Trace determination of antifungal drugs in biological fluids through a developed approach of hydrogel-based spin-column micro-solid-phase extraction followed by LC-MS/MS analysis

In the present research, a blended polyacrylamide-chitosan hydrogel was synthesized. For the first time, the prepared sorbent was efficiently employed in a hydrogel-based spin-column setup as a promising format. The proposed method was applied for monitoring the trace amounts of ketoconazole, clotrimazole, and miconazole in blood samples. Effective adsorption and desorption parameters were optimized using a central composite design and the one-variable-at-a-time method. Under the optimal conditions, the calibration curves were linear in the range of 15.0–1000.0, 1.0–1000.0, and 2.0–1000.0 ng mL^–1 for ketoconazole, clotrimazole, and miconazole, respectively, along with intra- and interday precision less than 8.4%. Limits of detection were obtained between 0.2 and 5.0 ng mL^–1. The preconcentration factors were found in the range of 5.9–7.8. The introduced method was successfully applied for micro-solid-phase extraction of trace amounts of target antifungal drugs in blood samples, followed by liquid chromatography-tandem mass spectrometry. Satisfactory relative recoveries of 94.5–103.5% were obtained, implying method reliability. Overall, the proposed method provides good accuracy and repeatability, high reusability, and good applicability to determine antifungal drugs in complex biological matrices.     

Determination of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography–tandem mass spectrometry

A sensitive and robust method using solid-phase extraction and ultrasonic extraction for preconcentration followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) has been developed for determination of 19 biocides: eight azole fungicides (climbazole, clotrimazole, ketoconazole, miconazole, fluconazole, itraconazole, thiabendazole, and carbendazim), two insect repellents (N,N-diethyl-3-methylbenzamide (DEET), and icaridin (also known as picaridin)), three isothiazolinone antifouling agents (1,2-benzisothiazolinone (BIT), 2-n-octyl-4-isothiazolinone (OIT), and 4,5-dichloro-2-n-octyl-isothiazolinone (DCOIT)), four paraben preservatives (methylparaben, ethylparaben, propylparaben, and butylparaben), and two disinfectants (triclosan and triclocarban) in surface water, wastewater, sediment, sludge, and soil. Recovery of the target compounds from surface water, influent, effluent, sediment, sludge, and soil was mostly in the range 70–120?%, with corresponding method quantification limits ranging from 0.01 to 0.31?ng?L^?1, 0.07 to 7.48?ng?L^?1, 0.01 to 3.90?ng?L^?1, 0.01 to 0.45?ng?g^?1, 0.01 to 6.37?ng?g^?1, and 0.01 to 0.73?ng?g^?1, respectively. Carbendazim, climbazole, clotrimazole, methylparaben, miconazole, triclocarban, and triclosan were detected at low ng?L^?1 (or ng?g^?1) levels in surface water, sediment, and sludge-amended soil. Fifteen target compounds were found in influent samples, at concentrations ranging between 0.4 (thiabendazole) and 372?ng?L^?1 (methylparaben). Fifteen target compounds were found in effluent samples, at concentrations ranging between 0.4 (thiabendazole) and 114?ng?L^?1 (carbendazim). Ten target compounds were found in dewatered sludge samples, at concentrations ranging between 1.1 (DEET) and 887?ng?g^?1 (triclocarban).     

High-performance thin-layer chromatography determination of some antimycotic imidazole derivatives and preservatives in medicinal creams and a gel

Simple and reliable thin-layer chromatography-densitometry methods for determination of antimycotics (bifonazole, clotrimazole, and miconazole) and preservatives (benzyl alcohol and benzoic acid) were developed. The pairs bifonazole/benzyl alcohol, clotrimazole/benzyl alcohol, and miconazole/benzoic acid were determined simultaneously. The following mobile phases were used: ethyl acetate-n-heptane-methanoldiethylamine (3 + 4.5 + 1 + 0.2, v/v/v/v) for bifonazole and benzyl alcohol; n-butyl acetate-n-heptane-methanol-dietylamine (3 + 4.5 + 1 + 0.2, v/v/v/v) for clotrimazole and benzyl alcohol; and n-butyl acetate-carbon tetrachloride-methanol-diethylamine (3 + 6 + 2.5 + 0.5, v/v/v/v) for miconazole and benzoic acid. The chromatographic zones on silica gel plates were scanned in the reflectance/absorbance mode at 230 nm (bifonazole, benzyl alcohol, miconazole, and benzoic acid) and 210 nm (clotrimazole and benzyl alcohol). The recovery for all substances ranged from 98.7 to 100.7%. The limits of detection and quantitation were 0.03 to 0.2 microg and 0.1 to 0.5 microg/spot, respectively. The proposed methods were applied for determination of antimycotics and preservatives in commercially available pharmaceuticals.     

