Bioanalytical method for in vitro metabolism study of repaglinide using 96-blade thin-film solid-phase microextraction and LC-MS/MS.

BACKGROUND: A high-throughput bioanalytical method using 96-blade thin film microextraction (TFME) and LC-MS/MS for the analysis of repaglinide (RPG) and two of its main metabolites was developed and used for an in vitro metabolism study. RESULTS: The target analytes were extracted from human microsomal medium by a 96-blade-TFME system employing the low-cost prototype 'SPME multi-sampler' using C18 coating. Method validation showed recoveries around 90% for all analytes and was linear over the concentration range of 2-1000 ng ml(-1) for RPG and of 2-500 ng ml(-1) for each RPG metabolite. CONCLUSION: The method was applied to an in vitro metabolism study of RPG employing human liver microsomes and proved to be very useful for this purpose.

Hollow-fiber liquid-phase microextraction and chiral LC-MS/MS analysis of venlafaxine and its metabolites in plasma.

BACKGROUND: An enantioselective analytical method was developed and validated for determination of venlafaxine and its metabolites O-desmethylvenlafaxine and N-desmethylvenlafaxine in plasma samples. The method employed LC-MS/MS analysis and hollow-fiber liquid-phase microextraction (HF LPME) for sample preparation. RESULTS: After HF LPME optimization the following condition was established: sample volume of 4 ml, sample agitation at 1750 rpm, 20 min of extraction, 0.1 mol/l acetic acid as acceptor phase, 1-octanol as organic phase and donor phase pH adjustment to 10. Under these conditions, the method was linear over the concentration range of 5-500 ng/ml with quantification limits of 5 ng/ml. CONCLUSION: The use of HF LPME for sample preparation provided suitable recoveries, efficient clean-up and low consumption of organic solvent.

Enantioselective analysis of ranolazine and desmethyl ranolazine in microsomal medium using dispersive liquid-liquid microextraction and LC-MS/MS.

BACKGROUND: An enantioselective bioanalytical method using dispersive liquid-liquid microextraction (DLLME) and LC-MS/MS was developed for the chiral analysis of ranolazine (RNZ) and one of its metabolites (desmethyl ranolazine [DRNZ]). RESULTS: The analytes were extracted from microsomal medium by DLLME, using chloroform as extractor solvent and acetone as dispersive solvent. The enantiomers of RNZ and DRNZ were analyzed simultaneously for the first time using a Chiralcel OD-H(®). Method validation showed recoveries in the order of 55 and 45%, and LLOQ of 25 and 10 ng ml(-1) for the enantiomers of RNZ and DRNZ, respectively. Linearity was established in the concentration range of 10 to 1000 and 25 to 2500 ng ml(-1) for each DRNZ and RNZ enantiomer, respectively. CONCLUSION: The unprecedented use of DLLME was demonstrated to be very useful for sample preparation of microsomal matrix. Furthermore, the in vitro metabolism of RNZ was enantioselective.

Combination of hollow-fiber liquid-phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver microsomal fraction.

Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver microsomal fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 µm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver microsomal fraction.

HPLC analysis of midodrine and desglymidodrine in culture medium: evaluation of static and shaken conditions on the biotransformation by fungi.

A high-performance liquid chromatography (HPLC) method is presented for the simultaneous determination of midodrine and desglymidodrine (DMAE) in Czapek-Dox culture medium, to be used in biotransformation studies by fungi. The HPLC analysis was conducted using a Lichrospher 100 RP18 column, acetonitrile-40 mmol/L formic acid solution (60:40, v/v) as mobile phase, and ultraviolet detection at 290 nm. The sample preparation was conducted by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.4-40.0 µg/mL for midodrine (r ≥ 0.9997) and DMAE (r ≥ 0.9998). Within-day and between-day precision and accuracy were evaluated by relative standard deviations (≤ 8.2%) and relative errors (-7.3 to 7.4%), respectively. The validated method was used to assess midodrine biotransformation by the fungi Papulaspora immersa Hotson SS13, Botrytis cinerea UCA 992 and Botrytis cinerea 2100 under static and shaken conditions. Under shaken conditions, the biotransformation of midodrine to DMAE was more efficient for all studied fungi, especially for the fungus Botrytis cinerea 2100, which converted 42.2% of midodrine to DMAE.

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry.

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.

Capillary electrophoretic enantioselective determination of zopiclone and its impurities.

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-ß-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.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies.

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r ≥ 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r ≥ 0.9977) and 6-ODD (r ≥ 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Methods for the analysis of nonbenzodiazepine hypnotic drugs in biological matrices.

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.

Glutaraldehyde release from heat-polymerized acrylic resins after disinfection and chemical and mechanical polishing.

