Desmethyl Metabolite Research Articles

High-performance liquid chromatographic method for a clozapine analogue, CGS 13429, and its N-oxide and desmethyl metabolites

High-performance liquid chromatographic method for a clozapine analogue, CGS 13429, and its N-oxide and desmethyl metabolites

Results compared for tricyclic antidepressants as assayed by liquid chromatography and enzyme immunoassay.

The tricyclic antidepressants amitriptyline, nortriptyline, imipramine, and desipramine in serum of patients taking one of the drugs were quantified in two laboratories by high-performance liquid chromatography (HPLC) and enzyme-multiplied immunoassay (EMIT; Syva). Results for split samples were highly correlated, but EMIT gave higher results in most cases, and the slopes of the correlation lines for each analyte were greater than 1. Detection limits for the two procedures were such that 18% of the EMIT results for the drug(s) were considered negative, as compared with 4% of the HPLC results. Additional assay of desmethyl or hydroxy antidepressant metabolites by HPLC did not explain the higher EMIT results. The relatively high detection limit for EMIT greatly limits its use in therapeutic drug monitoring, where low concentrations of tricyclic antidepressants are as important as high ones for dose adjustment or determination of compliance. Other problems with EMIT measurement of tricyclic antidepressants are discussed.

The toxicity and mechanism of action of bromethalin: A new single-feeding rodenticide

Bromethalin is a new rodenticide for the control of commensal rodents. Doses in excess of the LD50 (2 mg/kg in rats) will cause death within 8–12 hr and it is preceded by one to three episodes of clonic convulsions with death usually due to respiratory arrest. Multiple low doses or sublethal intoxication yields hind leg weakness and loss of tactile sensation in rodents. Histopathology of the brain and spinal cord of these animals revealed a spongy degeneration of the white matter which was shown upon ultramicroscopic examination to be intramyelenic edema. No inflammation or cellular destruction of neuronal tissue was noted. LD50 values ranged from 1.8 mg/kg in the cat to approximately 13 mg/kg in rabbits. The only apparent nonsusceptible species was the guinea pig which could tolerate doses in excess of 1000 mg/kg without effect. Identification of the desmethyl metabolite was demonstrated in the blood and liver of treated animals by comparison of chromatographic retention times to that of a reference standard, but direct mass spectral identification was unsuccessful in part due to the low dose which could be administered. There-fore, the metabolism of bromethalin was studied by indirect means. Animals were pretreated with three inducers of microsomal drug metabolism: phenobarbital, 3-methylcholanthrene (3MC), and Aroclor 1254 (Aroclor) and one inhibitor, SKF-525A. Pretreated mice or rats were given an LD50 dose of bromethalin or the desmethyl analog and the percentage of surviving animals was determined. Phenobarbital and SKF-525A were protective when bromethalin was given, but SKF-525A increased lethality when the desmethyl analog was administered. Aroclor and 3MC both promoted lethality. It was concluded that SKF-525A could block the conversion of bromethalin to the desmethyl analog and beyond but each of the inducers promoted conversion of bromethalin to the desmethyl analog. Induction of subsequent pathways also must have occurred so that the net level of the desmethyl metabolite was higher than control when Aroclor and 3MC were given but lower than control when phenobarbital was given. Mechanistic studies showed that bromethalin is rapidly converted to the desmethyl analog which is an extremely potent uncoupler of oxidative phosphorylation. It was theorized that if this occurs in the central nervous system, a fluid imbalance may ensue due to insufficient ATP. Fluid buildup in the cranium was determined by measuring cerebrospinal fluid pressure (CSFP), brain and spinal cord moisture, and cation concentrations. These studies demonstrated that rats had mean CSFP of 251 ± 23 mm water 24 hr after being treated with 1 mg/kg bromethalin compared to a control value of 68 ± 5 mm water. Pressures could be reduced within 1 to 2 hr by infusion of an osmotic diuretic such as mannitol or urea; however, CSFP would rise again when the infusion was stopped. CSFP returned to normal values within 7 days after a single dose of 1 mg/kg bromethalin. Subcutaneous injections of dexamethasone hastened reduction of CSF pressure.

