Neil J Creamer

Iryna P Mikheenko

Clive Johnson

Stephen P Cottrell

Lynne E Macaskie
email: l.e.macaskie@bham.ac.uk

Local magnetism in palladium bionanomaterials probed by muon spectroscopy

Keywords: Desulfovibrio desulfuricans, electronic magnetism, muon spin rotation spectroscopy, nanoparticles, palladium bionanoparticles

come

Introduction

There is substantial current interest in nanoparticles as they often exhibit properties at odds with those of the bulk material [START_REF] Hori | Anomalous magnetic polarization effect of Pd and Au nano-particles[END_REF][START_REF] Eberhardt | Clusters as new materials[END_REF]. Palladium nanoparticles have been extensively studied both experimentally and theoretically mainly due to the excellent performance of the metal as a catalyst in hydrogenation and hydrogenolysis [START_REF] Schuth | Hydrodechlorination and hydrogenation of aromatic compounds over palladium on alumina in hydrogen-saturated water[END_REF][START_REF] Nishimura | Handbook of Heterogeneous Catalytic Hydrogenation for Organic Synthesis[END_REF]. A recent development is the production of Pd nanoparticles supported on bacterial cells which show excellent potential as catalysts [START_REF] Creamer | A novel hydrogenation and hydrogenolysis catalyst using palladized biomass of Gram negative and Gram positive bacteria[END_REF]) in applications such as remediation of Cr(VI) [START_REF] Mabbett | Biorecovered precious metals from industrial wastes: single step conversion of mixed metal liquid waste to bioinorganic catalyst ["MMBio-Pd(0)"] with environmental application[END_REF], chlorinated aromatic compounds [START_REF] Vs | Sulphate-reducing bacteria, palladium and the reductive dehalogenation of chlorinated aromatic compounds[END_REF][START_REF] Redwood | Biomass-supported catalysts on Desulfovibrio desulfuricans and Rhodobacter sphaeroides[END_REF], pesticides [START_REF] Mertens | Biocatalytic dechlorination of lindane by nanoscale particles of Pd(0) deposited on Shewanella oneidensis[END_REF]) and chlorinated organophosphates [START_REF] Deplanche | Versatility of a new bioinorganic catalyst: palladized cells of Desulfovibrio desulfuricans and application to dehalogenation of flame retardant materials[END_REF], as well as major potential applications in "green chemistry" [START_REF] Bennett | Pd supported on bacteria and biomass as a novel heterogeneous catalyst: a comparison of Pd 2 AlO 3 and Bio-Pd in the hydrogenation of 2-pentyne[END_REF][START_REF] Wood | Hydrogenation of 2-butyne-1,4-diol using novel bio-palladium catalysts[END_REF] and in clean energy [START_REF] Yong | From bio-mineralisation to fuel cells: biomanufacture of Pt and Pd nanocrystals for fuel cell electrode catalyst[END_REF]2010;[START_REF] Orozco | Towards an integrated system for bio-energy: Hydrogen production by Escherichia coli and use of palladium-coated waste cells for electricity generation in a fuel cell[END_REF]). The nanoparticulate nature of catalytically-active "Bio-Pd" was shown by surface area measurements [START_REF] Bennett | Pd supported on bacteria and biomass as a novel heterogeneous catalyst: a comparison of Pd 2 AlO 3 and Bio-Pd in the hydrogenation of 2-pentyne[END_REF]) while, using a magnetic method, [START_REF] Mikheenko | Magnetic testing of Pdloaded bacteria[END_REF] showed the presence of nanoparticles of size ~ 5 nm.

The free Pd atom is non-magnetic and, in bulk, no spontaneous ferromagnetic order has been observed [START_REF] Staunton | Spin fluctuations in nearly magnetic metals from ab initio dynamical spin susceptibility calculations: Application to Pd and Cr 95 V 5[END_REF][START_REF] Vega | Electronic structure calculations of low-dimensional transition metals[END_REF]. However, studies of palladium nanoparticles formed by gas evaporation demonstrated ferromagnetism in a population of particles with mean diameter 5.9 nm [START_REF] Taniyama | Ferromagnetism of Pd fine particles[END_REF]) which has been attributed to non-typical metal-metal bonding due to the constraints of the particle size [START_REF] Vitos | Size-dependent paramagnetic-ferromagnetic phase transition in palladium clusters[END_REF].

The early finding that Bio-Pd particles are magnetically active [START_REF] Mikheenko | Magnetic testing of Pdloaded bacteria[END_REF]) together with the observation that the magnetic moment, nanoparticle size and hence catalytic activity are related [START_REF] Macaskie | Applications of bacterial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy[END_REF]) is important because these can be controlled by the choice of preparation conditions. In general mid-logarithmic and early stationary-phase cells give the smallest nanoparticles [START_REF] Mikheenko | Nanoscale palladium recovery[END_REF]). This degree of control makes the material very appropriate for this examination. In order to visualise the magnetic components we used two preparations of the same mass loading of Pd(0), made using two cultures of the same strain. These provide a common background biomatrix on which to evaluate the potential of μSR as a tool for characterisation of metallic bionanoparticles.

The muon is an unstable lepton with a mean lifetime of ~2.20 s. It has a magnetic moment approx. three times that of the proton and, in studies of condensed matter, is essentially a sensitive microscopic magnetometer (Dalmas de Reotier and Yaouanc 1997). For the systems studied here, positive muons typically thermalise at an interstitial location and thus probe the local magnetic fields in the regions between the atoms. The ISIS synchrotron produces a beam of positive muons with a unique momentum (29.8 MeV/c) which is 100% spin-polarised (ISIS web page). Unlike other particle probes, muons stop within the sample before decaying into a positron and two neutrinos. Crucially, the positron is emitted preferentially in the direction of the muon spin enabling the time evolution of the muon polarisation ( or decay asymmetry) to be followed by detecting the time dependence of the positron distribution. Thus it is possible to measure the time dependent depolarisation of the muon signal and characterise the distribution and dynamics of internal fields in the sample.

The µSR experiment is sensitive to both nuclear and electronic magnetism (Dalmas de Reotier and Yaouanc 1997). Where implanted muons interact with the static, dipolar fields associated with nuclei, the asymmetry decay function reflects the distribution of fields and is typically Gaussian in form. For an ordered electronic system a coherent precession of the muon polarisation may be seen provided muons thermalise into a limited number of unique sites. At ISIS the instrument pass-band (~6 MHz) will also limit the frequencies that can be measured, and instead an ordered state may be revealed by the loss of 2/3 of the initial asymmetry. Fluctuating electronic moments (due to unpaired spins) may also be measured, when the decay is typically described by a Lorentzian function.

In insulating materials, the implanted muon may bind an electron to form muonium, an exotic light hydrogen isotope with the positive muon as the nucleus [START_REF] Cox | Implanted muon studies in condensed matter science[END_REF]. This species may, in turn, react with organic systems to form radical states: in both cases the muon-electron hyperfine coupling complicates the signal measured and determining the fractions of the various muonic states present in the system is an important aspect of the experiment.

Prior to this study, [START_REF] Kemali | Trapped muon in heavily dislocated palladium[END_REF] had applied µSR to the study of heavily dislocated hydrogencontaining bulk Pd. [START_REF] Kaiser | µSR investigation of a cluster of monodisperse Pd nanoparticles[END_REF] investigated monodisperse, ligand-capped Pd nanoparticles of diameter 1.1 nm, smaller than most of the particles studied here (see [START_REF] Mikheenko | Magnetic testing of Pdloaded bacteria[END_REF]) but close to the 1.2 to 6 nm critical size range within which Pd is expected to demonstrate superparamagnetic/ferromagnetic behaviour (Taniyama et al. 2007;[START_REF] Staunton | Spin fluctuations in nearly magnetic metals from ab initio dynamical spin susceptibility calculations: Application to Pd and Cr 95 V 5[END_REF]. In that study, the Pd particles were capped by a ligand shell of diameter ~2.1 nm, while here they are supported in a biomatrix which extends to the micron scale. The current study is the first application of µSR as a probe for catalytic bionanoparticles.

Materials and Methods

Manufacture of palladium on bacterial cells (Bio-Pd)

Palladium bionanomaterial was manufactured by exposing resting cells of Desulfovibrio desulfuricans NCIMB 8307 (cultures harvested at different stages of growth to give samples of different magnetic activity, e.g. see [START_REF] Macaskie | Applications of bacterial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy[END_REF]) to an anaerobic solution (pH 2.3) of Na 2 PdCl 4 (Aldrich, 99.995 %) such that the total palladium available was 25% of the calculated dry cell weight [START_REF] Lloyd | Enzymatic recovery of elemental palladium by using sulfate -reducing bacteria[END_REF].

