title: A new and fast DLLME-CE method for the enantioselective analysis of zopiclone and its active metabolite after fungal biotransformation.
abstract: Zopiclone (ZO) is a chiral drug that undergoes extensive metabolism to N-desmethylzopiclone (N-Des-ZO) and zopiclone-N-oxide (N-Ox-ZO). Pharmacological studies have shown (S)-N-Des-ZO metabolite presents anxiolytic activity and a patent for this metabolite was requested for anxiety treatment and related disorders. In this context, biotransformation employing fungi may be a promising strategy to obtain N-Des-ZO. To perform the biotransformation study in this work, an enantioselective method based on capillary electrophoresis (CE) and dispersive liquid-liquid microextraction (DLLME) was developed. CE analyses were carried out in sodium phosphate buffer (pH 2.5; 50mmolL(-1)) containing 0.5% (w/v) carboxymethyl-β-CD, at a constant voltage of +25kV. DLLME was conducted using 2mL of liquid culture medium pH 9.5. Chloroform (100μL) and methanol (300μL) were employed as extraction and disperser solvent, respectively. After CE and DLLME optimization, the analytical method was fully validated. The method was linear over a concentration range of 90-6000ngmL(-1) for each ZO enantiomer (r>0.999) and 50-1000ngmL(-1) for each N-Des-ZO enantiomer (r>0.998). Absolute recovery of 51 and 82% was achieved for N-Des-ZO and ZO, respectively. The accuracy and precision results agreed with the EMA (European Medicines Agency) guideline, and so did the stability study. Application of the developed method in a biotransformation study was conducted in order to investigate the ability of fungi, belonging to the genus Cunninghamella, in metabolizing ZO chiral drug. Fungi Cunninghamella elegans ATCC 10028B and Cunninghamella echinulata var elegans ATCC 8688A demonstrated to be able to enantioselectively biotransform ZO to its active metabolite, N-Des-ZO. Therefore, the proposed goals of this work, i.e. a fast DLLME-CE method and an outstanding strategy to obtain N-Des-ZO, were successfully attained.
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Nayara Cristina Perez
authors.initials: NC
authors.lastname: de Albuquerque
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Cristiane Masetto
authors.initials: CM
authors.lastname: de Gaitani
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: deoliveira@usp.br.
authors.forename: Anderson Rodrigo Moraes
authors.initials: AR
authors.lastname: de Oliveira
mesh_terms: D053961:Azabicyclo Compounds / Q000032:analysis* / Q000378:metabolism*; D001711:Biotransformation; D020093:Cunninghamella / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D006993:Hypnotics and Sedatives / Q000032:analysis* / Q000378:metabolism*; D007202:Indicators and Reagents; D059627:Liquid Phase Microextraction; D010879:Piperazines / Q000032:analysis* / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D053961:Azabicyclo Compounds; D006993:Hypnotics and Sedatives; D007202:Indicators and Reagents; D010879:Piperazines; C513824:desmethylzopiclone; C515050:zopiclone
keywords: Biotransformation; Dispersive liquid–liquid microextraction; Enantiomeric separation; N-desmethylzopiclone; Zopiclone