Determination of pollutants, antibiotics, and drugs in surface water in Italy as required by the third EU Water Framework Directive Watch List: method development, validation, and assessment.

In this paper, we report a study concerning the quantification of new emerging pollutants in water as a request from the third European Watch List mechanism. The EU Watch List compound was investigated by an internal method that was validated in terms of detection limits, linearities, accuracy, and precision in accordance with quality assurance criteria, and it was used to monitor several rivers from 11 Italian regions. The methodology developed was satisfactorily validated from 5 to 500 ng L-1 for the emerging pollutants studied, and it was applied to different river waters sampled in Italy, revealing the presence of drugs and antibiotics. Rivers were monitored for 2 years by two different campaigns conducted in 2021 and 2022. A total of 19 emerging pollutants were investigated on 45 samples. The most detected analytes were O-desmethylvenlafaxine and venlafaxine. About azole compounds, sulfamethoxazole, fluconazole, and Miconazole were found. About antibiotics, ciprofloxacin and amoxicillin were found in three and one samples, respectively. Moreover, statistical analyses have found a significant correlation between O-desmethylvenlafaxine with venlafaxine, sulfamethoxazole with venlafaxine, and fluconazole with venlafaxine.

Water Quality, Heavy Metals, and Antifungal Susceptibility to Fluconazole of Yeasts from Water Systems.

Aquatic environments could be reservoirs of pathogenic yeasts with acquired antifungal resistance. The susceptibility to antifungal agents of yeasts present in the wastewater and natural waters of the city of Cali was evaluated. Samples were taken from two types of water: drinking water (Meléndez River, drinking water treatment plant "Puerto Mallarino" in the Cauca River) and wastewater (South Channel of the Cauca River, "Cañaveralejo-PTAR" wastewater treatment plant). Physico-chemical parameters, heavy metal concentration, and yeast levels were determined using standard procedures. Yeasts were identified using API 20 C AUX (BioMérieux) and sequence analysis of the ITS1-5.8S-ITS2 and D1/D2 regions of the large subunit of the ribosome. Susceptibility assays against fluconazole and amphotericin B using the minimum inhibitory concentration (MIC) test were determined using the microdilution method. The influence of physico-chemical parameters and heavy metals was established using principal component analysis (PCA). Yeast counts were higher at WWTP "PTAR" and lower at Melendez River, as expected. A total of 14 genera and 21 yeast species was identified, and the genus Candida was present at all locations. Susceptibility tests showed a 32.7% resistance profile to fluconazole in the order DWTP "Puerto Mallarino = WWTP "PTAR" > South Channel "Navarro". There were significant differences (p < 0.05) in the physico-chemical parameters/concentration of heavy metals and yeast levels between the aquatic systems under study. A positive association was observed between yeast levels and total dissolved solids, nitrate levels, and Cr at the "PTAR" WWTP; conductivity, Zn, and Cu in the South Channel; and the presence of Pb in the "Puerto Mallarino" DWTP. Rhodotorula mucilaginosa, Candida albicans, and Candida sp. 1 were influenced by Cr and Cd, and Diutina catelunata was influenced by Fe (p < 0.05). The water systems explored in this study showed different yeast levels and susceptibility profiles, and, therefore, possible genetic differences among populations of the same species, and different physico-chemical and heavy metals concentrations, which were probably modulating the antifungal-resistant yeasts. All these aquatic systems discharge their content into the Cauca River. We highlight the importance to further investigate if these resistant communities continue to other locations in the second largest river of Colombia and to determine the risk posed to humans and animals.

Dissipation dynamics and comparative dietary exposure assessment of mefentrifluconazole in rice.

