Quantitation of flupirtine and its active acetylated metabolite by reversed-phase high-performance liquid chromatography using fluorometric detection

A simple, selective and sensitive procedure is described for the quantitation of flupirtine maleate (FLU) and its active acetylated metabolite (Met. 1) in plasma and urine. Using a 0.5-ml sample, a sensitivity of 10 ng/ml is easily achieved with a reversed-phase octadecylsilane (C18) column, and a high-performance liquid chromatographic system with fluorescence detection. Quantitation from plasma involves addition of an internal standard, protein precipitation with acetonitrile and a sample concentrating step, while for urinalysis the samples are taken through a single extraction with methylene chloride. Analytical recoveries of FLU and Met. 1 from plasma averaged greater than or equal to 95%, while from urine only 60 and 50%, respectively, could be recovered. The overall, inter- and intra-day variability for both FLU and Met. 1 averaged 6, 5 and 3%, in plasma, respectively. Standard calibration plots in plasma were linear (r greater than or equal to 0.99) for FLU (range: 0.01-10.0 micrograms/ml) and Met. 1 (range: 0.5-25 micrograms/ml) over the extended range. A slightly modified elution system was employed for quantitation of FLU and Met. 1 in urine.     

Determination of the new fluoroquinolone fleroxacin and its N-demethyl and N-oxide metabolites in plasma and urine by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of the new fluoroquinolone fleroxacin and its metabolites in plasma and urine. Plasma samples are deproteinized with acetonitrile, and, after evaporation and reconstitution of the supernatant, samples are analysed on a reversed-phase column. The limit of quantification is 10-20 ng/ml for the parent drug and 10 ng/ml for the metabolites, using a 0.2-ml sample. Urine samples are diluted with the mobile phase. An aliquot is then injected directly onto the column. The limits of quantification are 1 micrograms/ml for the parent drug and 0.5 micrograms/ml for the metabolites, using a 0.1-ml sample. The method has been successfully applied to pharmacokinetic studies of human volunteers and patients.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection

A liquid chromatographic method using a solid-phase extraction procedure for the quantification of sotalol in plasma and urine is described. Sotalol is eluted from an extraction column with ethyl acetate-acetonitrile (1:2) and, after separation by reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column, is quantified by fluorescence detection at excitation and emission wavelengths of 240 and 310 nm, respectively. The method has been demonstrated to be linear over the concentration ranges 10-6000 ng/ml in plasma and 0.5-100 micrograms/ml in urine. Mean inter-assay accuracy of the method for plasma ranged from 93 to 100% and for urine from 102 to 114%; precision ranged from 0.5 to 1.6% for plasma over a concentration range of 200-4000 ng/ml and for urine from 0.7 to 2.0% at concentrations of 2-50 micrograms/ml. Mass spectrometry confirmed the presence of sotalol in isolated chromatographic fractions of plasma and urine extracts from subjects given sotalol orally.     

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

Simultaneous quantitation of phenobarbitone and p-hydroxyphenobarbitone and their perdeuteroethyl analogues in biological fluids by combined gas chromatography-mass spectrometry

In order to develop 5-pentadeuteroethyl-5-phenyl barbituric acid as an alternative tracer in pharmacokinetic and metabolic studies of phenobarbitone, and to search for possible isotope effects associated with such labelling, we propose a gas chromatographic-mass spectrometric assay for simultaneous measurement of phenobarbitone, p-hydroxyphenobarbitone and their perdeuteroethyl analogues, using [1,3-15N2,2-13C] phenobarbitone as internal standard. These compounds were extracted from plasma (50 microliter) or urine (500 microliter) and pentylated according to Greeley's method. Linear calibration curves were obtained in the concentration range from 0.5 to 3 micrograms/ml. The interday precision, mean accuracy and detection limit were 0.77-5.28%, 99.99-100.80% and 0.03-0.05 microgram/ml, respectively. Results for plasma and urine concentrations, and pharmacokinetic parameters in humans, are presented to illustrate this method.     