Determination of the antidepressant fluoxetine and its metabolite norfluoxetine in serum by reversed-phase HPLC with ultraviolet detection.

A procedure has been developed for measuring fluoxetine and its desmethyl metabolite, norfluoxetine, in serum by reversed-phase high-performance liquid chromatography (HPLC), with ultraviolet detection at 226 nm. Fluoxetine and norfluoxetine are isolated from serum by liquid-liquid extraction. They are then separated by HPLC and quantified, with reduced haloperidol as the internal standard. Fluoxetine, norfluoxetine, and the reduced haloperidol are separated from all interfering peaks in about 15 min. The standard curve is linear (r = 1.000) for both fluoxetine and norfluoxetine concentrations over the range of 25 to 800 micrograms/L. Between-run CVs for 60 and 200 micrograms/L controls (n = 8) were 6.8 and 4.1% for fluoxetine, and 8.8 and 6.2% for norfluoxetine, respectively. In a study of 24 patients with depression who were being treated with 20-60 mg of fluoxetine per day, fluoxetine and norfluoxetine concentrations in serum, measured during the last three weeks of treatment, were 47-469 micrograms/L and 52-446 micrograms/L, respectively.

Metabolic studies on the nigrostriatal toxin MPTP and its MAO B generated dihydropyridinium metabolite MPDP+.

The metabolic fates of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its two-electron oxidation product, the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), have been examined in mouse brain and liver tissue fractions. Incubations of MPTP (50 microM and 1 mM) with mouse brain preparations result in the expected MAO B catalyzed formation of MPDP+. The four-electron oxidation product, the 1-methyl-4-phenyl-pyridinium species (MPP+), was the only other metabolite detected. The oxidation of 50 microM MPDP+ to MPP+ in the same preparations appears to be mediated by unidentified components present in membrane containing structures. The behavior of MPTP in mouse liver subcellular fractions is considerably more complex. NADPH-supplemented liver microsomes convert MPTP to the desmethyl and N-oxide metabolites. At high (1 mM) initial concentrations of MPTP there is evidence that NADPH-dependent, pargyline-insensitive liver microsomal enzymes also catalyze the oxidation of MPTP to MPDP+. Incubations of MPDP+ with mouse liver preparations containing the cytosolic fraction led to the rapid disappearance of the substrate and the quantitative formation of a metabolic product with mass spectral and diode array UV characteristics expected for a lactam structure. Menadione, an inhibitor of the cytosolic enzyme aldehyde oxidase, inhibits the formation of this product. Unambiguous characterization of this metabolite as 1-methyl-4-phenyl-5,6-dihydro-2-pyridone was achieved by comparison of its high-resolution 1H NMR and high-resolution EI mass spectra with the corresponding spectra of a synthetic standard.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatograpic method for the determination of carvedilol and its desmethyl metabolite in body fluids

High-performance liquid chromatograpic method for the determination of carvedilol and its desmethyl metabolite in body fluids

Determination of azelastine and desmethylazelastine in human plasma by high-performance liquid chromatography

A manual-injection liquid chromatographic method using fluorescence detection permitted determination of a new antiasthmatic drug, azelastine, and its desmethyl metabolite extracted from human plasma. Reliable quantitation was achieved to at least 0.3 ng/ml for each analyte. No interference was seen in co-chromatography of sixteen other substances, which were potential co-medications (or their metabolites) as used in standard asthma or allergy treatment.

High sensitivity assay for the alpha 2 antagonist 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine and its desmethyl metabolite in plasma using gas chromatography mass spectrometry with ammonia/CCl4 chemical ionization.

A rapid and highly sensitive assay for quantifying the alpha 2-adrenoceptor antagonist SK&F 86466 and a desmethyl metabolite has been developed, using capillary column gas chromatography/chemical ionization mass spectrometry. This assay uses deuterium-labeled analogs of the analytes as internal standards, and has a lower limit of quantification of 100 pg ml-1. A novel reagent gas, consisting of carbon tetrachloride in anhydrous ammonia, was used to achieve maximal sensitivity.

