Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2`,3`-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2`,3`-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry

Polydimethylsiloxane/polypyrrole stir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples

A new polymeric coating consisting of a dual-phase, polydimethylsiloxane (PDMS) and polypyrrole (PPY) was developed for the stir bar sorptive extraction (SBSE) of antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and sertraline) from plasma samples, followed by liquid chromatography analysis (SBSE/LC-UV). The extractions were based on both adsorption (PPY) and sorption (PDMS) mechanisms. SBSE variables, such as extraction time, temperature, pH of the matrix, and desorption time were optimized, in order to achieve suitable analytical sensitivity in a short time period. The PDMS/PPY coated stir bar showed high extraction efficiency (sensitivity and selectivity) toward the target analytes. The quantification limits (LOQ) of the SBSE/LC-UV method ranged from 20 ng mL(-1) to 50 ng mL(-1), and the linear range was from LOQ to 500 ng mL(-1), with a determination coefficient higher than 0.99. The inter-day precision of the SBSE/LC-UV method presented a variation coefficient lower than 15%. The efficiency of the SBSE/LC-UV method was proved by analysis of plasma samples from elderly depressed patients. (C) 2008 Elsevier B.V. All rights reserved.

Improving the Detectability of Sequential Injection Chromatography (SIC): Determination of Triazines by Exploiting Liquid Core Waveguide (LCW) Detection

Coupling a liquid core waveguide cell to a sequential injection chromatograph improved the detection limits for determination of triazine herbicides without compromising peak resolution. Separation of simazine, atrazine, and propazine was achieved in water samples by a 25mm long C18 monolithic column. Detection was made at 238nm using a type II LCW (silica capillary coated with Teflon (R) AF2400) cell with 100cm of optical path length. Detection limits for simazine, atrazine, and propazine were 2.3, 1.9, and 4.5 mu g L-1, respectively. Reduced analysis time and low solvent consumption are other remarkable features of the proposed method.

Biocompatible in-tube solid phase microextraction coupled with liquid chromatography-fluorescence detection for determination of interferon alpha in plasma samples

The present work demonstrates the successful application of automated biocompatible in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC) for determination of interferon alpha(2a) (IFN alpha(2a)) in plasma samples for therapeutic drug monitoring. A restricted access material (RAM, protein-coated silica) was employed for preparation of a lab-made biocompatible in-tube SPME capillary that enables the direct injection of biological fluids as well as the simultaneous exclusion of macromolecules by chemical diffusion barrier and drug pre-concentration. The in-tube SPME variables, such as sample volume, draw/eject volume, number of draw-eject cycles, and desorption mode were optimized, to improve the sensitivity of the proposed method. The IFN alpha(2a) analyses in plasma sample were carried out within 25 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range, from 0.06 to 3.0 MIU mL(-1), with correlation coefficient equal to 0.998. The interday precision of the method presented coefficient of variation lower than 8%. The proposed automated method has adequate analytical sensitivity and selectivity for determination of IFN alpha(2a) in plasma samples for therapeutic drug monitoring. (C) 2010 Elsevier B.V. All rights reserved.

Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Nickel hydroxide electrodes as amperometric detectors for carbohydrates in flow injection analysis and liquid chromatography

The present paper describes the utilization of nickel hydroxide modified electrodes toward the catalytic oxidation of carbohydrates (glucose, fructose, lactose and sucrose) and their utilization as electrochemical sensor. The modified electrodes were employed as a detector in flow injection analysis for individual carbohydrate detection, and to an ionic column chromatography system for multi-analyte samples aiming a prior separation step. Kinetic studies were performed on a rotating disk electrode (RDE) in order to determine both the heterogeneous rate constant and number of electrons transferred for each carbohydrate. Many advantages were found for the proposed system including fast and easy handling of the electrode modification, low cost procedure, a wide range of linearity (0.5-50 ppm), low detection limits (ppb level) and high sensitivities. (C) 2009 Elsevier B.V. All rights reserved.

Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS

in this work, a simple method for the simultaneous determination of cocaine (COC) and five COC metabolites (benzoylecgonine, cocaethylene (CET), anhydroecgonine, anhydroecgonine methyl ester and ecgonine methyl ester) in human urine using CE coupled to MS via electrospray ionization (CE-ESI-MS) was developed and validated. Formic acid at 1 mol/L concentration was used as electrolyte whereas formic acid at 0.05 mol/L concentration in 1:1 methanol:water composed the coaxial sheath liquid at the ESI nozzle. The developed method presented good linearity in the dynamic range from 250 ng/mL to 5000 ng/mL (coefficient of determination greater than 0.98 for all compounds). LODs (signal-to-noise ratio of 3) were 100 ng/mL for COC and CET and 250 ng/mL for the other studied metabolites whereas LOQ`s (signal-to-noise ratio of 10) were 250 ng/mL for COC and CET and 500 ng/mL for all other compounds. Intra-day precision and recovery tests estimated at three different concentration levels (500, 1500 and 5000 ng/mL) provided RSD lower than 10% (except anhydroecgonine, 18% RSD) and recoveries from 83-109% for all analytes. The method was successfully applied to real cases. For the positive urine samples, the presence of COC and its` metabolites was further confirmed by MS/MS experiments.

