Enantioselective Analysis of Fluoxetine and Norfluoxetine by LC in Culture Medium for Application in Biotransformation Studies Employing Fungi

A liquid chromatography method is described for the analysis of fluoxetine and norfluoxetine enantiomers in fungi cultures. The analytes were separated simultaneously by LC employing a serial system. The resolution was performed using a mobile phase of ethanol: 15 mM ammonium acetate buffer solution, pH 5.9: acetonitrile (77.5:17.5:5, v/v/v). UV detection was at 227 nm. Hexane: isoamyl alcohol (98:2, v/v) was used as extractor solvent. The calibration curves were linear over the concentration range of 12.5-3,750 ng mL(-1) (r a parts per thousand yen 0.996). The values for intra- and inter-day precision and accuracy were a parts per thousand currency sign10% for all analytes. The validated method was used to evaluate fluoxetine biotransformation to its mammalian metabolite, norfluoxetine, by selected endophytic fungi. Although the desired biotransformation was not observed in the conditions used here, the method could be used to evaluate the biotransformation of fluoxetine by other fungi or to be extended to other matrices with adequate procedures for sample preparation.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

Two-step liquid-phase microextraction and high-performance liquid chromatography for the simultaneous analysis of the enantiomers of mefloquine and its main metabolite carboxymefloquine in plasma

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase micro-extraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35-38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (-)-(SR)-MQ after oral administration of the racemic drug to rats.

Enantiomeric resolution of (+/-)-licarin A by high-performance liquid-chromatography using a chiral stationary phase

(+/-)-Licarin A (1), a neolignan obtained by the oxidative coupling reaction of isoeugenol, had in this study its enantiomers resolved. A novel, quick and efficient enantiomeric resolution of 1 was directly performed by chiral high-performance liquid chromatography (HPLC-PDA) protocol (CHIRALPACK (R) AD column; 9:1 (v/v) n-hexane:2-propanol; 1.0 mL/min). This method provided a chromatogram profile with a well-resolved peak separation. After isolation of each enantiomer with ee >99.9%, they were analysed in a polarimeter. Compound 2, which showed a retention time (t(r)) of 12.13 min, was the (+)-enantiomer and compound 3 (t(r) =18.90 min) was the (-)-enantiomer. (C) 2011 Elsevier B.V. All rights reserved.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there area limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 mu L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCI was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 mu g L(-1) and 0.1 mu Lg L(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. (C) 2009 Elsevier B.V. All rights reserved.

A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS

A simple method for mercury speciation in hair samples with a fast sample preparation procedure using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry is proposed. Prior to analysis, 50 mg of hair samples were accurately weighed into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of inorganic mercury (Ino-Hg), methylmercury (Met-Hg) and ethylmercury (Et-Hg) was accomplished in less than 8 min on a C18 reverse phase column with a mobile phase containing 0.05% v/v mercaptoethanol, 0.4% m/v L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% v/v methanol. The method detection limits were found to be 15 ng g(-1), 10 ng g(-1) and 38 ng g(-1), for inorganic mercury, methylmercury and ethylmercury, respectively. Sample throughput is 4 samples h(-1) (duplicate). A considerable improvement in the time of analysis was achieved when compared to other published methods. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Finally, the proposed method was successfully applied to the speciation of mercury in hair samples collected from fish-eating communities of the Brazilian Amazon.

Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

Solid-phase microextraction using poly(pyrrole) film and liquid chromatography with UV detection for analysis of antidepressants in plasma samples

Poly(pyrrole) (PPY) coating was prepared on a stainless-steel (SS) wire for solid-phase microextraction (SPME) by electrochemical deposition (cyclic voltammetric). The PPY was evaluated by analyzing new-generation antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline) in plasma sample by SPME and liquid chromatography with UV detection (LC-UV). The effect of electrolyte Solution (lithium perchlorate or tetrabutylammonium perchlorate) and the number of cycles (50, 100 or 200) applied during the polymerization process on the SPME performance was evaluated. Important factors in the optimization of SPME efficiency such as extraction time, temperature, pH, influence of plasma proteins on sorption mechanisms, and desorption conditions are discussed. The SPME-PPY/LC method showed to be linear in concentrations ranging from the limit of quantification (LOQ) to 1200 ng mL(-1). The LOQ values range from 16 to 25 ng mL-1. The inter-day precision of the SPME-PPY/LC method presented coefficient of variation (CV) lower than 15%. Based on analytical validation results, the SPME-PPY/LC methodology showed to be adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the SPME-PPY/LC method was applied to the analysis of plasma samples from elderly depressed patients. (c) 2009 Elsevier B.V. All rights reserved,

Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high-performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Separation was obtained using a reverse phase C-18 column with UV detection (210 nm). The mobile phase consisted of water: acetonitrile (78:22, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.08-40.0 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125-40.0 mu g mL(-1) for phenytoin, The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.0, 4.0 and 20.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125 mu g mL(-1) for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. Based on figures of merit results, the SBSE/HPLC-UV proved adequate for antiepileptic drugs analyses from therapeutic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (c) 2008 Elsevier B.V. All rights reserved.

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirrazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50 ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500 ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants. (c) 2007 Elsevier B.V. All rights reserved.

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 mu L.), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. (C) 2010 Elsevier B.V. All rights reserved.

Simultaneous analysis of parabens in cosmetic products by stir bar sorptive extraction and liquid chromatography

A sensitive and precise stir bar sorptive extraction (SBSE) combined with LC (SBSE/LC) analysis is described for simultaneous determination of methyl, ethyl, propyl, and butyl parabens in commercial cosmetic products in agreement with the European Union Cosmetics Directive 76/768/EEC. Important factors in the optimization of SB SE efficiency are discussed, such as time and temperature of extraction, pH, and ionic strength of the sample, matrix effects, and liquid desorption conditions by different modes (magnetic stirring, ultrasonic). The LOQs of the SBSE/LC method ranged from 30 to 200 ng/mg, with linear response over a dynamic range, from the LOQ to 2.5 mu g/mg, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%. The effectiveness of the proposed method was proven for analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens.

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C(8) column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125-50.0 mu g mL(-1). The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 mu g mL(-1). Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (C) 2009 Elsevier B.V. All rights reserved.

Automated determination of rifampicin in plasma samples by in-tube solid-phase microextraction coupled with liquid chromatography

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1-100 mu g/mL range, with a linear coefficient value (r(2)) of 0.998. The inter-assay precision presented coefficient of variation <= 1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. (C) 2011 Elsevier B.V. All rights reserved.

Gingival capillary changes and oral motor weakness in juvenile dermatomyositis

Objective. We assessed the orofacial involvement in JDM, and evaluated the possible association of gingival and mandibular mobility alterations with demographic data, periodontal indices, clinical features, muscle enzyme levels, JDM scores and treatment. Methods. Twenty-six JDM patients were studied and compared with 22 healthy controls. Orofacial evaluation included clinical features, dental and periodontal assessment, mandibular function and salivary flow. Results. The mean current age was similar in patients with JDM and controls (P > 0.05). A unique gingival alteration characterized by erythema, capillary dilation and bush-loop formation was observed only in JDM patients (61 vs 0%, P = 0.0001). The frequencies of altered mandibular mobility and reduced mouth opening were significantly higher in patients with JDM vs controls (50 vs 14%, P = 0.013; 31 vs 0%, P = 0.005). Comparison of the patients with and without gingival alteration showed that the former had lower values of median of cementoenamel junction (-0.26 vs -0.06 mm, P = 0.013) and higher gingival bleeding index (27.7 vs 14%, P = 0.046). This pattern of gingival alteration was not associated with periodontal disease [plaque index (P = 0.332) and dental attachment loss (P = 0.482)]. The medians for skin DAS and current dose of MTX were higher in JDM with gingival alteration (2.5 vs 0.5, P = 0.029; 28.7 vs 15, P = 0.012). A significant association of lower median manual muscle testing with a reduced ability to open the mouth was observed in patients with JDM than those without this alteration (79 vs 80, P = 0.002). Conclusions. The unique gingival pattern associated with cutaneous disease activity, distinct from periodontal disease, suggests that gingiva is a possible target tissue for JDM. In addition, muscle weakness may be a relevant factor for mandibular mobility.