Biotransformation of rifampicin by Aspergillus niger and antimicrobial activity of proposed metabolites.

Drug biotransformation studies emerges as an alternative to pharmacological investigations of metabolites, development of new drug candidates with reduced investment and most efficient production. The present study aims to evaluate the capacity of biotransformation of rifampicin by the filamentous fungus Aspergillus niger ATCC 9029. After incubation for 312 h, the drug was metabolized to two molecules: an isomer (m/z 455) and the rifampicin quinone (m/z 821). The monitoring of metabolite formation was performed by high-performance liquid chromatography, followed by their identification through ultra-high-performance liquid chromatography coupled to tandem mass spectrometer. In vitro antimicrobial activity of the proposed metabolites was evaluated against Staphylococus aureus microorganism, resulting in the loss of inhibitory activity when compared with the standards, with minimum inhibitory concentration of 7.5 µg/ml. The significant biotransformation power of the ATCC 9029 strain of A. niger was confirmed in this study, making this strain a candidate for pilot studies in fermentation tanks for the enzymatic metabolization of the antimicrobial rifampicin. The unprecedented result allows us to conclude that the prospect of new biotransforming strains in species of anemophilic fungi is a promising choice.

Polyphenolic Composition and in Vitro Antihypertensive and Anti-Inflammatory Effects of Cuphea lindmaniana and Cuphea urbaniana.

The present study investigates the chemical composition, anti-inflammatory, and antihypertensive activities, in vitro, from extracts of Cuphea lindmaniana and Cuphea urbaniana leaves. The extraction was performed ultrasound-assisted, and UHPLC/MS analysis was in positive mode ionization. The anti-inflammatory activity of the extracts and miquelianin were assayed at concentrations 0.001-10 µg/mL by chemotaxis on rat polymorphonuclear neutrophils. The antihypertensive activity was performed by angiotensin-converting enzyme (ACE) inhibition. From the nineteen proposed compounds, six of them are described for the first time in this genus. The extracts displayed antichemotactic effect with a reduction of 100 % of the neutrophil migration, in vitro, in most concentrations. The ACE-inhibition presented results ranging from 19.58 to 22.82 %. In conclusion, C. lindmaniana and C. urbaniana extracts contain a rich diversity of flavonoids and display in vitro anti-inflammatory and antihypertensive potential. Thus, this study could serve as a scientific baseline for further investigation, on developmental novel products with therapeutic actions.

Stability and degradation products of imipenem applying high-resolution mass spectrometry: An analytical study focused on solutions for infusion.

Carbapenems show recognized instability in aqueous solutions; therefore some care must be taken in their handling and preparation and their use in the hospital environment. The stability and degradation products of imipenem were investigated from conditions that simulate its clinical use. For this, a simple stability-indicating method by HPLC-DAD was validated with a focus on the quantitation of drug concentration remaining from infusion solutions (sodium chloride 0.9% and glucose 5%). The degradation products formed were identified by high-resolution mass spectrometry (ESI-Q-TOF-MS/MS), with detection of the [M + H]+ ions at m/z 318 (DP-1), m/z 599 (DP-2) and m/z 658 (DP-3). The most probable elemental compositions were obtained with a high degree of confidence, where the error between the masses observed and calculated was 1.25 ppm for DP-1, -0.33 ppm for DP-2 and 1.82 ppm for DP-3. The DP-1 degradation product resulted from cleavage of the ß-lactam ring; DP-2 corresponded to the drug dimer; and DP-3 was generated from the interaction between imipenem and cilastatin. The proposed method provides a safe and reliable alternative for the quantitation of imipenem, and the stability data obtained by ESI-Q-TOF help in understanding the drug behavior under the conditions of clinical use.

Microbial transformation of ambrisentan to its glycosides by Cunninghamella elegans.

The purpose of this paper is to describe the glycosylation of ambrisentan (AMB) by cultures of Cunninghamella elegans ATCC 9245. AMB is an endothelin receptor antagonist, which is used to treat pulmonary arterial hypertension. Filamentous fungi are morphologically complex and may exhibit different forms depending on the species and the nature of the culture medium. A biotransformation study was conducted to investigate the ability of C. elegans to metabolize AMB. Parameters were optimized by testing on different culture media and concentrations, pH, drug concentration, static and shaking conditions. Ambrisentan's metabolite, obtained after 240 h of incubation as a result of glycosylation pathway, was separated by HPLC and determined by high-resolution mass spectrometry. The method showed linearity over 300-1000 µg mL-1 (r = 0.998). Accuracy, precision, robustness and stability studies agree with international guidelines. Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and used little solvent.

