Keyller Bastos Borges

Endophytic Fungi: Natural Products, Enzymes and Biotransformation Reactions

Endophytes exhibit a complex web of interactions with host plants and with other endophytic microorganisms and therefore they have been intensively studied over the last several years as prolific sources of new and bioactive natural products. In fact, an impressive number of natural products have been produced by endophytic microorganisms. In addi- tion, some studies have shown endophytes to be good producers of useful enzymes to improve industrial processes. More recently, endophytes have also received attention as biocatalysts in the chemical transformation of natural products and drugs. Results have shown their ability to modify chemical structures with a high degree of stereospecificity. Some reac- tions are similar to those catalyzed by mammal phase I metabolism; therefore endophytes could be used as models for drug metabolism studies. This paper summarizes recent data on endophytes research as a source of novel and bioactive natural products, as producers of enzymes and their use on biotransformation processes.

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 microm) at 35 degrees C under isocratic conditions using 5mM 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 0.1M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1M 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>or=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>or=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.

Assessment of the stereoselective fungal biotransformation of albendazole and its analysis by HPLC in polar organic mode

A high-performance liquid chromatographic method using polar organic mode was developed to analyze albendazole (ABZ), albendazole sulfone (ABZSO(2)) and the chiral and active metabolite albendazole sulfoxide (ABZSOX, ricobendazole) that was further applied in stereoselective fungal biotransformation studies. The chromatographic separation was performed on a Chiralpak AS column using acetonitrile:ethanol (97:3, v/v) plus 0.2% triethylamine and 0.2% acetic acid as the mobile phase at a flow rate of 0.5 mL min(-1). The present study employed hollow fiber liquid-phase microextraction as sample preparation. The method showed to be linear over the concentration range of 25-5000 ng mL(-1) for each ABZSOX enantiomer, 200-10,000 ng mL(-1) for ABZ and 50-1000 ng mL(-1) for ABZSO(2) metabolite with correlation coefficient (r)>0.9934. The mean recoveries for ABZ, rac-ABZSOX and ABZSO(2) were, respectively, 9%, 33% and 20% with relative standard deviation below 10%. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. This study opens the door regarding the possibility of using fungi in obtaining of the active metabolite ricobendazole. Nigrospora sphaerica (Sacc.) E. W. Mason (SS67), Pestalotiopsis foedans (VR8), Papulaspora immersa Hotson (SS13) and Mucor rouxii were able to stereoselectively metabolize ABZ into its chiral metabolite. Among them, the fungus Mucor rouxii was the most efficient in the production of (+)-ABZSOX.

Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4‐hydroxypropranolol (4‐OH‐Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused‐silica capillary, 4% w/v carboxymethyl‐β‐CD in 25 mmol/L triethylamine/phosphoric acid (H3PO4) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid–liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25–10.0 μg/mL for each 4‐OH‐Prop enantiomer and 0.10–10.0 μg/mL for each Prop enantiomer (r≥0.995). Within‐day and between‐day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4‐OH‐Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (−)‐(S)‐Prop to (−)‐(S)‐4‐OH‐Prop with no formation of (+)‐(R)‐4‐OH‐Prop in 72 h of incubation.

Molecularly imprinted pipette-tip solid phase extraction for selective determination of fluoroquinolones in human urine using HPLC-DAD.

A simple method using HPLC-DAD was developed for the determination of fluoroquinolones in human urine including ciprofloxacin (CIPRO), enrofloxacino (ENRO), marbofloxacino (MARBO) and norfloxacin (NOR). In addition, it was studied the extraction of fluoroquinolones in human urine samples using pipette tip-based molecularly imprinted polymers solid phase extraction (PT-MIPs-SPE). With the goal of finding the best procedure for extraction of four fluoroquinolones in human urine, several parameters that are likely to affect the efficiency of extraction during sample preparation, including the washing solvent, type and volume of eluent, amount of material, the volume of the sample, pH and the ionic strength were systematically optimized. Chromatographic separations of fluoroquinolones were hit within 10min using a Synergi(®) C18 (250×4.6mm, 4μm) column and mobile phase consisting of water (10mM of phosphoric acid, the pH adjusted at 3.29 with triethylamine) : acetonitrile (85.7: 14.3, v/v) at a flow rate of 1.5mLmin(-1). Detection was performed at 290nm. The average extraction recoveries/standard deviation relative to ENRO, CIPRO, NOR and MARBO were 96.40±5.51%, 42.47±4.81%, 41.82±7.99% and 87.49±4.70, respectively. The method was liner from 39 to 1260ngmL(-1) for each fluoroquinolone with correlation coefficient of 0.9904, 0.9910, 0.9914 and 0.9919, to ENRO, CIPRO, NOR and MARBO, respectively. The assays of within-day and between-day precision and accuracy for all analytes were studied at three concentration levels and were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of CIPRO to a healthy volunteer.

