Multi-matrix assay of cortisol, cortisone and corticosterone using a combined MEPS-HPLC procedure.

The development and validation of a bioanalytical assay for the simultaneous determination of cortisol, cortisone and corticosterone levels in several matrices, such as saliva, plasma, blood and urine samples have been described. The method is based on a rapid test which combines a microextraction by packed sorbent procedure and liquid chromatography-diode array technique. Chromatographic separation of the analytes (cortisol, cortisone and corticosterone) and the internal standard (methylprednisolone) was achieved in less than 10min on a reversed-phase pentafluorophenyl column using a mobile phase composed of phosphate buffer and acetonitrile. The assay was performed after an innovative microextraction procedure by means of C8 sorbent which guaranteed good clean-up of the matrices and satisfactory extraction yield of the analytes. Moreover, the method gave linear results over a range of 5-100ngmL(-1) and showed good selectivity and precision. This method was successfully applied for quantifying corticosteroids in specific matrices derived from some healthy volunteers in comparison to two socially diversified groups, namely former heroin addicts undergoing opioid replacement therapy and poly-drug abusers.

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

D-penicillamine is a thiol drug mainly used for Wilson's 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.

Chiral analysis of amphetamines, methadone and metabolites in biological samples by electrodriven methods.

Amphetamines and methadone are synthetic chiral drugs with a high potential for abuse. As such, several analytical methods have been developed for their enantioseparation and analysis in biological tissues, and some of these are based on electrodriven techniques. In this review, the most important and recent of these latter methods are reviewed and their main advantages and disadvantages are discussed. Particular attention is paid to the suitability of each method for the application to the biological matrix of interest: while all methods have been successfully applied for one or more biological tissues, to reach this goal they must overcome the sensitivity problem that is common to almost all capillary electrophoretic techniques. Most methods use one or more cyclodextrin derivatives as the chiral selector, thus the separation mechanism is not particularly complicated or unusual.

Fast analysis of amino acids in wine by capillary electrophoresis with laser-induced fluorescence detection.

A fast analytical method has been developed for the determination of nine amino acids, together with serotonin, in wine samples of different origin and vintage. The method is based on capillary electrophoresis coupled to laser-induced fluorescence detection. Separation was obtained by using a fused-silica capillary (75 µm id, 74.0 cm total length, 60.0 cm length to detector) and a background electrolyte composed of carbonate buffer (20 mM, pH 9.2), applying a 20 kV voltage. Direct hydrodynamic injection of wine samples was made after an original microwave-assisted derivatisation step with 5-(4,6-dichlorotriazinyl)aminofluorescein. Fluorescence was induced by an Ar-Ion laser, exciting at 488 nm. Good linearity (r(2) >0.9990) was obtained for all considered analytes and sensitivity was also good, with limits of detection in the 7-50 ng/mL range. The method was successfully applied for the analysis of commercial Italian wines and thus seems to be suitable for the determination of the relevant amino acids and serotonin, providing good results in terms of accuracy and precision, together with the advantage of a very fast, microwave-assisted derivatisation procedure. Future applications of the method are planned to check for wine adulterations and commercial frauds.

Promising medications for cocaine dependence treatment.

Cocaine dependence is characterized by compulsive drug seeking and high vulnerability to relapse. Overall, cocaine remains one of the most used illicit drugs in the world. Given the difficulty of achieving sustained recovery, pharmacotherapy of cocaine addiction remains one of the most important clinical challenges. Recent advances in neurobiology, brain imaging and clinical trials suggest that certain medications show promise in the treatment of cocaine addiction. The pharmacotherapeutic approaches for cocaine dependence include medications able to target specific subtypes of dopamine receptors, affect different neurotransmitter systems (i.e. noradrenergic, serotonergic, cholinergic, glutamatergic, GABAergic and opioidergic pathways), and modulate neurological processes. The systematic reviews concerning the pharmacological treatment of cocaine dependence appear to indicate controversial findings and inconclusive results. The aim of future studies should be to identify the effective medications matching the specific needs of patients with specific characteristics, abandoning the strategies extended to the entire population of cocaine dependent patients. In the present review we summarize the current pharmacotherapeutic approaches to the treatment of cocaine dependence with a focus on the new patents.

Advances in the enantioseparation of second-generation antidepressant drugs by electrodriven methods.

Stereochemistry is steadily increasing in importance in the development of new drugs, and the availability of pure enantiomer drugs can make therapy safer and more efficacious. In particular, almost all second-generation antidepressant drugs possess one or more chiral centres; however, only some of them are administered as single enantiomers. A fundamental part of the quality control of pharmaceutical formulations is the determination of enantiomeric excess and enantiomeric purity; this is also important for the therapeutic drug monitoring of depressed patients. For this purpose, efficient and reliable analytical methods are needed and electrodriven techniques (most of all CE, CEC and MEKC) are very efficient and inexpensive candidates for the role. In this review, the enantioselective electrodriven methods available for the analysis of second-generation antidepressant are presented and discussed. In particular, the following pharmacological classes of antidepressants will be considered: selective serotonin reuptake inhibitors (fluoxetine, citalopram, paroxetine, sertraline); norepinephrine reuptake inhibitors (reboxetine); serotonin and norepinephrine reuptake inhibitors (venlafaxine, milnacipran, duloxetine); and noradrenergic and specific serotonergic antidepressants (mirtazapine).