Following biosorption of soluble Pd(II) ions the suspension was sparged with H 2 for 30 minutes resulting in the formation of palladium particles in the periplasmic space and at the cell surface (e.g. [START_REF] Vs | Sulphate-reducing bacteria, palladium and the reductive dehalogenation of chlorinated aromatic compounds[END_REF]Macaskie 2005;[START_REF] Mikheenko | Bioaccumulation of palladium by Desulfovibrio fructosovorans and hydrogenase deficient mutants[END_REF]. Cells were washed in distilled water three times then once in acetone, air dried and ground to a fine powder. For the purposes of this study, two separate batches were prepared: "Sample 1" was from an early stationary phase culture while "Sample 2" was harvested in the mid-growth phase.

Magnetic measurements using a superconducting quantum interference device (SQUID)

The magnetic moments (emu/g) of the two biological palladium powders and, for reference, high purity Pd powder (Johnson Matthey, 99.998 %) were measured in a superconducting quantum interference device (SQUID: Quantum Design, USA) against the applied magnetic field (H, Oersted). The magnetic field was varied between zero and 10,000 Oe. All measurements are expressed per g of palladium and accuracy is limited by mass determination to ±10%. SQUID data were collected as described previously [START_REF] Mikheenko | Magnetic testing of Pdloaded bacteria[END_REF]) by measuring the dependence of the magnetic moment on the applied field.

Magnetic measurements using Muon Spin Rotation Spectroscopy (μSR)

The SR experiments were carried out using the EMU spectrometer at the ISIS pulsed muon source (ISIS web page). This instrument is configured with detectors forward (upstream) and backward (downstream) of the sample, and is ideal for the zero and longitudinal field (<4,500 G) measurements carried out in this work. For this instrument, the muon spin polarisation is co-linear with the beam momentum. The samples were mounted in a 24 mm diam.-recessed aluminium sample holder with a silver mask placed around the sample to ensure a predictable response from muons stopping outside the sample area. Temperature control was achieved using a closed-cycle refrigerator with the sample attached by a suitable cold-finger.

Data is initially reduced using Equation 1 to form the time dependent decay asymmetry for the counts recorded in the forward (N F ) and backward (N B ) detectors for the 32 s measured following muon implantation:

a(t) = [N F (t) -αN B (t)] / [N F (t) + αN B (t)] (1),
where α is a parameter describing the relative efficiencies of the forward and backward detector arrays that is calibrated for each sample mounted on the spectrometer by applying a small external field transverse to the muon polarisation.

The time dependent behaviour of the decay asymmetry takes the form:

a(t) = a 0 G z (t) (2),
where G z (t) is a function describing the time evolution of the muon spin polarisation due to spin interactions and a 0 , the full asymmetry for the EMU spectrometer (~23%). Analysis is completed by least squares fitting of the appropriate form of G z (t) to the reduced data.

Results and discussion

The magnetisation of samples 1 and 2 was determined by SQUID measurements to provide a reference for the μSR study. The bulk Pd control gave a linear response of magnetic moment against the applied field, whereas the two samples showed a non-linearity (Fig 1A ,B). From our results, we calculate that the average moment per nanoparticle in Sample 1, 1 to 3 10 2 B , is consistent with electronic magnetism in Pd grains of size ~ 4-6 nm assuming either that all atoms in the particle contribute equally to the overall magnetism each having a moment of 0.11 μ B [START_REF] Vitos | Size-dependent paramagnetic-ferromagnetic phase transition in palladium clusters[END_REF] or that only the surface atoms contribute and each atom has a moment of 0.23 μ B [START_REF] Taniyama | Ferromagnetism of Pd fine particles[END_REF]). These estimates also lie within the size range of 1.2 to 6 nm for which ferromagnetism has previously been observed (Taniyama et al. 2007;[START_REF] Staunton | Spin fluctuations in nearly magnetic metals from ab initio dynamical spin susceptibility calculations: Application to Pd and Cr 95 V 5[END_REF].

As shown in Figs. 1A and1B, Sample 2 had a significantly larger non-linear component than Sample 1, suggesting a larger proportion of small particles within the range of 1.2 to 6 nm. [START_REF] Skibar | Novel precious metal-based bionanocatralsyst from scrap[END_REF] showed that for a given microbial support (D. desulfuricans) the non-linear component can range from being practically zero to almost 100 % according to how the samples are prepared (e.g. by using the Pd amine salt instead of the chloride salt and a neutral instead of acidic pH during Pd-challenge) as well as the growth phase from which the culture was harvested. Examples were shown previously [START_REF] Macaskie | Applications of bacterial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy[END_REF]. For the present study, and from the SQUID characterisation shown in Figs. 1A and1B, Samples 1 ("magnetic") and 2 ("non-magnetic") were chosen as providing suitable references with which to evaluate the scope for using μSR to provide specific information about local magnetic effects within the samples.

The system under study comprises hydrogen-containing palladium nanoparticles (25% by mass) supported in dried biomatrix (75% by mass); implanted muons may stop in either the nanoparticles or the biomatrix approximately according to their mass fractions although additional stops in the metal particles cannot be ruled out [START_REF] Ma | Energy loss and straggling of MeV ions through biological bamples[END_REF]). To allow comparison, care was taken to ensure Samples 1 and 2 had the same mass ratio of nanoparticle to biomatrix. For muons thermalising in the biomass muonium or radical states may be formed, while the biological material also contains trace Fe associated with redox enzymes that might also give rise to local electronic fields. Muons stopping in (or close to) the Pd particles may interact with both the electronic or nuclear fields associated with the metal atoms or the trapped hydrogen. The latter has a significant nuclear magnetic moment as evidenced by its usefulness as a target for NMR Spectroscopy [START_REF] Harris | Encyclopedia of Nuclear Magnetic Resonance[END_REF]. Measurements of the nonmagnetic Sample 1 can provide a good indication as to the various muonic states forming in this system and, accordingly, this was the focus of the initial study.

Zero applied magnetic field measurements were carried out for Sample 1 between 10 K and 300 K.

Throughout this temperature range the form of the muon spin relaxation observed could be modelled by a two-component function comprising a Gaussian decay and a non-relaxing background, as described by equation (3):

a(t) = a 0 exp [-( t) 2 ] + a b (3).
In the present analysis, the background term, a b , subsumed both a large non-relaxed fraction originating from the sample and a much smaller signal arising from muons stopping in the surrounding silver mask. The relaxation rate, , reflects the distribution of local fields at the muon site.

The total asymmetry of the zero field signal is roughly constant at ~ 22 % over the full temperature range, with corresponding 100 G transverse field data confirming this to be diamagnetic in origin. This result suggests muonium formation in the biomatrix is minimal (< 1 % asymmetry)a fortunate outcome that greatly simplifies analysis of this system. The asymmetry of the Gaussian component remains constant, however, over a broad temperature range at ~6 %, suggesting that ~25% of the muons are consistently implanted into a similar environment. The temperature dependence of the relaxation rate, , is shown in Figure 2. Intriguingly, at low temperatures, the rate is similar to the value of 0.24 s - 1 reported for muons stopping in the hydrogen-saturated dislocated Pd metal (Kemali et al.1997).

Furthermore, the peak in the rate measured at ~75 K is consistent with the onset of mobility and subsequent trapping, although the precise nature of the trapping sites (whether in the bulk or at the surface) remains to be determined.

Considering these results, it seems unlikely that the Gaussian component could originate from the biomatrix, with the signal probably due to muons stopping in the Pd nanoparticles. While the moment on the Pd nucleus is small, and in itself will make only a small contribution to the measured depolarisation, hydrogen is known to be incorporated into the nanoparticle structure during manufacture and, assuming suitable traps, the interaction of its large nuclear moment with the implanted muon can explain the form of the measured signal. As the temperature is increased above 75 K there is a gradual reduction in the measured linewidth that continues to the highest temperature measured (300 K), a result suggesting the onset of mobility of the muon relative to the hydrogen in the nanoparticle structure. The weak relaxation of muons stopping in the biomatrix is surprising as the organic material is also likely to contain hydrogen; we assume that the preferred muon site and/or dynamics contrive to limit the local field on the muon.

Similar SR measurements were carried out on Sample 2 which was shown from SQUID measurements to have an electronic magnetic signature. Immediately apparent is the substantial missing fraction present in both the zero field (evident in the first points plotted in Fig 4B ) and the 100 G transverse field data (see Fig 3A). The measured transverse asymmetry of ~14.5 % at room temperature suggests a missing fraction of ~7 % in asymmetry. Since the mass fraction and composition of the biomatrix is maintained compared to the non-magnetic Sample 1, a near identical stopping distribution is expected with almost all muons thermalising into a diamagnetic state. Although Fe impurities are present in the biomatrix, there was no evidence from the measurements on Sample 1 that these are magnetically active and contributing to the signal. Futhermore, other studies using element-specific X-ray magnetic circular dichroism confirmed that the magnetic signal of the specimen was specifically attributable to Pd (N.J. Creamer, I. Mikheenko and L.E. Macaskie, unpublished work).

