Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-beta-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

Nonaqueous capillary electrophoretic separation and thermo-optical absorbance detection of five tricyclic antidepressants and metabolism of amitriptyline by Cunninghamella elegans

We developed a technique based on nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection to assay five antidepressants with similar structures and mass-to-charge ratios. A mixture of methanol and acetonitrile with ammonium acetate was essential to achieve baseline resolution of these compounds. We investigated the effects of ammonium acetate concentration, temperature, applied voltage, and capillary length on separation efficiency. The nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection technique was used to study the metabolism of amitriptyline by Cunninghamella elegans. Sample preparation procedures were simplified for fast screening of the parent drug and its metabolites. Reproducible electropherograms were obtained from replicate cultures of C. elegans growing in the presence of amitriptyline.     

On-line nonaqueous capillary electrophoresis and electrospray mass spectrometry of tricyclic antidepressants and metabolic profiling of amitriptyline by Cunninghamella elegans

An on-line nonaqueous capillary electrophoresis-electrospray mass spectrometry (ESI-MS) technique was developed using a commercial ion spray interface. The nonaqueous capillary electrophoresis ESI-MS system was used to profile tricyclic antidepressants of similar structures and mass-to-charge ratios. We found that pure methanol can be used as a sheath liquid to obtain stable ion spray from nonaqueous capillary electrophoresis. The flow rate of the coaxial nebulizing gas affected baseline signals, separation efficiency, and migration times. Other nonaqueous capillary electrophoresis operating conditions and electrospray parameters were optimized for enhanced baseline separation and high sensitivity detection. The effect of sample stacking on separation and detection was evaluated. The calculated detection limits were approximately 3 pg injected onto the capillary. ESI mass spectra of tricyclic antidepressants from a single quadrupole MS were obtained and elucidated. The information was used to propose fragmentation pathways of the tricyclic antidepressants. The method was also used to analyze the metabolites of amitriptyline produced by the fungus Cunninghamella elegans. Sixteen metabolites were detected and most of them were tentatively identified as demethylated and/or hydroxylated, and/or N-oxidized products.     

Analysis of the components of Lycopus europaeus L. in body fluids during metabolism studies. Comparison of capillary electrophoresis and high-performance liquid chromatography

During pharmacokinetic studies with extracts obtained from medicinally used plants, analysis in body fluids is mainly performed by HPLC, an established separation method. In this paper high-performance capillary electrophoresis (HPCE) is investigated for its ability to separate such complex extracts. Crude extracts of Lycopus europaeus L. (Lamiaceae) are traditionally used against mild forms of hyperthyroidism. The metabolism of a 70% ethanolic extract with respect to some of its individual main components (rosmarinic and caffeic acid, luteolin-7-glucoside) and a mixture of the pure compounds were investigated using isolated perfused rat liver. After solid-phase extraction metabolites were determined using HPCE and HPLC separation techniques. A buffer solution composed of 0.05 mol l-1 Na2HPO4 at pH 7.0 with 30% acetonitrile was found to be the most suitable electrolyte for HPCE separation. The best mobile phase for isocratic HPLC was 0.03% TFA-acetonitrile (82:18, v/v). Data obtained with HPCE are in good accordance with those from HPLC; HPCE, however, is clearly more rapid and simple to perform.     

Pharmacokinetic study of praziquantel administered alone and in combination with cimetidine in a single-day therapeutic regimen

A brief therapeutic regimen of praziquantel, reduced to a single day, has been effective for treatment of neurocysticercosis. To study its pharmacokinetic characteristics, levels of praziquantel in plasma were determined for eight healthy volunteers after the administration of three oral doses of 25 mg/kg of body weight given at 2-h intervals, alone and with the simultaneous administration of cimetidine. Each volunteer received both regimens in a randomized crossover design. Blood samples were taken during a period of 12 h, and praziquantel concentration was measured by high-performance liquid chromatography. Levels of praziquantel in plasma remained above 300 ng/ml during a period of 12 h; they increased 100% when cimetidine was jointly administered. Compared with other regimens, the high levels obtained and the longer duration of action seem to be advantageous in prolonging the exposure of the parasites to the drug and support previous clinical experience showing that the treatment of neurocysticercosis with praziquantel can be reduced from 2 weeks to 1 day with the drug still retaining its cysticidal properties. Moreover, simultaneous administration of praziquantel and cimetidine could improve further the efficacy of the single-day therapy for cysticercosis and other parasitic diseases, such as schistosomiasis.     

