Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract

A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C₁₈, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C₁₈ column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C₁₈ insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine steady-state concentrations of the drug and its metabolites in paediatric patients.     

Improved high-performance liquid chromatographic procedure for the determination of chlormethiazole levels following solid-phase extraction from plasma

An improved high-performance liquid chromatographic method for the determination of chlormethiazole levels in plasma is described. The drug is extracted from plasma using commercially available reversed-phase extraction columns; recovery values obtained using Sep-Pak C18 and Bond Elut C1, C2, C4, C6, C8, C18 columns are compared. Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of 0.025 M sodium acetate buffer, pH 4.5-acetonitrile (67:33) at a flow-rate of 1.6 ml/min in conjunction with a 15-cm Jones Chromatography Apex ODS column. The analytical column was protected by a Waters Assoc. Guard-Pak module containing a Guard-Pak CN insert. Using ultraviolet detection at 254 nm chlormethiazole levels in the region of 50 ng/ml can be measured with only 500 microliter of plasma.     

Improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma using solid-phase extraction

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Excellent resolution of all components is provided by reversed-phase chromatography using a mobile phase consisting of 1% acetic acid-methanol (83:17) at a flow-rate of 2.7 ml/min, in conjunction with a Waters Assoc. Nova-Pak C18 column which was protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak CN cartridge. Rapid extraction of caffeine and the dimethylxanthines from plasma was achieved using reversed-phase octadecylsilane bonded-silica columns (Bond-Elut C18). With only 100 microliters of sample, plasma levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined using ultraviolet detection at 273 nm. The method has been used for measuring umbilical cord plasma samples to provide information regarding foetal exposure to caffeine and its metabolites and is also suitable for therapeutic drug monitoring of caffeine and theophylline levels in the treatment of neonatal apnoea.     

Simultaneous determination of carbamazepine and its epoxide metabolite in plasma and urine by high-performance liquid chromatography

A simple procedure for the simultaneous determination of carbamazepine and its major metabolite, carbamazepine epoxide, in plasma and urine is described. The assay involves two extractions of the drugs and an internal marker, clonazepam, from the alkalinized sample. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (30 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile-water (40:60, v/v) on a C18 pre-column linked to a 5-microns C8 reversed-phase column. The eluent is detected at 215 nm. The method has been used to investigate the steady-state concentrations of carbamazepine and carbamazepine epoxide in the plasma and urine of a manic-depressive patient.     

Simultaneous determination of the free and total carbamazepine concentrations in human plasma by high-performance frontal analysis using an internal-surface reversed-phase silica support

High-performance frontal analysis (HPFA) was applied to simultaneous determinations of the free and total carbamazepine (CBZ) concentrations in human plasma. When 1.5ml of human plasma containing CBZ at a clinical therapeutic level (free fraction, about 30%) was directly injected into an internal-surface reversed-phase silica column, the CBZ was separated from the plasma blank and was eluted as a zonal peak with a plateau height corresponding to the free CBZ concentration in protein binding equilibrium. Slow and continuous introduction of the plasma sample and the use of titanium filters permitted us to inject the sample repeatedly while avoiding a rapid increase in column pressure. The free and total CBZ concentrations were determined simultaneously from the peak height at the plateau region and the area of the CBZ peak, respectively. The within-run and day-to-day reproducibilities were satisfactory (C.V. less than or equal to 1.63%, n = 5).     

Simultaneous quantitation of salivary carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbitone by high-performance liquid chromatography

A rapid, sensitive and accurate high-performance liquid chromatographic method for the simultaneous quantitation of phenobarbitone, phenytoin, carbamazepine and carbamazepine-10,11-epoxide in saliva is described. Only small volumes of saliva (100 microliters) are required. Separation of the drugs is achieved by reversed-phase chromatography on a Nova-Pak C18 column, with a mobile phase of acetonitrile-phosphate buffer at a flow-rate of 2.0 ml/min. Detection is effected by ultra-violet absorption at 215 nm. The total run time is under 12.5 min per assay. A precipitation but no extraction step is involved, simplifying the assay method. Salivary concentrations in the range 0.25-25 micrograms/ml for carbamazepine, 0.5-20 micrograms/ml for phenytoin and phenobarbitone and 0.4-20 micrograms/ml for carbamazepine-10,11-epoxide can be measured. Recovery varies from 94 to 108%. The method has been used for routine measurements of anticonvulsants in saliva collected daily from patients with intractable epilepsy.     

