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Echinacea purpurea is a traditional medicinal plant widely used as adjuvant for the treatment of respiratory and urinary infections. Caffeic acid derivatives are considered the main active markers, such as chicoric acid, caftaric acid and chlorogenic acid. An analytical method using ultra performance liquid chromatography (UPLC) and diode array detector was developed and validated, to quantify caffeic acid derivatives in commercial dried extracts of EP. UPLC method was developed using a C18 column (50 × 2.1 mm, 1.8 μm), at 30°C. Mobile phase was composed of acetonitrile and 0.05% (v/v) formic acid aqueous solution (10:90), flow rate 0.2 mL/min. Injection volume was 10 μL and detection was performed at 300 and 330 nm. The developed method complied with all required validation parameters, and showed to be linear, precise, accurate, selective and robust for all caffeic acid derivatives. Using the validated method, the levels of caftaric acid (0.110–0.507%w/w), chicoric acid (0.040–0.179%w/w) and chlorogenic acid (0.013–0.084%w/w) were determined in five commercial dried extracts of E. purpurea, with significant variation in the contents between different samples, indicating the need of standardization and control of individual caffeic acid derivatives in commercial extracts.

Background: Punica granatum L. (pomegranate) is one of the oldest edible fruits used for the treatment of countless diseases. The bioactivity of this fruit is mainly related to tannins, especially elagitannins. P.granatum fruit peel dried extracts (PPDEs) are widely marketed today and the lack of accessible quality control methods is a key to the variable and questionable quality of these products. Objective: To develop a miniaturized spectrophotometric method for quantification of tannins in commercial PPDEs. Method: Spectrophotometric method using Folin-Ciocalteu reagent was developed and validated for quantification of total polyphenols and polyphenols not adsorbed by hide powder in 17 samples of commercial PPDEs. Tannins content was calculated by the difference between total polyphenols and polyphenols not adsorbed by hide powder contents and expressed as pyrogallol. The UV/Visible microplate spectrophotometer was operated at 760 nm. Results: The developed miniaturized method was precise, accurate, selective and linear, and has no rotational or translational matrix effect. This method significantly reduced the cost of the analysis: 82.2% with reagents, 84.7% in electricity consumption, and 56.3% in the total analysis time. The wide variability in tannins contents in commercial PPDEs ( Conclusion: A miniaturized spectrophotometric method was successfully developed and fully validated, being appropriate for quantification of tannins of PPDEs in quality control routine analysis, as it showed to be a simple and cheaper method.

The therapeutic regimen for the treatment of AIDS using efavirenz, lamivudine and tenofovir disoproxil fumarate has demonstrated efficacy and safety. In this work, an analytical method was developed and validated for simultaneous quantitation of efavirenz (EFV), lamivudine (3TC) and tenofovir disoproxil fumarate (TDF) in fixed-dose combination tablet. Analysis by high performance liquid chromatography was performed using a reversed phase column Sun Fire® C18 (250 mm x 4,6 mm; 5 μm) at 30 °C, 20 μL injection volume and detection at 260 nm. The mobile phase, composed of acetate buffer pH 5.4 and methanol, was employed at 1.0 mL/min flow rate and gradient elution. Adequate linearity was obtained in the range from 66 to 198 μg/mL for EFV, 66 to 198 μg/mL from 3TC and 33 to 99 μg/mL for TDF. Relative standard deviation values were lower than 5% and mean recoveries were within 98% to 102%, confirming the precision and accuracy of the method, respectively. In addition, the developed method showed to be selective and robust and can be successfully applied for routine quality control analysis.

Introduction: Malaria, an infectious disease caused by protozoa of the genus Plasmodium, is highly prevalent in the Brazilian Amazon. Chloroquine is the first-choice drug for the treatment of malaria caused by P. vivax and P. malariae. The humid and hot climate characteristic of the Brazilian endemic region favors drug degradation and modification of its biopharmaceutical properties, which may result in subtherapeutic dosage, formation of degradation products that can be toxic to humans and appearance of parasitic resistance. Thus, it is necessary to monitor the quality of chloroquine tablets. Materials and Methods: An analytical method was developed and validated to determine chloroquine content in tablets by ultraviolet spectrophotometry. The diluent consisted of 0.06 M monosodium phosphate buffer pH 6.8 and detection was performed at 343 nm. Results and Conclusion: The method proved to be linear in the range of 7.2 to 19.2 µg.mL-1, precise, accurate, selective, robust, and statistically equivalent to a liquid chromatographic method by the United States Pharmacopeia. The developed method was applied to determine chloroquine content in six batches of the drug. The evaluated batches were considered adequate for identification, assay, dissolution, disintegration and uniformity of dosage units, and were found to be inadequate in terms of friability.

Hypertension and dyslipidemias are among the main risk factors for the development of cardiovascular diseases, which are responsible for the death of approximately 17 million people each year. There are several drugs available for the treatment of these diseases. Therefore, methods for the simultaneous analysis of several of these drugs are useful in a wide range of situations. In this context, this study aimed to develop a modern method for the simultaneous determination of eight cardiovascular drugs in human plasma. A vortex-assisted liquid-liquid microextraction (VALLME) procedure, combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Mass spectrometry conditions, chromatographic separation, and sample preparation were optimized. For VALLME optimization, pH, sodium chloride concentration, volume of buffer solution, extraction solvent (type and volume), and vortex stirring time were evaluated. The method proved to be simple, fast, and environmentally friendly since low volumes of organic solvent were employed. Furthermore, the VALLME procedure required small sample volume, which is desirable when large volumes are scarce. Suitable recoveries and lower limits of quantification were achieved with a chromatographic run of only 8 min. The method was validated, showing to be selective, precise, and accurate. Furthermore, the analytical curves were well fitted to the selected models and the matrix effect did not affect method reliability. The developed method was successfully applied for the analysis of plasma samples obtained from volunteers attending a hospital service.

