Gas chromatographic assay for oxcarbazepine and its main metabolites in plasma

The new anti-epileptic drug oxcarbazepine is temperature-labile and decomposes under the conditions of gas chromatography, even when injected into a cooled, inert, fused-silica capillary column. In contrast, the trimethylsilyl derivative of oxcarbazepine is stable. The bis-trimethylsilyl derivatives of the enol of oxcarbazepine and of its active metabolite, 10-hydroxycarbazepine, and the tris-trimethylsilyl derivative of carbazepine-10,11-trans-diol can be synthesized easily at room temperature. Using the readily available carbamazepine as internal standard, a simple gas chromatographic assay was developed for the simultaneous routine measurement of these three compounds at therapeutic levels. This assay is ten times more sensitive to oxcarbazepine than the previously described high-performance liquid chromatographic assays. It involves a single-step solvent extraction, uses a fused-silica capillary column and a flame ionization detector. On processing 0.5 ml of plasma, limits of detection of 10 ng/ml were obtained for oxcarbazepine and 10-hydroxycarbazepine and a limit of detection of 25 ng/ml for carbazepine-10,11-trans-diol.     

Sequential palladium-catalysed C- and N-arylation reactions as a practical and general protocol for the synthesis of the first series of oxcarbazepine analogues

The first series of oxcarbazepine analogues, starting from readily-available materials and through a high-yielding five-step sequence based on palladium catalysis, is reported. The so-obtained compounds incorporate not only a variety of substituents in both of the aryl rings comprising the framework of an oxcarbazepine, but also involve the more challenging palladium-catalysed coupling of a number of heteroaromatic substrates. The addition of small amounts of water in some of the metal-catalysed processes showed a beneficial effect, highly increasing the selectivity of such reactions.     

Enantioselective HPLC-UV method for determination of eslicarbazepine acetate (BIA 2-093) and its metabolites in human plasma

Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug undergoing clinical phase III trials for epilepsy and phase II trials for bipolar disorder. A simple and reliable chiral reversed-phase HPLC-UV method was developed and validated for the simultaneous determination of eslicarbazepine acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma. The analytes and internal standard were extracted from plasma by a solid-phase extraction using Waters Oasis HLB cartridges. Chromatographic separation was achieved by isocratic elution with water-methanol (88:12, v/v), at a flow rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodextrin, 5 microm) column at 30 degrees C. All compounds were detected at 225 nm. Calibration curves were linear over the range 0.4-8 microg/mL for eslicarbazepine acetate and oxcarbazepine, and 0.4-80 microg/mL for each licarbazepine enantiomer. The overall intra- and interday precision and accuracy did not exceed 15%. Mean relative recoveries varied from 94.00 to 102.23% and the limit of quantification of the assay was 0.4 microg/mL for all compounds. This method seems to be a useful tool for clinical research and therapeutic drug monitoring of eslicarbazepine acetate and its metabolites S-licarbazepine, R-licarbazepine and oxcarbazepine.     

Simultaneous HPLC Determination of Oxcarbazepine,Carbamazepine and Their Metabolites in Serum

Abstract

We propose a simple procedure for the simultaneous determination of the anticonvulsants oxcarbazepine, carbamazepine and three of their metabolites (10-hydroxy-10, 11-dihydro-carbamazepine, trans-10, 11-dihydroxy-10, 11-dihydro-carbamazepine and 10, 11-epoxy-carbamazepine) in serum or plasma. The alkalinized sample is extracted with ethyl acetate. The extract is evaporated to dryness and taken up with the mobile phase. An aliquot is injected into the liquid chromatograph and eluted with water/methanol/acetonitrile (55/40/5, by vol.) on a 5-μm C-18 reversed-phase column. Eluent is monitored at 254 nm. No interference by other anticonvulsants or by endogenous constituents from the sample is observed. Owing to its good precision, specificity, sensitivity, and selectivity, this method is well adapted to the therapeutic monitoring of oxcarbazepine or carbamazepine treated patients, as well as for pharmacokinetic studies.     

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.     

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.     

Rapid HPTLC Analysis of Oxcarbazepine in Human Plasma

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive high-performance thin-layer chromatographic method has been developed for analysis of oxcarbazepine (OXC) in human...     

