Separation and identification of phase I and phase II [14C]antipyrine metabolites in rat and dog urine.

A simple and accurate HPLC procedure was developed to quantify, in a single run, all phase I and phase II [14C]antipyrine metabolites that occur in rat and dog urine. All metabolites were subjected to thermospray-LC-MS and EI-MS in order to establish their structure. The rat metabolizes antipyrine to eight major metabolites, six of which are conjugated; 1.4% of the dose was excreted unchanged, 18.9% in a free form, 30.6% as sulfates and 21.1% as glucuronides. The dog metabolizes antipyrine to four metabolites, all as sulfate (61.0% of the dose) or glucuronide conjugates (16.2% of the dose).

Metabolism of the calcium antagonist gallopamil in man.

The metabolism of gallopamil (5-[(3,4-dimethoxyphenyl)methylamino]-2-(3,4,5-trimethoxyphenyl) -2- isopropylvaleronitrile hydrochloride, Procorum, G) was studied after single administration (2 mg i.v., 50 mg p.o.) of unlabelled and labelled G (14G, 2H). TLC, HPLC, GLC, MS and RIA were used for assessment of G and its metabolites in plasma, urine and faeces. G clearance is almost completely metabolic, with only minimal excretion of unchanged drug. Metabolites represent most of the plasma radioactivity after p.o. administration. They are formed by N-dealkylation and O-demethylation with subsequent N-formylation, or glucuronidation, respectively. Compound A, derived by loss of the 3,4-dimethoxyphenethyl moiety of G is the main metabolite in plasma and urine (about 20% of the dose). This metabolite is accompanied by its N-formyl derivative (C), by the N-demethylated compound (H) and the acid (F), formed by oxidative deamination of A. Only 3 unconjugated monphenoles from several O-demthylated products showed distinct plasma levels which were nevertheless lower than metabolite A. These metabolites had no relevance to the pharmacodynamic action. Conjugated monophenolic and diphenolic products represented the major part in plasma and were excreted predominantly via the bile: they represented almost the whole faecal metabolite fraction. Less than 1% of the dose was recovered unchanged in the urine. About 50% of the dose is excreted by urine and 40% by faeces.

Synthesis of glucuronides of propafenone and 5-hydroxypropafenone by Sepharose-bound uridine 5'-diphosphoglucuronyltransferase.

Propafenone (Rytmonorm) and its in vivo metabolite 5-hydroxypropafenone were glucuronidated by agarose-bound uridine 5'-diphospho (UDP)-glucuronyltransferase from bovine liver in the presence of UDP-glucuronic acid. The identification of the synthesized glucuronides was based on the specific enzymatic hydrolysis, by mass spectrometry and by chromatographic comparison with isolated metabolites from dog and man. The glucuronidation of propafenone and 5-hydroxypropafenone followed Michaelis-Menten kinetics up to substrate concentration of 0.4 mmol/l (propafenone) and 0.3 mmol/l (5-hydroxypropafenone). The pH optima were 7.5 and 8.0, respectively. The reaction was linear up to 30 min and for a protein concentration between 1 and 5 mg per assay. 5-Hydroxypropafenone showed the best affinity with a Km value of 1.4 mmol/l, in contrast to 11.6 mmol/l for propafenone. The use of agarose-bound UDP-glucuronyltransferase offers the possibility to synthesize propafenone glucuronide and 5-hydroxypropafenone glucuronide which are needed for kinetic and pharmacological studies in man and animal.

Studies on the metabolism of propafenone. 3rd Comm.: Isolation of the conjugated metabolites in the dog and identification using fast atom bombardment mass spectrometry.

The metabolism and urinary and biliary excretion of propafenone (2-(2'-hydroxy-3'-propylamino-propoxy)-omega-phenyl-propiophenone hydrochloride) were studied in the dog. Approximately 20% of the dose was excreted in 24 h with the urine and about 65% with the bile. Propafenone was extensively metabolized. Less than 4% of the dose was recovered unchanged in urine and bile. The metabolites were mainly excreted as conjugates. Free metabolites accounted for less than 20% of the dose. Separation methods were developed to isolate and purify the conjugated metabolites. Fractionation on an Al2O3-column yielded a sulphate and a glucuronide fraction, further separation and purification was done by TLC and HPLC. Positive and negative ion fast atom bombardment mass spectra (FAB/MS) were obtained of the purified glucuronides. The structures of two hydroxylated propafenone derivatives and two O-methylated catechol-like derivatives were definitely proven by FAB/MS, the glucuronic acid moiety being conjugated to the hydroxyl function in the different aromatic rings. Two isomeric propafenone glucuronides were found in the bile, probably diastereomeric forms of the O-glucuronide. Thus FAB/MS proved to be a complementary method to electron impact ionization mass spectrometry (EI/MS) for studying drug metabolism. The structures of free and conjugated metabolites can be defined from the combination of both mass spectrometric techniques.

Studies on the metabolism of propafenone. 1st Comm.: synthesis and chromatographic/mass spectrometric properties of the labelled compound and of the reference substances.

