In-depth limitation of the Lamma 500 for the in situ localization of organic compounds in biological embedded tissue samples.

The objective of this study was exploration of the potential, offered by the laser microprobe mass spectrometer (LAMMA), for the in situ localization of organic targets in embedded tissues by means of structurally relevant ions. A series of model systems was designed to evaluate stepwise the analytical problems involved. A preliminary screening pointed to the position of the target in comparison to the actual sample surface as a determining parameter. Refined simulations were carried out with sandwich samples, consisting of an epon carrier (thickness 1 micron), coated with microcrystalline targets and covered with a layer between 5 and 50 nm of different materials (epon, carbon, formvar). In addition to the stimulated conversion of molecular into fragment ions, the presence of a barrier leads to a drastic loss of sensitivity (20-50 x) and unacceptable degradation of the mass spectrometric quality (resolution, calibration).

Determination of (4-chlorophenyl)thiomethylene bisphosphonic acid, a new bisphosphonate, in biological fluids by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the assay of the bisphosphonate (4-chlorophenyl)thiomethylene bisphosphonic acid in plasma and urine is described. It requires selective precipitations and dissolutions of calcium salts prior to reversed-phase chromatography with UV detection. This method used semi-micro scale material and 200-microliters biological aliquots. The limit for accurate quantification is 50 ng/ml. Data on reliability criteria and application to a pharmacokinetic study are presented.

Identification of urinary metabolites of penticainide in the rat, dog, baboon and man.

[14C]-Penticainide, 2-[2-(diisopropylamino) ethyl]-4-methyl-2-(2-pyridyl)pentanamide, a new antiarrhythmic agent, was administered as a single oral dose to rats, dogs, baboons (30 mg kg-1) and to healthy, informed volunteers (300 mg). Excretion of radioactivity was followed for 3 days in urine and faeces. In man, about 95% of the administered radioactivity was eliminated in the urine and levels ranging from 56 to 86% were observed in animals. The radioactivity that did not appear in the urine was almost quantitatively recovered in the faeces. Metabolites in urine were isolated by thin-layer chromatography and identified by mass spectrometry and nuclear magnetic resonance. In addition to the unchanged drug, nine metabolites and an artifact compound resulting from the partial degradation of one metabolite, were identified among the thirteen radioactive compounds detected. The major metabolites resulted from N-dealkylation of the diisopropyl moiety, oxidation of the isobutyl side-chain and hydrolytic cleavage of the amide. Comparison of the excretion and metabolic patterns of animals with those of man revealed that the dog should be a most suitable model for predicting the pharmacological and toxicological effects of penticainide in man.

Determination of circulating ethyl loflazepate metabolites in the baboon by radio-high-performance liquid chromatography with injection of crude plasma samples: comparison with solvent extraction and thin-layer chromatography.

Circulating ethyl loflazepate metabolites in the baboon were determined, following a single oral administration of the 14C-labelled drug, by radio-high-performance liquid chromatography with injection of crude plasma samples and by selective extraction with thin-layer chromatographic analysis of the radioactive components. Metabolites identified by comparing their chromatographic behaviour with synthetic standards were loflazepate, descarboxyloflazepate and 3-hydroxydescarboxyloflazepate. Loflazepate represented about 70% of the circulating radioactivity; the two other metabolites were present in amounts too small to allow accurate quantification. The parent drug was not present in the blood. Comparison of high-performance liquid chromatography with solvent extraction demonstrated the inaccuracy of the latter to be caused by the conversion of loflazepate to descarboxyloflazepate.

Circulating and brain metabolites of minaprine in the baboon.

A mixture of 15N-labelled, 14C-labelled and unlabelled minaprine was administered orally to three baboons, and metabolites in blood, urine and brain investigated. Biological samples were extracted with dichloromethane and the radioactive components extracted were analysed by t.l.c. and autoradiography. Compounds identified by comparing their physiochemical properties with those of synthetic standards and by g.l.c.-mass spectrometry were minaprine, 3-[2-(3-oxo)morpholino-ethylamino]-4-methyl-6-phenylpyridazine, 3-amino-4-methyl-6-phenylpyridazine, 3-[2-(aminoethyl)ethylamino]-4-methyl-6-phenylpyridazine, p-hydroxyminaprine and minaprine N-oxide. In addition to the urinary metabolites, two circulating metabolites were detected: metabolite A, 3-[2-(3-oxo)morpholino-ethylamino]-4-methyl-6-phenylpyridazine, and metabolite B (unidentified). All circulating metabolites appeared very early in blood, confirming the rapid and extensive metabolism of the drug. Metabolites A, B and 3 (p-hydroxyminaprine) were the major metabolites present in plasma. The parent drug was not the major circulating form, and was present in a higher concentration in erythrocytes than in plasma. Erythrocytes might act as a reservoir of the drug and could explain the relatively slow blood clearance of minaprine despite its rapid metabolism. The qualitative metabolic profile in brain tissue was similar to that in blood.

