Simultaneous determination of glucuronides of trimetozine in human urine by gas chromatography

A gas chromatographic method for the simultaneous determination of four glucuronides (metabolites) of trimetozine excreted in human urine is described. The methodinvolves pretreatment of the urine specimen [i.e. removal of interfering substances by solvent extraction, desalting on an ion-exchange (Amberlite XAD-2) column], and permethylation of glucuronides by reaction with methylsulfinyl carbanion and methyl iodide. The permethylated derivatives were submitted to gas chromatographic separation on an OV-17 column, and their structures were investigated by subsequent gas chromatographic—mass spectrometric analysis. The minimum detectable concentration of each glucuronide is 5 μg/ml when 1 ml of urine is used. The utility of the present method is successfully demonstrated by determining the urinary concentration of four glucuronides following oral administration of trimetozine to human subjects.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.     

Gaschromatographische analyse von sauren urinmetaboliten nach trennung auf kieselgelfertigsäulen

Gas chromatographic estimation of acidic urinary metabolites after separation on prepacked silica gel columnsThe acidic ethylacetate extracts of 24-h urine specimens are evaporated and redissolved in chloroform—methanol—acetic acid. The resulting solution is transferred to a prepacked silica gel column. Elution takes 160 min using a specially designed chloroform—methanol—acetic acid gradient. The eluate is divided into fractions (16 min each) which are evaporated to dryness. The residues are silylated and determined quantitatively by gas chromatography. The capacity of the silica gel column allows analysis of 30% of a 24-h urine specimen. In consequence, metabolites can be quantitated at concentrations less than 1 mg per 24 h. The method is suitable to obtain more detailed metabolic profiles of the carboxylic acids in urine.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

Identification and determination of the enantiomers of moprolol and their metabolites in human urine by high-performance liquid chromatography and gas chromatography—mass spectrometry

A simple and sensitive high-performance liquid chromatographic (HPLC) method using chiral derivatization was developed to screen and determine the enantiomers of moprolol and their metabolites in human urine. The recovery of (+)- and (−)-moprolol from urine was 70.8–81.1% at different concentrations. The coefficients of variation (C.V.) were less than 3.2 and 6.5% for intra- and inter-assays, respectively. Moprolol could be detected in urine up to 24 h after oral administration of a 50-mg dose of moprolol. Unconjugated and conjugated enantiomers of moprolol and their metabolites were analyzed by gas chromatography (GC). A gas chromatographic—mass spectrometric (GC—MS) confirmatory method was established to identify the metabolites of moprolol. The double derivatization procedure for moprolol and their metabolites with S-(−)-menthyl chloroformate [(−)-MCF] and N-methyl(trimethylsilyl)trifluoroacetamide (MSTFA) gave very good GC—MS properties of the derivatized compounds and provided reliable structural information for their confirmation analysis. This is the first published report on the use of a GC—MS method for the detection of the enantiomers of moprolol and their metabolites in human urine.     

Disposition of 17β-trenbolone in humans

The urinary excretion and metabolic pattern of 17β-trenbolone, a synthetic anabolic steroid hormone used as a growth promotor for beef cattle in several countries, has been studied in a human subject. For the separation of the metabolites of 17β-trenbolone, a reversed-phase high-performance liquid chromatographic method was established. The method was tested with metabolites obtained from incubation of 17β-trenbolone with rat liver microsomes. Fifteen metabolites could be well separated in one run by using a concave acetonitrile—water—methanol gradient. After ingestion of the tracer-labelled hormone at a dose of 0.04 mg/kg body weight 54% of the administered radioactivity was found in the urine after 26 h and 63% after 72 h. Of the urinary material 54% was present as glucuronides, which contained mostly 17α-trenbolone, 17β-trenbolone and trendione. At least five other polar metabolites, presumably hydroxylated products, were found in smaller amounts, mostly in the unconjugated and sulphated fractions. Thus, the disposition of 17β-trenbolone in humans differs significantly from that in rats, which may have a bearing on the toxicological evaluation of the hormone.     

Capillary gas chromatography as a tool for characterization of urinary steroid excretion in patients with congenital adrenal hyperplasia

Urinary steroid excretion was studied by capillary gas chromatography in 23 patients with congenital adrenal hyperplasia. In 5 patients the estimated excretion rates of pregnanetriol were in or below the normal range and 7 patients presented supranormal excretion rates of tetrahydro-cortisone and/or other glucocorticoid metabolites. Deficiency of 21-hydroxylase was nevertheless demonstrated in each patient by an increased ratio of excreted precursors vs products of 21-hydroxylase, e.g. of pregnanetriol/tetrahydro-cortisone. Due to this relative deficiency of glucocorticoids the patients' steroid excretion was further characterized by a predominance of 5 alpha-hydrogenated C19O3 metabolites (11-keto-androsterone, 11-hydroxy-androsterone) over their 5 beta-hydrogenated homologues (11-keto-etiocholanolone, 11-hydroxy-etiocholanolone). An apparent preponderance in the excretion of pregnenetriol over that of pregnanetriol was found in 4 patients, but the presence of pregnenetriol was not confirmed by mass spectrometry following prepurification of the urine samples by thin-layer chromatography indicating interference of an unidentified steroid metabolite with the initial gas chromatographic analysis. The simultaneous determination of steroids serving as precursors or products of 21-hydroxylase by capillary gas chromatography helps to establish the diagnosis of 21-hydroxylase deficiency and to characterize the pattern of steroid excretion in this syndrome even in patients where the estimation of single urinary steroids may lead to erroneous conclusions.     