The asymmetry loss is similar to the signal amplitude measured from the Pd nanoparticles in Sample 1, and it therefore seems likely that muons stopping in and adjacent to the nanoparticles are experiencing a substantial magnetic field that depolarises the muon signal between implantation and the start of measurement.

To investigate the internal field strength of the magnetic nanoparticles and the associated field on the implanted muon, a longitudinal field decoupling measurement was carried out. Here, large external fields are applied and the initial muon asymmetry is recovered as these dominate the internal fields of the sample. The result of such a measurement is shown in Figure 3A. Even applying 4000 G, the strongest field available on the instrument, the field was insufficient to fully recover the full initial asymmetry; however, its dependence on the applied field is clear and the mid-point of the decoupling curve enables us to estimate the internal field at the muon site to be ~1000 G. While these measurements suggest the nanoparticles as the source of the bulk fields measured by the SQUID, a more detailed understanding of muon trapping in this system is required to properly correlate the data.

The overall shape of the curve suggests that muons are sampling a broad distribution of internal fields; however, this is a reasonable result given the highly inhomogeneous nature of the sample and the absence of a unique muon site. As expected, the longitudinal relaxation, obtained from fitting an exponential decay, is quenched as the applied field is increased (Figure 3B). For comparison, a similar measurement is shown for Sample 1 where there is clearly no dependence of the signal asymmetry on the applied field.

Since the fraction of muons implanted into the Pd nanoparticles is rapidly depolarised by strong internal fields, the zero field asymmetry measured for Sample 2 must originate from muons stopping in the biomatrix and sample holder. In contrast to the non-magnetic sample, where the signal from the biomatrix was non-relaxed, a significant decay was measured over the full temperature range for the magnetic system, with representative spectra shown in Figure 4A. Relaxation of the muon signal is not surprising since the magnetic nanoparticles are effectively forming a dilute spin system within the biomatrix. SQUID measurements demonstrate the nanoparticles to have a substantial magnetic moment

(1 -3 10 2 B , see above), and therefore there will be a field profile associated with each nanoparticle falling as r -3 to leave a region of field inhomogeneity between the nanoparticles. The temperature dependence of the relaxation signal is probably the result of dynamics, a conclusion supported by parameterising the data using the stretched exponential form defined in equation 4:

a(t) = a 0 exp [-( t) β ] (4).
From fitting the data we find that the exponent, β ( apply models, such as the root exponential form, used in the analysis of other dilute magnetic systems (e.g. [START_REF] Kaiser | µSR investigation of a cluster of monodisperse Pd nanoparticles[END_REF][START_REF] Bewley | Muon spin relaxation in a superparamagnet: Field dynamics in Cu 98 Co 2[END_REF][START_REF] Lord | Muon methods for studying nanomagnetism[END_REF] has not been successful, possibly because of the potential for multiple muon sites and complex dynamics within the present material.

Further work is clearly required to establish the nature of the dynamics observed in this material.

Conclusions

SQUID measurements have demonstrated bulk magnetism in palladium nanoparticles supported in a biomatrix. This paper reports the first application of the local magnetic field probe, SR, to identify the source of electronic magnetism within this system. Comparative measurements between two samples showing strong and weak bulk electronic magnetism have enabled us to identify a fraction of muons stopping within the palladium nanoparticles. The behaviour of this fraction is indicative of local coupling between the implanted muon and strong magnetic fields associated with the individual palladium nanoparticles present in a magnetic sample, a result consistent with measurements using element-specific X-ray Circular Magnetic Dichroism (XMCD) to be published at a later date.

Analysis of the muon signal has shown that almost 75% of the implanted muons thermalise in the biomatrix and are sensitive to the dynamics within the system. Measurements on Sample 2 suggest that electronic spin fluctuations persist throughout the temperature range studied. We tentatively conclude that these are associated with the electronic magnetism observed in the individual palladium nanoparticles. Further experimental work using SR is needed to properly characterise the nature and source of these dynamics.

Legends to Figures

  Fig 4B, open circles), changes from ~1.6 (tending towards a Gaussian form) at low temperature to ~1 (a simple exponential) at 300 K while the relaxation rate, (Fig 4B, closed circles), shows a slow reduction with increasing temperature. Attempting to

Fig 1 :

 1 Fig 1: Magnetic field dependence of the magnetic moment component for Sample 1 (A) and Sample 2 (B). : Total magnetisation : Non-linear. Note the magnitude difference of the y-axis in Fig. 1B

Fig 2

 2 Fig 2 Temperature dependence of the zero-field relaxation rate measured in Sample 1 for the Gaussian

Fig 4 .

 4 Fig 4. A: Representative zero field measurements for Sample 2 shown as a function of temperature.  15 K;  105 K;  195 K;  300 K. B: Stretched exponential analysis of data measured for Sample 2

  Figure 1

Acknowledgements

We acknowledge with thanks the financial support of the EU (Contract No. G5RD-CT-2002-0075), the BBSRC (Grant Nos BB/C516195/2 and BB/E003788/1) and the EPSRC (Grant Nos EP/D05768X/1 and EP/E034888/1).


Viktor ; Konakovsky

Margarete Focke

Karin Hoffmann-Sommergruber

Rainer Schmid

Otto Scheiner

Peter Moser

Reinhart Jarisch

Wolfgang Hemmer
email: hemmer@faz.at

Levels of histamine and other biogenic amines

Keywords: HPLC, Immunoassays Additives/Contaminants: Process contaminants Food Types: Beverages, Wine biogenic amines, histamine, HPLC, phenylethylamine, red wine, tyramine, wine intolerance

acting as regulators of growth, neuronal transmitters or inflammatory mediators. The interest for biogenic amine formation during wine production arises from the necessity to better control and understand the complexity of the wine-making process as well as from concerns related to human health since some amines commonly found in wines, such as histamine, tyramine and phenylethylamine, may cause unwanted side effects [START_REF] Wantke | Histamine-free diet: treatment of choice for histamineinduced food intolerance and supporting treatment for chronic headaches[END_REF][START_REF] Kalač | A review of dietary polyamines: formation, implications for growth and health and occurrence in foods[END_REF]. High histamine levels are acutely toxic and implicated in scombroid fish poisoning [START_REF] Lehane | Histamine fish poisoning revisited[END_REF]. In experimental settings, a single oral dose of 75 mg histamine, a quantity reasonably ingestible during a meal composed of histamine-rich food, provoked acute diarrhea in as much as 50% of healthy subjects [START_REF] Wöhrl | Histamine intolerance-like symptoms in healthy volunteers by oral provocation with liquid histamine[END_REF]). However, there might exist a population of susceptible individuals reacting to even much lower quantities of biogenic amines [START_REF] Wantke | Histamine-free diet: treatment of choice for histamineinduced food intolerance and supporting treatment for chronic headaches[END_REF].

Wine is a common cause for food adverse reactions in the population and may elicit a range of allergy-like symptoms including flushing, itching, headaches, rhinitis, meteorism, diarrhea, as well as urticaria and asthma [START_REF] Wantke | Histamine-free diet: treatment of choice for histamineinduced food intolerance and supporting treatment for chronic headaches[END_REF]Wantke 1996;Jarisch 1996;[START_REF] Vally | Allergic and asthmatic reactions to alcoholic drinks[END_REF]. The pathomechanisms underlying wine intolerance appear to be manifold. Symptoms may arise from hypersensitivity to sulphites added to wines [START_REF] Wüthrich | Asthma due to disulfites[END_REF][START_REF] Vally | Changes in bronchial hyperresponsiveness following high-and low-sulphite wine challenges in wine-sensitive asthmatic patients[END_REF] or, in rare cases, represent true allergic reactions to grape lipid transfer proteins or other allergens surviving in low amounts in the fermented product [START_REF] Pastorello | Identification of grape and wine allergens as an endochitinase 4, a lipid-transfer protein, and a thaumatin[END_REF][START_REF] Schad | Wine anaphylaxis in a German patient: IgE-mediated allergy against a lipid transfer protein of grapes[END_REF]. Other studies identified acetaldehyde, a metabolite of ethanol with the capacity to release histamine from lung mast cells, as a major cause for wine-induced asthma in Japanese patients [START_REF] Kawano | Acetaldehyde induces histamine release from human airway mast cells to cause bronchoconstriction[END_REF][START_REF] Vally | Allergic and asthmatic reactions to alcoholic drinks[END_REF].

