Frozen storage of high-pressure- and heat-induced gels of blue whiting (Micromesistius poutassou) muscle: rheological, chemical and ultrastructure studies

This paper examines the influence of frozen storage over 34 weeks on the rheological properties as well as the chemical and microstructural characteristics of gels made from muscle of blue whiting (Micromesistius poutassou) subjected to different gelling treatments entailing three combinations of pressure, temperature and time: 200 MPa, <10°C, 10 min (lot L), 375 MPa, 38°C, 20 min (lot H) and atmospheric pressure, 37°C, 30 min and then 90°C, 50 min (lot T). Freezing at –40°C caused certain changes in rheological parameters. In heat-induced gels, breaking deformation, elasticity and cohesiveness increased. Of the high-pressure-induced gels, breaking force increased and cohesiveness decreased in the gel formed at lower pressures, while the only change in the gel formed at higher pressure was some loss of elasticity. There was a general fall in water holding capacity (WHC) values. Lightness remained stable. In terms of protein solubility, there was an increase in covalent bonds in lot L. As for the ultrastructure, all gels matrixes were more disorganized as a result of freezing. In the course of frozen storage, the greatest changes in rheological parameters generally took place during the first 8 weeks, and in all the gels there was a decrease in WHC. In the heat-induced gel the changes were less marked over the storage period compared with those in the high-pressure-induced gels, but the heat-induced gel was more brittle and did not maintain maximum folding test scores. Covalent bonds increased and hydrophobic interactions decreased in all lots. The general appearance of the structure of gel T remained more homogeneous, while the pressurized gels exhibited more and larger cavities.     

Lipid oxidation in high-pressure processed chicken breast muscle during chill storage: critical working pressure in relation to oxidation mechanism

 The oxidative stability of chicken breast muscle subjected to high-pressure treatment at 300, 400, 500, 600, 700 or 800 MPa for 5 min or 10 min, or to heat treatment (80  °C for 10 min) and subsequent storage at 5  °C was evaluated over a 2-week period. Lipid oxidation in pressure-treated chicken breast muscle monitored as formation of thiobarbituric acid reactive substances depended to a high degree on the working pressure and less on the pressurizing time. The pressure treatment at 800 MPa for 10 min was found to enhance lipid oxidation to the same extent as the heat treatment. Pressure treatment at 600 MPa and 700 MPa resulted in less oxidation. Chicken breast muscle exposed to pressure at or below 500 MPa showed no indication of rancidity, similar to what was found for untreated meat during chill storage; accordingly 500 MPa is a critical pressure for pressure treatment of chicken breast muscle. Analysis of non-heme iron in pressure-treated chicken breast muscle revealed that the notable increase in lipid oxidation caused by high pressure above 500 MPa did not result from the release of iron ions during high-pressure treatment. Furthermore, no influence of high-pressure treatment on the catalytic activity of metmyoglobin on lipid oxidation was observed in a model system, and it is concluded that increased lipid oxidation is probably related to membrane damage. Received: 23 August 1999     

The effect of rosemary extract and omega-3 unsaturated fatty acids on the properties of gels made from the flesh of mackerel (Scomber scombrus) by high pressure and heat treatments

Gels made from the flesh of mackerel (Scomber scombrus) and fortified with rosemary extract and omega-3 unsaturated fatty acids were studied in connection with high pressure/thermal treatments. Elasticity and breaking deformation were significantly higher in pressure-induced gels (300 MPa, 25 °C, 15 min), while hardness was considerably lower. For gels without ingredients the fraction solubilised with 8 M urea and 2% β-mercaptoethanol was larger in the pressurised samples, indicating more covalent bonding in heat-induced gels which could not be disrupted by the solubilising agent. Scanning electron microscopy showed that pressure-induced gels generally presented a structure more compacted than heat-induced gels. The high pressure activated lipid oxidation, the antioxidant effect of rosemary being evident in all samples.     