Sulphonylureas are widely used in the treatment of Diabetes mellitus, one of the main causes of death in human population. Their determination is essential in pharmacological research and in the development of new drugs. Generally, determination of sulphonylureas in biological matrices is performed using conventional sample preparation techniques, which frequently leads to an increase of analysis time and errors. In this context, a bioanalytical method for the simultaneous determination of sulphonylureas by direct injection of human plasma was developed and optimized. An automated column-switching high performance liquid chromatographic system with a restricted access media (RAM) column coupled to a fused-core column was employed. At the first dimension, a RAM column with mobile phase of ultrapure water pH 6.0 at a flow-rate of 1.0 mL min-1 was used. The valve switching time was 3 minutes. At the second dimension, a C18 guard-column coupled to a C18 fused core column with mobile phase of acetonitrile and 10 mM phosphate buffer pH 3.0 (54:46 v/v) at a flow-rate of 0.8 mL min-1 were employed. The column switching system was performed in backflush configuration with an analyte elution time of 1 minute. Flufenamic acid was used as the internal standard. The mean plasma protein exclusion percentage by the RAM-column was 104.5%. The developed and optimized method showed to be fast and simple, allowing the direct injection of biological sample into the chromatographic system and the simultaneous determination of three sulphonylureas in only 12 minutes, including the sample treatment, separation and detection.

Biodegradable polymeric nanofibers containing mometasone furoate can be a new approach to drug delivery to treat chronic rhinosinusitis, providing controlled steroid delivery to the sinonasal mucosa. This study aimed to develop biodegradable polymeric nanofibers and explore the safety of these fibers in an in vivo rabbit model. The nanofibers' development has been optimized using the Response Surface Methodology (RSM) obtained with Design of Experiments (DoE) with the best conditions related to the polymer concentration and proportion of solvents used in the electrospinning process. The nanofibers were prepared, operating as a determinant factor, the nanofiber formation and its diameter evaluated by Scanning Electron Microscopy (SEM). The ideal system obtained was assessed by SEM, thermogravimetric analysis (TGA), X-ray diffraction (XRD), differential scanning calorimetry (DSC), assay, and drug delivery by UHLPC validated method. The results showed that the drug is dispersed in the polymeric matrix, is stable, and showed sustained release kinetics in a bio-relevant nasal environment (Higuchi model kinetics). In vivo tests, the level of inflammation at the animals' mucosa which received the nanofiber with the mometasone furoate was lower than those that received the nanofibers without the drug (α = 0.05). Histopathology analysis showed that the polymeric nanofibers containing mometasone are safe when topically applied on the sinonasal mucosa, opening a new horizon in chronic rhinosinusitis treatment.

Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant widely used for the treatment of anxiety and insomnia. The aim of this study was to investigate new therapeutic applications of this plant. Nociceptive response induced by heat (hot-plate) was used as pain model. Susceptibility of different strains to kava ethanolic dried extracts was evaluated by broth microdilution method. Acute oral toxicity was performed according to Organisation for Economic Cooperation and Development (OECD) guideline. Administration of kava dried extracts and kavain inhibited the nociceptive response in the hot-plate model and did not affect the time mice spent in the rota-rod apparatus. The samples showed no significant antibacterial activity, however slight antifungal activity was verified. The extracts may be considered of low oral acute toxicity. Kava extracts exhibited promising antinociceptive activity in model of nociceptive pain, which should be deeper explored as a new therapeutic application of kava.

Introduction Dried extracts of Piper methysticum G. Forst, also known as kava, has been widely used due to its anxiolytic and sedative properties. In order to assure the quality of these extracts, it is essential to accurately quantify kavalactones, known as the active principle. Objectives To develop and validate an analytical method for the simultaneous quantification of six major kavalactones (kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin and demethoxyyangonin) in kava extracts, comparing multi-standards and single standard validation approaches. Material and methods Separation was performed using a C18 column, water/methanol/acetonitrile/2-propanol (66:07:09:18 v/v/v/v) and detection at 245 and 350 nm. A full method validation was performed, employing analytical standards for each compound. Commercial kava dried extracts were assayed and the results obtained using the method validated for six kavalactone standards were compared with those obtained when only kavain was used as standard. Results Baseline resolution for all kavalactones was obtained in short run time (15 min). Although the total kavalactone content varied between samples, a similar distribution profile was observed. When the method validated with all six analytical standards was compared to the calibration using only kavain standard, kavalactone contents were considerably different (from 7.57 to 36.53%). Conclusion The obtained results demonstrate the importance of a validated method using individual kavalactone standards for the effective quality control of kava extracts. In a next step, the method needs to be adapted to also include flavokavin B (FKB), as an important authentication marker to distinguish between the accepted variety "noble Kava" and the toxic "two-day Kava".