A fast and sensitive analytical method based on UHPLC coupled with tandem mass spectrometry was established to investigate the dissipation and final residual amounts of mefentrifluconazole in rice, and dietary risk to consumers was evaluated. The method provided good linearity (R2 ≥ 0.9979), accuracy (recovery range, 79.0-101.5%), precision (relative standard deviation range, 1.3-13.9%), and sensitivity (limit of quantification, 0.005 mg/kg). The dissipation dynamics of mefentrifluconazole in rice followed first-order kinetics, with half-lives of 2.8-16.6 days. The final residues of mefentrifluconazole in various samples of harvested brown rice ranged from less than the limit of quantification to 0.092 mg/kg, the latter value being higher than the maximum residue limit recommended by the European Union. Comparative dietary exposure of mefentrifluconazole was assessed using field data and Chinese dietary patterns for different genders, regions, and age data. Although the results showed acceptable levels of risk for both acute exposure (the percentage of the acute reference dose ≤ 0.7483%) and chronic dietary intake (the percentage of acceptable daily intake ≤ 31.8516%), more studies of children are needed because they are at higher risk than other groups. This work provides the necessary data for registering and establishing the maximum residue limit for mefentrifluconazole in rice in China and reveals the potential risks to different groups of long-term application of mefentrifluconazole to rice and other crops.

Implementation of HILIC-UV technique for the determination of moxifloxacin and fluconazole in raw materials and pharmaceutical eye gel.

Hydrophilic interaction liquid chromatography (HILIC) has inherent merits over RP-HPLC in the analyzing of hydrophilic substances. Accordingly, an innovative HILIC-UV methodology is proposed for the simultaneous estimation of ethyl paraben (PRN), fluconazole (FLZ) and moxifloxacin hydrochloride (MOX) in raw materials and pharmaceutical eye gel. The separation process was conducted using Waters XBridge™ HILIC column (100 mm × 4.6 mm, 3.5 µm particle size) at room temperature. Isocratic mobile phase containing acetonitrile: 0.1% triethylamine buffer (90:10, v/v, pH 5.0), was pumped at flow rate 1.0 mL/min and detected at 260 nm. Under these optimized conditions, PRN, FLZ and MOX showed rectilinear relationships with the concentration ranges (0.5-6.0), (5.0-50.0) and (5.0-60.0) µg/mL, respectively. The developed method offered at least fivefold increase in sensitivity within shorter time than the reported methods. Three greenness assessment tools namely: Analytical eco-scale, GAPI and AGREE were exploited to investigate the method's impact on the environment and conduct a comparative study with the reported methods. International council of Harmonization (ICH) guidelines have been followed to calculate validation parameters. The statistical comparison between results of the suggested method and the comparison method showed no discrepancy confirming accuracy of the method.

Comparator product issues for biowaiver implementation: the case of Fluconazole

The aim of this work was to assess if the commercially available Fluconazole drug products (Reference, Generic and Similar) would meet the biowaiver criteria from Food and Drug Administration (FDA) and Brazilian Agency for Health Surveillance (ANVISA) agencies. All formulations were evaluated considering the dissolution profile carried out in Simulated Gastric Fluid (SGF) pH 1.2, Acetate Buffer (AB) pH 4.5 and Simulated Intestinal Fluid (SIF) pH 6.8. The results demonstrated that all formulations fulfilled the 85% of drug dissolved at 30 min criterion in SGF pH 1.2. However, in AB pH 4.5 and SIF pH 6.8, some formulations, including the comparator, did not achieve this dissolution percentage. The discrepant dissolution profiles also failed the ƒ2 similarity factor analysis, since none of the formulations showed values between 50 and 100 in the three dissolution media. Comparative dissolution profiles were not similar, considering that the main issues concerning the dissolution were evidenced for the comparator product. Hence, a revision in the regulatory norms in order to establish criteria to switch the comparator could result in an increased application of drugs based on biowaiver criteria

Validation of an innovative analytical method for simultaneous quantification of curcumin and fluconazole using high-performance liquid chromatography from nanostructured lipid carriers.

Vulvovaginal candidiasis is a public health problem with a high incidence among female patients. Currently, there is an increase in the identification of Candida spp. resistant to current therapy, making it necessary to search for new therapeutic alternatives. The synergistic potential of curcumin with fluconazole is described in the literature. However, due to its high lipophilicity, it is necessary to use drug-delivery systems to adequately explore its potential, among which is the nanostructured lipid carrier. However, to date, there is no validated method of high-performance liquid chromatography for simultaneous determination of fluconazole and curcumin in the literature. Thus, the present work developed a high-performance liquid chromatography method for simultaneous determination of fluconazole and curcumin co-encapsulated in nanostructured lipid carrier which was validated according to the International Conference on Harmonization (Technical Requirements for Registration of Pharmaceuticals for Human Use) - Q2 (R1) and the Food and Drug Administration - Guidance for Bioanalytical Method. The method was applied to determine the encapsulation efficiency and drug-loading of curcumin and fluconazole in nanostructured lipid carriers. The developed method proved to be selective, precise, accurate, and robust for the simultaneous determination of both drugs, enabling the quantification of encapsulation efficiency and drug-loading of curcumin and fluconazole in nanostructured lipid carriers.

