Chemical Imaging of Pharmaceuticals in Biofilms for Wastewater Treatment Using Secondary Ion Mass Spectrometry.

The occurrence of pharmaceuticals in the aquatic environment is a global water quality challenge for several reasons, such as deleterious effects on ecological and human health, antibiotic resistance development, and endocrine-disrupting effects on aquatic organisms. To optimize their removal from the water cycle, understanding the processes during biological wastewater treatment is crucial. Time-of-flight secondary ion mass spectrometry imaging was successfully applied to investigate and analyze the distribution of pharmaceuticals as well as endogenous molecules in the complex biological matrix of biofilms for wastewater treatment. Several compounds and their localization were identified in the biofilm section, including citalopram, ketoconazole, ketoconazole transformation products, and sertraline. The images revealed the pharmaceuticals gathered in distinct sites of the biofilm matrix. While citalopram penetrated the biofilm deeply, sertraline remained confined in its outer layer. Both pharmaceuticals seemed to mainly colocalize with phosphocholine lipids. Ketoconazole concentrated in small areas with high signal intensity. The approach outlined here presents a powerful strategy for visualizing the chemical composition of biofilms for wastewater treatment and demonstrates its promising utility for elucidating the mechanisms behind pharmaceutical and antimicrobial removal in biological wastewater treatment.

Physicochemical characterization and thermal behavior of hexosomes containing ketoconazole as potential topical antifungal delivery system.

OBJECTIVE: The main purpose of this article is to show the valuable characteristics that liotropic liquid crystal systems possess to be employed as new drug delivery systems. SIGNIFICANCE: Colloidal aqueous dispersions of lyotropic liquid crystal mesophases such as the identified as cubosomes and hexosomes, and so on, have received considerable attention due to their unique nanostructures and their thermodynamic properties, which provide the potential as a sustained drug release matrix. Additionally, their large surface area and similarity with the liquid crystal structures of intercellular lipids of stratum corneum enhances the interaction with the skin and mucous, increasing the potential for topical drug delivery efficiency of biopharmaceutical class II drugs as the antifungal ketoconazole. METHODS: This article presents the results in morphological characteristics, particle size, ζ potential, flow, thermal behavior and drug release studies of hexosomes containing ketoconazole (LHLC-K) obtained with glycerol monooleate, propylene glycol monolaurate, poloxamer, and water mixtures. RESULTS: This colloidal system exhibits a Newtonian-type flow and a hexagonal nanostructure with a median particle size of 107 ± 20 nm and ζ potential of +4.45 ± 0.50 mV. Through differential scanning calorimetry studies, the LHLC-K demonstrated physical and chemical stability for more than six months and mesophasic thermal reversibility between 10 and 50 °C. Finally, LHLC-K releases ketoconazole following a kinetics described by the first order model. CONCLUSIONS: Physicochemical properties of the hexosomes containing ketoconazole are important for topical mycosis treatment administration, conditions of storage, and for its incorporation into the formulation of semi-solid dosage forms.

Identification of endocrine disrupting chemicals acting on human aromatase.

Human aromatase is the cytochrome P450 catalysing the conversion of androgens into estrogens playing a key role in the endocrine system. Due to this role, it is likely to be a target of the so-called endocrine disrupting chemicals, a series of compounds able to interfere with the hormone system with toxic effects. If on one side the toxicity of some compounds such as bisphenol A is well known, on the other side the toxic concentrations of such compounds as well as the effect of the many other molecules that are in contact with us in everyday life still need a deep investigation. The availability of biological assays able to detect the interaction of chemicals with key molecular targets of the endocrine system represents a possible solution to identify potential endocrine disrupting chemicals. Here the so-called alkali assay previously developed in our laboratory is applied to test the effect of different compounds on the activity of human aromatase. The assay is based on the detection of the alkali product that forms upon strong alkali treatment of the NADP+ released upon enzyme turnover. Here it is applied on human aromatase and validated using anastrozole and sildenafil as known aromatase inhibitors. Out of the small library of compounds tested, resveratrol and ketoconazole resulted to inhibit aromatase activity, while bisphenol A and nicotine were found to exert an inhibitory effect at relatively high concentrations (100µM), and other molecules such as lindane and four plasticizers did not show any significant effect. These data are confirmed by quantification of the product estrone in the same reaction mixtures through ELISA. Overall, the results show that the alkali assay is suitable to screen for molecules that interfere with aromatase activity. As a consequence it can also be applied to other molecular targets of EDCs that use NAD(P)H for catalysis in a high throughput format for the fast screening of many different compounds as endocrine disrupting chemicals. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

The effect of polymeric excipients on the physical properties and performance of amorphous dispersions: Part I, free volume and glass transition.

