Quantitation of the anticonvulsant cinromide (3-bromo-n-ethylcinnamamide) and its major plasma metabolites by thin-layer chromatography

A quantitative thin-layer chromatography (TLC) procedure is described for the analysis of cinromide (3-bromo-N-ethylcinnamamide) and its two major metabolites, 3-bromocinnamamide and 3-bromocinnamic acid in plasma of the dog. These compounds were recovered from acidified plasma by extraction into benzene with a recovery of 95 ± 5%. All three compounds were quantitated directly on a TLC plate by ultraviolet absorbance densitometry at 270 nm. The linear dynamic range for the quantitation of the compounds on a TLC plate ranged between 10 and 1000 ng. The complete procedure is useful in the working range of 50 ng/ml to 100 μg/ml of plasma with a coefficient of variability of about 10%. Specificity of the method for parent drug and each of its plasma metabolites was confirmed by high-performance liquid chromatography. The method was used to determine the pharmacokinetics of cinromide and its two major plasma metabolites in dogs following a single oral dose of the drug.     

Liquid chromatographic/mass spectrometric procedure for measurement of NAD catabolites in human and rat plasma and urine

Monitoring level of the metabolites of the coenzyme NAD such as nicotinamide and its oxidized and methylated derivatives is important due to therapeutic applications of these compounds and monitoring of oxidative stress. We evaluated feasibility of using HPLC with electrospray ion-trap mass detection for single run separation and quantitation of all the NAD metabolites. We achieved good separation and retention of all the metabolites of interest using reversed-phase with ion-pairing. Single ion monitoring or tandem MS were used for detection and quantitation of the specific compounds with good linearity. The method was able to detect all the physiological metabolites in plasma samples of rats and humans or in urine. However, full validation is necessary before this method could be routinely applied.     

Liquid Chromatographic/Mass Spectrometric Procedure for Measurement of NAD Catabolites in Human and Rat Plasma and Urine

Monitoring level of the metabolites of the coenzyme NAD such as nicotinamide and its oxidized and methylated derivatives is important due to therapeutic applications of these compounds and monitoring of oxidative stress. We evaluated feasibility of using HPLC with electrospray ion-trap mass detection for single run separation and quantitation of all the NAD metabolites. We achieved good separation and retention of all the metabolites of interest using reversed-phase with ion-pairing. Single ion monitoring or tandem MS were used for detection and quantitation of the specific compounds with good linearity. The method was able to detect all the physiological metabolites in plasma samples of rats and humans or in urine. However, full validation is necessary before this method could be routinely applied.     

Elucidation of the structure of talinolol metabolites in man determination of talinolol and hydroxylated talinolol metabolites in urine and analysis of talinolol in serum

The objective of this study was to determine the structure of talinolol metabolites formed and the amounts excreted in urine. Talinolol metabolites in urine were identified by comparing their HPLC retention times and their GC—MS profile with those of previously characterized reference compounds. The metabolites were quantified by HPLC with a normal-phase silica column, a single chloroform extraction and UV detection. Less than 1% of an administered dose was found in urine as hydroxylated talinolol. Other metabolites could be excluded. A sensitive method to determine talinolol in serum and a simple method for analysis of talinolol in urine are described. These methods were found to be precise and accurate for the measurement of talinolol in samples obtained from patients during chronic talinolol treatment as well as from healthy volunteers after a single dose of talinolol.     

Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction-liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol-glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol-water (75:25, v/v) containing 0.025% NH(4)OH. Chromatography of glucuroconjugate metabolites and phenyl-beta-d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10-1500 ng mL(-1) for propofol and 1-QG, 20-3000 ng mL(-1) for PG and 25-3750 ng mL(-1) for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias < or =6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.     

Synthesis and analysis of selenomethionine metabolites

This paper describes the enzymatic synthesis of selenomethionine metabolites of the transmethylation and polyamine synthesis pathways: adenosylselenomethionine, adenosylselenohomocysteine, decarboxylated adenosylselenomethionine, and methylselenoadenosine. These compounds and the corresponding methionine metabolites were simultaneously separated by a single HPLC run. The sensitivity of the HPLC method is about 20 pmol per compound. The method may be used for direct analysis of the metabolite levels in tissues or cells treated with selenomethionine and it provides an assay method for the pulse-chase type of analysis of relative flows for both selenium- and sulfur-containing compounds in transmethylation and polyamine pathways.     

Simultaneous determination of ebastine and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry

We developed a method for determining ebastine, a new generation of antihistamines, and its three metabolites (hydroxyebastine, carebastine and desalkylebastine) in plasma simultaneously using LC/MS/MS. Four compounds and terfenadine, an internal standard, were extracted from plasma using a mixture of diethylether and dichloromethane in the presence of 1 M HCl. After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:5 mM ammonium acetate, 50:50, v/v) and injected onto a reversed-phase C(18) column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 470.7-->167.1, 486.7-->167.1, 500.6-->167.1, 268.4-->167.1 and 472.7-->436.0 for ebastine, hydroxyebastine, carebastine, desalkylebastine and terfenadine, respectively. The coefficient of variation of the assay precision was less than 12.5%, and the accuracy exceeded 88%. The limit of detection was 0.5 ng/ml for desalkylebastine; 0.2 ng/ml for ebastine, hydroxyebastine and carebastine, respectively. This method was used to measure the plasma concentration of ebastine and its three metabolites from healthy subjects after a single 20 mg oral dose of ebastine. This analytic method is a very simple, sensitive, and accurate to determine the pharmacokinetic profiles of ebastine including its metabolites.     

Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4'-hydroxy (OH), 3'-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4'-OH diclofenac and 3'-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9*3/*3 subject had the highest diclofenac/4'-OH ratios. However no difference was found between CYP2C9*2/*2 and *1/*1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.     

Metabolomics of a single vacuole reveals metabolic dynamism in an alga Chara australis

Metabolomics is the most reliable analytical method for understanding metabolic diversity in single organelles derived from single cells. Although metabolites such as phosphate compounds are believed to be localized in different organelles in a highly specific manner, the process of metabolite compartmentalization in the cell is not thoroughly understood. The analysis of metabolites in single organelles has consequently presented a significant challenge. In this study, we used a metabolomic method to elucidate the localization and dynamics of 125 known metabolites isolated from the vacuole and cytoplasm of a single cell of the alga Chara australis. The amount of metabolites in the vacuole and the cytoplasm fluctuated asynchronously under various stress conditions, suggesting that metabolites are spatially regulated within the cell. Metabolite transport across the vacuolar membrane can be directly detected using the microinjection technique, which may reveal a previously unknown function of the vacuole.     

Simultaneous quantitation of chlormethiazole and two of its metabolites in blood and plasma by gas—liquid chromatography

A simple and rapid gas chromatographic method for the quantitation of chlormethiazole and two of its pharmacologically active metabolites, 5-acetyl-4-methylthiazole and 5-(1-hydroxyethyl)-4-methylthiazole, in plasma and blood is described. The total analysis time is less than 20 min for a single sample. The method requires 50–500 μl of plasma or 1 ml of blood. The compounds are detected with a nitrogen—phosphorus detector. An internal standard technique is used for the quantitation. Calibration data are linear over the range 32–2376 ng of chlormethiazole and a similar range of the metabolites in plasma. The method may be used for pharmacokinetic studies.     

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-β-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2μg/ml. The calibration curves are linear from 0.4 to 16 μg/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection

A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidiized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The metthod was applied to the determination of AJ and the metabolites in rat plasma samples.     

Mass fragmentography. Identification of chlorpromazine and its metabolites in human blood by a new method

Chlorpromazine and some of its metabolites have been identified in human blood with the combination of gas chromatography and mass spectrometry. A new method, mass fragmentography has been elaborated. It is based upon a continuous recording of up to three mass numbers characteristic of a single substance or a group of compounds. With this technique both a high sensitivity and a high selectivity which can be changed according to wish are achieved. Compounds are identified by their retention times and the fact that all mass numbers are represented in characteristic relative intensities. Refocusing on other characteristic fragments and/or the molecular ion confirms the identity. Through repeated refocusing “a partial mass spectrum” of a compound can be established even when the amounts present are too small for the scanning of a complete spectrum.With this method chlorpromazine, and its desmethylated and didesmethylated metabolites have been identified in plasma. The latter two metabolites were obtained also after treatment of the plasma with β-glucuronidase, as was 2-chlorophenothiazinylpropionic acid, which was found in quantities that allowed the scanning of a complete mass spectrum. In red blood cells the two desmethylated metabolites could be identified with mass fragmentography only after treatment with β-glucuronidase. The use of the method is discussed, particularly with regard to blood levels of drugs as related to therapeutic effects and side effects.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

Specific assay for radiolabelled digoxin and its known apolar metabolites in biological fluids. I.

A specific assay method for radiolabelled digoxin and its known apolar metabolites in plasma, urine and saliva was developed. The assay permits the delineation of the pharmacokinetics of digoxin and its metabolites after single-dose administration of the drug to humans. Column chromatographic and solvent extraction procedures were used for the separation of apolar and polar compounds. Thin-layer chromatography was applied for the individual and specific assessment of digoxin and its apolar metabolites. Apolar and polar standards were used for quantitative assessments of all the procedures used. Accuracy and precision of the assay developed were evaluated in plasma, urine and saliva using biological samples spiked with known amounts of standards and by measuring replicates of biological samples obtained from pharmacokinetic studies with digoxin administration to humans.     

Determination of chlordiazepoxide,diazepam, and their major metabolites in blood or plasma by spectrophotodensitometry

An analytical procedure was developed for the determination of chlordiazepoxide, diazepam and their major metabolites in blood or plasma. Demoxepam, a metabolite of chlordiazepoxide, is determined by spectrofluorometry after selective extraction. The remaining compounds are determined by spectrophotodensitometry after thin-layer chromatographic separation.The sensitivity limit of the spectrofluorometric determinationn of demoxepam is 0.1 to 0.2 μg while that of the spectrophotodensitometric determination of chlordiazepoxide, diazepam and their N-desmethyl metabolites is 0.05 to 0.2 μg. The sensitivity and specificity of the assay renders it suitable for monitoring plasma levels of chlordiazepoxide and its major metabolites following single or chronic oral administration of chlordiazepoxide hydrochloride. The sensitivity limit for diazepam and nordiazepam, its major metabolite, renders the assay useful only for the determination of plasma concentrations resulting from high dosage of diazepam. The assay was used to determine chlordiazepoxide and its metabolites following oral administration of Librium. The data showed a significant correlation to those obtained on the same specimens by differential pulse polarography and by radio-immunoassay.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.