Detection Method of Falsified Medicines by Using a Low-Cost Raman Scattering Spectrometer Combined with Soft Independent Modeling of Class Analogy and Partial Least Squares Discriminant Analysis.

There are many reports of falsified medicines that may cause harm to patients. A rapid and simple method of identifying falsified medicines that could be used in the field is required. Although Raman scattering spectroscopy has become popular as a non-destructive analysis, few validation experiments on falsified medicines that are actually distributed on the market have been conducted. In this study, we validated a discriminant analysis using an ultra-compact, portable, and low-cost Raman scattering spectrometer combined with multivariate analysis. The medicines were three types of erectile dysfunction therapeutic tablet and one type of antifungal tablet: tadalafil (Cialis), vardenafil hydrochloride (Levitra), sildenafil citrate (Viagra), and fluconazole (Diflucan), which is sometimes advertised as female Viagra. For each medicine, the authentic standard product and products obtained by personal import via the internet (genuine or falsified) were used. Discriminant analyses were performed on the Raman spectra combined with soft independent modeling of class analogy (SIMCA) and partial least squares discriminant analysis (PLS-DA). It was possible to identify all falsified samples by SIMCA using the standard product model for all four products. Using the PLS-DA using the PLS models of the four standard products, falsified Levitra and Diflucan samples were classified correctly, although some falsified Cialis and all Viagra samples also belonged to the standard class. In this study, SIMCA might be more suitable than PLS-DA for identifying falsified medicines. A spectroscopic module that combines the low-cost Raman scattering spectroscopy with SIMCA might contribute to the rapid identification of falsified medicines in the field.

A fast and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method for determining mefentrifluconazole in plant- and animal-derived foods.

A rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method, in conjunction with multiwalled carbon nanotube purification, was developed to determine the mefentrifluconazole levels in grapes, cucumbers, tomatoes, peppers, wheat, maize, eggs, milk, pork, chicken, and fish. After purification, tandem mass spectrometry of mefentrifluconazole required <3.0 min. Matrix-matched external standard curves were used to quantify the residual mefentrifluconazole. The method meets the requirements of the European Union Document SANTE/11813/2017. Quantification was linear between 5 and 500 µg/kg (R2 ≥ 0.9988), and both the intra- and interday relative standard deviations were ≤13.7%. Analyte recovery ranged from 81.5% to 107.6%. The limit of mefentrifluconazole quantification was 5 µg/kg for all matrices. The method successfully detected and quantified mefentrifluconazole that had been applied to cucumbers and tomatoes grown in a test field. These results imply that the proposed method is effective and reliable for detecting mefentrifluconazole residues in plant- and animal-derived foods.

Photogeneration of hydroxyl radical in Fe(III)-citrate-oxalate system for the degradation of fluconazole: mechanism and products.

The photochemical role of Fe(III)-citrate complex is significant in natural waters due to its ubiquitous existence and excellent photoreactivity at near neutral pH. Although there are many reports on the photoinduced degradation of pollutants in the Fe(III)-citrate system, the optimum pH for its photoreactivity is yet not clearly understood. Here, for the first time, we demonstrated that the optimum pH was 5.5 for the photoproduction of •OH in the Fe(III)-citrate system via kinetics modeling based on the steady-state approximation. According to the experimental results, the •OH photoproduction increased with increasing pH until 5.5 and then decreased in Fe(III)-citrate solution, which agreed well with the prediction trend of kinetic modeling. The effect of the common ligand oxalate on the photoreactivity of Fe(III)-citrate system was also investigated. The addition of oxalate promoted the photoproduction of •OH in Fe(III)-citrate solutions, and the measured [•OH]ss increased with oxalate concentration under a fixed Fe(III)-to-citrate ratio. Little synergistic effect exists in Fe(III)-citrate-oxalate system at pH 4.0-5.5. In contrast, an appreciable synergistic effect was observed at near neutral pH (6.0-8.0). Higher oxalate-to-citrate ratio facilitated the synergistic effect. Furthermore, antifungal drug fluconazole could be removed efficiently in the Fe(III)-citrate-oxalate system. The photodegradation kinetics also verified the optimum pH of Fe(III)-citrate system and synergistic effect of oxalate. By LC-ESI-MS/MS analyses, the photoproducts of fluconazole in the Fe(III)-citrate-oxalate system were identified and the reaction mechanism involving hydroxylation substitution and subsequent cleavage of heterocyclic amine was proposed. These findings suggest that Fe(III)-citrate exhibits best photoreactivity at pH 5.5, and the coexistence of reactive ligands will enhance its photoreactivity at circumneutral pH, indicating potential application in wastewater treatment via addition of appropriate citrate and co-ligands.

