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.     

Determination of Carbamazepine and its Main Metabolite Carbamazepine-10,11-Epoxide in Rat Brain Microdialysate and Blood Using ESI–LC–MS (Ion Trap)

A new analytical method has been developed and validated for determination of the anti-epileptic drug carbamazepine (CBZ) and its main metabolite carbamazepine-10,11-epoxide (CBZ-E) using ESI–LC–MS (ion trap). The compounds were separated on a C18 (150 × 2.1 mm I.D., 3 μm particles) column and were isocratically eluted in the mobile phase consisting of water–acetonitrile–acetic acid (74.5:25:0.5, v/v) using the flow rate of 0.4 mL min⁻¹. The other anti-epileptic drug oxcarbamazepine (OXC) was used as an internal standard. The retention times for CBZ-E, OXC and CBZ were 5.6, 6.8, 12.8 min. Signals of the compounds were monitored under multi-reaction monitoring mode (MRM) of ESI–LC–MS (ion trap) for the quantification. Selected ions of CBZ-E, OXC and CBZ in MRM were m/z 253→210, m/z 253→180 and m/z 237→194. The method was validated over the concentration range of 5.0–500.0 ng mL⁻¹ and was applied to rat brain microdialysate and blood samples for the determination of CBZ and main metabolite. The brain microdialysate and the blood sample were collected simultaneously after intra-peritoneal injection of CBZ (12 mg kg⁻¹) during a period of 10 h. No interference from endogenous substances and matrix effect were found on the separation of microdialysates and blood samples. The consequent signals of the compounds were resolved and integrated clearly. The LC–MS method was presented as an alternative to investigate pharmacokinetic parameters of CBZ and CBZ-E in blood and brain studies.

     

A chiral HPLC-UV method for the quantification of dibenz[b,f]azepine-5-carboxamide derivatives in mouse plasma and brain tissue: eslicarbazepine acetate, carbamazepine and main metabolites

For the first time, a selective and sensitive chiral HPLC-UV method was developed and fully validated for the simultaneous quantification of eslicarbazepine acetate (ESL), carbamazepine (CBZ), S-licarbazepine (S-Lic), R-licarbazepine (R-Lic), oxcarbazepine (OXC) and carbamazepine-10,11-epoxide (CBZ-E), in mouse plasma and brain homogenate supernatant. After the addition of chloramphenicol as the internal standard, samples were processed using an SPE procedure. The chiral chromatographic analysis was carried out on a LiChroCART 250-4 ChiraDex column, employing a mobile phase of water and methanol (88:12, v/v) pumped at 0.9 mL/min and the UV detector set at 235 nm. The assay was linear (r(2) ≥0.995) for ESL, CBZ, OXC, S-Lic, R-Lic and CBZ-E in the range of, respectively, 0.2-4, 0.4-30, 0.1-60, 0.2-60, 0.2-60 and 0.2-30 μg/mL, in plasma, and of 0.06-1.5 μg/mL for ESL, 0.12-15 μg/mL for CBZ and CBZ-E and 0.06-15 μg/mL for OXC and both licarbazepine (Lic) enantiomers in brain homogenate supernatant. The overall precision was within 8.71% and accuracy ranged from -7.55 to 8.97%. The recoveries of all the compounds were over 92.1%. Afterwards, the application of the method was demonstrated using real plasma and brain samples obtained from mice administered simultaneously with ESL and CBZ.     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography.

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C18 column with isocratic elution with acetate buffer (25 mM, pH 4.5)-methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35 degrees C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.     

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.     

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.     

Quantitative Estimation of Urinary Metabolites in Ruminants by High-Performance Liquid Chromatography

Abstract

A reversed-phase high-performance liquid chromatographyc method for a simultaneous determination of cretinine (C); Uric Acid (A); Hipoxanthine (HX); Xanthine (X); Hippuric Acid (HA); Benzoic Acid (BA) and Phenylacetic Acid (PAA) in urine of ruminants is described.

Separation was achieved on a Nova-PaK column by-gradient elution. Quantitation and detection limits were determined Detection is effected by UV absorption at 254 nm with a total analysis time of less than 30 min. An aliquot of diluted urine is injected directly into the liquid chromatographic column.

The proposed HPLC method was verified for linearity, accuracy, precision and applicability.     

High-performance liquid chromatographic determination of carbamazepine and carbamazepine 10,11-epoxide in plasma and saliva following solid-phase sample extraction

A rapid, sensitive and simple to operate high-performance liquid chromatographic method for the simultaneous determination of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZ-EP) in plasma and saliva is described. The drug and its metabolite are extracted from both plasma and saliva using commercially available reversed-phase octadecylsilane bonded silica columns (Bond-Elut C18, 2.8 ml capacity). Separation of CBZ and CBZ-EP 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 Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Z-module RCSS and protected by a Guard-Pak precolumn module containing a Guard-Pak mu Bondapak C18 insert. Using ultraviolet detection at 214 nm, levels in the region of 50-100 ng/ml for CBZ and CBZ-EP can be measured with only 250 and 500 microliters of plasma and saliva, respectively. The method, which has been used to determine steady-state concentrations of the drug and its metabolite in paediatric patients receiving CBZ monotherapy, is also suitable for pharmacokinetic studies.     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

Determination of Ethosuximide,Phenobarbital, Carbamazepine,Carbamazepine Epoxide and Phenytoin in Serum by high performance liquid chromatography using Radially compressed Columns and an Aqueous methanolic mobile phase

Abstract

A liquid chromatographic method for the determination of antiepileptic drugs in serum is described. The drugs were adsorbed on activated charcoal from which they were recovered by extraction with methylene chloride. The organic extracts were evaporated to dryness and the residues were dissolved in small volumes of the mobile phase. The extracts were clean. Only trace amounts of endogenic compounds were detected. The chromatographic separation was performed with a radially compressed column (C18) that was used daily for several months.     