Single and multiple dose kinetics of a new antiepileptic drug, Org 6370, and its desmethyl metabolite, Org 6363.

The pharmacokinetic parameters of the new antiepileptic drug, Org 6370, and its desmethyl metabolite, Org 6363, were studied in healthy male volunteers. Plasma concentrations of the compounds were determined by a new method using liquid-solid extraction and capillary gas-chromatographic separation with a nitrogen-selective detector. The kinetic parameters obtained after a single oral dose of Org 6370 were not good predictors of multiple-dose parameters. With long-term treatment, there was unanticipated accumulation of the parent drug and especially the metabolite. The clinical implication of these findings is that caution must be exercised in clinical trials of Org 6370.

Determination of metapramine, imipramine, trimipramine and their major metabolites in plasma by reversed-phase column liquid chromatography

The determination of metapramine, imipramine, trimipramine and their desmethyl metabolites after alkaline diethyl ether extraction from plasma is achieved by column liquid chromatography using two internal standards and a μBondapak C 18 column. Elution is carried out isocratically at 1 or 0.6 ml min −1 with two mixtures of acetonitrile—potassium dihydrogen phosphate—distilled water (45:55:10 for metapramine and its metabolites; 45:50:5 for imipramine, trimipramine and their metabolites). Detection is monitored by absorption at 254 nm. The detection limit is < 5 ng ml −1 for each compound. The coefficients of variation (within-day and day-to-day) for the eight compounds are > 11.3%. Interference from several possible co-medications is discussed. The technique can be used for routine therapeutic monitoring of the antidepressants as well as analytical toxicology. However, the three antidepressants cannot be analysed simultaneously by this method because metapramine requires a different elution system and imipramine interferes with monodesmethyltrimipramine (retention times 8.90 and 8.60 min, respectively).

Femoxetine and cimetidine: interaction in healthy volunteers.

The possibility of a pharmacokinetic interaction between femoxetine and cimetidine has been evaluated in 8 healthy volunteers. Two volunteers received single doses of femoxetine, and 6 were given multiple doses of femoxetine for 7 days with and without concurrent cimetidine. No influence of cimetidine was observed on the kinetics of single doses of femoxetine, but after multiple doses the plasma concentration of femoxetine was significantly increased. Similarly, the AUC at steady state tended to be increased, but not to a significant extent. Concurrent cimetidine did not cause a reduction in the AUC of the active desmethyl metabolite. It is recommended that femoxetine is given in reduced doses (e.g. 400 mg) when administered with cimetidine.

Determination of fezolamine and its desmethyl metabolite in human plasma and urine by high-performance liquid chromatography : Intravenous pharmacokinetics in the beagle hound

Sensitive and selective high-performance liquid chromatographic methods for the quantitation of the experimental antidepressantfezolamine and its desmethyl metabolite in plasma and urine have been developed. Both assays are linear between 0 and 500 ng/ml in both plasma and urine and have calculated minimum quantifiable levels of less than 10 ng/ml. Statistical evaluation of analytical parameters under single-blind conditions demonstrated an overall precision within ±4% of nominal for both compounds in either biological medium. The overall accuracies of the assays were within ±5% of nominal values in urine and ±10% of nominal values in plasma. Following intravenous administration of fezolamine fumarate to beagle hounds, a biexponential decline in drug plasma levels was observedwith the first phase having a halflife of about 11 min and the second phase about 2.6 h. Peak plasma levels of the metabolite were observed at 2 h. Recovery of the parent drug in urine was less than 5% of the administered dose and less than 1% for the desmethyl metabolite.

Determination of citalopram, amitriptyline and clomipramine in plasma by reversed-phase high-performance liquid chromatography

The determination of citalopram, amitriptyline, clomipramine and their desmethyl metabolites after alkaline diethyl ether extraction from plasma is achieved by high-performance liquid chromatography using two internal standards and μBondapak C 18 as stationary phase. Elution is carried out isocratically at 0.5 or 1 ml/min with mixture of acetonitrile- potassium dihydrogen phosphate-distilled water (45:50:5). Detection is monitored by absorption at 254 nm. The detection limit is less than 5 ng/ml for each compound. The coefficients of variation are between 1.3% and 9.4% for 8-360 ng/ml. Interference from 22 possible co-medications is discussed. The technique can be used for therapeutic monitoring of these antidepressants as well as in analytical toxicology.

