Antibiotics and antimycotics in waste water treatment plants: Concentrations, removal efficiency, spatial and temporal variations, prediction, and ecological risk assessment.

For investigating the spatial, temporal variations and assessing ecological risk of 10 antibiotics and 6 antimycotics, influent sewage water and treated effluent were collected during three different seasons in 19 waste water treatment plants of Tianjin. High performance liquid chromatography tandem mass spectrometry was used to analyze 16 substances. The concentration range of influent samples was not detected (nd) -547.94 ng/L and the concentration range of effluent samples was nd-52.97 ng/L. By calculating the removal efficiency, it was found that Ciprofloxacin (CIP), Ofloxacin (OFL) and Clotrimazole (CTR) were effectively removed. There were significant spatial and temporal differences, the concentration in the dry season was evidently higher than that in the wet and normal seasons, and the northeast was lower than that in the northwest and southeast. By establishing a data set of influent and effluent, the priority features were extracted by feature engineering, which were temperature and NH3-N. Under the condition of ensuring the best performance of the models, the influent model with 9 features and the effluent model with 4 features were established, and the quantitative relationship between the above features and concentration was obtained through partial dependence analysis. Except for Moxifloxacin (MOX), Norfloxacin (NOR) and OFL in the influent samples, the RQ values for other antibiotics and antimycotics were less than 0.1. Among the effluent samples, only NOR had an RQ value greater than 0.1, and OFL, MOX, and Pefloxacin (PEF) had RQ values between 0.01 and 0.1. Comparing the observations and predictions individual RQ values, the predictions were ideal and matched the observations. This work effectively assessed environmental impact and provided a valuable reference for evaluating antibiotics and antimycotics ecological toxicity.

Development and validation of a simultaneous analytical method for non-steroidal therapeutic compounds in cosmetics using liquid chromatography-tandem mass spectrometry.

Atopic dermatitis is a typical chronic inflammatory skin disease that affects all age groups and requires basic skin care for treatment. Anti-inflammatory and antiallergy steroids are the most frequently used treatments but they are limited due to their side effects caused by a weakening of the immune system. Many consumers focus on performance as a criterion for selecting cosmetics. However, steroids have been illegally used to improve the performance of cosmetics, and consumers have been adversely affected by the corresponding side effects. In this paper, we propose a simple and rapid method using liquid chromatography-tandem mass spectrometry to simultaneously analyze ten non-permitted atopic therapeutic compounds in cosmetic products: chlorpheniramine maleate, ketotifen fumarate, doxepin hydrochloride, azelastine hydrochloride, bufexamac, clotrimazole, tranilast, fusidic acid, tacrolimus, and pimecrolimus. Additionally, the major characteristic fragment ions for tacrolimus, pimecrolimus, and clotrimazole were identified by time-of-flight mass spectrometry. The specificity, linearity, limit of detection, limit of quantification, recovery, precision, accuracy, and stability of the proposed method were validated. The limit of detection and quantification were in the ranges of 5.05-203.30 pg/mL and 15.15-609.90 pg/mL, respectively. The proposed analysis method could help improve the safety management of cosmetics.

Sequential Injection Analysis for Automation and Evaluation of Drug Liberation Profiles: Clotrimazole Liberation Monitoring.

A fully automated sequential injection system was tested in terms of its application in liberation testing, and capabilities and limitations were discussed for clotrimazole liberation from three semisolid formulations. An evaluation based on kinetic profiles obtained in short and longer sampling intervals and steady-state flux values were applied as traditional methods. The obtained clotrimazole liberation profile was faster in the case of Delcore and slower for Clotrimazol AL and Canesten cream commercial formulations. The steady-state flux values for the tested formulations were 52 µg cm-2 h-1 for Canesten, 35 µg cm-2 h-1 for Clotrimazol AL, and 7.2 µg cm-2 h-1 for Delcore measured in 4 min sampling intervals. A simplified approach for the evaluation of the initial rate based on the gradient between the second and third sampling points was used for the first time and was found to correspond well with the results of the conventional methods. A comparison based on the ratio of the steady-state flux and the initial rate values for Canesten and Clotrimazol AL proved the similarity of the obtained results. The proposed alternative was successfully implemented for the comparison of short-term kinetic profiles. Consequently, a faster and simpler approach for dissolution/liberation testing can be used.

