Optimisation of asymmetric flow field-flow fractionation for the characterisation of nanoparticles in coated polydisperse TiO2 with applications in food and feed.

Titanium dioxide (TiO2) has various applications in consumer products and is also used as an additive in food and feeding stuffs. For the characterisation of this product, including the determination of nanoparticles, there is a strong need for the availability of corresponding methods of analysis. This paper presents an optimisation process for the characterisation of polydisperse-coated TiO2 nanoparticles. As a first step, probe ultrasonication was optimised using a central composite design in which the amplitude and time were the selected variables to disperse, i.e., to break up agglomerates and/or aggregates of the material. The results showed that high amplitudes (60%) favoured a better dispersion and time was fixed in mid-values (5 min). In a next step, key factors of asymmetric flow field-flow fraction (AF4), namely cross-flow (CF), detector flow (DF), exponential decay of the cross-flow (CFexp) and focus time (Ft), were studied through experimental design. Firstly, a full-factorial design was employed to establish the statistically significant factors (p < 0.05). Then, the information obtained from the full-factorial design was utilised by applying a central composite design to obtain the following optimum conditions of the system: CF, 1.6 ml min-1; DF, 0.4 ml min-1; Ft, 5 min; and CFexp, 0.6. Once the optimum conditions were obtained, the stability of the dispersed sample was measured for 24 h by analysing 10 replicates with AF4 in order to assess the performance of the optimised dispersion protocol. Finally, the recovery of the optimised method, particle shape and particle size distribution were estimated.

Validation of a near infrared microscopy method for the detection of animal products in feedingstuffs: results of a collaborative study.

The performance characteristics of a near infrared microscopy (NIRM) method, when applied to the detection of animal products in feedingstuffs, were determined via a collaborative study. The method delivers qualitative results in terms of the presence or absence of animal particles in feed and differentiates animal from vegetable feed ingredients on the basis of the evaluation of near infrared spectra obtained from individual particles present in the sample. The specificity ranged from 86% to 100%. The limit of detection obtained on the analysis of the sediment fraction, prepared as for the European official method, was 0.1% processed animal proteins (PAPs) in feed, since all laboratories correctly identified the positive samples. This limit has to be increased up to 2% for the analysis of samples which are not sedimented. The required sensitivity for the official control is therefore achieved in the analysis of the sediment fraction of the samples where the method can be applied for the detection of the presence of animal meal. Criteria for the classification of samples, when fewer than five spectra are found, as being of animal origin needs to be set up in order to harmonise the approach taken by the laboratories when applying NIRM for the detection of the presence of animal meal in feed.

Inter-laboratory comparison of an analytical method for the determination of the feed additive semduramicin in poultry feed at authorised level by liquid chromatography single quadrupole mass spectrometry.

A collaborative study was carried out according to internationally recognised guidelines in order to establish the performance characteristics of an LC/MS method for the determination of the feed additive semduramicin (SEM) in poultry feed at the level (20-25 mg kg(-1)) authorised within the European Union. Fifteen laboratories participated in the validation study, and all reported results. The content of SEM in the tested materials, provided as blind duplicates, ranged from 11.5 mg kg(-1), which corresponds to half the mean authorised level, to 45.0 mg kg(-1), which corresponds to twice the mean authorised level. All the materials were analysed by the participating laboratories using two different quantification approaches: standard addition and external standard calibration. The relative standard deviation of reproducibility (RSD(R)) for both quantification approaches varied from 8% to 18%, corresponding to HORRAT values ranging from 0.8 to 1.5, which were therefore in all cases below the critical value of 2.0. Consequently, the proposed analytical method and both quantification approaches can be considered to be fully validated and transferable to the control laboratories and applied for the determination of SEM in poultry compound feed at authorised level within the frame of official control. Further steps in the administrative procedure aiming to adopt the method as part of an ISO/CEN standard are currently ongoing.

Key parameters for the development of a NIR microscopic method for the quantification of processed by-products of animal origin in compound feedingstuffs.

