Determination of O-dealkylation products of R-(−)- and S-(+)-10,11-methylenedioxy- N-n-propylnoraporphine in Cunninghamella elegans cultures by liquid chromatography

Abstract

A rapid and selective liquid chromatographic method is described for the simultaneous determination of 10,11-methylenedioxy- N-n-propylnoraporphine (MDO-APO) and N-n-propylnorapomorphine (NPA) in Cunninghamella elegans (ATCC 9245) cultures. Isolation of substrate R-(−)- or S-(+)-MDO-NPA, metabolite R-(−)- or S-(+)-NPA and internal standard R-(−)-10,11-methylenedioxyaporphine (MDO-APO) was achieved via liquid-liquid extraction from buffered fermentation samples (pH 7.5), and were confirmed by the addition of chemically pure compounds. Aliquots were separated on a 3-μm Supelcosil LC-CN column using acetonitrile-phosphate (pH 3.0) (19+81) as eluent and quantified by monitoring the ultraviolet absorbance at 280 nm. A single chromatographic run could be completed in ca. 13 min; no interference from other metabolites or endogenous compounds was noted. The detection limit for aporphines was 0.33 nmol ml −1; the within-assay and day-to-day variation were consistently lower than ca. 7%. The time course of MDO-NPA metabolism by the fungus was also studied. The bioconversion studies showed that (+)-MDO-NPA was O-dealkylated to a slightly higher extent to (+)-NPA by C. elegans compared with the (−)-enantiomer; after 4 days of incubation the (+)-NPA/(−)-NPA product ratio was calculated to be 3.2:1.