Photodegradation of Antibiotics by Noncovalent Porphyrin-Functionalized TiO2 in Water for the Bacterial Antibiotic Resistance Risk Management.

Antibiotics represent essential drugs to contrast the insurgence of bacterial infections in humans and animals. Their extensive use in livestock farming, including aquaculture, has improved production performances and food safety. However, their overuse can implicate a risk of water pollution and related antimicrobial resistance. Consequently, innovative strategies for successfully removing antibiotic contaminants have to be advanced to protect human health. Among them, photodegradation TiO2-driven under solar irradiation appears not only as a promising method, but also a sustainable pathway. Hence, we evaluated several composite TiO2 powders with H2TCPP, CuTCPP, ZnTCPP, and SnT4 porphyrin for this scope in order to explore the effect of porphyrins sensitization on titanium dioxide. The synthesis was realized through a fully non-covalent functionalization in water at room conditions. The efficacy of obtained composite materials was also tested in photodegrading oxolinic acid and oxytetracycline in aqueous solution at micromolar concentrations. Under simulated solar irradiation, TiO2 functionalized with CuTCPP has shown encouraging results in the removal of oxytetracycline from water, by opening the way as new approaches to struggle against antibiotic's pollution and, finally, to represent a new valuable tool of public health.

Rare-Earth Metal Complexes of the Antibacterial Drug Oxolinic Acid: Synthesis, Characterization, DNA/Protein Binding and Cytotoxicity Studies.

"Drug repositioning" is a current trend which proved useful in the search for new applications for existing, failed, no longer in use or abandoned drugs, particularly when addressing issues such as bacterial or cancer cells resistance to current therapeutic approaches. In this context, six new complexes of the first-generation quinolone oxolinic acid with rare-earth metal cations (Y3+, La3+, Sm3+, Eu3+, Gd3+, Tb3+) have been synthesized and characterized. The experimental data suggest that the quinolone acts as a bidentate ligand, binding to the metal ion via the keto and carboxylate oxygen atoms; these findings are supported by DFT (density functional theory) calculations for the Sm3+ complex. The cytotoxic activity of the complexes, as well as the ligand, has been studied on MDA-MB 231 (human breast adenocarcinoma), LoVo (human colon adenocarcinoma) and HUVEC (normal human umbilical vein endothelial cells) cell lines. UV-Vis spectroscopy and competitive binding studies show that the complexes display binding affinities (Kb) towards double stranded DNA in the range of 9.33 × 104 - 10.72 × 105. Major and minor groove-binding most likely play a significant role in the interactions of the complexes with DNA. Moreover, the complexes bind human serum albumin more avidly than apo-transferrin.

Oxolinic Acid Binding at Goethite and Akaganéite Surfaces: Experimental Study and Modeling.

Oxolinic acid (OA) is a widely used quinolone antibiotic in aquaculture. In this study, its interactions with synthetic goethite (α-FeOOH) and akaganéite (ß-FeOOH) particle surfaces were monitored to understand the potential fate of OA in marine sediments where these phases occur. Batch sorption experiments, liquid chromatography (LC) analyses of supernatants, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and multisite complexation (MUSIC) modeling were used to monitor OA binding at these particle surfaces. Both LC and ATR-FTIR showed that adsorption did not degrade OA, and that OA adsorption was largely unaffected by NaCl concentrations (10-1000 mM). This was explained further by ATR-FTIR suggesting the formation of metal-bonded complexes at circumneutral to low pHc = -log [H(+)] and with a strongly hydrogen-bonded complex at high pHc. The stronger OA binding to akaganéite can be explained both by the higher isoelectric point/point-of-zero charge (9.6-10) of this mineral than of goethite (9.1-9.4), and an additional OA surface complexation mechanism at the (010) plane. Geminal sites (≡Fe(OH2)2(+)) at this plane could be especially reactive for metal-bonded complexes, as they facilitate a mononuclear six-membered chelate complex via the displacement of two hydroxo/aquo groups at the equatorial plane of a single Fe octahedron. Collectively, these findings revealed that Fe-oxyhydroxides may strongly contribute to the fate and transport of OA-type antibacterial agents in marine sediments and waters.

Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

Synthesis of amino acid derivatives of quinolone antibiotics.

Optically pure conjugates of quinolone antibiotics with naturally occurring amino acids are synthesized in 40-98% yields.

Oxolinic acid photo-oxidation using immobilized TiO(2).