High-Performance Liquid Chromatographic Determination of Clavulanic Acid in Human Serum and Urine Using a Pre-Column Reaction with 1, 2, 4-Triazole

Abstract

A reversed-phase high-performance liquid chromatographic (RP-HPLC) assay for the determination of clavulanic acid in serum and urine is described. The clavulanic acid was assayed by reacting the sample with 1, 2, 4-triazole reagent which rapidly produces a derivative that has its UV absorption maximum at 317 nm. The resulting product was separated in a RP-18 column (μ-Bondapak, 10 μm) and detected at 313 nm. The method was applied to assays of clavulanate in serum and urine, and is reproducible with a lower limit of quantitation of 0.1 μ/Ml.     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

Quinidine oxidative metabolism. Identification and biosynthesis of quinidine 10,11-dihydrodiol stereoisomers

The isocratic reversed phase high performance liquid chromatographic method proposed for quinidine metabolic studies facilitates particularly the separation of 10(R) and (S) isomers of quinidine 10,11-dihydrodiols. The finding of each of these forms following a new synthetic pathway allows us to identify and quantify them in biological fluids. These two isomers have especially been observed in rat bile and hepatocyte secretions. The metabolic inducing effect of phenobarbital on the oxidative metabolism of quinidine is verified in rat isolated hepatocytes. Simultaneous secretion of the two dihydrodiols is also verified in human urine by a gas chromatography/mass spectrometry procedure.     

An Improved Microanalytical Procedure for the Quantitation of Procainamide and N-Acetyl Procainamide in Serum by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for analysis of procainamide (PA), and N-acetyl procainamide (NAPA) is presented. Sample preparation employs a simple base-acid double extraction procedure and analysis is carried out on a reverse phase chromatographic system using a μBondapak C₁₈ column and buffered aqueous acetonitrile as the mobile phase. The extraction procedure gives quantitative recovery of both PA and NAPA, and chromatographic results show that drug levels of as low as 0.3 mg per liter of serum can be conveniently analyzed without significant background interferences. The small volume (0.2 ml) of serum needed to perform an analysis makes this method suitable for pharmacokinetic studies in humans and animals as well as for clinical therapeutic drug monitoring studies.     

Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition

An improved protocol has been developed to isolate homovanillic acid (HVA) and vanilmandelic acid (VMA) from urine with strong anion-exchange resin. The sample is diluted with acetate buffer and passed through a disposable column. HVA, uric acid, and many hydrophobic organic acids are removed with 1.0 M acetic acid--ethanol. Then VMA is eluted with 0.5 M phosphoric acid. Two isocratic mobile phases allow rapid high-performance liquid chromatographic measurement of VMA (5 min) and HVA (8 mins) on a 5-micron ODS column. Selective conditions were developed with dual-electrode coulometric detection to permit specific measurement of VMA, HVA, and internal standards, with less than 5% between-run variation.     

Simultaneous Determination of Carbamazepine and its Active Metabolite Carbamazepine-10,11-epoxide in Human Plasma by UPLC

A simple method for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide by ultra performance liquid chromatography (UPLC) with ultraviolet absorbance detection (TUV) was developed. The method involves a two-step protein precipitation by liquid–liquid extraction. Phenytoin sodium was used as the internal standard. The separation was carried out on Acquity C18 column with acetonitrile:methanol:KH₂PO₄ buffer (adjusting pH to 4.6 with 85% o-phosphoric acid) (180/180/170, v/v/v) as the mobile phase at a flow rate of 0.4 mL min⁻¹. Linear detection response was obtained for concentrations ranging from 50 to 5,000 ng mL⁻¹. The limit of quantification (LOQ) was 50 ng mL⁻¹. The method was validated successfully for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide, which can be applied through pharmacokinetics and bioequivalence studies.

     

Ion Exchanger Liquid Chromatography of N-Acetylaspartic Acid and Some N-Acetylaspartyl Peptides

Abstract

A high performance liquid chromatographic (HPLC) procedure for the rapid separation of N-acetylaspartic acid, N-acetyl-aspartyl-glutamic acid and N-acetylaspartyl-glutamyl-aspartic acid is described. The procedure utilizes a pellicular ion-exchange column support, and ionic strength gradient with mobile phase solutions buffered to pH 5.0 and a UV detector operated at 210 nm. Reproducibility and quantitative capabilities are also discussed. The method has been used for a tentative estimation of N-acetylaspartic acid and N-acetylaspartyl peptides in a rat brain synaptosomal extract.     

Simultaneous Determination of Carbamazepine,Phenobarbital and Their Major Metabolites in Serum,Brain Tissue and Urine by High-Performane Liquid Chromatographya

Abstract

A high-performance liquid chromatographic method is described for the simultaneous measurement of carbamazepine, phenobarbital and their major metabolites in small samples of serum, brain tissue and urine. This involves solvent extraction, reversed-phase chromatography and ultraviolet detection at 195 nm. Glucuronides in urine are hydrolyzed by enzymatic cleavage with β-glucuranidase. Quantitation is based on the peak-height ratio of the analyte to its internal standard (10, 11-dihydrocarbamaze-pine or p-methylphenobarbital). The results obtained show that the method is precise and reproducible.