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.     

Charge-Transfer Complexation for Spectrophotometric Assay of Certain Imidazole Antifungal Drugs

Abstract

A spectrophotometric method is described for the assay of some antifungal agents containing an imidazole ring: clotrimazole, econazole, ketoconazole and miconazole. The method is based on the formation of a charge-transfer complex between the drug as n-electron donor and iodine as [sgrave]-acceptor. The product exhibited two absorption maxima at 290 and 377 nm; measurements are made at 290 nm. Beer's law is obeyed in a concentration range of 1-40 μg/ml. The method is rapid, simple and sensitive and can be applied to the analysis of some commercial dosage forms without interference. A detailed investigation of the formed complex was made with respect to its composition, association constant and free energy change.

     

Determination of methylparaben,propylparaben, clotrimazole and its degradation products in topical cream by RP-HPLC

Summary Reversed-phase, high-performance liquid chromatographic (RP-HPLC) methods with UV detection were developed and validated for determination of compounds in a topical cream. The first method describes determination of the active component clotrimazole and two preservatives present in the cream; methylparaben and propylparaben. The second method describes determination of two degradation products of clotrimazole, imidazole and (2-chlorophenyl) diphenylmethanol, in a topical cream after long-term stability tests. Chromatographic separation was on a Purospher RP-18e column; the mobile phase in Method1 for separation of clotrimazole, methylparaben and propylparaben comprises acetonitrile and water (70:30 v/v). For determination of degradations products-imidazole and (2-chlorophenyl) diphenylmethanol—the optimum composition of mobile phase in Method2 was acetonitrile and water (75:25 v/v) apparent pH^* 2.7. Analysis time was <10 min for both methods. The methods were found to be applicable for routine analysis of the active compound clotrimazole, preservatives and degradation products in the pharmaceutical product: topical cream 1% Clotrimazol Cream. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Quantitative Determination of Miconazole in Creams by Second Order Derivative Spectrophotometry

ABSTRACT

A derivative spectrophotometry was developed to determine miconazole in cream formulations that contain benzoic acid as preservative. The procedure was based on the linear relationship in the range 100-500 μg ml^?1 between the drug concentration and the second derivative amplitudes at 276 nm. Results of the recovery experiments performed on various amounts of benzoic acid and of the determination of miconazole in cream confirmed the applicability of the proposed method to complex formulations.     

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.     

Analysis,fate studies and monitoring of the antifungal agent clotrimazole in the aquatic environment

The analysis and presence of clotrimazole, an antifungal agent with logK _OW > 4, was thoroughly studied in the aquatic environment. For that reason analytical methods based on gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry were developed and validated to quantify clotrimazole with limits of quantification down to 5 and 1 ng/L, respectively. Both methods were compared in an intercalibration exercise. The complete mass-spectrometric fragmentation pattern could be elucidated with the aid of quadrupole time of flight mass spectrometry. Since clotrimazole tends to adsorb to laboratory glassware, studies on its adsorption behaviour were made to ensure the appropriate handling of water samples, e.g. pH, storage time, pretreatment of sampling vessels or material of the vials used for final extracts. The phenomena of adsorption to suspended matter were investigated while analysing different waste-water samples. Application of the methods in various investigated wastewater and surface water samples demonstrated that clotrimazole could only be detected in the low nanogram per litre range of anthropogenic influenced unfiltered water samples after acidification to pH 2.     

High Performance Liquid Chromatographic Determination of Nordihydroguaiaretic Acid

Abstract

A high performance liquid chromatography (HPLC) method was developed for the detection and quantitation of nordihydroguaiaretic acid (NDGA). Very low concentrations of NDGA in various extracts are detectable, thus making the method more sensitive than other previously reported analytical techniques. NDGA was extracted from leaves of Larrea divaricata as well as from rodent food containing NDGA. Since rats are fed NDGA in studies that examine the development of renal cystic disease, we modified extraction procedures to permit isolation of NDGA from tissue and serum samples.     

