Rapid method for the simultaneous analysis of hydrocortisone and clioquinol in topical preparations by high-performance liquid chromatography.

Reversed-phase high performance liquid chromatographic methods for the analysis of ointments containing hydrocortisone and clioquinol have been investigated. A successful method using a C18 column and methanol--0.05 M phosphoric acid (80:20) as eluting solvent has been developed which allows both compounds to be determined simultaneously. The high-performance liquid chromatographic procedure is rapid and sensitive whereas the assay described in the 1980 BP involves a different method for the analysis of each component of the ointment. The method has also been further applied to the analysis of ointments containing hydrocortisone combined with other halogenated hydroxyquinolines.

Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of aldehyde oxidase.

Molybdenum(V) e.p.r. spectra from reduced forms of aldehyde oxidase were obtained and compared with those from xanthine oxidase. Inhibited and Desulpho Inhibited signals from aldehyde oxidase were fully characterized, and parameters were obtained with the help of computer simulations. These differ slightly but significantly from the corresponding parameters for the xanthine oxidase signals. Rapid type 1 and type 2 and Slow signals were obtained from aldehyde oxidase, but were not fully characterized. From the general similarities of the signals from the two enzymes, it is concluded that the ligands of molybdenum must be identical and that the overall co-ordination geometries must be closely similar in the enzymes. The striking differences in substrate specificity must relate primarily to structural differences in a part of the active centre concerned with substrate binding and not involving the catalytically important molybdenum site.

Analysis of azanaphthalenes and their enzyme oxidation products by high-performance liquid chromatography, infrared spectroscopy and mass spectrometry.

Adsorption and reversed-phase high-performance liquid chromatography (HPLC) have been successfully used to separate metabolites from the parent heterocycles (isoquinoline, 3-methylisoquinoline, phthalazine, quinazoline, quinoxaline and cinnoline). Retention data are reported. The metabolites, hydroxyazanaphthalenes, which arise as a result of aldehyde oxidase catalysed oxidation, could be extracted in microgram quantities from incubation mixtures by n-butanol. Complete identification of the oxidation products was achieved by collecting fractions corresponding to each compound from the HPLC eluate and subjecting these to infrared and mass-spectral analysis.

The oxidation of azaheterocycles with mammalian liver aldehyde oxidase.

1. Isoquinoline, cinnoline, quinoxaline, quinazoline and phthalazine were incubated with preparations of rabbit liver aldehyde oxidase. 2. The oxidation products, 1-hydroxyisoquinoline, 4-hydroxycinnoline, 2-hydroxy- and 2,3-dihydroxy-quinoxaline, 4-hydroxy- and 2,4-dihydroxy-quinazoline, and 1-hydroxyphthalazine were identified by comparison of their spectral and chromatographic characteristics with those of authentic compounds. 3. Michaelis-Menten constants are reported for the action of the parent heterocycles with aldehyde oxidase. The compounds reported in this study are among the most efficient substrates yet described for rabbit liver aldehyde oxidase. 4. The compounds in 1 above were incubated with bovine milk xanthine oxidase: only quinazoline and phthalazine yielded significant amounts of metabolites. Km values were calculated for these compounds. 5. Incubation of the heterocycles with rat liver preparations gave qualitatively the same results as those obtained using rabbit liver, but smaller amounts of the oxidation products were detected from rat liver incubations.