Showing papers in "Journal of AOAC International in 2002"

Gravimetric Determination of Amylase-Treated Neutral Detergent Fiber in Feeds with Refluxing in Beakers or Crucibles: Collaborative Study

Abstract: As an important constituent of animal feeds, fiber represents the portion of feeds that is bulky and difficult to digest. The neutral detergent fiber (NDF) method, developed over 30 years ago, is the method of choice for measuring total fiber in forages and other feeds. Several modifications that were made to improve its general applicability to all feeds and others developed in individual laboratories often resulted in variability among laboratories in measuring NDF. The amylase-treated NDF (aNDF) method, therefore, was developed as an accurate and precise method of measuring total insoluble fiber in feeds. A collaborative study was conducted to evaluate the repeatability and reproducibility of the aNDF method over the full range of animal feed materials. Twelve laboratories representing research, feed company, regulatory, and commercial feed testing laboratories analyzed 11 materials as blind duplicates. The materials represented feed matrixes, including animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; and legume and grass hays and silages. Materials selected varied in chemical composition and contained 0-90% aNDF, 1-16% ash, 1-20% crude fat, 1-40% crude protein, and 0-50% starch. Correcting results for changes in blanks and reporting results as ash-free aNDF organic matter (aNDFom) improved the repeatability and reproducibility of results when aNDF was 10% fat. However, standard deviations of repeatability and reproducibility for feeds with >10% fat were similar to those of other materials. It is recommended that the aNDF method be accepted for Official First Action status.

Measurement of resistant starch.

Abstract: A robust and reliable method was developed to measure resistant starch (RS), ie, starch that enters the large intestine In vivo conditions were reflected as much as possible while a user-friendly format was maintained Parameters investigated included a-amylase concentration, pH of incubation, maltose inhibition of alpha-amylase, the need for amyloglucosidase inclusion, the effect of shaking and stirring on determined values, and problems in recovering and analyzing the RS-containing pellet The RS values obtained were in good agreement with published in vivo data An interlaboratory evaluation of the method has been completed (First Action Method 200202)

AOAC International methods committee guidelines for validation of qualitative and quantitative food microbiological official methods of analysis.

Abstract: Responding to a need for a guide for conducting Official Method validation studies of microbiological methods, AOAC utilized the experience of three microbiologists who have been active in the field of method validation. In collaboration, a document was prepared which covered the following areas: terms and their definitions associated with the Official Methods program (e.g., reference methods, alternative methods, and ruggedness testing), protocols and validation requirements for qualitative methods versus those for quantitative methods, the concept of the precollaborative study, ruggedness testing, tests for significant differences, performance indicators, and the approval process. After its preparation, this document was reviewed by the members of the Methods Committee on Microbiology and Extraneous Materials and by members of the Official Methods Board. Herein is presented the approved version of that document.

Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean

Abstract: New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.

Determination of crude protein in animal feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam distillation into boric acid: collaborative study.

Abstract: A collaborative study was conducted to evaluate the repeatability and reproducibility of an extension of AOAC Official Method 991.20, Nitrogen (Crude) in Milk, to animal feed, forage (plant tissue), grain, and oilseed materials. Test portions are digested in an aluminum block at 420 degrees C in sulfuric acid with potassium sulfate and a copper catalyst. Digests are cooled and diluted, and concentrated sodium hydroxide is added to neutralize the acid and make the digest basic; the liberated ammonia is distilled by using steam distillation. The liberated ammonia is trapped in a weak boric acid solution and titrated with a stronger standardized acid, hydrochloric acid; colorimetric endpoint detection is used. Fourteen blind samples were sent to 13 collaborators in the United States, Denmark, Sweden, Germany, and the United Kingdom. Recoveries of nitrogen from lysine, tryptophan, and acetanilide were 86.8, 98.8, and 100.1%, respectively. The within-laboratory relative standard deviation (RSDr, repeatability) ranged from 0.40 to 2.38% for crude protein. The among-laboratories (including within-) relative standard deviation (RSD(R), reproducibility) ranged from 0.44 to 2.38%. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL. A lower concentration (1% H3BO3) of trapping solution was compared with the concentration specified in the original protocol (4% H3BO3) and was found comparable for use in an automatic titration system in which titration begins automatically as soon as distillation starts. The Study Directors recommend that 1% H3BO3 as an optional alternative to 4% boric acid trapping solution be allowed for automatic titrators that titrate throughout the distillation.

