Use of post-column fluorescence derivatization to develop a liquid chromatographic assay for ranitidine and its metabolites in biological fluids

Ranitidine and its main metabolites, ranitidine N-oxide and ranitidine S-oxide, were determined in plasma and urine after separation using reversed-phase liquid chromatography. The mobile phase consisted of an initial isocratic step with 7:93 (v/v) acetonitrile–7.5 mM phosphate buffer (pH 6) for 8 min, followed by a linear gradient up to a 25:75 (v/v) mixture over 1 min. Detection was carried out by a post-column fluorimetric derivatization based on the reaction of the drugs with sodium hypochlorite, giving rise to primary amines that reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent products. The calibration graphs, based on peak area, were linear in the range 0.1–4 μg/ml for all drugs. The detection limits were 30, 41 and 32 ng/ml (8.6, 12.5 and 9.1 pmol) for ranitidine S-oxide, ranitidine N-oxide and ranitidine, respectively. Chromatographic profiles obtained for plasma and urine samples showed no interference from endogenous compounds.     

Phenylalanine hydroxylase: possible involvement in the S-oxidation of S-carboxymethyl-l-cysteine

Activated phenylalanine 4-monooxygenase, phenylalanine hydroxylase (PAH), is known to be involved in the S-oxidation of a number of sulfide compounds. One of these compounds, S-carboxymethyl-l-cysteine (SCMC), is currently used for the treatment of chronic obstructive pulmonary disease and otitis media with effusion as a mucolytic agent, and the S-oxides are the major metabolites found in urine. However, the enzyme catalyzing the S-oxidation of SCMC has yet to be identified. Here we report on the role of nonactivated phenylalanine 4-monooxygenase activity in rat liver cytosol in the S-oxidation of SCMC. Linearity of the enzyme assays was seen for both time (0-16 min) and cytosolic protein concentration (0.1-0.5mg/ml). The calculated K(m) and V(max) values for the formation of SCMC (S) S-oxide were 3.92+/-0.15 mM and 1.10+/-0.12 nmol SCMC (S) S-oxide formed/mg protein/min, respectively. The calculated K(m) and V(max) values for the formation of SCMC (R) S-oxide were 9.18+/-1.13 mM and 0.46+/-0.11 nmol SCMC (R) S-oxide formed/mg protein/min, respectively. These results indicate that in the female Wistar rat, nonactivated PAH showed a stereospecific preference for the formation of the (S) S-oxide metabolite of SCMC against the (R) S-oxide metabolite of SCMC.     

Testosterone metabolites in dog bile

Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours; only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one), etiocholanlone (3alpha-hydroxy-5beta-androstan-17-one), 5alpha-androstan-3beta, 17beta-diol, 5beta-androstan-3alpha, 17beta-diol and 5beta-androstan-3beta,17beta-diol.     

Biochemical studies of toxic agents. The metabolism of 1- and 2-bromopropane in rats

1. (+)-n-Propylmercapturic acid sulphoxide, i.e. (+)-N-acetyl-S-n-propyl-l-cysteine S-oxide, was prepared as the dicyclohexylammonium salt, (-)-n-propyl-mercapturic acid sulphoxide was prepared as the free acid, and S-isopropyl-l-cysteine and isopropylmercapturic acid were also prepared. 2. The metabolism of 1- and 2-bromopropane was studied by radiochromatographic examination of the urine excreted by rats that had been fed with a diet containing (35)S-labelled yeast and then injected subcutaneously with these compounds. In addition to n-propyl-mercapturic acid and 2-hydroxypropylmercapturic acid, the excretion of which has already been reported, n-propylmercapturic acid sulphoxide was shown to be a metabolite of 1-bromopropane. Sulphur-containing metabolites of 2-bromopropane, if present in the urine at all, were there in very small amounts. 3. n-Propylmercapturic acid and isopropylmercapturic acid were isolated from the urine of rats that had been injected subcutaneously with S-n-propyl-l-cysteine and S-isopropyl-l-cysteine respectively.     

The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.     

