Quantification of uronic acids in tea polysaccharide conjugates and their antioxidant properties

A technique of high-performance liquid chromatography (HPLC) was described for the measurement of total uronic acids in tea polysaccharide conjugates. This method was applied to polysaccharide conjugate extracts obtained from green tea after most of the components that produce interference were removed. The preliminary extraction process was according to the procedure of isolation of polysaccharide conjugates. The uronic acid content of different polysaccharide conjugate fractions was quantified by HPLC on a Sugar-Pak I column with a 1.0 x 10(-)(4) mol x L(-)(1) calcium disodium ethylenediaminetetraacetic acid solution as the mobile phase and refractive index detection. The validation study showed high recoveries (>97.0%) and low coefficients of variance (<3.0%). The minimum detectable limit concentration of uronic acid was 10 microg x mL(-)(1). The analysis of a standard range of galacturonic acid concentrations (100-4000 microg x mL(-)(1)) yielded linear results. The use of the method on different polysaccharide conjugate fraction samples confirmed its effectiveness. With the high content of uronic acids in polysaccharide conjugates, the stronger reactive oxygen species scavenging activities were found.     

Bioactive compounds and 1,3-Di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol from Apium graveolens L. seeds

Bioassay-directed isolation and purification of the hexane extract of Apium graveolens L. seeds led to the characterization of three compounds: beta-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-methoxyphenol (3). The structures of these compounds were established by using (1)H and (13)C NMR spectral methods. Compounds, 1-3 demonstrated 100% mortality on fourth-instar Aedes aegyptii larvae at 50, 25, and 200 microg mL(-)(1), respectively, in 24 h. Also, 2 inhibited the growth of Candida albicans and Candida kruseii at 100 microg mL(-)(1). It inhibited both topoisomerase-I and -II enzyme activities at 100 microg mL(-)(1). Compound 2 displayed 100% mortality at 12.5 and 50 microg mL(-)(1), respectively, when tested on nematodes, Panagrellus redivivus and Caenorhabditis elegans. The triglyceride, 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol (4) and 3 were isolated for the first time from A. graveolens seeds, although 4 was not biologically active.     

Determination of selenium in human milk by hydride cold-trapping atomic absorption spectrometry and calculation of daily selenium intake.

A technique of hydride cold-trapping atomic absorption spectrometry following microwave digestion was developed and optimized for the determination of selenium in human milk. The method was validated by the analysis of two standard reference materials (CRM milk powder). The detection limit was 0.5 ng mL(-)(1). The method was then used to analyze 78 milk samples from 38 Austrian mothers throughout their first 10 months of lactation. The mean concentration of selenium in the mother's milk decreased with the days postpartum from 23.9 +/- 12.0 microg L(-)(1) in colostrum to a plateau of 11.4 +/- 3.0 microg L(-)(1) in mature milk. On the basis of the milk selenium concentrations, the selenium intakes of the fully breast-fed infants and the lactating mothers were calculated. The selenium intake of the infants during their first 3 months of life was >8.2 microg day(-)(1). The selenium intake of the lactating mothers was 48 microg day(-)(1). Compared to the recommended dietary allowance, the fully breast-fed infants received sufficient selenium but the lactating mothers obtained less than the recommended.     

Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA)

A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect a broad range of (fluoro)quinolones in various matrices. In the optimized generic test, anti-sarafloxacin antibodies in combination with norfloxacin conjugate showed 50% binding inhibition at 0.21 ng mL(-)(1) for sarafloxacin in buffer. Screening for this class of antibiotics is accomplished using a simple, rapid extraction carried out with a 1:1 mixture of methanol and phosphate-buffered saline adjusted to pH 7.4. This common extraction was able to detect 15 (fluoro)quinolone residues such as sarafloxacin, norfloxacin, difloxacin, ciprofloxacin, pefloxacin, ofloxacin, cinoxacin, danofloxacin, enrofloxacin, marbofloxacin, lomefloxacin, enoxacin, flumequine, oxolinic acid, and nalidixic acid in pig kidney, poultry muscle, egg, fish, and shrimp. The assay's detection capabilities (CCbeta) for most of these compounds were <10 microg kg(-)(1) except for the sarafloxacin-, oxolinic acid-, flumequine-, and cinoxacin-spiked matrices, the estimated CCbeta values of which were <4, <25, <100, and <200 microg kg(-)(1), respectively.     