Sida tuberculata extract reduces the nociceptive response by chemical noxious stimuli in mice: Implications for mechanism of action, relation to chemical composition and molecular docking.

Sida tuberculata R.E.Fr. (Malvaceae) is a medicinal plant widely found in Southern Brazil, and popularly used for inflammatory disorders and to pain relief. A phytochemical analysis followed by an investigation about antinociceptive potential and mechanism of action were performed with leaves and roots extracts. Methanolic extracts, designated as S. tuberculata leaves extract (STLE) and S. tuberculata roots extract, were analyzed both by UHPLC­MS. The in vivo antinociceptive potential of STLE (10­300 mg kg−1) was assessed in mice subjected to the acetic acid­induced abdominal writhes and formalin model. Agonist/antagonist tests and computational docking suggest the involvement of opioid and adenosinergic systems. The main chemical class detected on extracts was the ecdysteroids, and 20­hydoxyecdysone (20HE) was confirmed as the major phytoconstituent. The pretreatment with STLE (100 mg kg−1) reduced more than 70% abdominal contortions induced by acetic acid model and produced significant inhibition on formalin­induced licking response. The mechanism of action study revealed STLE might act through opioid and adenosine systems. Molecular docking suggested kaempferol derivative and 20HE might interacting with µ­opioid receptor. Thus, the results suggest the existence of antinociceptive potential from S. tuberculata extracts being in accordance to the traditional use.

Stability, degradation impurities and decomposition kinetics for paliperidone from osmotic tablets.

The antipsychotic paliperidone was investigated with a focus on stability, degradation impurities and kinetics reaction profile. Osmotic tablets 3 mg (OROS® ) were subjected to extraction in an ultrasonic bath and the resulting acidic solution was stressed by forced conditions. Degraded samples were monitored by HPLC-DAD in different storage times for acidic and alkaline hydrolysis, oxidation, heat and photolysis. Photolysis was shown to be a strong degradation factor, with a drug content of 24.64% remaining after 24 h. Oxidation (H2 O2 18%) caused a slow decomposition, with a drug content of 83.49% remaining after 72 h. Through kinetics graphics, first-order reactions were found for oxidation, heat and photolysis. By UPLC-MS analysis, the degraded matrix could be investigated for identification of impurities with m/z 445.3128, m/z 380.8906, m/z 364.9391, m/z 232.9832 and m/z 217.0076, allowing the identification of derivatives N-oxide and with modifications in the lactam, benzisoxazole and pyrimidine rings. Paliperidone in liquid state, like analytical solutions or formulation, must be carefully handled to avoid drug exposure, specially in storage conditions.

Stability of doripenem in reconstituted solution - thermal and oxidative decomposition kinetics and degradation products by LC-MS.

A ultra-fast liquid chromatography method applied to quantitation of doripenem in powder for injection was validated. Validation parameters were assayed and a rapid analysis was established by a reversed-phase system comprising a C18 column endcapped (50 × 4.0 mm, 2.0 µm), mobile phase consisting of phosphoric acid 0.01% (pH 3.8) and acetonitrile (98:02, v/v) and a flow rate of 0.4 mL min-1 . Drug stability was studied through submission to forced conditions, allowing the major degradation products to be detected and the kinetics parameters to be established. Thermal and oxidative degradation were determined, and indicated a kinetic decomposition following first and second order, respectively. The main degradation products were identified by LC-MS analysis, and the results were evaluated in order to suggest the chemical structures corresponding to respective masses and fragmentations. Six compounds were identified, with m/z 411, 427, 437, 634, 650 and 664. All of them resulted from cleavage of ß-lactam ring and alcoholic chain and/or dimerization. These experimental results provide valuable information about the stability of doripenem reconstituted solution and procedures for its handling and storage.

Blueberry (Vaccinium ashei Reade) extract ameliorates ovarian damage induced by subchronic cadmium exposure in mice: Potential δ-ALA-D involvement.