Evaluation of Packed GLC Columns for Nanomolar Analysis of Unknowns Lincluding a New Type of Packed Column

Abstract

Fatty acid methyl esters, 2,6-dimethylaniline, and 2,6-xylenol were used for rapid evaluation of the usefulness of packed GLC columns for analysis of unknowns at the nanomolar level. Three types of column construction with three liquid phases of widely varying polarity were tested by the above procedure. A new type of GLC column, a dynamically coated packed column prepared with a surface active agent, was found to yield better overall performance for less polar liquid phases than packed silanized glass or packed stainless steel columns.     

HPLC Quantitation of Levobunolol and Its Metabolite,Dihydrolevobunolol in Biological Fluids

Abstract

In order to quantitate the concentrations of levobunolol and its major metabolite (dihydrolevobunolol) in aqueous humor and in blood after ophthalmic doses, HPLC procedures were developed. The direct injection procedure, after plasma protein precipitation and reduction of levobunolol to dihydrolevobunolol, was found to be suitable for clinical samples. A second procedure, involving extraction with ethyl ether, employed a uv/fluorescence dual detection system to simultaneously monitor levobunolol and dihydrolevobunolol. The sensitivity for levobunolol was 5 ng, and for dihydrolevobunolol, 1 ng. There was no observable interference from the biological fluids, blood and aqueous humor. Both procedures offer high reproducibility, selectivity, and sensitivity, which are essential for pharmacokinetic and metabolism studies.     

HPLC‐DAD for the determination of three different classes of antifungals: method characterization,statistical approach,and application to a permeation study

This study describes and characterizes methods for high‐performance liquid chromatography diode array detection (HPLC‐DAD) analysis of formulations containing molecules with antifungal activity of three different classes: terbinafine and butenafine (allylamines), miconazole and fluconazole (azoles), and geraniol, neral and geranial (monoterpenes). All methods used the same chromatographic column (RP₁₈), enabling the analysis to be performed in a single batch. The specificity was extensively discussed through the establishment of purity peak methods. The analytical parameters (linearity, precision and accuracy) were calculated and discussed in detail using specific statistical approaches. All substances showed satisfactory results for chromatographic and analytical parameters. Limits of 1.3% to mean repeatability and 2.0% for intermediate precision are suggested as acceptance criteria in validation of methods by HPLC‐DAD, in situations where there is no extensive pretreatment of the samples. The methods proved to be robust and significant factors were discussed regarding their influence on chromatographic parameters (retention time, resolution, tailing factor and column efficiency). Finally, the application of the developed methods was demonstrated by the results of a permeation study of the antifungal agents through bovine hoof membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

A Clinical Method for the Analysis of Amygdalin in Human Plasma

Abstract

A rapid, simple and sensitive method for the analysis of amygdalin in human plasma has been developed. The method is based on the hydrolytic action of the enzyme 6-glucosidase on amygdalin to yield a molar equivalent amount of benzaldehyde (along with 2 moles of glucose and a mole of HCN). The benzaldehyde is extracted from plasma with chloroform and determined by GLC using a flame ionization detector. The benzaldehyde recovered, when the method was applied to human plasma spiked with amygdalin, was found to be 9.5% of the added amygdalin. Such recovery was highly reproducible (± 1.5%) for amygdalin concentrations ranging from 2 to 20 μg/ml.     

Application of Supercritical Carbon Dioxide for the Preparation of Drug-Cyclodextrin Inclusion Compounds

Inclusion complexes of drugs into cyclodextrins (CDs) can be obtained at the solid state by means of supercritical dioxide (SCCO₂). A successful inclusion with a yield >98.5% has been achieved with piroxicam and -CD. The temperature and the time of exposure to SCCO₂ have a significant effect on the inclusion yield while the pressure has a negative effect. However, there is a strong interaction between temperature and pressure and this interaction has a positive influence. The molar ratio piroxicam--CD and the addition of ternary alkaline agents were also found to be significant factors. The dissolution rate of the complexes formed using SCCO₂ was found to be significantly higher than that of the physical mixture. Inclusion complexes have also been obtained with miconazole treating mixtures of miconazole, CDs and citric acid by SCCO₂. This new technique of inclusion of poorly soluble drugs into CDs allows the preparation of solid complexes without using organic solvents and thus without residues.     

