Chiral HPLC methods using macrocyclic glycopeptide-based chiral stationary phases have been widely used and reported; however, the development of efficient methods to separate and quantify the ...analytes with high resolution is a challenging task. Therefore, the knowledge regarding the optimization of chromatographic parameters regarding this type of chiral chromatography is essential. This review presents and discusses the optimization of HPLC conditions and parameters for the chiral resolution of racemic drugs on macrocyclic glycopeptide-based chiral stationary phases. Strategies for chiral method development are presented, using polar ionic, reversed phase, normal phase and polar organic modes. The effect of the most important chromatographic parameters, such as mobile phase composition, flow rate and temperature on the enantioseparation are discussed aiming the adequate screening and optimization protocol for each mode.
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”.
A fast and effective analytical method has been developed and validated for the simultaneous quantification of six kavalactones in kava dry extracts. When comparing the method developed using the six kavalactone standards with the kavaína‐only validated method, significant difference between the content values was observed. Although the total kavalactone content varied between samples, a similar distribution profile was observed. The results demonstrated the importance of a validated method using individual kavalactone standards for the effective quality control of kava extracts.
Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant native from South Pacific islands and widely used to treat anxiety, depression and stress. The psychoactive ...properties are related to the kavalactones, mainly kavain.
To evaluate the biopharmaceutical properties of synthetic kavain and when present in kava dried extracts by means of equilibrium solubility and intestinal permeability studies in the Caco-2 cell model.
The equilibrium solubility of kavain was performed using a shake flask incubator at 37 °C in different media at physiological pH range (1.2–6.8). The intestinal permeability of kavain evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Kavain concentrations were determined by reversed phase high performance liquid chromatography (HPLC).
HPLC methods were developed and fully validated for kavain quantitation. Kavain demonstrated low solubility and the pH of the aqueous media did not affect its solubility. Kavain was found to be highly permeable and efflux of kavain mediated by P-glycoprotein was not significant during intestinal permeation.
The results of biopharmaceutical studies provided useful information for predicting availability of kavain from the gastrointestinal tract and this compound was ranked as BCS Class II, exhibiting dissolution rate-limited absorption.
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Evaluating lumefantrine enantioselectivity and properly quantifying its enantiomers is essential for a deeper understanding of the impact of the enantioselectivity in the pharmacodynamic and ...pharmacokinetic properties of the drug. For this, a chiral chromatographic method was developed and validated for the separation of lumefantrine enantiomers. The method developed employed a Chiralpack AD-H column (150 × 4.6 mm, 5 μm), with amylose tris-3,5-dimethylphenylcarbamate stationary phase, at 25 °C, mobile phase composed of hexane and isopropanol (97:3), flow rate of 1.0 mL/min and detection at 335 nm. The method showed adequate resolution for lumefantrine enantiomers and was validated for linearity (48–72 µg/ml for each enantiomer), precision, accuracy, selectivity and robustness. The proposed method was successfully applied for determination of lumefantrine enantiomers in tablet formulations and may be useful to future researchers for chiral evaluation of lumefantrine.
Graphic Abstract
To describe and analyze the healthiness of formal and informal food establishments in bus terminals of the metropolitan region of the state of Rio de Janeiro.
An audit was conducted in 156 formal and ...127 informal food establishments located in 14 bus terminals of the five most populous cities of the metropolitan region of Rio de Janeiro. Proportions of types of establishments and means (95%CI) of food availability indicators in formal and informal settings were calculated. For the formal setting, prices, proportions of accepted payment methods, days and hours of operation, and food categories with displayed advertising were described.
The healthiness of food establishments in bus terminals was low (less than 36%). On average, ultra-processed food subgroups were 250% more available for purchase than fresh or minimally processed food. Purchasing food at these places was convenient because several forms of payment were available, and the opening hours of the establishments followed the peaks of movement. In addition, 73.3% of the advertising referred to ultra-processed drinks, and the cost-benefit of buying ultra-processed food was better than fresh or minimally processed food.
