Enhanced chemiluminescence determination of paracetamol.

Due to the severe consequences of potential overdoses of paracetamol (PCM) on the human body, the measurement of PCM in pharmaceutical and biological samples is essential. Therefore, a simple and rapid technique with a high detection limit and a wide linear range is presented here which plays a crucial role in detecting PCM's overdose leading to drug poisoning. This contribution illustrates a novel chemiluminescence (CL) system for the detection of PCM based on the chemiluminescence reaction between PCM and KMnO4. An enhanced CL was observed by the addition of rhodamine·6G within an SDS surfactant. The system shows good analytical performance over the linear range of 0.12 µM to 0.185 mM with a detection limit of 7.8 × 10-8 M. In addition, good precision was observed with a RSD of 0.81%. This system was successfully applied to the detection of PCM in pharmaceutical tablets and drops as well as in human urine samples with average % recoveries ranging from 95.5 to 105.7%. The possible interferents from the major excipients in pharmaceuticals and other related compounds as well as biological interferents were also studied. This highlights the feasibility of the proposed system for application in real sample analysis in both chemical and biological matrices.

Vortex-assisted liquid-liquid extraction combined with field-amplified sample injection and sweeping micellar electrokinetic chromatography for improved determination of ß-blockers in human urine.

A new micellar electrokinetic chromatography (MEKC) method was developed and validated for the analysis of carvedilol and propranolol in human urine samples. In this study, vortex-assisted liquid-liquid extraction (VALLE) coupled with field-amplified sample injection and sweeping was employed for biological sample clean-up and sensitivity enhancement in MEKC. After VALLE, the urine samples were analyzed by MEKC. Tris-phosphate buffer (60mmolL(-1), pH 2.0) containing 40% (v/v) methanol was first filled into an uncoated fused-silica capillary (56cm, 50µm i.d.). The pretreated urine sample was loaded by electrokinetic injection (10kV, 250s). The stacking and separation were performed using Tris-phosphate buffer (30mmolL(-1), pH 3.0) containing 30% (v/v) methanol and 50mmolL(-1) sodium dodecyl sulfate (SDS) at -25kV. Detection was carried out at 195 and 214nm for carvedilol and propranolol, respectively. The suggested method is linear (r(2)≥0.997) over a dynamic range of 0.005-1µgmL(-1) in urine. The intra- and inter-day relative standard deviation and relative error values of the method were below 20%, which shows good precision and accuracy. Finally, this method was successfully applied to the analysis of real urine samples.

Determination of five antiarrhythmic drugs in human plasma by dispersive liquid-liquid microextraction and high-performance liquid chromatography.

A fast and sensitive high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for the simultaneous quantitation of five antiarrhythmic drugs (metoprolol, propranolol, carvedilol, diltiazem, and verapamil) in human plasma samples. It involves dispersive liquid-liquid microextraction (DLLME) of the desired drugs from 660 µL plasma and separation using isocratic elution with UV detection at 200 nm. The complete separation of all analytes was achieved within 7 min. Acetonitrile (as disperser solvent) resulting from the protein precipitation procedure was mixed with 100 µL dichloromethane (as an extraction solvent) and rapidly injected into 5 mL aqueous solution (pH 11.5) containing 1% (w/v), NaCl. After centrifugation, the sedimented phase containing enriched analytes was collected and evaporated to dryness. The residue was re-dissolved in 50 µL de-ionized water (acidified to pH 3) and injected into the HPLC system for analysis. Under the optimal conditions, the enrichment factors and extraction recoveries ranged between 4.4-10.8 and 33-82%, respectively. The suggested method was linear (r(2) ≥0.997) over a dynamic range of 0.02-0.80 µg mL(-1) in plasma. The intra- and inter-days relative standard deviation (RSD%) and relative error (RE%) values of the method were below 20%, which shows good precision and accuracy. Finally, this method was applied to the analysis of real plasma samples obtained from the patients treated with these drugs.

Detection limit enhancement of antiarrhythmic drugs in human plasma using capillary electrophoresis with dispersive liquid-liquid microextraction and field-amplified sample stacking method.

BACKGROUND: A new capillary zone electrophoresis (CZE) with ultraviolet detection method has been developed and validated for the analysis of four antiarrhythmic drugs in human plasma samples. METHODS: In this study, a dispersive liquid-liquid microextraction (DLLME) coupled with field-amplified sample stacking (FASS) was employed for biological samples clean-up and sensitivity enhancement in CZE. RESULTS: Under optimum DLLME-FASS-CZE conditions, enhancement factors were in the range of 157-314. The method was validated over the concentration range of 20-800 ng/ml in human plasma. Inter- and intra-day precision and the accuracy were less than 20%; the detection limits ranged from 2.5 to 4.7 ng/ml. Furthermore, the validated method was successfully applied to the detection of studied drugs in patients' plasma samples.

