Iacopo Giannini

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.

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)-beta-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.

Microemulsion electrokinetic chromatography: An application for the simultaneous determination of suspected fragrance allergens in rinse-off products

A mixture of 18 neutral UV-active compounds with different characteristics of polarity was determined by capillary electrophoresis using a pseudostationary phase constituted by a microemulsion. The test analytes were volatile fragrance compounds, included in a list of 24 chemicals classified as suspected allergens according to Directive 2003/15/CE. The considered compounds were detected at 195 nm and p-anisaldehyde was chosen as internal standard. The background electrolyte consisted of a standard microemulsion made of 90.95% 10mM borax buffer, pH 9.2, 1.05% n-heptane, 8.00% SDS/n-butanol in 1:2 ratio, to which 40 mM methyl-β-cyclodextrin was added. Temperature and voltage were set at 20 °C and 25 kV, respectively. These experimental conditions allowed separation of the compounds to be obtained in about 20 min. The method was applied to real samples made up of rinse-off scented products. The results obtained using the standard microemulsion as pseudostationary phase showed its high resolution power, capable of effectively separating a complex mixture of analytes. Microemulsion electrokinetic chromatography was confirmed to have a great potential for different analytical challenges, holding up the possibility of using this technique as a good and complementary alternative to HPLC methods for routine analysis.

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.

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.

Pitfalls and success of experimental design in the development of a mixed MEKC method for the analysis of budesonide and its impurities

A mixed MEKC method for the analysis of budesonide and its related substances is presented. The micelles were formed from sodium cholate (CHOL) and 3‐(N,N‐dimethylmyristylammonio)propanesulfonate (MAPS). A multivariate optimisation was carried out with the aim of obtaining a baseline separation of all compounds. The influence of voltage, borate concentration, cholate concentration, MAPS concentration and pH was evaluated on the responses, corresponding to critical resolution values. Problems with the investigated experimental design were encountered due to the complexity of the separation process. As a consequence, a first design was not sufficient to reach the optimal conditions, but was needed in order to obtain the necessary information to successfully plan a second in‐depth study by means of response surface methodology. The optimal conditions were as follows: capillary total and effective lengths of 48.5 and 40.0 cm, respectively, with 50 μm id; 70 mM borate buffer (pH 8.8) containing 65 mM CHOL and 10 mM MAPS; temperature 20°C and voltage 16 kV. Separation of all the compounds, including R‐ and S‐epimers of budesonide, was obtained in a reasonable time. Validation of the method was performed for both drug substances and drug product.

Dual CD system-modified MEEKC method for the determination of clemastine and its impurities

A dual system of CDs was used for the first time in MEEKC with the aim of determining clemastine and its three main related impurities in both drug substances and tablets. The addition of methyl‐β‐cyclodextrin and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin to the microemulsion pseudo‐stationary phase was essential to increase the resolving power of the system to obtain a baseline separation among the compounds. The best microemulsion composition was identified by mixture design and the effects of the factors concentrations of CDs and voltage were investigated by a response surface study applying a Central Composite Design. In both cases, Derringers desirability function made it possible to find the global optimum, which corresponded to the following combination: microemulsion, 89.8% 10 mM borate buffer pH 9.2, 1.5% n‐heptane and 8.7% of SDS/n‐butanol in 1:2 ratio; 18 mM methyl‐β‐cyclodextrin, 38 mM heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin, 17 kV. By applying these conditions, the separation was completed in about 5.5 min. The method was validated following International Conference on Harmonisation guidelines and was applied to a real sample of clemastine tablets.

Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling.

Electrokinetic chromatography (EKC) allows the separation of closely related substances by the detection of fine effects in analyte–separation system interactions. With the goal of understanding the fine effects involved in separation using a dual cyclodextrin–microemulsion EKC system, an integrated study of NMR and molecular modeling was carried out. The above dual cyclodextrin–microemulsion system was previously used in the separation of clemastine and its related substances and was prepared by the addition of methyl‐β‐cyclodextrin (MβCD) and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DMβCD) to an oil‐in‐water microemulsion. The use of DMβCD was shown to be essential in the separation of clemastine from one of its related substance (IB). A molecular modeling study allowed the different affinities of clemastine and IB for the two cyclodextrins to be explained. Furthermore, rotating‐frame Overhauser effect spectroscopy NMR experiments clearly indicated that besides the primary pseudostationary phase, namely the ionic microemulsion, cyclodextrins acted as a secondary pseudostationary phase. In addition, it was shown that inclusion complexation of sodium dodecyl sulfate (SDS) monomers into the cyclodextrins cavity occurs; differently, the oil (n‐heptane) used in the preparation of microemulsion system resulted to be not included into the macrocycle cavity. These experimental results were supported by molecular modeling, which highlighted the preferential inclusion of SDS into DMβCD. On the basis of these results, it was confirmed that, besides its primary role as the ionic carrier in EKC, SDS is involved in inclusion equilibria toward CDs, which can be effective in increasing the system selectivity.