Detection of D-penicillamine in skin lesions in a case of dermal elastosis after a previous long-term treatment for Wilson's disease.

BACKGROUND: Skin adverse events associated with D-Penicillamine (DPA) are common and multi-faceted, although the presence of DPA or its metabolites has never been documented in the skin, because of inherent difficulties in determining its tissue levels. Thus, the association between DPA and DPA-related dermatoses has been only hypothesized on the basis of careful history, clinical observation and typical histopathological findings. OBJECTIVE: To detect DPA in biopsy specimens in a unique case of 25-year-late-onset elastosis perforans serpiginosa and pseudo-pseudoxanthoma elasticum associated with a history of long-term high dose DPA, by applying a recently described analytical method to assess the presence of DPA in skin. METHODS: We used a reliable analytical method based on high-performance liquid chromatography coupled with amperometric detection to look for the presence of DPA in skin biopsy specimens. RESULTS: A chromatographic peak corresponding to DPA was evidenced in some affected skin samples collected from the patient. CONCLUSION: We documented the effective presence and the persistence after 25 years of DPA in the skin of a woman affected by elastotic cutaneous change due to a long-term therapy with DPA. This report provides further evidence of the relationship between DPA deposit in affected skin and clinical manifestation.

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.

Analysis of reboxetine, a novel antidepressant drug, in pharmaceutical tablets by capillary electrophoresis and derivative spectrophotometry.

The recent antidepressant drug reboxetine was quantified in pharmaceutical tablets by derivative spectrophotometry and capillary zone electrophoresis. The feasible sample pretreatment consists of a single extraction with a pH 2.5 phosphate buffer, centrifugation and dilution. For the spectrophotometric assay, the fourth derivative of the absorbance was used which gave satisfactory results in terms of accuracy (mean recovery 99.7%) and precision (mean RSD 3.4%). The electrophoretic experiments were carried out using the shortest effective length of the capillary (8.5 cm) in order to obtain a very rapid separation of reboxetine and dibenzepine used as the internal standard. Using a pH 2.5, 50 mM phosphate buffer as the background electrolyte, each analysis lasted less than 2.5 min. Accuracy (101.3%) and precision (1.5%) were very good.

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)).

Comparison of three analytical methods for quality control of clozapine tablets.

Three different analytical methods for the quality control of clozapine in commercial formulations were developed and compared: a liquid chromatographic (LC) method with UV detection, a capillary zone electrophoretic (CZE) method, and a linear scan voltammetric (LSV) method. The isocratic LC procedure used a C18 reversed-phase column; the CZE method used an uncoated fused-silica capillary and phosphate buffer containing polyvinylpyrrolidone as the background electrolyte; the LSV method analyzed clozapine solutions with acidic phosphate buffer as the supporting electrolyte. The 3 methods gave similar and satisfactory results, in terms of precision and accuracy. Repeatability and intermediate precision were good (RSD% < 2.2) and accuracy, resulting from recovery studies, was between 98 and 102%. The rapidity of analysis was high for all 3 methods, especially for the LSV.

Simultaneous determination of all-trans-retinoic acid, beta-carotene, and vitamin A in galenic preparations by liquid chromatography.

The concentrations of vitamin A, beta-carotene, and all-trans-retinoic acid in oral preparations were determined in a single analysis by a method based on isocratic, reversed-phase liquid chromatography (LC). The LC system consisted of a C18 column, a mobile phase of acetonitrile, dichloromethane, methanol, and water and a UV detector set at 330 nm. The linearity ranges were 25-250 ng/mL for trans-retinoic acid and vitamin A, and 100-1,000 ng/mL for beta-carotene. This LC method for the determination of retinoids is simple, precise, and accurate. No extraction procedure is required before the chromatographic analysis; only a suitable dilution is necessary. The method proved to be reliable, fast, and economical. Furthermore, this method is indicative of stability, because it allows for the determination of degradation products such as 13-cis-retinoic acid.

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.

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.

Spectrophotometric determination of thiols in human lymphocytes.

A spectroscopic method for thiol analysis, based on the complexation reaction with Pd(II), is described. The proposed method is simple and sensitive and can be used for a rapid analysis of thiols in human lymphocytes.

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.

Extractive spectroscopic insulin determination in pharmaceutical formulations.

Two methods are proposed, one spectrophotometric and one spectrofluorimetric, for the determination of insulin in several pharmaceutical formulations. The methods were found to be fairly simple, sensitive and accurate, and thus suitable for this purpose. Both methods involve an extractive step with diethyl ether for the elimination of excipient interference, and subsequent direct spectrometric analysis. Spectrophotometric determinations were carried out at lambda = 276 nm; spectrofluorimetric determinations were carried out at lambda em = 306 nm with lambda exc = 277 nm.