The aim of this work is to show new advances in the analytical methods developed in the frame of the ban of processed animal by-products in compound feed that is currently applied within the European Union. With this aim, studies to develop a quantitative near infrared microscopy (NIRM) approach have been undertaken in order to fulfil future requirements of European legislation like the introduction of tolerance levels that would require for official control purposes the availability of specific quantitative methods. The capabilities of the NIRM method have been improved; no sample preparation is required and the acquisition parameters are optimised. Both the gross and the fine fractions of the samples are considered; the reflexion mode was used to analyse the gross raw fraction and the transmission mode was chosen to analyse the fine raw fraction. Parameters for reflexion analyses were already fixed in our previous studies while those of transmission mode have been determined in the present study. Because particles are too small, it is difficult to mark them; spectra were collected using the mapping technique. Quantitative analyses have been carried out for different percentages of adulteration (0.5, 1, 2 and 5%). Results were depending on the particle size distribution of the feed and of the fish meal which led to experimental values of adulteration varying between 0.13-0.92%, 0.93-3.7%, 2.42-5.83% and 1.95-9.39% for theoretical percentages of adulteration equal to 0.5, 1, 2 and 5%, respectively. The established protocol with the key parameters proposed has to be considered for the development of an accurate method of quantification.

Validation of an analytical method for the determination of glyceroltriheptanoate (GTH) in processed animal by-products: results of a collaborative study.

The performance characteristics of a method for the determination of the marker substance glycerol triheptanoate (GTH) in processed animal by-products (ABPs) based on gas chromatography (GC) coupled to mass spectrometry (MS) were determined via a collaborative study. Within the European Union, GTH needs to be added to the portion of processed ABPs that must not enter the feed and food chain (Categories 1 and 2) at a minimum concentration of 250 mg kg(-1) related to the fat fraction of the test samples analysed. The test materials included in the validation study consisted of three meat and bone meal (MBM) and three fat samples that contained GTH at different concentrations ranging from 61 to 455 mg kg(-1). The relative standard deviation of repeatability (RSD(r)) varied from 3.4 to 7.8% and the relative standard deviation of reproducibility (RSD(R)) varied from 9.0 to 16.5%, corresponding to HORRAT values that were, in all cases, equal or below the critical value of 2.0. The estimated trueness expressed in terms of average concentration compared to the target concentrations of GTH in all test materials varied from 95 to 107%, confirming acceptable values for the trueness of the method. Based on the acceptable values for precision and trueness, the method is fit for the intended purpose and can be used for official control purposes to determine GTH in processed animal by-products from Category 1 and Category 2.

Use of glyceroltriheptanoate as marker for processed animal by-products: development and validation of an analytical method.

A recently published European Regulation requires that the artificial marker, glycerol triheptanoate (GTH), be added to processed animal by-product (ABPs) prohibited from entering the food chain. The objective of this new requirement is to allow full traceability and ensure that these materials are disposed of in a proper way. Here, we report the development and single-laboratory validation of an analytical method for the determination of GTH in meat and bone meal plus animal fat. The method comprises three steps: (1) extraction of GTH from the samples with petroleum ether when analysing meat and bone meal or dissolving the sample in n-hexane when analysing fat; (2) clean-up of the extract using commercially available SPE cartridges; (3) determination of GTH by GC/MS or GC with flame ionisation detection (FID). The results of the validation study demonstrated that the relative standard for intermediate precision varied between 2.5 and 8.2%, depending on GTH concentration and the detector utilised. In all cases, the relative recovery rate was above 96%. The limit of quantification was 16 mg kg(-1) (GTH/fat content of the sample) with MS as detector and 20 mg kg(-1) with FID. Moreover, the method has been successfully applied in a second laboratory, indicating its transferability. Considering the minimum GTH concentration in ABPs of 250 mg kg(-1), the method is considered suitable for the intended purpose and can be utilised by EU Member States laboratories for official control and monitoring.

Validation of an analytical method for the determination of carbadox and olaquindox in feedstuff by liquid chromatography coupled to UV and/or diode array detection.

