Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-beta-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon.     

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.     

Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.     

Determination of clozapine and its metabolites in serum and urine by reversed phase HPLC

A rapid, specific and sensitive method using reversed phase HPLC for the simultaneous determination of clozapine and its two metabolites in serum and urine has been developed. The mobile phase was a mixture of 67% (v/v) methanol in water containing 0.4% tetramethylethylenediamine and 0.32% acetic acid (pH 5.5). The influence of methanol content, the pH of the mobile phase and the effect of adding alkylammonium ions as peak tailing reducer in the mobile phase have been investigated. The solvent for extracting clozapine from serum and urine was ether. 50 microliters of 0.25 M H2SO4 solution was used to redissolve the dry residue to eliminate the endogenous compounds which could otherwise be eluted together with clozapine from the HPLC column. The analysis of a single sample was accomplished within half an hour. The identities of the chromatographic peaks of clozapine and its N-demethyl metabolite collected from the patient urine sample were confirmed by mass spectrometry. The method is sufficiently sensitive (5 ng/ml) and reproducible (CV 2.9%-6.7%) for clinical and pharmacokinetic studies, and preliminary results in these respects are presented.     

Detection and separation of mitoxantrone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method using ion-pair chromatography on reversed-phase C18 material was developed. After sample clean-up on XAD columns, mitoxantrone at concentrations below 1 ng/ml in serum and 0.2 ng/ml in urine were measurable with a coefficient of variation of less than 9.3% at a wavelength of 658 nm. Four metabolites were separated in urine. The two major metabolites co-chromatographed with the synthesized mono- and dicarboxylic acid derivatives of mitoxantrone. The method allowed the measurement of mitoxantrone and its metabolites in serum up to more than one week and in urine up to four weeks after administration of the drug.     

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.     

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 glibenclamide and its two major metabolites in human serum and urine by column liquid chromatography

A simple reversed-phase liquid chromatographic method for the measurement of low concentrations of glibenclamide (glyburide) and its two major metabolites, 4-trans- and 3-cis-hydroxyglibenclamide, in human serum and urine has been developed. The compounds were extracted with n-hexane-dichloromethane (1:1). The UV detection wavelength was 203 nm. The minimum detectable serum level of glibenclamide was 1 ng ml (2 nM), and the relative standard deviation was 8.9% (n = 9). When maximum sensitivity was desired the metabolites were chromatographed separately. Metabolites in urine were measured by the same method after five-fold sample dilution. The utility of the method was tested on a healthy volunteer who ingested 3.5 mg of glibenclamide. The parent drug was present in the serum for at least 18 h, and the metabolites in the urine for at least 24 h.     

High-performance liquid chromatography followed by radioimmunoassay for the determination of a luteinizing hormone-releasing hormone analogue, leuprorelin, and its metabolite

A sensitive method for the determination of leuprorelin (TAP-144), a luteinizing hormone-releasing hormone analogue, and its C-terminal metabolite, M-I, in serum and urine has been developed. Leuprorelin and M-I were extracted from serum or urine samples with Sep-Pak C18 cartridges, and separated completely by high-performance liquid chromatography and determined by radioimmunoassay using [125I]leuprorelin as the labelled antigen. The detection limit of the method was 0.05 ng/ml for leuprorelin and M-I, and the recovery of the compounds added to serum and urine was over 88% with a coefficient of variation (within-assay) of less than 5%. The method was applied to the determination of leuprorelin and M-I-like immunoreactivity in serum or urine after administration of once-a-month injectable microspheres of leuprorelin acetate (TAP-144-SR) to patients with prostate cancer.     

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.     

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.     

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.     