PURPOSE: To investigate the structural effect of polymeric excipients on the behavior of free volume of drug-polymer dispersions in relation to glass transition. METHODS: Two drugs (indomethacin and ketoconazole) were selected to prepare amorphous dispersions with PVP, PVPVA, HPC, and HPMCAS through spray drying. The physical attributes of the dispersions were characterized using SEM and PXRD. The free volume (hole-size) of the dispersions along with drugs and polymers was measured using positron annihilation lifetime spectroscopy (PALS). Their glass transition temperatures (Tgs) were determined using DSC and DMA. FTIR spectra were recorded to identify hydrogen bonding in the dispersions. RESULTS: The chain structural difference-flexible (PVP and PVPVA) vs. inflexible (HPC and HPMCAS)-significantly impacts the free volume and Tgs of the dispersions as well as their deviation from ideality. Relative to Tg, free volume seems to be a better measure of hydrogen bonding interaction for the dispersions of PVP, HPC, and HPMCAS. The free volume of polymers and their dispersions in general appears to be related to their conformations in solution. CONCLUSIONS: Both the backbone chain rigidity of polymers as well as drug-polymer interaction can impact the free volume and glass transition behaviors of the dispersions.

Investigation of phase diagrams and physical stability of drug-polymer solid dispersions.

Solid dispersion technology has been widely explored to improve the solubility and bioavailability of poorly water-soluble compounds. One of the critical drawbacks associated with this technology is the lack of physical stability, i.e. the solid dispersion would undergo recrystallization or phase separation thus limiting a product's shelf life. In the current study, the melting point depression method was utilized to construct a complete phase diagram for felodipine (FEL)-Soluplus® (SOL) and ketoconazole (KTZ)-Soluplus® (SOL) binary systems, respectively, based on the Flory-Huggins theory. The miscibility or solubility of the two compounds in SOL was also determined. The Flory-Huggins interaction parameter χ values of both systems were calculated as positive at room temperature (25 °C), indicating either compound was miscible with SOL. In addition, the glass transition temperatures of both solid dispersion systems were theoretically predicted using three empirical equations and compared with the practical values. Furthermore, the FEL-SOL solid dispersions were subjected to accelerated stability studies for up to 3 months.

Magnetic field assisted centrifugal acceleration thin-layer chromatography: An approach to investigate the magnetic field effects on separation parameters including retention factor difference, selectivity factor, and resolution.

The effect of internal and external magnetic fields on the separation of antifungal drugs by centrifugal acceleration thin-layer chromatography was reported for the first time. External and internal magnetic fields were applied using neodymium magnets and CoFe2O4@SiO2 ferromagnetic nanoparticles. Separation of ketoconazole and clotrimazole was performed using a mobile phase consisting of n-hexane, ethyl acetate, ethanol, and ammonia (2.0:2.0:0.5:0.2, v/v). The influence of the magnetic field on the entire chromatographic system led to changes in the properties of the stationary and mobile phases and the analytes affecting the retention factor, shape, and width of the separated rings. The extent of this impact depended on the structure of the analyte and the type and intensity of the magnetic field. In the presence of the external magnetic field, there were more significant changes in the chromatographic parameters of the drugs, especially the width of the separated rings, and ketoconazole was more affected than clotrimazole. The changes are conceivably due to the effect of the magnetic field on the analyte distribution between the stationary and mobile phases, which is also caused by the possibility of the magnetic field affecting the viscosity, surface tension, and surface free energy between the stationary and mobile phases.

Differential pulse polarographic investigation of the antifungal drugs itraconazole, ketoconazole, fluconazole and voriconazole using a dropping mercury electrode.

The electrochemical reactions of the antifungal drugs itraconazole, ketoconazole, fluconazole and voriconazole have been investigated by differential pulse polarography (DPP) using a dropping mercury electrode (DME). All investigations were carried out in Britton-Robinson buffer solutions and methanol with varying pH values. Ketoconazole and itraconazole both showed a reduction peak with a potential between -1.5V and -1.6 V. Stable and reproducible conditions for the determination of itraconazole (c = 1 x 10(-7) M) were found within the pH range of 6.0 to 8.0 and for the determination of ketoconazole (c = 5 x 10(-8) M) within pH 6.0 to 7.0. Voriconazole showed a reduction peak with a peak potential of -1.7 V (c = 1 x 10(-5) M) within the pH range of 8.0 to 10.0. In the case of fluconazole no electrochemical activity was found.