Females are born with a finite number of oocyte-containing follicles and ovary damage results in reduced fertility. Cadmium accumulates in the reproductive system, damaging it, and the cigarette smoke is a potential exposure route. Natural therapies are relevant to health benefits and disease prevention. This study verified the effect of cadmium exposure on the ovaries of mice and the blueberry extract as a potential therapy. Blueberry therapy was effective in restoring reactive species levels and δ-aminolevulinate dehydratase activity, and partially improved the viability of cadmium-disrupted follicles. This therapy was not able to restore the 17 ß-hydroxysteroid dehydrogenase activity. Extract HPLC evaluation indicated the presence of quercetin, quercitrin, isoquercetin, and ascorbic acid. Ascorbic acid was the major substance and its concentration was 620.24 µg/mL. Thus, cadmium accumulates in the ovaries of mice after subchronic exposure, inducing cellular damage, and the blueberry extract possesses antioxidant properties that could protect, at least in part, the ovarian tissue from cadmium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 188-196, 2017.

Effects of Red Wine Tannat on Oxidative Stress Induced by Glucose and Fructose in Erythrocytes in Vitro.

The literature indicates that red wine presents in its composition several substances that are beneficial to health. This study has investigated the antioxidant effects of Tannat red wine on oxidative stress induced by glucose and fructose in erythrocytes in vitro, with the purpose to determine some of its majoritarian phenolic compounds and its antioxidant capacity. Erythrocytes were incubated using different concentrations of glucose and fructose in the presence or absence of wine. From these erythrocytes were determined the production of thiobarbituric acid reactive species (TBARS), glucose consumption, and osmotic fragility. Moreover, quantification of total phenolic, gallic acid, caffeic acid, epicatechin, resveratrol, and DPPH scavenging activity in wine were also assessed. Red wine showed high levels of polyphenols analyzed, as well as high antioxidant potential. Erythrocytes incubated with glucose and fructose had an increase in lipid peroxidation and this was prevented by the addition of wine. The wine increased glucose uptake into erythrocytes and was able to decrease the osmotic fragility of erythrocytes incubated with fructose. Altogether, these results suggest that wine leads to a reduction of the oxidative stress induced by high concentrations of glucose and fructose.

Phytochemistry Profile, Antimicrobial and Antitumor Potential of the Methanolic Extract of Tabernaemontana catharinensis A DC and Eragrostis plana NEES.

Natural compounds that have the potential to act as antimicrobials and antitumors are a constant search in the field of pharmacotherapy. Eragrostis plana NEES (Poaceae) is a grass with high allelopathic potential. Allelopathy is associated with compounds generated in the primary and secondary metabolism of the plant, which act to protect it from phytopathogens. Tabernaemontana catharinensis A DC (Apocynaceae), a tree in which its leaves and bark are used for the preparation of extracts and infusions that have anti-inflammatory and antinociceptive effects, is attributed to its phytochemical constitution. The objective of this study was to elucidate the phytochemical constitution, the antibacterial potential, the toxicity against immune system cells, hemolytic potential, and antitumor effect of methanolic extracts of E. plana and T. catharinensis. The phytochemical investigation was carried out using the UHPLC-QTOF MS equipment. The antibacterial activity was tested using the broth microdilution plate assay, against Gram-negative and Gram-positive strains, and cytotoxicity assays were performed on human peripheral blood mononuclear cells (PBMC) and in vitro hemolysis. Antitumor activity was performed against the colon cancer cell line (CT26). Results were expressed as mean and standard deviation and analyzed by ANOVA. p < 0.05 was considered significant. More than 19 possible phytochemical constituents were identified for each plant, with emphasis on phenolic compounds (acids: vanillic, caffeic, and quinic) and alkaloids (alstovenine, rhyncophylline, amezepine, voacangine, and coronaridine). Both extracts showed antibacterial activity at concentrations below 500 µg/mL and were able to decrease the viability of CT26 at concentrations below 2000 µg/mL, without showing cytotoxic effect on PBMCs and in vitro hemolysis at the highest concentration tested. This is the first report of the activity of E. plana and T. catharinensis extracts against colon cancer cell line (CT26). Studies should be carried out to verify possible molecular targets involved in the antitumor effect in vivo.

Fragmentation of Cannabinoids by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS).

BACKGROUND: The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered in cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of diverse origin. OBJECTIVE: In this work, flow injection analysis-tandem mass spectrometry with electrospray ionization (FIA-ESI-MS/MS) in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid, Δ8-tetrahydrocannabinol, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid and cannabinol. METHODS: By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). RESULTS: When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA-ESI-MS/MS data yielded satisfactory classification. CONCLUSION: Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding more accurate investigations in terms of chemical composition. HIGHLIGHTS: This study of the cannabinoid fragmentation pattern by flow injection MS showed that cannabinoids can be distinguished by their fragmentation spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed classification of different cannabis samples.