Development of a Method for the Quantification of Clotrimazole and Itraconazole and Study of Their Stability in a New Microemulsion for the Treatment of Sporotrichosis.

Sporotrichosis occurs worldwide and is caused by the fungus Sporothrix brasiliensis. This agent has a high zoonotic potential and is transmitted mainly by bites and scratches from infected felines. A new association between the drugs clotrimazole and itraconazole is shown to be effective against S. brasiliensis yeasts. This association was formulated as a microemulsion containing benzyl alcohol as oil, Tween® 60 and propylene glycol as surfactant and cosurfactant, respectively, and water. Initially, the compatibility between clotrimazole and itraconazole was studied using differential scanning calorimetry (DSC), thermogravimetric analysis (TG), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (PXRD). Additionally, a simple and efficient analytical HPLC method was developed to simultaneously determine the concentration of clotrimazole and itraconazole in the novel microemulsion. The developed method proved to be efficient, robust, and reproducible for both components of the microemulsion. We also performed an accelerated stability study of this formulation, and the developed analytical method was applied to monitor the content of active ingredients. Interestingly, these investigations led to the detection of a known clotrimazole degradation product whose structure was confirmed using NMR and HRMS, as well as a possible interaction between itraconazole and benzyl alcohol.

Occurrence and distribution of azole antifungal agents in eight urban Romanian waste water treatment plants.

Azole compounds are utilized to combat fungal infections in plants to protect them and also used for treating mycosis in humans. The LC-MS/MS method is a technique that combines liquid chromatography with tandem mass spectrometry for analysis of twelve azole compounds from wastewater (influent, effluent) and sewage sludge. The compounds were isolated from waste water using automatic extraction in the solid phase. Sludge samples were dried by lyophilization, after which they were subjected to ultrasound extraction with methanol. The quantification limits ranged from 0.3 ng/L (clotrimazole-CLO and prochloraz-PRO) to 1.5 ng/L (tetraconazole-TEB and penconazole-PEN), for wastewater samples and for sewage sludge, the LOQs ranged from 0.1 ng/g to 0.6 ng/g. High concentrations of climbazole-CLI (207-391 ng/L), tebuconazole (92-424 ng/L), and clotrimazole (6.9-93-ng/L) were observed in influent samples of the 8 urban wastewater treatment plants, followed by fluconazole (49.3-76.8 ng/L), and prochloraz (7.3-72 ng/L). The ∑Azoles had a maximum of 676 ng/L in the Galati effluent, followed by the Bucharest station 357 ng/L, and 345 ng/L in the Braila effluent. The highest value of the daily mass loading (input) level was observed for climbazole, 265 mg/day/1000 in Iasi station, followed by tebuconazole, 238 mg/day/1000 people in the Bucharest station, and 203 mg/day/1000 people for climbazole in the Targoviste station. The daily mass emission presented values between 0.7 and 247 mg/day/1000 people. The highest emissions were observed for climbazole, 247 mg/day/1000 people in Braila station; 174 mg/day/1000 people in the Iasi station and 129 mg/day/1000 people in the Bucharest station. The concentrations of climbazole detected in the effluent can present a high risk for the plants Lemna minor and Navicula pelliculosa. Clotrimazole may present a high risk to the plant Desmodesmus subspicatus and to the invertebrate Daphnia magna. PRO may present high risk to the invertebrate Mysidopsis Bahia.

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.

Application of dispersive liquid-liquid microextraction followed by HPLC-MS/MS for the trace determination of clotrimazole in environmental water samples.

A method for the analysis of clotrimazole was developed with dispersive liquid-liquid microextraction for sample pre-concentration and HPLC-MS/MS for analysis. A linear ion trap was used for the confirmation of clotrimazole identity in the samples. The developed method enables the analysis of clotrimazole in river water and sewage effluent from wastewater treatment plants with a LOQ of 0.7 ng/L. Environmental monitoring of clotrimazole was undertaken. Samples from river water and sewage effluents were analysed over a one-year period. Clotrimazole was found in every tested sample with concentration range from 1 to 31 ng/L. The amount of clotrimazole in tested samples was highly dependent on sampling season. The highest results were obtained in summer and autumn.

Monitoring of Clotrimazole Degradation Pathway in Presence of its Co-formulated Drug.

