New Metabolites of Coumarin Detected in Human Urine Using Ultra Performance Liquid Chromatography/Quadrupole-Time-of-Flight Tandem Mass Spectrometry.

Coumarin (1,2-benzopyrone) is a natural compound whose metabolism in humans was established in the 1970s. However, a new metabolite was recently identified in human plasma, indicating that the metabolism of coumarin has not been completely elucidated. To complement the knowledge of its metabolism, a rapid and sensitive method using UPLC-QTOF-MS was developed. A total of 12 metabolites was identified using MetaboLynxTM software, including eight metabolites not previously reported in human urine. The identified biotransformation included hydroxylation, glucuronidation, sulfation, methylation, and conjugation with N-acetylcysteine. The present work demonstrates that the metabolism study of coumarin was incomplete, possibly due to limitations of old techniques. The identification of eight inedited metabolites of such a simple molecule suggests that the information regarding the metabolism of other drugs may also be incomplete, and therefore, new investigations are necessary.

Applying liquid chromatography-tandem mass spectrometry to assess endodontic sealer microleakage.

The objective of this study was to describe a new method for the quantitative analysis of a microleakage of endodontic filling materials. Forty extracted single-rooted teeth were randomly divided into three experimental groups. After root canal shaping, the experimental groups were filled using the lateral condensation technique with the Epiphany system (G1), with gutta-percha + Sealapex (G2), and with gutta-percha + AH Plus (G3). Each root was mounted on a modified leakage testing device, and caffeine solution was used as a tracer (2000 ng mL-1, pH 6.0), applied in the coronal direction towards the tooth apex, creating a hydrostatic pressure of 2.55 kPa. Presence of caffeine in the receiving solution was measured after 10, 30, and 60 days, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). None of the groups presented microleakage at 10 days. At 30 days, G2 and G3 showed similar infiltration patterns (means: 16.0 and 13.9 ng mL-1, respectively), whereas G1 showed significantly higher values (mean: 105.2 ng mL-1). At 60 days, leakage values were 182.6 ng mL-1 for G1, 139.0 ng mL-1 for G2, and 53.5 ng mL-1 for G3. AH Plus showed the best sealing ability and HPLC-MS/MS showed high sensitivity and specificity for tracer quantification.

A kinetic study of the main guaco metabolites using syrup formulation and the identification of an alternative route of coumarin metabolism in humans.

For decades guaco species have been empirically used for the treatment of respiratory diseases. However, studies have shown that the toxic and therapeutic effects of the main guaco metabolites are dose-dependent, and none clinical study was done to evaluate the behavior of these substances in humans. In this work, a pilot study measuring the kinetic profile of the main guaco metabolites was performed leading to the knowledge of an alternative route of coumarin metabolism in humans. Initial screenings demonstrated that the administration of 60 mL of guaco syrup (single dose) did not provide sufficient levels of coumarin (COU), 7-hydroxycoumarin (7-HCOU), o-coumaric acid (OCA) and kaurenoic acid (KAU). The pharmacokinetic parameters were calculated by orally administering 60 mL of guaco syrup spiked with 1500 mg of COU. The kinetic study demonstrated that the plasmatic levels of 7-HCOU (considered the main metabolite of COU) were 10 times lower than the levels of COU, and the kinetic profile of 7-HCOU suggests sequential metabolism in the liver with low access of 7-HCOU to the systemic circulation. The study also demonstrated that OCA is one of the main bioavailable metabolites of COU. Therefore, the hydrolysis of the lactone ring forming a carboxylated compound is one of the possible routes of COU metabolism in humans. The half-lives of COU, 7-HCOU and OCA were approximately 4.0, 1.0 and 3.0 h, respectively and there was evidence that the recommended dosage of guaco syrup did not provide sufficient levels of COU, 7-HCOU or OCA to obtain a bronchodilation effect. Clinical studies are necessary to prove the efficacy and safety of products based on guaco.

Simultaneous determination of levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma by an HPLC-MS/MS method.