Introduction Arbutin is a phenol glucoside found in high concentrations in bearberry leaves and associated with the antimicrobial activity of the plant. Hydroquinone can also be found in leaves or be formed by degradation of arbutin. Lengthy exposure to free hydroquinone is associated with induction of toxicity in different organs. Objective To develop and validate a stability-indicating method by high-performance liquid chromatography diode array detector (HPLC-DAD) for simultaneous quantification of arbutin and hydroquinone in bearberry leaves and perform a comprehensive forced degradation study comparing synthetic arbutin and the arbutin in bearberry leaves. Methods Separation was performed using a C18 column, mobile phase with water-methanol (95:5), flow rate 1.0 mL/min and detection at 280 nm. Bearberry leaves were assayed and a forced degradation study of arbutin was performed in different conditions. Results The method complied with all required validation parameters. Contents varied from 1.19 to 4.15% (w/w) of arbutin and from 0.022 to 0.604% (w/w) of hydroquinone. Synthetic arbutin was susceptible to acid hydrolysis and oxidative degradation, forming hydroquinone as the main degradation product. The same study using bearberry leaves showed that constituents of the plant matrix may act as antioxidants, reducing the oxidative degradation of arbutin, however acid hydrolysis of arbutin occurred in higher intensity. Conclusion Analysis of bearberry leaves evidenced high variation in arbutin and hydroquinone levels, demonstrating the need for standardisation and control. The stability profiles of synthetic arbutin and the arbutin in bearberry leaves were considerably different and the results may be useful for determining the most appropriate conditions for extraction and production of bearberry-based formulations.

One of the highest incidences of illegal drug products is related to phosphodiesterase-5 inhibitors, used in treatment of erectile dysfunction, including those containing sildenafil citrate and tadalafil. In this context, comprehensive evaluation of the quality of genuine and illegal medicines was performed. A simple and rapid ultra-high performance liquid chromatography (UHPLC-UV) method to quantify sildenafil and tadalafil in the presence of six degradation products was developed and validated. Sildenafil and tadalafil were submitted to forced degradation. The separation was carried out on a Kinetex C18 (50 × 2.1 mm; 1.7 μm) column with mobile phase composed of acetonitrile and aqueous triethylamine solution. The calibration curves were linear in the range of 14–126 μg mL−1 for sildenafil citrate and 4–36 μg mL−1 for tadalafil and the method proved to be selective, precise, accurate and robust. Sildenafil degraded in oxidative media, whereas tadalafil degraded in acidic, alkaline and oxidative environment. The chemical structures and the mechanisms for the formation of the main degradation products were proposed by UHPLC coupled to tandem mass spectrometry. The UHPLC-UV method was applied in the pharmaceutical analysis of genuine and seized medicines. Some of them did not meet quality standards, mainly due to contents below specifications and the large variation on contents between units within a batch.

Lumefantrine is the first-choice treatment of Falciparum uncomplicated malaria. Recent findings of resistance to lumefantrine has brought attention for the importance of therapeutic monitoring, since exposure to subtherapeutic doses of antimalarials after administration is a major cause of selection of resistant parasites. Therefore, this study focused on the development of innovative, selective, less expensive and stable molecularly imprinted polymers (MIPs) for solid-phase extraction (SPE) of lumefantrine from human plasma to be used in drug monitoring. Polymers were synthesized by precipitation polymerization and chemometric tools (Box-Behnken design and surface response methodology) were employed for rational optimization of synthetic parameters. Optimum conditions were achieved with 2-vinylpyridine as monomer, ethylene glycol dimethacrylate as crosslinker and toluene as porogen, at molar ratio of 1:6:30 of template/monomer/crosslinker and azo-bisisobutyronitrile as initiator at 65 °C. The MIP obtained was characterized and exhibited high thermal stability, adequate surface morphology and porosity characteristics and high binding properties, with high affinity (adsorption capacity of 977.83 μg g−1) and selectivity (imprinting factor of 2.44; and selectivity factor of 1.48 and selectivity constant of 1.44 compared with halofantrine). Doehlert matrix and fractional designs were satisfactorily used for development and optimization of a MISPE-HPLC-UV method for determination of lumefantrine. The method fulfilled all validation parameters, with recoveries ranging from 83.68% to 85.42%, and was applied for quantitation of the drug in plasma from two healthy volunteers, with results of 1407.89 and 1271.35 ng mL−1, respectively. Therefore, the MISPE-HPLC-UV method optimized through chemometrics provided a rapid, highly selective, less expensive and reproducible approach for lumefantrine drug monitoring.

Malaria is the most incident parasite infection worldwide. Artemisinin based combination therapy (ACT) has been proposed as a promising treatment for malaria, and artemether + lumefantrine (20 + 120 mg) is the recommended association in endemic areas. Despite its widespread use, there is still scarce information about dissolution of artemether and lumefantrine, reflecting in the absence of a specific method in pharmacopoeias and international compendia. Because the of their low solubility, both artemether and lumefantrine are candidates for in vitro-in vivo correlation (IVIVC) studies. Previous equilibrium solubility studies have been carried out for both drugs using the shake-flask method and dissolution profiles. Experiments were conducted with a range of parameters such as medium composition, pH and surfactants. In vivo data obtained in a previous pharmacokinetic study was used to select the optimum conditions for dissolution test, based on IVIVC. For drug quantitation, a selective method by high performance liquid chromatography was optimized and validated. For this dosage form, the best dissolution conditions found for artemether were: paddles, 900 mL of dissolution medium containing phosphate buffer pH 6.8 with 1.0% sodium lauryl sulfate and rotation speed of 100 rpm. The same was obtained for lumefantrine, except the dissolution medium, which was pH 1.2 with 1.0% polysorbate 80. After obtaining the curve of in vitro dissolved fraction versus in vivo absorbed fraction, the calculated coefficient of determination (R squared) was close to 1.00 for both drugs, indicating a level A correlation. Therefore, a novel method for assessing dissolution of arthemeter and lumefantrine tablets was established and validated.