Two stability-indicating chromatographic methods for the determination of clotrimazole and its two acid induced degradation products, with dexamethasone acetate without prior separation. First method depends on RP-HPLC utilizing ODS-3 Inertsil C18 column. Mobile phase consists of acetonitrile:phosphate buffer (pH 6.0) in ratio (65:35, v/v) with flow rate 1.5 mL/min and UV-detection at 220 nm. Linearity range 1.0-75.0 µg/mL for clotrimazole and 2.0-75.0 µg/mL for dexamethasone with mean percentage recovery of 99.49 ± 1.10 for CLT and 99.60 ± 1.06 for DA. Second method depends on HP-TLC. Developing system is composed of chloroform:ethyl acetate in the ratio of (5:3.5, v/v), scanned at 220 nm. Linearity range 1.0-12.0 µg/band for clotrimazole and 1.0-20.0 µg/band for dexamethasone with mean R% of 99.33 ± 0.76 for clotrimazole and 99.77 ± 0.99 for dexamethasone. Conditions and parameters affecting the separation of the cited components without interference of the degradation products are tested and optimized. Suitability of the methods for quantization of the drugs concentrations is proven by validation as instructed from the ICH. Validation results and statistical treatment of the data demonstrate reliability of these methods. Kinetics of acid degradation process of clotrimazole are investigated by the proposed HPLC method and the order rate constant, half life and shelf life are computed.

Comparative study of the efficiency of computed univariate and multivariate methods for the estimation of the binary mixture of clotrimazole and dexamethasone using two different spectral regions.

Three methods of analysis are conducted that need computational procedures by the Matlab® software. The first is the univariate mean centering method which eliminates the interfering signal of the one component at a selected wave length leaving the amplitude measured to represent the component of interest only. The other two multivariate methods named PLS and PCR depend on a large number of variables that lead to extraction of the maximum amount of information required to determine the component of interest in the presence of the other. Good accurate and precise results are obtained from the three methods for determining clotrimazole in the linearity range 1-12 µg/mL and 75-550 µg/mL with dexamethasone acetate 2-20 µg/mL in synthetic mixtures and pharmaceutical formulation using two different spectral regions 205-240 nm and 233-278 nm. The results obtained are compared statistically to each other and to the official methods.

Spectrophotometric resolution of the severely overlapped spectra of clotrimazole with dexamethasone in cream dosage form by mathematical manipulation steps.

Several spectrophotometric techniques were recently conducted for the determination of binary mixtures of clotrimazole (CLT) and dexamethasone acetate (DA) without any separation procedure. The methods were based on generation of ratio spectra of mixture then applying simple mathematic manipulation. The zero order absorption spectra of both drugs could be obtained by the constant center (CC) method. The concentration of both CLT and DA could be obtained by constant value via amplitude difference (CV-AD) method depending on ratio spectra, Ratio difference (RD) method where the difference between the amplitudes at two wavelengths (ΔP) on the ratio spectra could eliminate the contribution of the interfering substance and bring the concentration of the other, and the derivative ratio (DD1) method where the derivative of the ratio spectra was able to determine the drug of interest without any interference of the other one. While the concentration of DA could be measured after graphical manipulation as concentration using the novel advanced concentration value method (ACV). Calibration graphs were linear in the range of 75-550 µg/mL for CLT and 2-20 µg/mL for DA. The methods applied to the binary mixture under study were successfully applied for the simultaneous determination of the two drugs in synthetic mixtures and in their combined form Mycuten-D cream. The results obtained were compared statistically to each other and to the official methods.

Silver-loaded graphene as an effective SERS substrate for clotrimazole detection: DFT and spectroscopic studies.

Vibrational infrared, Raman and surface-enhanced Raman scattering (SERS) spectra of clotrimazole (CTZ) were documented and evaluated. Density-functional theory, B3LYP/6-311++G(d,p), approach was implemented to identify the possible conformations, develop the electrostatic potential map, evaluate frontier molecular orbitals and calculate the vibrational spectra of the target compound. The silver-loaded graphene was shown to be an effective SERS substrate for CTZ trace detection. The SERS spectrum showed two enhanced bands at 670 cm-1 and 700 cm-1 which confirmed the absorption of the silver substrate through chlorine and nitrogen atoms. A detection limit as low as 5 nM could be reached with a determination coefficient of 0.9988 using the band at 670 cm-1. The protein-ligand interaction with Secreted Aspartic Proteinase 2 (SAP2) of C. albicans showed that the four stable forms of CTZ maintain a free energy of binding of 6-7 kcal/mol, which could give insights into the mode of action in treating Candidiasis.