Metabolism of ethyl loflazepate in the rat, the dog, the baboon and in man.

The metabolism of ethyl loflazepate (Victan), a new benzodiazepine used as an anxiolytic drug, was studied in man and several animal species. The urinary metabolites were determined and quantified following oral administration of the 14C-labeled drug. Further analysis of blood samples was carried out in order to get information on the circulating metabolites in man. Except the rat, from which only 25% of the administered dose were excreted via the kidneys, the urine appeared to be the main excretion route in man (63%) and the other animal species (80%). The major urinary metabolites were identified as being loflazepate, 3-hydroxyloflazepate, 3-hydroxydescarbethoxyloflazepate, 4'-hydroxydescarbethoxyloflazepate and 6-chloro-4-(2'-fluorophenyl)-2(1H)-quinazolinone. The observed differences on the excretion patterns between the species resulted from the presence of a large number of quantitatively minor metabolites. Analysis of human blood samples indicated that the radioactivity was almost entirely in the plasma. The main circulating metabolites were detected as descarbethoxyloflazepate, loflazepate, and 3-hydroxydescarbethoxyloflazepate.

Pharmacokinetics of CM 57755, a new histamine H2-receptor antagonist, after single oral doses in man.

The plasma pharmacokinetics and urinary excretion of CM 57755, an H2-receptor antagonist, were studied after administration of single oral doses in a range between a 100 and 700 mg in human volunteers. Pharmacokinetic parameters were calculated model-independent. Absorption of CM 57755 was bimodal and the maximum plasma concentration was reached between 2 and 4 h after dosing. The drug was widely distributed with an apparent volume of distribution between 140 and 200 l. The plasma clearance was between 56 and 69 L/h. The plasma concentrations declined following a monoexponential function with an elimination half-life of 2 h. No modification in the plasma clearance or other pharmacokinetic parameters with these doses was observed. Therefore, a linear pharmacokinetic profile of CM 57755 was proposed. About 40% of the parent drug was unchanged in urine excreted over the 24 h. The drug was compared with cimetidine and ranitidine, the three compounds seemed to exhibit a consistent pharmacokinetic profile.

Determination of glaucine in plasma and urine by high-performance liquid chromatography.

A sensitive method is described for the measurement of d-glaucine in pharmacokinetic studies using only 100-microliter plasma samples or 200-microliter urine samples. It requires a simple extraction clean-up on kieselguhr micro-columns and straight-phase high-performance liquid chromatography with fluorescence detection. Data on selectivity, sensitivity and precision demonstrate the reliability of this method. Its applicability is revealed by single and repeated oral administration pharmacokinetic studies in human subjects.

Biotransformation and pharmacokinetics of tetrazepam in man.

The pharmacokinetics and the metabolism of tetrazepam (Musaril) were studied in 12 healthy volunteers. Tetrazepam was given orally as a single dose of 50 mg in tablet form (commercially available formulation). Tetrazepam and nortetrazepam were measured in serum using a selective and sensitive HPLC method. Urinary metabolites were identified after acid hydrolysis and thin-layer chromatographic separation. Tetrazepam is rapidly absorbed after oral administration with a lag-time of 0.45 +/- 0.10 h and reaches a peak serum level of 0.57 +/- 0.06 mg/l at 1.92 +/- 0.19 h after administration. The drug is largely distributed in the organism with an apparent volume of distribution of 225 +/- 40 l. The substitution of the phenyl moiety in the 5-position by a cyclohexenyl ring results in a different metabolism when compared to other benzodiazepines. This different metabolic pathway is the reason why only very small levels of active metabolites are present in serum. Therefore it seems reasonable, at least from the pharmacokinetic point of view, to attribute pharmacologic activity of tetrazepam mainly to the parent drug. Tetrazepam is eliminated with a half-life of 14.9 +/- 4.4 h and can be classified as a benzodiazepine with medium half-life value. This medium half-life is the result of the high hepatic clearance of the drug in spite of its large distribution volume. Since in this study 6 male and 6 female volunteers were studied it was possible to compare the pharmacokinetic profile in the two groups. No significant differences were observed.

Butofilolol pharmacokinetics in chronic renal insufficiency.

The pharmacokinetics of butofilolol, a new beta-blocking drug used in the treatment of hypertension, were investigated in 9 patients with varying degrees of renal impairment (creatinine clearance ranging from 65 ml/min to 6.6 ml/min). The drug was administered as a single oral 100-mg dose. Plasma and urine concentrations of the parent drug were measured. The pharmacokinetic parameters of butofilolol calculated from examination of patients with renal failure were compared to those obtained from healthy subjects. Renal failure was found to be associated with a marked increase of the areas under the plasma concentration-time curves of the parent drug whereas its elimination rate constant tended to increase, especially in severe renal failure. A highly significant relationship between the amounts of unchanged drug excreted in urine and the creatinine clearance was observed (r = 0.90, p less than 0.001). The higher plasma levels of the parent drug observed in renal failure might be due to a decrease in the first-pass hepatic extraction of the drug. But these changes in plasma clearance were probably counteracted by modification of the volume of distribution, so that the net result of these alterations was a slight increase in the elimination half-life.