Identification of cereal alkylresorcinol metabolites in human urine-potential biomarkers of wholegrain wheat and rye intake

Alkylresorcinols, phenolic lipids present in high amounts in wholegrain wheat and rye, are of interest as potential biomarkers of the intake of these cereals. Alkylresorcinols are known to be absorbed by humans and animals, but little is known about their metabolism or resulting metabolites. A preliminary human study was carried out to identify alkylresorcinol metabolites in human urine. Urine samples, collected before and after a wheat-bran based meal, were deconjugated with beta-glucuronidase/sulphatase and then extracted with ethyl acetate. Extracts were separated by thin-layer chromatography, and fractions containing alkylresorcinols and possible metabolites were identified by retention on the plate compared to standard compounds, and staining with fast blue B. These fractions were further analysed by gas chromatography-mass spectrometry. Deconjugated human urine after the wheat-bran based meal contained two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-1-propanoic acid, as well as smaller amounts of unchanged alkylresorcinols, confirming the hypothesis that alkylresorcinols are metabolised in humans via beta-oxidation of their alkyl chain.     

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.     

Determination of ranitidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

A method using ion-pair high-performance liquid chromatography is presented for determining ranitidine, ranitidine N-oxide, ranitidine S-oxide and desmethyl ranitidine in the urine from four volunteers, given on separte occasions an intravenous and oral dose of 100 mg ranitidine. This method has been used to study the metabolism and pharmacokinetics of ranitidine by man. It was found that the elimination half-life of ranitidine ranged from 110–246 min. The mean renal clearance of ranitidine in these four volunteers was 512 ml/min.     

A revision of the metabolic disposition of amantadine

Amantadine is one of the most commonly used drugs for the control of tremor in Parkinson's disease. Additionally, it has an antiviral action in the prevention of type A influenza. It has been previously reported that amantadine is nearly completely eliminated in the urine. No metabolites have been detected. Surprisingly, in a case of amantadine overdose, several metabolites could be identified by gas chromatography/mas spectrometry. This finding prompted us to re-investigate the metabolism of amantadine under a therapeutic dosing regimen. The bulk of the dose was eliminated unchanged. However, eight metabolites could be identified. Besides N-acetylation which is the major metabolic pathway, several rather unusual metabolic pathways were observed: N-methylation, formation of Schiff bases and N-formiates. No metabolites with a hydroxylated adamantane ring system could be detected.     

Syntheses of s-Triazolo [4,3-a] Pyrimidine Derivatives

The metabolism in rats of dihydrocapsaicin, a pungent principle of hot pepper, was investigated in vivo and in vitro by thin-layer chromatography, high-performance liquid chromatography and combined gas chromatography-mass spectrometry. Within 48 hr of oral administration of dihydrocapsaicin (20 mg/kg body weight) to male adult rats, unchanged dihydrocapsaicin and eight of its metabolites were identified in urine; i.e., dihydrocapsaicin (8.7% of total dose), vanillylamine (4.7%), vanillin (4.6%), vanillyl alcohol (37.6%) and vanillic acid (19.2%) as free forms and/or their glucuronides. The proportions of free and glucuronide metabolites in urine were 14.5% and 60.5% of the total dose. Part of the unchanged dihydrocapsaicin (10% of total dose) was excreted into the feces within 48 hr. Cell-free extracts of rat liver catalyzed the hydrolysis of dihydrocapsaicin to vanillylamine and 8-methyl nonanoic acid. The former compound was further transformed to vanillin in situ. Dihydrocapsaicin-hydrolyzing enzyme activity was found in various organs of rat. The activity was located mainly in the liver. On the basis of the present data, the metabolic pathway of dihydrocapsaicin in rats was proposed.     

The metabolism of four sulphonamides in cows

1. The metabolism of sulphanilamide, sulphadimidine (4,6-dimethyl-2-sulphanilamidopyrimidine), sulphamethoxazole (5-methyl-3-sulphanilamidoisoxazole) and sulphadoxine (5,6-dimethoxy-4-sulphanilamidopyrimidine) given by intravenous injection has been examined in cows. 2. The sulphonamides were present mainly as unchanged drugs in blood samples collected 2h after administration. 3. The sulphonamides were excreted in the milk partly as unchanged drugs and partly as conjugated metabolites whereas only small amounts were excreted as the N(4)-acetyl derivatives. 4. The unchanged drug and the N(4)-acetyl derivative were the major constituents in urine samples after administration of sulphanilamide, sulphamethoxazole and sulphadoxine. 5. Besides the unchanged drug, the N(4)-acetyl derivative and the conjugated metabolites, three further metabolites of sulphadimidine were isolated from urine samples and identified. They were 5-hydroxy-4,6-dimethyl-2-sulphanilamidopyrimidine, 4-hydroxymethyl-6-methyl-2-sulphanilamidopyrimidine and sulphaguanidine.     

Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.     

Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.     