This work studied the photocatalysed oxidation of the antibiotic oxolinic acid (OA) in an annular reactor operated with immobilized TiO(2) on sintered glass cylinders (SGC). Experiments were carried out in 1l solution of OA (18 mg l(-1)) at pH 9 with oxygen bubbling. Irradiation was performed with black light (36 W). The reaction was monitored by COD, TOC and average oxidation state (AOS) calculations. The antibacterial activity of intermediates was followed using the inhibition halo technique on Escherichia coli cultures. The initial antibiotic concentration decreases in one order of magnitude after 60 min irradiation, and was completely eliminated at 100 min reaction. The TOC was reduced in 54% and the AOS reach values around +3 indicating the formation of low molecular weight carboxylic acids. The oxidation reaction fit well with the Langmuir-Hinshelwood kinetic model indicating the dependence of reaction rate with initial adsorption step. The antibacterial activity of the solution decreases with antibiotic removal, demonstrating that intermediates do not present antibiotic activity.

Structure and medium effects on the photochemical behavior of nonfluorinated quinolone antibiotics.

The photophysical behavior of the quinolone antibiotics, oxolinic (OX), cinoxacin (CNX) and pipemidic (PM) acids was studied as a function of pH and solvent properties. The ground state of these compounds exhibits different protonated forms, which also exist in the first excited states. Theoretical calculations of the Fukui indexes allowed to assigning the different protonation equilibria. The pK values indicate that the acidity of the 3-carboxylic and 4-carbonyl groups increases with the N-atom at position 2 in CNX. It has been found that fluorescence properties are strongly affected by pH, the more fluorescent species is that with protonated carboxylic acid, protonated species at the carbonyl group and the totally deprotonated form present very low fluorescence. The fluorescence behavior also depends on the chemical structure of the quinolone and on the solvent properties. The analysis of the solvent effect on the maximum and the width of the fluorescence band of OX, using the linear solvent-energy relation solvatochromic equation, indicates that the polarizability and hydrogen bond donor ability are the parameters that condition the spectral changes. The hydrogen bond acceptor ability of the solvents also contributes to the spectral shifts of CNX. The compound bearing the piperazinyl group at the position 7, PM only is fluorescent in high protic solvents. These results are discussed in terms of the competition between the intra- and intermolecular hydrogen bonds. The irradiation of OX, CNX and PM using 300 nm UV light led to a very low photodecomposition rate. Under the same conditions the nalidixic acid (NA), a structurally related quinolone, photodecomposes two orders of magnitude faster.

Synthesis, structure and biological activity of copper(II) complexes with oxolinic acid.

The neutral mononuclear copper complexes with the quinolone antibacterial drug oxolinic acid in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline, 2,2'-bipyridine or 2,2'-dipyridylamine have been synthesized and characterized with infrared, UV-visible and electron paramagnetic resonance spectroscopies. The experimental data suggest that oxolinic acid acts as a deprotonated bidentate ligand and is coordinated to the metal ion through the pyridone and one carboxylate oxygen atoms. The crystal structure of (chloro)(1,10-phenanthroline)(oxolinato) copper(II), 2, has been determined with X-ray crystallography. For all complexes a distorted square pyramidal environment around Cu(II) is suggested. The EPR (electron paramagnetic resonance) behavior of 2 in aqueous solutions indicates mixture of dimeric and monomeric species. The investigation of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and showed that the complexes are bound to calf-thymus DNA. The antimicrobial activity of the complexes has been tested on three different microorganisms. The complexes show a decreased biological activity in comparison to the free oxolinic acid.

Synthesis, characterization and biological evaluation of (99m)Tc/Re-tricarbonyl quinolone complexes.