Sida tuberculata: In vitro cytotoxicity and in vivo anti-inflammatory effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Sida tuberculata R. E. Fries (Malvaceae) is a pioneer species considered a weed in farm fields in Southern Brazil. Widely distributed in South Brazil, S. tuberculata is popularly used to treat inflammatory conditions. AIMS OF THE STUDY: The current study aimed to assess the in vitro cytotoxic and in vivo anti-inflammatory properties of S. tuberculata. MATERIALS AND METHODS: Initially, extracts obtained from leaves (STLE) and roots (STRE) were submitted to cytotoxicity tests using human leukocytes (non-malignant cell line) and HepG2 and MCF-7 (tumor cell lines). In sequence, anti-inflammatory properties were investigated against carrageenan-induced peritonitis model. RESULTS: In vitro analyses displayed a significant decrease in human leukocytes viability without genotoxic damage. IC50 results from tumor cells presented significant decrease in cell viability, slightly more pronounced for STRE. In addition, STLE significantly inhibited the inflammatory and oxidative parameters (TBARS, NPSH, SOD, MPO activity, cell influx, and cytokines release). CONCLUSION: Our findings indicate S. tuberculata extracts have cytotoxic potential more pronounced on tumor cell lines, as well as leaves extract shows a significant reduction in acute inflammation process, as already reported for Sida genus and specifically for this species.

Analytical Study of the Antifungal Posaconazole in Raw Material: Quantitative Bioassay, Decomposition Chemical Kinetics, and Degradation Impurities by LC-QTOF-MS.

BACKGROUND: Posaconazole is a triazole antifungal drug that was approved by the U.S. Food and Drug Administration in 2006. No bioassay of it is available in the literature nor official codes for potency determination in bulk. OBJECTIVE: To conduct an analytical study focused on posaconazole in bulk. METHODS: An alternative microbiological assay was validated for drug quantitation, applying agar diffusion technics (3 × 3 design), using Saccharomyces cerevisiae ATCC MYA 1942 as a test microorganism (2% inoculum). An isocratic HPLC-DAD method, with C8 Shim-pack column (250 × 4.6 mm, 5 µm) and methanol-water (75:25 v/v) mobile phase was used for stress stability by photolysis and oxidation, indicating the formation of degradation products, which were investigated by ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry. RESULTS: The established conditions for the bioassay were satisfactory. It was linear in the range evaluated (2.5-10.0 µg/mL), as well as precise, accurate, and robust. Stress tests showed drug susceptibility to the factors evaluated (60% of degradation after 120 min). Kinetics curves for photolytic decomposition followed first-order kinetics. From a photolytic and oxidative degraded matrix, three major degradation products were identified as being derivatives with modifications in the piperazine central ring and in the triazole and triazolone side chains, whose mass spectra results were m/z 683 (DP1), m/z 411 (DP2), and m/z 465 (DP3). CONCLUSIONS: The microbiological method was adequately validated and demonstrated to be equivalent to physico-chemical ones. The impurities found are described for the first time in studies with posaconazole raw material. HIGHLIGHTS: A microbiological bioassay was developed for posaconazole, first-order kinetics was determined for photolytic degradation, and structures for new degradation products were suggested.

Cuphea spp.: antichemotactic study for a potential anti-inflammatory drug.

Cuphea genus (Lythraceae) popularly known in Brazil as "sete-sangrias", it's described as antimicrobial, anti-inflammatory, diuretic and antihypertensive mainly. Investigating the chemotactic ability plays an important role in the identification of new anti-inflammatory agents. Thus, this research aims to assay the antichemotactic activity of hydroethanolic extracts of C. calophylla, C. carthagenensis, C. glutinosa, and C. racemosa as well as the compounds miquelianin and myricitrin. The antichemotactic activity of the hydroethanolic extracts, miquelianin, and myricitrin were assayed at concentrations 0.001 to 10 µg/mL in the lipopolysaccharide-induced chemotaxis on rat polymorphonuclear neutrophils. All the assayed samples displayed antichemotactic activity with reduction of the neutrophil migration in the range of 4.46-100%, and an IC50 value in the range of 0.30-1.24 µg/mL. Thus, this study demonstrates that the extracts hydroethanolic of Cuphea species, miquelianin, and myricitrin display a significant antichemotactic activity. Therefore, in future studies, extracts from Cuphea spp. could be used as anti-inflammatory drugs.