A porous graphitized carbon LC-ESI/MS method for the quantitation of metronidazole and fluconazole in breast milk and human plasma.

Information on drug transfer into the breast milk is essential to protect the infant from undesirable adverse effects of maternal consumption of drugs and to allow effective pharmacological treatment of breastfeeding mothers. Metronidazole and fluconazole are two drugs frequently used in nursing women to treat various infections, thus questioning infant's safety due to drug exposure through breast milk. In this article a porous graphitized carbon LC/ESI-MS assay was developed for the quantitation of metronidazole and fluconazole in breast milk and human plasma. The assay was based on the use of 150 µL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the LC/ESI-MS system. All analytes and the internal standard, ropinirole, were separated by using a porous graphitized carbon analytical column (150 × 2.1 mm i.d., particle size 5 µm) with isocratic elution. The mobile phase consists of 55% acetonitrile in water acidified with 0.1% concentrated formic acid and pumped at a flow rate of 0.25 mL min-1. The assay was linear over a concentration range of 0.1 to 15 µg mL-1 for all analytes in both biological samples. Intermediate precision was found to be <8.4% over the tested concentration ranges. A run time of <5 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application for the analysis of metronidazole and fluconazole in both breast milk and human plasma and it can be used to support a wide range of clinical studies.

Utilizing online-dual-SPE-LC with HRMS for the simultaneous quantification of amphotericin B, fluconazole, and fluorocytosine in human plasma and cerebrospinal fluid.

Amphotericin B (AMB), fluconazole (FZ), and fluorocytosine (FC) are recommended for HIV-associated cryptococcal meningitis (CM) patients as preferred antibiotics. This study presents a fast and automated online-dual-solid phase extraction (SPE)-LC coupled with high resolution mass spectrometer (HRMS) method to simultaneously measure the concentrations of AMB, FZ, and FC in human plasma and cerebrospinal fluid (CSF). Automated sample clean-up was performed on the human plasma and CSF samples with stop-flow heart-cutting two dimensional (2D) separation using a online-dual-SPE system, allowing retention and accumulation of AMB, FZ, and carbamazepine (CBZ, Internal standard (IS)) by the Oasis®HLB cartridge, and retention and accumulation of FC and 5-methylcytosine hydrochloride (MC, IS) by the HyperSep Hypercarb cartridge respectively. Followed by LC elution, quantification by Q-Exactive Hybrid Quadrupole-Orbitrap with targeted-selected ion monitoring (t-SIM) mode was applied to simultaneously determine the concentrations of AMB, FZ and FC. The bioanalysis was achieved in a total running time of 7min. The method was fully validated according to FDA guidelines. The lowest limit of quantification (LLOQ) was 0.04, 0.04, and 0.40µgmL-1 for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04-2.00µgmL-1, 0.04-2.00µgmL-1 and 0.40-20.00µgmL-1, respectively. The method showed good performance for human plasma and CSF samples with linearity (R2>0.99), intra-day and inter-day precision (relative standard deviation, RSD<4.32% and <4.06%, respectively), recovery (89.93-93.28% and 90.09-93.58%, respectively) and matrix effect (96.35-103.78% and 92.32-101.48%, respectively). The validated method was successfully applied in real samples of Chinese patients. Overall, our results indicate that this fully automated, sensitive, and reliable online-dual-SPE-LC-HRMS method is effective for therapeutic drug monitoring (TDM) of AMB, FZ, and FC levels.

Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

Ultrafiltration is a rapid and convenient method to determine the free concentrations of drugs in plasma. Several ultrafiltration devices based on Eppendorf cups are commercially available, but are not validated for such use by the manufacturer. Plasma pH, temperature and relative centrifugal force as well as membrane type can influence the results. In the present work, we developed an ultrafiltration method in order to determine the free concentrations of linezolid or fluconazole, both neutral and moderately lipophilic antiinfective drugs for parenteral as well as oral administration, in plasma of patients. Whereas both substances behaved relatively insensitive in human plasma regarding variations in pH (7.0-8.5), temperature (5-37°C) or relative centrifugal force (1000-10.000xg), losses of linezolid were observed with the Nanosep Omega device due to adsorption onto the polyethersulfone membrane (unbound fraction 75% at 100mg/L and 45% at 0.1mg/L, respectively). No losses were observed with Vivacon which is equipped with a membrane of regenerated cellulose. With fluconazole no differences between Nanosep and Vivacon were observed. Applying standard conditions (pH 7.4/37°C/1000xg/20min), the mean unbound fraction of linezolid in pooled plasma from healthy volunteers was 81.5±2.8% using Vivacon, that of fluconazole was 87.9±3.5% using Nanosep or 89.4±3.3% using Vivacon. The unbound fraction of linezolid was 85.4±3.7% in plasma samples from surgical patients and 92.1±6.2% in ICU patients, respectively. The unbound fraction of fluconazole was 93.9±3.3% in plasma samples from ICU patients.

Phytotoxicity of wastewater-born micropollutants--Characterisation of three antimycotics and a cationic surfactant.

Sewage sludge applied to soil may be a valuable fertiliser but can also introduce poorly degradable and highly adsorptive wastewater-born residues of pharmaceuticals and personal care products (PPCPs) to the soil, posing a potential risk to the receiving environment. Three azole antimycotics (climbazole, ketoconazole and fluconazole), and one quaternary ammonium compound (benzyldimethyldodecylammonium chloride, BDDA) that are frequently detected in municipal sewage sludge and/or treated wastewater were therefore characterised in their toxicity toward terrestrial (Brassica napus) and aquatic (Lemna minor) plants. Fluconazole and climbazole showed the greatest toxicity to B. napus, while toxicity of ketoconazole and BDDA was by one to two orders of magnitude lower. Sludge amendment to soil at an agriculturally realistic rate of 5 t/ha significantly reduced the bioconcentration of BDDA in B. napus shoots compared to tests without sludge amendment, although not significantly reducing phytotoxicity. Ketoconazole, fluconazole and BDDA proved to be very toxic to L. minor with median effective concentrations ranging from 55.7 µg/L to 969 µg/L. In aquatic as well as terrestrial plants, the investigated azoles exhibited growth-retarding symptoms presumably related to an interference with phytohormone synthesis as known for structurally similar fungicides used in agriculture. While all four substances exhibited considerable phytotoxicity, the effective concentrations were at least one order of magnitude higher than concentrations measured in sewage sludge and effluent. Based on preliminary hazard quotients, BDDA and climbazole appeared to be of greater environmental concern than the two pharmaceuticals fluconazole and ketoconazole.

Influence of sustained low-efficiency diafiltration (SLED-f) on interstitial fluid concentrations of fluconazole in a critically ill patient: Use of microdialysis.