Evaluation of monolithic columns for determination of formaldehyde and acetaldehyde in sugar cane spirits by High-Performance Liquid Chromatography

High-Performance Liquid Chromatography (HPLC) conditions are described for separation of 2,4-dinitrophenylhydrazone (2,4-DNPH) derivatives of carbonyl compounds in a 10 cm long C-18 reversed phase monolithic column. Using a linear gradient from 40 to 77% acetonitrile (acetonitrile-water system), the separation was achieved in about 10 min-a time significantly shorter than that obtained with a packed particles column. The method was applied for determination of formaldehyde and acetaldehyde in Brazilian sugar cane spirits. The linear dynamic range was between 30 and 600 mu g L-1, and the detection limits were 8 and 4 mu g L-1 for formaldehyde and acetaldehyde, respectively.

Rapid and direct determination of glyphosate and aminomethylphosphonic acid in water using anion-exchange chromatography with coulometric detection

A simple, rapid, and low-cost coulometric method for direct detection of glyphosate and aminomethylphosphonic acid (AMPA) in water samples using anion-exchange chromatography and coulometric detection with copper electrode is presented. Under optimized conditions, the limits of detection (LODs) (S/N = 3) were 0.038 mu g ml(-1) for glyphosate and 0.24 mu g ml(-1) for AMPA, without any preconcentration method. The calibration curves were linear and presented an excellent correlation coefficient. The method was successfully applied to the determination of glyphosate and AMPA in water samples without any kind of extraction, clean-up, or preconcentration step. No interferent was found in the water, like this, the recovery was, practically, 100%. (c) 2008 Elsevier B.V. All rights reserved.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C 18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 mu m of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 103 less sample than using conventional methods). (C) 2008 Elsevier B.V. All rights reserved.

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 min x 4.6 mm, 10 mu m) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 pm) analytical column in order to perform online sample preparation. Direct injection of 100 mu L, of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.

Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD

A method is reported for the quantification of isoorientin (using a standard addition method) and total flavonoids (expressed as rutin, using the external standard method) in passion fruit pulp (Passiflora edulis Sims f. flavicarpa Degener, Passifloraceae). Extraction of flavonoids was optimized by experimental design methodology, and quantitative analysis was performed by high-performance liquid chromatography with photo-diode array detection (HPLC-UV/DAD). The method was developed and validated according to ICH requirements for specificity, linearity, accuracy, precision (repeatability and intermediate precision). LOD and LOQ. Rutin was chosen as standard for the quantification of total flavonoids in order to propose a HPLC method feasible for routine analysis of the flavonoids in the passion fruit pulp. The passion fruit pulp contained 16.226 +/- 0.050 mg L(-1) of isoorientin and 158.037 +/- 0.602 mg L(-1) of total flavonoid, suggesting that P. edulis fruits may be comparable with other flavonoid food sources such as orange juice or sugarcane juice. (C) 2010 Elsevier B.V. All rights reserved.

Quantification of chlordesmethyldiazepam by liquid chromatography-tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam-test, Eurofarma Lab. Ltda and Olcadil (R)-reference, Novartis Biociencias S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUCO-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright (C) 2009 John Wiley & Sons, Ltd.

Ciprofibrate quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry for pharmacokinetic studies

A rapid, sensitive and specific method for quantifying ciprofibrate in human plasma using bezafibrate as the internal standard (IS) is described. The sample was acidified prior extraction with formic acid (88%). The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (diethyl ether/dichloromethane 70/30 (v/v)). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed using Genesis C18 4 mu m analytical column (4.6 x 150 mm i.d.) and a mobile phase consisting of acetonitrile/water (70/30, v/v) and 1 mM acetic acid. The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 0.1-60 mu g/mL (r > 0.99). The limit of quantification was 0.1 mu g/mL. The intra- and interday accuracy and precision values of the assay were less than 13.5%. The stability tests indicated no significant degradation. The recovery of ciprofibrate was 81.2%, 73.3% and 76.2% for the 0.3, 5.0 and 48.0 ng/mL standard concentrations, respectively. For ciprofibrate, the optimized parameters of the declustering potential, collision energy and collision exit potential were -51 V, -16 eV and -5 V, respectively. The method was also validated without the use of the internal standard. This HPLC-MS/MS procedure was used to assess the bioequivalence of two ciprofibrate 100 mg tablet formulations in healthy volunteers of both sexes. The following pharmacokinetic parameters were obtained from the ciprofibrate plasma concentration vs. time curves: AUC(last), AUC(0-168 h), C(max) and T(max). The geometric mean with corresponding 90% confidence interval (CI) for test/reference percent ratios were 93.80% (90% CI = 88.16-99.79%) for C(max), 98.31% (90% CI = 94.91-101.83%) for AUC(last) and 97.67% (90% CI = 94.45-101.01%) for AUC(0-168 h). Since the 90% Cl for AUC(last), AUC(0-168 h) and C(max) ratios were within the 80-125% interval proposed by the US FDA, it was concluded that ciprofibrate (Lipless (R) 100 mg tablet) formulation manufactured by Biolab Sanus Farmaceutica Ltda. is bioequivalent to the Oroxadin (R) (100 mg tablet) formulation for both the rate and the extent of absorption. (C) 2011 Published by Elsevier B.V.

Multimilligram enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography on polysaccharide-based chiral stationary phase

The enantiomers of sulfoxide proton pump inhibitors - omeprazole, lansoprazole, rabeprazole and Ro 18-5364 - were enantiomerically separated by liquid chromatography at multimilligram scale on a poly saccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantionteric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose. (C) 2007 Elsevier B.V. All rights reserved.