Analysis of carbamazepine in serum by liquid chromatography with direct sample injection and surfactant-containing eluents

Surfactant-containing eluents are evaluated for the analysis of carbamazepine in serum with conventional reversed-phase columns. Bovine serum was quantitatively eluted at the column void volume using surfactant concentrations in conventional reversed-phase eluents. The effect of pH, guard columns and column switching was evaluated with respect to separating and detecting clinical levels of the drug and its primary metabolite. Column lifetime was also investigated.     

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.     

Column-switching high-performance liquid chromatographic analysis of carbamazepine and its principal metabolite in human plasma with direct sample injection using an alkyl-diol silica (ADS) precolumn

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with high-performance reverse phase chromatography for the analysis of carbamazepine (CBZ) and carbamazepine-10,11-epoxide (CBZ-E) in human plasma samples is described. A precolumn packed with 25 mum C(18) alkyl-diol support is used for direct plasma injection. Using column-switching techniques, the analytes were enriched on the precolumn by a 5 mM phosphate buffer (pH 7) with 2% of methanol solution at a flow-rate of 0.8 ml min(-1), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C(18) column, separated by a mixture of 10 mM phosphate buffer (pH 7) acetonitrile (70:30 v/v) solution at a flow-rate of 1.0 ml min(-1) and detected by the ultraviolet absorbance set at 212 and 285 nm and without transfer loss. Linear calibration graphs were obtained for sample injection volumes of 50 (0.2-4.0 of mug of CBZ ml(-1) and 0.1-5.0 mug of CBZ-E ml(-1), respectively), and 20 mul (5.0-20.0 mug of CBZ ml(-1)); in either case the r-value was >0.9963. Recoveries from spiked plasma samples were quantitative for both analytes and the coefficients of variation were below 3.83%. The lowest samples concentrations that can be quantified with acceptable accuracy and precision was 0.2 mug CBZ ml(-1) and 0.1 mug CBZ-E ml(-1) when a sample volume of 50 mul was injected. Concentrations of 0.08 and 0.05 mug ml(-1) of CBZ and CBZ-E were considered the limit of detection for a signal-to-noise ratio of 3. Furthermore, the developed column-switching method was successfully applied to the determination of CBZ and CBZ-E in plasma samples of patients submitted to CBZ therapy.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Automated microanalysis of carbamazepine and its epoxide and trans-diol metabolites in plasma by column liquid chromatography.

A fully automated high-performance liquid chromatographic procedure for the simultaneous determination of carbamazepine and its main metabolites, epoxycarbamazepine and dihydroxycarbamazepine, in plasma is described. Liquid-solid extraction on disposable C18 columns and reversed-phase chromatography on a 3 microns particle size C18 column were combined and automated by using the Automatic Sample Preparation with Extraction Columns system. Ultraviolet detection was performed at 210 nm. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]azepine-5-carboxamide was used as internal standard. A small plasma volume (100 microliters) was required. The total run time for the assay of one sample was about 10 min. The assay demonstrated good reproducibility. The limit of quantitation was 0.1 mumol/l (about 25 ng/ml).     

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.     

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.     

Rapid and sensitive liquid chromatographic determination of carbamazepine suitable for use in monitoring multiple-drug anticonvulsant therapy.

A rapid, sensitive and selective method for the determination of carbamazepine and its major metabolite in plasma has been developed. Other commonly used anticonvulsants can be determined in the same procedure without interference. After extraction with dichloromethane, the components are separated by high-pressure liquid chromatography without further clean-up or concentration on a column packed with small-particle silica gel. The mean recovery from plasma is 98.6% with a relative standard deviation of 1.6%. The detection limit for carbamazepine is approximately 2 ng/ml, requiring 1 ml of plasma.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens.