Anyhow, biogenic amines are considered the most important reason for wine intolerance. Due to the predominant generation of biogenic amines during malolactic (Vidal-Carou 1990b;Vidal-Carou 1990a;[START_REF] Radler | Proceedings of the International Symposium on Nitrogen in Grapes and Wines[END_REF][START_REF] Soufleros | Correlation between the content of biogenic amines and other wine compounds[END_REF][START_REF] Romero | Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination[END_REF]), amine levels are usually higher in red wines than in white wines [START_REF] Zee | Biogenic amines in wines[END_REF][START_REF] Maxa | HPLC-Methode zur routinemäßigen Quantifizierung von biogenen Aminen in Wein[END_REF][START_REF] Romero | Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination[END_REF]. This fits well to the observation that among alcoholic beverages red wine is most often accused for eliciting adverse reactions [START_REF] Linneberg | Prevalence of selfreported hypersensitivity symptoms following intake of alcoholic drinks[END_REF]. Although red wines contain still moderate amine levels in comparison to cheese (up to 2500 mg/kg), salami (up to 600 mg/kg) and fish products (up to 4000 mg/kg) [START_REF] Diel | Histamine containing food: Establishment of a German Food Intolerance Databank (NFID)[END_REF], red wine intolerance might be a typical marker symptom and thus has been proposed as a model for histamine intolerance [START_REF] Wantke | The red wine provocation test: intolerance to histamine as a model for food intolerance[END_REF][START_REF] Wantke | The red wine maximization test: drinking histamine rich wine induces a transient increase of plasma diamine oxidase activity in healthy volunteers[END_REF]). Possibly, the pharmacologic properties of biogenic amines in wine are potentiated by some bystander effects of ethanol such as augmented gut permeability or potential interference with amine-metabolising pathways [START_REF] Draper | Effect of alcohol on the integrity of the intestinal epithelium[END_REF][START_REF] Sessa | Effect of acute ethanol administration on diamine oxidase activity in the upper gastrointestinal tract of rat[END_REF].

However, according to clinical experience not all red wines are able to elicit adverse reactions to the same degree. This might be reasonably explained by the variable amine content of different products and has prompted some attentive winemakers to specialize in the production of low-histamine wines expected to be tolerated even by wine-sensitive subjects [START_REF] Bodmer | Biogenic amines in foods: Histamine and food processing[END_REF]. Because high amine concentrations in wines may reflect poor hygienic conditions during wine-making [START_REF] Bodmer | Biogenic amines in foods: Histamine and food processing[END_REF][START_REF] Lüthy | Biogene Amine in Lebensmitteln: Zur Wirkung von Histamin, Tyramin und Phenyethylamin auf den Menschen[END_REF], we wondered whether carefully processed high-quality wines from top wineries will show below-average amine concentrations. The aim of the present study was to determine levels of histamine and other common biogenic amines in 100 high-quality red wines from Austria randomly selected from a set of wine samples submitted to professional wine tasters for enrolment in a wellacknowledged national wine guide.

Materials and methods

Wine Samples

One hundred Austrian red wines from 77 wineries made from seven different grape varieties grown locally or worldwide were analysed (Zweigelt n=25, Blaufränkisch n=25, Merlot n=10, St. Laurent n=10, Pinot noir n=10, Shiraz n=10, Cabernet-Sauvignon n=10). All wines were high quality red wines submitted to a national wine challenge and rated with a mean Parker score of 89.5 ± 1.69 (range 85-94). 90% of the wines reached a score of 88 or higher. Most wines were from vintage 2004 (n=84), 10 from 2005, and 6 from 2003.

Derivatization of wine samples

Biogenic amines were detected by HPLC after pre-column derivatization to fluorescent dansyl chloride conjugates (Figure 1). 1 mL of wine was mixed in a glass vial (≤5 mL) with 100 µL diaminoheptane (80 mg/L) as internal standard, 30 µL NaOH and 1 mL of Merck Titrisol ® boric acid/potassium chloride/sodium hydroxide buffer giving a final pH of 8. If pH was below 8 at the start, derivatization of some amines (e.g. histamine) was incomplete, leading to a striking loss of sensitivity and altered elution times.

To finish the reaction, 2 mL dansyl chloride were added, the tubes shaken vigorously and placed into a water bath at 55°C for 1 hour. After cooling for 10 min on ice, samples were centrifuged for 5 minutes and the supernatants were ready for analysis which was performed within the following 24 hours. All Chemicals were purchased from Sigma-Aldrich (Vienna, Austria). Preparation of amine standards 10 mL stock solutions with a concentration of 10,000 mg/L were made from the following eight amines: isoamylamine, tryptamine, phenylethylamine, putrescine, cadaverine, tyramine (all from Sigma-Aldrich, Vienna, Austria) and from histamine and spermidine (FLUKA, Vienna, Austria). For tryptamine and tyramine, methanol was added (1 part methanol + 9 parts H 2 O) to improve solubility. Dansyl chloride (FLUKA, Vienna, Austria) was prepared by dissolving 400 mg in 100 mL acetone and subsequent filtration through a 0.45 µm nylon filter to remove unsolved particles.

A standard mix containing all eight amines was prepared by mixing 1 mL of each stock solution together with 2 ml of water giving a final volume of 10 mL and a concentration of 1000 mg/L for each amine. From this concentration a 6-step dilution series was prepared.

To enable identification of individual biogenic amines and their potential matrix effects in real wine samples, selected wines were spiked with the standard mix.

After the first results obtained from spiked samples, the standard mix was optimized by changing amine concentrations to values close to those found in wines ("optimized STD").

For the different amines, the following maximum calibration points were determined: isoamylamine 10 mg/L, tryptamine 10 mg/L, phenylethylamine 10 mg/L, cadaverine 15 mg/L, tyramine 30 mg/L, spermidine 30 mg/L, spermine 30 mg/L, histamine 40 mg/L, and putrescine 150 mg/L. The results obtained from the two standard mixes established excellent linearity between area and concentration.

Recovery rates and detection limits

Recovery rates were assessed by spiking two selected red wines with the standard mix dilution series. Recovery rates were calculated separately from each of the two wines and expressed as the average of the recoveries obtained from the set of dilution experiments.

Detection limits were calculated only for histamine because it represents the most interesting amine with respect to effects on human health. To test the limits of quantification, histamine standard solutions with concentrations of 1.0, 0.5, 0.1, 0.05, 0.01 and 0.005 mg/L were injected. The limit of detection (LOD) was obtained by measuring the peak area from 6 blanks at the position of histamine, calculating the difference between the highest and the lowest value, and multiplying this difference by 3. For limit of quantification (LOQ), the difference between the highest and lowest blank was multiplied by 10.

HPLC running conditions

Derivatized samples were subjected to RP-HPLC chromatography (Shimadzu, Korneuburg, Austria). Samples were loaded onto a 20 mm guard column (Phenomenex, Aschaffenburg, Germany) followed by a Phenomenex Synergi 4u polar RP 80A column 150 x 4.60 mm employing a stepwise gradient within 43 minutes at room temperature. Mobile phase A consisted of 200 mL ethanol (Merck, Vienna, Austria), 300 mL acetonitrile (Sigma-Aldrich, Vienna, Austria), 470 mL water, and 30 mL tris buffer pH 8 (0.1 M trishydroxymethylaminomethan plus 0.1 M acetate plus water, 2+1+2). Mobile phase B consisted of 450 mL ethanol, 450 mL acetonitrile, 98 mL water, and 2 mL tris buffer pH 8. Gradient elution started with 5% phase B and was gradually increased to 13% over 15 min and subsequently to 100% at 28 min. After staying at 100% for 3 min, the column was regenerated to 5% within 13 min. The flow rate was 1mL/min and the injection volume was 50 µL. The time of analysis was 43 minutes. UV detection at 220 nm was chosen for best results. An advantage of this simple and fast method was that the derivatized samples were stable enough to be put in the autosampler for a day without cooling and there was no need for a column oven.

Determination of histamine by radioimmune assay

Histamine was determined in all wines also by a commercial radioimmune assay (Immunotech, Marseille, France).

Results

Recovery rates and detection limits

Mean recovery rates for the different biogenic amines obtained from spiking experiments in two different red wines are summarized in Table 1. For very high amine concentrations the recovery rates were often lower. Table 1 also shows minimum amine concentrations found in the investigated 100 red wine samples. Injecting histamine standards down to 0.005 mg/L, LOD and LOQ for histamine were calculated as 0.07 and 0.22 mg/L, respectively. The lowest histamine concentration found in our wine samples was 0.53 mg/L indicating that all histamine measurements were clearly above the calculated LOQ.