BACKGROUND: In this study, we developed and validated a HPLC-MS/MS method capable of simultaneously determining levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma. RESULTS & METHODOLOGY: Chromatographic separation was achieved using a C8 column with a mobile phase consisting of a gradient of water and acetonitrile:methanol (90:10 v/v), both containing 0.1% formic acid. The developed method was selective, sensitive (LD<7.0 ng ml(-1)), linear (r>0.99), precise (RSD<11.3%), accurate (RE<11.8%) and free of residual and matrix effects. The developed method was successfully applied in plasma patients with Parkinson's disease using Stalevo®. CONCLUSION: The new method can be used for the clinical monitoring of these substances and applied to adjustments in drug dosages.

Applying liquid chromatography-tandem mass spectrometry to assess endodontic sealer microleakage

The objective of this study was to describe a new method for the quantitative analysis of a microleakage of endodontic filling materials. Forty extracted single-rooted teeth were randomly divided into three experimental groups. After root canal shaping, the experimental groups were filled using the lateral condensation technique with the Epiphany system (G1), with gutta-percha + Sealapex (G2), and with gutta-percha + AH Plus (G3). Each root was mounted on a modified leakage testing device, and caffeine solution was used as a tracer (2000 ng mL-1, pH 6.0), applied in the coronal direction towards the tooth apex, creating a hydrostatic pressure of 2.55 kPa. Presence of caffeine in the receiving solution was measured after 10, 30, and 60 days, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). None of the groups presented microleakage at 10 days. At 30 days, G2 and G3 showed similar infiltration patterns (means: 16.0 and 13.9 ng mL-1, respectively), whereas G1 showed significantly higher values (mean: 105.2 ng mL-1). At 60 days, leakage values were 182.6 ng mL-1for G1, 139.0 ng mL-1 for G2, and 53.5 ng mL-1 for G3. AH Plus showed the best sealing ability and HPLC-MS/MS showed high sensitivity and specificity for tracer quantification.

Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC-MS/MS method.

The objective of this work was to develop and validate a HILIC-MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-µm particle size column maintained at 40°C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3mM ammonium formate. The flow rate was maintained at 400 µL min(-1). Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection <1.5 ng mL(-1)) and free of matrix and residual effects. The new method was also precise (RSD<9.0%), accurate (RE<11.2%) and linear (r ≥ 0.99) over the ranges of 5-500 ng mL(-1) for each compound. The developed method was successfully applied to determine metformin and vildagliptin in plasma volunteers who orally received a single dose of metformin (850 mg), vildagliptin (50mg) or drug association (metformin 850 mg+vildagliptin 50mg). The new method can thus also be used as a tool for the clinical monitoring of metformin and vildagliptin.

Development and validation of an HPLC-DAD method for simultaneous determination of cocaine, benzoic acid, benzoylecgonine and the main adulterants found in products based on cocaine.

Here, an HPLC-DAD method was developed and validated for simultaneous determination of cocaine, two cocaine degradation products (benzoylecgonine and benzoic acid), and the main adulterants found in products based on cocaine (caffeine, lidocaine, phenacetin, benzocaine and diltiazem). The new method was developed and validated using an XBridge C18 4.6mm×250mm, 5µm particle size column maintained at 60°C. The mobile phase consisted of a gradient of acetonitrile and ammonium formate 0.05M - pH 3.1, eluted at 1.0mL/min. The volume of injection was 10µL and the DAD detector was set at 274nm. Method validation assays demonstrated suitable sensitivity, selectivity, linearity, precision and accuracy. For selectivity assay, a MS detection system could be directly adapted to the method without the need of any change in the chromatographic conditions. The robustness study indicated that the flow rate, temperature and pH of the mobile phase are critical parameters and should not be changed considering the conditions herein determined. The new method was then successfully applied for determining cocaine, benzoylecgonine, benzoic acid, caffeine, lidocaine, phenacetin, benzocaine and diltiazem in 115 samples, seized in Brazil (2007-2012), which consisted of cocaine paste, cocaine base and salt cocaine samples. This study revealed cocaine contents that ranged from undetectable to 97.2%, with 97 samples presenting at least one of the degradation products or adulterants here evaluated. All of the studied degradation products and adulterants were observed among the seized samples, justifying the application of the method, which can be used as a screening and quantification tool in forensic analysis.

Malva sylvestris L. extract suppresses desferrioxamine-induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC-MS/MS method for prostaglandin quantification.