Manufacture of fermentable sugar solutions from sugar cane bagasse hydrolyzed with phosphoric acid at atmospheric pressure

Sugar cane bagasse, a renewable and cheap bioresource, was hydrolyzed at 100 degrees C using phosphoric acid at different concentrations (2, 4, or 6%) and reaction times (0-300 min) to obtain fermentable sugar solutions, which have a high concentration of sugars (carbon source for microorganism growth) and a low concentration of growth inhibitors (acetic acid and furfural). Xylose, glucose, arabinose, acetic acid, and furfural were determined following the hydrolysis. Kinetic parameters of mathematical models for predicting these compounds in the hydrolysates were obtained. Derived parameters such as efficiency of hydrolysis or purity of hydrolysates were considered to select as optimal conditions 6% phosphoric acid at 100 degrees C for 300 min. Using these conditions, 21.4 g of sugars L(-)(1) and <4 g of inhibitors L(-)(1) were obtained from the hydrolysis with a water/solid ratio of 8 g of water g(-)(1) of sugar cane bagasse on a dry basis.     

Multiresidue analysis of cotton defoliant, herbicide, and insecticide residues in water by solid-phase extraction and GC-NPD, GC-MS, and HPLC-diode array detection

A multiresidue procedure was developed for analysis of cotton pesticide and harvest-aid chemicals in water using solid-phase extraction and analysis by GC-NPD, GC-MS, and HPLC-DAD. Target compounds included the defoliants tribufos, dimethipin, thidiazuron; the herbicide diuron; and the insecticide methyl parathion. Three solid-phase extraction (SPE) media, octadecylsilyl (ODS), graphitized carbon black (GCB), and a divinylbenzene-N-vinyl pyrollidine copolymer (DVBVP), were evaluated. On GCB and ODS, recoveries varied depending on compound type. Recoveries were quantitative for all compounds on DVBVP, ranging from 87 to 115% in spiked deionized water and surface runoff. The method detection limit was less than 0.1 microg L(-)(1). SPE with DVBVP was applied to post-defoliation samples of surface runoff and tile drainage from a cotton research plot and surface runoff from a commercial field. The research plot was defoliated with a tank mixture of dimethipin and thidiazuron, and the commercial field, with tribufos. Dimethipin was detected (1.9-9.6 microg L(-)(1)) in all research plot samples. In the commercial field samples, tribufos concentration ranged from 0.1 to 135 microg L(-)(1). An exponentially decreasing concentration trend was observed with each successive storm event.     

Direct and combined methods for the determination of chromium, copper, and nickel in honey by electrothermal atomic absorption spectroscopy

In the present work, direct methods for the determination of chromium, copper, and nickel in honey by electrothermal atomic absorption spectroscopy were developed using experimental design as an optimization tool. Once the optimum conditions for the individual methods were established, a direct method for the combined determination of the three elements was optimized using the response surface tool. Palladium was used as chemical modifier in all cases. Honey was diluted in water, hydrogen peroxide, and nitric acid. Triton X-100 was added to minimize the matrix effect and the viscosity of the sample. The RSD (better than 10%) and the analytical recovery (98-103%) were acceptable for all of the developed methods. Calibration graphs were used in the four methods to determine the concentration of the analytes in the sample. The detection limits of the combined method (0.21, 0.35, and 0.37 microg L(-)(1) for Cr, Cu, and Ni, respectively) were similar to those obtained for the individual methods (LOD = 0.17, 0.21, 0.33 microg L(-)(1) for Cr, Cu, and Ni, respectively). The direct-combined proposed method has been applied to the determination of chromium, copper, and nickel content in representative honey samples from Galicia (northwestern Spain). The concentrations found in the analyzed samples were in the range of (5.75 +/- 0.64)-(26.4 +/- 0.38) ng g(-)(1) of Cr, (79 +/- 7.8)-(2049 +/- 80) ng g(-)(1) of Cu, and (12.6 +/- 1.36)-(172 +/- 6.88) ng g(-)(1) of Ni.     

Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.     

Metabolism of metamitron in goat following a single oral administration of a nontoxic dose level: a continued study

