Francesca Bugamelli

HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure.

An original analytical method has been developed for the determination of the antioxidants trans-resveratrol (t-RSV) and cis-resveratrol (c-RSV) and of melatonin (MLT) in red and white wine. The method is based on HPLC coupled to fluorescence detection. Separation was obtained by using a RP column (C8, 150 mm x 4.6 mm id, 5 mum) and a mobile phase composed of 79% aqueous phosphate buffer at pH 3.0 and 21% ACN. Fluorescence intensity was monitored at lambda = 386 nm while exciting at lambda = 298 nm, mirtazapine was used as the internal standard. A careful pretreatment of wine samples was developed, using SPE with C18 cartridges (100 mg, 1 mL). The calibration curves were linear over the following concentration ranges: 0.03-5.00 ng/mL for MLT, 3-500 ng/mL for t-RSV and 1-150 ng/mL for c-RSV. The LOD values were 0.01 ng/mL for MLT, 1 ng/mL for t-RSV and 0.3 ng/mL for c-RSV. Precision data, as well as extraction yield and sample purification results, were satisfactory. Thus, the method seems to be suitable for the analysis of MLT and resveratrol isomers in wine samples. Moreover, wine total polyphenol content and antioxidant activity were evaluated.

Separation and analysis of glycyrrhizin, 18β-glycyrrhetic acid and 18α-glycyrrhetic acid in liquorice roots by means of capillary zone electrophoresis

Abstract Glycyrrhizin is the main active compound of Glycyrrhiza glabra root extracts; according to recent studies, glycyrrhizin and its aglycon, glycyrrhetic acid, have interesting therapeutic properties. A new capillary electrophoretic method has been developed for the separation and quantification of glycyrrhizin, β-glycyrrhetic acid and its isomer α-glycyrrhetic acid. Separation of the analytes was achieved in less than 3min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% β-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254nm. Linearity was found over the 5–200 and 2.5–100μgmL−1 concentration ranges for glycyrrhizin and glycyrrhetic acid, respectively. This method has been applied to the determination of the analytes in different matrices (liquorice roots and commercial confectionery products), and to the purity control of β-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing β-glycyrrhetic acid and its epimer in roots, the samples were purified by means of a suitable solid-phase extraction (SPE) procedure with Oasis HLB cartridges, which granted good selectivity, eliminating matrix interference.

Determination of fluoxetine and norfluoxetine in human plasma by high-pressure liquid chromatography with fluorescence detection

Fluoxetine is an atypical antidepressant drug, which selectively inhibits the neuronal reuptake of serotonin, and is widely used in the treatment of depressive disorders. The aim of this research is the development of an HPLC method with fluorescence detection for the monitoring of fluoxetine plasma levels. The determination requires no more than 250 microl of plasma, which undergo solid phase extraction (SPE), then are injected in the HPLC. For the analytical separation a reversed phase C8 column (150 x 4.6 mm I.D.) was used, while the mobile phase was a mixture of acetonitrile and water containing perchloric acid and tetramethylammonium perchlorate (flow rate: 1 ml min(-1)). The very low levels of analytes in plasma required the employment of a fluorescence detector (lambda(exc) = 230 nm, lambda(em)=290 nm), which also granted a good selectivity. Fluoxetine is revealed as a single peak at a retention time of 9.7 min, while norfluoxetine, the main metabolite of fluoxetine, is revealed at a retention time of 8.1 min. Linearity was obtained over the concentration range 8-200 ng ml(-1) for both substances. The method seems suitable, in accuracy and precision, for the determination of fluoxetine plasma levels of patients; furthermore, it is rapid and sensitive.

Determination of l-dopa, carbidopa, 3-O-methyldopa and entacapone in human plasma by HPLC–ED

The aim of the study was the development of analytical methods suitable for the quantification of L-dopa, carbidopa and entacapone in plasma of Parkinsonian patients treated with Stalevo(®). The metabolite 3-O-methyldopa was also determined to obtain some indications on the pharmacokinetics of L-dopa. For the simultaneous analysis of L-dopa, 3-O-methyldopa and carbidopa, a RP C18 column as the stationary phase and a mixture of methanol and a pH 2.88 phosphate buffer (8:92, v/v) as the mobile phase were used. A feasible plasma pre-treatment based on protein precipitation was implemented, obtaining extraction yield higher than 94% for all the analytes. For the analysis of entacapone a RP C8 column and a mixture of methanol, acetonitrile and a pH 1.90 phosphate buffer as the mobile phase (17.5:22.5:60, v/v/v) were used. A plasma pre-treatment procedure was developed, based on solid phase extraction of entacapone using Oasis HLB cartridges. Extraction yields were good, being always higher than 96%. Both methods, based on HPLC-ED (V=+0.8V), have been fully validated. Good linearity was obtained over the following concentration ranges: 100-4000 ng mL(-1) for L-dopa, 200-10,000 ng mL(-1) for 3-O-methyldopa, 25-4000 ng mL(-1) for carbidopa and 20-4000 ng mL(-1) for entacapone. Precision data were satisfactory, being R.S.D.% values lower than 5.64%; accuracy also resulted very good with recovery data higher than 90%. The proposed methods have been successfully applied to the analysis of patient plasma samples and seem to be suitable for therapeutic drug monitoring purposes.