New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces

A new sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and validated for the simultaneous quantitation of vinorelbine, its main metabolite, 4-O-deacetylvinorelbine and two other minor metabolites, 20'-hydroxyvinorelbine and vinorelbine 6'-oxide. All these compounds, including vinblastine (used as internal standard) were deproteinised from blood, plasma and faeces (only diluted in urine), analysed on a cyano column and detected on a Micromass Quattro II system in the positive ion mode after ionisation, using an electrospray ion source. Under tandem mass spectrometry conditions, the specific product ions led one to accurately quantify vinorelbine and its metabolites in all biological fluids. In whole blood, linearity was assessed up to 200 ng/ml for vinorelbine and up to 50 ng/ml for the metabolites. The limit of quantitation was validated at 250 pg/ml for both vinorelbine and 4-O-deacetylvinorelbine. In the other biological media, the linearity was assessed within a same range and the limit of quantitation was adjusted according to the expected concentrations of each compound. This method was initially developed in order to identify the metabolite structures and to elucidate the metabolic pathway of vinorelbine. Thanks to its high sensitivity, this method has enabled the quantitation of vinorelbine and all its metabolites in whole blood over 168 h (i.e., 4-5 elimination half lives) whilst the previous liquid chromatographic methods allowed their measurement for a maximum of 48-72 h. Therefore, using this method has improved the reliability of the pharmacokinetic data analysis of vinorelbine.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Simultaneous determination of centbutindole and its hydroxy metabolite in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic assay has been developed and validated for the determination of centbutindole and its hydroxy metabolite in serum. The method involves extraction of serum samples with diethyl ether at pH greater than 8, back-extraction into 0.5 M hydrochloric acid and finally again with diethyl ether after addition of 2 M potassium hydroxide. Separation was accomplished by reversed-phase high-performance liquid chromatography on a cyano column with an acetonitrile-phosphate buffer system. The recovery of centbutindole and its metabolite was always greater than 80%. Calibration curves were linear over the concentration range 0.25-5 ng/ml for centbutindole and 0.05-1 ng/ml for the hydroxy metabolite. Although the lower limit of detection was 0.1 ng/ml for centbuntindole and 0.02 ng/ml for the hydroxy metabolite, the reliable limits of quantitation were 0.25 and 0.05 ng/ml, respectively, using 4 ml of serum.     

Capillary zone electrophoresis applied to the determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite in pharmaceuticals and urine

A capillary zone electrophoresis method has been developed for the quantitation of antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine. The separation of the compounds was performed in a fused-silica capillary filled with the running electrolyte, which consisted of a 60 mM borate buffer solution at pH 9.5. Under the optimized experimental conditions, the separation took less than 5 min. The analysis of urine samples required a previous solid-phase extraction step using C8 cartridges. The method was successfully applied to the determination of the drug and its metabolite in urine samples obtained from three hypertensive patients (detection limits of 115 ng ml(-1) for cilazaprilat and 125 ng ml(-1) for cilazapril) and to pharmaceutical dosage forms. The method was validated in terms of reproducibility, linearity and accuracy.     

Simultaneous determination of a new gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by high-performance liquid chromatography using automated column-switching.

A method based on high-performance liquid chromatography using column-switching is described for the simultaneous determination of HSR-803 and its metabolites in human serum and urine. The system uses a six-port valve with a Nucleosil CN pre-column for on-line sample clean-up, and direct injection of samples. The limits of quantitation in serum and urine were 5 and 20 ng/ml for HSR-803 and 50 and 200 ng/ml for the metabolites, respectively. The coefficients of variation for the intra- and inter-day accuracies were between 0.8 and 7.1% for each compound. This method was applied to the pharmacokinetic studies in humans after oral administration of HSR-803.     

Simultaneous Determinations of Cinobufagin and Its Metabolites by Reverse-Phase High Performance Liquid Chromatography in Rat Serum and Urine

Abstract

A high-performance liquid chromatography was developed for simultaneous determinations of Cinobufagin (CB) and its metabolites deacetylcinobufagin, 3-epideacetyl-cinobufagin, 3-ketodeacetylcinobufagin, 3-epicinobufagin and 3-ketodeacetylcinobufagin in serum and urine of rat. The biological samples were extracted with diethylether-ethylacetate (4:1v/v) in the presence of bufalin as the internal standard. Recoveries for CB and all other compounds were in the range of 80.8%–100.2%. Excellent resolution was obtained by reversed-phase chromatography on a 4.6 mm I.D.×150 mm ODS column using acetonitrile:water (50:50 v/v) as the mobile phase at a flow-rate of 0.7 ml/min with UV detector at 300 nm. Standard curve data revealed linearity over a range of 10–500 ng per ml serum and urine. The detection limits of CB and its metabolites were less than 10 ng/ml. Coefficients of variation for the analysis were less than 10. CB and its some metabolites were observed in     

Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.