Neuroendocrine and behavioural responses to opioid receptor-antagonist during heroin detoxification: relationship with personality traits.

The present study investigated clinical, cardiovascular and neuroendocrine consequences of rapid opioid detoxification (ROD) in heroin-dependent individuals, affected, or not, by comorbid antisocial personality disorder (ASPD). Thirty-two patients underwent ROD and subsequent treatment with daily naltrexone: 3 days detoxification procedures were performed utilizing clonidine, baclofen, oxazepam and ketoprofene, without anaesthesia. Withdrawal symptoms, mood changes, cardiovascular indexes (heart rate, blood pressure), norepinephrine (NE), epinephrine (EPI), adrenocorticotropic hormone (ACTH) and cortisol (CORT) were evaluated during naloxone-naltrexone administration on the second day of detoxification treatment. The patients were divided into two groups following DSM-IV criteria for ASPD. Group A comprised 14 ASPD patients and group B comprised 18 patients without ASPD. Slight and transient withdrawal symptoms and mood changes were demonstrated on the second day in the whole sample of patients, in association with a significant, but moderate, elevation of heart rate, blood pressure, NE (two-fold), EPI (five-fold), ACTH (two-fold) and CORT (two-fold) plasma levels, in response to opioid receptor-antagonist administration. When evaluated separately in ASPD (group A) and non-ASPD patients (group B), significantly higher withdrawal symptoms and mood changes, heart rate, blood pressure, NE, ACTH and cortisol levels were observed in ASPD subjects. By contrast, no differences were found in EPI responses to naloxone-naltrexone administration between group A and B patients. The significant differences demonstrated in clinical and neuroendocrine responses to opioid receptor-antagonist administration, in relation to personality traits, could be due to reduced alpha-adrenergic receptor sensitivity, which was previously reported in ASPD, with a possible impairment of clonidine action. Our study suggests that a detailed diagnostic assessment before detoxification procedure may help to predict treatment outcome.

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis.

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.

Determination of valproic acid (2-propylpentanoic acid) in human plasma by capillary electrophoresis with indirect UV detection.

A rapid capillary zone electrophoresis method with indirect UV detection was developed and validated for the determination of valproic acid (VPA) in human plasma. The analyses were carried out under optimized conditions, using a buffer system composed of 15 mM benzoate and 0.5 mM cetyltrimethylammonium bromide at pH 6.0, and 25% v/v methanol; 2-hydroxybutyric acid was selected as the internal standard (IS). The capillary electrophoresis (CE) separation was carried out at a negative potential of 30 kV and the indirect UV detection was operated at 210 +/- 20 nm for all assays. The influence of buffer pH, ionic strength, concentration of electroosmotic flow (EOF) modifier and organic modifier on indirect signal response and migration behavior of the organic acid was investigated. Isolation of VPA from plasma was accomplished by a carefully implemented procedure using methanol as the precipitant agent. Using a high ratio of methanol to plasma for deproteinization (4:1), good absolute recovery of the analyte and satisfactory selectivity was obtained. The calibration line for VPA was linear over the 1-100 microg/mL concentration range. Sensitivity was high; in fact, the limit of detection (LOD) of VPA was 150 ng/mL and 450 ng/mL the limit of quantitation (LOQ). The results obtained analyzing real plasma samples from schizophrenic patients under polytherapy with VPA as well as antipsychotic drugs were satisfactory in terms of precision, accuracy and sensitivity.

Separation of reboxetine enantiomers by means of capillary electrophoresis.

The novel antidepressant reboxetine, a selective norepinephrine reuptake inhibitor, is increasingly used in the treatment of different forms of major depression. Reboxetine is a chiral compound, and is marketed as a racemic mixture of (R,R)- and (S,S)-reboxetine; however, the pharmacokinetic and toxicological profiles of the two enantiomers are rather different. For this reason, a simple capillary electrophoretic method for the separation of reboxetine enantiomers has been developed. Sulfobutyl ether-beta-cyclodextrin was chosen as the chiral selector, and several parameters, such as cyclodextrin and buffer concentration, buffer pH and capillary temperature were investigated in order to obtain good separation and acceptable run times. Using an uncoated, fused-silica capillary (internal diameter 50 microm, total length 48.5 cm, effective length 40.0 cm) and a background electrolyte consisting of a pH 3.0, 100 mM phosphate buffer containing 1.25 mM cyclodextrin, reboxetine enantiomers were baseline separated (resolution > 4) with a voltage of 20 kV in less than 16 min. Since pure enantiomers of reboxetine were not available, they were obtained from the racemic powder by means of direct-phase, high-performance liquid chromatography and their identity confirmed by circular dichroism spectra.