Etoposide-loaded poly(lactic-co-glycolic acid) implants were developed for intravitreal application. Implants were prepared by a solvent-casting method and characterized in terms of content uniformity, morphology, drug-polymer interaction, stability, and sterility. In vitro drug release was investigated and the implant degradation was monitored by the percent of mass loss. Implants were inserted into the vitreous cavity of rabbits' eye and the in vivo etoposide release profile was determined. Clinical examination and the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method were performed to evaluate the implant tolerance. The original chemical structure of the etoposide was preserved after incorporation in the polymeric matrix, which the drug was dispersed uniformly. In vitro, implants promoted sustained release of the drug and approximately 57% of the etoposide was released in 50 days. In vivo, devices released approximately 63% of the loaded drug in 42 days. Ophthalmic examination and HET-CAM assay revealed no evidence of toxic effects of implants. These results tend to show that etoposide-loaded implants could be potentially useful as an intraocular etoposide delivery system in the future.

Malaria is the most common parasitic disease in humans. It is estimated that 3 billion people live under the risk of contracting this disease in the world. Chloroquine (CQ) is the drug of choice to treat cases of non-complicated malaria. Forced degradation studies are important to know the drug's potentials degradation products and to develop a stability indicating method. Thus, chloroquine active pharmaceutical ingredient (API), chloroquine tablets and placebo were submitted to a detailed forced degradation study, using several stressing agents. The results were used on the development of a stability indicating method, using high performance liquid chromatography. The method was validated showing selectivity, precision, accuracy, robustness and linearity in the range of 30-360μg/mL of chloroquine. Chloroquine API and tablets were susceptible to alkaline hydrolysis with NaOH 1mol/L, and to oxidation with H2O2 3.0%. Two degradation products were formed in oxidative test. Kinetics of chloroquine degradation in alkaline hydrolysis was performed for both API and tablets. The calculated decay constant (k1) was 0.223days-1 for API and 0.182days-1 for tablets, while the half-life (t1/2) was 3.1days for API and 3.8days for tablets. Chemical structures have been proposed for the two degradation products formed in the presence of H2O2, using an UHPLC-UV-MS/MS approach.

Diabetes mellitus is a chronic disease with high and growing prevalence worldwide. Therefore, the development of fast and efficient methods for the QC ofantidiabetic drugs is of fundamental importance. Twoultra-fast methods, using a conventional (C18 100 × 2.1 mm, 5 μm fully porous particle) column or a fused-core (C18 100 × 2.1 mm, 2.7 μm fused-core particle) column, were developedfor the simultaneous determination of four antidiabetic drugs (chlorpropamide, glibenclamide, gliclazide, and glimepiride). The developed methods were compared in terms of efficiency, speed of analysis, resolution, and peak symmetry. Both methods were validated with respect to selectivity, system suitability, linearity, precision, accuracy, LOD, LOQ, and robustness, using glibenclamide as model. Conventional and fused-core methods were shown to be appropriate for the determination of glibenclamide in tablets; however, the fused-core column presented higher efficiency, detectability, and resolution. Also, it enabled faster analysis, with separation of the four drugs inless than 1 min.

Tuberculosis is mainly treated by fixed dose combination therapy containing mainly rifampicin, isoniazid, pyrazinamide and ethambutol. The quality control of these drugs in these formulations is very important because of high incidence of resistance to these drugs. Most of the analytical methods developed for the quantification analyze these drugs individually and they are laborious, costly, and time consuming. The present work aims at developing an alternative analytical method addressing these limitations of the traditional methods A Raman spectroscopic method was developed to simultaneously estimate all these four first line antituberculosis drugs. This method has addressed all these limitations of traditional methods as it was very simple, rapid and cost effective. Calibration and validation samples of these drugs were prepared with a wider range and analyzed by both Raman and HPLC. Various figures of merit were determined for the developed method to evaluate the efficacy and quality of the method. Partial Least Squares regression (PLS) approach was used in Raman spectroscopic method to quantify the drugs. Correlation graphs and paired t test were used to compare these two analytical. Based on the results obtained, Raman spectroscopic method was found to be an efficient alternative for the simultaneous estimation of rifampicin, isoniazid, pyrazinamide and ethambutol. These results also indicate that the developed Raman method can be used as a screening technique in the quality control of fixed dose combination products for the therapy of tuberculosis.