Effect of alkalis on konjac glucomannan gels for use as potential gelling agents in restructured seafood products

Four dispersions of 3% glucomannan in water, deacetylated with 5% 0.6 N and 1 N KOH (lots L1 and L2) and 0.6 N and 1 N NaOH (lots L3 and L4) as gelling agents, were evaluated for use in raw restructured seafood products. Several properties (pH, moisture content, water binding capacity, cooking loss and lightness) together with puncture data (breaking force and breaking deformation) were determined after 1 and 10 days of chilled storage at 5 °C. All these data were analyzed together with different viscoelastic parameters obtained at small amplitude oscillatory strain (SAOS) after 1 day of chilled storage, showing that L1 and L4 samples were the most suitable gels for incorporation in raw restructured fish products. In both cases the highest stress (σ_max) and strain (γ_max) amplitude values were found in the linear viscoelastic (LVE) range; however, L1 showed both high strain amplitude and breaking deformation values. Moreover, creep and recovery (transient) data showed that L1 was the most time-stable gel with the highest elasticity and the lowest relaxation exponent (n). L4 gel showed strong rigidity, i.e. the highest values of breaking force and storage moduli (G′) and the highest n value, making it less gel-like. Both L1 and L4 gels became significantly less gel-like over 10 days of chilled storage due to the loss of gel strength (S) and a noticeable increase of n. These chilled storage effects were more intense in L4 than in L1.     

Rheological Properties of Heat-induced Gels from Egg Albumen Subjected to Freeze-thaw

A back-extrusion device was used to measure gel strength, elasticity, and viscosity index of heat-induced gels of albumen maintained at 80°C from 5 to 30 min. These rheological properties were the same in albumen refrigerated 24 hr at 3°C as those observed in gels from fresh albumen suspensions. Short term frozen storage (?10°C for 24 hr) significantly reduced each gel parameter compared with fresh (control) albumen gels. Incorporation of sucrose into fresh albumen protected rheological properties of the heat-induced gels from deleterious effects of freezing the albumen suspensions. Addition of 5 or 10% NaCl to albumen reduced or eliminated the ability of albumen to form heat-induced gels.     

Effect of high pressure processing on heat-induced gelling capacity of blue crab (Callinectes sapidus) meat

There has been increasing use of High pressure processing (HPP) in the fishery industry since this technology facilitates shellfish shucking. Nevertheless, there is limited information about the effect of HPP on protein functional properties of some shellfish. The aim of this study was to evaluate the effect of 100, 300 and 600 MPa/5 min on the gelling capacity of heat-induced (40 °C/30 min + 90 °C/20 min) blue crab (Callinectes sapidus) meat. HPP treatment resulted in crab meat gels with a lighter and reddish colour as compared to the control. HPP at 600 MPa induced the formation of high molecular aggregates from the denaturation-aggregation of myosin heavy chain. Pressurization at 100 MPa promoted the shift of α-helix structures to β-sheet and β-turn as compared with the other pressure levels. TPA values were higher in gels made at 100 MPa than at 300 or 600 MPa. Low pressure levels, then, increased the heat-induced gelling capacity of crab meat, improving the texture through modification of its protein structure.Industrial relevanceHigh pressure processing (HPP) technology has been successfully applied to several seafood products, both for processing and storage. However, in the case of blue crab meat it is important to study the effect of HPP on protein functional properties such as gelling capacity in order to optimize processing parameters for the preparation of high-quality restructured products. This paper reports the development of a HPP process (100, 300 and 600 MPa/5 min 40 °C/30 min + 90 °C/20 min) prior to thermal gelling for the preparation of crab meat gels. The application of 600 MPa produced considerable protein aggregation of gels, whereas with pressures below 300 MPa protein functionality can be modified to produce crab meat gels with adequate brightness, TPA values and a fresh, high-quality appearance. These results could provide a basis for further pressurization applications in the crab industry to create new seafood product analogues based on this kind of crab meat.     

Improving the keeping quality of pomegranate fruit by intermittent warming

 Mollar pomegranates (Punica granatum, Punicaceae) were stored at 0  °C or 5  °C and 95% relative humidity (RH) for 80 days. Intermittent warming (IW) cycles of 1 day at 20  °C every 6 days, during which time the fruit had been stored at 0  °C or 5  °C, followed by a shelf-life period of 7 days at 15  °C and 70% RH were applied. IW during storage at 0  °C was the best treatment for maintaining the red skin colour as at harvest. However the red colour of the arils (hue angle) was kept better under warming at 5  °C. Although significant changes in the individual anthocyanins were observed in all treatments, particularly after the shelf-life period, the total anthocyanin content of the juice at harvest was maintained. While storage at 0  °C avoided decay although increased the risk of chilling injuries such us pitting and husk scald, 5  °C-storage reduced these injuries but fungal attacks were not inhibited. After the shelf-life period, IW alleviated chilling injuries without any incidence of decay. Warming treatments gave very good results with respect to storage and the keeping quality of pomegranates, particularly when applied during storage at 0  °C. Received: 10 February 1998 / Revised version: 24 April 1998     

