This study expands and shows the versatility of applications in hydrophilic interaction chromatography coupled with triple quadrupole mass spectrometry (HILIC-MS/MS) using a FRULIC-N column to determine 31 phenolic compounds in beverage industry residues (coffee, apple juice, beer, and wine). Optimization, via Doehlert design, determined the acetonitrile proportion (50-90%) and pH (3.0-7.0) in the mobile phase (MP). A 22 factorial design assessed the concentration of ammonium acetate (10-110 mmol L-1) in the MP and column temperature (20-60 °C). Injection volume (5-45 µL) was univariately optimized. Separations at 60 °C reduced chromatographic run time to less than 11 min. Gradient elution (500 µL min-1) employed an MP comprising acetonitrile and ammonium acetate (110 mmol L-1, pH 7). Validation demonstrated average coefficient of determination (R2) 0.9707, limit of detection 0.001-0.503 mg L-1 and quantification 0.004-1.524 mg L-1, precision < 12.2%, recovery 92.4-110.8%. The method was applied to five residues, indicating its viability for determining phenolics in beverage production residues. HILICMS/MS HILICMSMS HILIC MS/MS MS (HILIC-MS/MS FRULICN FRULIC N 3 coffee, coffee (coffee juice beer wine. wine . wine) Optimization 5090% 5090 50 90% 90 (50-90% 3.07.0 3070 3.0 7.0 0 7 (3.0-7.0 MP. (MP) 2 10110 10 110 (10-11 L1 L 1 2060 20 (20-6 °C. C °C) 545 5 45 (5-4 optimized 6 min 500 (50 min1 min-1 (11 L1, 1, 7. 7) R2 R (R2 09707 9707 0.9707 0.0010.503 00010503 0.001 0.503 001 503 0.001-0.50 L- 0.0041.524 00041524 0.004 1.524 004 524 0.004-1.52 122 12 12.2% 92.4110.8%. 9241108 92.4 110.8%. 92 4 8 92.4-110.8% HILICMS MSMS 509 9 (50-90 07 3.07. 307 30 3. 70 (3.0-7. (MP 1011 (10-1 206 (20- 54 (5- (5 min- (1 (R 0970 970 0.970 0010 0.0010.50 0001050 0001 0.00 0503 0.50 00 0.001-0.5 0041 0.0041.52 0004152 0004 1524 1.52 52 0.004-1.5 12.2 4110 92.4110.8% 924110 924 92. 1108 110.8% 92.4-110.8 (50-9 3.07 (3.0-7 101 (10- (20 ( 097 97 0.97 0.0010.5 000105 000 0.0 050 0.5 0.001-0. 0.0041.5 000415 152 1.5 0.004-1. 12. 411 92.4110.8 92411 110.8 92.4-110. (50- (3.0- (10 (2 09 0.9 0.0010. 00010 0. 05 0.001-0 0.0041. 00041 15 1. 0.004-1 41 92.4110. 9241 110. 92.4-110 (3.0 0.0010 0.001- 0.0041 0.004- 92.4110 92.4-11 (3. 92.411 92.4-1 (3 92.41 92.4-

The tricarboxylic acid (TCA) cycle is responsible for the generation of energy in the human body and may inform about the energy status in different physiological and pathological conditions. It was developed a method to quantify the TCA cycle intermediates (α-ketoglutaric, citric, fumaric, lactic, malic, and succinic acids) by capillary zone electrophoresis with diode array detector (CZE-DAD) in indirect mode. The background electrolyte consisted of 24 mmol L–1 2,6-pyridine carboxylic acid, 76 mmol L–1 β-alanine, and 4 mmol L–1 hexadecyltrimethylammonium hydroxide (pH 4). The method was validated and presented a good linearity (R2 < 0.9922) and adequate limits of detection (0.24-1.84 mg L–1) and quantifcation (0.74-5.57 mg L–1). The method was successfully applied to samples of individuals with obesity before being submitted to bariatric surgery and one year after the surgery. The citric, lactic, and malic acids were detected in the analyzed samples. To evaluate the changes in concentrations of the TCA intermediates, principal component analysis was carried out, and there was no signifcant difference between the two sets of samples. Altogether, the data obtained after applying this new method suggest that CZE-DAD could be used to detect and quantify the TCA cycle metabolites in human urine. (TCA conditions αketoglutaric, αketoglutaric α ketoglutaric, ketoglutaric (α-ketoglutaric citric fumaric lactic CZEDAD CZE DAD (CZE-DAD mode 2 L1 L 1 L– 2,6pyridine 26pyridine pyridine 2,6 6 7 βalanine, βalanine β alanine, alanine β-alanine pH 4. . 4) R2 R (R 0.9922 09922 0 9922 0.241.84 024184 0.24 1.84 84 (0.24-1.8 0.745.57 074557 0.74 5.57 74 5 57 (0.74-5.5 L–1. out Altogether urine 6pyridine 26 2, 0.992 0992 992 241 0.241.8 02418 024 0.2 184 1.8 8 (0.24-1. 745 0.745.5 07455 074 0.7 557 5.5 (0.74-5. 0.99 099 99 0.241. 0241 02 0. 18 1. (0.24-1 0.745. 0745 07 55 5. (0.74-5 0.9 09 9 0.241 (0.24- 0.745 (0.74- (0.24 (0.74 (0.2 (0.7 (0. (0 (