Ultrasound-assisted extraction optimization and validation of ultra-performance liquid chromatographic method for the quantification of miquelianin in Cuphea glutinosa leaves.

Cuphea glutinosa is a medicinal species abundant in South of Brazil, known because of its flavonoids, which have pharmacological properties as antioxidant, anti-hypertensive, diuretic, and antimicrobial. The present study aimed to optimize the extraction and validate an ultra-performance liquid chromatographic method coupled to a photodiode array detector (UPLC-PDA) method for the quantification of a chemical marker miquelianin in C. glutinosa leaves. The optimum conditions for the extraction of miquelianin from leaves of C. glutinosa were determined using a fractional factorial design (FFD) and a central composite design (CCD). An UPLC-PDA method was validated, following the ICH guidelines and RDC 166/2017 of ANVISA (Brazil). The extraction-optimization methodology was obtained with the following parameters: plant:solvent 1:60 (w/v), percentage solvent 38% ethanol, 60 min time, five extractions and particle size ≤ 180 µm. The validation parameters of the quantification method were satisfactory. The results revealed a method with excellent selectivity, linearity, precision (repeatability and intermediate precision were below 2.18 and 1.40%, respectively) and accuracy (mean recovery 90.6%). The average content of miquelianin was 1.03%. Briefly, the optimization of the extractive method in the leaves of C. glutinosa increased the concentration of miquelianin in the crude extract and the method was validated according to the current legislation.

Polyphenols composition from leaves of Cuphea spp. and inhibitor potential, in vitro, of angiotensin I-converting enzyme (ACE).

ETHNOPHARMACOLOGICAL RELEVANCE: Cuphea is the largest genus of the Lythraceae family. It is popularly known as "sete-sangrias" in Brazil used in folk medicine as a diuretic, antipyretic, anti-inflammatory, laxative and antihypertensive agent. The raw material of Cuphea has shown promising results in the production of fitotherapics, which are chemically characterized by quercetin core flavonoids. AIMS OF THE STUDY: Present work aims to investigate the chemical composition of Cuphea calophylla, Cuphea carthagenensis, Cuphea glutinosa and Cuphea racemosa by UHPLC-MS using ESI-Q-TOF, and also to investigate the inhibition of angiotensin-converting enzyme (ACE) in vitro. MATERIALS AND METHODS: Leaves extraction was conducted by an ultrasound-assisted system under the following conditions: 40% ethanol, particle size ≤180 µm, plant:solvent ratio 1:20 (w/v) for 30 min. The leaf extracts were analyzed by UHPLC-MS positive mode ionization. For the inhibition of ACE, the leaf extracts used were obtained from different Cuphea species collected from several regions of Rio Grande do Sul (Brazil). RESULTS: In total 26 polyphenolic compounds were proposed, which were mostly derived from quercetin, myricetin, and kaempferol. Of these compounds, ten are described in the genus for the first time. The ACE-inhibiting activities are presented in descending order: miquelianin (32.41%), C. glutinosa 1 (31.66%), C. glutinosa 5 (26.32%) and C. carthagenensis 1 (26.12%). CONCLUSION: The obtained results suggest that the ACE-inhibiting potential may be increased by the interactions among the different phytoconstituents present in the crude extract. These results corroborate with the popular usage of Cuphea genus as diuretic and antihypertensive agents in folk medicine.

Stability in clinical use and stress testing of meropenem antibiotic by direct infusion ESI-Q-TOF: Quantitative method and identification of degradation products.

An ESI-MS/MS method through direct infusion was validated for quantitative analysis of meropenem powder for injection. The validation parameters were established in a rapid analysis of 30 s. Drug stability was studied through the submission to stress testing, resulting on four degradation products. Under hydrolytic conditions, in acid, neutral and alkaline media, the major degradation product was formed through the cleavage of the ß-lactam ring. Oxidation of the drug using H2O2 (3%) showed the formation of two degradation products from a decarboxylation reaction and N-oxide formation. Under high humidity conditions, there was detected a dimer product. The stability of meropenem after reconstitution was studied in conditions that simulate its clinical use. In samples reconstituted and diluted in infusion fluids, an extensive degradation was observed. At room temperature meropenem maintained its content > 90% for up to 4 h when prepared in 5% glucose and for up to 12 h when prepared in 0.9% NaCl. Through ESI-MS/MS analyzes it was observed a degradation product formed by ß-lactam ring cleavage, detected in all conditions studied. It was also identified a degradation product formed only in 5% glucose, generated by the hydrolysis of ß-lactam followed by the attachment of a glucose molecule to the nitrogen of the pyrrolidine ring. In general, all the results obtained in the stability studies contribute to the knowledge about this antibiotic and future candidates of this class.