Biogenic amine levels in red wine samples

Median values and range of amine concentrations found in the 100 included red wines are depicted in Table 2. Putrescine, histamine and tyramine were the most abundant biogenic amines. All amines showed substantial variability without consistent differences between different cultivars. One striking exception was found for tryptamine which was significantly elevated in Pinot noir wines (0.542 ± 0.42 mg/L; Mann-Whitney U-test p<0.0001) and, to a lesser extent, also in St. Laurent wines (0.100 ± 0.07 mg/L; p<0.05) as compared to wines from the other varieties (0.056 ± 0.06 mg/L) (Figure 2). Furthermore, also cadaverine levels were significantly higher in Pinot noir (1.25 ± 0.39 mg/L; p<0.0001) and St. Laurent wines (1.55 ± 0.95 mg/L; p<0.0001) as compared to wines made from other cultivars (0.57 ± 0.05).

Histamine levels were lowest on average in wines prepared from the local grape varieties Zweigelt and Blaufränkisch, but the difference was not statistically significant due to the pronounced variability of amine levels in both cultivars. Results for histamine correlated very well between HPLC (mean 8.50 ± 6.66 mg/L, range 0.52 -26.97) and radioimmune assay (mean 8.44 ± 7.49 mg/L, range 0.45 -27.54) with a correlation coefficient of r = 0.86.

Correlation between different biogenic amines

Each biogenic amine was analysed for a possible positive or negative correlation with any of the other amines (Table 3). A strong positive correlation was evident between phenylethylamine and isoamylamine levels (r = 0.87) whereas otherwise these two amines were not linked up with any of the other amines. Positive correlations were observed also between histamine, putrescine and tyramine (r = 0.55-0.66). All of the latter also correlated moderately with cadaverine (r = 0.36-0.42). No correlation with any other amine was found for spermidine and tryptamine.

Influence of vintage year on amine levels

To study the possible influence of the vintage year on amine production, wines from 2004 (n=84) and 2005 (n=10) were compared. Levels of isoamylamine (p<0.001, Mann-Whitney U-test) and phenylethylamine (p<0.00001) were significantly higher in wines from 2005 (a year delivering average red wine quality) than in wines from 2004 (a vintage with very good red wines), whereas histamine, putrescine and tyramine levels were higher in 2004 than in 2005 (p<0.05) (Table 4). The higher mean tryptamine concentration in 2004 wines was due to the higher number of Pinot noir wines in the 2004 group.

Discussion

The present study was undertaken to examine high-quality red wines for the presence of histamine and other potentially harmful biogenic amines. The dansyl chloride method used by us ensured very good selectivity and reproducibility for eight biogenic amines commonly found in wine, i.e. isoamylamine, phenylethylamine, histamine, putrescine, tyramine, cadaverine, spermidine and tryptamine. Due to optimised conditions (adjustment of pH) and the establishment of a high throughput method with low running and disposable costs highly reproducible amine values were obtained. An improvement of the dansyl chloride method as compared to the frequently used ortho-pthaldiadehyde (OPA) method is the higher stability of derivatized samples which may be stored for 24 hours without significant loss in signal intensity. As another practical advantage, the method does not require handling with toxic mercaptoethanol and therefore may be used also on systems without autosampler.

Furthermore, OPA derivatization does not allow measurement of the polyamines spermidine and spermine.

Putrescine, histamine and tyramine were identified as the most abundant biogenic amines in this study which is consistent with the results from previous studies (Vidal-Carou 1990a;[START_REF] Maxa | HPLC-Methode zur routinemäßigen Quantifizierung von biogenen Aminen in Wein[END_REF]Gloria 1998;[START_REF] Bodmer | Biogenic amines in foods: Histamine and food processing[END_REF][START_REF] Herbert | Free amino acids and biogenic amines in wines and musts from the Alentejo region. Evolution of amines during alcoholic fermentation and relationship with variety, sub-region and vintage[END_REF][START_REF] Landete | Biogenic amines in wines from three Spanish regions[END_REF]Garcia-Villar 2007). Contrasting with our expectations, histamine values were remarkably high in the tested red wines with a mean of 8.5 mg/L and maximum concentrations up to 27 mg/L. Although wines with histamine levels above 20 mg/L have been reported in the past [START_REF] Herbert | Free amino acids and biogenic amines in wines and musts from the Alentejo region. Evolution of amines during alcoholic fermentation and relationship with variety, sub-region and vintage[END_REF], most studies found much lower levels with upper limits rarely exceeding 10 mg/L (Subden 1979;[START_REF] Mafra | Evaluation of biogenic amines in some Portuguese quality wines by HPLC fluorescence detection of OPA derivates[END_REF][START_REF] Romero | Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination[END_REF][START_REF] Hernández-Orte | Determination of the biogenic amines in musts and wines before and after malolactic fermentation using 6aminoquinolyl-N-hydroxysuccinimidyl carbamate as the derivatizing agent[END_REF]Garcia-Villar 2007). Also in two previous studies of Austrian red wines, mean histamine levels were below 2 mg/L [START_REF] Maxa | HPLC-Methode zur routinemäßigen Quantifizierung von biogenen Aminen in Wein[END_REF][START_REF] Eder | Einfluss von Traubenfäulnis und Schönungsmitteln auf Gehalte biogener Amine in Mosten und Weinen[END_REF]. However, the good correlation between the histamine values obtained from HPLC and from the commercial radioimmunoassay strongly supports the reliability of our results. It must remain open for the time being if the observed above-average histamine levels are a real feature of these selected high quality red wines, e.g. resulting from longer aging in oak barrels which may be associated with increased histamine formation (Garcia-Villar 2007), or if the discrepancies are rather due to methodological differences.

In agreement with earlier papers (Gloria 1998;[START_REF] Mafra | Evaluation of biogenic amines in some Portuguese quality wines by HPLC fluorescence detection of OPA derivates[END_REF][START_REF] Landete | Biogenic amines in wines from three Spanish regions[END_REF]Garcia-Villar 2007) we could not observe consistent differences in major biogenic amines between different grape varieties. This supports the view that the strong variability in final amine showed the lowest mean histamine levels, but there was substantial overlap with other grape varieties and some representatives contained up to 20 mg/L histamine, so that these cultivars cannot generally be recommended to wine-sensitive persons as a convenient alternative.

An interesting exception concerning cultivar-specific amine profiles concerns Pinot noir and St. Laurent. High amounts of tryptamine were solely found in Pinot noir wines. This fits well to studies in Oregon wines where tryptamine was detected only in Pinot noirs but not in Cabernets (Gloria 1998). Remarkably, moderately elevated tryptamine concentrations were also found in St. Laurent, and both Pinot and St. Laurent contained significantly more cadaverine than other cultivars. Considering that St. Laurent has been recently identified as a descendent of the Pinot family [START_REF] Regner | Genetic relationship among Pinots and related cultivars[END_REF], these observations might suggest some genetic influence on the aberrant tryptamine and cadaverine profile in these two cultivars.

Analysing possible relationships between different biogenic amines revealed a marked positive correlation between histamine, putrescine and tyramine. Concordant associations have been previously described for histamine-tyramine [START_REF] Soufleros | Correlation between the content of biogenic amines and other wine compounds[END_REF], putrescine-tyramine [START_REF] Herbert | Free amino acids and biogenic amines in wines and musts from the Alentejo region. Evolution of amines during alcoholic fermentation and relationship with variety, sub-region and vintage[END_REF] or all three amines [START_REF] Romero | Characterization of selected Spanish table wine samples according to their biogenic amine content from liquid chromatographic determination[END_REF]. We could not confirm a close linkage between phenylethylamine, tryptamine and cadaverine (Garcia-Villar 2007). High putrescine levels have been suggested to indicate poor hygiene conditions during wine production [START_REF] Radler | Proceedings of the International Symposium on Nitrogen in Grapes and Wines[END_REF]. However, the red wines studied by us were high quality wines submitted by winemakers for being awarded by well-acknowledged wine connoisseurs. It seems reasonable to assume that particular care has been taken concerning selection and processing of grapes.

All in all, the close correlation between putrescine, histamine and tyramine found by us and other investigators suggests a common origin of these amines primarily during MLF, possibly Oenococcus oeni and other bacteria participating in MLF is highly variable and largely straindependent [START_REF] Coton | Histamine-producing lactic acid bacteria in wines: early detection, frequency, and distribution[END_REF][START_REF] Guerrini | Biogenic amine production by Oenococcus oeni[END_REF][START_REF] Moreno-Arribas | Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine[END_REF], high amine levels may be essentially determined by the specific local microflora participating in MLF and not necessarily indicate poor hygienic conditions.