New rhenium(I) tricarbonyl complexes with the quinolone antimicrobial agents oxolinic acid (Hoxo) and enrofloxacin (Herx) and containing methanol, triphenylphosphine (PPh3) or imidazole (im) as unidentate co-ligands, were synthesized and characterized. The crystal structure of complex [Re(CO)3(oxo)(PPh3)]∙0.5MeOH was determined by X-ray crystallography. The deprotonated quinolone ligands are bound bidentately to rhenium(I) ion through the pyridone oxygen and a carboxylate oxygen. The binding of the rhenium complexes to calf-thymus DNA (CT DNA) was monitored by UV spectroscopy, viscosity measurements and competitive studies with ethidium bromide; intercalation was suggested as the most possible mode and the DNA-binding constants of the complexes were calculated. The rhenium complex [Re(CO)3(erx)(im)] was assayed for its topoisomerase IIα inhibition activity and was found to be active at 100µM concentration. The interaction of the rhenium complexes with human or bovine serum albumin was investigated by fluorescence emission spectroscopy (through the tryptophan quenching) and the corresponding binding constants were determined. The tracer complex [(99m)Tc(CO)3(erx)(im)] was synthesized and identified by comparative HPLC analysis with the rhenium analog. The (99m)Tc complex was found to be stable in solution. Upon injection in healthy mice, fast tissue clearance of the (99m)Tc complex was observed, while both renal and hepatobiliary excretion took place. Preliminary studies in human K-562 erythroleukemia cells showed cellular uptake of the (99m)Tc tracer with distribution primarily in the cytoplasm and the mitochondria and less in the nucleus. These preliminary results indicate that the quinolone (99m)Tc/Re complexes show promise to be further evaluated as imaging or therapeutic agents.

Micellar electrokinetic chromatography for determination of drug partition in phospholipids.

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely L-alpha-dilauroyl phosphatidylcholine (DLPC) or L-alpha-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.

Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique.

At pH 9.75, the resonance light scattering (RLS) intensity of OA-Eu3+ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0x10(-9) to 1.0x10(-6)g/ml for herring sperm DNA, 8.0x10(-10) to 1.0x10(-6) g/ml for calf thymus DNA and 1.0x10(-9) to 1.0x10(-6) g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA-Eu3+ is also investigated.

Vibrational spectroscopic characterization of fluoroquinolones.

Quinolones are important gyrase inhibitors. Even though they are used as active agents in many antibiotics, the detailed mechanism of action on a molecular level is so far not known. It is of greatest interest to shed light on this drug-target interaction to provide useful information in the fight against growing resistances and obtain new insights for the development of new powerful drugs. To reach this goal, on a first step it is essential to understand the structural characteristics of the drugs and the effects that are caused by the environment in detail. In this work we report on Raman spectroscopical investigations of a variety of gyrase inhibitors (nalidixic acid, oxolinic acid, cinoxacin, flumequine, norfloxacin, ciprofloxacin, lomefloxacin, ofloxacin, enoxacin, sarafloxacin and moxifloxacin) by means of micro-Raman spectroscopy excited with various excitation wavelengths, both in the off-resonance region (532, 633, 830 and 1064 nm) and in the resonance region (resonance Raman spectroscopy at 244, 257 and 275 nm). Furthermore DFT calculations were performed to assign the vibrational modes, as well as for an identification of intramolecular hydrogen bonding motifs. The effect of small changes in the drug environment was studied by adding successively small amounts of water until physiological low concentrations of the drugs in aqueous solution were obtained. At these low concentrations resonance Raman spectroscopy proved to be a useful and sensitive technique. Supplementary information was obtained from IR and UV/vis spectroscopy.

Synthesis, characterization and antitumor activity of quinolone-platinum(II) conjugates.

A new series of quinolone-platinum(II) conjugates, [Pt(Q'-NH2)(dmso)X2] and cis-[Pt(Q"-en)X2], where Q' and Q" are quinolones (flumequine, nalidixic acid or oxolinic acid) linked to monodentate and bidentate amine ligands, respectively, and X2 is Cl2 or 1,1-cyclobutanedicarboxylate, have been synthesized with the aim of examining the synergetic antitumor activity of quinolone intercalation and platinum(II) chelation. The complexes were characterized by elemental analyses and IR and multinuclear (1H and 195Pt) NMR spectroscopies, and then subjected to in vitro and in vivo bioassays using the leukemia L1210 cell line.

1-octanol/water distribution coefficient of oxolinic acid: influence of pH and its relation to the interaction with dissolved organic carbon.

The distribution of oxolinic acid (OA) between 1-octanol and buffers at a broad range of pH values was studied and found to decrease with increasing pH. The distribution coefficient to dissolved organic carbon (DOC), log DDOC, was estimated and compared with an experimentally derived log DDOC, showing the experimental value to be almost three orders of magnitude higher. Because only the neutral molecule is assumed to distribute to 1-octanol, the interaction with DOC is considered to be electrostatic in character.

Insights into solar TiO2-assisted photocatalytic oxidation of two antibiotics employed in aquatic animal production, oxolinic acid and oxytetracycline.