Disposition kinetic behavior and metabolism studies of metamitron and its metabolite in terms of the parent compound were carried out in black Bengal goats after a single oral administration of a nontoxic oral dose at 30 mg kg(-1) of body weight. Metamitron was detected in the blood sample at 5 min (2.23 +/- 0.04 microg mL(-1)), maximum at 1 h (3.43 +/- 0.02 microg mL(-1)) and minimum at 12 h (0.41 +/- 0.01 microg mL(-1)), after a single oral administration. Metabolite [3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one] in terms of the parent compound was detected in the blood sample at 5 min (0.47 +/- 0.006 microg mL(-1)), maximum at 6 h (5.12 +/- 0.02 microg mL(-1)) and minimum at 96 h (1.06 +/- 0.016 microg mL(-1)), after a single oral administration. The t(1/2 K) and Cl(B) values of metamitron were 3.63 +/- 0.05 h and 1.36 +/- 0.016 L kg(-1) h(-1), respectively, whereas the t(1/2K)(m) and Cl(B)(m) values of the metabolite were 38.15 +/- 0.37 h and 0.091 +/- 0.001 L kg(-1) h(-1), respectively, which suggested long persistence of the metabolite in blood and tissues of goat. Metamitron was excreted through feces and urine for up to 48 and 72 h, whereas the metabolite was excreted for up to 168 and 144 h, respectively. Metabolite alone contributed to 96 and 67% of combined recovery percentage of metamitron and metabolite against the administered dose in feces and urine of goat, respectively. All of the goat tissues except lung, adrenal gland, ovary, testis, and mammary gland retained the metabolite residue for up to 6 days after administration.     

Isolation and acclimation of a microbial consortium for improved aerobic degradation of alpha-hexachlorocyclohexane

A microbial consortium that can utilize alpha-hexachlorocyclohexane (alpha-HCH) as a sole source of carbon and energy was isolated from soil and sewage through a novel technique involving an initial enrichment in a glass column reactor followed by a shake flask enrichment. This consortium took 14 days to completely mineralize 5 and 10 microg mL(-)(1) alpha-HCH in mineral salts medium in shake flasks. The degradative ability of this consortium improved very markedly on acclimation by successive and repeated passages through media containing increasing concentrations of alpha-HCH. The acclimated consortium could degrade 100 microg mL(-)(1) of alpha-HCH within 72 h at a degradation rate of 58 microg mL(-)(1) day(-)(1) with concomitant release of stoichiometric amounts of chloride. Accumulation of any intermediary metabolites was not detected in the culture broth as tested by TLC and GC, implying complete mineralization of the substrate. The acclimated consortium contained eight bacterial strains and a fungus. The individual strains and the different permutations and combinations of them, however, were able to utilize only 10 microg mL(-)(1) of alpha-HCH. Mesophilic temperatures (20-30 degrees C) and near-neutral pH (6.0-8.0) were most favorable for alpha-HCH degradation. Among the auxiliary carbon sources tested, ethanol, benzoate, and glucose (at higher concentrations) retarded the degradation of alpha-HCH, whereas the addition of cellulose, sawdust, and low concentrations of glucose (<200 microg mL(-)(1)) and acetone enhanced the rate of degradation.     

Simultaneous stopped-flow determination of butylated hydroxyanisole and propyl gallate using a T-format luminescence spectrometer

A simple and fast luminescent method is used for the first time to resolve a mixture of two synthetic antioxidants, propyl gallate (PG) and butylated hydroxyanisole (BHA), by the joint use of the stopped-flow mixing technique and a T-format luminescence spectrometer. The determination of these compounds involves two different and independent reactions. On the one hand, PG determination is based on an energy transfer process that involves the formation of a lanthanide chelate with terbium in the presence of Triton X-100 and tri-n-octylphosphine oxide. On the other hand, BHA is determined using a reaction between the oxidized form of Nile Blue and the antioxidant. Both systems are excited at the same excitation wavelength (310 nm), and the emission wavelengths are 545 and 665 nm for PG and BHA, respectively. The absence of overlap in the emission spectra makes it possible to measure separately the analytes in each channel of the instrument. Initial rate and equilibrium signal are used as analytical parameters and measured in 0.1 and 1 s for PG and BHA, respectively. Calibration graphs are linear over the range 0.09-3.5 microg mL(-)(1) for PG and 0.3-15 microg mL(-)(1) for BHA. The relative standard deviations of both systems are close to 2%. The proposed method is applied to the determination of these two antioxidants in several commercial food samples with recoveries ranging between 94.8 and 102.9% for PG and between 94.1 and 102.1% for BHA.     

Determination of whole-body rotenone residues in the brown tree snake (Boiga irregularis)

The brown tree snake (Boiga irregularis) is an introduced pest in Guam, responsible for extensive agricultural damage, the extinction of several bird species, and severe and frequent electrical power outages. Rotenone, a naturally occurring pesticide, has been investigated as a possible chemical control agent. An analytical method was developed to assess whole body rotenone residues ranging in concentration from 0.035 to 250 microg g(-)(1) in snakes. The method employed ethyl acetate extraction of 2 g samples of cryogenically frozen, pulverized snakes, followed by silica and Florisil solid-phase extraction cleanup. Extract analysis was performed using a high-performance liquid chromatography system employing a cyanopropyl analytical column. Tissues fortified to concentrations of 0.035, 4.82, and 250 microg g(-)(1) yielded analyte recoveries of 85.1, 85.6, and 83.5%, respectively. The linear response of rotenone standard solutions was assessed from 0. 025 to 0.25 microg mL(-)(1) (r(2) = 0.9968) and from 0.250 to 125 microg mL(-)(1) (r(2) = 0.9999). The method was simple, rugged, and reliable.     