Application of capillary electrophoresis and related techniques to drug metabolism studies

The use of capillary electrophoresis (CE) for the separation of small organic molecules such as pharmaceutical agents and drug/xenobiotic metabolites has become increasingly popular. This has arisen, at least in part, from the complimentary mode of separation afforded by CE when compared to the more mature technique of HPLC. Other qualities of CE include relative ease of method of development, rapid analysis, and low solvent consumption. The recent introduction of a variety of detector systems (including UV diode array, laser-induced fluorescence, conductivity) and the demonstrated coupling of CE to MS have also aided acceptance of this technology. In the present report, we review the role of CE coupled to various detector systems including a mass spectrometer for the characterization of both in vitro and in vivo derived drug metabolite mixtures. Attributes of CE for this application are demonstrated by discussion of metabolism studies of the neuroleptic agent haloperidol. Various aspects of the development and use of CE and CE-MS for the characterization of haloperidol metabolites, including criteria for selection of parameters such as pH, ionic strength, extent of organic modification, and the use of nonaqueous capillary zone electrophoresis are discussed. We also consider potential limitations of CE and CE-MS for drug metabolism research and describe the introduction of membrane preconcentration-CE (mPC-CE) and mPC-CE-MS as a solution that overcomes the rather poor concentration limits of detection of CE methods without compromising the resolution of analytes or separation efficiency of this technique.     

Representation of foveal visual fields in the ventral bank of the superior temporal sulcus in the posterior inferotemporal cortex of the macaque monkey

A fast and sensitive analytical method was developed to characterize artemether and its metabolites in small amounts in body fluids. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide, separated on an optimized capillary gas chromatographic system and identified by chemical ionization mass spectrometry by using ammonia as reagent gas. The analytical assay is demonstrated on samples extracted from bovine hemoglobin, rat blood and dog blood. Full mass spectra of artemether and three metabolites were obtained at a level of 1 x 10(-6) g/ml.     

Capillary electrophoresis for therapeutic drug monitoring of antiepileptics

We examined the use of capillary electrophoresis for therapeutic drug monitoring of antiepileptic drugs. Micellar electrokinetic capillary chromatography (MEKC) with a diode array detector simultaneously determined concentrations of zonisamide, a new type of antiepileptic drug, and phenobarbital, phenytoin and carbamazepine, typical antiepileptic drugs, in human serum. Zonisamide levels in human serum obtained by MEKC correlated well with levels obtained by high-performance liquid chromatography. The serum levels of phenobarbital, phenytoin and carbamazepine determined by MEKC were almost equal to those obtained by fluorescence polarization immunoassay. The reproducibility of separation and quantification with MEKC for intra- and inter-day assays were appropriate. This MEKC method could provide a simple and efficient therapeutic drug monitoring method for antiepileptic drugs, especially in patients treated with a combination of zonisamide and other antiepileptic drugs. MEKC may be an attractive method for therapeutic drug monitoring, because of its specificity of separation, automation of procedure, ease of method development, low cost, small aqueous buffer amounts, speed of analysis, small injection volume and high environment-directed performance.     

Strategies for the monitoring of drugs in body fluids by micellar electrokinetic capillary chromatography

Electrokinetic capillary techniques can exploit numerous separation principles, making them flexible and easily applicable to a variety of separation problems. In recent publications, this emerging technology has been shown to be well suited for monitoring drugs and metabolites in body fluids, including serum, saliva and urine. Most attention has been focused on micellar electrokinetic capillary chromatography (MECC) because it permits the separation and determination of drugs with discrimination being largely based on differences in hydrophobicity. An overview of literature data on the MECC of drugs in body fluids and recent data obtained with antiepileptics in serum and saliva, with model mixtures of illicit drugs, and with extracts from urine specimens that tested positively for opiates and cocaine metabolites are presented. Emphasis is focused on buffer selection and simple sample preparation procedures, including direct injection of body fluids, ultrafiltration and solid-phase extraction.     

