Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

Simultaneous Determination of Fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane—dichloromethane—butanol (55:40:5). The plasma extract is chromatographed on a 10-μm, C₁₈ reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration—response curve for all four compounds are linear from 0.05 μg/ml to at least 10 μg/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within ± 9% of the various amounts added with a standard deviation of ± 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Quantification of a decapeptide anticoagulant in rat and monkey plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed to quantify a decapeptide anticoagulant in rat and monkey plasma. The compound and internal standard, a nonapeptide analogue, were extracted from plasma with an amino solid-phase extraction column with an extraction efficiency in the range 75–90%. A C₁₈ analytical column was used to separate the analytes by gradient elution followed by ultraviolet detection at 215 nm. Quantification of the decapeptide over the concentration range 0.1–10.1 μg/ml resulted in an assay relative error and relative standard deviation both less than 10%. The anticoagulant decapeptide was stable in both rat and monkey plasma frozen at −20°C.     

Validation of high-performance liquid chromatographic assay methods for the analysis of carboplatin in plasma ultrafiltrate

Validation of two HPLC assays for the quantitation of carboplatin in human plasma ultrafiltrate is described. Both assay methods employed a YMC ODS-AQ 3.9×150 mm (3 μm) column for the chromatographic separation. The first method utilized direct UV detection, the second method utilized UV detection following post-column derivatization with sodium bisulfite. Structural analogues of carboplatin were synthesized and used as internal standards for the assays. With direct UV detection, sample clean-up using solid-phase extraction on amino cartridges was required prior to injection, with extraction recoveries ranging from 80 to 90%. This extraction procedure was not necessary with the post-column reaction method, which employed a more selective analytical wavelength. Unfortunately, instability of the post-column reagent was a problem and led to greater variability in predicted concentration values. For standard curves, a weighted (1/y²) regression approach was used for plots of peak area or peak height ratio (carboplatin/internal standard) vs. carboplatin concentration. The limit of detection of both assays was 0.025 μg/ml and both were validated for carboplatin concentrations from 0.05 to 40 μg/ml. Accuracy and precision data were generated using three batches of validation samples, each batch consisting of a standard curve and five sets of quality control samples. Stability of carboplatin in blood, plasma, plasma ultrafiltrate, and reconstituted extracts was evaluated. The assay methods were employed for the pharmacokinetic analysis of blood samples drawn from a pediatric patient that received a 400 mg/m² dose of carboplatin.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 μg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 μg/ml for acrylamide and from 0.5 to 100 μg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration–time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.     

Rapid and simultaneous determination of mycophenolic acid and its glucuronide conjugate in human plasma by ion-pair reversed-phase high-performance liquid chromatography using isocratic elution

A high-performance liquid chromatographic method has been developed for the simultaneous determination of mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) in human plasma. The method involves protein precipitation with acetonitrile, followed by ion-pair reversed-phase chromatography on C₁₈ column, with a 40 mM tetrabutyl ammonium bromide (TBA)–acetonitrile (65:35, v/v) mobile phase. A 20-μl volume of clear supernatant was injected after centrifugation, and the eluent was monitored at 304 nm. No interference was found either with endogenous substances or with many concurrently used drugs, indicating a good selectivity for the procedure. Calibration curves were linear over a concentration range of 0.5–20.0 μg/ml for MPA and 5–200 μg/ml for MPAG. The accuracy of the method is good, that is, the relative error is below 5%. The intra- and inter-day reproducibility of the analytical method is adequate with relative statistical deviations of 6% or below. The limits of quantification for MPA and MPAG were lower than 0.5 and 5.0 μg/ml, respectively, using 50 μl of plasma. The method was used to determine the pharmacokinetic parameters of MPA and MPAG following oral administration in a patient with renal transplantation.     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

Sensitive and specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of cocaine and its metabolites in rat plasma