The clinical response to donepezil in patients with mild and moderate dementia was investigated in relation to the drug plasma concentration and APOE and CYP2D6 polymorphisms. In a prospective naturalistic observational study, 42 patients with Alzheimer's disease (AD) and AD with cerebrovascular disease who took donepezil (10 mg) for 12 months were evaluated. Their DNA was genotyped, and the donepezil plasma concentrations were measured after 3, 6, and 12 months. Good responders scored ≥-1 on the Mini-Mental State Examination at 12 months in comparison to the baseline score. The study results indicated the good response pattern was influenced by the concentration of donepezil, but not by APOE and CYP2D6 polymorphisms.

Glaucoma is a heterogeneous group of multifactorial optic neuropathies and the leading cause of irreversible blindness and visual impairment. Epidemiological data has estimated that in 2020 there will be more than 80 million individuals affected by the disease worldwide. Nowadays, intraocular pressure (IOP) lowering is carried out mainly by pharmacotherapy, with different drugs. The study of ocular pharmacokinetics of antiglaucoma drugs, crucial for better understanding of ocular distribution, bioavailability, and pharmacodynamic parameters, can benefit the development of antiglaucoma drugs or formulations. Bioanalysis of drugs in ocular matrices is still underestimated, since it is challenging and rarely performed. Therefore, this review summarized the chromatographic methods employed for the quantification of several antiglaucoma drugs in different ocular matrices, discussing bioanalytical steps, such as sample preparation, separation, and detection. Animals and matrices as well as the challenges faced in ocular bioanalysis were also discussed. Ocular bioanalysis has been performed mainly in rabbits, the most adequate animal model for ocular studies. The matrix most used is aqueous humor, because it is cleaner and easier to sample. Sample preparation was carried out primarily employing classic techniques, such as liquid-liquid extraction, protein precipitation, and solid-phase extraction, with conventional solvents and sorbents. Chromatographic separation was achieved predominantly by reversed-phase liquid chromatography. Ultraviolet spectrophotometry and tandem mass spectrometry prevailed for detection, although other techniques, such as fluorimetry, have also been used. It was evidenced that more efforts must be directed towards miniaturized, eco-friendly, and non-terminal sampling for sample preparation. In its turn, ultra high-performance liquid chromatography and mass spectrometry should gain prominence in ocular bioanalysis for separation and detection, respectively, since it combines high separation capacity with selectivity and sensitivity, in addition to being an environmental friendly approach.

The emergence of fluconazole-resistant Cryptococcus gattii is a global concern, since this azole is the main antifungal used worldwide to treat patients with cryptococcosis. Although pharmacokinetic (PK) and pharmacodynamic (PD) indices are useful predictive factors for therapeutic outcomes, there is a scarcity of data regarding PK/PD analysis of antifungals in cryptococcosis caused by resistant strains. In this study, PK/PD parameters were determined in a murine model of cryptococcosis caused by resistant C. gattii. We developed and validated a suitable liquid chromatography-electrospray ionization tandem mass spectrometry method for PK studies of fluconazole in the serum, lungs, and brain of uninfected mice. Mice were infected with susceptible or resistant C. gattii, and the effects of different doses of fluconazole on the pulmonary and central nervous system fungal burden were determined. The peak levels in the serum, lungs, and brain were achieved within 0.5h. The AUC/MIC index (area under the curve/minimum inhibitory concentration) was associated with the outcome of anti-cryptococcal therapy. Interestingly, the maximum concentration of fluconazole in the brain was lower than the MIC for both strains. In addition, the treatment of mice infected with the resistant strain was ineffective even when high doses of fluconazole were used or when amphotericin B was tested, confirming the cross-resistance between these drugs. Altogether, our novel data provide the correlation of PK/PD parameters with antifungal therapy during cryptococcosis caused by resistant C. gattii.

Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, is a drug widely used in the treatment of Acquired Immunodeficiency Syndrome (AIDS). The evaluation of NVP stability is of fundamental importance in order to guarantee drug product efficacy, safety and quality. In this study, NVP active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, employing several degrading agents (acid, alkaline, water, metal ions, humidity, heat, light and oxidation agents). In order to determine NVP and the degradation products formed, a stability-indicating UHPLC method using fused core column was developed and validated. The separation was carried out using a Poroshell 120C18 column (100×2.1mm i.d.; 2.7μm particle size) and the mobile phase was composed of acetonitrile and water in a gradient elution, at a flow rate of 0.2ml/min. Chemical structures and mechanisms for the formation of three degradation products were proposed by means of LC/MS-MS. Also, NVP degradation kinetic was studied and its order of degradation evaluated. NVP was degraded in acidic and oxidative conditions and the degradation profile for NVP tablets and API were similar. The stability-indicating method proved to be selective for NVP and its degradation products. Calibration curve was linear in the range of 8-48μg/ml and the method showed to be precise, accurate and robust for both NVP API and tablets, with detection and quantification limits of 0.092μg/ml and 0.174μg/ml, respectively.

Angiotensin II receptor antagonists are one of the most widely used classes of antihypertensive drugs. In this study, an HPLC fluorescence method after protein precipitation (PPT) extraction was developed and validated for determination of olmesartan, losartan, irbesartan, and valsartan in human plasma. The separation was carried out on a Luna cyano (250 × 4.6 mm i.d.; 5 μm particle size) column and the mobile phase was composed of acetonitrile and 0.1 % phosphoric acid in gradient elution, at a flow rate of 1.2 mL min-1. A PPT method was optimized by a two-level factorial design with triplicate at the central point. The parameters that could affect the extraction (sample volume and acetonitrile/plasma volume ratio) were evaluated and the method was compared to microextraction by packed sorbent (MEPS) and liquid-liquid extraction (LLE). The developed method allowed the simultaneous quantification of the analytes employing a simple and cheap sample preparation method and a short chromatographic run (13 min). This method was fully validated showing selectivity, precision, accuracy, and linearity over the range of 25.0-1500.0 ng mL-1 for olmesartan and valsartan, 25.0-2500.0 ng mL-1 for irbesartan, and 35.0-2500.0 ng mL-1 for losartan. Finally, the method was successfully applied in the analysis of human plasma from volunteers.

