Octakis-6-sulfato-gamma-cyclodextrin as additive for capillary electrokinetic chromatography of dibenzoazepines: carbamazepine,oxcarbamazepine and their metabolites

Single isomer octakis-(2,3-dihydroxy-)6-sulfato-gamma-cyclodextrin used as pseudostationary phase of the background electrolyte interacts with dibenzo[b,f]azepines (consisting of a condensed 3-ring system) and forms negatively charged complexes. Hydroxygroups in position 2 and 3 at carbamazepine increase the extent of interaction, whereas substitution by oxygen at position 10 and/or 11 reduces it. The complex constants for the analytes are ranging from few tens L/mol (10-hydroxycarbamazepine, 10,11-dihydroxycarbamazepine, 10,11-epoxycarbamazepine, oxcarbazepine) to several hundreds L/mol (carbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine), and are much larger than those of the analytes with octakis-(2,3-dimethyl-)-6-sulfato-gamma-cyclodextrin. Full enantiomeric separation of the chiral metabolites of carbamazepine and oxcarbazepine is obtained at octakis-(2,3-dihydroxy-)-6-sulfato-gamma-cyclodextrin concentrations of about 10 mM (3 mM borate buffer, pH 8.5). Compared to heptakis-6-sulfato-beta-cyclodextrin, selectivity differs and stereoselectivity is more pronounced.     

A PLS-based extractive spectrophotometric method for simultaneous determination of carbamazepine and carbamazepine-10,11-epoxide in plasma and comparison with HPLC

Carbamazepine (CBZ) undergoes enzyme biotransformation through epoxidation with the formation of its metabolite, carbamazepine-10,11-epoxide (CBZE). A simple chemometrics-assisted spectrophotometric method has been proposed for simultaneous determination of CBZ and CBZE in plasma. A liquid extraction procedure was operated to separate the analytes from plasma, and the UV absorbance spectra of the resultant solutions were subjected to partial least squares (PLS) regression. The optimum number of PLS latent variables was selected according to the PRESS values of leave-one-out cross-validation. A HPLC method was also employed for comparison. The respective mean recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 (+/-0.25)% and 103.00 (+/-0.09)% for PLS and 99.40 (+/-0.15)% and 102.20 (+/-0.02)%. The concentrations of CBZ and CBZE were also determined in five patients using the PLS and HPLC methods. The results showed that the data obtained by PLS were comparable with those obtained by HPLC method.     

Approach to the Dynamic of Carbamazepine and its Main Metabolites in Soil Contamination through the Reuse of Wastewater and Sewage Sludge

The release of pharmaceutically active compounds to the soils through the application of sewage sludge and the irrigation with wastewater, or even with surface water, is constant. The adsorption of these compounds onto the soil is one of the key factors affecting their fate in the environment and their potential environmental risks. In this work, the adsorption of carbamazepine (CBZ) and its metabolites, 3-hydroxy-carbamazepine (3OH-CBZ), carbamazepine-10,11-dihydro-10,11-epoxide (EP-CBZ), and 10,11-dihydro-10-hydroxycarbamazepine (10OH-CBZ), in three Mediterranean soils was evaluated using single-solute and four-solute experiments. The highest adsorptions were measured for 3OH-CBZ, followed by CBZ, EP-CBZ, and 10OH-CBZ, in that order. A high influence of the physicochemical characteristics of the compounds, pH, and soil characteristics in the adsorption of the studied compounds was observed and corroborated by the statistical analysis of the results. Moreover, a good fit was observed in the three isotherm models evaluated (linear, Freundlich, and Langmuir) in single-solute experiments (R² > 0.90). However, a decrease of the measured adsorptions and a worse fit to the isotherm models were observed in the case of multiple-solute experiments. This could be mainly due to the competition established between the studied compounds for the active sites of the soils.     