Determination of amifloxacin and two of its principal metabolites in plasma and urine by high-performance liquid chromatography using automated column switching

The automated determination of amifloxacin and two of its principal metabolites in human plasma and urine by column-switching high-performance liquid chromatography is described. Plasma or urine samples, diluted 1:1 with 0.5 M sodium citrate buffer pH 2.5, were directly injected onto a cation-exchange pre-column. Following a 2.0-min wash of the pre-column with water at a flow-rate of 1.1 ml/min, the effluent from the pre-column was directed to the analytical column by a column-switching device. The precision of the plasma and urine methods ranged from a +/- 1.9 to +/- 3.6% for all compounds. The accuracies of the methods were within a range of -3.3% to 6.4% of the nominal values for all compounds. Linear responses were observed for all the standards in the range 0.10-5.0 micrograms/ml for plasma and 0.50-100 micrograms/ml for urine for all three compounds. The minimum quantifiable levels were 0.10 and 0.50 micrograms/ml for plasma and urine, respectively. The analytical methods may be used to quantify amifloxacin and the piperazinyl-N-desmethyl and piperazinyl-N-oxide metabolites in plasma and urine samples obtained from humans, monkeys, dogs and rats.     

Development of a gas chromatographic/mass spectrometric method to quantify R(-)-apomorphine, R(-)-apocodeine and R(-)-norapomorphine in human plasma and urine

A method was developed and validated for the analysis of R(-)-apomorphine, (R-)-apocodeine and R(-)-norapomorphine in human plasma and urine with N-propylnorapomorphine as internal standard using gas chromatography/mass spectrometry (GC/MS) and single-ion monitoring after a single liquid-liquid extraction and silylation of compounds. The quantification limits were 1 ng/ml for apomorphine and apocodeine and 25 ng/ml for norapomorphine. Calibration curves were linear, within the range 1-100 ng/ml. Variation in intraday and interday precision was below 10%. This method was applied to study apomorphine bioavailability in nine patients with Parkinson's disease before and after coadministration of a catechol-O-methyl transferase inhibitor.     

High-performance liquid chromatographic method for the quantitation of bupivacaine, 2,6-pipecoloxylidide and 4'-hydroxybupivacaine in plasma and urine.

A high-performance liquid chromatographic method with ultraviolet detection at 210 nm for quantitation of bupivacaine and two of its metabolites from plasma and urine is described. The compounds are extracted into n-hexane-isopropanol (5:1), evaporated and the reconstituted residue injected onto a reversed phase C18 column. Standard curves for all compounds were linear (r2 greater than 0.999) in the range 20-2000 ng/ml, with a limit of detection of 10 ng/ml. The inter-day coefficients of variation ranged between 2.7 and 12.2%. The method was applied to analyse bupivacaine and metabolite concentrations in patients on long-term epidural bupivacaine-fentanyl infusions.     

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-micron, C18 reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration--response curves for all four compounds are linear from 0.05 micrograms/ml to at least 10 micrograms/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.     

Headspace gas chromatography of stimulants in urine by in-column trifluoroacetyl derivatization method

The analysis of stimulants in urine using a headspace gas chromatography system equipped with an in-column sample trifluoroacetylation unit was investigated. A 5-ml aliquot of urine containing stimulants was pipetted into a 20-ml autosampler vial together with 3.5 g of potassium carbonate. The vial was sealed and heated for 20 min at 80 degrees C, then 0.8 ml of the headspace gas and N-methylbis(trifluoroacetamide) gas were injected simultaneously into the gas chromatograph equipped with a flame ionization detector and a fused-silica capillary column (DB-1, 30 m x 0.32 mm I.D., film thickness 0.25 micron), with a gas-tight syringe. Calibration graphs prepared by the absolute calibration curve method showed good linearity over the concentration range of 0.04 to 50 micrograms/ml for methamphetamine hydrochloride and amphetamine sulphate. The detection limits were 0.03 micrograms/ml for both of these compounds.     

Determination of a new calcium antagonist and its main metabolite in plasma by thermospray liquid chromatography-mass spectrometry.