The food environment of bus terminals in the metropolitan region of Rio de Janeiro promotes unhealthy eating. Regulatory public policies should focus on initiatives to limit the wide availability and advertising of ultra-processed food in spaces of great circulation of people.
Abstract
Ravuconazole (RAV) is a triazole antifungal with broad spectrum and a novel alternative in the treatment of systemic fungal infections. A stability-indicating method by high-performance ...liquid chromatography–diode array detection was developed and fully validated to assay ravuconazole in the presence of its degradation products. Separation was achieved with a Sunfire C18 column (250 mm × 4.6 mm id, 5 μm), mobile phase composed of acetonitrile and water (80:20), at 1 mL/min. The volume of injection was 5 μL and DAD detection was performed at 287 nm. RAV was well resolved from its degradation products and the method proved to be linear, selective, accurate, precise and robust. A forced degradation study was conducted on the pure drug under oxidative conditions in presence of H2O2 and metallic ions and under acid, alkaline and neutral hydrolysis. RAV was degraded mainly under alkaline hydrolysis, forming two main degradation products. The chemical structures were proposed according to the data obtained by liquid chromatography coupled to mass spectrometry (LC-MS) analysis. This study provided a new and selective stability-indicating method to evaluate the intrinsic stability of ravuconazole in active pharmaceutical ingredients. The developed method was found to be suitable for quality control routine analysis and to stability studies of ravuconazole.
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.
The analysis of different samples of bearberry leaves by a new stability‐indicating HPLC method evidenced high variation in arbutin and hydroquinone levels, demonstrating the need for standardization of these contents. The stability of arbutin under different forced degradation conditions was evaluated both isolated and in bearberry leaves. Isolated arbutin showed to be labile to acid hydrolysis and oxidation, however the bearberry matrix prevented its oxidative degradation.
The compound 2-hydrazinyl-4-(4-methoxyphenyl)thiazole (PD76) is a novel thiazolyl hydrazine derivative with proven antifungal activity against different fungal species, mainly Candida and ...Cryptococcus. Considering the advantages of oral route for clinical therapy, the aim of this work was to evaluate the potential intestinal permeability of this new antifungal drug. For the quantitation of PD76, a high-performance liquid chromatography method was developed and fully validated. The cytotoxicity of the compound in Caco-2 cells was analyzed and intestinal permeability of PD76 was assessed by means of the comparison of in vitro assay in Caco-2 cells and in silico platforms ADMETlab and admetSAR. Cell viability above 70% was obtained at all PD76 studied concentrations. Using Caco-2 cell model, the compound showed apparent permeability coefficients (Papp) of 5.25 × 10-6 and 23.28 × 10-6 cm s-1 in apical-basolateral and basolateral-apical directions, respectively. Experiments performed using verapamil as P-gp inhibitor demonstrated that PD76 is slightly susceptible to active efflux. Both in silico platforms inferred that PD76 presents permeability in Caco-2 cells, with Log P values of 2.82 (ADMETlab) and 2.10 (admetSAR). The results obtained in permeability studies showed that PD76 presents moderate intestinal permeability and a promising profile for clinical application.
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.
Opportunistic fungal infections represent a global health problem, mainly for immunocompromised individuals. New therapeutical options are needed since several fungal strains show resistance to ...clinically available antifungal agents. 2-Thiazolylhydrazones are well-known as potent compounds against Candida and Cryptococcus species. A scaffold-focused drug design using machine-learning models was established to optimize the 2-thiazolylhydrazone skeleton and obtain novel compounds with higher potency, better solubility in water, and enhanced absorption. Twenty-nine novel compounds were obtained and most showed low micromolar MIC values against different species of Candida and Cryptococcus spp., including Candida auris, an emerging multidrug-resistant yeast. Among the synthesized compounds, 2-thiazolylhydrazone 28 (MIC value ranging from 0.8 to 52.17 μM) was selected for further studies: cytotoxicity evaluation, permeability study in Caco-2 cell model, and in vivo efficacy against Cryptococcus neoformans in an invertebrate infection model. All results obtained indicate the great potential of 28 as a novel antifungal agent.
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