Antibacterial and antioxidant effects of Rosmarinus officinalis L. extract and its fractions.

The production of reactive species over physiological levels associated to pathogenic bacteria could represent a high risk for many diseases. The Rosmarinus officinalis L. is used around the world due its pharmacological proprieties. So, in this study our aim is to test for the first time if R. officinalis L. extract (eeRo) and its fractions (DCM, EA, ButOH) could have better or similar antioxidant action to standars and among themselves in vitro or ex vivo, in brain, stomach and liver of rats. Moreover, we intend to clarify their possible effects on pathogenic bacteria. The eeRo was obtained from the dried leaves subjected to an alcoholic extraction and fractioned. The quantification of the constituents of eeRo and fractions were done by HPLC. The antioxidant proprieties of R. officinalis was analyzed by DPPH•- radical scavenging, total antioxidant, dichlorofluorescein, lipid peroxidation and sodium nitroprusside -induced lipid peroxidation assays. The Minimum inhibitory concentrations of R. officinalis L. were tested with standard strains of danger bacteria. The eeRo, DCM, EA had significant total antioxidant and DPPH•- radical scavenging activities. The DCM and eeRo got significant effects against basal levels of reactive species in liver, stomach and brain. The eeRo and DCM protected the liver and brain against lipid peroxidation. The eeRo, DCM, EA and ButOH had inhibitory effect in the Gram-positive and Gram-negative bacteria. In general way, the DCM and eeRo had the best antioxidant and antibacterial effects among all tested fractions.

Thermal and alkaline stability of meropenem: degradation products and cytotoxicity.

The stability of the broad-spectrum antibiotic meropenem was investigated in order to isolate and elucidate the mean degradation products involved in thermal and alkaline decomposition of meropenem in solution. The purification of thermal degradation product (45 degrees C) involved a combination of preparative chromatographic techniques. The degradates were characterized by NMR and ESI-MS. The thermal degradation product was a result of several chemical reactions, with modification of side chain and beta-lactam ring, resulting in a pyrrolic derivative. Under alkaline conditions (NaOH 0.1N), meropenem was converted totally to the corresponding beta-lactam ring-opened derivative, in sodium salt state. The degraded samples of meropenem reconstituted solution, powder for injection and alkaline solution was also studied in order to determine the preliminary cytotoxicity in vitro against mononuclear cells. The results obtained indicated that samples could be toxic in high concentration (2.0mg/mL) after 48h of incubation. The present study confirms the lability of the drug in aqueous solution, specially when submitted to thermal and alkaline conditions. Thus, it is necessary attention during the handling and storage of this antibiotic.

Extraction optimization and UHPLC method development for determination of the 20-hydroxyecdysone in Sida tuberculata leaves.

Sida tuberculata (ST) is a Malvaceae species widely distributed in Southern Brazil. In traditional medicine, ST has been employed as hypoglycemic, hypocholesterolemic, anti-inflammatory and antimicrobial. Additionally, this species is chemically characterized by flavonoids, alkaloids and phytoecdysteroids mainly. The present work aimed to optimize the extractive technique and to validate an UHPLC method for the determination of 20-hydroxyecdsone (20HE) in the ST leaves. Box-Behnken Design (BBD) was used in method optimization. The extractive methods tested were: static and dynamic maceration, ultrasound, ultra-turrax and reflux. In the Box-Behnken three parameters were evaluated in three levels (-1, 0, +1), particle size, time and plant:solvent ratio. In validation method, the parameters of selectivity, specificity, linearity, limits of detection and quantification (LOD, LOQ), precision, accuracy and robustness were evaluated. The results indicate static maceration as better technique to obtain 20HE peak area in ST extract. The optimal extraction from surface response methodology was achieved with the parameters granulometry of 710 nm, 9 days of maceration and plant:solvent ratio 1:54 (w/v). The UHPLC-PDA analytical developed method showed full viability of performance, proving to be selective, linear, precise, accurate and robust for 20HE detection in ST leaves. The average content of 20HE was 0.56% per dry extract. Thus, the optimization of extractive method in ST leaves increased the concentration of 20HE in crude extract, and a reliable method was successfully developed according to validation requirements and in agreement with current legislation.