Kinetics of sequential metabolism. Contribution of parallel, primary metabolic pathways to the formation of a common, secondary metabolite

Interpretation of rate constants in the sequential metabolism of two different primary metabolites (MIA and MIB) for formation of a common, secondary metabolite (MII) after drug administration requires theoretical development of formulations that govern mass transfer during intravenous and oral administrations. Two cases (a and b) were presently considered for metabolism occurring only in the first-pass organs (intestine and liver) for flow-limited drugs and primary and secondary metabolites: (case a) wherein drug formed only the two primary metabolites, with the fractions of total body clearance that formed MIA and MIB, being denoted by f1 and f2, respectively, and (case b) wherein other additional elimination pathways for drug were present. MIA and MIB only partially formed MII (denoted by fMIA and fMIB, respectively), because provision was made for alternate elimination pathways; the fractional clearance in the formation of MII from MIA and MIB were fMIA and fMIB, respectively. Drug was metabolized to MIA than MII within the gut lumen with oral drug administration; the MIA and MII formed were further absorbed. Triangular matrices were found to result from mass transfer equations for first-order conditions with oral and intravenous administrations. Upon inversion of the matrices, the areas under the curve for drug and metabolite species were obtained after multiplication by the administered dose and division by the volume of the species considered. However, the dose-corrected area under the curve was used as the basis for comparison. Case-independent solutions were obtained for the fractions absorbed (Fa, FaMIA, FaMIB) and the availabilities (F, F(MIA), F(MIB)) of drug and the primary metabolites, and for f1, f2, f1/f2, fMIA/fMIB, and (f1fMIA)/(f2fMIB) (ratio of effective clearances of MII formation from D via MIA and MIB). Case-dependent solutions also existed. For case a (f1 + f2 = 1), the fraction of total body clearance that formed MIA (f1) or MIB (f2) was solved with the area under the curve of MII after intravenous D, MIA, and MIB administrations. For case b, however, the same constants were obtained after greater manipulation, and entailed oral administration of the metabolites. Although solutions for the ratios of f1/f2 and (f1fMIA)/(f2fMIB) were found, the fractional clearances in formation of MII from MIA (fMIA) and MIB (fMIB) were, however, not provided in both cases unless MII was completely absorbed.     

Determination of the enantiomeric purity of (-) terbutaline by capillary electrophoresis using cyclodextrins as chiral selectors in a polyethylene glycol gel

A method was developed for determination of the enantiomeric purity of the therapeutic-pharmacological active (-)-enantiomer of terbutaline using cyclodextrins as a chiral selector dissolved in a removable liquid polyethylene glycol gel by use of capillary electrophoresis. The effect of temperature, type and concentration of polyethylene glycol and cyclodextrins was studied on the resolution between the two enantiomers. Best results were obtained with 10 mM hydroxyethyl-beta-cyclodextrin dissolved in a 10% polyethylene glycol-2000 solution at 15 degrees C. Under these conditions, an impurity of 0.1% (distomer/eutomer) can be readily detected.     

Separation and detection of phencyclidine in urine by gas chromatography

A rapid, sensitive and selective analytical procedure for phencyclidine and one of its metabolites in urine has bee developed. Three techniques have been studied for extraction of the drug from the biological matrix: (a) reversed-phase XAD resin, (b) charcoal absorption, and (c) solvent extraction using chloroform. Temperature-programmed gas chromatography was used to quantitate the illicit drug. Solvent extraction appears to offer the most efficient separation of the drug and its metabolite, as the recovery was 94% and the technique required only 7-8 min per sample. Reversedphase column extraction is also quite useful; although more time-consuming for an indivisual sample, it would be useful for screening purposes.     