Effects of high pressure and temperature on buckwheat starch characteristics

Pressure-induced gelatinisation of buckwheat starch suspensions (25% w/w) was studied and compared to heat-induced gelatinisation. Starch suspensions were treated at increased pressure (200–600 MPa) or temperature (60–95 °C) for 10 min. The degree of gelatinisation and the temperature and pressure ranges of gelatinisation were determined using differential scanning calorimetry, changes in birefringence and pasting behaviour. Furthermore, the structural changes during gelatinisation were investigated using microscopy. The pressure-induced as well as the temperature-induced gelatinisation curves were sigmoid shaped. Gelatinisation occurred between 300 and 500 MPa or between 60 and 70 °C. Scanning electron microscopy images showed retention of the granular structure after treatment with 600 MPa. However, when heated at temperatures above 65 °C, the formation of a “sponge-like” structure was observed. Better preservation of the granular structure for pressure treatment compared to temperature treatment resulted in stronger gels for the former. Pre-treatment with pressure as well as temperature made the buckwheat starch granules more resistant to swelling and disintegration under the influence of additional heat.     

High pressure-induced tapioca starch gels: physico-chemical characterization and stability

Gelatinization of tapioca starch (25% dry basis) was induced by high hydrostatic pressure processing (HPP) at 600 MPa under different time and temperature regimes (30 °C for 10, 20 and 30 min; 50 °C for 10 min; 80 °C for 10 min). Textural, thermal and structural properties of the gels were studied and their stability was evaluated after 28 days of refrigerated (4 °C) and frozen (−18 °C) storage. Thermally induced gels (90 ± 1 °C, 20 min, gel-T) were used as controls. HPP resulted in the formation of harder gels than thermal processing (more significantly at lower processing temperatures) partially preserving the granular structure of the native starch. Longer HPP treatments caused only a slight decrease in hardness that was significant only at longer processing times (30 min). DSC thermograms of high pressure-induced samples showed a more asymmetrical ice-melting peak than that of thermally induced gels. Asymmetry of the peak of HP treated samples was more pronounced in samples processed at lower than at higher temperature. A different starch–water and/or starch/starch interaction may be hypothesized. During storage, all samples became stiffer and the amylopectin recrystallization increased, more extensively in thermally induced than in HPP samples where a stronger starch–starch and/or starch/water interactions may have hindered the recrystallization process.     

Modification of rheological properties of whey protein isolates by limited proteolysis

Whey protein isolate (WPI) was subjected to limited tryptic hydrolysis and the effect of the limited hydrolysis on the rheological properties of WPI was examined and compared with those of untreated WPI. At 10% concentration (w/v in 50 mM TES buffer, pH 7.0, containing 50 mM NaCl), both WPI and the enzyme-treated WPI (EWPI) formed heat-induced viscoelastic gels. However, EWPI formed weaker gels (lower storage modulus) than WPI gels. Moreover, a lower gelation point (77 °C) was obtained for EWPI as compared with that of WPI which gelled at 80 °C only after holding 1.4 min. Thermal analysis and aggregation studies indicated that limited proteolysis resulted in changes in the denaturation and aggregation properties. As a consequenece, EWPI formed particulated gels, while WPI formed fine-stranded gels. In keeping with the formation of a particulate gel, Texture Profile Analysis (TPA) of the heat-induced gels (at 80 °C for 30 min) revealed that EWPI gels possessed significantly higher (p < 0.05) cohesiveness, hardness, gumminess, and chewiness but did not fracture at 75% deformation. The results suggest that the domain peptides, especially β-lactoglobulin domains released by the limited proteolysis, were responsible for the altered gelation properties.     

Principal component analysis to study the effect of temperature fluctuations during storage of frozen potato

 The effects of temperature fluctuation ranges, number of fluctuations carried out, and packaging during frozen storage on the texture of potato tissue in terms of compression, shear, and tension rheological parameters were assessed through data generated according to a factorial design using principal component analysis (PCA). Five ranges of fluctuation (–24  °C to –18  °C, –18  °C to –12  °C, –12  °C to –6  °C, –24  °C to –12  °C and –18  °C to –6  °C) applied 2, 4, 8, 16, 24, and up to 32 times on unpacked and pre-packed frozen potatoes, were considered. The controls were unpacked and prepacked frozen tissues thawed immediately without undergoing any fluctuation. In addition, several geometrical, technological, and chemical parameters were determined. PCA showed that maximum shear force, F_s was the best rheological parameter for differentiation of the structural damage and softening occurring in the tissue at each treatment, which was closely related to its duration, TT _d . PCA did not permit complete discrimination between the five fluctuation ranges, but it clearly separated samples subjected to –18  °C/–6   °C from those subjected to –24  °C/–18  °C. Frozen samples undergoing up to four fluctuations formed a separate cluster from those undergoing a higher number. Analysis also clearly separated unpacked from pre-packed samples in response to slower freezing rates reached in the latter. Received: 17 December 1999     

