Gas chromatographic method for determination of a piperazine derivative (Trelibet) and its metabolites in human plasma and urine

A sensitive gas chromatographic method was developed for the determination of Trelibet, 1-benzyl-4-(2'-pyridinecarbonyl)piperazine, and of its major metabolites in biological fluids. The compounds were extracted as bases into dichloromethane, and the extracts were analysed by a dimethylsilicone capillary column with a nitrogen-phosphorus flame-ionization detector. The lower limit of detection was 1 ng/ml for Trelibet and 5 ng/ml for the metabolites. Peak-area ratios of the compounds and internal standard were linearly correlated to their plasma concentrations between 1 and 1000 ng/ml. The method was used for quantification of Trelibet and two of its metabolites in depressed patients after oral administration of a single dose of 200 mg of Trelibet. Concentration data measured in plasma and urine showed that the method is sensitive enough to monitor concentrations both for pharmacokinetic studies and for plasma steady-state levels daily.     

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.     

Determination of lansoprazole and its metabolites in plasma by high-performance liquid chromatography using a loop column.

A high-performance liquid chromatographic method for the simultaneous determination of lansoprazole, a new proton pump inhibitor, and its metabolites in human plasma is described. Lansoprazole, its metabolites and an internal standard were extracted with tert.-butyl methyl ether. Samples were injected using an automatic injector via a loop column, and separation was obtained using a reversed-phase column under isocratic conditions. The absorbance was monitored at 285 and 303 nm. The quantification limit was 2 ng/ml for lansoprazole and 3 or 5 ng/ml for the metabolites. No endogenous compounds were found to interfere. The mean overall recovery was between 75 and 95% for lansoprazole and its metabolites. This method is suitable for pharmacokinetic studies.     

Assay of nicergoline and three metabolites in human plasma and urine by high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry.

A method for the simultaneous determination of nicergoline and three of its metabolites in human plasma and urine has been developed using high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry. Nicergoline and its metabolites were extracted from the plasma and urine samples with chloroform and separated on a reversed-phase ODS column. The eluents were led to the atmospheric pressure ionization interface and then analysed in the selected-ion monitoring mode. The detection limits of nicergoline and three of its metabolites were ca. 2 ng/ml in plasma and ca. 10 ng/ml in urine, at a signal-to-noise ratio of 4.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography-mass spectrometry.

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane-dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography-electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a single-to-noise ratio of greater than 3 and greater than 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Determination of oxprenolol in human plasma by high-performance liquid chromatography, in comparison with gas chromatography and gas chromatography-mass spectrometry

A high-performance liquid chromatographic method for the quantitative assay of oxprenolol in human plasma is described. After addition of alprenolol as internal standard, the compounds are extracted from plasma at alkaline pH into an organic phase and back-extracted into an acidic aqueous phase. Separation of the plasma components and metabolites was achieved on a reversed-phase column. Concentrations down to 66 nmol/l (20 ng/ml) can be determined with UV detection at 222 nm. This technique compares favourably with gas chromatographic and gas chromatographic-mass spectrometric methods.     

High-performance liquid chromatographic evaluation of 2-(alpha-thenoylthio)propionylglycine and its two metabolites in biological fluids

A high-performance liquid chromatographic (HPLC) method for determining 2-(alpha-thenoylthio)propionylglycine (TTPG) and its two main metabolites, thiophenecarboxylic acid and thiopronine, in biological samples was developed. TTPG and its metabolites were extracted by solvent partition and then determined by reversed-phase HPLC with UV detection at 245, 295 and 360 nm. This procedure was validated in order to allow the assay of these compounds in plasma and urine samples with sufficiently low detection limits (50 ng/ml for TTPG and TCA and 100 ng/ml for thiopronine) and with good linearity within the concentration range investigated. It was applied to a comprehensive pharmacokinetic investigation of TTPG in healthy volunteers.     

Plasma quantification of quazepam and its 2-oxo and N-desmethyl metabolites by capillary gas chromatography

The authors have developed a gas chromatographic method for the simultaneous quantification of quazepam in plasma and its two main metabolites, 2-oxoquazepam and N-desmethylquazepam. This method involves an extraction from plasma using butyl acetate, and an analysis by electron-capture detection on a CP-Sil 5 WSCOT capillary column. Intra- and inter-day precision and accuracy were better than 10% for each of these three compounds, even near their detection limit estimated at 0.2 ng/ml. Linearity proved satisfactory between 0.2 and 60-70 ng/ml. For endogenous plasma components, adequate specificity was achieved. Despite some inconveniences, a long analysis time, a progressive saturation of the column owing to a low oven temperature, and a relatively short life-span of the CP-Sil 5 columns, this method was the only one available in the literature for the quantification of quazepam and its metabolites from the same plasma sample. It was successfully applied to phase I studies in healthy volunteers.     