Simultaneous Estimation of Ofloxacin, Clotrimazole, and Lignocaine Hydrochloride in Their Combined Ear-Drop Formulation by Two Spectrophotometric Methods.

Two sensitive, accurate, and precise spectrophotometric methods have been developed and validated for the simultaneous estimation of ofloxacin (OFX), clotrimazole (CLZ), and lignocaine hydrochloride (LGN) in their combined dosage form (ear drops) without prior separation. The derivative ratio spectra method (method 1) includes the measurement of OFX and CLZ at zero-crossing points (ZCPs) of each other obtained from the ratio derivative spectra using standard LGN as a divisor, whereas the measurement of LGN at the ZCP of CLZ is obtained from the ratio derivative spectra using standard OFX as a divisor. The double divisor-ratio derivative method (method 2) includes the measurement of each drug at its amplitude in the double divisor-ratio spectra obtained using a standard mixture of the other two drugs as the divisor. Both methods were found to be linear (correlation coefficients of >0.996) over the ranges of 3-15, 10-50, and 20-100 µg/mL for OFX, CLZ, and LGN, respectively; precise (RSD of <2%); and accurate (recovery of >98%) for the estimation of each drug. The developed methods were successfully applied for the estimation of these drugs in a marketed ear-drop formulation. Excipients and other ingredients did not interfere with the estimation of these drugs. Both methods were statistically compared using the t-test.

Separation and determination of clotrimazole, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography.

In this study, micellar electrokinetic chromatography (MEKC) method was developed for the determination of clotrimazole (CLO), methylparaben (MP) and propylparaben (PP) in a pharmaceutical preparation. Separation was carried out in a fused silica capillary (60 cm x 75 microm i.d.) at 25 kV with UV detection at 212 nm. Optimized background electrolyte (BGE) was 15 mM phosphate buffer (pH 7.2) containing 30 mM sodium dodecyl sulfate (SDS) as a surfactant. Rectilinear calibration ranges were 50-500 mg l(-1) for CLO, 10-100 mg l(-1) for MP and 2.5-25 mg l(-1) for PP. The total analysis time was < 12 min.

Full spectrum and selected spectrum based multivariate calibration methods for simultaneous determination of betamethasone dipropionate, clotrimazole and benzyl alcohol: Development, validation and application on commercial dosage form.

Five different chemometric methods were developed for the simultaneous determination of betamethasone dipropionate (BMD), clotrimazole (CT) and benzyl alcohol (BA) in their combined dosage form (Lotriderm® cream). The applied methods included three full spectrum based chemometric techniques; namely principal component regression (PCR), Partial Least Squares (PLS) and Artificial Neural Networks (ANN), while the other two methods were PLS and ANN preceded by genetic algorithm procedure (GA-PLS and GA-ANN) as a wavelength selection procedure. A multilevel multifactor experimental design was adopted for proper construction of the models. A validation set composed of 12 mixtures containing different ratios of the three analytes was used to evaluate the predictive power of the suggested models. All the proposed methods except ANN, were successfully applied for the analysis of their pharmaceutical formulation (Lotriderm® cream). Results demonstrated the efficiency of the four methods as quantitative tool for analysis of the three analytes without prior separation procedures and without any interference from the co-formulated excipient. Additionally, the work highlighted the effect of GA on increasing the predictive power of PLS and ANN models.

Application of hollow fiber membrane mediated with titanium dioxide nanowire/reduced graphene oxide nanocomposite in preconcentration of clotrimazole and tylosin.