An even stronger linkage was found between phenylethylamine and isoamylamine levels (r = 0.86). This is consistent with the findings of Eder and co-workers who made similar observations also in white wines [START_REF] Eder | Einfluss von Traubenfäulnis und Schönungsmitteln auf Gehalte biogener Amine in Mosten und Weinen[END_REF]. In their study, high phenylethylamine and isoamylamine levels were evidently associated with wine or must spoilage but not with MLF. Interestingly, phenylethylamine and isoamylamine were the amines showing the most striking differences between vintage years in our study. We speculate that the elevated levels in the 2005 wines were related to unfavourable weather conditions (cool and rainy summer) promoting bursting, infection and subsequent spoilage of crops. In fact, red wines from the vintage 2005 were of inferior quality (median Parker score 88) compared to the much better rated wines from 2004 (median Parker score 90). Vintage year might thus have little influence on histamine and other MLF-associated biogenic amines but a significant effect on phenylethylamine and isoamylamine levels, although admittedly the number of 2005 wines investigated in this study was very low. With a clinical view to wine intolerance, phenylethylamine and isoamylamine might be expected to occur in comparable amounts in red and in white wines, whereas amines associated with MLF appear to be more typical for red wines. It remains to be elucidated whether patient-reported intolerance to red wines alone vs. intolerance to all types of wine (including sparkling wines) reflects susceptibility to different biogenic amines.

Currently, histamine levels in foodstuffs are regulated in the USA and in Europe only for certain sea fish products but not for wine, cheese and other histamine-containing foods. No thresholds at all exist for other pharmacologically active amines like tyramine and phenylethylamine. While this may suggest a need for action for public health care authorities concerning enhanced regulation, it has to be emphasized that the toxicological knowledge about critical amine levels in foodstuffs and the scientific evidence demonstrating a significant health effect of moderate biogenic amine doses as found in red wines is still insufficient and often contradictory [START_REF] Jansen | Intolerance to dietary biogenic amines: a review[END_REF][START_REF] Panconesi | Alcohol and migraine: trigger factor, consumption, mechanisms. A review[END_REF]. For instance, patients with phenylethylamine-induces headaches, confirmed by double-blind placebo-controlled provocation test, paradoxically reacted more often to commercial wines samples with low amine content than to those with high concentrations [START_REF] Lüthy | Biogene Amine in Lebensmitteln: Zur Wirkung von Histamin, Tyramin und Phenyethylamin auf den Menschen[END_REF]. Likewise, in a more recent French study in wine-intolerant subjects, no clear relationship was found between the amine content of test wines and the frequency and severity of symptoms elicited by these wines [START_REF] Kanny | No correlation between wine intolerance and histamine content of wine[END_REF] suggesting that wine components other than biogenic amines might be even more important in wine intolerance.

To our knowledge, Switzerland is the only country having temporarily introduced a legal histamine threshold of 10 mg/L for wines (abandoned in 2008 when adjusting regulations to current EU standards). Legal limits down to 2 mg/L have been recommended in early papers and been reclaimed during recent European COST research programs [START_REF] Lüthy | Biogene Amine in Lebensmitteln: Zur Wirkung von Histamin, Tyramin und Phenyethylamin auf den Menschen[END_REF]. Applying these recommendations to the red wines investigated by us, as much as 84% would have been excluded from the market in case of a 2 mg/L limit, and still 34% when sticking to the 10 mg/L threshold. For comparison, the maximum allowed histamine concentration in fish products according to EU regulation 2073/2005 is 200 mg/kg. In the USA, the corresponding toxicity level defined by the FDA is 500 mg/kg. A recent risk assessment study based on simulated real life situations proposed a possible threshold of 500 mg/kg for cheeses, and 400 mg/kg for fermented sausages [START_REF] Rauscher-Gabernig | Assessment of alimentary histamine exposure of consumers in Austria and development of tolerable levels in typical foods[END_REF]. In view of the substantially higher limits for solid foods, the justification of the recommended threshold levels for wines must be challenged. However, once reliable pharmacological data

  low molecular substances generated by decarboxylation of amino acids

  et al. / Biogenic amines in red wine page 7

  et al. / Biogenic amines in red wine page 9

  et al. / Biogenic amines in red wine page 10

  on factors such as crop health, fermenting yeasts and bacterial strains involved in MLF which together override possible minor variety-specific differences. With regard to histamine, wines from the locally prevailing cultivars Zweigelt and Blaufränkisch

  same set of bacteria. Taking into account that decarboxylase activity in

  et al. / Biogenic amines in red wine page 15

  et al. / Biogenic amines in red wine page 16 on critical amine levels in wine are available, mandatory food labelling of biogenic amine content may be helpful for consumers suffering from wine intolerance. It is evident from this and previous studies that the production of wines largely devoid of biogenic amines is technically possible.

LegendsFigure 1 :

 1 Figure 1: Derivatization of biogenic amines to dansyl chloride conjugates. Dansyl chloride

Figure 2 :

 2 Figure 2: Tryptamine (left panel) and cadaverine levels (right panel) in Pinot noir and St.

  et al. / Biogenic amines in red wine Table1

Table 1 :

 1 Mean recovery rates for the analysed biogenic amines according to spiking experiments and minimum amine concentrations found in the tested 100 red wine samples. n.a. not analysed.

			Lowest value measured in
	Biogenic amine	Recovery rates (%)	red wines samples (mg/L)
	Isoamylamine	94.5 -110.1	0.02
	Tryptamine	82.3 -102.1	0.004
	Phenylethylamine	80.6 -102.5	0.04
	Putrescine	80.4 -98.4	2.93
	Cadaverine	67.6 -90.4	0.03
	Histamine	89.4 -104.6	0.52
	Tyramine	65.6 -110.8	1.07
	Spermidine	69.4 -108.5	0.03
	Spermine	n.a.	n.a.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Spermidine 1.9 ± 0.9 1.7 ± 0.9 n.s. Tryptamine 0.12 ± 0.22 0.05 ± 0.07 n.s.


S A Ordoudi

M Z Tsimidou

Consideration of fluorescence properties for the direct determination of erythrosine in saffron in the presence of other synthetic dyes

Keywords: saffron, erythrosine, fluorimetry, derivative spectrometry, food colour analysis

Consideration of fluorescence properties for the direct determination of erythrosine in saffron in the presence of other synthetic dyes

Stella A Ordoudi, Maria Z Tsimidou

Introduction

Since 2003, in international transactions, certification of saffron authentication according to the ISO 3632-1, 2 trade standards includes verification of absence of synthetic water-soluble acid dyes. The recommended analytical procedure in the version issued in 2003 proposed retention of these potential adulterants on polyamide columns or solid phase extraction (SPE) cartridges after pH adjustment of aqueous solutions of the test samples at ~2.5; removal of endogenous pigments, i.e. crocetin glycosides (crocins) by successive washing with water, methanol, acetone, and once more with methanol before elution of the bound synthetic dyes with a mixture of methanol: ammonia solution. After solvent evaporation the dyes, re-dissolved in methanol, were separated by different chromatographic protocols. TLC systems were proposed as screening procedures for all of the tested dyes. Ion-pair RP-HPLC protocols were then used as confirmatory procedures. The latter proved to be effective for azo-dyes but not for erythrosine (2′,4′,5′,7′-tetraiodo-3′,6′-dihydroxy-spiro[3Hisobenzofuran-1,9′-xanthen]-3-one disodium salt, E127, FD&C Red No 3, Food Red 14) due to the weak acidic character of the latter [START_REF] Kiseleva | Optimization of conditions for the HPLC determination of synthetic dyes in food[END_REF]. As a result the recovery of erythrosine was lower than that reported for the other dyes. The recently issued revised ISO 3632-2 version (2010) does not address the above mentioned analytical limitations regarding erythrosine. On the contrary, this dye is excluded from the list of synthetic dyes that can be detected using the methods therein, not because such an adulteration is of less importance but because the recommended protocols cannot cover its determination sufficiently.

To our knowledge, publications on the detection of synthetic dyes in saffron are rather limited. Lozano et al. (1999) tested tartrazine, ponceau S and methyl orange after addition to saffron extracts at a rather high level (2 % w/w). The synthetic dyes were separated from crocins on a RP-HPLC column (C 18 ) by gradient elution with methanol-water (20 -70 % in 50 min). Subsequently Zougagh et al. (2005) used an automated extraction system to pre-concentrate tartrazine, ponceau 4R and methyl orange on a cotton column from which the tested dyes were then eluted with a dilute ammonia solution. Identification at selected wavelengths was by means of an on-line photometer whereas quantification was achieved using partial least squares regression analysis. Another screening procedure was reported at that time for the detection of eleven synthetic dyes in saffron. It involved removal of crocins by acid hydrolysis (Zalacain et al., 2005). Under the sample pre-treatment conditions (pH 0.1, 90 o C) all of the tested compounds including those referred in the relative ISO specification plus allura red AC, naphthol yellow and red 2G were stable except for erythrosine. Our experimentation with the ion-pair RP-HPLC protocols described in ISO 3632-2 (2003) using a fluorimetric detector instead of DAD did not improve quantification limit as the acid ion-pair environment quenched its fluorescence severely (Ordoudi et al., 2010).