Simultaneous determination of some common food dyes in commercial products by digital image analysis.

A simple and relatively fast image-analysis method using digital images, obtained with a flatbed scanner, has been described. The method was used for the simultaneous determination of four common food dyes, namely, carmoisine, brilliant blue, sunset yellow, and quinoline yellow, in binary mixtures in commercial products without a need for any prior separation steps. The results obtained were validated against a standard high-performance liquid chromatography method and a good agreement was obtained. The parameters affecting the experimental results were optimized. Under the optimal conditions, the method provided acceptable linear ranges (20-250 mg/L) with correlation coefficients higher than 0.998, suitable precision (relative standard deviation ≤ 4.5%), and limits of detection between 4.82 and 8.05 mg/L.

Determination of valproic acid in human plasma using dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection.

OBJECTIVES: Dispersive liquid-liquid microextraction coupled with gas chromatography (GC)-flame ionization detector was developed for the determination of valproic acid (VPA) in human plasma. MATERIALS AND METHODS: Using a syringe, a mixture of suitable extraction solvent (40 µl chloroform) and disperser (1 ml acetone) was quickly added to 10 ml of diluted plasma sample containing VPA (pH, 1.0; concentration of NaCl, 4% (w/v)), resulting in a cloudy solution. After centrifugation (6000 rpm for 6 min), an aliquot (1 µl) of the sedimented organic phase was removed using a 1-µl GC microsyringe and injected into the GC system for analysis. One variable at a time optimization method was used to study various parameters affecting the extraction efficiency of target analyte. Then, the developed method was fully validated for its accuracy, precision, recovery, stability, and robustness. RESULTS: Under the optimum extraction conditions, good linearity range was obtained for the calibration graph, with correlation coefficient higher than 0.998. Limit of detection and lower limit of quantitation were 3.2 and 6 µg/ml, respectively. The relative standard deviations of intra and inter-day analysis of examined compound were less than 11.5%. The relative recoveries were found in the range of 97 to 107.5%. Finally, the validated method was successfully applied to the analysis of VPA in patient sample. CONCLUSION: The presented method has acceptable levels of precision, accuracy and relative recovery and could be used for therapeutic drug monitoring of VPA in human plasma.

Salt-assisted LLE combined with field-amplified sample stacking in CE for improved determination of beta blocker drugs in human urine.

BACKGROUND: A simple and sensitive CE method was developed and validated for the analysis of some beta blockers in human urine. METHODS: In this study, salting-out assisted LLE combined with field-amplified sample stacking method was employed for biological sample clean-up and sensitivity enhancement in CE. RESULTS: Under the optimal conditions good linearity (r(2) ≥0.998) was obtained, within 0.025-1 µg/ml for propranolol and metoprolol, and within 0.05-1 µg/ml for carvedilol in urine samples. LODs and LLOQs ranged from 0.005 to 0.015 µg/ml, and from 0.025 to 0.05 µg/ml, respectively. The RSDs of intra- and inter-day analysis of examined compounds were less than 4.0%. The recoveries were in the range of 98-119%. CONCLUSION: The validated method is successfully applied to determine propranolol, metoprolol and carvedilol in human urine samples obtained from the patients who received these drugs.

Gold nanorods-enhanced rhodamine B-permanganate chemiluminescence and its analytical application.

A novel enhanced chemiluminescence system was developed by applying gold nanorods (Au NRs) as catalysts in rhodamine B-permanganate reaction. Au NRs with three different aspect ratios were synthesized by seed mediated growth method and characterized by UV-Vis spectra and transmission electron microscopy. It was demonstrated that Au NRs have much higher catalytic effect than spherical nanoparticles on rhodamine B-permanganate chemiluminescence reaction. Among various sizes of Au NRs, those with average aspect ratio of 3.0 were found to have the most remarkable catalytic activity. As an analytical application of the new chemiluminescence system, albumin as a model protein was quantified based on its interaction with NRs. Albumin binds to Au NRs active surfaces and inhibits their catalytic action and therefore decreases the intensity of chemiluminescence. This diminution effect is linearly related to the concentration of the human and bovine serum albumin over the ranges of 0.45-90 and 0.75-123 nmol L(-1), respectively with the corresponding limits of detection of 0.18 and 0.30 nmol L(-1). The method was successfully applied to the determination of albumin in human and bovine serum samples.

