Analysis of lamotrigine and its metabolites in human plasma and urine by micellar electrokinetic capillary chromatography

A reliable micellar electrokinetic capillary chromatographic method was developed and validated for the determination of lamotrigine and its metabolites in human plasma and urine. The variation of different parameters, such as pH of the background electrolyte (BGE) and Sodium dodecyl sulfate (SDS) concentration, were evaluated in order to find optimal conditions. Best separation of the analytes was achieved using a BGE composed of 10 mM borate and 50 mM SDS, pH 9.5; melatonin was selected as the internal standard. Isolation of lamotrigine and its metabolites from plasma and urine was accomplished with an original solid-phase extraction procedure using hydrophilic-lypophilic balance cartridges. Good absolute recovery data and satisfactory precision values were obtained. The calibration plots for lamotrigine and its metabolites were linear over the 1-20 microg/mL concentration range. Sensitivity was satisfactory; the limits of detection and quantitation of lamotrigine were 500 ng/mL and 1 microg/mL, respectively. The application of the method to real plasma samples from epileptic patients under therapy with lamotrigine gave good results in terms of accuracy and selectivity, and in agreement with those obtained with an high-performance liquid chromatography (HPLC) method.     

Determination of S-carboxymethyl-L-cysteine and some of its metabolites in urine and serum by high-performance liquid chromatography using fluorescent pre-column labelling.

Pre-column labelling techniques are described for the determination of S-carboxymethyl-L-cysteine (CMC) and its metabolites in urine and plasma samples by high-performance liquid chromatography (HPLC) without prior extraction. All substances containing an amino group were converted into fluorescent fluorenylmethyl derivatives with 9-fluorenylmethyloxycarbonyl chloride (FMOC). Deaminated or N-acetylated carbocysteine metabolites were coupled with 1-pyrenyldiazomethane (PDAM) to give fluorescent PDAM esters. Similar results were obtained with the two commercially available and stable diazomethane derivatives PDAM and 9-anthryldiazomethane (ADAM). Following double derivatization with PDAM and FMOC, in a single chromatographic run with two fluorescence detectors connected in series, amines and amino(carboxylic) acids could be detected by their FMOC residues and, simultaneously, carboxylic acids were detected as fluorescent PDAM esters. The (R) and (S) enantiomers of the sulphoxides of CMC, of methylcysteine and of N-acetyl CMC were separated, although the reversed-phase HPLC system did not contain a chiral additive or stationary phase designed for the separation of enantiomers. The methods do not include liquid extraction steps and can therefore be performed either manually or automatically using an HPLC autosampler. These methods were used for the investigation of a disputed pharmacogenetic polymorphism of S-oxidation of CMC in humans, which until now has most often been studied using paper chromatography. The described techniques were applied to the determination of CMC and its metabolites in human urine and plasma samples.     

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.     

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.     

Validation of liquid chromatography-electrospray ionization ion trap mass spectrometry method for the determination of mesocarb in human plasma and urine

Mesocarb metabolism in humans is the target of this investigation. A high-performance liquid chromatographic (LC) method with electrospray ionization (ESI)-ion trap mass spectrometric (MS) detection ion trap "SL" for the simultaneous determination of mesocarb and its metabolites in plasma and urine is developed and validated. Ten metabolites and the parent drug are detected in human urine, and only four in human plasma, after the administration of a single oral dose of 10 mg of mesocarb (Sydnocarb, two 5-mg tablets). Seven of this metabolites have been found for the first time. The confirmation of the results and identification of all the metabolites except amphetamine is performed by LC-MS, LC-MS-MS, and LC-MS3. In the case of doping analysis, the reliable detection time for mesocarb (long-life dihydroxymesocarb metabolites of mesocarb) is approximately 10-11 days after the administration of the drug, which is a significant increase over the existing data. The detection of amphetamine in plasma and urine is made using simple flow-injection analysis without a chromatographic separation. The addition-calibration method is used with plasma and urine. The mean recoveries from plasma are 49.2% and 57.4% for mesocarb concentrations of 33.0 and 66.0 ng/mL, respectively, whereas the recoveries from human urine are 76.9% and 81.4% for concentrations of 1 and 2 ng/mL, respectively. Calibration curves (using an internal standard method) are linear (r2>0.9969) for concentrations 0.6 to 67 ng/mL and from 0.05 to 5 ng/mL in plasma and urine, respectively. Both intra- and interassay precision of plasma control samples at 3, 40, and 55 ng/mL are lower than 6.2%, and the concentrations do not deviate for more than -3.4% to 7.3% from their nominal values. In urine, intra- and interassay precision of control samples at 0.08, 1.5, and 3.0 ng/mL is lower than 14.1%, with concentrations not deviating for more than -11.3% to 13.7% from their nominal values. The plasma disappearance curve of the parent drug is obtained. The major pharmacokinetic parameters are calculated.     

