Determination of denaverine and its metabolites in urine samples by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed for the determination of the neurotropic-musculotropic spasmolytic agent denaverine and five of its metabolites in urine. In a first step beta-glucuronidase was used to cleave glucuronides in the human urine. After that samples containing denaverine and its phase I metabolites were extracted and cleaned up using an automated solid phase extraction method. An external calibration was used. The analytes were measured employing the multiple reaction-monitoring mode (MRM). The linear dynamic range for denaverine and its five metabolites determination was demonstrated from lower limit of quantification (8.0 ng/ml) to at least 500 ng/ml. The presented method is suitable for pharmacokinetic or toxicokinetic studies. With the help of reference substances some additional potential metabolites could be excluded in the urine samples. To look for additional unknown metabolites the LC-MS-MS system operated on one hand in the precursor ion mode using typical product ions of denaverine and of its metabolites and on the other hand in the product ion mode using postulated protonated molecules [M+H](+). With the help of the chromatographic behaviour and typical fragment ions of the unknown metabolites it was possible to elucidate their structures. Nine until now unknown metabolites were found in the urine samples. However, without reference substances a quantification of these analytes was not possible.     

High-performance liquid chromatography and preliminary pharmacokinetics of rufloxacin and its metabolites, N-desmethylrufloxacin and rufloxacinsulfoxide, in urine of rhesus monkey Macaca mulatta.

A gradient high-performance liquid chromatographic method for the quantification of rufloxacin and two of its metabolites in urine, N-desmethylrufloxacin and rufloxacinsulfoxide, has been developed and validated. Monkey urine samples were diluted ten times with distilled water and 20 microliters were injected onto a Cp Spher 5-ODS column, 5 microns particle size. The mobile phase was a mixture of 4% acetonitrile and 96% buffer at time 0, which changed linearly over 37 min to 26% acetonitrile and 74% buffer. Detection was achieved at 246 nm. The limit of detection of the three compounds was 0.50 microgram/ml. An example of a pharmacokinetic study of rufloxacin and its metabolites in monkeys is shown.     

Determination of propiverine and its metabolites in rat samples by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of the anticholinergic and antimuscarinc drug propiverine and eight of its metabolites in serum, urine, faeces and different tissue samples of rats. Samples containing propiverine and its metabolites in serum and urine and in the supernatants of faeces and tissue homogenates were extracted and cleaned up using an automated solid phase extraction (SPE) method. An external calibration was used. The analytes were measured employing the multiple reaction monitoring mode (MRM). A sufficient response over the range of 10-1000 ng/ml was demonstrated. The lower limit of quantification of the nine substances was 10 ng/ml. The presented method is suitable for pharmacokinetic or toxicokinetic studies. To look for additional unknown metabolites, the LC-MS-MS system operated in the precursor ion mode using typical product ions of propiverine and of its metabolites. With the help of the chromatographic behaviour and typical fragment ions of the unknown metabolites, it was possible to elucidate their structure. Five until now unknown metabolites were found in the urine and faeces samples. However, without reference substances, a quantification of these analytes was not possible.     

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract

A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg^–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSⁿ spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.     

Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system

Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid), a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibrozil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.     

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.     

Determination of some benzodiazepines and metabolites in serum, urine and saliva by high-performance liquid chromatography

The performance of a number of liquid--solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated. A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent. The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).     

High-performance liquid chromatographic analysis of tiaprofenic acid and its metabolites in plasma and urine by direct injection

A rapid, convenient, sensitive and selective reversed-phase high-performance liquid chromatographic method was developed to measure tiaprofenic acid, its reduced and oxidized metabolites and their conjugates in biological fluids. The method involved direct injections of plasma and urine samples into the chromatograph before and after alkaline hydrolysis of the conjugates. Concentrations as low as 0.5 micrograms/ml of the drug in plasma and urine were quantifiable. The method was suitable for analysis of tiaprofenic acid and its metabolites in biological fluids after administration of therapeutic doses. Several other non-steroidal anti-inflammatory drugs which were applied to the system did not interfere with the assay.     

HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.     

Determination of clobazam, N-desmethylclobazam and their hydroxy metabolites in plasma and urine by high-performance liquid chromatography

Owing to the pharmacological and clinical importance of the determination of plasma and urine levels of the hydroxy metabolites of clobazam and N-desmethylclobazam in healthy volunteers and in epileptic patients, a high-performance liquid chromatographic (HPLC) method was developed that permits the determination of all these compounds in the same plasma or urine sample. The method involved ether extraction at pH 13 with diazepam as internal standard for the measurement of clobazam and N-desmethylcobazam, followed by ether extraction at pH 9 with nitrazepam as internal standard for the measurement of the hydroxy derivatives. The limit of detection was about 10-20 ng/ml for each of these compounds. Applications to patients were limited by chromatographic interferences between the hydroxy metabolites and some medications currently associated with clobazam in the treatment of epilepsy. The only interference in clobazam and N-desmethylclobazam analysis was from N-desmethyldiazepam. Despite these inconveniences, this HPLC procedure appears to be the only available method for the simultaneous quantification of clobazam and its three main metabolites.     

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies.

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.     

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.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

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.     

Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

High-performance liquid chromatographic determination of ibuprofen, its metabolites and enantiomers in biological fluids

An isocratic high-performance liquid chromatographic method to determine racemic ibuprofen (assay I) and its major metabolites (assay II) in biological fluids (plasma, urine, bile) using a conventional reversed-phase column is described. A third assay using beta-cyclodextrin as stationary phase (Cyclobond I) for the separation of the ibuprofen enantiomers is also described. A wavelength of 220 nm was used to monitor the substances. The sensitivity of the method was 0.1 microgram/ml for all three assays. The method was demonstrated to be suitable for stereoselective pharmacokinetic studies of ibuprofen in humans and animals.     

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.     

Determination of piroxicam and its major metabolites in the plasma, urine and bile of humans by high-performance liquid chromatography.

A simple and sensitive liquid chromatographic method with ultraviolet detection is described for the determination of the nonsteroidal anti-inflammatory drug piroxicam and its major metabolites in human plasma, urine and bile. Separation of these components occurs on a reversed phase C10CN column with a mobile phase consisting of acetonitrile-water-sodium dihydrogenphosphate solution. The detection limit of the assay was 50 ng/ml with intra- and inter-assay coefficients of variation for piroxicam of the order of 2 and 5%, respectively. The assay linearity was good (typically r = 0.9999). This method can be readily utilised for clinical pharmacokinetic and mass-balance studies.     

Topical administration of ibuprofen in man. Simultaneous determination of the drug and its metabolites in urine by high resolution gas chromatography

A selective and sensitive high resolution gas chromatography assay for simultaneous determination of Ibuprofen and its major metabolites in urine is described. Biological samples were collected from healthy volunteers after a single topical administration of the drug in gel form. The chromatographic system, developed on a WCOT OV-1 glass capillary column, ensured a clear separation of Ibuprofen and its metabolites and their quantitative evaluation.