Acute kidney injury is a common complication in critically ill patients, and hybrid techniques including sustained low-efficiency dialysis/diafiltration (SLED-f) are being increasingly utilised in intensive care units. Most fungal infections occur in the interstitial fluid (ISF) of tissues and successful treatment of a fungal infection relies on the ability of an antifungal agent to achieve adequate concentrations at the site of infection. Tissue distribution of antimicrobials is impaired in critically ill patients owing to a variety of disease-related physiological changes, e.g. sepsis. Fluconazole is a widely used antifungal agent used to treat Candida spp. infections in critically ill patients. The implications for ISF concentrations of enhanced elimination during renal replacement therapy have not yet been reported for fluconazole. The aim of this single-patient case report was to describe the influence of SLED-f on subcutaneous (SC) ISF concentrations of fluconazole and the implications for achieving pharmacokinetic/pharmacodynamic targets. Serial blood and ISF samples were collected at pre- and post-filter ports within the SLED-f circuit and subcutaneously inserted microdialysis probe, respectively. Fluconazole concentrations were measured using a validated chromatography method. The SC ISF-to-plasma partition coefficient of fluconazole in this patient was 0.91, indicating rapid equilibrium. SC ISF fluconazole concentrations consistently decreased after initiating SLED-f. The majority of the fluconazole was eliminated from the SC ISF as a result of redistribution. Considering the extensive tissue re-distribution of fluconazole and observed elimination from tissue compartments, higher doses may be required to treat deep-seated fungal infections.

Molecularly imprinted solid phase extraction of fluconazole from pharmaceutical formulations.

This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

Detection and quantification of fluconazole within Candida glabrata biofilms.

Candida infections are often associated with biofilms and consequent high resistance to most common drugs (e.g. azoles). These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a diffusional barrier imposed by the biofilm matrix; however, the real biochemical role of the biofilm components remains very unclear. So, in order to further clarify this issue, we intend to determine, for the first time, fluconazole in biofilms within both supernatants and matrices. Candida biofilms were formed in the presence of fluconazole, and it was recovered from both supernatant and matrix cell-free fractions. Then, high-pressure liquid chromatography was used to identify and quantify the amount of drug that was present in the two fractions. Moreover, this study also showed that the presence of fluconazole in both fractions indicated that the drug administrated did not completely reach the cells, so this phenomena can easily be associated with lower biofilm susceptibility, since the drug administered did not completely reach the cells.

Triclosan as a surrogate for household biocides: an investigation into biocides in aquatic environments of a highly urbanized region.

Biocides are widely formulated in household and personal care products. We investigated the distribution and ecological risks of 16 household biocides in aquatic environments of a highly urbanized region in South China, evaluated triclosan as a chemical indicator for this group of household chemicals, and proposed a novel approach to predict the environmental occurrence and fate of these household biocides by using triclosan usage data and a level-III fugacity model. Eleven biocides were quantitatively detected at concentrations up to 264 ± 15.3 ng/L for climbazole in surface water, and up to 5649 ± 748 ng/g for triclocarban in sediment of four rivers in the region. The distribution of biocides in the aquatic environments was significantly correlated with environmental variables such as total nitrogen, total phosphorus and population. Domestic sewage in the region was the dominant pollution source for most biocides such as azole fungicides (fluconazole, climbazole, clotrimazole, ketoconazole, miconazole, and carbendazim) and disinfectants (triclosan and triclocarban). Preliminary risk assessment showed high ecological risks posed by two biocides carbendazim and triclosan in river waters. Mostly important, triclosan was found to be a reliable chemical indicator to surrogate household biocides both in water and sediment based on the correlation analysis. In addition, the fugacity modeling could provide simulated concentrations comparable to the monitoring results. Therefore, with the usage data of the chemical indicator triclosan and correlation formula with other biocides, this model can be applied for predicting the occurrence and fate of various household biocides in a catchment.

Micellar HPLC and derivative spectrophotometric methods for the simultaneous determination of fluconazole and tinidazole in pharmaceuticals and biological fluids.

Micellar high-performance liquid chromatography (HPLC) and first-derivative ultraviolet spectrophotometry were used to simultaneously determine fluconazole (FLZ) and tinidazole (TNZ) in combined pharmaceutical dosage forms. The derivative procedure is based on the linear relationship between the drug concentration and the first derivative amplitudes at 220 and 288 nm for FLZ and TNZ, respectively. The calibration graphs were linear in the range of 1.5-9.0 µg/mL for FLZ and 10.0-60.0 µg/mL for TNZ. Furthermore, an HPLC procedure with ultraviolet detection at 210 nm was developed. For the HPLC procedure, good chromatographic separation was achieved using an ODS C18 column (250 × 4.6 mm i.d.). The mobile phase containing 0.15M sodium dodecyl sulphate, 0.3% triethylamine and 12% n-propanol in 0.02M orthophosphoric acid at pH 5.5 was pumped at a flow rate of 1 mL/min. Indapamide was used as an internal standard. The method showed good linearity over the concentration ranges of 1.5-30.0 and 10.0-200.0 µg/mL, with limits of detection of 0.36 and 2.70 µg/mL and limits of quantification of 1.1 and 8.2 µg/mL for FLZ and TNZ, respectively. The suggested methods were successfully applied for the simultaneous analysis of the drugs in their laboratory prepared mixture, co-formulated tablet and single dosage forms. Moreover the second method was also extended to the determination of the drugs in biological fluids.

