High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Simultaneous quantitation of buspirone and 1-(2-pyrimidinyl)piperazine in human plasma and urine by capillary gas chromatography-mass spectrometry

Buspirone and a buspirone metabolite, 1-(2-pyrimidinyl)piperazine (1-PP), are extracted from matrix using C18 extraction columns. The metabolite and its internal standard (d4-1-PP) are derivatized with pentafluorobenzoyl chloride to the corresponding amides. The 1-PP derivatives, buspirone and the buspirone internal standard (5-fluorobuspirone) are co-chromatographed. Chromatography and detection are performed using capillary gas chromatography with a fused-silica column and selected-ion monitoring-mass spectrometry. Linear range of the standard curves in plasma is 0.1-14 ng/ml for buspirone and 0.2-25 ng/ml for 1-PP with lower limits of quantitation of 0.1 and 0.2 ng/ml, respectively. In urine the linear range of the standard curves is 0.2-14 ng/ml for buspirone and 8-500 ng/ml for 1-PP with lower limits of quantitation of 0.2 and 8.0 ng/ml, respectively. Intra-assay accuracies were within 14% for buspirone and 1-PP in plasma and urine. Intra-assay precision was within 12% for both compounds in both matrices.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

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.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

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.     

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.     

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.     

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 cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Reversed-phase chromatography of fenflumizole, a new potential anti-inflammatory agent, and its application in pharmacokinetic studies in rat, dog and man

Two high-performance liquid chromatographic methods for quantitation of fenflumizole in biological fluids are described. Fenflumizole is a new agent with demonstrable anti-inflammatory properties in laboratory rodents. The major difference between the two methods lies in the utilization of fluorescence detection of the column eluate in assay II as compared to the less selective ultraviolet-visible detection used in assay I. Assay II appeared suitable for monitoring metabolites in addition to the parent compound. The quantitation limit of plasma fenflumizole was below 10 ng/ml for assay I and approximately 100 pg/ml for assay II. The reproducibility within a plasma concentration range of 50-150 ng/ml was below 7% (standard deviation) in both assays. Fenflumizole was found to be subject to photodecomposition. The impact of this on the analytical performance was evaluated and precautionary measures were assessed. The reliability of the analytical methods is examined and their applicability in in vivo studies conducted in rat, dog, and man.     

Analysis of 5-fluorouracil in plasma and urine by high-performance liquid chromatography.

A relatively simple, sensitive and rapid high-performance liquid chromatographic method is described for measuring the anticancer drug 5-fluorouracil (5-FU) in human plasma and urine. The procedure includes liquid-liquid extraction using ethyl acetate-methanol (95:5) and preparative column chromatography to separate 5-FU from constituents normally occurring in these biological samples. The columns contained a specially modified form of diatomaceous earth, which requires no pre-conditioning washes. Reversed-phase high-performance liquid chromatography was performed on a C18 column (70 mm x 4.6 mm I.D.) with a mobile phase of water-methanol (95:5) and ultraviolet detection (268 nm). The overall recovery from plasma and urine was 91 and 94%, respectively, at the concentration of 50 ng/ml. The determination limit of the assay for 5-FU was 10 ng/ml of plasma and urine. Concentrations of 5-FU between 10 and 500 ng/ml were measured in plasma and urine with a relative standard deviation of 6.8%. In order to evaluate the procedure, plasma and urine samples from three patients treated with 5-FU by continuous intravenous perfusion, were investigated.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. III. Determination of the arotinoid sumarotene and its Z-isomer in human and animal plasma.

A fully automated and sensitive high-performance liquid chromatographic method, using on-line solid-phase extraction, automated column switching and ultraviolet detection, was developed for the third-generation retinoid (arotinoid) sumarotene (methyl p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl]phe nyl sulphone; Ro 14-9706) and its Z-isomer. Nearly quantitative recoveries for human, rat and dog plasma were obtained by addition of acetonitrile (final content ca. 17%) to the plasma sample prior to injection. No isomerization was observed when the samples were stored in the autosampler for more than 20 h. The injection volume was 0.5 ml, resulting in quantification limits of 1 ng/ml for sumarotene and 2 ng/ml for the Z-isomer. More than 40 injections could be made on to one precolumn, allowing routine overnight injections. Using a 1-ml injection volume, the limit of quantification for sumarotene could be improved to 0.5 ng/ml. The method was applied to toxicokinetic studies in rats and dogs, and was used to monitor human plasma samples after repeated topical application. The method could also be adapted to etarotene (Ro 15-1570), which was used as an internal standard, and which is at present in clinical development.     

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.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) and chromatographed on a column packed with Spherosil XOA 600 (5 micrometers) using a 7:3 (v/v) mixture of diisopropyl either--isooctane (1:1, v/v + 0.2% triethylamine and diisopropyl ether--methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. less than 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography.

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6 ,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid-liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate-acetonitrile-methanol-nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C18 reversed-phase column. The standard curves were linear in the range 50-5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2-14%. In the range 50-5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

Determination of nicorandil in plasma using high-performance liquid chromatography with photoconductivity and ultraviolet detection. Application to pre-clinical pharmacokinetics in beagle dogs

A sensitive and precise method for the determination of nicorandil, a new anti-anginal medication, is reported. The method involves solid-phase extraction of the drug and internal standard using Bond-Elut C18 extraction columns, reversed-phase high-performance liquid chromatography on a Zorbax-Phenyl column and detection with photoconductivity and ultraviolet detection in series. Photoconductivity, performed with the Tracor 965 photoconductivity detector, provided a limit of detection of 2 ng/ml in plasma (between-day coefficient of variation of 15%) but the linear range of response was limited to only about 100 ng/ml. Ultraviolet detection in series with the photoconductivity detector extended the linear range of the analytical system to 1000 ng/ml (coefficient of variation 4.4%). The utility of the method is demonstrated in a dog pharmacokinetic study in which a 5-mg intravenous dose was compared to a 10-mg oral solution dose in six beagle dogs. The oral solution was absorbed rapidly, achieving an average maximum concentration of 857 ng/ml in 11.2 min. The absolute bioavailability of nicorandil in dogs in this study was determined to be 84.2%.