Considering the economic potential of secondary metabolites, researches have been focusing several strategies in order to explore and enhance the accumulation of these metabolites in plant matrices in a broad context. One of those strategies is the development of suitable methodologies for the determination of some elicitors, such as salicylic acid (SA). Since literature presents only a few analytical methods for the quantification of SA in plants, this study developed, validated and evaluated these metabolite contents in Polygala species using capillary zone electrophoresis with ultraviolet-visible detection. The background electrolyte (BGE) consisted of 15 mmol L−1 of hydroxymethyl-aminomethane and 30 mmol L−1 of 2-hydroxy-isobutyric acid, pH = 3.9. The BGE was chosen using a rational procedure to increase selectivity and reduce migration time. The method presented rapid performance, migration time less than 0.76 min for salicylic and phthalic acid (internal standard), precision results for peak area were better than 2.9% (intra-day) and 2.8% (inter-day) and the responses for the analytes were linear (range of 1-5 mg L−1), with limits of detection and quantification of 0.2 and 0.6 mg L−1, respectively. The developed method was applied for 39 samples of plant extracts.

This work aims to develop a rapid and reliable method by HPLC-QqQ-MS/MS for identification and quantification of Kaurenoic acid (KA) in extract, tincture and syrup from Mikania glomerata. The developed method allows identifying and quantifying KA in complex samples obtained from extract marketed in pharmaceutical industry, a tincture prepared with the leaves from plant and two syrups purchased in the local market. The quantification was performed using standard addition calibration curve. An accurate method for Kaurenoic acid determination in M. glomerata samples was developed. The concentrations of KA found in M. glomerata were 0.19 mg mL-1 in the commercial extract, 0.93 mg mL-1 in the tincture, 3.00 x 10-3 mg mL-1 in the syrup B and in syrup A was not possible to quantify due to the concentration is below the detection limit. Since most reports describe the quantification of the main active constituent (coumarin) and do not provide relevant information on the quantification of KA in these sample types (syrup and tincture), the developed method represents an excellent alternative for agile and efficient analysis of the KA in complex matrices from simple dilution.

Extracts of black mulberry leaves present phytochemicals such as phenolic compounds and minerals. These substances have been studied because they show several health benefits. In this work we report the influence of drying temperature, infusion temperature and extraction time by means of factorial design monitoring the total phenolic content (CFT) of aqueous extracts of leaves of Morus nigra L. The concentrations of CFT, flavonoids, flavonols and antioxidant activity of the extracts were determined by spectrophotometry. The identification and quantification of phenolic compounds were performed by LC-ESI-MS / MS. The determination of the mineral concentration in the black mulberry leaves was performed by ICP-MS. The Pareto and surface response graphs suggested that the most efficient extractions occurred for leaves drying at lower temperatures and using higher infusion temperatures, extraction time did not show significant influence. The extracts presented concentration of CFT from 16.96 ± 0.15 to 5.73 ± 0.33 mg GAE g-1. The analyzes by LC-ESI-MS / MS allowed to identify phenolic substances not yet reported in the literature to date. In addition 10 mineral elements were determined in the black mulberry leaves at mg kg-1 concentrations range.