Enantioselective fungal biotransformation of risperidone in liquid culture medium by capillary electrophoresis and hollow fiber liquid-phase microextraction

Knowing that microbial transformations of compounds play vital roles in the preparation of new derivatives with biological activities, risperidone and its chiral metabolites were determined by capillary electrophoresis and hollow fiber liquid‐phase microextraction after a fungal biotransformation study in liquid culture medium. The analytes were extracted from 1 mL liquid culture medium into 1‐octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 100 mmol/L sodium phosphate buffer pH 3.0 containing 2.0% w/v sulfated‐α‐CD and carboxymethyl‐β‐CD 0.5% w/v with a constant voltage of −10 kV. The method was linear over the concentration range of 100–5000 ng/mL for risperidone and 50–5000 ng/mL for each metabolite enantiomer. Within‐day and between‐day assay precisions and accuracies for all the analytes were studied at three concentration levels, and the values of relative standard deviation and relative error were lower than 15%. The developed method was applied in a pilot biotransformation study employing risperidone as the substrate and the filamentous fungus Mucor rouxii. This study showed that the filamentous fungus was able to metabolize risperidone enantioselectively into its chiral active metabolite, (−)‐9‐hydroxyrisperidone.

Capillary electrophoresis and hollow fiber liquid-phase microextraction for the enantioselective determination of albendazole sulfoxide after biotransformation of albendazole by an endophytic fungus.

Hollow fiber liquid‐phase microextraction and CE were applied for the determination of albendazole sulfoxide (ASOX) enantiomers in liquid culture medium after a fungal biotransformation study. The analytes were extracted from 1 mL of liquid culture medium spiked with the internal standard (rac‐hydroxychloroquine) and buffered with 0.50 mol/L phosphate buffer, pH 10. The analytes were extracted into 1‐octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 0.05 mol/L tris(hydroxymethyl)aminomethane buffer, pH 9.3, containing 3.0% w/v sulfated‐β‐CD (S‐β‐CD) with a constant voltage of +15 kV and detection at 220 nm. The method was linear over the concentration range of 250–5000 ng/mL for each ASOX enantiomer. Within‐day and between‐day assay precision and accuracy for the analytes were studied at three concentration levels and the values of RSD% and relative error % were lower than 15%. The developed method was applied for the determination of ASOX after a biotransformation study employing the endophytic fungus Penicillium crustosum (VR4). This study showed that the endophytic fungus was able to metabolize the albendazole to ASOX enantioselectively. In addition, it was demonstrated that hollow fiber liquid‐phase microextraction coupled to CE can be an excellent and environmentally friendly technique for the analysis of samples obtained in biotransformation studies.

Self-assembly pipette tip-based cigarette filters for micro-solid phase extraction of ketoconazole cis-enantiomers in urine samples followed by high-performance liquid chromatography/diode array detection

A simple stereoselective HPLC/DAD method was developed for the determination of ketoconazole (KTZ) cis-enantiomers in human urine. In addition, the potential use of pipette tip-based cigarette filters for micro-solid phase extraction (PT-CFs-μ-SPE) of KTZ cis-enantiomers in human urine samples prior to HPLC/DAD was studied in this paper. In order to find a suitable procedure for extraction of KTZ cis-enantiomers in human urine, various parameters that probably affect the extraction efficiency including the washing solvent, eluent kind and volume, pH, quantity of the material, sample volume and ionic strength were systematically optimized. Optimum chromatographic separations among the KTZ cis-enantiomers have been achieved within 10 minutes using a Chiralpak® IA column (100 × 4.6 mm, 3 μm) as the stationary phase with a mobile phase consisting of 100% ethanol at a flow rate of 1 mL min−1. Detection was performed at 250 nm. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, robustness and stability have been assessed and were within the recommended guidelines. The mean extraction recoveries/relative standard deviation for (+)-(2R,4S)-KTZ and (−)-(2S,4R)-KTZ were 100.74 ± 9.52% and 106.90 ± 9.26%, respectively. The method showed to be linear over the concentration range of 12.5 to 400 ng mL−1 for each enantiomer with the correlation coefficients of 0.9946 and 0.9910, for (+)-(2R,4S)-KTZ and (−)-(2S,4R)-KTZ, respectively. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of racemic cis-KTZ to a healthy volunteer.

On-line restricted access molecularly imprinted solid phase extraction of ivermectin in meat samples followed by HPLC-UV analysis.

A new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) was synthesized, characterized and used for direct analysis of ivermectin from bovine meat samples, in a two-dimensional liquid chromatography system with UV detection. Ivermectin, 4-vynilpiridine and ethylene glycol dimethacrylate were employed as template, functional monomer and cross-linker, respectively. A BSA layer was cross-linked around the polymer, resulting in a biocompatible chemical barrier able to eliminate about 100% of protein from the samples. Ivermectin was extracted from the minced meat samples through a solvent extraction using methanol:water (70:30, v:v), and the extracts were directly injected into the two-dimensional liquid chromatography system, without any other treatment. Samples, fortified with ivermectin from 50 to 500 μg kg(-1), were used to build the analytical calibration curve (r=0.996). The limit of quantification was 50 μg kg(-1). Precision and accuracy presented variation coefficients, as well as relative errors lower than 17.0% and within -18.5% and 22.0%, respectively.

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)‐β‐CD as running electrolyte. The applied voltage and temperature used were 15 kV and 15°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 μg/mL for each enantiomer of midodrine and desglymidodrine (r≥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.