Pharmaceuticals and other contaminants of emerging concern in Admiralty Bay as a result of untreated wastewater discharge: Status and possible environmental consequences.

Considering how the impact of human activity in Antarctica is growing, the aim of this study was to conduct the first assessment of pharmaceuticals and personal care products (PPCPs), other emerging contaminants (ECs), and antibiotic resistance genes present in the western shore of the Admiralty Bay region of King George Island. In total, more than 170 substances were evaluated to assess the potential environmental risks they pose to the study area. The major evaluated source of pollutants in this study is discharged untreated wastewater. The highest PPCP concentrations in wastewater were found for naproxen (2653 ngL-1), diclofenac (747 ngL-1), ketoconazole (760 ngL-1), ibuprofen (477 ngL-1) and acetaminophen (332 ngL-1). Moreover, the concentrations of benzotriazole (6340 ngL-1) and caffeine (3310 ngL-1) were also high. The Risk Quotient values indicate that azole antifungals (ketoconazole), anti-inflammatories (diclofenac, ibuprofen) and stimulants (caffeine) are the main groups responsible for the highest toxic burden. In addition, antibiotic resistance genes integrons (int 1) and sulphonamide resistance genes (sul 1-2) were detected in wastewater and seawater. These results indicate that regular monitoring of PPCPs and other ECs is of great importance in this environment. Additionally, the following mitigation strategies are suggested: (1) to create a centralised record of the medications prescribed and consumed in situ (to improve knowledge of potential contaminants without analysis); (2) to use more environmentally friendly substitutes both for pharmaceuticals and personal care products when possible (limiting consumption at the source); and (3) to apply advanced systems for wastewater treatment before discharge to the recipient (end-of-pipe technologies as a final barrier).

High-performance thin-layer chromatographic determination of ketoconazole in pharmaceutical formulations.

A high-performance thin-layer chromatographic method was developed for the determination of ketoconazole. The sample was separated on a silica gel 60 F254 plate and developed in ethanol-acetone-1.0 mol l-1 H2SO4 by means of an automatic multiple-development system. The area of the spot was quantified by a TLC scanner at a wavelength of 298 nm. A linear calibration curve was established over the range of 3-20 µg/ml of ketoconazole, with a correlation coefficient of 0.9992. The relative standard deviations for intraday and interday precisions, for three replicate determinations, were found to be 1.72% and 0.69% for 5 µg/ml and 2.18% and 0.94% for 10 µg/ml of ketoconazole, respectively. The average percentage recoveries of ketoconazole shampoos (Nora, Kenalyn, and Nizoral) and ketoconazole creams (Nizoral, Fungasin, and Ketazon) were found to be 96.10, 97.06, and 99.58, and 96.77, 97.26, and 95.74, respectively. This method has been applied to the determination of ketoconazole in various pharmaceutical dosage forms. Common excipients in formulations do not interfere. This method is simple, precise, accurate, and inexpensive. It should be used for routine analysis.

Electron paramagnetic resonance method for the quantitative assay of ketoconazole in pharmaceutical preparations.

In this study, electron paramagnetic resonance (EPR) is used, for the first time, as an analytical tool for the quantitative assay of ketoconazole (KTZ) in drug formulations. The drug was successfully characterized by the prominent signals by two radical species produced as a result of its oxidation with 400 microg/mL cerium(IV) in 0.10 mol dm(-3) sulfuric acid. The EPR signal of the reaction mixture was measured in eight capillary tubes housed in a 4 mm EPR sample tube. The radical stability was investigated by obtaining multi-EPR scans of each KTZ sample solution at time intervals of 2.5 min of the reaction mixing time. The plot of the disappearance of the radical species show that the disappearance is apparently of zero order. The zero-time intercept of the EPR signal amplitude, which should be proportional to the initial radical concentration, is linear in the sample concentration in the range between 100 and 400 microg/mL, with a correlation coefficient, r, of 0.999. The detection limit was determined to be 11.7 +/- 2.5 microg/mL. The method newly adopted was fully validated following the United States Pharmacopeia (USP) monograph protocol in both the generic and the proprietary forms. The method is very accurate, such that we were able to measure the concentration at confidence levels of 99.9%. The method was also found to be suitable for the assay of KTZ in its tablet and cream pharmaceutical preparations, as no interferences were encountered from excipients of the proprietary drugs. High specificity, simplicity, and rapidity are the merits of the present method compared to the previously reported methods.