The performance characteristics of an analytical method based on high-performance liquid chromatography (HPLC) for the detection of the banned growth promoters, carbadox and olaquindox, in feedstuff were determined via a collaborative study. The relative standard deviation of repeatability (RSDr) ranged 1.1-5.5% for carbadox and 2.5-6.2% for olaquindox. The relative standard deviation of reproducibility (RSDR) ranged 6.4-10.7% for carbadox and 12.8-20.0% for olaquindox. In all cases, the HORRAT values were equal or below the critical value of 1.5. Moreover, trueness in all cases was between the acceptance limits of 80 and 110%. Consequently, it was concluded that the method is suitable for quantitative evaluation. The method was also qualitatively assessed in terms of correct identification of the target analytes by examination of the UV spectrum when the more specific diode array detector was coupled to HPLC. In all cases, the percentage of correct identifications was >or=94% for olaquindox and carbadox, while the percentage of false negatives was

Determination of processed animal proteins in feed: The performance characteristics of classical microscopy and immunoassay methods.

Species-specific detection and detection of groups of species such as ruminants is required according to European legislation dealing with the safe use of animal by-products in animal nutrition. Various methods are applied to the analysis of feed samples for the presence of banned processed animal proteins (PAPs) including meat and bone meal (MBM). Classical microscopy as described in the Commission Directive EC/2003/126 is the only official method to detect the presence of constituents of animal origin in feed, nevertheless some deviating protocols allowed under the old Directive (EC/88/1988) claim to gain comparable results. An inherent limitation of the microscopic method is the lack of species specificity. Immunoassays showed the most promising potential in research projects or intercomparison studies being able to detect ruminant PAPs at a concentration level of 0.5%. The aim of this paper is to present the results of the intercomparison study conducted on behalf of European Commission's Directorate General for Health and Consumer Protection (SANCO) in 2004 to establish whether the two-solvent method would gain comparable results to the current European Method and to evaluate the current capability of immunoassays of determining the species in PAPs present in feed.

Determination of dioxins (PCDDs/PCDFs) and PCBs in food and feed using the DR CALUX bioassay: results of an international validation study.

Maximum levels for dioxins in food and feedstuffs have been recently established by the European Commission through two regulations. Dioxin-monitoring programmes of food and feedstuffs will therefore be undertaken by the European Member States to implement these regulations, which would be facilitated by fast and low-cost screening methods. Commission Directives 2002/70/EC and 2002/69/EC describe specific characteristics for such screening methods. In the present study, the performance characteristics of the DR CALUX method from BioDetection Systems were established in a validation study with 14 participants. The study was based on two materials (fish oil and feed), each containing four different levels of dioxins and dioxin-like PCBs around the current limits. The results demonstrate that the test is very promising but that in particular the clean-up procedure was a source of variation and requires further optimization and standardization. In addition the quantification is improved by the use of control samples to correct for background contamination, recovery and differences between the TEF values and REP (relative potency) factors in the test.

An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy.

Enforcing the ban on meat-and-bone meal in feed for farmed animals, and especially ruminants, is considered an important measure to prevent the spread of bovine spongiform encephalopathy. The authors describe current analytical methods for the detection and identification of animal tissues in feed. In addition, recently approved requirements, such as the ban of intra-species recycling (practice of feeding an animal species with proteins derived from the bodies, or parts of bodies, of the same species) are described. In principle, four different approaches are currently applied, i.e. microscopic analysis, polymerase chain reaction, immunoassay analysis and near infrared spectroscopy or microscopy. The principal performance characteristics of these methods are presented and compared, and their specific advantages and disadvantages described. Special emphasis is also placed on the impact of rendering conditions, particularly high temperatures and on the use of molecular biology techniques.

Correction of analytical results for recovery: a comparison of the method performance characteristics from recent collaborative trials studies for aflatoxin quantification using conventional and robust statistics.

Results from recently conducted collaborative trials on the determination of aflatoxin B(1) in various matrices have been evaluated to establish whether the use of recovery data would result in a distinct change of the relative between-laboratory standard deviation (RSD(R)) of the corrected data compared with the uncorrected data. In addition, we applied conventional and robust statistics to evaluate whether the impact of the use of recovery data on the estimation of RSD(R) depended on the statistical method applied for data analysis. This investigation was based on means before and after correction for recovery. The method performance characteristics were calculated using results from naturally contaminated test materials, while the results from test materials fortified with the target analytes were used to estimate the recovery. The study revealed that applying conventional and robust statistics in general led to comparable estimates for RSD(R). The comparison about the use of recovery data showed that in most cases, the RSD(R) obtained from the analysis of aflatoxin B(1) decreased after correction of the results for recovery. This tendency was similar when the comparison was done using robust or conventional statistics. However, in three cases, conventional statistics yielded a higher RSD(R) for the corrected data, whereas robust statistics showed the opposite. Looking carefully at the data, the treatment of conventional statistics indicated that the way outliers are detected and removed could result in an under- or overestimation of RSD(R). Applying the law of error propagation revealed that most likely the correlation between the uncorrected data and the recovery rate led to a reduced variability of the data corrected for recovery.