In this study, solar driven TiO2-assisted heterogeneous photocatalytic experiments in a pilot-plant with compound parabolic collectors (CPCs) were carried out to study the degradation of two authorized veterinary antibiotics with particular relevance in finfish aquaculture, oxolinic acid (OXA) and oxytetracycline (OTC), using pure solutions of individual or mixed antibiotics. Firstly, the influence of natural solar photolysis was assessed for each antibiotic. Secondly, photocatalytic degradation kinetic rate constants for individual and mixed antibiotics were compared, using a catalyst load of 0.5 g L(-1) and an initial pH around 7.5. Thirdly, for individually photocatalytic-treated OXA and OTC in the same conditions, the growth inhibition of Escherichia coli DSM 1103 was followed, and the mineralization extent was assessed by the residual dissolved organic carbon (DOC), low-molecular-weight carboxylate anions and inorganic ions concentration. Finally, the effect of inorganic ions, such as chlorides, sulfates, nitrates, phosphates, ammonium and bicarbonates, on the photocatalytic degradation of individual solutions of OXA and OTC was also evaluated and the formation of different reactive oxygen species were probed using selective scavengers. The removal profiles of each antibiotic, both as single component or in mixture were similar, being necessary 2.5 kJ L(-1) of solar UV energy to fully remove them, and 18 kJ(UV) L(-1) to achieve 73% and 81% mineralization, for OXA and OTC, respectively. The remaining organic carbon content was mainly due to low-molecular-weight carboxylate anions. After complete removal of the antibiotics, the remaining degradation by-products no longer showed antibacterial activity. Also, 10% and 55% of the nitrogen content of each antibiotic was converted to ammonium, while no conversion to nitrite or nitrate was detected. The presence of phosphates hindered considerably the removal of both antibiotics, whereas the presence of other inorganic ions did not substantially altered the antibiotics photocatalytic degradation kinetics.

First- and second-generation quinolone antibacterial drugs interacting with zinc(II): structure and biological perspectives.

Interaction of equimolar quantities of ZnCl2 with the quinolone antibacterial drugs flumequine (Hflmq), oxolinic acid (Hoxo) or enrofloxacin (Herx) and the N,N'-donor heterocyclic ligands 1,10-phenanthroline (phen) or 2,2'-bipyridine (bipy) results in the formation of 1:1 drug to metal complexes with the general formula [Zn(quinolone)(N,N'-donor)Cl], while excess of the quinolone leads to 1:2 metal to drug [Zn(quinolone)2(N,N'-donor)] complexes. In all complexes, the deprotonated bidentate quinolonato ligands are coordinated to zinc ion through the pyridone oxygen and a carboxylato oxygen. The crystal structures of [Zn(oxo)(phen)Cl], [Zn(flmq)(phen)Cl] and [Zn(flmq)2(phen)] have been determined by X-ray crystallography. All complexes exhibit good binding propensity to human or bovine serum albumin protein showing relatively high binding constant values. Interaction of the complexes with calf-thymus (CT) DNA, studied by UV spectroscopy, has shown that they bind to CT DNA, while [Zn(flmq)(phen)Cl] and [Zn(flmq)2(phen)] complexes exhibit the highest binding constants to CT DNA. Competitive study with ethidium bromide (EB) has shown that all complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. Intercalative binding mode is proposed for the interaction of the complexes with CT DNA and has also been verified by DNA solution viscosity measurements. DNA electrophoretic mobility experiments suggest that all complexes bind to linearized pDNA and supercoiled pDNA by intercalative manner resulting in catenanes formation as well as in double-stranded cleavage reflecting (or ending) in the formation of linear DNA. The complexes exhibit significant antimicrobial activity tested on five different microorganisms.

Structure, antimicrobial activity, DNA- and albumin-binding of manganese(II) complexes with the quinolone antimicrobial agents oxolinic acid and enrofloxacin.