The analytical limitations described above along with legislative restrictions on the uses of erythrosine because of its low ADI value (0-0.1 mg/kg body weight/day) motivated us to focus on the direct selective detection of this additive in mixture with yellow or red synthetic dyes utilizing its fluorescence properties. The results were compared with those obtained by using derivative UV-Vis spectrometry as the identification means in conjunction to the same sample preparation process.

Our effort deserves attention by the ISO Technical Committee for "Herbs, culinary spices and condiments" responsible for ISO 3632-1 and 2 revisions.

Materials and methods

Reagents and materials

Authentic saffron samples were donated by Saffron Cooperative of Kozani (Greece). Amaranth (80%, CI 16185), allura red AC (80 %, CI 16035), erythrosine (ER) (90%, CI 45430:2), naphthol yellow (99%, CI 10315), orange II (85%, CI 15510), ponceau 4R (75%, CI 16255), quinoline yellow (95%, CI 47005), sunset yellow FCF (90%, CI 15985) and tartrazine (85%, CI 19140) were supplied by Sigma-Aldrich Chemie (Steinhem, Germany). Azorubine (85%, CI 14720) and red 2G (60%, CI 18050) were from Aldrich (Steinhem, Germany). Rocelline (99 %, CI 15620) was from Acros Organics (USA). All chemicals were of analytical reagent grade. 3 mL/200 mg) were from Macherey-Nagel (Düren, Germany). PTFE filters (11 mm, 0.45 mm) were from Millipore (Bedford, MA, USA). Stock and working dye solutions were prepared in methanol according to the ISO 3632-2 test method for water-soluble acid synthetic dyes (2003; 2010). Details for the preparation of spiked samples are given below: saffron powder (0.5 g) was weighed into a glass centrifuge tube to which water (10 mL, 60 o C) was added. The mixture was vortexed (1 min), kept in the dark for 10-12 min, vortexed again (1 min) and finally centrifuged (4080 × g, 10 min). The supernatant was spiked with the synthetic dye (10, 25, 62.5, 87.5, 112.5 µL from 1 µM solution) and the mixture was homogenized. The pH of the mixture was adjusted to ~2.5 by addition of glacial acetic acid (2 mL). The extract was transferred onto a polyamide SPE cartridge preconditioned with 1 mL of water. The cartridge was successively washed with water (10 x 3 mL), methanol (10 x 3 mL), acetone (10 x 3 mL) and finally methanol1 (10 x 3 mL) under vacuum (15 mm Hg) and the eluates were discarded. The bound colourants were then eluted using a mixture of methanol: ammonia solution, 95:5, v/v (1 x 5 mL) in a heart-shaped flask and the solvent was evaporated at room temperature under vacuum. The residue was re-dissolved in 0.5 mL methanol and vortexed for 1 min. The extraction and purification procedure was carried out in triplicate.

Sample preparation

Fluorimetric examination

An aliquot of the methanol extract was transferred into a volumetric flask and the volume was made up with methanol (dilution factor = 10 or 100). Fluorescence excitation (exc) maximum was found to be at 532 nm. The corresponding emission (em) maximum was then recorded at 548 nm on a Shimadzu RF-1501 spectrofluorimeter (Kyoto, Japan) using the PC-1501 software facilities of the instrument. Scan speed was 2775 nm min -1 and instrument response time was automatically set. The system was thermostated at 25 o C via an outer watercirculating bath. Spectra were corrected for background as described by Papoti and Tsimidou (2009). For each sample, measurements were made in triplicate.

Method validation

Intra-laboratory validation of the fluorimetric method was performed according to the Decision 2002/657/EC. Parameters such as method linearity, limit of value and b, the slope of the calibration graph), limit of quantification (LOQ, calculated as 10s/b), intra and inter day precision, specificity and minimum required performance limit (MRPL) 2 were examined. These parameters were checked using standard solutions of ER or mixtures with other synthetic dyes. Recovery studies were performed using saffron samples spiked with five levels of ER. For each level, triplicate measurements were obtained.

Spectrophotometric examination

An aliquot of the methanol extract (0.1 mL) was placed in a quartz micro cell (1x 1x 0.1 cm) from Starna Brand (Essex, Germany) and visible spectra were then recorded on a Shimadzu UV 1601 spectrophotometer equipped with the UV-1601PC software (v. 3.9, Shimadzu). The system was thermostated at 25 o C via an outer water-circulating bath. Second order spectra derivatives were calculated according to the procedure described by Zalacain et al. (2005). For each sample, measurements were made in triplicate.

Statistical analysis

Statistical comparisons of the mean values were performed by one-way analysis of variance (ANOVA), followed by the multiple Duncan test (p < 0.05 confidence level) using the SPSS 14.0 software (SPSS Inc., Chicago, IL).

Results and Discussion

Background work

The spectral characteristics of ER as well as those of the other water soluble acid dyes referred in the relevant ISO methodology were tested in methanol so that through calculation of molar absorption coefficients (ε) at λ max of ER (Fery-Forgues and Lavabre 1999), to estimate the respective quantum yield (Φ F532 ) values not available in literature. Absorption maxima, molar absorption coefficients, excitation and emission maxima as well as quantum yields of the tested compounds at 532 nm in methanol are shown in Table 1. The first interesting extrapolation from these data 2 Minimum Required Performance Limit: the minimum content of an analyte in a sample, which at least has to be detected and confirmed. 6 concerned the visible properties of all of the analytes. Azorubine, amaranth, ponceau 4R, red 2G, allura red AC and rocelline, with λ max greater than 500 nm, exhibited ε 532 values that were four to six-fold lower than that of ER. As a consequence, to achieve a certain absorption value at 532 nm the required amount of extractable ER is expected to be many folds lower than that of any other tested dye. Concerning the estimated Φ F532 value of ER in methanol, it was in good agreement with that reported in strongly alkaline phosphate buffer (Ryvolova et al. 2007). The corresponding quantum yield values of yellow dyes (tartrazine, quinoline yellow, sunset yellow FCF, naphthol yellow and orange II) that absorb light in the region below 500 nm were found negligible. None of these compounds is expected to interfere with fluorescence detection at 532 nm (ex) due to the lack of a fluorescein moiety in their structure. The Φ F532 values of azorubine, amaranth, ponceau 4R, red 2G, allura red AC and rocelline were three to four orders of magnitude lower than that of ER pointing out a rather weak fluorescence at 532 nm. Based on the quantum yield values of Table 1, allura red AC, azorubine and red 2G could be considered as the only potential interferences so that these dyes were used for further experimentation toward the selective detection of ER in saffron.

Method development and validation procedure

Linearity and sensitivity. The linearity of fluorescence response for ER was tested in the range 1 to 22.5 nM. The regression equation was y = a + bx (R 2 = 0.993), where a = 861.4 ± 171.0 and b = 1008.7 ± 12.6. These results demonstrate the linearity of the fluorescence response in the assessed range. LOD was found to be 0.56 nM and LOQ was 1.70 nM. It should be noted that our data are of the same order of magnitude with the respective ones found for this dye by other researchers using other techniques (e.g. Minioti et al. 2006;Ryvolova et al. 2007;Rongjie 2008).

Precision. Measurement precision was satisfactory for the determination of ER at nM levels with regard to performance criteria for quantitative methods adopted in the Decision 2002/657/EC. Intra-day precision was checked by the same analyst on three random days. Obtained percent coefficient of variation (% CV) values of pooled data for 7.5, 12.5, and 22.5 nM of the analyte were 8.4%, 4.8% and 3.1%, respectively (n = 9). The inter-day precision was also examined at different levels; % CV values were below 8.4 % showing that the procedure is reproducible. Fluorescence response of allura red AC, azorubine and red 2G at all working concentrations (12.5 -625 nM) was found to be close to instrumental noise (data not shown). Our findings did not verify dependence on concentration that could be attributed to the low range of concentrations used. Such dependence was observed by Ryvolova et al. (2007) for azorubine and red 2G at much higher concentrations (10 x 10 4 -120 x 10 4 nM). The present data imply that fluorescence response of ER is not expected to be influenced by the presence of allura red AC, azorubine or red 2G as suggested in the background work.