Alternative extraction methods for Zearalenone: Microwave Assisted Extraction and Accelerated Solvent Extraction.

Zearalenone (ZON) was extracted from wheat and corn by using Microwave Assisted Extraction and Accelerated Solvent Extraction. A factorial design approach was applied to evaluate the influence of the most important extraction parameters such as temperature, time and solvent extraction mixture on fortified cereals. ZON was determined by LC-MS without performing any clean-up after the extraction to better evaluate the extraction efficiency. The selected extraction conditions were tested on samples which had been previously used in an international proficiency test. Applying the selected extraction conditions both alternative extraction procedure provided satisfactory results comparable to the most commonly used method.

Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples.

Five types of fat retainers were investigated for the lipid-free extraction of PCBs from fat-containing matrixes using accelerated solvent extraction: florisil, basic alumina, neutral alumina, acidic alumina, and sulfuric acid-impregnated silica. All of the fat retainers generated fat-free extracts when the fat/fat retainer ratio was 1:40. Sulfuric acid-impregnated silica and florisil were the only retainers that gave completely clear extracts, and the former was the only not to show any reaction when treated with sulfuric acid after the extraction. Using sulfuric acid-impregnated silica as fat retainer, on-line cleanup of fat-containing matrixes was possible, as demonstrated for naturally contaminated fish meal as well as certified cod liver oil (CRM 349).

On-line clean-up of pressurized liquid extracts for the determination of polychlorinated biphenyls in feedingstuffs and food matrices using gas chromatography-mass spectrometry.

This paper describes a fast and simple pressurized liquid extraction method for the determination of polychlorinated biphenyls (PCBs) in feedingstuffs and food matrices. The method is based on a simultaneous extraction/clean-up step requiring a minimum of sample handling. The final analysis was performed with gas chromatography-mass spectrometry. Seven PCBs (28, 52, 101, 118, 138, 153 and 180) were analyzed, all of which are indicator congeners that, according to European legislation should be included in the analytical monitoring program. The extracted matrices were spiked feed for poultry and two certified reference materials naturally contaminated with PCBs (cod-liver oil and milk powder), which showed excellent conformity with certified data.

Pesticide residues in products of plant origin in the European Union. Sampling strategy and results from the co-ordinated EU monitoring programmes in 1996 and 1997.

The results of a co-ordinated monitoring programme for pesticide residues in the European Union and Norway carried out in 1996 and 1997 are presented. The aim of this programme is to work towards a system, which makes it possible to estimate actual dietary pesticide intake for the population of the European Union. Based on a statistically dérived sampling plan and within the limited number of pesticides/commodities analysed, the most critical pesticides (benomyl group and dithiocarbamates) and commodities (mandarine and lettuce) were identified. In case of detected non-compliances, repeated sampling and, if necessary, enforcement actions are to be taken by national authorities. The programme will be continued in the next years.

Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach.

An experimental design procedure was used to investigate the effects of some operating parameters on the supercritical fluid extraction of carotenoids beta-carotene, beta-cryptoxanthin and zeaxanthin from Spirulina Pacifica algae, a carotenoid-rich dietary product. Variables tested were temperature and pressure of the supercritical fluid, dynamic extraction time and percentage of ethanol added as the modifier. Each variable was tested at three levels; 31 experiments were performed in random order. Analyses of the extracts were performed by high-performance liquid chromatography with UV-Vis photodiode array detection. Analytical responses (chromatographic peak areas) were processed by using a stepwise multiple regression analysis, in order to find polynomial functions describing the relationships between variables and responses. For all the analytes the experimental conditions providing the highest extraction yield inside the experimental domain considered were found. Supercritical fluid extraction results obtained in these conditions were compared with those obtained by performing solvent extraction in order to evaluate the effectiveness of the supercritical fluid extraction procedure.

Use of an immunoassay as a means to detect polychlorinated biphenyls in animal fat.