The reaction of MnCl2 with the quinolone antibacterial drug oxolinic acid (Hoxo) results to the formation of [KMn(oxo)3(MeOH)3]. Interaction of MnCl2 with the quinolone Hoxo or enrofloxacin (Herx) and the N,N'-donor heterocyclic ligand 1,10-phenanthroline (phen) results in the formation of metal complexes with the general formula [Mn(quinolonato)2(phen)]. The crystal structures of [KMn(oxo)3(MeOH)3] and [Mn(erx)2(phen)], exhibiting a 1D polymeric and a mononuclear structure, respectively, have been determined by X-ray crystallography. In these complexes, the deprotonated bidentate quinolonato ligands are coordinated to manganese(II) ion through the pyridone oxygen and a carboxylato oxygen. All complexes can act as potential antibacterial agents with [Mn(erx)2(phen)] exhibiting the most pronounced antimicrobial activity against five different microorganisms. Interaction of the complexes with calf-thymus DNA (CT DNA), studied by UV spectroscopy, has shown that they bind to CT DNA. Competitive study with ethidium bromide (EB) has shown that all complexes can displace the DNA-bound EB indicating their binding to DNA in strong competition with EB. Intercalative binding mode is proposed for the interaction of the complexes with CT DNA and has also been verified by DNA solution viscosity measurements and cyclic voltammetry. DNA electrophoretic mobility experiments suggest that [Mn(erx)2(phen)] binds strongly to supercoiled pDNA and to linearized pDNA possibly by an intercalative manner provoking double-stranded cleavage reflecting in a nuclease-like activity. The complexes exhibit good binding propensity to human or bovine serum albumin protein showing relatively high binding constant values. The binding constants of the complexes towards CT DNA and albumins have been compared to their corresponding zinc(II) and nickel(II) complexes.

Synthesis of oligodeoxynucleotides with 5'-caps binding RNA targets.

Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5'-O-terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L-prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step-wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap-bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis.

Nickel-quinolones interaction. Part 5-Biological evaluation of nickel(II) complexes with first-, second- and third-generation quinolones.

The nickel(II) complexes with the quinolone antibacterial agents oxolinic acid, flumequine, enrofloxacin and sparfloxacin in the presence of the N,N'-donor heterocyclic ligand 2,2'-bipyridylamine have been synthesized and characterized. The quinolones act as bidentate ligands coordinated to Ni(II) ion through the pyridone oxygen and a carboxylato oxygen. The crystal structure of [(2,2'-bipyridylamine)bis(sparfloxacinato)nickel(II)] has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA with [(2,2'-bipyridylamine)bis(flumequinato)nickel(II)] exhibiting the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the [Ni(quinolonato)(2)(2,2'-bipyridylamine)] complexes have been evaluated in comparison to the previously reported Ni(II) quinolone complexes [Ni(quinolonato)(2)(H(2)O)(2)], [Ni(quinolonato)(2)(2,2'-bipyridine)] and [Ni(quinolonato)(2)(1,10-phenanthroline)]. The quinolones and their Ni(II) complexes have been tested for their antioxidant and free radical scavenging activity. They have been also tested in vitro for their inhibitory activity against soybean lipoxygenase.

Supramolecular solvents in solid sample microextractions: application to the determination of residues of oxolinic acid and flumequine in fish and shellfish.

Supramolecular solvents are here proposed firstly as extractants in solid sample microextractions. The approach was evaluated by extracting flumequine (FLU) and oxolinic acid (OXO), two widely used veterinary medicines, from fish and shellfish muscle using a supramolecular solvent made up of decanoic acid (DeA) reverse micelles. The antibiotics were extracted in a single step (approximately 15 min), at room temperature, using 400 microL of solvent. After centrifugation, an aliquot of the extract was directly analyzed by liquid chromatography and fluorescence, without the need of clean-up or solvent evaporation. Contrary to the previously reported methods, both OXO and FLU were quantitatively extracted from fish and shellfish, independently of sample composition. The high extraction efficiencies observed for these antibiotics were a consequence of their amphiphilic character which resulted in the formation of DeA-OXO and DeA-FLU mixed aggregates. The quality parameters of this quantitative method including sensitivity, linearity, selectivity, repeatability, trueness, ruggedness, stability, decision limit and detection capability were evaluated according to the 2002/657/EC Commission Decision. Quantitation limits in the different samples analyzed (salmon, sea trout, sea bass, gilt-head bream, megrim and prawns) ranged between 6.5 and 22 microg kg(-1) for OXO and, 5 and 15 microg kg(-1) for FLU. These limits were far below the current maximum residue limits (MRLs) set by the European Union (EU) (i.e. 100 and 600 microg kg(-1), for OXO and FLU, respectively). The trueness of the method was determined by analyzing a Certified Reference Material (CMR, BCR-725) consisting of a lyophilised salmon tissue material. Recoveries for fortified samples (50-100 microg kg(-1) of OXO and 50-600 microg kg(-1) of FLU) and their relative standard deviations were in the intervals 99-102% and 0.2-5%, respectively. The repeatability, expressed as relative standard deviation, was 3.6% for OXO and 2.3% for FLU ([OXO]=[FLU]=200 microg kg(-1) and n=11).