A new and fast method was developed to determine the iodine number of biodiesel samples by capillary zone electrophoresis (CZE). In this procedure, the biodiesel sample reacts with Wijs solution in the presence of a catalyst and all the remaining iodine monochloride is converted to the iodide ion, which is quantified by CZE. Only small amounts of sample and reactant are required and µL level amounts of chloroform and Wijs solution. The reaction time was reduced to 3 min and the total analysis time is less than 15 min. The simplicity and speed of the method can be further enhanced by using single-point calibration. The limits of quantification (LOQ) and determination (LOD) values were better than 0.6 and 0.18 g per 100 g of biodiesel. Instrumental, intraday, and interday precisions were better than 1.13, 1.04 and 6.02% relative standard deviation (RSD). The accuracy was statistically verified by comparison with the standard method EN 14111.

This paper describes the use of a multiple-injection capillary electrophoresis method as a fast strategy to determine the antinitrosating capacity of nine commercial teas through nitrite quantification. The method consists of the injection of the sample followed by the injection of the control solution, employing a fused-silica capillary of 32.0 cm total length (23.5 cm effective length, 50 µm internal diameter) with background electrolyte composed of 4.0 g L-1 β-alanine and 1.5 g L-1 perchloric acid (pH 3.79) and sodium thiocyanate was used as the internal standard. Before the injections the tea samples were maintained by 1 h of incubation, at 37 °C, with sodium nitrite in perchloric acid medium (pH 2.3). In order to avoid nitrite oxidation and nitrate formation, ultra-pure nearly oxygen-free water was used to prepare the solutions. Black tea, green tea and white tea, obtained from Camellia sinensis, showed greater antinitrosating capacity (96, 93 and 89%, respectively).

ABSTRACT Many essential oils (EOs) of different plant species possess interesting antimicrobial effects on buccal bacteria and antioxidant properties. Eugenia klotzschiana Berg (pêra-do-cerrado, in Portuguese) is a species of Myrtaceae with restricted distribution in the Cerrado. The essential oils were extracted through the hydrodistillation technique using a modified Clevenger apparatus (2 hours) and chemically characterized by GC-MS. The major compounds were α-copaene (10.6 %) found in oil from leaves in natura, β-bisabolene (17.4 %) in the essential oil from dry leaves and α-(E)-bergamotene (29.9 %) in oil from flowers. The antioxidant activity of essential oils showed similarities in both methods under analysis (DPPH and ABTS˙+) and the results suggested moderate to high antioxidant activity. The antibacterial activity was evaluated by determining minimum inhibitory concentrations (MICs), using the microdilution method. MIC values below 400 µg/mL were obtained against Streptococcus salivarius (200 µg/mL), S. mutans (50 µg/mL), S. mitis (200 µg/mL) and Prevotella nigrescens (50 µg/mL). This is the first report of the chemical composition and antibacterial and antioxidant activities of the essential oils of E. klotzschiana. These results suggest that E. klotzschiana, a Brazilian plant, provide initial evidence of a new and alternative source of substances with medicinal interest.

ABSTRACT Eugenia brasiliensis Lam., Myrtaceae, is used in folk medicine for anti-inflammatory diseases such as arthritis and rheumatism. This study investigated the anti-inflammatory activity and phenolic profile of the crude hydroalcoholic extract and ethyl acetate fraction from E. brasiliensis leaves. Crude hydroalcoholic extract and the ethyl acetate fraction were analyzed by HPLC-ESI-MS/MS in comparison to standard phenolic compounds. The anti-inflammatory activity of the crude hydroalcoholic extract (1, 10 and 25 mg kg-1) and the ethyl acetate fraction (10, 25 and 50 mg kg-1) was evaluated in a swiss mouse model of acute pleurisy induced by carrageenan, being the total cell count, exudation and analysis of nitrite/nitrate the inflammation parameters. HPLC-ESI-MS/MS analysis revealed apigenin, catechin, galangin, isoquercetin, myricetin, quercetin and rutin. Crude hydroalcoholic extract and ethyl acetate fraction were effective in inhibiting cell migration in all tested doses. Crude hydroalcoholic extract was effective in inhibiting exudation only at the 10 mg kg-1 dose; ethyl acetate fraction was effective in all tested doses. Results for nitrite/nitrate levels reveals that only the ethyl acetate fraction was effective at the tested doses. This is the first report of the presence of isoquercetin, galangin and apigenin in this species. Results from the phytochemical analysis enhance the chemical knowledge of this species. In the future, together with more studies, validation of its popular use in inflammatory diseases is possible.