Characterization of the metabolites of rosmarinic acid in human liver microsomes using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Rosmarinic acid (RA) is a phenolic acid originally isolated from the herb medicine Rosmarinus officinalis. The purpose of this study was to identify the metabolites of RA. RA was incubated with human liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate tetrasodium salt and/or uridine diphosphate glucuronic acid using glutathione (GSH) as a trapping agent. After 60-min incubation, the samples were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Under the current conditions, 14 metabolites were detected and identified. Our data revealed that RA was metabolized through the following pathways: the first pathway is the oxidation of catechol to form ortho-quinone intermediates, which react with GSH to form mono-GSH adducts (M1, M2, and M3) and bis-GSH adducts (M4 and M5); the second pathway is conjugation with glucuronide to yield acylglucuronide (M7), which further reacts with GSH to form RA-S-acyl-GSH adduct (M9); the third pathway is hydroxylation to form M10, M11, and M12, which further react with GSH to form mono-GSH adducts (M13 and M14); the fourth pathway is conjugation with GSH through Michael addition (M6); the fifth pathway is conjugation with glucuronidation, forming M8, which is the major metabolic pathway of RA.     

Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.     

Determination of carbamazepine and carbamazepine-10,11-epoxide in human serum and plasma by micellar electrokinetic capillary chromatography in the absence of electroosmosis

Therapeutic drug monitoring of carbamazepine (CBZ), a widely used antiepileptic drug, is required for optimization of pharmacotherapy with this drug and for assessment of the patient's compliance to therapy. The suitability of employing micellar electrokinetic capillary chromatography (MEKC) in the absence of electroosmosis for the determination of CBZ and its main metabolite carbamazepine-10,11-epoxide (CBZE) in extracts of human serum and plasma is reported. Using micelles formed by dodecyl sulfate, analyses performed in untreated fused-silica capillaries at acidic pH and in commercially available coated capillaries under application of reversed polarity are compared. Uncoated and polyvinyl alcohol coated capillaries proved to be unsuitable for this purpose, whereas capillaries coated with linear polyacrylamide and N-acryloylaminoethoxyethanol and operated at pH 7.6 are shown to provide high-quality and reliable data on a short time scale. Assay performance is discussed via statistical analysis of the data produced from a set of quality control sera that contain up to 14 different drugs and via analysis of patient samples. Intraday and interday imprecision data for concentrations between 4.0 and 84 microM are demonstrated to be < 10%. Run times are shown to be < 50% compared to those observed in conventional MEKC at alkaline pH (i.e., in the presence of electroosmosis).     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

Application of thermospray liquid chromatography-mass spectrometry to the simultaneous quantification of tracer concentrations of isotopically labelled carbamazepine epoxide and steady-state levels of carbamazepine and carbamazepine epoxide

A thermospray high-performance liquid chromatography-mass spectrometry method for the separation and quantification of tracer concentrations of isotopically labelled carbamazepine epoxide ([15N, 13C]CBZE) in the presence of steady-state levels of the anticonvulsant carbamazepine (CBZ) and its epoxide metabolite (CBZE) has been developed. The technique does not require derivatization, demonstrates little or no thermal degradation of the analytes, provides increased specificity not available from conventional high-performance liquid chromatography, and has a detection limit of 500 pg for CBZE on-column. The method, incorporating d4-CBZ and d4-CBZE as internal standards, allows precise and accurate determination of the analytes with good reproducibility and stability.     

Liquid chromatographic analysis of oxcarbazepine and its metabolites in plasma and saliva after a novel microextraction by packed sorbent procedure

A rapid and reliable analytical method suitable for the simultaneous determination of the antiepileptic drug, oxcarbazepine and its metabolites in human plasma and saliva by means of liquid chromatography with diode array detection (DAD) has been developed. Oxcarbazepine and its metabolites (10,11-dihydro-10-hydroxycarbamazepine, trans-10,11-dihydro-10,11-dihydroxycarbamazepine and 3-hydroxycarbamazepine) were baseline separated within 6.5 min on a reversed-phase C18 column with a phosphate buffer-acetonitrile-triethylamine mixture as the mobile phase. The DAD detector was set at 240 nm. A sample preparation method for biological samples using a microextraction by packed sorbent technique has been implemented, employing a C18 sorbent inserted into a microvolume syringe and using only a small volume (25 μL) of plasma or saliva. The extraction yield values were satisfactory for all analytes (>86.5%) as well as the precision data, which were always in the low percentage of relative standard deviation values (<4.6%). The method was successfully applied to both plasma and saliva samples drawn from psychiatric and neurological patients undergoing treatment with oxcarbazepine (Tolep^®) tablets.     

Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex

The dinuclear RuII arene complexes [{(eta6-arene)RuCl}2(mu-2,3-dpp)](PF6)2, arene=indan (1), benzene (2), p-cymene (3), or hexamethylbenzene (4) and 2,3-dpp=2,3-bis(2-pyridyl)pyrazine, have been synthesized and characterized. Upon irradiation with UVA light, complexes 1 and 2 readily underwent arene loss, while complexes 3 and 4 did not. The photochemistry of 1 was studied in detail. In the X-ray structure of [{(eta6-indan)RuCl}2(mu-2,3-dpp)](PF6)2 (1), 2,3-dpp bridges two RuII centers 6.8529(6) A apart. In water, aquation of 1 in the dark occurs with replacement of chloride with biexponential kinetics and decay constants of 100+/-1 min-1 and 580+/-11 min-1. This aquation was suppressed by 0.1 M NaCl. UV or visible irradiation of 1 in aqueous or methanolic solution led to arene loss. The fluorescence of the unbound arene is approximately 40 times greater than when it is complexed. Irradiation of 1 also had a significant effect on its interactions with DNA. The DNA binding of 1 is increased after irradiation. The non-irradiated form of 1 preferentially formed DNA adducts that only weakly blocked RNA polymerase, while irradiation of 1 transformed the adducts into stronger blocks for RNA polymerase. The efficiency of irradiated 1 to form DNA interstrand cross-links was slightly greater than that of cisplatin in both 10 mM NaClO4 and 0.1 M NaCl. In contrast, the interstrand cross-linking efficiency of non-irradiated 1 in 10 mM NaClO4 was relatively low. An intermediate amount of cross-linking was observed when the sample of DNA already modified by non-irradiated 1 was irradiated. DNA unwinding measurements supported the conclusion that both mono- and bifunctional adducts with DNA can form. These results show that photoactivation of dinuclear RuII arene complexes can simultaneously produce a highly reactive ruthenium species that can bind to DNA and a fluorescent marker (the free arene). Importantly, the mechanism of photoreactivity is also independent of oxygen. These complexes, therefore, have the potential to combine both photoinduced cell death and fluorescence imaging of the location and efficiency of the photoactivation process.     

Cetirizine as pH-dependent cross-reactant in a carbamazepine-specific immunoassay

High performance liquid chromatography (HPLC) was hyphenated with a previously reported carbamazepine-specific enzyme-linked immunosorbent assay (ELISA) as a screening approach to water analysis in order to identify possible interferences from transformation products. Treated wastewater was analysed and three substances were recognized by the antibody besides carbamazepine: the metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 2-hydroxycarbamazepine plus the structurally not obviously related antihistamine cetirizine. The molar cross-reactivity against cetirizine was found to be pH-dependent and assessed to be 400% at pH 4.5 and 22% at pH 10.5. Performing the ELISA at pH 10.5 greatly improved the accuracy when carbamazepine was determined in surface and wastewater samples.     

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.     

Analysis of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma using high-performance liquid chromatography

A sensitive method based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of carbamazepine (CBZ) and one of its active metabolites, carbamazepine-10,11-epoxide (CBZ-E) in human plasma. CBZ, CBZ-E and the internal standard (IS) 10,11-dihydrocarbamazepine were extracted from human plasma into methyl tert-butyl ether. CBZ, CBZ-E and the IS were successfully separated on an RP C18 column with a mobile phase of acetonitrile:methanol:water (18:19:63, v/v/v) and monitored via UV detection at 210 nm. The calibration curves were linear over the concentration ranges of 0.01–10 μg/mL for CBZ and 0.005–5 μg/mL for CBZ-E in human plasma, respectively. The method displayed excellent sensitivity, precision and accuracy, and was successfully applied to the quantification of CBZ and CBZ-E in human plasma after oral administration of a single 200 mg CBZ CR tablet. This method is suitable for bioequivalence studies following single doses given to healthy volunteers.     

