Multivariate optimisation and validation of a capillary electrophoresis method for the analysis of resveratrol in a nutraceutical

A rapid and simple method based on capillary electrophoresis was developed for the quality control of nutraceuticals containing resveratrol. Setting the UV detector at 280nm, the optimisation involved the separation of 11 effervescent tablet components, including the active compounds vitamin C, vitamin B(2), flavanones and hydroxycinnamic acids. Flufenamic acid was employed as internal standard. The effects of background electrolyte concentration, acetonitrile percentage and voltage were investigated by means of response surface methodology, considering as responses the critical resolution values and analysis time. The optimum conditions were found by Derringer desirability function. The background electrolyte consisted of 23mM borate buffer, adjusted to pH 10.0 with 1M sodium hydroxide, containing 7% (v/v) acetonitrile. Temperature and voltage were set at 25 degrees C and 26kV, respectively. Applying these conditions, the analysis time was below 7min. The performances of the method were tested in terms of selectivity, robustness, linearity and range, accuracy and precision and system suitability, following ICH guidelines.     

Identification and determination of mainstream and sidestream smoke components in different brands and types of cigarettes by means of solid-phase microextraction-gas chromatography-mass spectrometry

The qualitative and quantitative determination of components of mainstream and sidestream smoke has been performed by solid-phase microextraction-gas chromatography-mass spectrometry. Several brands and types of cigarettes sold in Italy were considered: normal, mild, light, extra light, some with filter and some without. Extraction of the analytes was performed by means of solid-phase microextraction (SPME) and the optimisation of the extraction procedure was performed by experimental design, taking into consideration type of fiber polymer, exposure temperature and time. Sixty-seven components of mainstream and sidestream smoke were identified. The quantified compounds (by means of deuterium-labelled isotopologues) were benzene, toluene, p-xylene, m-xylene, pyridine, o-xylene, limonene, naphthalene, phenol and nicotine. Finally, a comparison between the chemical profile of smoke from the different cigarettes was made.     

High-performance liquid chromatographic assay of erythromycin from biological matrix using electrochemical or ultraviolet detection

Two chromatographic methods were developed for the determination of erythromycin A (EA) residues in animal tissues (muscle, liver, kidney and fat of cattle, pigs and poultry) and cow's milk. In addition to a more traditional method using electrochemical detection, we developed an original alternative method based on UV detection at 236 nm, by pretreating to create a chromophore in the molecule. An internal standard was used with both methods to check the variability of the analytical system. Analysis times and performance were compared. The recovery of EA from various matrices was greater than 95%. For both methods the quantification limit for EA was 0.25 microgram ml-1 for plasma, 0.025 microgram g-1 for milk and 0.125 microgram g-1 for the other biological matrices. The methods can be used to check for EA residues in these matrices; in fact, the statutory maximum residue limits (MRLs) of EA are 0.4 microgram g-1 in muscle, kidney, liver and fat of beef cattle, sheep, pigs and poultry, and 0.04 microgram g-1 in cow's and sheep's milk.     

Application of Experimental Design Methodologies in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: A Review

Chirality is one of the major issues in pharmaceutical research and industry. Capillary electrophoresis (CE) is an interesting alternative to the more frequently used chromatographic techniques in the enantioseparation of pharmaceuticals, and is used for the determination of enantiomeric ratio, enantiomeric purity, and in pharmacokinetic studies. Traditionally, optimization of CE methods is performed using a univariate one factor at a time (OFAT) approach; however, this strategy does not allow for the evaluation of interactions between experimental factors, which may result in ineffective method development and optimization. In the last two decades, Design of Experiments (DoE) has been frequently employed to better understand the multidimensional effects and interactions of the input factors on the output responses of analytical CE methods. DoE can be divided into two types: screening and optimization designs. Furthermore, using Quality by Design (QbD) methodology to develop CE-based enantioselective techniques is becoming increasingly popular. The review presents the current use of DoE methodologies in CE-based enantioresolution method development and provides an overview of DoE applications in the optimization and validation of CE enantioselective procedures in the last 25 years. Moreover, a critical perspective on how different DoE strategies can aid in the optimization of enantioseparation procedures is presented.     

Development of a capillary electrophoresis method for the assay of ramipril and its impurities: an issue of cis-trans isomerization

The development of a rapid and selective capillary electrophoresis method for the quantitation of ramipril and its eight main impurities in pharmaceutical dosage form is described. Ramipril and three of its impurities contain a proline-similar moiety which causes in solution the presence of interconverting cis-trans isomers with respect to the amide bond. The interplay between electrophoretic migration and isomerization may yield the presence of an undesired interconversion zone between the two isomer peaks in the electropherogram, depending on the experimental conditions. Different capillary electrophoresis operative modes and pseudostationary phases were evaluated, both in normal and reverse polarity, in order to find the essential analytical parameters which could make it possible to overcome this issue and thus accurately quantify the analytes. The best results were obtained by using microemulsion electrokinetic chromatography in reverse polarity, where all the compounds which undergo cis-trans interconversion migrate as a single narrow peak. Experimental design led to identification of the following optimised conditions: background electrolyte, microemulsion made by 88.95% of 90 mM phosphate pH 2.5, 1.05% of n-heptane and 10.00% of SDS/n-butanol in 1:2 ratio; voltage, -26 kV; temperature, 17°C. Applying these conditions, the baseline separation of the analytes was obtained in about 10 min. Validation of the method following ICH guidelines was carried out and the procedure was applied to a real sample of ramipril tablets.     