Spectrofluorimetric Determination of Chlorpromazine Hydrochloride and Thioridazine Hydrochloride

Abstract

Fluorescence spectroscopy was applied to the development of a sensitive and simple method for the determination of chlorpromazine HCl and thioridazine-HCl. The method is based upon development of an intense fluorescence using N-bromosuccinimide as the fluorogenic reagent. The produced fluorescence has very characteristic excitation and emission spectra and was stable for at least one hour. The results were reproducible and as little as 5 ng/ml chloropromazine HCl and 1 ng/ml thioridazine-HCl could be determined. The method was applied successfully to the analysis of various commercially available dosage forms. The obtained results were in good agreement with those of the official BP 93 procedures.     

Determination of commonly used azole antifungals in various waters and sewage sludge using ultra-high performance liquid chromatography–tandem mass spectrometry

Sensitive and reliable methods have been developed and validated for determination of commonly consumed azole antifungal pharmaceuticals (clotrimazole, econazole, ketoconazole, and miconazole) and biocides (propiconazole and tebuconazole) in various waters and sewage sludge. Solid phase extraction (SPE) combined with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used to determine the azole antifungals in waters. Azole antifungals in sewage sludge were extracted with ultrasonic-assisted extraction, followed by SPE cleanup and UHPLC–MS/MS detection. Quantification was performed by internal standard calibration in multiple reaction monitoring mode. Recoveries were mostly in the range of 52–110% with relative standard deviations generally within 20%. Method quantification limits were 0.5–6 ng L⁻¹ in waters and 3–9 ng g⁻¹ dry weight (dw) in sewage sludge, respectively. The methods were applied to determine the azole antifungals in wastewater, river water, sediment, and sewage sludge sampled from the Pearl River Delta, China. Clotrimazole, ketoconazole, and miconazole were widely detected at low ng L⁻¹ in waters, low ng g⁻¹ dw in river sediment, and low μg g⁻¹ dw in sewage sludge. The methods can provide valuable tools for investigating occurrence and fate of the azole antifungals in the environment.     

Thermoanalytical studies on complexes of clotrimazole with cyclodextrins

γ-Cyclodextrin and dimethyl-β-cyclodextrin were used as solubilizing agents for a very poorly water-soluble drug, an imidazole derivative antifungal agent, clotrimazole; with the aim of improving the physicochemical properties of the drug. Solid products were prepared by physical mixing, kneading, precipitation and spray-drying methods in 1:1 and 1:2 drug:cyclodextrin molar ratios. Drug interactions were studied by thermoanalytical methods such as DSC, DTA, TG and DTG, X-ray diffractometry and Fourier transformation-infrared spectroscopy. The results demonstrated the formation of inclusion complexes in some products. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Direct Spectrofloorimetric Method for the Determination of B-Naphthoxyacetic Acid Residues in Strawberries

Abstract

β-naphthoxyacetic acid (NOA) residues in strawberries have been determined in acetone extracts using direct, first and second derivatives with spectrofluorimetric detection, without clean-up procedures needed. Solvent effects on the spectral characteristics of NOA solutions and their influences on the sensitivity of its variables and instrumental parameters are also studied. A detection limit of 1.14 ng/ml was achieved using the first derivative approach. Strawberries were fortified at different NOA concentrations (10 to 90 ng/ml). Recoveries averaged 87.2% (range 80.16–98.53) at the lower fortification level and 98.36% (range 97.54–99.63) for the higher fortification level.     

Determination of exemestane and 17-hydroxyexemestane by high-performance liquid chromatography coupled with tandem mass spectrometry and high-resolution mass spectrometry

An approach was developed for determining and confirming the presence of exemestane and its metabolite 17-hydroxyexemestane in urine. It is based on the application of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/MS) and atmospheric pressure chemical ionization high-resolution mass spectrometry (HPLC-HRMS). To detect hydroxyexemestane, the analysis of the hydrolyzed fraction of urine is preferable. The recovery rates of exemestane and 17-hydroxyexemestane were 83 and 91%, respectively. The detection limits were 1 ng/mL for HPLC-MS/MS and 2.5 ng/mL for HPLC-HRMS. In spite of a considerable effect of ionization suppression, the sensitivity and selectivity of the determination are affected by the selection of the optimal detection conditions in HPLC-MS/MS and by the high accuracy of mass determination in mass spectrometry with orbitrap detection, enabling resolution at a level of 5 ppm. The procedures can be used for screening and confirmatory analysis.