A simple and fast bioanalytical method for the quantification of kavain in mice plasma was developed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A full method validation was performed, according to regulatory guidelines, employing isotopically labeled kavain as the internal standard (racemic-kavain-d3). For the quantification, [M+H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) was employed using a quadrupole-linear ion trap (4000 QTRAP®) instrument. The monitored MRM transitions were 231.0 → 115.1 and 231.0 → 152.8 for kavain; and 234.2 → 199.2 for the internal standard. A linear response was obtained at the concentration range of 10 to 200 ng/mL with intra- and inter-day variations within the acceptable criteria for all quality control samples. After validation, the method was successfully applied for the quantification of kavain in mice plasma after oral administration of the kavain standard and Kava-kava extract. The plasma concentration over time results were applied for a pharmacokinetics study. The obtained pharmacokinetic parameters indicated a considerably higher bioavailability for kavain when Kava-kava extract was administered due to a pharmacokinetic synergism between the analyte and the other compounds present in the extract.

The activities of 2-phthalimidethyl nitrate (PTD-NO) and 2-phthalimidethanol (PTD-OH) were recently demonstrated in models of pain and inflammation. We expanded our investigation by evaluating their activities in models of nociceptive and inflammatory pain and inflammatory edema, the preliminary pharmacokinetic parameter for PTD-NO and the role of opioid and cannabinoid pathways in the activity of analogs. Per os (p.o.) administration of PTD-NO or PTD-OH, 1h before intraplantar injection of formaldehyde, inhibited both phases of the nociceptive response (500 and 750 mg/kg) and paw edema (125, 250, 500 and 750 mg/kg). After p.o. administration of PTD-NO, peak plasma concentrations of PTD-NO and PTD-OH were found 0.92 and 1.13 h, respectively. The plasma concentrations of PTD-NO were higher than those of PTD-OH. Intraperitoneal (i.p.) administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists (4 or 8 mg/kg, -30 min) or opioid antagonist naltrexone (5 or 10mg/kg, -30 min) did not affect the antinociceptive activities of the analogs. AM251 (8 mg/kg, i.p., -30 min) attenuated the antiedematogenic activity of both analogs, while naltrexone (10mg/kg, i.p., -30 min) only attenuated the antiedematogenic activity of PTD-NO. The antiedematogenic activities of both analogs were not affected by the CB2 cannabinoid antagonist AM630 (4 or 8 mg/kg, i.p., -30 min). Concluding, we expanded the knowledge on the activities of PTD-NO and PTD-OH by showing that these phthalimide analogs also exhibit marked activity in models of nociceptive and inflammatory pain and inflammatory edema. Opioid and cannabinoid mechanisms partially mediate the anti-inflammatory, but not the antinociceptive activity.

A stability indicating HPLC method to determine diltiazem hydrochloride (DTZ) in tablets and compounded capsules was developed and validated according to Brazilian and the International Conference on Harmonization (ICH) guidelines. The separation was carried out on a Purospher Star® C18 (150 x 4.6 mm i.d., 5 µm particle size, Merck Millipore) analytical column. The mobile phase consisted of a 0.05% (v/v) trifluoroacetic acid aqueous solution and a 0.05% trifluoroacetic acid methanolic solution (44:56, v/v). The flow rate was 1.0 mL.min-1 with a run time of 14 minutes. The detection of DTZ and degradation products (DP) was performed at 240 nm, using a diode array detector. The method proved to be linear, precise, accurate, selective, and robust, and was adequate for stability studies and routine quality control analyses of DTZ in tablets and compounded capsules. Um método de HPLC indicador de estabilidade para determinação de cloridrato de diltiazem (DTZ) em comprimidos e cápsulas manipuladas foi desenvolvido e validado de acordo com as diretrizes brasileiras e da Conferência Internacional de Harmonização (ICH). A separação foi realizada em uma coluna analítica Purospher Star® C18 (150 x 4,6 mm i.d., tamanho de partícula de 5 µm, Merck Millipore). A fase móvel consistiu de uma solução aquosa de ácido trifluoroacético a 0,05% (v/v) e uma solução metanólica de ácido trifluoroacético a 0,05% (44:56, v/v). A vazão foi de 1,0 mL.min-1 com tempo de corrida de 14 minutos. A detecção de DTZ e produtos de degradação (DP) foi realizada em 240 nm, utilizando um detector de arranjo de diodos. O método mostrou-se linear, preciso, exato, seletivo e robusto, sendo adequado para estudos de estabilidade e análises de rotina de controle de qualidade de DTZ em comprimidos e cápsulas manipuladas.