Molecular Subtyping Methods for Listeria monocytogenes

Abstract: Conventional, phenotypic, and DNA-based subtyping methods allow differentiation of Listeria monocytogenes beyond the species and subspecies level. Bacterial subtyping methods not only improve our ability to detect and track human listeriosis outbreaks, but also provide tools to track sources of L. monocytogenes contamination throughout the food system. The use of subtyping methods also provides an opportunity to better understand the population genetics, epidemiology, and ecology of L. monocytogenes. The last 5 years have seen tremendous advancements in the development of sensitive, rapid, automated, and increasingly easy-to-use molecular subtyping methods for L. monocytogenes. This review highlights key aspects of different L. monocytogenes subtyping methods and provides examples of their application in public health, food safety, population genetics, and epidemiology. A significant focus is on the application of subtyping methods to define L. monocytogenes subtypes and clonal groups, which may differ in phenotypic characteristics and pathogenic potential.

Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: collaborative study.

Abstract: Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high.

Determination of Dissolved Naphthenic Acids in Natural Waters by Using Negative-Ion Electrospray Mass Spectrometry

Abstract: Naphthenic acids (NAs) have been implicated as some of the most toxic substances in oil sands leachates and identified as priority substances impacting on aquatic environments. As a group of compounds, NAs are not well characterized and comprise a large group of saturated aliphatic and alicyclic carboxylic acids found in hydrocarbon deposits (petroleum, oil sands bitumen, and crude oils). Described is an analytical method using negative-ion electrospray ionization mass spectrometry (ES/MS) of extracts. Preconcentration was achieved by using a solid-phase extraction procedure utilizing a crosslinked polystyrene-based polymer with acetonitrile elution. Recovery of the Fluka Chemicals NA mixture was highly pH-dependent, with 100% recovery at pH 3.0, but only 66 and 51% recoveries at pHs 7 and 9, respectively. The dissolved phase of the NA was very dependent on sample pH. It is thus critical to measure the pH and determine the appropriate mass profiles to identify NAs in natural waters. The ES/MS analytical procedure proved to be a fast and sensitive method for the recovery and detection of NAs in natural waters, with a detection limit of 0.01 mg/L.

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules.

Abstract: Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.

Molecular methods for identification and detection of bacterial food pathogens.

Abstract: The polymerase chain reaction (PCR) shortens conventional microbiological methods for the detection of food pathogens either by replacing the conventional biochemical and serological identification or by its direct use on pre-enrichment media or food products. PCR allows fast and highly reliable identification of bacterial taxa, particularly phenotypically atypical bacterial strains. For reliablity, PCR primers and reaction conditions must be thoroughly optimized and evaluated, appropriate sample preparations must be developed, and a stringent laboratory protocol must be followed. Positive control systems are used to monitor possible inhibition of the reaction and negative controls are needed to monitor for contamination. The most recent developments involve messenger RNA-based (mRNA-based) detection of viable bacterial pathogens and real-time PCR quantitation of pathogens.

Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations.

Abstract: Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits Using existing US Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws

Cleanup procedure for determination of aflatoxins in major agricultural commodities by liquid chromatography.

Abstract: A simple, fast, reliable, and inexpensive chemical cleanup procedure was developed for quantitation of aflatoxins in major important agricultural commodities by liquid chromatography (LC). Aflatoxins were extracted from a ground sample with methanol-water (80 + 20, v/v), and after a single cleanup step on a minicolumn packed with basic aluminum oxide, they were quantitated by LC equipped with a C18 column, photochemical reactor, and fluorescence detector. Water-methanol-1-butanol (1,400 + 720 + 25, v/v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5.0, 2.5, 7.5, and 2.5 microg/kg were 87.2 +/- 2.3, 82.0 +/- 0.8, 80.0 +/- 1.8, and 80.4 +/- 2.8%, respectively. Similar recoveries, precision, and accuracy were achieved for corn, cottonseed, almonds, Brazil nuts, pistachios, and walnuts. The quantitation limit for aflatoxin B1 was 1 microg/kg. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.

Quantitative LC/MS-MS determination of sulfonamides and some other antibiotics in honey.

Abstract: A simple and rapid method was developed for the determination of 20 antibiotics (sulfonamides, tetacyclines, and flumequine) in honey by liquid chromatography tandem mass spectrometry. The proposed method is sensitive (limit of detection 0.5 to 10 ppb for the various antibiotics) and selective. A hydrolysis step ensures the liberation of sugar-bound sulfonamides. The approach has been used to analyze some 300 honey samples. A number of them were found to have exceeded the Swiss limit of 50 ppb.

Polymerase chain reaction-based methods for detection of Listeria monocytogenes: toward real-time screening for food and environmental samples.