This study evaluated the release of glutaraldehyde from heat-polymerized acrylic resins subjected to disinfection followed by chemical and mechanical polishing. Ninety disc-shaped specimens (15 x 4 mm), 30 per resin (Lucitone 550, QC-20 and Classico), were made and assigned to 2 groups according to the type of polishing. One side of each specimen was not polished and the other was either mechanically (n = 45) or chemically (n = 45) polished, and immersed in water at 50 °C for 1 h to allow the release of intrinsic substances and then kept in distilled water for 7 days. The specimens were disinfected by immersion in 2% glutaraldehyde for 10 min. After this period, 3 specimens from each group were immersed in water for 15, 30, 60, 120 and 240 min. For the 15-, 30-, 60-min immersions, 4 water exchanges were done at the end of period. High performance liquid chromatography (HPLC) was used to detect and quantify the glutaraldehyde released after each period. Data were analyzed statistically by two-way ANOVA and multiple comparisons were done by Tukey's and Scheffé's tests (α = 0.05). No glutaraldehyde release was observed from the specimens with chemical polishing at any of the immersion periods, while the mechanically polished specimens released glutaraldehyde. In the groups with water exchanges, Lucitone released more disinfectant in the 15-min period (0.040 µg/mL), Classico in the 30-min (0.021 µg/mL) and 60-min (0.018 µg/mL) periods, and QC-20 the same amount (-1.760 µg/mL) in all periods. In the groups without water exchanges, Lucitone released the highest amount of disinfectant (-1.370 µg/mL), differing significantly from QC-20 (0022 g/mL) and Classico (0019 g/mL), which were similar. The findings of this showed that chemically polished specimens from the 3 resin brands did not release glutaraldehyde after different periods of immersion, while glutaraldehyde release was observed from the mechanically polished specimens, especially from those made of Lucitone resin.

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry.

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.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction.

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and ρ-hydroxy rosiglitazone (ρ-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and ρ-OH-R, respectively. Michaelis-Menten constant (K(m)) values were of 58.12 and 78.52 µM for N-Dm-R and ρ-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of ρ-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (ο-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

Young investigator.

Supervisor's supporting comments Keyller B Borges carried out his MS and PhD studies in toxicology under my supervision. During the time he spent in my laboratory, he worked on the development of analytical methods and studied the biotransformation of several drugs by fungi. Characteristics such as dedication, ability, initiative and dynamism had been the basis for the success of his projects developed under my supervision, resulting in several publications in prestigious periodics of the area. Therefore, I consider Dr Keyller highly capable for the development of activities in the area of bioanalysis.

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study.

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak(®) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 ± 2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.

Assessment of ametryn contamination in river water, river sediment, and mollusk bivalves in São Paulo state, Brazil.

São Paulo state, Brazil, is one of the main areas of sugar cane agriculture in the world. Herbicides, in particular, ametryn, are extensively used in this extensive area, which implies that this herbicide is present in the environment and can contaminate the surface water by running off. Thereby, residues of ametryn were analyzed in samples of river water an river sediment and in freshwater bivalves obtained from the rivers Sapucaí, Pardo and Mogi-Guaçu in São Paulo State, Brazil. Samples were taken in the winter of 2003 and 2004 in two locations in each river. The specimens of freshwater bivalves collected and analyzed were Corbicula fluminea, an exotic species, and Diplodon fontaineanus, a native species. Additionally, the evaluation of the ability of bioconcentration and depuration of ametryn by the freshwater bivalve Corbicula fluminea was also performed. Ametryn concentrations in the samples were measured by liquid chromatography coupled to mass spectrometry. Residues of ametryn in water (50 ng/L) and in freshwater bivalves (2-7 ng/g) were found in the Mogi-Guaçu River in 2004, and residues in river sediments were found in all rivers in 2003 and 2004 (0.5-2 ng/g). The observation of the aquatic environment through the analysis of these matrixes, water, sediment, and bivalves, revealed the importance of the river sediment in the accumulation of the herbicide ametryn, which can contaminate the biota.

LC-MS-MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi.

The purpose of this study was the development and validation of an LC-MS-MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 × 4.6 mm, 5 µm particle size), column temperature 8 °C, and the mobile phase hexane-isopropanol-trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min(-1). Post-column infusion with 10 mmol L(-1) ammonium acetate in methanol at a flow rate of 0.3 mL min(-1) was performed to enhance MS detection (positive electrospray ionization). Liquid-liquid extraction was used for sample preparation with hexane-ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1-20 µg mL(-1) for IBP, 0.05-7.5 µg mL(-1) for each 2-OH-IBP enantiomer, and 0.025-5.0 µg mL(-1) for each COOH-IBP stereoisomer (r ≥ 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at -20 °C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.

