical reported signs in humans. This study aimed to understand the toxicity of krokodil using Wistar male rats as experimental model. METHODS: Animals were divided into seven groups and exposed subcutaneously to NaCl 0.9% (control), krokodil mixture free of psychotropic substances (blank krokodil), pharmaceutical grade desomorphine 1 mg/kg, and four different concentrations of krokodil (containing 0.125, 0.25, 0.5, and 1 mg/kg of desomorphine) synthesized accordingly to a "domestic" protocol followed by people who inject krokodil (PWIK). Daily injections for five consecutive days were performed, and animals were sacrificed 24 hr after the last administration. Biochemical and histological analysis were carried out. RESULTS: It was shown that the continuous use of krokodil may cause injury at the injection area, with formation of necrotic zones. The biochemical results evidenced alterations on cardiac and renal biomarkers of toxicity, namely, creatine kinase, creatine kinase-MB, and uric acid. Significant alteration in levels of reduced and oxidized glutathione on kidney and heart suggested that oxidative stress may be involved in krokodil-mediated toxicity. Cardiac congestion was the most relevant finding of continuous krokodil administration. CONCLUSIONS: These findings contribute notably to comprehension of the local and systemic toxicological impact of this complex drug mixture on major organs and will hopefully be useful for the development of appropriate treatment strategies towards the human toxicological effects of krokodil.

Characterization of the variation of carbonyl compounds concentrations before, during, and after the renovation of an apartment at Niterói, Brazil.

The present work reports the variation of 31 carbonyl compounds (CC) in an apartment located at Niterói City, Rio de Janeiro State, Brazil. Eight sampling campaigns were conducted through a 1-year period, and three areas (living room, kitchen, and bedroom) were evaluated before, during, and after the renovation activities and reoccupation of the apartment. Samples were collected using SEP-PAK cartridges impregnated with 2,4-dinitrophenylhydrazine, and the hydrazones were analyzed using rapid resolution liquid chromatography with UV detection. The lowest total concentration of CC (19.0 ± 1.5 µg m(-3)) was found before the renovation when the apartment was empty, but door varnishing resulted in highest contamination of the apartment (1386 ± 384 µg m(-3)); however, an important dispersion of CC was observed in the subsequent sampling (148 ± 1.8 µg m(-3)). After apartment reoccupation, the indoor contamination seemed to depend on the routine activities taken there, such as household product use and cooking activities, but apparently, local temperature increase favored the vaporization of the volatile CC from the building materials in the apartment. As far as we are concerned, this is the first study comparing the concentrations of 31 CC in residential areas before, during, and after renovation activities taken in Brazil.

Data analysis of "krokodil" samples obtained by street-like synthesis.

The data described in this work is related to be the subject of an article in the Forensic Science International, titled: "The harmful chemistry behind "krokodil": street-like synthesis and product analysis" (http://dx.doi.org/10.1016/j.forsciint.2015.07.042) [1]. The data presented here provides additional description of the chemical profile of "krokodil". Physicochemical and organoleptic characteristics, TLC profile, UV/Vis, (1)H NMR and FTIR spectrum are presented. These data validate the proposed synthetic procedure and pathway and give further information about the contaminants present in "krokodil".

Early changes in system [Formula: see text] and glutathione in the retina of diabetic rats.

Diabetic retinopathy (DR), the main cause of blindness among diabetic patients, affects both neuronal and vascular cells of the retina. Studies show that neuronal cell death begins after 4 weeks of diabetes and could be related with an increase in oxidative stress. System [Formula: see text] is a glutamate/cystine exchanger, formed by a catalytic subunit called xCT and a regulatory subunit 4F2hc, whose activity is crucial to the synthesis of glutathione, which is a key antioxidant molecule for cells. Although some studies have shown that glutamate transport mediated by excitatory amino acid transporters (EAATs) in diabetic rats is downregulated, there are no studies investigating system [Formula: see text] in this context. To evaluate whether system [Formula: see text] is modified by early onset of diabetes, primary retinal cell culture exposed to high glucose and retinas of rats 3 weeks after streptozotocin injection were used. We observed that xCT subunit protein expression both in cultures and in vivo were diminished. Furthermore, system [Formula: see text] activity and GSH levels were also decreased whereas oxidative stress was increased in retinas of diabetic animals. Therefore, this study raises the possibility that alterations in system [Formula: see text] expression and activity could occur during early onset of diabetes. In that way, system [Formula: see text] modifications could be related to increased ROS in diabetic retinopathy.