Simultaneous determination of four antiepileptic drugs in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin. After protein precipitation by acetonitrile, the supernatant was analysed on a C18 reversed-phase HPLC column. Antiepileptic drugs and oxazepam (internal standard) were detected by ultraviolet absorbance at 240 nm. Linearity was established for the whole concentration range for each compound. Quantitation limits of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin were 0.58, 3.5, 2.35, 0.66, 1.02 and 3.13 microg/mL, respectively, and mean recoveries added to serum were 105.15, 84.76, 94, 45, 96.52, 98.62 and 95.08%, respectively. This method has been used for the simultaneous determination of steady-state serum concentration of antiepileptic drugs in patients treated by one or more anticonvulsive treatment.     

Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography.

A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simulataneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 microns C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 microL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.     

Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for levetiracetam

A high-performance liquid chromatography (HPLC) assay using UV detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10- hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for the measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders.     

Accurate and simple determination of oxcarbazepine in human plasma and urine samples using switchable-hydrophilicity solvent in GC–MS

This work presents a sensitive and rapid analytical method for the determination of oxcarbazepine in human plasma and urine samples. A vortex-assisted switchable hydrophilicity solvent-based liquid phase microextraction (VA–SHS–LPME) was used to preconcentrate oxcarbazepine from the samples before the determination by gas chromatography mass spectrometry. The switchable hydrophilicity solvent was synthesized by protonating N,N-dimethylbenzylamine with carbon dioxide to make it totally miscible with an equivalent volume of water. Parameters of the VA–SHS–LPME method including volume of switchable hydrophilicity solvent, concentration/volume of sodium hydroxide and vortex period were systematically optimized. Under the optimum conditions, good linearity ranging from 27.03 to 353.47 μg/kg was obtained for the analyte. Limit of detection and quantitation values were found to be 6.2 and 21 μg/kg (mass base), respectively. The relative standard deviation was calculated as 6.9% for six replicate measurements of the lowest concentration of the calibration plot. Satisfactory recovery results were calculated in the range of 97–100% for human plasma and urine samples spiked at five different concentrations.     

A novel microextraction by packed sorbent–gas chromatography procedure for the simultaneous analysis of antiepileptic drugs in human plasma and urine

A simple, accurate, and sensitive microextraction by packed sorbent–gas chromatography‐mass spectrometry method has been developed for the simultaneous quantification of four antiepileptic drugs; oxcarbazepine, carbamazepine, phenytoin, and alprazolam in human plasma and urine as a tool for drug monitoring. Caffeine was used as internal standards for the electron ionization mode. An original pretreatment procedure on biological samples, based on microextraction in packed syringe using C₁₈ as packing material gave high extraction yields (69.92–99.38%), satisfactory precision (RSD < 4.7%) and good selectivity. Linearity was found in the 0.1–500 ng/mL range for these drugs with limits of detection (LODs) between 0.0018 and 0.0036 ng/mL. Therefore, the method has been found to be suitable for the therapeutic drug monitoring of patients treated with oxcarbazepine, carbamazepine, phenytoin, and alprazolam. After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. A comparison of the detection limit with similar methods indicates high sensitivity of the present method over the earlier reported methods. The present method is applied for the analysis of these drugs in the real urine and plasma samples of the epileptic patients.     

Quantitation of oxcarbazepine and its metabolites in human plasma by micellar electrokinetic chromatography

A reliable micellar electrokinetic chromatographic method for the determination of oxcarbazepine and its two main metabolites, 10-hydroxycarbamazepine and 10,11-trans-dihydroxy-10,11-dihydroxycarbamazepine, in human plasma was developed. The separation and determination of the analytes was achieved using a system consisting of 60 mM SDS in phosphate buffer (30 mM, pH 8.0), to which 20% (v/v) methanol was added. Separation was carried out in an uncoated fused-silica capillary with a separation voltage of 25 kV and currents typically less than 40 microA. Spectrophotometric detection was at 205 nm. Isolation of oxcarbazepine and its metabolites from plasma was accomplished by a solid-phase extraction procedure. The mean extraction yield of the analytes from plasma was higher than 94%. The linear correlation coefficients were better than 0.994 for all analytes. The limit of detection was 0.05 microg/mL, the limit of quantitation 0.15 microg/mL. The repeatability for the spiked blank plasma samples was lower than 1.9% and the intermediate precision lower than 2.1%, both expressed as RSD%. The results obtained analysing real plasma samples from epileptic patients under therapy with Tolep were satisfactory in terms of precision, accuracy and detectability.     