Efeitos de compostos naturais, sintéticos e da fototerapia antifúngica sobre Candida tropicalis resistente ao fluconazol / Effects of natural compounds, synthetic and herbal medicine on Candida tropicalis antifungal fluconazole-resistant

A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16...

Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16...

Validation of high-performance liquid chromatographic method for analysis of fluconazole in microemulsions and liquid crystals

In recent decades, there has been a significant increase in the incidence of fungal diseases. Certain fungal diseases cause cutaneous lesions and in the usual treatment, generally administred orally, the drug reaches the site of action with difficulty and its concentration is too low. An approach much explored in recent years is the development of nanotechnology-based drug delivery systems, and microemulsions (ME) and liquid crystals (LC) are promising. ME and LC were developed with oleic acid or copaiba oil as the oil phase, propoxyl (5OP) ethoxyl (20 OE) cetyl alcohol as surfactant and water. An analytical method to assess the incorporation of fluconazole (FLU) in the systems under study was validated according to guidelines of the International Conference on Harmonization (ICH) guidelines and the Brazilian Food, Drug and Sanitation Agency (ANVISA). The method was conducted on a C18-RP column (250 × 4.6 mm i.d.), maintained at room temperature. The mobile phase consisted of acetonitrile and water (50:50, v/v), run at a flow rate of 1.0mL/min and using ultraviolet detection at 210nm. The chromatographic separation was obtained with a retention time of 6.3min, and was linear in the range of 20-400 µg/mL (r2=0.9999). The specificity showed no interference of the excipients. The accuracy was 100.76%. The limits of detection and quantitation were 0.057 and 0.172 µg.mL-1, respectively. Moreover, method validation demonstrated satisfactory results for precision and robustness. The proposed method was applied for the analysis of the incorporation of FLU in ME and LC, contributing to improve the quality control and to assure the therapeutic efficacy.

Nas últimas décadas, houve aumento significativo na incidência de doenças fúngicas. Certas doenças fúngicas provocam lesões cutâneas, sendo que no tratamento usual, geralmente administrado por via oral, o medicamento chega ao local de ação com dificuldade, em concentração muito baixa. Uma abordagem muito explorada nos últimos anos é o desenvolvimento de sistemas de administração de fármacos baseados em nanotecnologia, como as microemulsões (ME) e cristais líquidos (LC). ME e LC foram desenvolvidos com o ácido oleico ou óleo de copaíba como fase oleosa, álcool cetílico propoxilado (5 OP) e etoxilado (20 OE) como tensoativo e água. Método analítico para avaliar a incorporação de fluconazol (FLU) nos sistemas em estudo foi validado de acordo com as diretrizes da Conferência Internacional de Harmonização (ICH) e Agência Nacional de Vigilância Sanitária (ANVISA). O método foi desenvolvido empregando coluna C18-RP (250 x 4,6 mm id), mantida à temperatura ambiente. A fase móvel consistiu de acetonitrila e água (50:50, v/v), executado a uma taxa de fluxo de 1,0 mL/min e com detecção ultravioleta a 210 nm. A separação cromatográfica foi obtida com o tempo de retenção de 6,3min, e mostrou-se linear no intervalo de 20-400 µg/mL (r2=0,9999). Pelo estudo de especificidade, observou-se não interferência dos excipientes. A precisão foi 100,76%. Os limites de detecção e de quantificação foram 0,057 e 0,172 µg.mL-1, respectivamente. Além disso, a validação do método demonstrou resultados satisfatórios para a precisão e robustez. O método proposto foi aplicado para a análise da incorporação do FLU em ME e cristais líquidos, contribuindo para aumentar o controle de qualidade e garantir a eficácia terapêutica.