Indeed, calculated values for erythrosine content in 1:1 binary mixtures (Table 2) showed acceptable deviations, expressed as percentage (positive or negative) within the range 5-22.5 nM. A possible interference due to the co-presence of red 2G was observed only at the lowest tested concentration (5 nM). The presence of the second analyte did not affect significantly quantification of ER within the 95 % confidence limits of the regression equation in line with suggestions for specificity recommended in the Decision 2002/657/EC.

Accuracy and matrix effect. Recovery studies were then conducted for standard ER solutions as well as for spiked saffron samples to evaluate matrix effect and overall accuracy of the fluorimetric method. Analyses were carried out on two different days by two different analysts for one spiking level corresponding to the mid of the fluorescence regression line (12.5 nM standard solution or 1 mg kg -1 saffron). These data are shown in Table 3. The recovery of ER from standard solutions was 61 -73.5 % in accordance with that reported by [START_REF] Kirschbaum | Liquid chromatographic quantification of synthetic colours in fish roe and caviar[END_REF] for the recovery rates of standard ER solution (0.28 mM) retained on various types of polyamide adsorbents. These authors had noticed that the performance of the adsorbent may vary depending on the manufacturing process or/and lot-to-lot variability of the polymeric material. In our study, for the same lot of polyamide and one spiking level, the matrix effect to both mean recovery values and measurement precision was obvious. To further examine the effect of endogenous saffron pigments on the determination of 4. Recovery rates from spiked saffron samples ranged from 62.9 ± 6.6 % (for 0.2 mg kg -1 saffron) to 141.0 ± 26.7 % (for 0.04 mg kg -1 saffron). These values are close to the minimum acceptable ones according to the Codex Alimentarius requirements for residue recovery in foods (80-110 %). Analysis of samples containing ER at 0.02 mg kg -1 resulted in overestimation of recovered amounts (227.8 ± 15.6 %). Based on all abovementioned results the MRPL value for ER was 0.04 mg kg -1 . Further efforts to detect the dye at lower levels are out of the aim of the study, which is the control of saffron adulteration with this particular dye.

Comparison with other direct detection protocols.

An inherent advantage of our proposal with regard to the screening method by Zalacain et al. (2005) is the avoidance of harsh sample pretreatment conditions, which eliminate crocins but also decompose erythrosine. Replacement of UV-Vis spectrometry by fluorimetry also proved to be advantageous as sensitivity, specificity and accuracy of the former were inferior. Indeed, after spectrophotometric examination of standard ER solutions and linear regression analysis [a five-point regression line (2.5 to 12.5 µM) was described by the equation y = a + bx (R 2 = 0.999), where a = 0.043 ± 0.005 and b = 0.085 ± 0.001], the LOD and LOQ values were found to be 0.19 µM and 0.57 µM, respectively. Upon findings sensitivity of the spectrophotometric determination was 3 x 10 3 -fold lower than that evidenced by means of fluorimetry. Additionally, evaluation of calculated second derivative spectra data of both ER and its binary mixtures (1:50, 1:10, 1:5, 1:1, mol/mol) with allura red AC, azorubine or red 2G (C=12.5 µM) did not assist specificity. In particular, the characteristic wavelengths corresponding to minimum and maximum Y-ordinate values in the derivative spectrum of each binary mixture (Figure 1) were found almost identical to those of standard ER. Only when the latter was present at a much lower level than that of the second analyte (e.g. 1:50, mol/mol) a shift in λ min was observed.

The accuracy of the spectrophotometric method examined at one level of addition (1 mg kg -1 ) was 98.9 ± 19.2 % for the standard solution and 57. 3).

Matrix effect due to visible spectra overlapping was pronounced.

Conclusion

Summarizing current and previous findings it can be suggested that 'complete absence' of ER in saffron can be certified by the direct fluorimetric approach. The proposed procedure offers high selectivity and sensitivity (MRPL=0.04 mg kg -1 ) indicating that it is fit for the determination of traces of erythrosine in this precious spice in the presence of other synthetic dyes. Our method deserves attention by the ISO Technical Committee for "Herbs, culinary spices and condiments"

  -concentration of the dyes on polyamide cartridges and direct detection by means of derivative UV-Vis spectrometry

  , calculated as 3.3 s/b, where s, the standard deviation of the intercept

  Taking into account the results of our background work, specificity of the fluorimetric method was assessed for binary mixtures of ER with allura red AC, azorubine or red 2G at different molar ratios (1:50, 1:10, 1:5, 1:1, mol/mol).

  studies were performed before and after spiking a saffron sample with very low levels of the dye (ca. 0.02 -0.2 mg kg -1 saffron). The calculated values are presented in Table

  2 ± 25.8 % for the spiked sample. The corresponding % CV values indicated poor repeatability (% CV = 19.4 respectively), worse than those recorded for fluorescence (see Table

Figure 1 .

 1 Figure Captions

  Second derivative visible spectra of ER solutions and the corresponding binary mixtures (1:50, 1:10, 1:5, 1:1, mol/mol) with (a) allura red AC, (b) azorubine or (c) red 2G (C=12.5 µM) 204x140mm (96 x 96 DPI)

Table 1 .

 1 Absorption maxima, molar absorption coefficients, excitation and emission maxima and quantum yields of the tested dyes in methanol at 532 nm.

			Food Additives and Contaminants
	E value	Dye	λ max a	ε λmax	ε 532	λ exc λ em Φ F532	b
				(10 4	(10 4			(x 10 4 )
			(nm)	L mol -1 cm -1 )	L mol -1 cm -1 ) (nm) (nm)
	E102	Tartrazine	426	1.73	0.00				-
	E104	Quinoline Yellow	426	1.88	0.00				-
	E110	Sunset Yellow FCF 480	1.60	0.00				-
	E122	Azorubine	516	1.85	1.65			2.63
	E123	Amaranth	520	2.12	2.04			0.84
	E124	Ponceau 4R	510	1.85	1.61			0.47
	E127	Erythrosine	532	9.06	9.06	532	548 1058.90
	E128	Red 2G	534	2.02	1.99			1.87
	E129	Allura red	506	2.18	1.96	474	551	2.91
	-	Naphthol Yellow	428	1.47	0.35 x 10 -4				-
	-	Orange II	480	1.78	0.44 x 10 -4				-
	-	Rocelline	509	1.47	1.27	329	408	0.32

a Methanol solutions (4 x 10 -5 M, 25 o C); b F 532 : area under curve at 532 nm excitation/548 nm emission; Rhodamine 6G was used as standard.

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Table 2 .

 2 Percent deviation of erythrosine content* for 1:1 (mol/mol) binary mixtures with allura red, azorubine or red 2G from the calibration curve based on calculation of area under curve at 532 nm excitation/548 nm emission (y= 861.4 + 1008.7x, R 2 =0.993)

	Concentration		% Deviation (positive or negative)
	(nmol L -1 )		Mixture with
		allura red AC	azorubine	red 2G
	5.0	+12.7	-3.2	+46.4
	7.5	+ 6.2	-6.1	+16.4
	12.5	-1.4	-8.7	-3.2
	17.5	+ 9.7	-13.4	-11.5
	22.5	-8.2	+ 6.7	-5.6

*

Table 3 .

 3 Accuracy of the fluorimetric method in the mid of the fluorescencre regression line (12.5 nM standard solution or 1 mg kg -1 saffron) a . from the calibration curve based on calculation of area under curve at 532 nm excitation/548 nm emission; b y= 861.4 + 1008.7x, R 2 =0.993; c y= 1453.3 + 846.0x, R 2 =0.990; Mean value ± standard deviation (n = 3)

		Food Additives and Contaminants	Page 15 of 17
		% Recovery of ER
		Standard solution	Spiked saffron sample
	Day 1 Analyst 1 b	67.6 ± 5.9 (CV= 8.8%)	78.6 ±14.3 (CV=18.2%)
	Day 2 Analyst 2 c	65.4 ± 3.8 (CV= 5.7%)	62.9 ±10.4 (CV=16.6%)

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Table 4 .

 4 Recovery rates of ER at very low spiking levels*

	Spiking level	% Recovery of ER
	(mg kg -1 )
	0.02	227.8 ± 15.6 (CV= 6.9%)
	0.04	141.0 ± 26.7 (CV= 18.9%)
	0.11	71.7 ± 11.3 (CV=15.7%)
	0.15	67.4 ± 0.7 (CV=1.1%)
	0.20	62.9 ± 6.6 (CV=10.5%)

The use of acidic methanol (recommended in the

version of ISO 3632-2) was avoided as it affects erythrosine stability

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Acknowledgement:

Mrs. K. Tsiogka, undergraduate student, is acknowledged for her participation in the recovery study. We also thank the unknown reviewers for useful remarks.