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.     

Development and validation of a reversed‐phase HPLC method for licarbazepine monitoring in serum of patients under oxcarbazepine treatment

Licarbazepine is the pharmacologically active metabolite of oxcarbazepine, a drug indicated for the treatment of partial seizures and bipolar disorders. Several HPLC methods have been developed thus far but there is lack of control for interferences from antipsychotic drugs. The aim of the present study was to develop a simple, low‐cost and reliable HPLC‐UV method for the determination of licarbazepine in human serum in the presence of co‐administered antiepileptic, antipsychotic and commonly prescribed drugs. Sample preparation consisted of a single protein precipitation step with methanol. Separation lasted ~9 min on a reversed‐phase C₁₈ column using a mobile phase composed of 50 mm sodium‐dihydrogen‐phosphate‐monohydrate/acetonitrile (70:30, v/v) delivered isocratically at 0.9 mL/min and 30°C. Wavelength was 210 nm and calibration curve was linear with r ² 0.998 over the range 0.2–50.0 μg/mL. Coefficient of variation was <5.03% and bias <−4.92%. Recovery ranged from 99.49 to 104.52% and the limit of detection was 0.0182 μg/mL. No interferences from the matrix or from antiepileptic, antipsychotic and commonly prescribed drugs were observed. The method was applied to serum samples of patients under oxcarbazepine treatment and proved to be a useful tool for the therapeutic drug monitoring of licarbazepine during monotherapy or adjunctive treatment of seizures or affective disorders.     

Simultaneous determination of four antiepileptic drugs in human plasma samples using an ultra‐high‐performance liquid chromatography tandem mass spectrometry method and its application in therapeutic drug monitoring

Therapeutic drug monitoring of antiepileptic drugs is widely practiced to achieve optimal efficacy and avoid adverse side effects. We describe an ultra‐high‐performance liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method developed for the monitoring of four frequently prescribed antiepileptic drugs – lamotrigine, levetiracetam, oxcarbazepine and topiramate. The main pharmacologically active metabolite of oxcarbazepine (mono‐hydroxy‐derivative metabolite, MHD) was also quantified. After addition of internal standards and a simple stage of protein precipitation, plasmatic samples were analyzed on a C₁₈ column. All antiepileptic drugs were separated and quantified in 6 min, without interference. A good linearity was observed all over the calibration range (r² > 0.99), up to 20 μg/mL (40 μg/mL for MHD). The limit of quantification was 0.20 μg/mL (0.40 μg/mL for MHD) with precision and accuracy ranging from 1.0 to 2.1% and from 96.7 to 110.8%, respectively. Intra‐ and inter‐day precision and accuracy values were within 15%. No significant matrix effect was observed for all analytes. Clinical application was successfully evaluated in 259 samples from patients treated for epilepsy or bipolar disorders. In conclusion, a rapid, specific and sensitive UHPLC/MS/MS method was developed and validated for simultaneous quantification of antiepileptic drugs, suitable for therapeutic drug monitoring in neurology and psychiatry.     

Development and validation of an HPLC-UV method for the simultaneous quantification of carbamazepine, oxcarbazepine, eslicarbazepine acetate and their main metabolites in human plasma