Analytical methods for the quality control of Prozac® capsules

Some analytical methods (two spectrophotometric and two chromatographic procedures) for the determination of fluoxetine in Prozac capsules are described. All of them are applied to the samples after extracting the drug with a methanol water mixture. The direct and derivative spectrophotometric methods are simple and reliable; the derivative method gives better recovery and lessens interference. Both methods show linearity in the 5-30 microg ml(-1) range of the fluoxetine concentration range. Both HPLC methods (spectrophotometric and spectrofluorimetric detection) use a tetramethylammonium perchlorate buffer-acetonitrile mixture as the mobile phase and a C8 reversed phase column. The UV detection is performed at 226 nm, while the fluorimetric detection is performed by exciting at 230 nm and revealing the emission at 290 nm. The HPLC method with UV detection is more precise, but the procedure with fluorimetric detection is more sensitive.

Controlled insulin release from chitosan microparticles

This study deals with the production of chitosan microparticles containing insulin by interfacial crosslinkage of chitosan solubilized in the aqueous phase of a water/oil dispersion in the presence of ascorbyl palmitate. The use of ascorbyl palmitate as interfacial crosslinker is based on its amphiphilic properties allowing its disposition at the water/oil interface of the preparative dispersion, thus permitting covalent bond formation with the amino groups of chitosan when its oxidation to dehydroascorbyl palmitate takes place during microparticle preparation. This preparation method produced microparticles characterized by high loading levels of insulin, completely releasing the drug in about 80 h at an almost constant release rate as determined by spectrophotometric and spectrofluorimetric methods. In contrast, the replacement of ascorbyl palmitate by dehydroascorbyl palmitate provided microparticles incompletely releasing the incorporated drug and characterized by a non‐constant release rate over time due to the higher lipophilicity of dehydroascorbyl palmitate which hinders its disposition at the water/oil interface and thus decreases the crosslinking efficiency and increases the lipophilicity of the microparticle surface. The efficiency of the spectrofluorimetric and spectrophotometric methods used for determination of the stability and release of the insulin from the chitosan microparticles is also discussed.

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products

Abstract Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18β‐glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1–50 and 0.05–2.50 µg/mL for 1 and 2, respectively. A simple and rapid sample pre‐treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices. Copyright

DEVELOPMENT OF AN HPLC METHOD FOR THE TOXICOLOGICAL SCREENING OF CENTRAL NERVOUS SYSTEM DRUGS

A simple and sensitive HPLC (high performance liquid chromatography) method has been developed for the qualitative and quantitative analysis of several CNS (central nervous system) drugs in clinical and forensic toxicology. The leading conditions were studied, namely parameters such as mobile phase pH, organic modifier percentage, and salt concentration. An isocratic HPLC elution, using a mobile phase composed of acetonitrile and pH 2.8 aqueous tetramethylammonium perchlorate and a C8 reversed phase column as the stationary phase, was found to be convenient for the separation of several CNS drugs, and for their detection and quantitation. The identification of the drugs was assured using their relative retention times, together with the peak area ratios at two different wavelengths (230 and 270 nm). A quick pre-treatment of the plasma samples, based on a SPE (solid phase extraction) procedure, with good extraction efficiency and satisfactory selectivity was developed. Under these conditions, a mixture of fifteen CNS drugs (including antipsychotics, antidepressants and antiepileptics) and some selected active metabolites, was well separated for identification and quantitative determination purposes.

Rapid capillary electrophoretic method for the determination of clozapine and desmethylclozapine in human plasma

A rapid and sensitive high-performance capillary electrophoretic method for the determination of clozapine and its main metabolite desmethylclozapine in human plasma was developed. The separation of the two analytes was carried out in an untreated fused-silica capillary [33 cm (8.5 cm effective length) x 50 microm I.D.] filled with a background electrolyte at pH 2.5 containing beta-cyclodextrin. Baseline separation of clozapine and desmethylclozapine was recorded in less than 3 min. An accurate sample pretreatment by means of solid-phase extraction and subsequent concentration allows for reliable quantitation of clozapine in the plasma of schizophrenic patients under treatment with the drug. The method showed good precision (mean RSD = 4.0%) as well as satisfactory extraction yields (approximately 88%) and a good sensitivity (limit of quantitation = 0.075 microg ml(-1), limit of detection = 0.025 microg ml(-1)).

A novel HPLC-electrochemical detection approach for the determination of D-penicillamine in skin specimens.

D-penicillamine is a thiol drug mainly used for Wilsons disease, rheumatoid arthritis and cystinuria. Adverse effects during normal use of the drug are frequent and may include skin lesions. To evaluate its toxic effects in clinical cases an original method based on high performance liquid chromatography coupled to amperometric detection in a specific biological matrix such as skin has been developed. The chromatographic analysis of D-penicillamine was carried out on a C18 column using a mixture of acid phosphate buffer and methanol as the mobile phase. Satisfactory sensitivity was obtained by oxidizing the molecule at +0.95 V with respect to an Ag/AgCl reference electrode. A chemical reduction of D-penicillamine-protein disulphide bonds using dithioerythritol combined with microwaves was necessary for the determination of the total amount of D-penicillamine in skin specimens. A further solid-phase extraction procedure on C18 cartridges was implemented for the sample clean-up. The whole analytical procedure was validated: high extraction yield (>91.0%) and satisfactory precision (RSD<6.8%) values were obtained. It was successfully applied to skin samples from a patient who was previously under a long-term, high-dose treatment with the drug and presented serious D-penicillamine-related dermatoses. Thus, the method seems to be suitable for the analysis of D-penicillamine in skin tissues.