Studies of the metabolism of the antihistaminic drug dimethindene by high-performance liquid chromatography and capillary electrophoresis including enantioselective aspects

A reversed-phase high-performance liquid chromatography method for the determination of dimethindene and its main metabolites N-demethyldimethindene, 6-hydroxydimethindene and 6-hydroxy-N-demethyldimethindene in human urine was developed. The assay was also applied to the quantification of dimethindene-N-oxide in rat urine. Conjugates of the hydroxylated metabolites were determined after enzymatic deconjugation. Moreover the direct determination of dimethindene and its metabolites without prior extraction from urine was performed by capillary electrophoresis. The direct simultaneous determination of the enantiomers of dimethindene and N-demethyldimethindene was achieved on a Chiralcel OD column. Urinary data after oral administration of dimethindene are presented. The assays were used to study dimethindene and it metabolites in urine upon oral administration of the drug to rats and human volunteers.     

Metabolism and pharmacokinetic studies of propionylpromazine in horses

The propionylpromazine concentrations in plasma after intramuscular administration to horses were determined using gas chromatography with nitrogen-phosphorus detection. After hydrolysis by β-glucuronidase/arylsulphatase, the parent drug and three metabolites were detected in urine. The metabolites were identified as 2-(1-hydroxypropyl)promazine, 2-(1-propenyl)promazine and 7-hydroxypropionylpromazine by gas chromatography-mass spectrometry. No N-demethylated or sulphoxidated metabolites of propionylpromazine were observed in the horse urine.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Species differences in the metabolism of norephedrine in man, rabbit and rat

1. (+/-)-2-Amino-1-phenyl[1-(14)C]propan-1-ol ([(14)C]norephedrine) was administered orally to man, rat and rabbit and the metabolites excreted in the urine were identified and measured. Pronounced species differences in the metabolism of the drug were found. 2. Three male human subjects, receiving 25mg each of [(14)C]norephedrine hydrochloride, excreted over 90% of the (14)C in the first day. The main metabolite was the unchanged drug (86% of the dose) and minor metabolites were hippuric acid and 4-hydroxynorephedrine. 3. In rats given 12mg of the drug/kg almost 80% of the (14)C administered was excreted in the first day. The major metabolites in the urine were the unchanged drug (48% of the dose), 4-hydroxynorephedrine (28%) and trace amounts of side-chain degradation products. 4. Rabbits given 12mg of the drug/kg excreted 85-95% of the dose of (14)C in the urine in the first 24h after dosing. The major metabolites in the urine were conjugates of 1,2-dihydroxy-1-phenylpropane (31% of the dose) and of 1-hydroxy-1-phenylpropan-2-one (27%) and hippuric acid (20%). The unchanged drug was excreted in relatively small amounts (8%).     

Methodology for the identification of the urinary metabolites of the analgesic-narcotic antagonist, codorphone, by gas chromatography mass spectrometry

Methodology is presented for the identification of codorphone and its metabolites in urine samples using gas chromatography mass spectrometry. The procedure focuses on the clean-up of biological samples and a derivatization technique suitable for these samples. Sep-Pak C-18 cartridges were employed in the clean-up procedure permitting the biological sample to be derivatized in a relatively small volume of reagents. The derivatization procedure incorporated a one-step trimethylsilyloxime reaction to prevent enol formation while simultaneously derivatizing free hydroxyl groups with the excess trimethylsilylimidazole present in the reaction mixture. This was followed by the addition of BSTFA directly to this reaction mixture to complete derivatization of any metabolites possessing dealkylation of the nitrogen. Using this derivatization scheme, synthetic metabolites were analyzed by gas chromatography mass spectrometry, and their mass spectra were characterized emphasizing the diagnostic fragment ions observed in the spectra. To illustrate the usefulness of this methodology, a urine sample obtained from a dog that had been dosed with codorphone was analyzed by gas chromatography mass spectrometry, and the metabolites were identified by comparison to the mass spectra of the synthetic derivatives.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Excretion of corticosteroid metabolites in urine and faeces of rats.

Stress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioactive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 +/- 1.9 h and in faeces after 16.7 +/- 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC), several peaks of radioactive metabolites were found. Some metabolites were detected by enzyme immunoassay (EIA) using two different antibodies (corticosterone, 11beta-OH-aetiocholanolone). There was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. This diurnal variation was influenced neither by the intraperitoneal injection of isotonic saline nor by ACTH. However, the administration of dexamethasone eliminated the morning peak for 2 days.