Kinetics of softening of potato tissue by temperature fluctuations in frozen storage

 Pre-packed and unpacked potato tissues were frozen, subjected to temperature fluctuations and then thawed. Temperature fluctuations ranged from –24  °C to –18  °C and from –18  °C to –6  °C. The number of fluctuacions ranged from 0 (that is to say, only freezing and thawing processes) to 32 at each above fluctuation range, simulating practical frozen storage conditions. Compression, shear and tension tests were carried out to measure the extent of structural damage caused to the potato tissue. Plots of log (rheological parameters and moisture content) versus number of temperature fluctuations showed two distinct regions; the first was a rectilinear plot with a steep negative slope up to four fluctuations. The second was also a rectilinear plot with a shallow negative slope beyond four fluctuations. For higher number of fluctuations, most of rheological parameters reached a value almost constant. These two-stage softening rate curves are consistent with the biphasic model and qualitatively similar to those for thermal softening of the vegetables. This study shows that two substrates S_a and S_b may be involved in providing firmness to potato tissue in freezing and frozen storage conditions. By analogy with earlier works, the term "frozen storage firmness" can be proposed to describe the amount of firmness that is resistant to degradation by freezing with temperature fluctuations during frozen storage and final thawing of the product. Received: 30 April 1999     

Effects of Hydrolysates from Silver Carp (Hypophthalmichthys molitrix) Scales on Rancidity Stability and Gel Properties of Fish Products

This study aimed to evaluate the effectiveness of hydrolysates, which were obtained from the scales of silver carp (Hypophthalmichthys molitrix) by papain, flavourzyme, and Alcalase 2.4 L, as natural antioxidants in silver carp mince and surimi gels during storage at 4 °C. The hydrolysates that possess greater in vitro antioxidant activities (DPPH radical-scavenging activity, Fe²⁺-chelating activity, and reducing power), including hydrolysates catalyzed by papain at 10 min (HP), flavourzyme at 5 min (HF), and Alcalase 2.4 L at 5 min (HA), were chosen as additives. Color, cooking loss, conjugated dienes (CDs), thiobarbituric acid reactive substances (TBARS), fatty acids, and sensory scores of mince were measured on days 0, 2, 4, 6, and 8 during 4 °C storage; additionally, whiteness, breaking force, deformation, gel strength, and sensory score of surimi gels were measured on days 1, 3, 5, 7, 9, and 11 during 4 °C storage. The results indicate that HA was conducive to lowering the cooking loss of mince and that HF significantly (P?<?0.05) reduced the CDs value of mince. For surimi gels, HF improved whiteness, deformation, and gel strength. Hence, HF could serve as a natural antioxidant during early oxidation and improve gel formation of silver carp products.     

Freeze- and bake-stable glazing for confectionery products characterized by differential scanning calorimetry

 A freeze- and bake-stable glazing for pastry has been developed based on an optimization procedure in which starches and carrageenans as thickening agents were combined with sugar alcohols and sodium tripolyphosphate. Instrumental gloss measurement on test biscuits showed that glazing consisting of 3% ι-carrageenan and 15% sorbitol (w/w) had maximal gloss. This was confirmed on pastry products. In contrast to glazing based on mannitol, no crystallization problems were encountered for this glazing, for which differential scanning calorimetry showed that some water freezes at –3  °C followed by freezing of supercooled encapsulated water at –20  °C and by a glass transition at –61  °C. During normal frozen storage of preglazed, nonbaked pastry, the glazing is thus in a nonstable rubbery state with a limited resistance to water migration and ice crystal formation, which can be substantially improved by storage at temperatures below –61  °C. Received: 1 March 1999 / Revised version: 19 April 1999     

Characteristics of stirred low-fat yoghurt as affected by high pressure

The effects of high pressure on the physicochemical, chemical, microbiological and sensory characteristics of stirred low-fat yoghurt were studied. Laboratory-made yoghurts were treated at high pressure (100–400 MPa) for 15 min at 20°C. No significant changes in pH and total organic acids were observed after pressuring the yoghurt. Pressures over 200 MPa prevented post-acidification of the yoghurt during chilled storage. Pressurized yoghurts exhibited higher viscosity and amino acid contents than did the untreated controls, and the differences were maintained after chilled storage. High-pressure treatments at 300 and 400 MPa reduced the number of viable cells of lactobacilli to below the legal minimum permitted in many countries. Significant differences in sensory characteristics between untreated and pressurized yoghurts (200–300 MPa) were detected after chilled storage.     