Cycloaddition of Cyclopentadiene to 3-Deoxy-1,2:5,6-di-O-isopropylidene-alpha-D-erythro-hex-3-enofuranose. Synthesis and Representative Chemistry of 1,6-Anhydro-2,3-dideoxy-beta-D-glycero-hex-2-enopyran-4-ulose ("Isolevoglucosenone")

Treatment of D-glucose-derived alkene 4 with cyclopentadiene in the presence of a Lewis acid results in the formation of cycloaddition products 8-11. Evidence is presented to show that these 1,6-anhydro sugar-cyclopentadiene adducts do not arise from rearrangement of 4 to isolevoglucosenone (5) followed by cycloaddition but are the result of Lewis acid-catalyzed rearrangement of alkene 4 to acyclic dienophile 12 followed by addition of cyclopentadiene. Major cycloadduct 8 has been utilized as a source of the enantiomerically pure carbocycles 14-25 by manipulation of the alkene and ketone functions and cleavage of the 1,6-anhydro bridge. In the absence of diene, alkene 4 undergoes rearrangement to enone 5 in 32% yield. Reaction of 5 with several dienes results only in the formation of "bottom-face" adducts 10,11, 28, and 29, and conjugate addition of either HN(3) or Me(3)COOH is found to be completely stereoselective to afford 30 and 31, respectively. Subsequent manipulation of azide 30 leads to precursors of several naturally occurring 2-amino-2,3-dideoxy sugars.     

Isocratic Reversed-Phase HPLC for Simultaneous Separation and Determination of Seven Antiepileptic Drugs and Two of their Active Metabolites in Human Plasma

A simple reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of the antiepileptic drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine (CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD) and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL) was pretreated by deproteinization with 300 μL methanol containing 20 μg mL⁻¹ propranolol hydrochloride as internal standard. HPLC was performed on a C₈ column (4.6 mm × 250 mm; particle size 5 μm) with methanol–acetonitrile–0.1% trifluoroacetic acid, 235:120:645 (v/v), as mobile phase at a flow rate of 1.5 mL min⁻¹. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm, and PRI, LTG, MHD, PB, PHT, and CBZE by UV detection at 215 nm. Relationships between response and concentration were linear over the concentration ranges 1–80 μg mL⁻¹ for ZNS, 5–50 μg mL⁻¹ for PRI, 1–25 μg mL⁻¹ for LTG, 1–50 μg mL⁻¹ for MHD, 5–100 μg mL⁻¹ for PB, 1–10 μg mL⁻¹ for CBZE, 0.5–25 μg mL⁻¹ for OXC, 1–50 μg mL⁻¹ for PHT, and 1–25 μg mL⁻¹ for CBZ. Intra-day and inter-day reproducibility were adequate (coefficients of variation were ≤11.6%) and absolute recovery ranged from 95.2 ± 6.13 to 107.7 ± 7.76% for all the analytes; for the IS recovery was 98.69 ± 1.12%. The method was proved to be accurate, reproducible, convenient, and suitable for therapeutic monitoring of the nine analytes.     

Mechanistic Pathways and Identification of the Electrochemically Generated Oxidation Products of Flavonoid Eriodictyol in the Presence of Glutathione

The metabolic oxidation pathways of dietary flavonoid eriodictyol (Er) are not very well‐probed. In the present work, the electrochemical oxidation behavior of Er was studied in aqueous Britton‐Robinson (B‐R) buffer solution using cyclic voltammetry (CV), chronoamperometry (CA), and bulk‐electrolysis (BE). The oxidation products and reaction pathways of Er in the absence and the presence of glutathione (GSH) were proposed and identified in view of the results obtained by ultra‐high‐performance liquid chromatography coupled with mass spectrometry (UPLC‐MS). In the absence of GSH, eriodictyol shows one quasi‐reversible oxidation process at E_1/2=0.305 V, followed by a totally irreversible anodic peak at a more positive potential (E_p^a=1.05 V vs. Ag/AgCl, 3 M KCl). Putatively, the first process corresponds to the oxidation of the catechol moiety on the B ring of Er while the second one is attributed to the oxidation of the resorcinol moiety on the A ring. In the presence of GSH, however, the anodic oxidation of Er was proposed to be an ECEC‐type mechanism. The Er molecule first underwent a two‐electron oxidation coupled with loss of two‐proton to generate the corresponding quinone, which was either reduced to the original Er molecule by GSH, or further interacted with GSH to produce mono‐ and bi‐ glutathione conjugates of Er. The proposed mechanism was confirmed by digital simulation of the cyclic voltammograms.     

