Laura Mercolini

Benzodiazepine Metabolism : An Analytical Perspective

Benzodiazepines are currently among the most frequently prescribed drugs all over the world. They act as anxiolytics, sedatives, hypnotics, amnesics, antiepileptics and muscle relaxants. Despite their common chemical scaffold, these drugs differ in their pharmacokinetic and metabolic properties. In particular, they are biotransformed by different cytochrome P450 isoforms and also by different UDP-glucuronosyltransferase subtypes. The most important studies on the metabolic characteristics of several 1,4-benzodiazepines, carried out from 1998 onwards, are reported and briefly discussed in this review. Moreover, the analytical methods related to these studies are also described and commented upon and their most important characteristics are highlighted. Most methods are based on liquid chromatography, which provides wide applicability and good analytical performance granting high precision, accuracy and feasibility. Mass spectrometry is gaining widespread acceptance, particularly if the matrix is very complex and variable, such as human or animal blood. However, spectrophotometric detection is still used for this purpose and can grant sufficient selectivity and sensitivity when coupled to suitable sample pre-treatment procedures. A monograph is included for each of the following benzodiazepines: alprazolam, bromazepam, brotizolam, clotiazepam, diazepam, etizolam, flunitrazepam, lorazepam, midazolam, oxazepam and triazolam.

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.

Novel mucoadhesive nasal inserts based on chitosan/hyaluronate polyelectrolyte complexes for peptide and protein delivery

OBJECTIVESnThe purpose of this study was the preparation and characterisation of mucoadhesive nasal inserts based on chitosan/hyaluronate polyelectrolyte complexes prepared at various pHs and at different molar ratios.nnnMETHODSnA suspension of chitosan/hyaluronate complexes with or without the model drugs (vancomycin or insulin) was lyophilised into small inserts. Complexation yield, FT-IR spectra and thermogravimetric analysis were used to study the degree of interactive strength between polyions. In-vitro swelling, mucoadhesion and release tests were performed in order to investigate delivery of vancomycin and insulin in the nasal cavity.nnnKEY FINDINGSnThe results indicated that the selection of complex preparative conditions allows modulation of insert swelling and mucoadhesion ability. Nasal inserts containing vancomycin or insulin had showed completely different drug release behaviour.nnnCONCLUSIONSnChitosan/hyaluronate polyelectrolyte complexes can be used for the formulation of mucoadhesive nasal inserts for the delivery of peptide and protein drugs.

Simultaneous analysis of diazepam and its metabolites in rat plasma and brain tissue by HPLC-UV and SPE

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to normal rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

Quantitative analysis of cocaine in human hair by HPLC with fluorescence detection

Cocaine is currently one of the most widespread abuse drugs in the world. Since hair cocaine concentrations are a reliable marker of exposition to the drug, an original liquid chromatographic method has been developed for the determination of cocaine in human hair. The chromatographic analysis was carried out on a Hydro-RP C18 column, using a mobile phase containing a phosphate buffer (pH 3.0)-acetonitrile-methanol (75:15:10, v/v/v). Native cocaine fluorescence was monitored at 315nm while exciting at 230nm. Mirtazapine was used as the internal standard. Sample pre-treatment was carried out by incubative extraction with 0.1M HCl followed by solid-phase extraction with C2 cartridges. Good linearity was obtained over a working range of 0.3-100.0ng/mg. Both extraction yield (>89%) and precision values (R.S.D.<6.2%) were highly satisfactory. The method was successfully applied to hair samples collected from cocaine users. Thus, the method is suitable for the long-term monitoring of cocaine use by means of hair testing.

Fast analysis of catecholamine metabolites MHPG and VMA in human plasma by HPLC with fluorescence detection and a novel SPE procedure.

A fast and sensitive high-performance liquid chromatographic method has been developed for the determination in human plasma of MHPG (3-methoxy-4-hydroxyphenylethylenglycol) and VMA (vanillyl mandelic acid), the main metabolites of epinephrine and norepinephrine. Analyses were carried out at 325 nm while exciting at 285 nm on a reversed-phase column (Atlantis C18, 150 mm x 4.6 mm I.D., 5 microm) using a mobile phase composed of 2% methanol and 98% aqueous citrate buffer at pH 3.0. A careful solid-phase extraction procedure, based on mixed-mode reversed-phase - strong anion exchange Oasis cartridges (MAX, 30 mg, 1 mL), was developed for the pre-treatment of plasma samples. Extraction yields were satisfactory, always higher than 90%. Calibration curves were linear over the 0.2-40.0 ng mL(-1) concentration range for MHPG and over the 0.5-40.0 ng mL(-1) concentration range for VMA. The method was successfully applied to plasma samples of former drug users undergoing detoxification therapy and subjects at risk of developing drug addiction.