The choice of an appropriate background electrolyte (BGE) and its components for capillary electrophoresis analysis is the main step in capillary electrophoresis method development. The use of an inadequate co-ion component could lead to asymmetrical peaks and selecting an inappropriate counter-ion could affect the buffer capacity and the pH of the BGE, leading to unreliable analysis. In this paper, we describe a systematic procedure for the development of a capillary electrophoresis method, based on the effect of varying pH on the ion effective mobility, to optimize the BGE composition. The method was applied to the separation of L-ascorbic acid in different samples. The optimized background electrolyte composition was 40 mmol L-1 tris(hydroxymethyl)aminomethane and 20 mmol L-1 2-morpholinoethanesulfonic acid, at pH 8.1. Sorbic acid was used as the internal standard and separation was carried out in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 µm inner diameter), with a short-end-injection configuration and direct ultraviolet (UV) detection at 266 nm. The separation was performed in 26 s. The method shows good linearity (R2 > 0.999), excellent values for inter-day and intra-day precision and good recovery (in the range of 94-107%). The values obtained for limit of detection (LOD) and limit of quantification (LOQ) were 0.14 and 41 mg L-1, respectively. The systematic procedure applied shows to be a very useful tool for the first step method development for capillary electrophoresis.

The main objective of the present work was to validate a chromatographic method to determine herbicides commonly applied in the irrigated rice farming. For this, matrix-matched calibration was employed along with the extraction and clean-up of the samples by quick, easy, cheap, effective, rugged and safe (QuEChERS) method and determination of the analytes by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray ionization in the positive mode. By this method, it was possible to achieve the ionization and detection of a total of 18 herbicides, with quantification of 12 of them. The method presented adequate precision and accuracy according to the European Commission and the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for analytes in low concentrations. The limits of quantification ranged from 0.015 μg g-1 for oxadiazon to 0.165 μg g-1 for imazapyr. The method showed good linearity with R2> 0.99 and recovery values from 92 to 103%. The proposed protocol is adequate for monitoring bispyribac-sodium, cyclosulfamuron, cycloxydim, clomazone, ethoxysulfuron, fenoxaprop-p-ethyl, imazapic, imazapyr, imazethapyr, metsulfuron-methyl, oxadiazon and thiobencarb in rice grains in concentrations up to 109 times lower than the maximum residue limits established by the Brazilian Health Surveillance Agency (ANVISA) for these compounds in rice samples.

Two new synthetic melanin-like oligomers inspired by the synthesis of pheomelanin (cys-DOPA) were prepared using L-DOPA as a precursor and the amino acids serine (ser-DOPA) and threonine (thr-DOPA) for modifications. The products obtained exhibited appreciable antioxidant activity. The compounds were characterized by liquid chromatography-mass spectrometry (LC-MS), elemental analysis and infrared spectroscopy. The pKa values of the groups present in each melanin suspension were determined in aqueous solution by potentiometric and spectrophotometric titration. Cytotoxicity assays showed an IC50 > 500 mg L-1 resulting in non-toxic compounds for living cells. Finally, cys-DOPA, ser-DOPA and thr-DOPA exhibited antioxidant activity determined by the DPPH scavenging assay, with EC50 = 7.69, 1.02 and 27.60 mg L-1, respectively.

Biodegradable nanoparticles (NPs) have received considerable attention because of their possible use in the development of strategies for the topical delivery of oils and therapeutic drugs, particularly when drug penetration in dermis is desired. Zein is a prolamine and is a promising material for the design of drug delivery systems. In this study, NPs were prepared with zein and were used to encapsulate and release terpinen-4-ol, which is a therapeutic agent for the treatment of melanoma. The results show that the zein NPs are promising nanostructured systems for the prolonged delivery of T4OL with potential applications in anti-melanoma therapy.