Effect of temperature on the cell wall composition of raisins during storage under a modified atmosphere

 Raisins obtained from seedless grapes ("Flame" variety) were kept under a modified atmosphere (MA) composed of CO₂ (60%) and N₂ (40%), and stored at 10  °C (10MA), 20  °C (20MA), 30  °C (30MA) and 40  °C (40MA). An additional sample was stored under air at 20  °C (20A). Colour, and changes in cell wall components were monitored during storage. At the end of the storage period, the 40MA and 20A samples showed a significant decrease (∼18–19%) in the yield of cell wall material (CWM), whereas less than 6% of CWM had been degraded in the 10MA sample. The decrease in CWM was mainly due to pectic polysaccharide degradation, although for 20A and 40MA samples, hemicelluloses were also affected. Throughout storage, 10MA, 20MA and 30MA samples exhibited similar CWM solubility; however, that of the 40MA sample underwent a significant decrease, from 10% to 4.5%, probably due to the formation of new pectic chains of higher molecular weight. In contrast, the CWM solubility of sample 20A increased from 10% to 15%, suggesting that MA may have promoted the inhibition of pectic-polysaccharide-degrading enzymes. In general, the combined use of relatively low temperatures and a MA helped to preserve both the colour of raisins and maintain the levels of their CWMs at values similar to initial concentrations. Received: 5 October 1998     

Effect of high-pressure treatment on lipid oxidation in turkey thigh muscle during chill storage

 Formation of secondary lipid oxidation products during chill storage of vacuum-packed (99% vacuum), pressure-treated turkey thigh muscle was found to depend on working pressure (pressure range up to 500 MPa at 10°C) and to a lesser degree on pressurization time (10 and 30 min). Pressure treatment at 400 MPa and lower pressures for 30 min (and for 10 min) resulted in less formation of thiobarbituric-acid-reactive substances (TBARS) during 6 days of storage at 5°C compared to heat treatment at 100°C for 10 min, while pressure treatment at 500 MPa for 30 min gave similar development of TBARS as did the heat treatment. The formation of TBARS during storage at 5°C was found to depend exponentially on the pressure used for treatment at both 10 min and 30 min, and apparent volumes of activation are proposed as a parameter for quantification of the effects of pressure on lipid oxidation in meat during subsequent storage. Received: 18 November 1996     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Inactivation of peroxidase and lipoxygenase in carrots, green beans, and green peas by combination of high hydrostatic pressure and mild heat treatment

The efficiency of high hydrostatic pressure (HHP) with the combination of mild heat treatment on peroxidase (POD) and lipoxygenase (LOX) inactivation in carrots, green beans, and green peas was investigated. In the first part of the study, the samples were pressurized under 250–450 MPa at 20–50 °C for 15–60 min. In the second part, two steps treatments were performed as water blanching at 40–70 °C for 15 and 30 min after pressurization at 250 MPa and 20 °C for 15–60 min. Carrot POD was decreased to 16% residual activity within the first 30 min at a treatment condition of 350 MPa and 20 °C and then it decreased to 9% at 60 min. When the carrots were water blanched at 50 °C for 30 min after HHP treatment of 250 MPa at 20 °C for 15 min, 13% residual POD activity was obtained. For green beans, the most effective results were obtained by two steps treatment and approximately 25% residual POD activity was obtained by water blanching at 50 °C for 15 min after pressurization at 250 MPa and 20 °C for 60 min. An effective inactivation of POD in green peas was not obtained. For carrots, LOX activity could not be measured due to very low LOX activity or the presence of strong antioxidants such as carotenoids. After pressurization at 250 MPa and 20 °C for 15 or 30 min, water blanching at 60 °C for 30 min provided 2–3% residual LOX activity in green beans. The treatment of 250 MPa for 30 min and then water blanching at 50 °C for 30 min provided 70% LOX inactivation in green peas.     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998