• Home
  • Search Results
  • A new and fast DLLME-CE method for the enantioselective analysis of zopiclone and its active metabolite after fungal biotransformation.

A new and fast DLLME-CE method for the enantioselective analysis of zopiclone and its active metabolite after fungal biotransformation.

Journal of pharmaceutical and biomedical analysis (2015-03-18)
Nayara Cristina Perez de Albuquerque, Cristiane Masetto de Gaitani, Anderson Rodrigo Moraes de Oliveira
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
N,N-Dimethylformamide, anhydrous, 99.8%
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Acetone, ACS reagent, ≥99.5%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%
Sigma-Aldrich
Dichloromethane, anhydrous, ≥99.8%, contains 40-150 ppm amylene as stabilizer
Sigma-Aldrich
2-Propanol, BioReagent, for molecular biology, ≥99.5%
Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Supelco
Methanol, analytical standard
Sigma-Aldrich
2-Propanol, suitable for HPLC, 99.9%
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Dichloromethane, suitable for HPLC, ≥99.8%, contains amylene as stabilizer
Sigma-Aldrich
2-Propanol, ACS reagent, ≥99.5%
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Chloroform, contains 100-200 ppm amylenes as stabilizer, ≥99.5%
Sigma-Aldrich
Dichloromethane, contains 40-150 ppm amylene as stabilizer, ACS reagent, ≥99.5%
Sigma-Aldrich
Acetone, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Acetone, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Sodium hydroxide, reagent grade, ≥98%, pellets (anhydrous)
Sigma-Aldrich
Sodium hydroxide, ACS reagent, ≥97.0%, pellets
Sigma-Aldrich
Dichloromethane, puriss. p.a., ACS reagent, reag. ISO, ≥99.9% (GC)
Sigma-Aldrich
Tris(hydroxymethyl)aminomethane, ACS reagent, ≥99.8%