Simultaneous measurement of various antidepressants in the plasma of depressed patients by high performance liquid chromatography.

A simultaneous analytical method was reported for measuring the plasma levels of amitriptyline, imipramine, clomipramine, maprotiline, nortriptyline, desipramine, desmethylclomipramine, desmethylmaprotiline and amoxapine by high performance liquid chromatography (HPLC). The total plasma levels of each parent drug plus its desmethyl metabolite were monitored in 29 depressed patients administered with amitriptyline, maprotiline or amoxapine using the present analytical method. There were significant linear correlations between the dose per kg body weight and the total plasma levels with amitriptyline and maprotiline, but no such correlation was found with amoxapine. The ratios of total plasma levels to dose per kg body weight of these three drugs were lower in outpatients than in inpatients. These results indicate that the monitoring of plasma levels of antidepressants is useful in treating depression.

The metabolic fate of [14C]oxaprotiline.HCl in man. I. Disposition and preliminary pharmacokinetics.

Absorption, biotransformation and elimination of [14C]oxaprotiline.HCl have been studied after oral administration of 50 mg doses to two human subjects. Absorption was complete, and peak blood concn. of total 14C were 590 and 297 ng equiv./ml after 3-6 h in the two subjects. After 11 days, 84 and 90% dose was excreted in urine, and a total of 98% was excreted. Peak blood concn. of unchanged oxaprotiline were 16 and 19 ng/ml before, and 167 and 207 ng/ml after enzymic hydrolysis. The blood half-life in the two subjects was 23 and 29 h. The blood concn. of the desmethyl metabolite was low (2 ng equiv./ml), but also increased after hydrolysis (11-19 ng equiv./ml). Oxaprotiline was bound (83%) in vitro to serum proteins. Sixty per cent was bound to serum albumin and 20% to alpha 1-acid glycoprotein. In urine only 1% of total 14C was present as unchanged oxaprotiline, and 0.2% as the desmethyl metabolite. After enzymic hydrolysis these increased to 48 and 6%, respectively, and after acid hydrolysis to 85 and 10%.

Determination of tiapamil and of its two main metabolites in plasma and in urine by high-performance liquid chromatography

Selective high-performance liquid chromatographic methods for the determination of tiapamil and its two main metabolites in plasma and urine are described. Tiapamil together with its metabolites is extracted at alkaline pH into dichloromethane. Separation is carried out using normal-phase high-performance liquid chromatography with ultraviolet detection (278 nm). The unchanged drug and the desmethyl metabolite are analysed simultaneously. The second metabolite is analysed separately under more polar conditions. The sensitivity limits are 50 ng/ml for tiapamil, 100 ng/ml for the desmethyl metabolite and 75 ng/ml for the second metabolite, using 0.5 ml of plasma. The sensitivity limits in urine are 100 ng/ml for all three compounds using a 0.5 ml specimen. The method has been applied to the analysis of human plasma and urine after intravenous (70 mg) and oral (400 mg) administration of tiapamil.

High-performance liquid chromatographic method for the determination of indomethacin and its two primary metabolites in urine