A highly sensitive thermospray liquid chromatographic-mass spectrometric method has been developed for the simultaneous determination of FRC-8653 (I), a new calcium antagonist, and its main metabolite (M-4) in plasma. A deuterated analogue of I was added to the plasma as the internal standard. After the purification and concentration of the plasma sample on bonded-phase disposable columns, the extract was injected into the thermospray liquid chromatograph and analysed by selected-ion monitoring mass spectrometry. The calibration curves obtained were linear over the concentration range 0.5-100 ng/ml. The limits of quantification are 0.5 ng/ml for I and 1 ng/ml for M-4 in plasma, which are sufficient to evaluate plasma concentrations after oral administration to rats.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.     

Determination of the monocyclic beta-lactam antibiotic carumonam in plasma and urine by ion-pair and ion-suppression reversed-phase high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatography method has been developed for the determination of the new monocyclic beta-lactam antibiotic carumonam in plasma and urine. The method for plasma involves protein precipitation with acetonitrile and removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantification are achieved using a mobile phase based on either ion-suppression or ion-pair chromatography on a reversed-phase column with UV detection. The limit of determination is 0.5 micrograms/ml plasma, using a 0.5-ml specimen, and 25 micrograms/ml urine, using a 50-microliter specimen. The inter-assay reproducibility is generally better than 4% when an internal standard is used. Since beta-lactam antibiotics may degrade on storage, close attention must be paid to the stability of these drugs in biological fluids; novel measures to prevent degradation on storage are described. The assay has been successfully applied to the analysis of several thousand samples from pharmacokinetic studies, including a study involving patients with impaired renal function.     

Determination of a novel steroidal androgen receptor antagonist (Win 49596) in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection.

A rapid, sensitive and selective method was developed for the determination of a novel steroidal androgen receptor antagonist (Win 49596, I) in human plasma. The procedure involved extraction from plasma using a solid-phase phenyl support and elution directly onto a reversed-phase C8 column using a mobile phase consisting of 0.2 mol/l sodium acetate buffer at pH 7-acetonitrile (45:55, v/v). Drug was monitored by ultraviolet detection at a wavelength of 238 nm. Linear responses were observed for standards over the range 0.01-5.0 micrograms/ml. The minimum quantifiable level was 0.02 microgram/ml, using a 0.5-ml plasma sample. The precision was 5.5% and the accuracy ranged from -9.4% to 0.23%. The analytical method has been used to quantify I in plasma from dogs and rats and is projected for use with human plasma from clinical trials.     

Determination of p-methylthiobenzamide and p-methylthiobenzamide-S-oxide from rat plasma using solid-phase extraction and high-performance liquid chromatography

p-Methylthiobenzamide (PMTB) is a thiocarbonyl compound exhibiting marked hepatotoxicity and nephrotoxicity. We describe a high-performance liquid chromatographic method for analyzing PMTB and a metabolite, p-methylthiobenzamide-S-oxide (PMTBSO), from rat plasma using a solid-phase extraction technique. In this way, PMTB and PMTBSO can be extracted from 0.5 ml of plasma and separation achieved by an ODS analytical column in as little as 9 min. The mobile phase used was methanol-water (55:45, v/v) and the wavelength for detection was 290 nm. The limits of detection in plasma were 15 ng/ml for PMTB and 33 ng/ml for PMTBSO; the absolute recovery from spiked plasma samples was greater than 84.4% for both compounds and the internal standard. The method was linear throughout the range used with correlation coefficients greater than 0.969. The intra-day accuracy ranged from 1.52 to 15.23% relative error for the PMTB concentration range 151-3025 ng/ml; accuracy of 4.97% or less was obtained for PMTBSO concentrations of 1672-20,068 ng/ml. The intra-day precision (coefficient of variation) of the procedure was found to be no greater than 5.28% for PMTB and 7.9% for PMTBSO. Inter-day accuracy and precision measurements were similar.     

A Validated Ion-Pairing High Performance Liquid Chromatographic Method for the Determination of Enoxacin and its Metabolite Oxo-Enoxacin in Plasma and Urine

Abstract

A rapid, sensitive, and specific determination of enoxacin and its principal metabolite, oxo-enoxacin, in plasma and urine is described. the method, which employs the structurally related compound ciprof loxac in as internal standard, involves a protein precipitation step for plasma and solid-phase extraction for urine. Liquid chromatographic analysis is carried out on a C-18 bonded silica column; the mobile phase consists of 0.1 M citric-acid/acetonitrile employing ammonium perchlorate and tetrabutyl-ammonium hydroxide as ion-pairing agents. Quantitation is performed by UV-detection at 340 nm.