Tuberculosis is the second most deadly infectious disease, surpassed only by HIV/AIDS, and has resulted in over 1 billion deaths in the last 200 years. The World Health Organization estimates that in 2014, 9.6 million people were infected by this disease and 1.5 million had died. First-choice treatment consists of fixed-dose combination tablets containing rifampicin, isoniazid, pyrazinamide, and ethambutol hydrochloride (4-FDC). There are pharmacopeial protocols available to test 4-FDC, but they are prolonged, two-step methods. One single-step method in the literature performs the simultaneous determination by HPLC, but requires a long acquisition time. In this context, an ultra-HPLC (UHPLC) method was developed based on the HPLC method with the objective of reducing analysis time. A C18 column (1.9 µm particle size) was used with UV–diode-array detection at 238 and 282 nm. The method was found to be selective, linear, exact, precise, and robust. Samples from two batches were analyzed and the results compared with those obtained by the HPLC method, with no statistically significant differences observed (P > 0.05). This UHPLC method reduced the analysis time from 17 to 4 min, with a more than 90% reduction in sample and reagent consumption and a financial economy of almost 50-fold.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico An historical review indicated that the provision of proficiency testing (PT) in the pharmaceutical area in Brazil is critical. Three providers were identified in the period between 2002 and 2015; only one provider was regular, but none was accredited according to the requirements of ISO 17043. A PT was organized without registration fees for educational purposes for the determination of mefenamic acid (2-[(2,3-dimethylphenyl)amino]benzoic acid) in raw materials. Proficiency test items consisted of a mixture of mefenamic acid and polyvinylpyrrolidone in the mass ratio 90:10. This proportion was used to simulate an adulterated raw material. Homogeneity and stability testing was performed and indicated that the materials were suitable for use in the PT scheme. First, 68 pharmaceutical laboratories, of which five were ISO 17025 accredited, were contacted to disseminate information about the specific PT and the benefits of this important quality assurance tool. Then, the proficiency test items were sent to the 13 registered participants, of which two were ISO 17025 accredited. In addition, the participating laboratories were surveyed regarding the technical and management aspects of their quality management system (QMS), such as the status of the accreditation process according to the ISO 17025 requirements, staff training, equipment calibration and participation in other PT schemes. All 11 participants that reported results in the study adopted the United States Pharmacopoeia Official Method. Only one laboratory, representing 9.1 % of the participants, had an unsatisfactory z-score result. Factors such as the reduced number of ISO 17025 accredited laboratories, the low number of participants in the educational PT, the high proportion of laboratories that participated for the first time and the portion of the participants who did not know about PT and its importance were considered as determinants for the restricted availability of the PT supply for medicines, as evidenced in the present study in Brazil. Thus, an urgent need for nationwide actions was identified to promote the critical consciousness of managers regarding QMS in pharmaceutical laboratories, including the importance of participation in PT schemes, as well as the importance of encouraging the providers already structured to broaden their scope in this area.

Prednisone is an anti-inflammatory steroid drug widely used in clinical practice. However, no high-performance liquid chromatographic (HPLC) method has been described in the literature for the determination of prednisone in capsules until now. Thus, an HPLC method was developed using a C18 (250x4.0, 5 µm) column, with methanol:water (70:30) as mobile phase at a flow rate of 1 mL/min and detection at 240 nm. The developed method was validated following current Brazilian legislation. Additionally, linearity was assessed by evaluating the assumptions of normality, homoscedasticity, and independency of residuals, and the fit to the linear model. The method showed linearity (r2>0.99) over the range of 14.0-26.0 µg/mL, selectivity, precision (RSD

Nicorandil (N-(2-hydroxyethyl)nicotinamide nitrate) is an antianginal drug, which activates guanylyl cyclase and opens the ATP-dependent K(+) channels, actions that have been suggested to mediate its vasodilator activity. We synthesized nicorandil and its two isomers, which vary in the positions of the side chain containing the nitric oxide (NO) donor, and also their corresponding denitrated metabolites. The activities of these compounds were evaluated in an experimental model of pain in mice. Pharmacokinetic parameters of nicorandil and its isomers, as well as the plasma concentrations of the corresponding denitrated metabolites and also nicotinamide and nitrite were determined. Nicorandil exhibited the highest antinociceptive activity, while the ortho-isomer was the least active. Nicorandil and para-nicorandil, which induced higher plasma concentrations of nitrite, exhibited higher antinociceptive activity, which suggests that the release of NO may mediate this activity.

An improved LC-MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide-d3 was used as internal standard (IS) and liquid–liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP-column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 m/z 188.9 and m/z 367.0 m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25–50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration–time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratorios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption. Copyright © 2014 John Wiley & Sons, Ltd.

The present study developed and validated an HPLC method for the simultaneous determination of artesunate (AS) and mefloquine hydrochloride (MQ) in fixed-dose combination tablets, according to ICH guidelines. The chromatographic separation was carried out on an XBridge C18 (250 x 4.6 mm i.d., 5 µm particle size, Waters) analytical column. The mobile phase included a 0.05 M monobasic potassium phosphate buffer (pH adjusted to 3.0 with phosphoric acid) and acetonitrile (50 + 50, v/v). The flow rate was 1.0 mL/min, and the run time was 13 minutes. A dual-wavelength approach was employed: AS detection was performed at 210 nm and MQ was detected at 283 nm, using a diode array detector. Stability of sample solutions was evaluated for 8 hours after preparation, during which time the solutions remained stable. Youden's test was employed to evaluate robustness. The method proved to be linear (r²>0.99), precise (RSD0,99), preciso (DPR