Comparison of SSI with APCI as an interface of HPLC-mass spectrometry for analysis of a drug and its metabolites

Sonic spray ionization (SSI) was compared with atmospheric pressure chemical ionization (APCI) as an interface of high-performance liquid chromatography (HPLC)-mass spectrometry (MS) for sensitive analyses of a neuroleptic drug, haloperidol and its two metabolites, such as reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), in biological samples. For both SSI and APCI interfaces, HPLC-MS-MS gave higher sensitivity than HPLC-MS. The sensitivities by HPLC-SSI-MS-MS for haloperidol and reduced haloperidol were 100 and 30 times higher, respectively, than those by HPLC-APCI-MS-MS; no spectrum with recognizable peaks was obtained for CPHP with the APCI interface. Therefore, detection limits and regression equations were examined by the HPLC-SSI-MS-MS for human plasma and urine samples spiked with the above drug and its metabolites. Haloperidol, reduced haloperidol, and CPHP showed good linearity in the ranges of 5-800, 10-800, and 100-800 ng/mL, respectively, for both human plasma and urine; their detection limits were 2.5, 5, and 75 ng/mL, respectively, using a new polymer HPLC column which enabled direct application of biological samples.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

High-performance liquid chromatography of pefloxacin and its main active metabolites in biological fluids

We describe a high-performance liquid chromatographic (HPLC) method for the analysis of pefloxacin, a new antibacterial agent, in plasma and urine following administration of a therapeutic dose in humans. HPLC assay of pefloxacin and its two main active metabolites in urine is also described. The applicability of the methods to pharmacokinetic studies of pefloxacin in humans is demonstrated.     

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.     

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.     

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.     

Determination of imipenem (N-formimidoyl thienamycin) in human plasma and urine by high-performance liquid chromatography, comparison with microbiological methodology and stability

High-performance liquid chromatographic (HPLC) methods using ultraviolet (UV) detection have been developed for the assay of the antibiotic imipenem (N-formimidoyl thienamycin) in human plasma and urine. A reversed-phase analytical column is employed in the plasma assay method and a cation-exchange column is used in the urine assay method. Both methods use borate buffer in the mobile phase. The method of preparation of human fluid samples for HPLC injection has been optimized with respect to the stability of imipenem in aqueous buffers, in morpholine buffer--ethylene glycol stabilizer, and in urine and plasma. Preparation of the samples before injection into the HPLC systems involves deproteination/filtration of the plasma/urine samples. The open lactam metabolite and the coadministered dehydropeptidase inhibitor, cilastatin sodium, do not interfere with the 313-nm detection of imipenem in either the plasma or the urine assay. Thienamycin, the precursor of imipenem and an impurity in imipenem formulations, is separated from the drug using both of these methods. Concentrations generated from the HPLC analysis of plasma and urine samples from two healthy volunteers compare favorably with results using a microbiological assay method. Correlation of the two methods gives r greater than or equal to 0.990 for both fluids.     

Bestimmung von Clanobutin und Metaboliten in Plasma und Urin durch Hochdruck-Flüssigkeits-Chromatographie

Zusammenfassung Die Analyse und quantitative Bestimmung von Clanobutin und seinen Metaboliten aus Plasma- und Urinproben mittels HPLC an einem Reversed-phase-System wird beschrieben.