In this paper we describe the validation and utilization of the Hybrinzme DELFIA immunoassay for detecting polychlorinated biphenyls (PCBs) in animal fat. The immunoassay incorporates a rapid sample processing protocol that has been optimized to detect concentrations of PCBs in animal fat at or above 200 ng/g. The limit of detection of the assay was less than 50 ng/g. When the action level for the DELFIA assay was set at 100 ng/g to detect samples having 200 ng/g or more PCBs, the false negative rate was estimated at 0.2%. The false positive rate depends on the concentration of PCBs in the sample population and was estimated at 4% and 7% for samples having 10 ng/g and 20 ng/g PCBs, respectively. By utilizing the DELFIA assay system to select for positive samples and GC/MS analysis for confirmation analysis, an accurate and cost-effective programme has been established for the high throughput detection of PCBs in animal products.

Comparison of different extraction and clean-up procedures for the determination of fumonisins in maize and maize-based food products.

In order to optimize the analytical method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in different maize products, five materials (maize flour, corn flakes, extruded maize, muffins and infant formula) were investigated under a variety of experimental conditions organized in a ruggedness test according to a factorial design. The influence of five factors (extraction solvent, extraction mode, volume of extraction solvent, test sample size and clean-up) on method performances was tested by four laboratories using spiked materials (0.5 microgram/g and 1.5 micrograms/g FB1 + FB2) and naturally contaminated materials (ca 1.5 micrograms/g with FB1 + FB2). The end determination step was performed by high-performance liquid chromatography analysis of the o-phthaldialdehyde derivatized extracts. The ruggedness test permitted identification of two critical factors in the analysis of fumonisins in the above products, namely 'extraction solvent' and 'clean-up procedure'. In particular, the use of acetonitrile (ACN)-water (1 + 1, v + v) as extraction solvent and immunoaffinity column for clean-up provided better recovery of fumonisins and chromatographic resolution as compared with methanol (MeOH)-water (3 + 1, v + v) and strong anion exchange (SAX), respectively. However, phase separation occurring after extraction with ACN-water may have given incorrect results. Based on the information obtained with the present study it was possible to develop a new method horizontally applicable to all the above mentioned maize-based food matrices.

Intercomparison study for the determination of selected polychlorinated biphenyls (PCBs) in feed matrices.

Analytical methodology currently employed for the determination of seven indicator PCBs in three compound feeds and fish meal has been evaluated in a collaborative study. The majority of the obtained relative standard deviations of the PCBs varied from 20 to 30%. On assuming a target relative standard deviation of 22% for the analytical results, statistical evaluation showed that about 80% of the participating laboratories delivered data within an acceptable range of +/- 44% of the assigned concentration in the test materials. However, between 10 and 20% of the participating laboratories reported unacceptable results. Major problems seemed to arise from insufficient separation of PCB congeners, low extraction efficiency, and calculation errors. Correct identification of the target PCB congeners was a prevalent problem if only one capillary column in combination with an electron capture detector (ECD) was employed. The correct preparation of the calibration solution by the laboratories turned out to be only a minor problem. The laboratories participating in this study employed quite different techniques at all stages of the analytical procedure. Principal component analysis indicated that laboratories using an internal standard tended to report higher values for the target analytes. If the PCB concentrations were related to the fat content of the sample, the variability of the reported results decreased for compound feed but increased for fish meal. These inconsistent results are probably due to the fact that fat is not an objective parameter but is defined by the analytical technique employed. It is assumed that harmonizing analytical methods for the determination of this parameter could improve the precision of the PCB results.

Post process product control of rendering plant sterilization conditions by ELISA.

Experiments were conducted in a laboratory autoclave and in a commercial rendering plant to set a limit for the R-value of an enzyme-linked immunosorbent assay (ELISA) to prove that animal meal had been sufficiently heat-treated. The immunoassay method is currently the only technique used to check for appropriate sterilization of animal meal. The results of these experiments were statistically assessed using operation characteristic curves and confirmed that insufficiently heat-treated animal meal has an R-value > 2. Results of the trials also demonstrated that the main parameters of sterilization, temperature, and duration strongy influence the R-value, thus indicating that deviation from the target sterilization conditions is verifiable by the ELISA method.