Glutaraldehyde release from heat-polymerized acrylic resins after disinfection and chemical and mechanical polishing

This study evaluated the release of glutaraldehyde from heat-polymerized acrylic resins subjected to disinfection followed by chemical and mechanical polishing. Ninety disc-shaped specimens (15 x 4 mm), 30 per resin (Lucitone 550, QC-20 and Classico), were made and assigned to 2 groups according to the type of polishing. One side of each specimen was not polished and the other was either mechanically (n=45) or chemically (n=45) polished, and immersed in water at 50°C for 1 h to allow the release of intrinsic substances and then kept in distilled water for 7 days. The specimens were disinfected by immersion in 2% glutaraldehyde for 10 min. After this period, 3 specimens from each group were immersed in water for 15, 30, 60, 120 and 240 min. For the 15-, 30-, 60-min immersions, 4 water exchanges were done at the end of period. High performance liquid chromatography (HPLC) was used to detect and quantify the glutaraldehyde released after each period. Data were analyzed statistically by two-way ANOVA and multiple comparisons were done by Tukey’s and Scheffé’s tests (α=0.05). No glutaraldehyde release was observed from the specimens with chemical polishing at any of the immersion periods, while the mechanically polished specimens released glutaraldehyde. In the groups with water exchanges, Lucitone released more disinfectant in the 15-min period (0.040 μg/mL), Classico in the 30-min (0.021 μg/mL) and 60-min (0.018 μg/mL) periods, and QC-20 the same amount (-1.760 μg/mL) in all periods. In the groups without water exchanges, Lucitone released the highest amount of disinfectant (-1.370 μg/mL), differing significantly from QC-20 (0022 g/mL) and Classico (0019 g/mL), which were similar. The findings of this showed that chemically polished specimens from the 3 resin brands did not release glutaraldehyde after different periods of immersion, while glutaraldehyde release was observed from the mechanically polished specimens, especially from those made of Lucitone resin.

Este estudo determinou a liberação de glutaraldeído de resinas acrílicas termopolimerizáveis submetidas a polimento químico e mecânico e desinfetadas. Noventa corpos-de-prova circulares (15 x 4 mm), 30 de cada tipo de resina (Lucitone, QC-20 e Clássico), foram confeccionados e divididos em 2 grupos referentes ao tipo de polimento. Um dos lados de cada corpo-de-prova não foi polido e o outro foi polido mecanicamente (n=45) ou quimicamente (n=45), e imersos em água aquecida a 50°C por 1 h para liberação de substâncias intrínsecas e mantidos em água destilada por 7 dias. A seguir era realizada a desinfecção por imersão em solução de glutaraldeído a 2% por 10 min. Decorrido este período, três corpos-de-prova de cada grupo eram imersos em água por 15, 30, 60, 120 e 240 min. Nos períodos de 15, 30 e 60 min foram realizadas até 4 trocas de água após cada período. As amostras eram analisadas por meio de cromatografia líquida de alta eficiência (HPLC) após cada período. Os dados foram analisados estatisticamente pela Análise de Variância e testes complementares de Tukey e Scheffé (α=0,05). Os corpos-de-prova com polimento químico, de todas as marcas comerciais de resina, não liberaram glutaraldeído em qualquer um dos períodos de imersão em água, enquanto os com polimento mecânico liberaram. Nos grupos com trocas de água, a resina Lucitone liberou maior quantidade de desinfetante nas trocas de 15 min (0,040 μg/mL), a resina Clássico nas de 30 (0,021 μg/mL) e 60 min (0,018 μg/mL) e a QC-20 liberou a mesma quantidade (-1,760 μg/mL), em todos os períodos de imersão em água. Nos grupos sem trocas de água, a resina Lucitone liberou maior quantidade de desinfetante (-1,370 μg/mL), sendo diferente estatisticamente das resinas QC-20 (0,022 μg/mL) e Clássico (0,019 μg/mL), que são similares. Pelos resultados conclui-se que corpos-de-prova polidos quimicamente, das três marcas comerciais de resina, não liberaram glutaraldeído após os diferentes períodos de imersão. Contudo, nos corpos-de-prova polidos mecanicamente houve liberação do desinfetante, com Lucitone liberando maior quantidade em relação às demais resinas estudadas.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation.

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

Stereoselective determination of midodrine and desglymidodrine in culture medium: application to a biotransformation study employing endophytic fungi.

A CE method was developed and validated for the stereoselective determination of midodrine and desglymidodrine in Czapek culture medium to be applied to a stereoselective biotransformation study employing endophytic fungi. The electrophoretic analyses were performed using an uncoated fused-silica capillary and 70 mmol/L sodium acetate buffer solution (pH 5.0) containing 30 mmol/L heptakis (2, 3, 6-tri-O-methyl)-beta-CD as running electrolyte. The applied voltage and temperature used were 15 kV and 15 degrees C, respectively. The UV detector was set at 200 nm. The sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.1-12 microg/mL for each enantiomer of midodrine and desglymidodrine (r> or =0.9975). Within-day and between-day precision and accuracy evaluated by RSDs and relative errors, respectively, were lower than 15% for all analytes. The method proved to be robust by a fractional factorial design evaluation. The validated method was used to assess the midodrine biotransformation to desglymidodrine by the fungus Phomopsis sp. (TD2), which biotransformed 1.1% of (-)-midodrine to (-)-desglymidodrine and 6.1% of (+)-midodrine to (+)-desglymidodrine.