In this paper, TiO2 nanowires and TiO2 nanoparticles have been successfully anchored on graphene oxide (GO) nanosheets by a facile one-step hydrothermal method. The synthesized TiO2 NWs/RGO and TiO2 NPs/RGO nanocomposites were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. After comparatively studying of the as-made nanocomposites, TiO2 NWs/RGO nanocomposite showed the best adsorbing performance and applied as an attractive efficient sorbent reinforced with microporous hollow fiber membrane through the sol-gel technology. In the following, the selected nanocomposite was utilized for simultaneous preconcentration and determination of clotrimazole and tylosin using high performance liquid chromatography (HPLC)-UV detection, respectively. In order to optimize the extraction conditions through affecting parameters (pH, stirring rate, salt addition, extraction time and volume of donor phase), response surface methodology (RSM) was employed as a powerful statistical technique. Under the optimal conditions, the limit of detection (S/N=3) of proposed HFSPME method, was 0.67 µg L(-1) for clotrimazole and 0.91 µg L(-1) for tylosin with good linear ranges of 1.7-8000.0 µg L(-1) and 4.0-6000.0 µg L(-1). The inter-day and intra-day relative standard deviations (RSD%) at 100 µg L(-1) concentration level were in the ranges of 2.10-3.58% for clotrimazole and 3.45-7.80% for tylosin (n=5), respectively. The proposed microextraction device was extended for determination of ultra trace amounts of target analytes in milk and urine samples with satisfactory results.

HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations.

Reversed-phase HPLC on different column packing materials (Hypersil C-18, Spherisorb-CN, Chromspher-B) is used to obtain selective separations of imidazole antimycotic drugs, such as ketoconazole, clotrimazole, tioconazole, bifonazole, isoconazole, econazole, miconazole and fenticonazole. The use of a post-column on-line photochemical reactor is shown to be useful for the enhancement of the sensitivity of the HPLC analysis with UV detection. The proposed HPLC methods are applied to the analysis of commercial dosage forms (creams) with solid-phase extraction (SPE) procedure, using a diol sorbent, being found to be a convenient technique for the sample preparation giving quantitative drug recovery.

Differential pulse polarographic determination of clotrimazole after derivatization with Procion Red HE-3B.

Clotrimazole was shown to react at room temperature in Britton Robinson buffer pH 2 with the reactive dye Procion Red HE-3B. The product exhibited a differential pulse polarographic peak at -0.38 V, which was well separated from the peaks of the reactive dye at -0.08, -0.80 and -0.95 V, and this allowed the indirect determination of clotrimazole in the presence of excess of the reactive dye. The method has been applied satisfactorily to the determination of clotrimazole in pharmaceutical formulations, calibration graphs are rectilinear up to at least 40 microg ml(-1). The detection limit was calculated to be 2.6 microg ml(-1) (3 sigma).

Triiodide ion and alizarin red S as two new reagents for the determination of clotrimazole and ketoconazole.

The reactions of a triiodide ion and alizarin red S with two important antifungal drugs containing an imidazole ring (ketoconazole (KC) and clotrimazole (CT)) have been studied for the development of two simple, rapid, sensitive and accurate indirect titrimetric and extractive-spectrophotometric methods for determining the concentration of these drugs. Spectroscopic studies and chemical analysis showed that the protonated forms of KC and CT react with triiodide ion forming highly stable and insoluble ion-pair products such as (KCH(2))(I(3))(2) and (CTH)I(3). Formation of ion-association complexes have been applied to the development of an indirect visual titrimetric method for the determination of KC and CT over the range 10(-5)-10(-2) M. The extractive-spectrophotometric method is based on the formation of (1:1) ion-association complexes between drugs and alizarin red S as chromogenic reagent in acidic citrate buffer that are extractable into chloroform with an absorption maximum at 425 nm. The system obeyed Beer's law in the concentration range 2.5-50 and 2.7-80 microgram ml(-1) for CT and KC, respectively. The proposed methods were applied for the analysis of the studied drugs in pure forms and their commercial preparations. Results are in good agreement with those obtained by official methods.

Comparison between micellar electrokinetic chromatography and HPLC for the determination of Betamethasone Dipropionate, Clotrimazole and their related substances.

The complete separation of a composite mixture that consisted of Betamethasone Dipropionate (BMD), Clotrimazole and their derivatives in a pharmaceutical dosage form was achieved within 15 min using sodium dodecyl sulfate (SDS) micellar electrokinetic chromatography (MEKC). For the MEKC separations, electrophoretic media consisting of SDS-phosphate buffer and various concentrations of alcohols or acetonitrile were used. The optimal condition for separating BMD, Clotrimazole and their analogues was found to be 50 mM SDS-15% acetonitrile-5% butanol at pH 7.2. The results demonstrated that the method was valid for the quantitation of BMD, Clotrimazole and analogues with selectivity and precision comparable to that of High-Performance Liquid Chromatography (HPLC).

On-line post-column photochemical derivatization in liquid chromatographic--diode-array detection analysis of binary drug mixtures.

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.