Preparation of (R)-N-methylsalsolinol via reversed-phase high-performance liquid chromatography using beta-cyclodextrin as a mobile-phase additive

A new preparation method was devised using beta-cyclodextrin as a mobile-phase additive in a reversed-phase high-performance liquid chromatography system. Preparative separation of the biologically active (R)-enantiomer was achieved from racemic N-methylsalsolinol. Beta-cyclodextrin was removed completely in good yield by acid extraction and solid-phase extraction. By a slight modification, this method will be applicable to the isolation of various types of biologically important enantiomers.     

Simultaneous determination of dextromethorphan and its metabolites in human plasma by capillary electrophoresis

A sensitive capillary electrophoretic method was developed and validated for the simultaneous determination of dextromethorphan and its metabolites, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan, in human plasma. After cleavage of conjugates by enzymatic hydrolysis with beta-glucuronidase, dextromethorphan and its metabolites were extracted from 1.5 ml of plasma by a liquid liquid extraction procedure using heptane-ethylacetate (50:50, v/v) and re-extracted to aqueous phase. The compounds were separated within 8 min on a fused silica capillary, 75 microm internal diameter using sodium borate (pH 9.4; 50 mM) as running buffer, and measured by UV-detection at 195 nm using a detection cell with a path length of 1.2 mm. The method was accurate and precise. Linear relationships were observed between the peak response and the concentration in the range of 1-400 ng ml(-1) plasma with correlation coefficients above 0.998. The limit of detection was 0.5-1 ng ml(-1) plasma for all compounds. The method was used for determination of plasma levels of dextromethorphan and its metabolites after transdermal and oral administration of dextromethorphan.     

Analysis of beta-agonists in urine and tissues by capillary gas chromatography-mass spectrometry: in vivo study of salbutamol disposition in calves

The fate of salbutamol sulphate given orally has been investigated in calves. The urinary excretion rate and the tissue distribution of this beta-agonistic drug were studied by capillary gas chromatography coupled to low resolution mass spectrometry (GC-LRMS) under electron impact (EI) ionization mode, using an hexadeuterated salbutamol analogue as the internal standard. The parent drug and metabolites were extracted via solid phase extraction (SPE) mixed-phase-containing disposable columns and analysed as their trimethylsilyl derivatives. A more efficient clean-up had to be carried out for tissue samples. An acidic precipitation followed by a liquid-liquid extraction were therefore performed before the SPE. Moreover, the problem of tissue digestion was elucidated by means of an ultrasonic probe. Samples were also analysed before and after enzymic hydrolysis using purified beta-glucuronidase and a mixture of beta-glucuronidase and arylsulphatase, to obtain evidence of phase II conjugation mechanisms. Both free salbutamol and conjugated metabolites were detected in urine and tissue samples. Except for liver or kidney, salbutamol was rapidly cleared from most tissues after a withdrawal period. The possible excretion of some phase I metabolites was also investigated, using further analyses under positive chemical ionization LRMS and high resolution mass spectrometry (HRMS).     

A theoretical framework for sex-biased parental care

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5beta-Pregnan-3alpha-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC-ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography-mass spectrometry with an electron impact ion source (GC-MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Pharmacological effects of SA96 (bucillamine) and its metabolites as immunomodulating drugs--the disulfide structure of SA-96 metabolites plays a critical role in the pharmacological action of the drug

SA96 (generic name, bucillamine) is a disease-modifying anti-rheumatoid arthritis (RA) drug with immunological effects. This compounds has two sulfhydryl groups in its molecule, and the differences and similarities between this drug and D-penicillamine, which is also a sulfhydryl group-containing anti-rheumatic drug, have frequently been discussed. To clarify the pharmacological differences between these two drugs, we examined the concentrations of the compounds and its metabolites in serum and synovial fluid, paying special attention to the metabolites of SA96 produced in vivo. SA96 was metabolized in a very short time to SA981 which is a disulfide compound formed by intramolecular binding of two sulfhydryl groups, and transferred to synovial fluid. In addition SA981 had significant suppressive effects on IL-6 and IL-8 production by synovial cells in vitro. These results demonstrate that SA96, which has two sulfhydryl groups, exhibits anti-rheumatic effects via a pharmacological action clearly different from that of D-penicillamine.     

