Floie M. Vane

Determination of isotretinoin or etretinate and their major metabolites in human blood by reversed-phase high-performance liquid chromatography

A method is described for the quantitative analysis of isotretinoin and its 4-oxo metabolite, or of etretinate and its principal metabolites, in human blood in the range 10-2000 ng/ml. Following a simple one-step extraction, the compounds are determined by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution and detection at 365 nm. This highly specific method separates the cis and trans isomers of the parent compounds and their metabolites. Examples are given of the application of this method to clinical studies of these two therapeutically important retinoids.

Diazepam metabolites in the rat: Characterization by high-resolution mass spectrometry and nuclear magnetic resonance

This report illustrates the usefulness of combining thin-layer chromatography for metabolite separation and purification with high-resolution mass spectrometry and nuclear magnetic resonance for metabolite characterization. Four metabolites of diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) were identified in the intestinal tract of one rat that had been injected intraperitoneally with 25 mg of3H-diazepam. These are the glucuronide and/or sulfate conjugates of 3-hydroxy diazepam (metabolite A), and the conjugates of the 5-hydroxyphenyl analogues of diazepam, 3-hydroxy diazepam, and N-desmethyl diazepam (metabolites B, C, and D, respectively). In each of the phenolic metabolites (B, C, and D) the hydroxyl function is in the para position.

Determination of 13-cis-retinoic acid and its major metabolite, 4-oxo-13-cis-retinoic acid, in human blood by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10--2000 and 50--2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.

Chlordiazepoxide metabolites in the rat. Characterization by high resolution mass spectrometry

Abstract Four rat urinary metabolites of chlordiazepoxide (7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine 4-oxide) labeled at C-2 with 14 C were separated by solvent extraction and thin-layer chromatography (TLC) and characterized by high resolution mass spectrometry. Each metabolite was found to have a hydroxyl function in the C-5 phenyl ring, and two-dimensional TLC of metabolites with reference compounds indicated that the most probable location of the phenolic function was para to the diazepine ring. The four metabolites were: 7-chloro-1,3-dihydro-5-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one 4-oxide (Metabolite 1); 2-amino-7-chloro-5-(4-hydroxyphenyl)-3H-1,4-benzodiazepine 4-oxide (Metabolite 2); 7-chloro-5-(4-hydroxyphenyl)-2-mwthylamino-3H-1,4-benzodiazepine 4-oxide (Metabolite 3); and 7-chloro-1,3-dihydro5-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one (Metabolite 4), which differs from the others in that it no longer retains the N-oxide function).

Human biliary metabolites of isotretinoin: identification, quantification, synthesis, and biological activity

1. The metabolites of isotretinoin (13-cis-retinoic acid, Accutane) were investigated in the bile of two patients with biliary T-tube drainage after administration of a single, oral, 80-mg dose of 14C-isotretinoin. Radioactivity measurements showed that the two patients excreted 22.7 and 17.1% of the dose in their bile in 4 days. 2. The two major drug-related components in the bile were identified as the glucuronide conjugates of 4-oxo-isotretinoin and 16-hydroxy-isotretinoin. Two minor components were identified as the glucuronide conjugates of isotretinoin and 18-hydroxy-isotretinoin. 3. H.p.l.c. analyses of Glusulase-treated bile samples indicated that the glucuronides of isotretinoin and the two major metabolites accounted for about 48% and 44% of the total radioactivity in the bile of the two patients. 4. Racemic 16-hydroxy-isotretinoin was synthesized and evaluated for its effect on human sebocytes in vitro. This metabolite and the other major metabolites of isotretinoin were less active than isotretinoin in inhibiting the proliferation of the sebocytes.

Studies on the Metabolism of 3-O-tert.-Butylmorphine, a New Morphine Derivative, by the Rat

1. The O-dealkylation of 3-O-tert.-butylmorphine, a new derivative of morphine, has been compared to the O-dealkylation of codeine by the rat. Rats were given either 3-O-tert.-butylmorphine or codeine intraperitoneally and 24 h urine samples were collected and analysed for morphine by a quantitative gas chromatographic procedure. With codeine, about 7% of the dose was excreted as morphine. In contrast, about 0.3% of a dose of 3-O-tert.-butylmorphine was recovered from urine as morphine. These results indicate that, unlike codeine, 3-O-tert.-butylmorphine is not O-dealkylated to any appreciable extent by the rat.2. Three metabolites of 3-O-tert.-butylmorphine have been recovered from rat urine, resolved by gas chromatography and identified by mass spectroscopy as 3-O-tert.-butylnormorphine, 3-O-(2-hydroxymethyl-2-propyl)morphine and 3-O-(2-hydroxymethyl-2-propyl)normorphine. The identities of the last two metabolites were confirmed by comparison of their mass spectra with the mass spectra of synthetic refe...

Concentrations of Isotretinoin and the Arotinoid Ro 15-0778 in Plasma and Tissues of Hamsters and Rats

The arotinoid Ro 15-0778 is a potent inhibitor of sebum secretion in rodents but not in humans, while isotretinoin (13-cis-retinoic acid, Accutane) inhibits sebum secretion in rodents and humans. The concentrations of Ro 15-0778 and isotretinoin were determined in plasma and target tissues of castrated, testosterone-stimulated hamsters after oral and topical dosing and in castrated, testosterone-stimulated rats after oral dosing. Comparison of this concentration data to that obtained in the plasma and sebum of humans after dosing with Ro 15-0778 and with isotretinoin did not explain why Ro 15-0778 was a potent inhibitor of sebum production in rodents but not in humans.

IN VITRO METABOLISM OF 13‐cis‐RETINOIC ACID BY 9,000 ×g RAT LIVER SUPERNATANT

13-cis-Retinoic acid (RA) was incubated for 1 hr at 37°C with 9,000 xg liver supernatant prepared from normal rats and fortified with an NADPH-generating system. Extracts of the incubation mixture were analyzed by reverse-phase high pressure liquid chromatography (HPLC). Metabolites isolated by HPLC were converted to methyl esters (ME) for mass spectrometry (MS) and nuclear magnetic resonance (NMR) studies. A typical HPLC profile is shown in FIGURE 1. The major peak was shown by MS and NMR to be intact 13-cis-RA. The MS and NMR of the methyl esters of I, 11, and IIIc were identical to the spectra of the reference compounds: methyl alltrans-retinoate, methyl 4-0x013-cis-retinoate and methyl 4-oxo-all-trans-retinoate, respectively. IIIb-ME was shown by MS to have a molecular weight of 330, which suggested a hydroxy metabolite. The NMR of IIIb-ME was very similar to

The Application of Various Spectroscopies to the Identification of Drug Metabolites

It has long been recognized that structural elucidation of drug metabolites contributes greatly to the understanding of the mechanism of drug action. However, in the past, the difficult and expensive nature of this task restricted studies to relatively few drugs.