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.     

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.     

Gas chromatographic determination of ifosfamide in microvolumes of urine and plasma.

In oncology, particularly in pediatric malignancies, high doses (5-10 g/m2) of the oxazaphosphorine ifosfamide play an important role in the treatment of sarcomas. Pharmacokinetic data of ifosfamide and its metabolites in these cases are scanty. Considering the special demands of the determination of ifosfamide in plasma of young children, a very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma, has been developed. This bioanalysis of ifosfamide shows good linearity and accuracy in the concentration range 10 ng to 100 micrograms per ml of plasma and 25 ng to 1 mg per ml of urine. The absolute limits of detection in plasma and urine are 2 ng/ml and 5 ng/ml, respectively. The stability of various solutions of ifosfamide and trofosfamide was tested and proved to be satisfactory, except for ifosfamide in plasma and urine kept in the refrigerator. The validity of the method for pharmacokinetic purposes is shown in the case of one patient.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

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.     

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.     

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.     

Quantitation of pancuronium, 3-desacetylpancuronium, vecuronium, 3-desacetylvecuronium, pipecuronium and 3-desacetylpipecuronium in biological fluids by capillary gas chromatography using nitrogen-sensitive detection

A sensitive and specific capillary gas chromatographic (GC) assay was developed for the quantitation of the quaternary ammonium steroidal neuromuscular blocking drugs pancuronium (PANC), vecuronium (VEC) and pipecuronium (PIP), as well as the metabolites 3-desacetylpancuronium (3-desPANC) and 3-desacetylvecuronium (3-des VEC) in plasma, bile and urine; the putative metabolite 3-desacetylpipecuronium (3-des PIP) was extracted and quantitated only in urine. The procedure employed a single dichloromethane extraction of the iodide ion-pairs of the monoquaternary or bisquaternary ammonium compounds (including internal and external standards) from acidified, ether-washed biological fluid followed by the formation of stable O-tert.-butyldimethylsilyl derivatives at the 3-hydroxy steroidal position of the metabolites. An automated capillary GC system fitted with a nitrogen-sensitive detector and an integrator was then used to analyze and quantitate both parent compounds and their derivatized metabolites. Optimal extraction, derivatization and GC conditions, as well as short-term stability and recoveries of these drugs and metabolites in plasma, are reported. Electron ionization mass spectrometry combined with GC was used to confirm the identities of compounds eluted from the column. The assay demonstrated a 10(3)-fold linear range up to 5000 ng/ml for PANC, VEC, 3-des VEC and PIP, and lower limits of detection with adequate precision of 2 ng/ml for PANC, VEC and PIP, and 4 ng/ml for 3-des VEC; 3-des PANC was linear from 8 to 500 ng/ml while 3-des PIP was linear from 25 to 1000 ng/ml. The precision (coefficient of variation) of the calibration curves for underivatized drugs and their derivatized metabolites over the linear ranges was 2-20% and the reproducibility of the assay over a range of clinical concentrations of these drugs found in human plasma was 5-16% for PANC, 2-4% for VEC and 6-11% for PIP. No interferences were detected in the assay of plasma samples from 106 surgical patients.     

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

Sensitive methods were developed for the analysis of dextromethorphan (I) and two metabolites, (+)-17-methyl-morphinan-3-ol (II) and (+)-morphinan-3-ol (III), in plasma as well as dextromethorphan and three metabolites II, III and (+)-3-methoxymorphinan (IV) in urine using high-performance liquid chromatography followed by detection with a fluorometer. Dextromethorphan and its metabolites were extracted from plasma and urine and separated in the reversed-phase mode. The practical lower limits of determination for I, II, and III in plasma were 0.5, 5, and 5 ng/ml, respectively; for I, II, III, and IV in urine, the limits were 20 ng/ml, 0.6 microgram/ml, 0.5 microgram/ml, and 15 ng/ml, respectively. The linearity of the calibration graphs was excellent (r varied from 0.9994 to 0.9999) over concentration ranges of two orders of magnitude.     

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.     

Quantification of amitriptyline, nortriptyline, and 10-hydroxy metabolite isomers in plasma by capillary gas chromatography with nitrogen-sensitive detection

A selective, sensitive method for the determination of amitriptyline and its metabolites is described. This method involves liquid-liquid extraction and capillary gas chromatography with nitrogen-sensitive detection. The detection limits of amitriptyline, nortriptyline, 10-hydroxy(E)amitriptyline, 10-hydroxy(E)nortriptyline, and 10-hydroxy(Z)nortriptyline were slightly less than 0.5 ng/ml in 1.0-ml plasma samples. The coefficients of variation for within-run and between-run analyses of samples containing 100 ng/ml were less than 12% and 9%, respectively. The method offers rapid analysis of individual isomers, increased sensitivity over high-performance liquid chromatographic methodology and the conveniences of the gas chromatographic technique.     