For the first time, a simple, selective and accurate high-performance liquid chromatography method with ultraviolet detection was developed and validated to quantify simultaneously three structurally related antiepileptic drugs; carbamazepine, oxcarbazepine, and the recently launched eslicarbazepine acetate and their main metabolites, carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine, and licarbazepine. The method involves a solid-phase extraction and a reverse-phase C18 column with 5 cm length. The mobile phase consisting of water, methanol, and acetonitrile in the ratio 64:30:6 was selected as the best one and pumped at 1 mL/min at 40 °C. The use of this recent column and an aqueous mobile phase instead of buffers gives several advantages over the method herein developed; namely the fact that the chromatographic analysis takes only 9 min. The method was validated according to the guidelines of the Food and Drug Administration, showing to be accurate (bias within ±12%), precise (coefficient variation <9%), selective and linear (r ² > 0.997) over the concentration range of 0.05–30 μg/mL for carbamazepine; 0.05–20 μg/mL for oxcarbazepine; 0.15–4 μg/mL for eslicarbazepine acetate; 0.1–30 μg/mL for carbamazepine-10,11-epoxide; 0.1–10 μg/mL for 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine, and 0.1–60 μg/mL for licarbazepine. It was also shown that this method can adequately be used for the therapeutic drug monitoring of the considered antiepileptic drugs, carbamazepine, oxcarbazepine, eslicarazepine acetate, and their metabolites.     

A chiral liquid chromatography method for the simultaneous determination of oxcarbazepine, eslicarbazepine, R-licarbazepine and other new chemical derivatives BIA 2-024, BIA 2-059 and BIA 2-265, in mouse plasma and brain

Recently, in silico models have been developed to predict drug pharmacokinetics. However, before application, they must be validated and, for that, information about structurally similar reference compounds is required. A chiral liquid chromatography method with ultraviolet detection (LC‐UV) was developed and validated for the simultaneous quantification of BIA 2–024, BIA 2–059, BIA 2–265, oxcarbazepine, eslicarbazepine (S‐licarbazepine) and R‐licarbazepine in mouse plasma and brain. Compounds were extracted by a selective solid‐phase extraction procedure and their chromatographic separation was achieved on a LiChroCART 250–4 ChiraDex column using a mobile phase of water–methanol (92:8, v/v) pumped at 0.7 mL/min. The UV detector was set at 235 nm. Calibration curves were linear (r² ≥ 0.996) over the concentration ranges of 0.2–30 µg/mL for oxcarbazepine, eslicarbazepine and R‐licarbazepine; 0.2–60 µg/mL for the remaining compounds in plasma; and 0.06–15 µg/mL for all the analytes in brain homogenate. Taking into account all analytes at these concentration ranges in both matrices, the overall precision did not exceed 9.09%, and the accuracy was within ±14.3%. This LC‐UV method is suitable for carrying out pharmacokinetic studies with these compounds in mouse in order to obtain a better picture of their metabolic pathways and biodistribution. Copyright © 2011 John Wiley & Sons, Ltd.     

A new synthesis of oxcarbazepine using a Friedel-Crafts cyclization strategy

A novel, simple, and straightforward process for the large-scale synthesis of oxcarbazepine, the active ingredient of Trileptal^®, a medicine for the treatment of epilepsy, has been developed. Starting from readily available 1,3-dihydro-1-phenyl-2H-indol-2-one, a Friedel-Crafts cyclization strategy provides a direct route to the tricyclic framework of the target molecule. Crucial to the success of the strategy was the choice of the proper nitrogen-protecting group.     

Rapid ultraperformance liquid chromatography–tandem mass spectrometry method for quantification of oxcarbazepine and its metabolite in human plasma

A rapid and sensitive ultraperformance liquid chromatography tandem mass spectrometry assay was developed for the simultaneous analysis of oxcarbazepine and its main metabolite in human plasma. The assay involves a simple solid‐phase extraction procedure of 0.3 mL of human plasma and analysis was performed on a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode via electrospray ionization. Separation was achieved on an Acquity UPLC™ BEH C₁₈ column (50 × 2.1 mm, i.d., 1.7 µm) with isocratic elution at a flow‐rate of 0.25 mL/min and imipramine was used as the internal standard. The standard calibration curve was linear over the range 9.580–5070.205 ng/mL for oxcarbazepine (OXC) and 19.444–10290.800 ng/mL for 10,11‐dihydro‐10‐hydroxycarbamazepine (MHD), expressed by the linear correlation coefficient r², which was better than 0.995 for OXC and MHD. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recoveries were 81.0, 89.6 and 66.6% for OXC, MHD and imipramine, respectively. The total run time was 1.5 min only for each sample, which makes it possible to analyze more than 350 samples per day. Copyright © 2010 John Wiley & Sons, Ltd.