Maurizio Fiori

Phytotoxicity to and uptake of enrofloxacin in crop plants

Phytotoxicity of enrofloxacin on crop plants Cucumis sativus, Lactuca sativa, Phaseolus vulgaris and Raphanus sativus was determined in a laboratory model: the effect of 50, 100 and 5000 microgl(-1) were evaluated after 30 days exposure by measuring post-germinative growth of primary root, hypocotyl, cotyledons and leaves. Concentrations between 50 and 5000 microgl(-1) induced both toxic effect and hormesis in plants, by significantly modifying both length of primary root, hypocotyl, cotyledons and the number/length of leaves. A toxic effect is induced by high concentration (5000 microgl(-1)), while hormesis occurs at low concentrations (50 and 100 microgl(-1)). A continuum between toxic effect and hormesis is found in the four plant species. Both toxic effect and hormesis can be related to an efficient plant drug uptake, in the order of microgg(-1). Plants are able to metabolize enrofloxacin into ciprofloxacin, as also happens in animals; Cucumis, Lactuca and Phaseolus biologically convert about one quarter of stored enrofloxacin. The ecological implication of enrofloxacin contamination in terrestrial environments is discussed.

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.

Phytotoxicity to and uptake of flumequine used in intensive aquaculture on the aquatic weed, Lythrum salicaria L.

Phytotoxicity of Flumequine on the aquatic weed Lythrum salicaria L. was determined by two laboratory models: a single concentration test, by which the effects of 100 mg l-1 were evaluated after 10, 20, 30 days and a multiple concentration test, by which the effects of 5000-1000-500-100-50 micrograms l-1 were evaluated after 35-day exposure. 100 mg l-1 are highly toxic and significantly decrease the growth of plants; this effect increases with time. Concentrations between 5000 and 50 micrograms l-1 induced hormesis in plants, by significantly increasing mean number and dimension of leaves and secondary roots. The effect is the highest at 50 micrograms l-1 and decreases with increase in concentration. Both toxic effect and hormesis can be related to plant drug uptake, quite high, in the order of micrograms g-1. The ecological implication of Flumequine contamination in aquatic environments and the possible use of Lythrum salicaria for bioremediation and/or monitoring technique are discussed.

Identification of main corticosteroids as illegal feed additives in milk replacers by liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry

Corticosteroids were proposed as growth promoting agents to improve commercial quality of meat. We developed a liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS) method able to identify the presence in milk replacers, when given by mouth, of dexamethasone, betamethasone, flumethasone, triamcinolone, predinisotone, prednisolone, methylprednisolone, fludrocortisone and beclomethasone, at levels in the range of 20-100 ppb. C18 solid-phase extraction, LC-RP C8 column separation, data acquisition (positive ions) in the scan range m/z 200-550 allowed us to differentiate and identify compounds by protonated molecules, their methanolic adducts and fragmentation patterns.

Sulphadimethoxine and Azolla filiculoides Lam.: a model for drug remediation.

Plants can be an interesting tool for in situ remediation of drug contaminated waters. In a laboratory model Azolla filiculoides Lam., an aquatic fern known to absorb pollutants, has been exposed to an environmental persistent antibiotic commonly used in intensive farming, sulphadimethoxine (S), to test its bioremediation capability. In a 5 week experiment, plants were cultivated outdoor at four drug concentrations (50, 150, 300 and 450 mg l(-1)) in N-free mineral medium. Drug affects growth rate (as biomass yield per week), N2-fixation, heterocyst frequency, but plants are able to survive. Notwithstanding, at all concentrations tested drug was actively removed from the medium and the accumulation in the biomass is in order of magnitude up to mg g(-1) plant dry weight (1000 ppm). Drug uptake and degradation rates increase with S concentrations in the culture medium. The efficacy of the model was very high. These results demonstrated that Azolla can be taken into consideration as a tool for sulphonamides environmental monitoring and decontamination.

Confirmatory identification of sixteen non-steroidal anti-inflammatory drug residues in raw milk by liquid chromatography coupled with ion trap mass spectrometry

The European Council Decision 2002/657/EC established that group B substances detected in foods must be identified and confirmed on the basis of their molecular structure. To this aim, we have developed a panel of methods for unambiguous determination of sixteen non-steroidal anti-inflammatory drugs (NSAIDs) in cattle and buffalo raw milk. A multi-residue reversed-phase high-performance liquid chromatography method with photodiode array detection is described for quantitative screening analysis. For confirmatory purposes, two multi-residue reversed-phase ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) methods were developed: the former to identify salicylic acid, naproxen, carprofen, flurbiprofen, ibuprofen, meclofenamic acid, niflumic acid, flunixin and its metabolite 5-hydroxyflunixin in the negative ion mode; the latter to identify ketoprofen, suxibutazone, diclofenac, mefenamic acid, tolfenamic acid, phenylbutazone and its metabolite oxyphenbutazone in the positive ion mode. These drugs are representative of different subclasses of NSAIDs not chemically related. The methods were in-house validated, evaluating specificity and calculating the mean recoveries, repeatability, within-laboratory reproducibility, and limits of quantification. For all the NSAIDs, apart from salicylic acid and 5-hydroxyflunixin, mean recoveries ranging between 69.0% and 96.7% were measured. The qualitative identification of all drugs was attained by their MS/MS spectra in the concentration range studied. Similarly, at 5 microg/kg all NSAIDs, apart from flurbiprofen, were unambiguously confirmed.