A highly sensitive RP-HPLC-fluorescence method to study aldehyde oxidase activity.

Aldehyde oxidase is a widely distributed enzyme that is involved in the metabolism of an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance. In the present study, a highly sensitive RP-HPLC-fluorescence method based on the oxidation of phenanthridine to phenanthridinone has been developed and validated to assay aldehyde oxidase activity in biological samples. Determination of phenanthridinone was achieved on a C18 column using 10 mmol/L phosphate buffer (pH 5.0) containing 0.1 mmol/L EDTA-acetonitrile (40 + 60, v/v) as the mobile phase. The fluorescence intensity of phenanthridinone was measured at 364 nm with excitation at 236 nm. The proposed method was precise, accurate, specific and rapid (analysis time, approximately 8 min) with a mean RSD of 2.54%. Peak responses were linear from 0.5 to 100 nmol/L, with an LOD of 0.125 nmol/L. The applicability of the method was demonstrated by measurement of aldehyde oxidase activity in rat liver, kidney, ovary, and heart fractions.

Study of aldehyde oxidase-catalyzed metabolic pathway of phenanthridine using MCR-ALS method.

Evaluation of metabolic pathways is one of the challenging areas in biological and pharmaceutical sciences. Phenanthridine oxidation to phenanthridinone is used commonly to study aldehyde oxidase activity. This reaction could pass through phenanthridine N-oxide intermediate. In the present study, the application of multivariate curve resolution, optimized by alternating least squares (MCR-ALS) to investigate this metabolic pathway has been described. The results obtained from MCR-ALS analysis along with those obtained from the use of potassium ferrocyanide method indicated that phenanthridine is directly oxidized to phenanthridinone by rat liver aldehyde oxidase without passing through phenanthridine N-oxide intermediate. It was also found that the later compound is not metabolized by this enzyme.

Simultaneous Determination of 6-Mercaptopurine and its Oxidative Metabolites in Synthetic Solutions and Human Plasma using Spectrophotometric Multivariate Calibration Methods.

INTRODUCTION: 6-Mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). It is catabolized to 6-thiouric acid (6TUA) through 8-hydroxo-6-mercaptopurine (8OH6MP) or 6-thioxanthine (6TX) intermediates. METHODS: High-performance liquid chromatography (HPLC) is usually used to determine the contents of therapeutic drugs, metabolites and other important biomedical analytes in biological samples. In the present study, the multivariate calibration methods, partial least squares (PLS-1) and principle component regression (PCR) have been developed and validated for the simultaneous determination of 6MP and its oxidative metabolites (6TUA, 8OH6MP and 6TX) without analyte separation in spiked human plasma. Mixtures of 6MP, 8-8OH6MP, 6TX and 6TUA have been resolved by PLS-1 and PCR to their UV spectra. RESULTS: Recoveries (%) obtained for 6MP, 8-8OH6MP, 6TX and 6TUA were 94.5-97.5, 96.6-103.3, 95.1-96.9 and 93.4-95.8, respectively, using PLS-1 and 96.7-101.3, 96.2-98.8, 95.8-103.3 and 94.3-106.1, respectively, using PCR. The NAS (Net analyte signal) concept was used to calculate multivariate analytical figures of merit such as limit of detection (LOD), selectivity and sensitivity. The limit of detections for 6MP, 8-8OH6MP, 6TX and 6TUA were calculated to be 0.734, 0.439, 0.797 and 0.482 µmol L-1, respectively, using PLS and 0.724, 0.418, 0783 and 0.535 µmol L-1, respectively, using PCR. HPLC was also applied as a validation method for simultaneous determination of these thiopurines in the synthetic solutions and human plasma. CONCLUSION: Combination of spectroscopic techniques and chemometric methods (PLS and PCR) has provided a simple but powerful method for simultaneous analysis of multicomponent mixtures.

A new multi-wavelength model-based method for determination of enzyme kinetic parameters.

Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method.

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1-30.0 microM and the absorption spectra of the solutions were recorded in the range of 210-280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis-Menten constants from a Lineweaver-Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04+/-0.01 and 0.03+/-0.01 microM (mean+/-SD, n=5), respectively. Using this method, the Km value for the oxidation of phenanthridine was calculated as 1.72+/-0.09 microM (mean+/-SD, n=3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.