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid-liquid extraction. Separation was achieved on a C18 reversed-phase column. The limit of detection of ciprofloxacin is 0.05 microgram/ml and that of its three metabolites is 0.25 microgram/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Simultaneous determination of itraconazole and hydroxyitraconazole in human plasma by high-performance liquid chromatography

The development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of itraconazole and its metabolite, hydroxyitraconazole, in human plasma is described. The method involved liquid-phase extraction of itraconazole and hydroxyitraconazole using a hexane-dichloromethane (70:30) mixture, after addition of loratidine as an internal standard (IS). Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing fluorescence detection (excitation: 264 nm, emission: 380 nm). The mobile phase consisted of [0.01% triethylamine solution adjusted to pH 2.8 with orthophosphoric acid-acetonitrile (46:54)]-isopropanol (90:10, v/v) at a flow rate of 1.0 ml/min. For both the drug and metabolite, the standard curve was linear from 5.0 to 500 ng/ml with goodness of fit (r2) greater than 0.98 observed with four precision and accuracy batches during validation. An observed recovery was more than 70% for drug, metabolite and internal standard. The applicability of this method to pharmacokinetic studies was established after successful application during 35 subjects bioavailibity study. The method was found to be precise, accurate and specific during the study.     

Determination of tenoxicam in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of tenoxicam in plasma has been developed. Tenoxicam was extracted from buffered plasma (pH 3 or 4, respectively) with dichloromethane and the evaporated extracts were analysed on a C18 reversed-phase column using a methanol-phosphate buffer mobile phase and with UV detection at 371 nm. The detection limit was 20 ng/ml using a 0.5-ml sample. The method is selective with respect to the 5'-hydroxy metabolite, which is present in plasma after multiple administration of tenoxicam; this metabolite may also be determined using this procedure.     

Quantification of carbamazepine and its 10,11‐epoxide metabolite in rat plasma by UPLC‐UV and application to pharmacokinetic study

A rapid, selective and sensitive UPLC‐UV method was developed and validated for the quantitative analysis of carbamazepine and its epoxide metabolite in rat plasma. A relatively small volume of plasma sample (200 μL) is required for the described analytical method. The method includes simple protein precipitation, liquid–liquid extraction, evaporation, and reconstitution steps. Samples were separated on a Waters Acquity UPLC BEH C₁₈ column (1.7 µm, 2.1 × 100 mm) with a gradient mobile phase consisted of 60:40 going to 40:60 (v/v) water–acetonitrile at a flow rate of 0.5 mL/min. The total run time was as low as 6 min, representing a significant improvement in comparison to existing methods. Excellent linearity (r² > 0.999) was achieved over a wide concentration range. Close to complete recovery, short analysis time, high stability, accuracy, precision and reproducibility, and low limit of quantitation were demonstrated. Finally, we successfully applied this analytical method to a pre‐clinical oral pharmacokinetic study, revealing the plasma profiles of both carbamazepine and carbamazepine‐10,11‐epoxide following oral administration of carbamazepine to rats. The advantages demonstrated in this work make this analytical method both time‐ and cost‐efficient approach for drug and metabolite monitoring in the pre‐clinical/clinical laboratory. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of the pyridinium metabolite derived from haloperidol in brain tissue, plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method for the determination of the pyridinium metabolite (HPP+) derived from the antipsychotic drug haloperidol (HP) in brain tissue, plasma and urine using high-performance liquid chromatography with fluorescence detection is described. The HPP+ present in biological samples was extracted using a Sep-Pak C18 cartridge. Recoveries of HPP+ ranged from 78 to 90%. Final separation and quantitative estimations of HPP+ were achieved on a C18 reversed-phase column employing a mobile phase of acetonitrile-30 mM ammonium acetate (40:60, v/v) containing 10 mM triethylamine and adjusted to pH 3 with trifluoroacetic acid. The fluorescence detection utilized an excitation wavelength of 304 nm and an emission wavelength of 374 nm. Standard curves were linear in the range of 2.5-100 ng/ml for brain tissue homogenate and plasma samples and 10-500 ng/ml for urine samples. The detection limit of HPP+ was about 1 ng/ml in all biological samples. The concentrations of HPP+ in brain tissue, plasma and urine from HP-treated rats were determined using this method.     

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.