Simplified method for monitoring tricyclic antidepressant therapy using gas—liquid chromatography with nitrogen detection

A simplified gas chromatographic method for the rapid measurement of tricyclic anti-depressant drugs in plasma using a nitrogen-sensitive detector is described. All drugs are extracted and chromatographed under identical conditions. Tertiary amines are separated from their secondary amine metabolites, which are determined simultaneously without the need for derivatisation. The lower limit of accurate determination for most drugs is 10 μg/1.

The method has been applied to the routine measurement of amitriptyline and nortriptyline in plasma from patients receiving antidepressant treatment. Large and important interindividual differences in plasma concentrations in the patients investigated have been found, and the significance of these results is discussed.     

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography—mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

An Improved Isolation Procedure for the Gas Chromatographic Analysis of Urinary Polyamines

The isolation of polyamines from urinary hydrolysates in a sufficiently pure state for subsequent analysis by gas chromatography has proved to be difficult. However, by using columns of Porapak-Q and ion-exchange resins, urinary hydrolysates are readily purified and formation of trifluoroacetyl derivatives of polyamines proceeds in high yield without carryover of artifacts in the gas chromatographic elution profile. Good yields from the trifluoroacetylation reaction are not achieved if large quantities of salts or urinary pigments are present. By obtaining the polyamine carbonates in the final stages of the method described, the trifluoroacetylation reaction yields excellent derivatives of nanogram or microgram amounts, particularly after standing over-night at room temperature. The procedure described in detail should permit routine urinary polyamine analysis where rapidity, ease of handling many samples, freedom from complications and artifacts are a consideration. The recent reports by Russell^{1, 2} that the urinary excretion of polyamines are greatly elevated in cancer patients has stimulated interest in these compounds as possible biological “markers” for the diagnosis of cancer. The polyamines usually considered are: putrescine, 1, 4-diaminobutane; cadaverine, 1, 5-diaminopentane; spermidine, and spermine. An extensive literature has developed over the last 50 years concerning the isolation and determination of polyamines including many excellent reviews. ^3–5 However, the isolation and determination of small quantities of polyamines from biological sources has proven to be difficult. This has led to conflicting conclusions among investigators as to which polyamine is the major excretion product in the urine of cancer patients. ^{2, 6, 7, 8} The following report presents in detail a new procedure of isolation of urinary polyamines in high yield and pure state that facilitates quantitation of these amines by gas chromatography.     

Simultaneous analysis of phenylglycols and phenylethanols in human urine by gas chromatography—mass spectrometry

A method is described for the determination of the neutral metabolites formed from catecholamines and various other structurally related phenylethylamines by using gas chromatography—chemical ionization—mass spectrometry. These metabolites (phenylglycols and phenylethanols) were extracted from urine specimens and converted to pentafluoropropionyl derivatives which were separated on either 3% OV-1, 3% SP-2250, or 3% QF-1 packed columns. Our results demonstrate the presence in human urine of p-hydroxyphenylglycol, a metabolite of octopamine. One patient excreted 13 and 91 μg/day of free and total (free + conjugated) p-hydroxyphenylglycol, respectively. Treatment with a monoamine oxidase inhibitor reduced the excretion of total p-hydroxyphenylglycol to 30% of baseline level.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

Excretion of non-conjugated androstenedione and testosterone in human urine.

A method for the measurement of unconjugated testosterone and androstenedione in human urine is described. The method uses chromatographic separation followed by radioimmunoassay and has been examined for reliability. The mean 24-hour excretion of androstenedione by adult male subjects was 2.5 mug and of testosterone was 0.8 mug. For women, the mean excretion was 2.9 mug of androstenedione and 0.25 mug of testosterone. In pregnancy, androstenedione excretion was occasionally elevated above the normal range, but testosterone excretion was quite commonly increased. Some hirsute subjects exhibited an increase in androstenedione excretion, which was decreased by administration of dexamethasone. The results suggest that the amount of unconjugated testosterone in urine is not a direct reflection of the plasma free testosterone, but urinary androstenedione may be a useful reflection of plasma androstenedione levels.     

Improved assay for the quantification of 11-dehydrothromboxane B2 by gas chromatography—mass spectrometry

Endogenous thromboxane production is best assessed by the measurement of its excreted metabolites, of which 11-dehydrothromboxane B₂ (11-dehydro-TxB₂) is most abundant. Gas chromatographic—mass spectrometric assays have been developed for this compound but suffer from the presence of co-eluting impurities which make the measurement of 11-dehydro-TxB₂ difficult. Furthermore, these assays are often time-consuming. We now report a modified assay for the measurement of this compound employing gas chromatography—mass spectrometry which alleviates the problem of co-eluting impurities primarily through modification of extraction and chromatographic methods. Furthermore, the time to complete the assay is significantly shortened. It is adaptable to both urine and plasma. Precision of the assay is ± 7% and accuracy is 90%. The lower limit of sensitivity in urine is approximately 20 pg/mg creatinine. Normal levels of urinary excretion of this metabolite were found to be 370 ± 137 pg/mg creatinine (mean ± 1 S.D.) and normal plasma levels were found to be 1.5 ± 0.4 pg/ml (mean ± 1 S.D.). Urinary excretion of 11-dehydro-TxB₂ is markedly altered in situations associated with abnormalities in thromboxane generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous thromboxane production and to further explore the role of this compound in human disease.     

Metabolic analysis of four phenolic acids in rat by liquid chromatography-tandem mass spectrometry

A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC-DAD-ESI-MS(n)) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid-liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C(18) reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.