Synthesis of Substituted Methyl Pyridazine-4-carboxylates via Cycloaddition of Diazomethane to 2,3-Disubstituted 2-Cyclopropenecarboxylic Acids

A three-step procedure has been developed for the synthesis of 3,5-disubstituted methyl pyridazine-4-carboxylates from accessible 2,3-disubstituted 2-cyclopropenecarboxylates. In the first step, cyclopropene derivatives react with diazomethane to give adducts having 2,3-diazabicyclo[3.1.1]hex-2-ene structure. The regio- and stereoselectivity of the cycloaddition has been determined. The second step is isomerization of the bicyclic adducts into 1,4-dihydropyridazine derivatives by the action of sodium methoxide. Finally, oxidation with potassium permanganate yields the target pyridazine-4-carboxylates.     

Investigation of the regio- and stereo-selectivity of deoxyguanosine linkage to deuterated 2-hydroxyestradiol by using liquid chromatography/ESI-ion trap mass spectrometry

From previous studies on the reactivity of estradiol 2,3-quinone towards deoxyribonucleosides, it was demonstrated that several isomeric adducts were formed. Although adduction on steroid ring A or B has been evidenced using sequential MS(n) experiments, in some cases attachment positions are difficult to identify unambiguously. In this work, 2-hydroxyestradiol labeled with deuterium at various positions [6beta (1); 6alpha-7alpha (2); 6alpha-6beta-7alpha (3)] have been used. Isomeric adduct differentiation could be achieved using LC-ESI-MS(n). The m/z shift of the quasi-molecular ions as well as the fragmentation pathways suggested that adduction could occur on both C6 and C9 sites of the steroid B ring: Nucleophilic attack of the base on the C6 position of the steroid led to major adducts and addition of the base on the activated C9 site gave minor adducts that were found to be unstable. LC-MS(n) experiments carried out under deuterated medium provided information about some fragmentation processes by studying the m/z shift of fragment ions: (1) the loss of deoxyribose from the quasi-molecular ions took place according to a process involving a deuterium transfer from the deoxyribose alcohol function; (2) the cleavage of the steroid-base linkage involved a deuterium transfer from the hydroxy group of the catechol and likely occurred via the formation of an ion-dipole complex. The model studies conducted in this work provide new information on the fragmentation mechanisms of covalent adducts formed from estrogen quinones and deoxyguanosine, the most reactive DNA base. Besides, the first unequivocal characterization of adducts involving the steroid C9 position is shown by using deuterium labeled estrogen quinones.     

Simultaneous separation and determination (in serum) of phenytoin and carbamazepine and their deuterated analogues by high-performance liquid chromatography--ultraviolet detection for tracer studies

The use of stable isotope-labeled tracer compounds is the safest and most effective method to perform many steady state pharmacokinetic and drug interaction studies. We describe a method by which the heavily deuterated 2H10 analogues of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromatographically separated by high-performance liquid chromatography from unlabeled CBZ and PHT. All compounds are quantitated against an internal standard (IS) (10,11-dihydrocarbamazepine) and measured using conventional UV detection rather than mass spectrometry. Baseline resolution of extracted serum containing 2H10 CBZ, CBZ, 2H10 PHT, PHT and IS is achieved on a heated (55 degrees C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5 microns ODS column with an isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran (80:16:4, v/v/v) at 1.2 ml/min. Eluting compounds were monitored at a UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were linear (R greater than or equal to 0.999) over a range of 0.5-14 micrograms/ml for 2H10 CBZ, 0.5-20 micrograms/ml for CBZ, 0.5-20 micrograms/ml for 2H10 PHT, and 0.5-30 micrograms/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.     

Evaluation of GSH adducts of adrenaline in biological samples

The sustained high release of catecholamines to circulation is a deleterious condition that may induce toxicity, which seems to be partially related to the products formed by oxidation of catecholamines that can be further conjugated with glutathione (GSH). The aim of the present study was to develop a method for the determination of GSH adducts of adrenaline in biological samples. Two position isomers of the glutathion-S-yl-adrenaline were synthesized and characterized by HPLC using diode array, coulometric and mass detectors. A method for the extraction of these adducts from human plasma was also developed, based on adsorption to activated alumina, which showed adequate recoveries and proved to be crucial in removing interferences from plasma. The selectivity, precision and linearity of the method were all within the accepted values for these parameters. Furthermore, the sensitivity of this method allows the detection of adduct amounts that are within the range of the expected concentrations for these adducts under certain pathophysiological conditions and/or drug treatments. In conclusion, the development of this method allows the direct analysis of GSH adducts of adrenaline in human plasma, providing a valuable tool for the study of the catecholamine oxidation process and its related toxicity.