Foi proposta a determinação de ácidos graxos (FA) no fígado de ratos Wistar de três grupos diferentes (seis ratos por grupo) submetidos à dieta (AIN-93G) utilizando a técnica de eletroforese capilar de zona (CZE). Cada grupo recebeu a mesma dieta, sendo o óleo de soja a fração lipídica da dieta (7% m/m). O primeiro grupo foi alimentado com ração contendo óleo de soja fresco, o segundo foi alimentado com a dieta cuja fração lipídica foi óleo de soja utilizado durante 7 dias em processo de fritura por imersão e, finalmente, o terceiro grupo foi alimentado com dieta cuja fração lipídica foi óleo de soja utilizado durante 15 dias em processo de fritura. Após 45 dias consumindo essas dietas, os ratos foram submetidos à eutanásia e o teor de FA no fígado foi monitorado por CZE de uma forma simples, rápida e eficiente. Os resultados obtidos foram comparados com o método oficial por cromatografia à gás e não foram observadas diferenças significativas no intervalo de confiança de 95%. Os eletroferogramas para análise de FA foram submetidos à análise de componentes principais (PCA), sendo possível discriminar o grupo final dos grupos intermediários e controle. Além dos resultados da PCA, a lipoproteína de baixa densidade (LDL), foi avaliada, indicando que o tempo de exposição total de óleo submetido a processos de fritura profunda pode ser considerado relevante para a avaliação da qualidade do óleo.

The determination of fatty acids (FA) in liver from three different groups of Wistar rats (six rats by group) submitted to diet (AIN-93G) by capillary zone electrophoresis (CZE) was proposed. Each group received the same diet. However, the soybean oil used to prepare the feed, whose amount was 7% w/w, had different origins. The first group was fed with feed containing soybean oil fresh; the second one was fed with soybean used during 7 days in deep frying process; finally, the third was fed with soybean used during 15 days in deep frying process. After 45 days the rats were submitted to euthanasia and the FA amount in liver was successful monitored by CZE in a simple, fast and efficient way. The results obtained were compared to gas chromatography official method and no significant differences were observed within 95% confidence interval. The electropherograms to FA analysis were submitted to principal component analysis (PCA) being possible to discriminate the final from the control and intermediate groups. In addition to PCA results, the low density lipoprotein (LDL) was evaluated indicating that the total time exposure of the oil submitted to deep frying processes can be considered relevant to evaluating the oil quality.

Uma investigação fitoquímica de Polygala molluginifolia (raiz, caule, folhas e flores) resultou no isolamento de quatro piranoisoflavonas das quais a isoflavona1é desconhecida na literatura. As isoflavonas 2-4: , embora sejam compostos sintéticos conhecidos são descritos neste trabalho como novos produtos naturais. Os compostos isolados foram avaliados em termos do seu efeito antioxidante e o potencial para inibir a enzima acetilcolinesterase. As isoflavonas 1 e 4 inibiram a acetilcolinesterase, exibindo valores de IC50 de 68 µmol L-1 e 84 µmol L-1, respectivamente. No ensaio de DPPH, os mesmos compostos e a rutina 5 apresentaram valores de EC50 de 61 µmol L-1, 55 µmol L-1 e 16 µmol L-1, respectivamente. Além disso, a permeabilidade das compostos 1-4 foi testada através do ensaio de permeabilidade em membrana artificial paralela (PAMPA) com as isoflavona 3 (9.25 × 10-6 cm s-1) e 4 (3.48 × 10-6 cm s-1) exibindo as maiores permeabilidades.

A phytochemical investigation of Polygala molluginifolia (root, stem, leaves and flowers) resulted in the isolation of four pyranoisoflavones, of which isoflavone 1 was previously unknown in the literature. Isoflavones 2-4: , although they are known synthetic compounds, are described in this work as new natural products. The isolated compounds were evaluated for their antioxidant effects and potential for inhibiting the acetylcholinesterase enzyme. Isoflavones 1 and 4 inhibited acetylcholinesterase, displaying IC50 values of 68 µmol L-1 and 84 µmol L-1, respectively. In the DPPH assay, the same compounds and rutin 5 exhibited EC50 values of 61 µmol L-1, 55 µmol L-1 and 16 µmol L-1, respectively. Moreover, the permeability of compounds 1-4: was evaluated using parallel artificial membrane permeability assay (PAMPA) with isoflavones 3 (9.25 × 10-6 cm s-1) and 4 (3.48 × 10-6 cm s-1) exhibiting the highest permeabilities.