Determination of a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite in plasma and urine by gas chromatography-mass spectrometry using negative ion chemical ionization

A sensitive and specific gas chromatographic-mass spectrometric method for the simultaneous determination of angiotensin-converting enzyme inhibitor (I, CS-622) and its active desethyl metabolite (II, RS-5139) in plasma and urine was developed. Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative ion chemical ionization. Extraction from plasma and urine was carried out using Sep-Pak C18 and silica cartridges. The extract of plasma or urine was treated with diazomethane followed by trifluoroacetic anhydride to convert I and II into their methyl ester trifluoroacetyl derivatives. The detection limit of I and II was 0.5 ng/ml in plasma and 5 ng/ml in urine. The proposed method was satisfactory for the determination of I and II in plasma and urine with respect to accuracy and precision. Thus it is suitable for measurement of bioavailability and pharmacokinetics of I and II in body fluids.     

Simultaneous determination of the sulphonylurea glimepiride and its metabolites in human serum and urine by high-performance liquid chromatography after pre-column derivatization

A sensitive and selective high-performance liquid chromatographic method has been developed for a new sulphonylurea, glimepiride, and its metabolites. The assay involves extraction with diethyl ether, thermolysis of the sulphonylureas at 100 degrees C and trapping of the resulting amines with 2,4-dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. Analogous compounds were used as internal standards. The detection limit was 5 ng/ml for glimepiride and metabolite II and 10 ng/ml for metabolite I using 1 ml of serum. The method has been applied to the analysis of serum and urine samples from pharmacokinetic studies in humans.     

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

A selective and sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma or urine has been developed. With glibornuride as internal standard, acid-buffered plasma or urine was extracted with benzene. The organic layer was evaporated and the residue was dissolved in equilibrated mobile phase (acetonitrile-phosphate buffer 0.01 M pH 3.5, 50:50). An aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column, and quantitation was achieved by monitoring the ultraviolet absorbance at 225 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents could be noted. The utility of the method was demonstrated by analysing glibenclamide in samples from diabetic subjects on therapeutic doses of the drug.     

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

A sensitive and selective high-performance liquid chromatographic method for determination of intact glipizide in human plasma or urine has been developed. The plasma and urine samples were acid-buffered, before tolbutamide was added as internal standard. The samples were extracted with benzene, and the organic layer was evaporated to dryness. The residue was dissolved in equilibrated mobile phase (acetonitrile-0.01 M phosphate buffer pH 3.5, 35:65), and an aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column. Quantitation was achieved by monitoring the ultraviolet absorbance at 275 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents were observed. The utility of the assay was demonstrated by determining glipizide in samples from a diabetic subject receiving a therapeutic dose of 5 mg of the drug.     

Highly sensitive method for the determination of 5-fluorouracil in biological samples in the presence of 2'-deoxy-5-fluorouridine by gas chromatography-mass spectrometry

A highly sensitive and convenient gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of 5-fluorouracil in the presence of 2'-deoxy-5-fluorouridine (which breaks down into 5-fluorouracil during ordinary GC derivatization) in biological samples such as plasma and urine. After extraction with ethyl acetate, 5-fluorouracil and 5-chlorouracil, the latter being used as an internal standard, were converted into their tert.-butyldimethylsilyl derivatives by allowing the mixture to stand for 30 min at room temperature and were assayed by electron-impact ionization GC-MS. Under these conditions, 2'-deoxy-5-fluorouridine did not decompose or interfere with the determination of 5-fluorouracil. The assay method, including the extraction and tert.-butyldimethylsilyl derivatization of 5-fluorouracil, showed good linearity in the range 0-100 ng/ml for 5-fluorouracil in plasma (detection limit 0.5 ng/ml) and urine (detection limit 1 ng/ml). The usefulness of this method was demonstrated by determining plasma concentrations of 5-fluorouracil in rats treated intravenously with 5-fluorouracil and 2'-deoxy-5-fluorouridine.     

Assay of isradipine and of its major metabolites in biological fluids by capillary gas chromatography and chemical ionization mass spectrometry

A method is described for the determination of isradipine, a dihydropyridine calcium antagonist, and five of its metabolites in plasma and urine. The neutral compounds were extracted in toluene and analysed in a wide-bore silica capillary column. The acidic compounds were extracted in two steps, then esterified with diazomethane and assayed separately using the same column. Detection was performed by negative-ion mass spectrometry with chemical ionization. The limit of detection of isradipine was 0.04 ng/ml when the compound was determined alone and 0.7 ng/ml when its oxidized metabolite was determined simultaneously. The limits of detection of the metabolites in plasma ranged from 0.15 to 2 ng/ml. The method was successfully used in conventional pharmacokinetic studies and in a multicentre study of population pharmacokinetics.     

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.     

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.