Determination of ketoconazole in capsules by high-performance liquid chromatography and microbiological assay.

The development and validation of an HPLC-UV method and a microbiological assay were performed for the analysis of ketoconazole in capsule formulations. The bioassay was developed using a specific agar diffusion technique with the strain of Candida albicans ATCC 18804 as the test organism. The effect of the mobile phase pH in the range of 2.5-7.5 on the retention and tailing factors of the ketoconazole peak was analyzed in the chromatography method and a pH value of 4.5 was considered to be adequate. A prospective validation of both methods showed adequate linearity (r2 > 0.99 for the two methods), precision, (RSD = 2.42% for intraday and 2.69% for interday precision for bioassay; RSD = 0.74% for intraday and 0.66% for interday precision for HPLC-UV), and accuracy (mean recoveries were 103 +/- 1.0% for bioassay and 99 +/- 1.0% for HPLC-UV). Student's t-test revealed no significant difference between the results obtained by the two methods (P < 0.05). The contents found for three capsule samples showed a strong correlation, as attested by Pearson's coefficient value (r = 0.9998), which also evidenced the concordance between the studied methodologies.

Investigation of dextrin-based synergistic system with chiral ionic liquids as additives for enantiomeric separation in capillary electrophoresis.

In this paper, two spiral structure CILs, 1-butyl-3-methylimidazolium(T-4)-bis[(2S)-2-(hydroxy-κO)-3-methyl-butanoato-κO]borate(BMIm+BLHvB-) and 1-butyl-3-methylimidazolium (T-4)-bis[(αS)-α-(hydroxy-κO)-4-methyl-benzeneacetato-κO]borate (BMIm+BSMB-)were applied to evaluate their potential synergistic effect with dextrin for CE enantiomeric separation. The established dextrin-based synergistic system with CILs as additives showed good separation performance towards four tested drugs, including duloxetine, ketoconazole, sulconazole and citalopram. It was also observed that significantly improved separation and selectivity for tested analytes were achieved in CILs/dextrin synergistic system compared to single dextrin system. Primary parameters, such as the concentration of CIL, dextrin concentration, buffer pH and applied voltage, were systematically investigated to optimize the enantiomeric separation with BMIm+BLHvB-/dextrin as model system. Finally, the method of Statistical Product and Service Solutions (SPSS) was exploited to further elucidate the influence of experimental parameters on the synergistic effect.

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).

Development and Validation of Stability-Indicating Method for the Simultaneous Determination of Ketoconazole and Beauvericin in Pharmaceutical Tablets.

In this work, a stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous analysis of ketoconazole (KCZ) and beauvericin (BEA) as well as their degradation products in the combination tablets. KCZ is a synthetic broad-spectrum antifungal agent with the risk of hepatoxicity. However, it was found that the combined use of KCZ and BEA in their low dose had not only maintained the antifungal activity of KCZ but also significantly reduced the liver toxicity. The method development was started from forced degradation studies including acidic, basic, oxidative, thermal and photolytic degradations in the solution mixtures of KCZ and BEA. The forced degradation study results indicate that hydrolysis and oxidation were the major degradation pathways for KCZ while BEA mainly decomposed under basic hydrolytic condition. The newly developed HPLC method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to specificity, linearity, precision, accuracy, limits of detection and quantification and robustness. The method validation results indicate that the new HPLC method could be successfully applied in the simultaneous detection and quantitation of KCZ and BEA and their degradation products. For example, the accuracy and the precision of the method were determined by a recovery study at 80, 100 and 120% of the tablet dosage levels. The recovery was found to be 99.6-100.2 for both analytes with a relative standard deviation of no more than 1.2% (n = 5) at any concentration level. This new method can be used for further development of various KCZ and BEA combination drug products.

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.

Microbiological assay of ketoconazole in shampoo.