Mycophenolic acid (MPA) is an immunosuppressive agent widely used in the treatment of solid organ transplant rejection. The success of MPA in the treatment of inflammatory intraocular diseases has been reported in recent literature. The treatment of inflammatory eye diseases in the posterior chamber is a challenge due to the anatomy of the eye, which presents certain barriers to drug access. Thus, the bioavailability of drugs in the eye is quite low, and successful drug delivery may well represent a key limiting factor to attaining a successful therapeutic strategy. Ophthalmic controlled drug delivery offers the potential to enhance the efficacy of treatment for pathological conditions. Thus, a novel delivery system based on a biodegradable polymeric device, which can be implanted inside the eye and deliver MPA directly to the target, is being developed. Specific analytical methods to determine the use of effective drugs within the eye are needed to characterize this device. A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the quantitation of MPA in the vitreous humor of rabbits was developed and validated. The vitreous was collected from rabbits, extracted by a protein precipitation extraction procedure and then separated on a C 18 column with a mobile phase comprised of 0.15% aqueous acetic acid and methanol (60:40, v/v). The calibration curve was constructed within the range of 3–10,000 ng/mL for MPA. The mean R.S.D. values for the intra-run and inter-run precision were 5.15% and 4.35%. The mean accuracy value was 100.16%. The validated method was successfully applied to determine the MPA concentration in the vitreous humor of rabbits treated with an ocular implantable device.

A new method was developed for the quantitation of 3-α-hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3-α-Hydroxy tibolone was extracted by a liquid–liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p-toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C18 column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3-α-hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 m/z 170.1 and m/z 417.0 m/z 357.1 for 3-α-hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100–30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3-α-hydroxy tibolone. No matrix effect or carry-over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers. Copyright © 2013 John Wiley & Sons, Ltd.

Nicorandil (2-nicotinamide ethyl nitrate), an antianginal drug characterized by the coupling of nicotinamide with a nitric oxide (NO) donor, activates guanylyl cyclase and opens ATP-dependent K(+) channels. In the present study, we investigated the effects induced by per os (p.o.) administration of nicorandil (12.5, 25 or 50 mg/kg) or equimolar doses (corresponding to the highest dose of nicorandil) of N-(2-hydroxyethyl) nicotinamide (NHN), its main metabolite, or nicotinamide in the model of nociceptive response induced by formaldehyde in mice. Nicorandil, but not NHN or nicotinamide, inhibited the second phase of the nociceptive response. This activity was observed when nicorandil was administered between 30 and 120 min before the injection of formaldehyde. Ipsilateral intraplantar injection of nicorandil (125, 250 or 500 μg/paw) did not inhibit the nociceptive response. After p.o. administration of nicorandil (50 mg/kg), peak plasma concentrations of this compound and NHN were observed 0.63 and 4 h later, respectively. Nicotinamide concentrations were not increased after administration of nicorandil. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 or 2 mg/kg), a guanylyl cyclase inhibitor, partially attenuated the antinociceptive activity of nicorandil. However, this activity was not changed by glibenclamide (30 or 60 mg/kg), an inhibitor of ATP-dependent K(+) channels. In conclusion, we demonstrated the antinociceptive activity of nicorandil in a model of pain that exhibits both a nociceptive and an inflammatory profile. This activity is not mediated by nicotinamide or NHN. The coupling of an NO-donor to nicotinamide results in a compound with an increased potency. The NO-cGMP pathway, but not ATP-dependent K(+) channels, partially mediates the antinociceptive activity of nicorandil.

Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii . Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii .

BACKGROUND A liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of endogenous uracil (U) and dihydrouracil (UH2) was developed and tested in a Brazilian population of patients with gastrointestinal cancer previously exposed to 5-fluorouracil (5FU). METHODS The analytes were extracted by a liquid-liquid method using 5-clorouracil as internal standard. The separation was performed on a reversed-phase XTerra C18 column with a mobile phase composed of methanol and aqueous 0.1% ammonium hydroxide (15:85). Mass spectrometry detection was carried out using negative electrospray ionization and selected reaction monitoring. Bovine serum albumin was employed as an alternative matrix to prepare the calibration standards, aiming to avoid the measurement of physiologic U and UH2. Calibration curves were constructed over the range of 5-200 ng/mL for U and 10-500 ng/mL for UH2. RESULTS The mean RSD values in the intrarun precision were 6.5% and 10.0% and in the interrun precision were 7.8% and 9.0% for U and UH2, respectively. The mean accuracy values were within the range of 90%-110% for both analytes. The analytes were stable in plasma under different conditions of temperature and time. The validated method was successfully applied to determine the plasma concentrations of U and UH2 in patients with gastrointestinal cancer (n = 32) previously treated with 5FU and for whom clinical toxicity was well documented. U concentrations varied from 21.8 to 56.6 ng/mL, whereas UH2 concentrations varied from 57.7 to 271.5 ng/mL. UH2/U ratio ranged from 1.56 to 6.18. CONCLUSIONS The method has proved to provide a quick, reliable, and reproducible quantitation of the plasma concentrations of U and its metabolite UH2. The UH2/U ratios did not discriminate patients previously exposed to 5FU with and without severe toxicities, possibly due to the small sample. Further studies in a larger population are desirable.