Abstract: A review is presented of nucleic acid amplification-based methodology, specifically polymerase chain reaction (PCR)-based assays, for the detection of Listeria monocytogenes in food and environmental samples. Until recently, developmental challenges including poor sensitivity, due in part to reaction inhibition by components of the sample matrix, and the potential for false-positive reactions have limited routine application of PCR-based screening assays. Commercial assays address these challenges while offering convenient, standardized protocols, a high level of automation, and results within 2 days after the sampling date. Although sample enrichment is necessary to achieve desired detection limits, continued efforts toward template purification will facilitate the development of assays offering real-time, quantitative results. The development of ribonucleic acid (RNA) amplification-based assays may increase in importance, particularly if end-product testing is prioritized by regulatory agencies, as messenger RNA appears to serve as an accurate indicator of cell viability. Further, the increase in target copy number may improve assay sensitivity. PCR-based screening methods offer efficient, reliable results and are ideal for monitoring the presence of L. monocytogenes in foods and in the food processing environment.

Validation of a method for the determination of multiclass pesticide residues in fruit juices by liquid chromatography/tandem mass spectrometry after extraction by matrix solid-phase dispersion.

Abstract: A multiresidue method was developed and validated for the determination of pesticide residues (omethoate, dimethoate, carbendazim, propoxur, thiabendazole, carbaryl, pirimicarb, azinphos-methyl, methidathion, and iprodione) in fruit juices. The samples were extracted by matrix solid-phase dispersion with diatomaceous earth and analyzed by liquid chromatography/tandem mass spectrometry. The method detection limits were <0.2 ppb for all pesticides; the relative standard deviations for analyses of samples fortified over the range of 2-50 ng/g were <9%, and the recoveries for each pesticide were all between 77 and 102%. The proposed method was used to analyze 21 commercial fruit juices; pesticide residues were found in 71 % of the samples.

Detection of genetically modified crops and their derivatives: critical steps in sample preparation and extraction.

Abstract: The detection of genetically modified crops in foodstuff relies on detection of transgenic DNA or protein material in the sample matrix. Purified DNA or proteins are used as analytical material for polymerase chain reaction technologies and immunodiagnostics. Successful sample preparation is critical to the validity of subsequent analysis. For routine analysis, a good sample preparation technique should be simple, safe, and inexpensive while reproducibly generating DNA/protein of sufficient quality and yield. The suitability of isolated DNA or protein as an analyte for a detection or characterization technique depends on amount or concentration, purity, and integrity, each of which may be influenced by sample matrix and the extraction technique, and, in turn, may impact the validity of analytical techniques. The key sample preparation steps of homogenization, pretreatment, extraction, and purification are discussed as well as typical analytical methods. Consideration is given to application of these steps for particular sample matrixes to maximize yield, reduce inhibition effects, and minimize contamination. The choice of the most appropriate and valid methods for sample preparation from particular foods is discussed with respect to DNA analysis. Attention is also given to ease of use, cost, and generic applicability of the procedures.

Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food.

Abstract: Quantitative detection methods are needed for en- forcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling thresh- old, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are ap- proved in the European Union: the insect-resistant Bt176 maize (Maximizer), Bt11 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link TM ) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymer- ase chain reaction detection methods were devel- oped for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system. the control of a labeling limit requires quantitative detection systems. The polymerase chain reaction (PCR) method has recently been shown to be the method of choice for detection of GMPs. First, qualitative methods that were able to detect genetic ele- ments present in almost all approved GMPs (so-called screen- ing systems) such as 35S-promoter, NOS-terminator, kanamycin-resistance gene, ampicillin-resistance gene, and others were developed (6, 7). GMP-specific qualitative detec- tion systems have also been developed: Roundup Ready TM soybean (8, 9), Bt176 maize (10, 11), Bt11 maize (12), Mon810 maize (13), Flavr Savr™ tomato (14), Zeneca to- mato (15), Liberty Link™ Canola (16), etc. At first, quantita- tive-competitive PCR (QC-PCR) was used to quantitate the genetically modified organism (GMO) content. For the 35S- and NOS-screening (17), the Roundup Ready soybean (18), the Bt176 maize (18, 19), and the Bt11 maize (12), quantita- tive-competitive systems were developed. Some of them were also successfully tested in ring trials (20-22). However, only highly specialized, experienced laboratories were able to quantitate the GMP content correctly. The easier handling (no agarose gels, closed systems, etc.) and more accurate results led to the use of real-time PCR, and most laboratories have now been equipped with real-time PCR devices. The first methods for the quantitative detection of GMPs by real-time PCR are already published (23, 24). The present study describes the quantitative detection of ge- netically modified maize. Detection methods used for the ap- proved genetically modified maize in the EU until the end of 2000 were investigated. Products derived from 4 genetically modified maize and approved in the EU (25; Table 1) included Bt176 (26) and Bt11 maize from Novartis (Basel, Switzer- land; 12), Mon810 maize from Monsanto (St. Louis, MO; 13), and T25 maize from Aventis (Lyon, France). Quantitative real-time PCR systems to determine the con- tent of the respective approved maize are discussed. To calcu- late the GMO content corresponding to the maize content, a detection system for the maize content was developed. As the housekeeping gene, the invertase gene was chosen. The TaqMan ® probe for the housekeeping gene assay was labeled with the fluorescent dye VIC™ instead of the normally used