Micellar electrokinetic capillary chromatography for therapeutic drug monitoring of zonisamide

The simultaneous determination of zonisamide, a new type of antiepileptic drug, and the typical antiepileptic drugs phenobarbital, phenytoin and carbamazepine in human serum was developed using micellar electrokinetic capillary chromatography (MECC) with a diode array detector. A high correlation was revealed between the zonisamide levels in human serum obtained by MECC and those obtained by high-performance liquid chromatography (r=0.981). The serum levels of phenobarbital, phenytoin and carbamazepine determined by MECC were almost equal to those obtained by fluorescence polarization immunoassay. The reproducibility of separation and quantification with MECC analysis was appropriate for the intra- and inter-day assay coefficients. Therefore, the MECC method established here could provide a simple and efficient therapeutic drug monitoring method for antiepileptic drugs in patients, especially those treated with a combination of zonisamide and other antiepileptic drugs.     

Capillary electrophoretic purity method for the novel metal chelator TMT-NCS

A capillary electrophoretic method (CE) was developed for determining the purity of the novel metal chelator TMT-NCS. The separation of TMT-NCS from its degradation products, synthetic intermediates and by-products was accomplished using free solution CE in an aqueous-organic solvent system. This compound exhibits a complex impurity profile with the potential for over 30 degradants/impurities. The CE separation was optimized with respect to buffer type and concentration, pH, organic solvent and competitive chelator additive, allowing the resolution of all impurities in under 20 min. The specificity was established by examining stressed samples and evaluating peak purity using a diode-array detector. The sensitivity for low level impurities was optimized using sample stacking. Preliminary validation data were accumulated to support the utility of this method for estimating the purity of this drug intermediate.     

Ropivacaine, a new amide-type local anesthetic agent, is metabolized by cytochromes P450 1A and 3A in human liver microsomes

Ropivacaine is a new amide-type local anesthetic agent. Unlike bupivacaine and mepivacaine, two structurally similar local anesthetic compounds, ropivacaine is exclusively the S-(-)-enantiomer. Ropivacaine is predominantly eliminated by extensive metabolism in the liver, with only 1% of the dose being excreted unchanged in the urine of humans. Four of the metabolites formed in human liver microsomes were identified as 3-OH-ropivacaine, 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and 2',6'-pipecoloxylidide (PPX). The enzymes involved in the human metabolism of ropivacaine have not been identified. To ascertain which forms of cytochrome P450 are involved, ropivacaine was incubated with human microsomes from 10 different livers having different cytochrome P450 activities. A strong correlation was found between the formation of 3-OH-ropivacaine and CYP1A (r = 0.87-0.89) and between the formation of 4-OH-ropivacaine, 2-OH-ropivacaine, and PPX and CYP3A (r = 0.97-1). Incubation of ropivacaine and human liver microsomes in the presence of alpha-naphthoflavone or furafylline, inhibitors of CYP1A, decreased the formation of 3-OH-ropivacaine by about 85%, without affecting the formation of the other metabolites. The formation of 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX was markedly inhibited in the presence of troleandomycin, an inhibitor of CYP3A. Microsomes from cells expressing CYP1A2 formed 3-OH-ropivacaine, whereas 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX were formed in microsomes from cells expressing CYP3A4. Inhibitors of CYP2C (sulfaphenazole), CYP2D6 (quinidine), and 2E1 (diethyldithiocarbamate) did not inhibit the formation of any metabolite from ropivacaine. In conclusion, CYP1A catalyzes the formation of 3-OH-ropivacaine, the main metabolite formed in vivo, whereas the formation of 4-OH-ropivacaine, 2-OH-methyl-ropivacaine, and PPX was catalyzed by CYP3A.