Cyclodextrin-MEEKC for the analysis of oxybutynin and its impurities

The development of a cyclodextrin-MEEKC method for the analysis of oxybutynin and five related impurities is described. Experimental design strategies were applied in order to reach baseline separation of the compounds in a short analysis time. Mixture design made it possible to find the best composition for the microemulsion acting as pseudostationary phase, which was constituted by 89.1% 10 mM borate buffer pH 9.2, 1.7% n-heptane, 9.2% SDS/n-butanol in 1:2 ratio. The addition of (2-hydroxypropyl)-β-cyclodextrin to the background electrolyte was found to improve analysis performance. A Doehlert design, for the factors cyclodextrin concentration and voltage, was carried out and Derringer desirability function led to the identification of 18 mM and 29 kV as the optimal values. Applying the optimum conditions, separation of all the compounds, including the enantiomers of impurity 1, was obtained in less than 12 min. The method was validated according to ICH guidelines for drug assay and determination of impurities and was applied to oxybutynin tablet analysis.     

Measuring the primordial deuterium abundance during the cosmic dark ages

We discuss how measurements of fluctuations in the absorption of cosmic microwave background photons by neutral gas at redshifts z approximately 7-200 could reveal the primordial deuterium abundance of the Universe. The strength of the cross-correlation of brightness-temperature fluctuations in the redshifted 21-cm line of hydrogen with those in the redshifted 92-cm line of deuterium is proportional to the value of the deuterium-to-hydrogen ratio [D/H] fixed during big bang nucleosynthesis. Although challenging, this measurement would provide the cleanest possible determination of [D/H], free from contamination by structure formation processes at lower redshifts. We additionally report our result for the thermal spin-change cross section in deuterium-hydrogen scattering.     

Determination of the antidepressant paroxetine and its three main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method has been developed for the determination in human plasma of the specific serotonin reuptake inhibitor (SSRI) antidepressant paroxetine and its three main metabolites (M1, M2, M3). Fluorescence detection was used, exciting at λ = 294 nm and monitoring emission at λ = 330 nm for paroxetine (λ_exc = 280 nm, λ_em = 330 nm for M1 and M2; λ_exc = 268 nm, λ_em = 290 nm for M3). Separation was obtained on a reversed-phase C18 column using a mobile phase composed of 66.7% aqueous phosphate at pH 2.5 and 33.3% acetonitrile. Imipramine (λ_exc = 252 nm, λ_em = 390 nm) was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C8 cartridges (50 mg, 1 mL). The calibration curves were linear over a working range of 2.5-100 ng mL⁻¹ for paroxetine and of 5-100 ng mL⁻¹ for all metabolites. The limit of detection (LOD) was 1.2 ng mL⁻¹ for PRX and 2.0 ng mL⁻¹ for the metabolites. The method was applied with success to plasma samples from depressed patients undergoing treatment with paroxetine. Hence, the method seems to be suitable for the therapeutic drug monitoring of paroxetine and its main metabolites in depressed patients’ plasma.     

Optimisation and validation of a capillary electrophoresis method for the simultaneous determination of diazepam and otilonium bromide.

A simultaneous assay of diazepam and otilonium bromide in coated tablets by capillary zone electrophoresis (CZE) was developed. The influence of various parameters (voltage, temperature, buffer concentration and pH, ethanol percentage) on analysis time and on the theoretical plates of the two peaks was investigated by means of experimental design. A response surface study was carried out by means of a 27-run D-optimal matrix. The best background electrolyte was found to be 0.13 M, pH 2.9 Britton-Robinson buffer, containing 10% v/v ethanol. Other optimised parameters were voltage (30 kV) and temperature (30 degrees C). The UV detector for quantitation of otilonium bromide and diazepam was set at 280 nm and 230 nm, respectively. Procaine hydrochloride was used as internal standard and run time was less than five minutes. Validation was performed, for drug substance and drug product, according to ICH3 guidelines. For drug product the recovery for otilonium bromide and diazepam ranged from 98.3% to 101.2% and from 97.1% to 99.0%, respectively; the RSD values found for otilonium bromide and diazepam ranged from 2.4% to 3.0% and from 1.1% to 4.5%, respectively.