Simultaneous determination of nitroglycerin and its dinitrate metabolites by capillary gas chromatography with electron-capture detection

A sensitive gas chromatographic-electron-capture detection method for the simultaneous determination of the antianginal drug nitroglycerin (GTN) and its dinitrate metabolites (1,2-GDN and 1,3-GDN) was developed. Human plasma samples (1 ml) spiked with 2,6-dinitrotoluene as the internal standard were extracted once with 10 ml of a methylene chloride-pentane mixture (3:7, v/v). Using this solvent system, less contaminants are extracted into the organic phase from plasma, resulting in cleaner chromatograms and prolonged column life. A break point was observed on the standard curves of GTN and GDNs. The two linear regions for the detectable concentrations of GTN are 0.025-0.3 and 0.3-3 ng/ml and for 1,2-GDN and 1,3-GDN they are 0.1-1 and 1-10 ng/ml. The limits of detection by this method for GTN, 1,2-GDN and 1,3-GDN in plasma are 0.025, 0.1 and 0.1 ng/ml, respectively.     

Determination of sulfinpyrazone and two of its metabolites in human plasma and urine by gas chromatography and selective detection

A selective and sensitive gas chromatographic method for simultaneous determination of sulfinpyrazone and two of its metabolites (the para-hydroxylated metabolite and the sulfone metabolite) in biological fluids using alkali flame ionization detection (AFID), electron capture detection (ECD) and mass fragmentographic detection is described. The compounds are extracted from the samples, methylated and separated on 2% OV-17 or 3% OV-225 columns. Phenylbutazone is used as internal standard. Standard curves are linear. The coefficient of variation at 10 microgram/ml of sulfinpyrazone in plasma was shown to be 1.8% (AFID), and the detection limits were 0.1 microgram/ml (AFID) and 10 ng/ml (ECD). Mass spectra of the methylated compounds are shown and serum concentration curves after oral administration of 100 mg sulfinpyrazone to two persons are determined together with the excreted amounts of drug and metabolites.     

Determination of nitrazepam and its main metabolites in urine by thin-layer chromatography and direct densitometry

A method for the direct quantitative densitometry of nitrazepam and its main metabolites (7-aminonitrazepam, 7-acetamidonitrazepam and 2-amino-5-nitrobenzophenone) in urine was developed. The unchanged drug and its metabolites were extracted with benzene-dichloromethane (4:1), subjected to thin-layer chromatography, and determined by direct ultraviolet densitometry. Recovery experiments showed that the method was quantitative. The limit of detection was 5 ng/ml for 2-amino-5-nitrobenzophenone and 10 ng/ml for other compounds. The method was applied to the determination of nitrazepam and its metabolites excreted in human urine after administration of 10 mg of the drug.     

Simultaneous determination of zopiclone and its two major metabolites (N-oxide and N-desmethyl) in human biological fluids by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of zopiclone and its main metabolites (N-oxide and N-desmethyl derivatives) in biological fluids. After selective extraction (dichloromethane-2-propanol) these compounds are chromatographed on a column packed with Spherisorb ODS-2 (5 micron) using monobasic sodium phosphate-methanol (45:55, v/v). The eluted compounds are measured by fluorescence detection. The limit of detection of the method is 5 ng/ml for zopiclone in plasma and urine and 10 ng/ml for its two main metabolites (coefficient of variation less than 10%). This method has been successfully applied to pharmacokinetic studies of zopiclone and its two main metabolites in healthy subjects and patients with chronic renal failure.     

A novel procedure for the determination of tricyclic antidepressants in plasma by use of high performance liquid chromatography.

A novel method for the determination of the antidepressant 3-(1-chloro-5-H-dibenzo [a,d] cycloheptene-5-ylidene)-N,N-dimethylpropylamine-N-oxide hydrochloride and its metabolites by use of high performance liquid chromatography was developed. The procedure is applicable to the assay of other similar drugs in biological samples. The method involves extraction of the unchanged drug and its metabolites from plasma, back-extraction into diluted phosphoric acid and re-extraction into an organic phase. Separation is performed on a silica gel column with an acidic mobile phase, containing sodium dodecyl sulfate as ion-pairing agent. The quantitation is carried out by UV detection. The procedure allows the determination of plasma levels down to about 5 ng/ml of the unchanged drug and its metabolites, respectively, when 1 ml of plasma is used. The plasma levels of two volunteers were determined after a single oral dose of the drug.     