Biodegradation of oxytetracycline by Pleurotus ostreatus mycelium: a mycoremediation technique

Oxytetracycline (OTC) is administered in high doses to livestocks and enters the environmental compartments as a consequence of animal waste disposal. As a first step in setting up a useful mycoremediation technique, an OTC lab degradation test was performed in liquid medium using the ligninolytic fungus Pleurotus ostreatus. OTC disappearance in culture medium was clearly evident as early as the third day of exposure onwards, with an almost complete removal after 14d. The drug removal was mediated by fungal absorption in the mycelia, where the OTC molecule underwent a degradation step, as demonstrated by mass spectrometry analyses. A putative degradation product, ADOTC (2-acetyl-2-decarboxamido-oxytetracycline) is proposed. Experimental conditions excluded OTC abiotic degradation; the degradation by extracellular laccase was also experimentally discarded.

Confirmatory analysis of non-steroidal anti-inflammatory drugs in bovine milk by high-performance liquid chromatography with fluorescence detection.

HPLC with fluorescence detection is considered for confirmatory analysis of group B veterinary drugs by the European Union legislation. A procedure for confirming the presence of anti-inflammatory non-steroidal drug (NSAID) residues in bovine milk by reversed phase high-performance liquid chromatography with fluorescence detection is herein described. The native fluorescence of nine drugs belonging to different NSAID sub-classes, namely flurbiprofen, carprofen, naproxen, vedaprofen, 5-hydroxy-flunixin, niflumic acid, mefenamic acid, meclofenamic acid and tolfenamic acid, allowed for detection in bovine milk down to 0.25-20.0 microg/kg. Confirmation of the nine NSAIDs is attained by fluorescence detection at characteristic excitation and emission wavelengths. The procedure described is simple and selective. Limits of quantification (LOQs) ranging between 0.25 and 20 microg/kg were measured; satisfactory trueness and within-laboratory reproducibility data were calculated at LOQ spiking levels, apart from 5-hydroxy-flunixin. The procedure developed is used in our laboratory for confirmation of each one of the above mentioned NSAIDs in bovine milk, to support results after HPLC quantitative analysis with UV-vis detection.

Phytotoxic antibiotic sulfadimethoxine elicits a complex hormetic response in the weed Lythrum salicaria L

In order to evaluate the hormetic response of the weed Lythrum salicaria to drug exposure we investigated the effects of the antibiotic Sulfadimethoxine by growing Lythrum plants for 28 days on culture media containing different drug concentrations (between 0.005 and 50 mg.L−1). The antibiotic was absorbed by plants and can be found in plant tissue. The plant response was organ-dependent: roots, cotyledons and cotyledon petioles, were always affected by a toxic effect, whilst internodes and leaves length, showed a variable dose-depending response, with an increased growth at the lower drug concentrations and toxic effects at the higher ones. This variable response was probably dependant on different levels of local contamination resulting from a balance between accumulation rate and drug dilution in the increasing plant biomass. As a consequence, drug toxicity or hormetic response varied according to concentration and were different in each of the examined plant organ/tissue. Thus, even if hormesis can be considered a general plant response, each plant organ/tissue responds differently, depending on the local drug concentration and exposure time.

α1-Acid glycoprotein affinity columns for the clean-up of adrenergic drugs

alpha 1-Acid glycoproteins (AAGs) have a structure resembling beta-adrenergic receptors and bind several basic drugs in plasma. Chromatographic columns were prepared by linking epsilon-NH2 groups of AAG lysines to a Sepharose 4B support, in order to purify by affinity chromatography adrenergic drugs of possible use in animal production. Loading capacities, binding efficiency, memory effects and matrix interferences from urine samples were studied. The method developed involves sample application in buffered media (pH 7.4), washing with 5 ml of PBS, and elution with 4 ml of 1% v/v acetic acid. Under these conditions no memory effect was observed. Loading capacity is correlated with the physiological plasma binding rate (PB) of the drug. For clenbuterol (PB 50%) and anilino-like related drugs, 5 mg of AAG were able to bind about 15 x 10(-6) g of drug, with a 100% recovery from the column. Repeatability and reproducibility, expressed as RSD, were 4.2 and 5.4%, respectively. The calculated AAG: drug molar ratio was 4.5:1, indicating 22% of the AAG bound to the column retained drug affinity. Among phenolic-like agonists, salbutamol (PB 5%), fenoterol and isoxsuprine hardly interacted, whereas nylidrin, ritodrine and bamethan showed more effective binding. We also checked binding of other drugs of possible use in veterinary medicine. Application of the AAG column to spiked bovine urine revealed a mean recovery of 97.8%; no matrix interferences were observed.