Linuron sorption-desorption in field-moist soils

Pesticide sorption or binding to soil is traditionally characterized using batch slurry techniques. The objective of this study was to determine linuron sorption in field-moist or unsaturated soils. Experiments were performed using low-density (i.e., 0.25 g mL(-)(1)) supercritical carbon dioxide to remove linuron from the soil water phase, thus allowing calculation of sorption coefficients (K(d)) at low water contents. Both soil water content and temperature influenced sorption. K(d) values increased with increased water content, if less than saturated. K(d) values decreased with increased temperature. K(d) values for linuron sorption on silty clay and sandy loam soils at 12% water content and 40 degrees C were 3.9 and 7.0 mL g(-)(1), respectively. Isosteric heats of sorption (DeltaH(i)) were -41 and -35 kJ mol(-)(1) for the silty clay and sandy loam soils, respectively. The sorption coefficient obtained using the batch method was comparable (K(f) for sandy loam soil = 7. 9 microg(1)(-)(1/)(n)() mL(1/)(n)() g(-)(1)) to that obtained using the SFE technique. On the basis of these results, pesticide sorption as a function of water content must be known to more accurately predict pesticide transport through soils.     

Application of micellar electrokinetic capillary chromatography to the analysis of uncharged pesticides of environmental impact

A test mixture of five pesticides and metabolites (naphthalene acetamide, carbaryl, 1-naphthol, thiabendazole, and carbendazime) has been investigated by capillary electrophoresis with an ultraviolet diode array detector. These compounds were separated in <10 min by micellar electrokinetic capillary chromatography (MEKC). MEKC was performed in 30 mM ammonium chloride/ammonia buffer (pH 9.0) containing 15 mM sodium dodecyl sulfate. The lowest detection limit was obtained for the insecticide carbaryl (0.22 microg mL(-)(1)) and the highest for its metabolite 1-naphthol (1.13 microg mL(-)(1)). This method was applied to the analysis of the pesticides in cultivated vegetables such as cucumbers, which were extracted with a liquid-liquid extraction procedure, obtaining recovery percentages ranging from 90.1 to 110.2%.     

Accurate determination of oosporein in fungal culture broth by differential pulse polarography

A simple and accurate differential pulse polarographic method has been developed for the determination of oosporein in the culture broth of the fungus Beauveria brongniartii. This hydroxybenzoquinone derivative is the only major secondary metabolite secreted by this entomopathogenic fungus, which is used as biological pest control agent (BCA) against Melolontha melolontha larvae. It can be found in the host organism as well as in the formulated product. The polarographic behavior of oosporein was examined in various buffer systems over the pH range 3-10. In Britton-Robinson buffer/methanol solution (3:7 v/v, pH 5.5) the differential pulse polarograms exhibited reproducible peaks at E(p) = -0.18 V vs silver/silver chloride/potassium chloride (3 M). Under these conditions, a plot of peak height vs concentration of oosporein was found to be linear over the range 5.9 x 10(-)(7) to 2.5 x 10(-)(5) M (0.18-7.74 microg mL(-)(1); r = 0.9998). The detection limit was calculated to be 54 ng mL(-)(1). To evaluate the concentration of oosporein, the standard addition method was applied. The analysis of oosporein in the culture broth led to a mean value of 524.9 microg mL(-)(1) broth with a relative standard deviation (S(rel)) of +/-2.6%. The proposed polarographic method is accurate, not time-consuming, and it is of low cost because no separation steps are necessary.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