Quantitation of total amounts (i.e., free compound plus glucuronide conjugate) of indomethacin (INDO) and its deschlorobenzoyl (DBI) and desmethyl (DMI) metabolites in human urine is described. An aliquot (0.4 ml) of urine is incubated with glucuronidase (1000 U, 2 h, 37° C) and extracted with 5 ml of dichloromethane containing the internal standards: the fluoro analogue of INDO, F-INDO, and indole-3-propionic acid (IPA). The organic phase is concentrated, dissolved in mobile phase and aliquots are injected onto the high-performance liquid chromatograph. INDO and DMI are measured with UV detection at 264 nm over a linear range of 0.25–125 μg/ml. Retention times for DMI, F-INDO and INDO are 4.0, 6.8 and 12.1 min, respectively, using a C, reversed-phase column with an acetonitrile-O.1 M acetate, pH 5.0 (30:70) mobile phase at a 2.5 ml/min flow-rate. DBI is measured using fluorescence detection (excitation = 305 nm, emission = 370 nm) over a linear range of 0.26–12.5 μg/ml. Retention times for DBI and IPA are 4.5 and 7.8 min, respectively, on the same C 8 column with an acetonitrile-0.025 M acetate, pH 4.0 (22:78) mobile phase at a 2.0 ml/min flow-rate. Inter- and intra-day precision were smaller than 10% for INDO, DMI and DBI over the concentration ranges indicated.

Thin-layer chromatographic determination of imipramine and desipramine in human plasma and urine at single—dose levels

Thin-layer chromatographic methods were up-dated for pharmacokinetic studies of imipramine in plasma and urine. The free parent compound and its free desmethyl metabolite desipramine are determined in plasma. Conjugates of both compounds in urine are cleaved on treatment with glucuronidase/arylsulfatase. Following chromatography, intense yellow derivatives are obtained overnight on standing or by exposure to nitrous gases. Detection is performed in the visible range at 405 nm (plasma) or 460 nm (urine). The methods are selective, accurate and sensitive, with detection limits for plasma of 2 ng/ml imipramine—HCl and 2 ng/ml desipramine—HCl, and 0.06 μg/ml total imipramine—HCl and 0.126 μg/ml total desipramine—HCl for urine. Pharmacokinetic data from plasma and urine results following single oral doses of 50 mg imipramine—HCl to eight volunteers were computed using one-compartment open models.

Determination of midazolam and two metabolites of midazolam in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring midazolam, a new anesthesia induction agent and hypnotic, and its hydroxymethyl and desmethyl metabolites in human plasma. Deuterated analogues of each compound are added to plasma as internal standards. The compounds are extracted from plasma with benzene containing 20% 1, 2-dichloroethane and after removal of the extracting solvent are dissolved in a solution of bis-(trimethylsilyl)acetamide and acetonitrile. An aliquot of this solution is analyzed by gas chromatography—mass spectrometry with the mass spectrometer set to monitor in the gas chromatographic effluent the M$$$ ions of drug, metabolites and internal standards generated by methane electron-capture negative chemical ionization. For all three compounds, the limit of quantitation is 1 ng ml −1, and the precision (relative standard deviation) at a concentration of 5 ng ml −1 is less than 6%. Measurable amounts of the hydroxymethyl, but not the desmethyl, metabolite of midazolam could be found in the plasma of humans given either an intravenous or an oral dose of midazolam maleate.

On the long acting gastric antisecretory activity of LM 24056 A non H 2 antagonist

LM 24056, a phenothiazine derivative with no central effects, can be classified as a non anti H 2 antisecretory agent with a long duration of action. Its activity was demonstrated orally at low dose in pentagastrin stimulated Shay rat and in Heidenhain pouch in dog against gastrin, pentagastrin, carbachol and test meal. LM 24056 was inactive in histamine induced secretion. LM 24056 possesses very weak affinity to muscarinic receptors in vitro and in vivo. It has negligible anticholinergic properties in rats and mice at the peripheral level but no effect at the central level. The long lasting antisecretory action of LM 24056 may be supported by the persistent presence in plasma of a desmethyl metabolite at higher concentrations tham that of LM 24056 at any time. Contrary to LM 24056 sulfoxide and LM 24056 sulfone, desmethyl LM 24056 is a more potent antisecretory drug than LM 24056. Desmethyl LM 24056 possesses more marked affinity to peripheral muscarinic receptor than LM 24056. As the administration of therapeutic doses of LM 24056 was not followed by anticholinergic side-effects, it may be suggested that LM 24056 activity is related to a “prodrug like effect”. Finally the activity of LM 24056 may be related to LM 24056 itself and/or a desmethyl metabolite.