Ketoconazole, an anti-fungal agent, is often incorporated in several pharmaceutical forms and in shampoo formulation it is known to be effective against fungal infection on the scalp. This paper describes a method to quantify ketoconazole in shampoo by comparing the cylinder plate assay and the HPLC method. The test organism used for the agar diffusion assay was Candida albicans ATCC 10231. Three different concentrations of ketoconazole were used for the diffusion assay. A mean zone diameter was obtained for each concentration. A standard curve was obtained by plotting the three values derived from the zone diameters. A prospective validation of the method showed that the method was linear (r = 0.9982), precise (R.S.D. = 2.57%) and accurate. The results obtained by the two methods were statistically evaluated by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference between these two methods.

[Determination of antidangdruff agent salicylic acid, zinc pyrithione, octopirox, climbazole and ketoconazole in shampoo by high performance liquid chromatography].

A high performance liquid chromatography method was established for determination of antidangdruff agent salicylic acid,zinc pyrithione, octopirox, climbazole and ketoconazole in shampoo on a C18 column using acetonitrile-metholaqueous solution (10 mmol/L KH2 PO4 and 5 mmol/L EDTANa2, pH is adjusted to 4.0 with H3 PO4) (50:10:40) as mobile phase at a flow rate of 1.0 ml/min, with the column temperature 25 degrees C and detection wave 230nm. The precision was less than 3.8% and recovery varied from 92.7% to 104.9%. The experimental results showed that the method was simple, precise and accurate.

Simple high-performance liquid chromatography method for the simultaneous determination of ketoconazole and piperine in rat plasma and hepatocyte culture.

Piperine, a major alkaloid of black and long peppers has been reported to act as bioavailability enhancer of several drugs by inhibiting drug metabolising enzymes and/or by increasing oral absorption. Ketoconazole is a well established potent inhibitor of CYP 3A4 and P-glycoprotein. A simple and rapid HPLC method has been developed for the simultaneous analysis of ketoconazole and piperine in rat plasma and hepatocyte culture. Analysis was performed using a Symmetry C18 column (150x4.6 mm, 5 microm) and isocratic elution with 25 mM KH2PO4 (pH 4.5)-acetonitrile (50:50) with a flow-rate of 1 ml/min. Photodiode array detection was used to simultaneously monitor piperine at 340 nm and ketoconazole at 231 nm in a single sample. Calibration plots in spiked plasma, hepatocytes and William's medium E were linear over the range studied (10-2000 ng for both drugs). The detection limits for piperine and ketoconazole are 2 and 4 ng, respectively, and the limits of quantitation are 10 and 12 ng, respectively. Intra- and inter-assay variations were less than 8%.

Simultaneous determination of ketoconazole and formaldehyde in a shampoo: liquid chromatography method development and validation.

Ketoconazole is an antifungal agent, which is the active ingredient in a shampoo primarily used for the treatment of seborrhatic dermatitis (anti-dandruff shampoo). The shampoo also contains imidazolidinylurea as a formaldehyde releasing preservative. The aim of this study was to develop a HPLC system that allows the determination of both ketoconazole and formaldehyde. The finally selected isocratic system consisted of an Interchrom Nucleosil (250 X 4.6 mm, 5 microm) C8 column and a mobile phase containing acetonitrile-phosphate buffer 0.025 M, pH 4.0, 45/55 (v/v). Ketoconazole could immediately be determined at 250 nm after injection of diluted shampoo. Formaldehyde was measured at 345 nm after derivatisation with a 2,4-dinitrophenylhydrazine solution. At the selected conditions, the other excipients of the shampoo did not interfere in the assays for both substances. Method validation was performed on both assays. Different selectivity towards ketoconazole and formaldehyde was observed when applying other C8 columns. This fact, however, did not affect the assays of both substances.

Quantitation of ketoconazole in biological fluids using high-performance liquid chromatography.

A rapid, specific procedure is described for the quantitation of ketoconazole in biological fluids using high-performance liquid chromatography (HPLC). The procedure involves sample preparation using a reverse-phase C-18 cartridge prior to chromatography and quantitation using peak height ratios (UV absorbance detection, 231 nm) of ketoconazole to the internal standard, phenothiazine. A sensitivity of 0.2 micrograms/ml was achieved using a 0.5-ml sample. The mean recovery was 86.2%, and overall coefficient of variation of the procedure was 7.1%. This procedure has been used to determine ketoconazole levels in human serum, plasma, CSF, and synovial fluid. A comparison with a microbiological assay is presented, and adaptability of this procedure to quantitation by fluorescence to increase the sensitivity fivefold is discussed.