Development of a validated stability-indicating HPLC- DAD method for dasabuvir and the characterization of its degradation products using LC-QToF-MS/MS

Abstract A stability-indicating HPLC-DAD method was developed and validated for the simultaneous determination of dasabuvir and its degradation products in the pharmaceutical formulation. The proposed method utilized a Symmetry® C18 (4.6 x 75 mm, 3.5 µm) column, and the mobile phase consisted of an isocratic elution of formic acid (0.1%) and acetonitrile (55:45, v/v), at a flow of 1 mL min-1; analytes were detected at 244 nm. Dasabuvir was submitted to different stress degradation conditions, such as acidic, alkaline, neutral, thermal, oxidative and photolytic, and the structural elucidation of degradation products was performed using LC-QToF-MS/MS. The HPLC-DAD stability-indicating method was validated for selectivity, linearity, limit of detection and quantification, accuracy, precision and robustness, according to ICH guidelines. Dasabuvir produced two degradation products (DP1 and DP2) from the alkaline stress conditions, which were characterized in negative ion mode. Dasabuvir was linear in the range 9.78 to 136.92 µg mL-1, and DP and DP were linear in the range 2.9 to 20.2 µg mL-1 and 1.3 to 14.9 µg mL-1, respectively. The 1 2 recovery ranged between 99.16 and 100.86%, while precision ranged from 1.02 to 2.89%. As the method can effectively separate the dasabuvir from its degradation products and quantitate them, it may be employed as a stability-indicating method for the pharmaceutical formulation.

Sensitivity and specificity of multibacillary and paucibacillary leprosy laboratory tests: A systematic review and meta-analysis.

This systematic review (number register: CRD42018112736) was performed to compare the sensitivity and specificity of leprosy diagnostic methods. The search was conducted in 3 electronic databases in January 2021. Studies evaluating leprosy diagnostic tests were included according the eligibility criteria. Meta-analysis was performed to calculate the sensibility and specificity of the groups. We included 36 studies. The test sensitivity for paucibacillary patients was 0.31 (95%CI: 0.29-0.33) and the specificity was 0.92 (95%CI: 0.92-0.93). In multibacillary patients, the sensitivity was 0.78 (95%CI: 0.77-0.80) and specificity was 0.92 (95%CI: 0.92-0.93). Comparing the sensitivity and specificity of the different techniques included, it should be noted that polymerase chain reaction (PCR) test presented the highest sensitivity for paucibacillary patients, while the western blot technique showed the highest sensitivity for multibacillary patients. However, further studies are needed to optimise the diagnosis of leprosy, requiring research with a larger number of samples and more uniform protocols.

Morpho-anatomy of the inflorescence of Musa × paradisiaca

ABSTRACT Bananas and plantains are herbaceous monocotyledonous plants belonging to the genus Musa, Musaceae, which has a widespread distribution around the world. Various parts of banana plant are commonly used in traditional medicines. Several species of Musa are reported to possess anti-inflammatory, anti-hyperglycemic and antidiabetic properties. This work is aimed at studying the morphological and anatomical characteristics of the inflorescences of Musa × paradisiaca L., that could contribute to the characterization of these species cultivated in Brazil. Plant materials were collected and prepared in accordance with standard optical microscopy techniques. Morphological characterizations were conducted using morphological descriptors for inflorescences, including some descriptors from International Plant Genetic Resources Institute for Musa spp. Microscope slides were prepared using glycol-methacrylate and were stained in toluidine blue. Main features observed for M. × paradisiaca inflorescence were amphistomatic bracts with tetracytic stomata, fiber caps next to the phloem, adaxial and abaxial uniseriate epidermis, and papillose on the abaxial face. Outer tepals have multilayer epidermis and vascular bundles aligned next to the abaxial face. Free tepal has unilayeredepidermis. Anthers are tetrasporangiate and the locules are separated by the septum. Ovary is inferior and trilocular with external unilayered and internal epidermis. The main morpho-anatomical characteristics of inflorescence of Musa × paradisiaca are highlighted in this study, contributing to provide more information about the characterization of this species cultivated in Brazil.

Effect of Different Tensoactives on the Morphology and Release Kinetics of PLA-b-PEG Microcapsules Loaded With the Natural Anticancer Compound Perillyl Alcohol.

Perillyl alcohol is a natural compound that has attracted a significant interest due to its potent antitumor activity. However, clinical trials have exhibited poor tolerance by oral administration, mainly due to gastrointestinal side effects. We propose the entrapment of perillyl alcohol into poly(D,L-lactic acid)-block-poly(ethylene glycol) (PLA-b-PEG) as delivery platform (entrapment efficiency of 63%-68%). The influence of different concentrations of the tensoactives poly(vinyl alcohol) and sodium cholate (SC) on shear strength and morphology was evaluated by confocal laser scanning microscopy and interfacial tension studies. Only the microcapsules formulated with SC maintained their sphericity when submitted to shear stress. These results indicate that the interface is better organized with SC, conferring mutual stacked packing that is able to better stabilize the organic drop. The in vitro release profile of the drug from the microcapsules was correlated with pore formation and polymer degradation, best fitted to the Baker-Lonsdale model. The loaded microcapsules showed an IC50 equivalent to that of the free drug (80 µg/mL) after 72 h of exposure. However, after 24 h of exposure, loaded microcapsules showed an IC50 almost two-fold higher (220 µg/mL) suggesting gradual release.

Simultaneous quantification of artesunate and mefloquine in fixed-dose combination tablets by multivariate calibration with middle infrared spectroscopy and partial least squares regression.

BACKGROUND: Malaria is one of the most lethal and life-threatening infectious diseases in the world, causing more than half a million deaths annually. Treatment with mefloquine and artesunate is currently recommended by the World Health Organization, and was historically the first artemisinin-based combination therapy used clinically for treatment of Plasmodium falciparum. The problem of poor-quality medicines, such as counterfeit and sub-standard anti-malarials, is a worldwide issue; therefore, it is essential to develop rapid, low cost, solvent-free, and reliable methods for routine quality control for these drugs. The aim of this study was to develop and validate a novel multivariate method for direct simultaneous quantification of mefloquine and artesunate in tablets by diffuse reflectance, middle infrared spectroscopy and partial least squares regression (MIR-PLS). METHODS: Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and partial least squares regression were applied for simultaneous quantification of artesunate and mefloquine in tablets provided by the Brazilian Government. The model was obtained with full spectra (4000-400 cm(-1)) preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centring, and built with three latent variables. The method was validated according to Brazilian and international guidelines through the measuring of figures of merit, such as trueness, precision, linearity, analytical sensitivity, selectivity, bias, and residual prediction deviation. The results were compared to an HPLC-MS/MS method. RESULTS: The MIR-PLS method provided root mean square errors of prediction lower than 2.0 mg per 100 mg of powder for the two analytes, and proved to be valid according to guidelines for analytical methods that use infrared (IR) spectroscopy with multivariate calibration. For the samples obtained from Brazilian healthcare units, the method provided results statistically similar to those obtained by HPLC-MS/MS. CONCLUSION: MIR-PLS was found to be suitable for the quality control of these drugs. It is fast, does not use solvents, and does not generate chemical waste. Furthermore, the proposed method may be transferred and developed for use in portable equipment, increasing the possibilities for assessing the quality of these drugs.

A new HILIC-MS/MS method for the simultaneous analysis of carbidopa, levodopa, and its metabolites in human plasma.

Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 µm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800 ng/mL for LEV, 65-800 ng/mL for CAR and 3-OMD, and 20-400 ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.