Malva sylvestris is a species used worldwide as an alternative to anti-inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti-inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro-inflammatory mediators PGE2 and PGD2 in desferrioxamine-stimulated phorbol 12-myristate 13-acetate-differentiated U937 cells. An HPLC-DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC-MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE2 and PGD2) and quantification (1.0 ng/mL for PGE2 and PGD2) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0-500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937-d cells. A significant dose-dependent reduction of PGE2 and PGD2 levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti-inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators.

Ethnobotanical and scientific aspects of Malva sylvestris L.: a millennial herbal medicine.

OBJECTIVES: Malva sylvestris L., known as common mallow, is native to Europe, North Africa and Asia. In the Mediterranean region, this species has a long history of use as food, and due to its therapeutic relevance, some parts of this plant have been employed in traditional and ethnoveterinary medicines. The leaves in particular have been reported to have potent anti-inflammatory, antioxidant, anti-complementary, anticancer and skin tissue integrity activity. Additionally, an anti-ulcerogenic effect was recently proven, demonstrating that the aqueous extract was more effective than cimetidine, a potent medicine used to treat gastric ulcers. Due to its wide use and medicinal importance, many studies have been conducted; however, the information in the literature is very extensive and disseminated, making it difficult to use. KEY FINDINGS: A complete review involving the ethnobotanical and scientific aspects of M. sylvestris has been made. The research has provided evidence that M. sylvestris has potential use as a medicinal plant and has highlighted a need for more studies involving clinical and toxicological aspects of its use. SUMMARY: This review can contribute to the field with its historical context, and by describing the progress made, new ideas for researchers can arise.

Análisis crítico de metodologías empleadas en la evaluación de selladores en endodoncia / Critical analysis of methodologies used in the evaluation of sealants in endodontics

La incompleta obturación del sistema de conductos radiculares es uno de los motivos del fracaso en el tratamiento endodóntico. La presencia de espacios en el material obturador puede permitir la filtración en los sentidos apical y coronario. A medida que nuevos materiales son propuestos, se deben realizar investigaciones para la evaluación de todas sus propiedades. Dentro de ellas se puede destacar la capacidad selladora del material obturador en el interior de los conductos radiculares. Varias metodologías han sido preconizadas para esta finalidad. La literatura indica que han sido cuestionables la falta de estandarización y correlación entre estos métodos y los resultados de estas investigaciones. La falta de métodos confiables hace que estos materiales sean empleados en la práctica clínica de forma enpírica, en lo que respecta a esta propiedad. El propósito de este trabajo fue analizar metodologías de evaluación de la capacidad selladora de materiales obturadores endodónticos, buscando estudiar detalladamente sus variables y correlacionar los resultados de estas investigaciones con la práctica clínica

The incomplete filling of the root canal systen is one of the reasons for endodontic treatment failure. The presence of gaps in the seal material can allow leakage into the apical and coronary senses. As new materials are proposed, investigations must be conducted for the evaluation of all its properties. Within then you can highlight the sealing capacity of the filling material inside the root canals. Several methods have been advocated for this purpose. Therefore, the literature points towards the lack of standardization and correlation between these methods and the results of these investigations have been questionable. The lack of reliable methods makes the clinical use of these materials enpirical. The goal of this paper is to discuss methodologies for assessing the sealing ability of endodontic filling materials, seeking to study in detail the variables and correlate the results of these researchs with the clinical practice

Avaliação da quantidade de carboidratos em xaropes infantis usados no tratamento da asma / Evaluation of the carbohydrate content in paediatric syrups used in asthma treatment

A identificação de possíveis fatores envolvidos no processo saúde-doença cárie dental é uma passo fundamental para a prevenção. O objetivo do trabalho foi verificar a concentração de carboidratos (glucose, frutose, sacarose e lactose) presentes em xaropes infantis usados no tratamento da asma. Foram selecionados quinze xaropes, os quais foram analisados quanto à sua composição por cromatografia de camada delgada e quantificados por densitometria óptica. Os resultados mostraram uma concentração total de carboidratos variando de 0% a 43%, sendo que apenas quatro medicamentos eram isentos de sacarose. Os resultados indicam que os xaropes utilizados no tratamento da asma podem ser um dos fatores associados à cárie dental na infância