Simultaneous HPLC-F analysis of three recent antiepileptic drugs in human plasma.

An original high-performance liquid chromatographic method with fluorescence detection is presented for the simultaneous determination of the three antiepileptic drugs gabapentin, vigabatrin and topiramate in human plasma. After pre-column derivatisation with dansyl chloride, the analytes were separated on a Hydro-RP column with a mobile phase composed of phosphate buffer (55%) and acetonitrile (45%) and detected at lambda(em)=500 nm, exciting at 300 nm. An original pre-treatment procedure on biological samples, based on solid-phase extraction with MCX cartridges for gabapentin and vigabatrin, and with Plexa cartridges for topiramate, gave high extraction yields (>91%), satisfactory precision (RSD<6.4%) and good selectivity. Linearity was found in the 0.2-50.0 microg mL(-1) range for gabapentin, in the 1.0-100.0 microg mL(-1) range for vigabatrin and in the 1.0-50.0 microg mL(-1) range for topiramate, with limits of detection (LODs) between 0.1 and 0.3 microg mL(-1). After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. Accuracy results were satisfactory (recovery >91%). Therefore, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients treated with gabapentin, vigabatrin and topiramate.

Separation and HPLC analysis of 15 benzodiazepines in human plasma

Benzodiazepines (BZDs) are often prescribed to schizophrenic or depressed patients, as a part of polypharmacy regimens. An HPLC method has been developed for the simultaneous determination of 15 BZDs in human plasma. Separation was obtained by using a C8 RP column and a mobile phase composed of 65% aqueous phosphate buffer at pH 3.0 and 35% ACN. The UV detector was set at 220 nm and clomipramine was used as the internal standard. A careful pretreatment procedure of plasma samples was developed, using SPE with C1 cartridges, which gives high extraction yields (> 97%). The LOQs were always lower than 7.6 ng/mL and the LODs always lower than 2.6 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed and schizophrenic patients undergoing polypharmacy with one or more BZDs. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients undergoing therapy with one or more BZDs.

Determination of plasma and urine levels of Δ9-tetrahydrocannabinol and its main metabolite by liquid chromatography after solid-phase extraction

Delta9-Tetrahydrocannabinol is the most widespread drug of abuse in the world and it is also currently available as the active principle of formulations for the treatment of chronic pain. Its main metabolite, 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid, is the most important marker of Delta9-tetrahydrocannabinol consumption. An original liquid chromatographic method has been developed for the determination of these two analytes in human plasma and urine. Separation was obtained on a C8 column using a mobile phase with 35% phosphate buffer at pH 2.7 and 65% acetonitrile. The UV detector was set at 220 nm and indomethacin was used as the internal standard. Sample pre-treatment was carried out by solid-phase extraction with C8 cartridges; urine samples were subjected to basic hydrolysis before extraction. Both extraction yields (>91%) and precision values were highly satisfactory. The method was successfully applied to biological samples collected from Cannabis users. Accuracy and selectivity results were satisfactory. This is the first HPLC-UV method developed for the simultaneous quantification of Delta9-tetrahydrocannabinol and 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in both plasma and urine for the monitoring of either therapeutic or recreational use.

Determination of insulin in innovative formulations by means of LC coupled to fluorescence detection

A fast and simple method based on LC with fluorescence detection has been developed for the determination of insulin in innovative formulations consisting of microparticles and inserts for oral and nasal drug administration, respectively. A reverse-phase C8 column and a mobile phase composed of pH 3.7, 40 mM sodium sulphate solution and acetonitrile (24%, v/v) were employed. Using isocratic elution at 1.0 mL/min flow, analysis is completed within 7 min. Three different kinds of spray-dried microparticles were analysed, consisting of an insulin loaded core composed of chitosan salts (chitosan succinate, chitosan adipate or chitosan suberate) coated with stearic acid. Nasal inserts consisted of chitosan/hyaluronate polyelectrolyte complexes which were loaded with insulin and freeze-dried. Insulin was extracted from both the oral and nasal formulations using pH 7.4 phosphate buffer. The employment of fluorescence detection (lambda(exc) = 276 nm, lambda(em) = 306 nm) granted high selectivity, with no interference from the matrix. Full method validation was performed with good results in terms of linearity (insulin concentration range 0.10-30.0 microg/mL), LOD (0.03 microg/mL) and LOQ (0.10 microg/mL), precision (R.S.D.%<3.6) and accuracy (recovery percentage>90.0%). Insulin content in innovative formulations, expressed as percentage w/w, resulted to be between 0.90 and 0.97 for oral innovative formulations, while an average value of 342 microg of insulin was found in a single nasal insert, in good agreement with preparative protocols.