11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide exhibits acyl-migration isomers


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Abstract

HIGHLIGHTSTHCCOOH-glucuronide undergoes isomerization by acyl migration.In-vitro, 6 isomers were detected and the degradation product THCCOOH.In urine and bile of cannabis users 1 major glucuronide and 4 isomers were detected.The major authentic THCCOOH-glucuronide differs from reference standards.Acyl migration does not lead to formation of the unknown THCCOOH-isomer.11-nor-Δ9-Tetrahydrocannabinol-9-carboxylic acid glucuronide (THCCOOH-glucuronide) is an 1-β-O-acyl glucuronide which degrades not only to 11-nor-9-carboxy-Δ9-THC (THCCOOH) but, additionally, to an isomer with a currently unknown structure. The present study was carried out to examine whether acyl glucuronide isomers are formed by acyl migration and if they are involved in formation of this isomer.THCCOOH-glucuronide was incubated in phosphate buffer (pH 7.4, 37 °C, 7 days) and a variety of glucuronide cleavage procedures were performed. Samples of the incubation mixture and of different biological specimens from cannabis users were analyzed using liquid chromatography-mass spectrometry (LC–MS/MS).A total of six chromatographically separated isomeric acyl glucuronides were detected during incubation of THCCOOH-glucuronide reference substance. In biological specimens of cannabis users, two additional isomers were found. However, the main glucuronide present in human specimens was different from that of a commercially available reference substance. Both, the commercial and the authentic glucuronide were cleaved by β-glucuronidases, the other formed isomers by alkaline hydrolysis only. Mass spectrometric investigations (i.e. product ion, precursor ion and neutral loss scans) confirmed identity. The THCCOOH isomer was detected in all authentic samples, but not in those after buffer incubation.By analyzing THCCOOH-glucuronide in authentic samples, it has to be taken into account that the authentic glucuronide is different from that of the commercial reference standard. THCCOOH-glucuronide undergoes acyl migration and some isomers occur to minor extents in biological specimens. Acyl migration does not lead to the formation of the THCCOOH isomer.

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