A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min(-1). Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL(-1). The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL⁻¹ for each zopiclone enantiomer, 0.8-1.6 μg mL⁻¹ for 2-amino-5-chloropyridine and 0.4-0.8 μg mL⁻¹ for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.

Zopiclone, zolpidem and zaleplon (Z-drugs) are nonbenzodiazepine hypnotic drugs that are used for the treatment of insomnia. These drugs were developed with the intent to overcome some disadvantages of benzodiazepines, such as dependence and next day sedation. In general, the nonbenzodiazepine hypnotic drugs are administered in oral doses daily and are widely biotransformed in the body. A large number of analytical methods based on chromatographic and electrophoretic techniques for the quantification of Z-drugs and their metabolites in biological matrices have been reported. In this review, the bioanalytical methods for Z-drugs were reviewed with the focus placed on sample preparation procedures and the separation techniques used. Furthermore, as these drugs are also reported as drugs of abuse or in drug-facilitated crime, screening methods that simultaneously cover these drugs and also other drugs of abuse were included in this review.

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL min(-1). Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7% for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29-344.8 ng g(-1), with quantification limits of 0.29 ng g(-1) for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15%). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.

AGRADECIMENTOSAgradeço a Deus pelo amor incondicional, pela vida, por todos os milagres que realizas na minha vida, por todas as pessoas maravilhosas que conheci, por me carregar no colo nas horas mais difíceis. Por me fazer enxergar, a cada dia, o arco íris da vida mesmo quando rodeada pela neblina dos problemas. "Ser Feliz é deixar de ser vítima dos problemas e se tornar autor da própria história", "Senhor que sempre seja feita a Sua vontade".Aos meus pais Tadeu e Rute e meu irmão Matheus pelo amor. Amor eterno e incondicional. Família unida no amor. Vencemos as batalhas juntos, vivenciamos todos os milagres de Deus com fé e gratidão. Vocês são meu apoio, meu exemplo, minha fortaleza. Obrigada meus eternos amores. Obrigada por cada abraço de retorno ou despedida, por cada ida à rodoviária, por acreditarem em mim, por sempre me apoiarem, enfim por serem a família que eu tanto amo.Ao meu querido Ricardo pelo amor, apoio e compreensão. Obrigada por estar sempre comigo nos momentos perfeitos e nas horas mais difíceis. Ao cogitar retornar a Ribeirão Preto para mais essa etapa você me olhou nos olhos e disse "Eu tenho orgulho de você, você é capaz, vai . A moclobemida foi utilizada como padrão interno. O método desenvolvido foi linear no intervalo de concentração plasmática de 7,5-500 ng mL -1 . As recuperações médias absolutas foram de 74,6 e 75,7; 61,6 e 56,9; 72,5 e 70,7 % para os enantiômeros da ZO, N-Des e N-Ox, respectivamente, e 75,9 % para o padrão interno. A precisão e a exatidão apresentaram resultados dentro de níveis aceitáveis (<15 %). A aplicação do método em um estudo piloto de disposição cinética da ZO em ratos mostrou que os níveis de ( . Pôde-se observar que os níveis de (+)-(S)-ZO, o enantiômero mais ativo, foram sempre superiores aos de (-)-(R)-ZO. Finalmente, um terceiro método foi desenvolvido para análise enantiosseletiva da ZO e das suas impurezas N-Ox e ACP em comprimidos, empregando a eletroforese capilar com detecção UV (CE-UV). Os analitos foram extraídos dos comprimidos utilizando acetonitrila e foram separados em um capilar não revestido de sílica fundida (50 um, 42 cm de comprimento efetivo, 50 cm de comprimento total), utilizando tampão fosfato de sódio 80 mmol L Zopiclone (ZO) is a non-benzodiazepine hypnotic drug of the cyclopyrrolone class, indicated for the treatment of insomnia. ZO is a chiral drug administered as a racemic mixture, however its pharmacological activity is mainly related to (+)-(S)-ZO, also known as eszopiclone. It is extensively metabolized and the main metabolites are Ndesmethyl zopiclone (N-Des) and zopiclone-N-oxide (N-OX). N-Ox is also found as an impurity in the raw material. Other impurities, coming from the synthetic procedure or due to degradation can also be found: impurity B (RP29307), impurity C (2-amine-5-chloropyridine, ACP or RP26695) and RP 48497. Therefore, the aim of this study was the development of methods for the enantioselective analysis of ZO, metabolites and impurities in pharmaceutical formulations and biological materials. A highperform...