The analytical method was validated by examining the performance characteristics specificity, linearity, precision, accuracy, sensitivity, and recovery. Enoxacin calibration curves were linear between 0.02 and 3.2 μg/ml of plasma and from 0.5 to 125 μg/ml of urine. Limits of quantitation in plasma and urine were 0.01 and 0.5 μg/ml, respectively. For oxo-enoxacin, linear of calibration curves were obtained i n the range 0.05 to 1.6 μg/ml (plasma) and 1 to 50 μg/ml (urine); the respective quantitation limits were approximately 0.02 and 1 μg/ml.

The present assay procedure has been applied to monitoring plasma and urine concentrations in several pharmacokinetic studies in humans and different animal species.     

Detection of trace levels of thiodiglycol in blood, plasma and urine using gas chromatography-electron-capture negative-ion chemical ionisation mass spectrometry

A sensitive method has been developed for the detection and quantitative determination of thiodiglycol in blood, plasma and urine. Samples were extracted from Clin Elut columns and cleaned up on C18 Sep-Pak cartridges (blood, plasma) or Florisil Sep-Pak cartridges (urine). Tetradeuterothiodiglycol was added to the sample prior to extraction as internal standard. Thiodiglycol was converted to its bis-(pentafluorobenzoate) derivative and analysed by capillary gas chromatography-electron-capture negative-ion chemical ionisation mass spectrometry using selected ion monitoring. Levels of thiodiglycol down to 1 ng/ml (1 ppb) could be detected in 1-ml spiked blood and urine samples; calibration curves were linear over the range 5- or 10-100 ng/ml. Blood and urine samples from a number of control subjects were analysed for background levels of thiodiglycol. Concentrations up to 16 ng/ml were found in blood, but urine levels were below 1 ng/ml.     

Determination of the new morpholino anthracycline MX2.HCl and its metabolites in biological samples by high-performance liquid chromatography.

Methods for determining concentrations of a new morpholino anthracycline MX2.HCl and its metabolites in biological samples using reversed-phase high-performance liquid chromatography and fluorescence detection are described. The limits of detection were less than 1 ng/ml for all compounds after extraction from 0.5 ml of plasma using C18 Sep-Pak cartridges and consecutive solvent extraction. The recoveries from rat plasma ranged from 72.0 to 89.3%. The peak-height ratio of the fluorescence intensities of these compounds versus internal standard showed a linear correlation for concentrations up to at least 500 ng/ml in the plasma (correlation coefficient r greater than 0.999). The within-day and between-day precisions of this assay were in the range 0.8-8.7% (n = 5) and 2.0-3.5% (n = 5), respectively. The concentrations of these compounds in the blood and urine can be also determined by a slight modification of the extraction procedure.     

Determination of glyphosate and its metabolite, (aminomethyl)phosphonic acid, in serum using capillary electrophoresis.

Capillary electrophoresis has been used to separate and quantitate glyphosate and its major metabolite, (aminomethyl)phosphonic acid (AMPA), in serum. The two compounds, after derivatization with p-toluenesulphonyl chloride, were clearly separated with 0.1 M boric acid-sodium hydroxide buffer (pH 9.6) containing 10% methanol. The separation was completed within 15 min at an applied potential of 30 kV. Calibration curves for the assay were linear over both the lower (0.5-10 micrograms/ml) and the higher (10-100 micrograms/ml) concentration ranges. The within-run and day-to-day coefficients of variation of peak area were 1.4-4.4 and 4.4-8.5%, respectively, for glyphosate and 1.8-2.9 and 1.8-2.9%, respectively, for AMPA. The within-run and day-to-day precisions of the migration time for both compounds were less than 1.8% and less than 2.5%, respectively. The detection limit of both derivatives was 0.1 microgram/ml in spiked sera, and the recoveries of glyphosate and AMPA were 87.9-88.8 and 78.4-86.9%, respectively. In this study, the reproducibility and the effect of pH changes on the electropherograms were especially examined.