Pharmacokinetics of butofilolol (CAFIDE) after repeated oral administration in man.

The pharmacokinetics of butofilolol, a new beta-blocking drug, was studied in 6 healthy subjects. Plasma concentrations and urinary excretion of the unchanged drug were determined after a single 100-mg oral administration, and also during chronic treatment (100 mg/day for a week) and after the last dose. Maximum plasma concentrations were observed 2 to 3 h after drug administration and varied between the subjects (120 to 430 ng/ml). The apparent volumes of distribution were large, ranging between 200 I and 500 I, and the apparent clearances of elimination appeared to be intermediate between 40 and 70 l/h. Drug elimination in the urine (about 4% of administered dose) occurred by filtration, tubular secretion and pH-sensitive reabsorption following a non-linear process. However, pharmacokinetic parameters remained constant during chronic treatment, since urinary elimination of the parent drug was too low to influence its pharmacokinetic profile. In addition, a high correlation was found between plasma levels of butofilolol and the effect of the drug on resting heart rate, while a slight effect on diastolic blood pressure could be discerned.

Analysis of the metabolites of ethyl loflazepate by gas chromatography with electron-capture detection.

A gas chromatographic assay with electron-capture detection (GC--EC) is described for the metabolites of ethyl loflazepate (Victan), a new benzodiazepine with a potent anti-anxiety activity, in biological fluids. Since the parent drug undergoes a first-pass effect, pharmacokinetic data may only be obtained by measuring the total levels of two of the major metabolites. Accurate data can not be obtained for the metabolites separately since one of them (M1) is chemically transformed to the other (M2) during plasma sampling, storage and extraction. A sensitive, specific and accurate GC--EC assay is developed using a synthetic analogue of M2 as an internal standard. The limit of detection in plasma is approximately 2 ng/ml and the precision about 3% (within-run and between-run). The method is applied to plasma samples collected after oral administration of 2 mg and 4 mg of the drug in tablet form to human volunteers. The results obtained are correlated with those from an existing gas chromatographic--mass spectrometric assay. A very good correlation between the results (inter-laboratory comparison) is obtained, validating both techniques.

The biotransformation of [14C]minaprine in man and five animals species.

1. [14C]Minaprine was administered as a single oral dose to five animal species and to a healthy and informed volunteer. Excretion of radioactivity was followed during 48 h in urine and faces; biliary excretion was followed only in rat. 2. Urinary metabolites were isolated and identified by mass spectrometry. 3. A quantitative comparison of metabolites in different species was made. On the basis of these data it it concluded that the dog is not a suitable model for man for pharmacological or toxicological studies. 4. The major metabolic route is 4-hydroxylation of the aromatic ring. The only unexpected metabolic route found was the biotransformation of the morpholino ring, probably by reductive ring-cleavage. 5. About 50% of the 14C was excreted in 0-48 h urine. The other 50% was excreted in the 0-48 h faces. In the rat, this was attribute entirely to biliary excretion. The drug is well absorbed after oral administration and is not accumulated in the body.

Quantitative analysis of CM 6912 (ethyl loflazepate) and its metabolites in plasma and urine by chemical ionization gas chromatography mass spectrometry. Application to pharmacokinetic studies in man.

A chemical ionization gas chromatographic mass spectrometric assay is described for ethyl loflazepate (CM 6912), a new benzodiazepine with a potent anti-anxiety activity, and two of its metabolites in plasma and urine. The parent drug was separated from the biological samples by simple extraction at pH 9. In all samples analysed, parent drug levels were below the detection limit, confirming its extensive biotransformation at the sites of absorption and during its first pass through the liver. Pharmacokinetic data were obtained by measuring the total levels of two of the major metabolites. Accurate data may not be obtained for both metabolites separately since one of them (M1) is chemically transformed to metabolite M2 during plasma sampling, storage and extraction. Their total levels were measured after complete degradation of the unstable metabolite and extraction of the sample at pH 9. The precision and accuracy of this method are better than 10% in the low ng range. The detection limit is 1 ng ml-1. Pharmacokinetic data were obtained from human volunteers given a single oral dose of 2 mg CM 6912 in tablet form. Cp max values ranged from 30 to 55 ng ml-1 and were reached within 1.5 to 3 h after administration. Following the achievement of peak levels the concentration declined with an apparent biexponential pattern showing an elimination half-life ranging from 51 to 103 hours in its terminal phase.