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.     

Determination of etretinate and its main metabolite in human plasma using normal-phase high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of astemizole and its primary metabolite in plasma and animal tissues. Both compounds and the internal standard were extracted from alkalinized plasma with heptane--isoamyl alcohol and analyzed using a reversed-phase column and UV monitoring at 254 nm. The detection limits for both compounds were 1 ng/ml of plasma and 5 ng/g of tissue and extraction recoveries were sufficiently high (71-84%). The method was applied to plasma and tissue samples from dogs after repeated oral administration, and to plasma samples from a volunteer taking a 300-mg oral dose of the drug. The results were compared with those obtained by a formerly developed radioimmunoassay.     

The Determination of a New Trifluorinated Quinolone,Fleroxacin, Its N-Demethyl,and N-Oxide Metabolites in Plasma and Urine by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method is described for the determination of the new fluoroquinolone Ro 23–6240 and its N-demethyl and N-oxide metabolites in plasma and urine. The three substances were extracted from aqueous solution with dichloromethane/isopropanol containing sodium dodecyl sulphate. After evaporation and reconstitution, samples were analysed on a reversed-phase column using ion pair chromatography and fluorescence detection. The limit of quantification was 10–20 ng/ml (RSD 4%) using a 0.5 ml plasma sample, and the inter assay precision was 3–10% over the concentration range 50 ng/ml to 20 μg/ml. Recovery from plasma was 81% (RSD 10%) over the range 10 ng/ml to 5 μg/ml. The method has been applied successfully to the analysis of several thousand samples from human pharmacokinetic studies. Care has to be taken to avoid exposure of samples to direct sunlight, and the use of opaque vessels for sample storage and handling is recommended.     

Determination of bromazepam in plasma and of its main metabolites in urine by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bromazepam in plasma and of its main metabolites in urine is described. The unchanged drug is extracted from plasma with dichloromethane, using Extrelut 1 extraction tubes. The residue from this extract is subsequently analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection (230 nm). The limit of detection is 6 ng/ml of plasma, using a 1-ml specimen. For the determination of the metabolites, the urine samples are incubated to effect enzymatic deconjugation and are then extracted with dichloromethane. Following two clean-up steps (back extractions), the final residue is analysed on the same reversed-phase system as the plasma samples. The limit of detection for the two metabolites is 200 ng/ml.     

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.     

Determination of three metabolites of a new angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine by gas chromatography-mass spectrometry using multiple ion detection.

A specific and sensitive gas chromatographic-mass spectrometric method for the determination of three metabolites of the angiotensin-converting enzyme inhibitor, imidapril, in plasma and urine was developed. The metabolites were isolated from plasma and urine using a Bond Elut C18 solid-phase extraction cartridge. The isolated metabolites were converted to sensitive derivatives by pentafluorobenzyl bromide and heptafluoro-n-butyric acid anhydride. Following derivatization, the sample solutions were analysed by wide-bore column gas chromatography-mass spectrometry with multiple ion detection. The detection limits of the three metabolites were each 1 ng/ml in plasma and 5 ng/ml in urine. Analysis of the spiked plasma and urine samples demonstrated the good accuracy and precision of the method. This method was very useful for use in pharmacokinetic and bioavailability studies of the three metabolites of imidapril in humans.     

Determination of pyrimethamine in human plasma after administration of fansidar of fansidar-mefloquine by means of high-performance liquid chromatography with fluorescence detection

A sensitive, rapid and selective high-performance liquid chromatographic (HPLC) method has been developed to measure plasma levels of pyrimethamine in human subjects dosed with the antimalarials Fansidar or Fansidar and mefloquine. The drug was extracted from plasma at basic pH with n-butyl chloride-dichloromethane (96:4, v/v) and quantified on a normal-phase HPLC column with fluorescence detection (excitation 290 nm, emission 345 nm). Pyrimethamine was almost quantitatively extracted from plasma in the concentration range 20-200 ng/ml. The sensitivity limit was about 10 ng/ml of plasma, using a 0.5-ml specimen. The method was shown to be specific with respect to the other two components in the antimalarial combinations, namely sulfadoxine and mefloquine, and their metabolites. The assay was applied to pharmacokinetic studies of pyrimethamine in man following the oral administration of Fansidar of Fansidar and mefloquine.