Toxicological assessment of liquorice: biliary excretion in rats.

Glycyrrhizin (G) and its aglycone, glycyrrhetic acid (GA) have been prescribed for several therapeutic purposes. However, side effects have pointed out the problem of the toxicity of G. On the contrary, it was recently shown that the pure aqueous liquorice extract (LE), which also contains G, produces reduced adverse effects in rat and human, as compared to pure G, this is likely be related to differences in G bioavailability and the resulting pharmacokinetics of G and GA. Using a sensitive HPLC procedure for the determination of G and GA in rat bile, pharmacokinetics of G and GA in bile have been determined. The results of the analysis showed significantly lower concentrations of G in bile samples from rats treated with LE compared to pure G. Furthermore, LE presented a significant choleretic effect after both oral and i.v. administration, which increases the excretion rate of G. In case of GA, all the concentrations were very low, often below the detection limit. The results prompted us to assess the risk associated with liquorice intake and to determine the daily amount of pure liquorice root extract that can be safely consumed.

[The choleretic effects of licorice: identification and determination of the pharmacologically active components of Glycyrrhiza glabra]. / Studio dell'effetto coleretico della liquirizia: identificazione e determinazione di componenti della Glycyrrhiza glabra farmacologicamente attivi.

Recent studies indicate that licorice extract, when administered per os or i.v., causes an evident choleretic effect in rats. Aim of this research is to identify and quantify those licorice constituents which are responsable for the observed choleresis. The quali-quantitative analysis of umbelliferon (7-idroxycoumarin), was at first performed by a fluorimetric method, subsequently by a more selective HPLC method. Moreover, this HPLC method allows the determination of glycyrrhizin, an important licorice constituent. Unlike the glycyrrhizin, which is present in a fairly large amount, umbelliferon resulted to be present at a very low concentration (at trace level), both in licorice and in bile. Research is in progress, aiming to determine the substances, beyond glycyrrhizin, which are responsable for the choleretic effect of licorice.

HPLC determination of glycyrrhizin and glycyrrhetic acid in biological fluids, after licorice extract administration to humans and rats.

Simple and sensitive HPLC methods were developed for the determination of glycyrrhyzin (G) and its main metabolite glycyrrhetic acid (GA) in biological samples, in order to investigate the pharmacokinetic behaviour of G after oral administration of licorice extract (LE) or G to humans and rats. The analysis have been carried out by HPLC with UV detector (251 nm), after a careful pretreatment of the samples. These methods are suitable in terms of precision and accuracy for the G and GA determination in plasma and urine of human volunteers and in bile, plasma ad urine of rats.

Interaction of licorice on glycyrrhizin pharmacokinetics.

The effects of components of aqueous licorice root extract (LE) on the pharmacokinetics of glycyrrhizin (G) and glycyrrhetic acid (GA) were investigated in rats and humans. The aim of this work was to define the role of pharmacokinetics in G toxicity. In the procedure, G and GA were detected in biological fluids by means of recently improved HPLC methods. Significantly lower G and GA plasma levels were found in rats and humans treated with LE compared to the levels obtained with those in which G alone was administered. The pharmacokinetic curves showed significant differences in the areas under the plasma-time curve (AUC), Cmax, and Tmax parameters. The data obtained from urine samples are in agreement with the above results and confirm a reduced bioavailability of G present in LE compared to pure G. This should be attributed to the interaction during intestinal absorption between the G constituent and the several components in LE. The modified bioavailability could explain the various clinical adverse effects resulting from the chronic oral administration of G alone as opposed to LE.

Bioavailability of glycyrrhizin and licorice extract in rat and human plasma as detected by a HPLC method.

The pharmacokinetic behaviour of glycyrrhizin (1) was investigated in order to evaluate the difference in bioavailability after oral administration of licorice extract (LE) or glycyrrhizin (1) to rats and humans. For this study, two reliable HPLC methods were developed for the dosage of the levels of 1 and of its metabolite, the glycyrrhetic acid (2) in plasma samples. The determinations were carried out by HPLC on a reversed phase column with UV detector (251 nm), after a careful extraction step of 1 and 2 from the biological matrix. These methods afford good accuracy and satisfactory precision and they allow the determination of both compounds at levels as low as 200 ng/ml. The analytical results improved the knowledge of 1 pharmacokinetics, showing a significantly reduced bioavailability, when administered as LE compared to 1 when administered as such.