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High-performance liquid chromatographic determination of clanobutine and its metabolites in biological samples

Summary This paper describes the separation and quantitative analysis of Clanobutine and its metabolites from plasma and urine samples by HPLC on a reversed phase system.

     

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.     

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.     

A novel reversed-phase HPLC method for the determination of urinary creatinine by pre-column derivatization with ethyl chloroformate: comparative studies with the standard Jaffé and isotope-dilution mass spectrometric assays

Creatinine is an important biomarker for renal function diagnosis and normalizing variations in urinary drug/metabolites concentration. Quantification of creatinine in biological fluids such as urine and plasma is important for clinical diagnosis as well as in biomonitoring programs and urinary metabolomics/metabonomics research. Current methods for creatinine determination either are nonselective or involve the use of expensive mass spectrometers. In this paper, a novel reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of creatinine of high hydrophilicity by pre-column derivatization with ethyl chloroformate is presented. N-Ethyloxycarbonylation of creatinine significantly enhanced the hydrophobicity of creatinine, facilitating its chromatographic retention as well as quantification by HPLC. Factors governing the derivatization reaction were studied and optimized. The developed method was validated and applied for the determination of creatinine in rat urine samples. Comparative studies with isotope-dilution mass spectrometric method revealed that the two methods do not yield systematic differences in creatinine concentrations, indicating the HPLC method is suitable for the determination of creatinine in urine samples.

Automated sample preparation and chromatographic analysis: determination of CGS 10787B and related compounds

An automated method is described for analyzing biological samples originating from CGS 10787B drug disposition studies. The method incorporates a laboratory robot to prepare plasma and urine samples and a high performance liquid chromatographic system to simultaneously analyze for CGS 10787B, as well as metabolites and drug-related compounds (CGS 12094, CGS 17000, and CGS 17001). The robot allquots the biological sample, adds an internal standard, and performs all the steps necessary for the liquid-liquid extraction and concentration of the drug and related components, while operating unattended around the clock. Recovery and reproducibility assessments indicate good accuracy and precision for the method. The limits of detection for the method are 0.2 microgram/mL in plasma and 0.5 microgram/mL in urine, for all components.     

Mass spectrometric and high-performance liquid chromatographic studies of medroxyprogesterone acetate metabolites in human plasma.

Medroxyprogesterone acetate (MPA) treatment has been shown to exert several beneficial effects in cancer patients. It has been suggested that such effects are due in part to the metabolites derived from MPA in vivo. The first results are reported on the identification of 2 alpha-hydroxy- and 21-hydroxy-MPA, 20-dihydro-MPA, 17 alpha-acetoxy-2 alpha,3 beta-dihydroxy-6 alpha-methylpregn-1,4-dien-20-one and two X,21-dihydroxy-MPAs, one of them presumably being 6 alpha-hydroxymethyl-21-hydroxy-MPA, in patient's plasma by high-performance liquid chromatographic (HPLC), gas chromatographic-mass spectrometric and NMR methods. Additionally, the presence of other metabolites such as di- and tetrahydro-MPAs and 6,21-dihydroxy-MPA, found in urine and other samples, was demonstrated in plasma. For routine clinical examinations an HPLC method is described for determination of, e.g., the unreduced MPA metabolite group in Sep-Pak-ODS column extracts of patients' plasma.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

HPLC Separation of Theophylline,Paraxanthine, Theobromine,Caffeine and Other Caffeine Metabolites in Biological Fluids

Abstract

An HPLC method is described for rapid analysis of caffeine and seven of its metabolites in plasma, urine, milk and saliva in a single operation using a 5 μ C₁₈ reverse phase column. The metabolites are extracted with chloroform - iso-propanol (85:15) from 100 μL samples added to NH₄HCO₃. No interference from normal blood, urine, milk or saliva constituents was observed. The method is accurate and precise and separates 1,7-dimethylxanthine (paraxanthine) from 1,3-dimethylxanthine (theophylline). Sensitivity for most metabolites is in the range of 0.1 to 0.3 μg/mL and the detectability is at the nanogram level.