The synthesis of 14C-propafenone and 2H-propafenone (propafenone: 2-(2'-hydroxy-3'-propylamino-propoxy)-omega-phenyl-propiophenone hydrochloride) and some reference compounds is described. The thin-layer chromatographic, high-performance liquid and gas chromatographic properties of the substances are described. Propafenone and the reference substances were studied by mass spectrometry and compared with each other, with respect to structural elucidation of the metabolites. The chromatographic and mass spectrometric data (key ions) enables the metabolites of propafenone to be identified in biological material.

Studies on the metabolism of propafenone. 2nd comm.: studies on the biotransformation in the dog.

Following administration of 14C- and 2H-labelled propafenone hydrochloride (2-(2'-hydroxy-3'-propylaminopropoxy)-omega-phenyl-propiophenone hydrochloride) to beagle dogs, the metabolites were isolated from urine and bile and analysed by mass spectrometry. Reference substances were synthesized on the basis of the structures postulated from the mass spectra and compared with the substances isolated from the biological material. Propafenone was absorbed completely following i.d. administration and eliminated mainly with the bile. Within 28 h 10% of the dose was excreted with the urine and 87% with the bile. Propafenone was extensively metabolized. Less than 1% of the dose was recovered as unchanged substance in urine and bile. The urinary and biliary metabolites were almost exclusively conjugated. The main metabolite, accounting for more than 30% of the dose, was propafenone glucuronide, followed by conjugates of hydroxylated propafenone derivatives with glucuronic acid and sulphuric acid. 5-Hydroxy-propafenone, a propafenone derivative hydroxylated in the middle aromatic ring, and a derivative hydroxylated in the omega-phenyl ring each accounted for about 15% of the dose. Besides these monohydroxy metabolites, two other O-methylated catechol-like derivatives, substituted in the different aromatic rings were recovered. The remainder of the metabolic products identified were mainly substances resulting from degradation of the propoxyamine side chain.

The metabolic fate of 2H-labelled propafenone in man.

The metabolism of propafenone was studied in three humans by using 300 mg of the deuterated compound given orally. The excretion of propafenone and its metabolites was assessed by measuring the deuterium content in the excretion products by means of a microwave plasma detector. At selected times the metabolic pattern in plasma, urine, bile and feces was determined. The metabolites were isolated and the structure of the main compounds was characterized. Propafenone is absorbed completely and quantitatively metabolized. Unchanged propafenone is excreted neither in the urine nor in the feces in amounts of more than 1% the dose. The percentage of free unconjugated propafenone of the total deuterium content in the plasma was about 10% 3 and 6 hours after application. Conjugates of hydroxylated derivatives of propafenone are present predominantly in the plasma. The excretion of the metabolites takes place mainly by way of the feces, 53% of the dose is excreted by this route within 48 hours. Urinary elimination accounted for 18.5 and 38% of the dose within 48 hours in two humans. It was possible to elucidate the structure of 11 metabolites, accounting for more than 90% of the dose administered. The major metabolites are conjugates of 5-hydroxypropafenone and hydroxy-methoxy-propafenone with glucuronic and sulphuric acid and propafenone glucuronide. Furthermore, metabolic products of oxydative deamination pathway were identified, i.e. a glycol and a lactic acid derivative. C-C splitting yields a relatively large amount of 3-phenyl-propionic acid, while cleavage of the ether group leads to a phenolic product and is only of minor importance.

[The determination of verapamil in maternal and fetal blood (author's transl)]. / Nachweis von Verapamil im mütterlichen und fetalen Blut des Menschen.

During the late first stage of labor and during the second stage of labor seven women in labor received an intravenous infusion during 60-110 minutes of verapamil at 2 g/k/per minute. Immediately following delivery of the infant, maternal and cord blood was drawn and the verapamil level measured by gas chromatography. The serum levels in the maternal blood were from 5.4 to 33.6 ng/ml. (Mean value 19.3 +/- 3.3 ng/ml) and the cord blood concentrations were 4.3-17.8 ng/ml (Mean value 8.5 +/- 1.9 ng/ml). The mean fetal serum level of verapamil was 51.4% of the mean maternal value.

[Diaplacental transfer of verapamil in guinea pigs (author's transl)]. / Untersuchungen zur Plazentapassage von Verapamil beim Meerschweinchen.

Verapamil (1--50 mcg/kg/min) was given to guinea pigs by intravenous infusion for 30 minutes. The average clinical dosage amounts to 1 (0,5--2) mcg/kg/min. At doses of 1--10 mcg/kg/min we could not find pharmacologically effective concentrations of verapamil in plasma, although concentrations in mother rose corresponding to administered doses. Only at dosage 50 mcg/kg/min we found comparatively to the mother low, but clearly measurable concentrations also in fetal plasma. That means: Verapamil, administered to guinea pigs--hemomonochorial placentation like man--in dosage, given for tocolysis does not appear in fetal plasma with pharmacologically effective concentration.

Gas chromatographic determination of verapamil in plasma and urine.

A gas-liquid chromatographic method for the determination of verapamil in body fluids has been developed. Verapamil and internal standard are extracted from alkalized fluid with heptane and then back extracted into 1 N HCl. After re-extraction into heptane, verapamil and internal standard are analysed by gas-liquid chromatography using a nitrogen-specific detector (N-FID). The method is specific for verapamil. Concentrations can be measured down to 4 ng ml-1 plasma. The coefficients of variation in plasma samples were 5--8% (within-day; range of concentration 5--30 ng ml-1). Recovery in this range was 100 +/- 6.2% during several weeks.