A sensitive, specific and precise HPLC–UV assay was developed to quantitate cocaine (COC) and its metabolites benzoylecgonine (BE), norcocaine (NC) and cocaethylene (CE) in rat plasma. After adding 50 μl of the internal standard solution (bupivacaine, 8 μg/ml) and 500 μl of Sørensen's buffer (pH 6) to 100 μl of rat plasma sample, the mixture was extracted with 10 ml of chloroform. The organic layer was transferred to a clean test tube and was evaporated under nitrogen. The residue was reconstituted in 100 μl of mobile phase and 35 μl was injected onto the HPLC column. The mobile phase consisted of methanol–acetonitrile–50 mM monobasic ammonium phosphate (5:7:63, v/v/v) and was maintained at a flow-rate of 0.4 ml/min. Separation of COC and its metabolites was achieved using a Supelcosil ABZ+plus deactivated reversed-phase column (250×2.1 mm I.D., 5 μm). Calibration curves were linear over the range of 25–5000 ng/ml for COC and its three metabolites. The absolute extraction efficiencies for BE, COC, NC, CE and bupivacaine were 56.6%, 78.6%, 61.1%, 76.4% and 67.0%, respectively. COC and its metabolites were stable in mobile phase for 24 h at room temperature and in rat plasma for 2 weeks at −20°C. The limits of detection for BE, COC, NC and CE were 20, 24, 15 and 12.9 ng/ml, respectively. These values correspond to 0.70, 0.84, 0.525 and 0.452 ng of the according compound being injected on column. The within-day coefficient of variation for the four compounds ranged from 3.0% to 9.9% while the between-day precision varied from 3.6% to 14%. This method was used to analyze rat plasma samples after administration of COC alone and in combination with alcohol. The pharmacokinetic profiles of COC and its metabolites in these rats are also described.     

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 μg/ml and that of its three metabolites is 0.25 μg/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of vigabatrin in human plasma and urine. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan, solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Aspartam was used as an internal standard. The assay was linear over the concentration range of 0.2–20.0 μg/ml for plasma and 1.0–15.0 μg/ml for urine with a lower limit of detection of 0.1 μg/ml using 0.1 ml of starting volume of the sample. Both the within-day and day-to-day reproducibilities and accuracies were less than 5.46% and 1.6%, respectively. After a single oral dose of 500 mg of vigabatrin, the plasma concentration and the cumulative urinary excretion of the drug were determined.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Liquid chromatographic determination of sodium mercaptoundecahydrododecaborate in rat urine and plasma after precolumn derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of disodium mercaptoundecahydrododecaborate (BSH) in biological fluids. Monobromobimane was used as a precolumn derivatizing agent. A stable derivative was obtained. The derivative was separated on a C₁₈ column using reversed-phase ion-pairing chromatography and detected by a spectrophotometric detector at 373 nm. The detection limit was 200 ng/ml (0.1 ppm boron). Calibration curves were prepared for rat urine and plasma samples. The calibration curves were linear in the range of 1 μg/ml to 100 μg/ml for urine samples and 0.2 μg/ml to 50 μg/ml for plasma samples.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

Quantitative determination of dipalmitoylphosphatidylcholine and palmitic acid in porcine lung surfactants used in the treatment of respiratory distress syndrome

A high-performance liquid chromatography (HPLC) method was developed that can separate and quantify dipalmitoylphosphatidylcholine and its degradation product, palmitic acid from various phospholipids contained in a porcine lung surfactant used in the treatment of respiratory distress syndrome, which was recently approved for use by the FDA. The method used a C₈ reversed-phase HPLC column with a (50:45:10) acetonitrile/methanol/acetic acid mobile phase, and refractive index detection. The active component of the lung surfactant, dipalmitoylphosphatidylcholine (DPPC) and palmitic acid (PA), could be quantified following a liquid-liquid extraction procedure along with an internal standard, dimyristoylphosphatidylcholine (DMPC). The assay was validated for linearity, accuracy, precision, reproducibility and ruggedness. Column stability was measured by performing the assay over time and monitoring the system suitability parameters. The extraction procedure has a 90% recovery and the assay is linear over a range of 5 μg/ml to 300 μg/ml. The assay is used to release commercial product and monitor stability of existing lots of material.     

Rapid and sensitive high-performance liquid chromatographic method for busulfan assay in plasma

A reversed-phase liquid chromatographic method with ultraviolet detection has been developed to determine busulfan concentrations in plasma of children undergoing bone marrow transplantation. Plasma samples (200 μl) containing busulfan and 1,6-bis(methanesulfonyloxy)hexane as an internal standard were prepared by a simple derivatization method with diethyldithiocarbamate followed by extraction with ethyl acetate and solid-phase purification on C₈ columns conditioned with methanol and water and eluted with acetonitrile (recovery 99%). Chromatography was accomplished using a Hypersil octadecylsilyl column (10 cm×4.6 mm I.D.) and a mobile phase of acetonitrile, tetrahydrofuran and distilled water (65:5:30, v/v). The limit of detection was 25 ng/ml (signal-to-noise ratio of 5). Calibration curves were linear up to 25 000 ng/ml. Intra-day and inter-day coefficients of variation of the assay were ≤5%. This method was used to analyse busulfan plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in children.