Short communication: Macrocyclic lactone residues in butter from Brazilian markets.

Macrocyclic lactones (ML) are commonly used in drug formulations for the treatment of parasites in cattle. In Brazil, except for drugs (or formulations) with long-term (half-life) effects, ML are registered for use in bovines. Indiscriminate use of ML may result in the presence of residues in milk and dairy products due to their lipophilic properties and thermal stability. This study applied a method of liquid chromatography with fluorimetric detection, recently developed and validated for the determination of residues of abamectin, doramectin, ivermectin, and moxidectin in butter. The method was applied to 38 samples of commercial butter purchased in the metropolitan area of Rio de Janeiro, Brazil, between June and September 2013, analyzed in triplicate. Ivermectin was detected in 89.5% of the samples, with concentrations between 0.3 and 119.4 µg/kg; 76.3% of the samples contained doramectin (0.6 to 64.7 µg/kg) and 55.2% contained abamectin (0.7 to 4.5 µg/kg). Most butter samples (76.3%) contained residues of more than 1 ML; however, no residues of moxidectin were detected. The results showed a high incidence of the presence of avermectins in butter samples. Butter is not included in the Brazilian National Plan for Control of Residues and Contaminants in Animal Products. As ML residues concentrate in lipophilic compounds, butter and other fatty dairy products should be screened for the presence of ML residues.

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.

Development and validation of a method for the determination of low-ppb levels of macrocyclic lactones in butter, using HPLC-fluorescence.

An analytical method was developed and validated for the simultaneous determination of four macrocyclic lactones (ML) (abamectin, doramectin, ivermectin and moxidectin) in butter, using liquid chromatography with fluorescence detection. The method employed heated liquid-liquid extraction and a mixture of acetonitrile, ethyl acetate and water, with preconcentration and derivatization, to produce stable fluorescent derivatives. The chromatographic run time was <12.5 min, with excellent separation. The method validation followed international guidelines and employed fortified butter samples. The figures of merit obtained, e.g. recovery (72.4-106.5%), repeatability (8.8%), within-laboratory reproducibility (15.7%) and limits of quantification (0.09-0.16 µg kg(-1)) were satisfactory for the desired application. The application of the method to real samples showed that ML residues were present in six of the ten samples evaluated. The method proved to be simple, easy and appropriate for simultaneous determination of ML residues in butter. To our knowledge, this is the first method described for the evaluation of ML in butter.

Evaluation of the concentrations and distribution of carbonyl compounds in selected areas of a Brazilian bus terminal.

This study describes the determination of 30 carbonyl compounds (CCs) in three areas (bus boarding platform, passenger circulation area, and a pastry shop) of the Presidente João Goulart Bus Terminal, located at Niterói City, RJ, Brazil, and in an open area 700 m distant from the terminal. Samples were collected using SEP-PAK cartridges impregnated with 2,4-dinitrophenylhydrazine, during May to July 2012. The hydrazones formed were analyzed using rapid resolution liquid chromatography with UV detection. The studied locations showed distinct profiles of distribution of CC. The circulation area, which is influenced by different pollution sources, presented an intermediate profile between that of the pastry shop and boarding platform. Formaldehyde and acetaldehyde were the most abundant CC, but acetaldehyde predominated in the pastry shop once it is a by-product of baking yeast fermentation. Samples taken in the pastry shop and circulation area showed significant concentrations of hexanaldehyde and nonanaldehyde emitted during cooking. The pastry shop showed the largest level of total CC among the studied areas followed by the circulation area, the boarding platform, and the open area.

PAHs and BTEX in groundwater of gasoline stations from Rio de Janeiro City, Brazil.

PAHs and BTEX were studied in groundwater from monitoring wells of gasoline stations of Rio de Janeiro City, Brazil during 2003 and 2004. Total PAH concentration ranged from 0.05 to 84.9 microg/L. Total BTEX concentration varied between not-detected to 3.6 x 10(3) microg/L. Some samples exceeded the maximum concentration limit of Brazilian regulations for benzene in fresh or drinking water. Carcinogenic PAHs were found in few samples but only one sample in each year exceeded the regulated concentrations for benzo[a]pyrene. The concentrations of PAHs and BTEX indicate that some places may be contaminated by gasoline leakage.