A kinetic model for the glucose-fructose-glycine browning reaction

The browning of glucose-fructose-glycine mixtures involves parallel glucose-glycine and fructose-glycine reactions, which share a common intermediate, the immediate precursor of melanoidins in the kinetic model. At pH 5.5, 55 degrees C glucose is converted into this intermediate in a two step process where k(1) = (7.8 +/- 1.1) x 10(-)(4) mol L(-)(1) h(-)(1) and k(2) = (1.84 +/- 0.31) x 10(-)(3) h(-)(1) according to established kinetics, whereas fructose is converted into this intermediate in a single step where k(4) = 5.32 x 10(-)(5)()()mol L(-)(1) h(-)(1). The intermediate is converted to melanoidins in a single rate limiting process where k(mix) = 0.0177 h(-)(1) and the molar extinction coefficient (based on the concentration of sugar converted) of the melanoidins so formed is 1073 +/- 4 mol(-)(1) L cm(-)(1). Whereas the value of k(mix) is the same when the individual sugars undergo browning, the value of the molar extinction coefficient is similar to that for melanoidins from the glucose-glycine reaction (955 +/- 45 mol(-)(1) L cm(-)(1)) but it is approximately double the value for melanoidins from the fructose-glycine reaction (478 +/- 18 mol(-)(1) L cm(-)(1)). This is the reason that the effects of glucose and fructose on the rate of browning are synergistic.     

Semiautomated determination of pesticides in water using solid phase extraction disks and gas chromatography-mass spectrometry

A method based on semiautomated solid phase extraction using octadecyl-bonded silica disks and gas chromatography-mass spectrometry, operated in selected ion monitoring mode, allows detection and quantification of approximately 100 pesticides and transformation products in drinking water. Samples (500 mL) were passed through the disk, and the retained pesticides were eluted with acetone and ethyl acetate. Typical recoveries for pesticides at 0.1 microg L(-1) in water were in the range of 72-120% with relative standard deviations less than 20%. Calibration curves were linear over the range of 0.025-0.5 microg mL(-1) (equivalent to a concentration range in drinking water of 0.05-1.0 microg L(-1)).     

Monitoring the benzene contents in soft drinks using headspace gas chromatography-mass spectrometry: a survey of the situation on the belgian market

Whenever benzoic acid is combined with ascorbic acid in acidic beverages such as soft drinks, benzene can be formed. To determine the current situation on the Belgian market, a headspace gas chromatographic-mass spectrometric method was developed, which needs little to no sample preparation. This method was then used to analyze 134 soft drinks sampled on the Belgian market by the Federal Agency for the Safety of the Food Chain. Thirty-three percent of the samples contained no detectable benzene, whereas the majority of the samples (47%) contained trace amounts below the limit of quantification of the method (0.3 microg L (-1)). Ten samples were above the European limit for benzene in drinking water of 1 microg L (-1), and one sample had a concentration of 10.98 microg L (-1), thereby exceeding the action limit for benzene in soft drinks of 10 microg L (-1) discussed at the Standing Committee on the Food Chain and Animal Health of the European Commission. Statistical analyses revealed that besides benzoic acid, ascorbic acid, and acidity regulators, the packing may also play an important role in benzene formation.     

Development of a novel and automated fluorescent immunoassay for the analysis of beta-lactam antibiotics

An automated immunosensor for the rapid and sensitive analysis of penicillin type beta-lactam antibiotics has been developed and optimized. An immunogen was prepared by coupling the common structure of the penicillanic beta-lactam antibiotics, i.e., 6-aminopenicillanic acid to keyhole limpet hemocyanin. Polyclonal antibodies raised in rabbits after immunization with this conjugate have been applied for the development of a competitive fluoroimmunoassay (FIA), using a novel fluorescent penicillin {[2S,5R,6R]-3,3-dimethyl-7-oxo-6-[(pyren-1ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxilic acid, PAAP} as the tracer and penicillin G as the reference antibiotic. Protein A/G covalently bound to an azlactone-activated polymeric support was used for the orientated capture of the antibody-antigen immunocomplexes. Upon desorption from the immunosupport, the emission signal generated by the PAAP-Ab complexes is related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 30 ng mL(-)(1) with a detection limit (10% binding inhibition) of 2.4 ng mL(-)(1) and a dynamic range from 6.0 to 191 ng mL(-)(1) (20-80% binding inhibition) penicillin G. The generic nature of the antiserum was shown by good relative cross-reactivities with penicillin type beta-lactam antibiotics such as amoxicillin (50%), ampicillin (47%), and penicillin V (145%) and a lower response to the isoxazolyl penicillins such as oxacillin, cloxacillin, and dicloxacillin. No cross-reactivity was obtained for cephalosporin type beta-lactam antibiotics (cephapirin), cloramphenicol, or fluoroquinolones (enrofloxacin and ciprofloxacin). The total analysis time was 23 min per determination, and the immunoreactor could be reused for more than 200 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of penicillin G and amoxicillin in spiked influent and effluent sewage treatment plant water samples with excellent recoveries (mean values for penicillin G and amoxicillin, 99 and 105%, respectively). Results displayed by comparative analysis of the immunosensor with a chromatographic procedure for penicillins showed excellent agreement between both methods.