Comparison of magnesium sulfate and sodium sulfate for removal of water from pesticide extracts of foods.

Abstract: Water-miscible solvents, such as acetone and acetonitrile, effectively extract both polar and nonpolar pesticide residues from nonfatty foods. The addition of sodium chloride to the resulting acetonitrile-water or acetone-water extract (salting out) results in the separation of the water from the organic solvent. However, the organic solvent layer (pesticide extract) still contains some residual water, which can adversely affect separation procedures that follow, such as solid-phase extraction and/or gas chromatography. Drying agents, such as sodium sulfate or magnesium sulfate, are used to remove the water from the organic extracts. In the present study, we used nuclear magnetic resonance spectroscopy to study the composition of the phases resulting from salting out and to compare the effectiveness of sodium sulfate and magnesium sulfate as drying agents. The study showed that considerable amounts of water remained in the organic phase after phase separation. Sodium sulfate was a relatively ineffective drying agent, removing little or no residual water from the organic solvent. Magnesium sulfate proved to be a much more effective drying agent.

Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury.

Abstract: Detection of L. monocytogenes is often limited by the performance of the enrichment media used to support bacterial growth to detectable levels. Because Listeria may exist at extremely low levels in foods, sample enrichment protocols must amplify these low initial populations to detectable limits. Listeria may also exist in an injured state in food products as a result of processing treatments such as heating, freezing, exposure to acids, or exposure to sanitizing compounds. Selective agents in enrichment media normally used for recovery of Listeria may inhibit repair and detection of sublethally injured Listeria, which may go on to repair, grow, and regain pathogenicity. Simple modifications to existing regulatory protocols, such as those that use more than one enrichment broth, raise sensitivity of detection to 90%. This review shows the efficacy of repair/enrichment strategies, which increase sensitivity of detection to 97.5-98.8% compared with 65-70% by standard regulatory protocols. Ribotype analysis of isolates obtained from meat samples reveals a complex microbial ecology, with striking differences in both number and distribution of distinct genetic types of Listeria, depending upon whether samples are enriched in selective or repair/enrichment media. In studies on enrichment of dairy environmental samples in University of Vermont medium and Listeria repair broth (UVM and LRB), combining these 2 primary enrichment media into a single tube of Fraser broth for dual secondary enrichment yielded a significantly higher percentage (p < 0.05) of Listeria-positive samples than did use of either LRB or UVM alone. Refinement of conventional Listeria recovery methods should consider the importance of the enrichment step, the nutritional needs of specific genetic types, and the physiological condition of Listeria isolates in foods.

Determination of Vitamin B12 in Milk Products and Selected Foods by Optical Biosensor Protein-Binding Assay: Method Comparison

Abstract: in arange of foods The analytical technique was config-ured as a biosensor-based, nonlabeled inhibitionprotein-binding assay using nonintrinsic R-proteinSample extraction conditions were optimized, andboth ligand specificity and nonspecific binding con-siderations were evaluated Performance parametersincluded a quantitation range of 008–240 ng/mL, re-coveries of 89–106 % , agreement against assignedreference values for 3 independent certified foodreference materials, and a mean between-laboratoryreproducibility relative standard deviation of 49% The proposed method was compared with referencemicrobiological and radioisotope protein-bindingmethods for a range of food samples A wide selec-tion of milks, infant formulas, meats, and liver wereevaluated for their vitamin B

Measurement of alpha-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study.

Abstract: This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 μmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.

Interlaboratory study of a multiresidue gas chromatographic method for determination of organochlorine and pyrethroid pesticides and polychlorobiphenyls in milk, fish, eggs, and beef fat.

Abstract: An interlaboratory study was conducted to validate a gas chromatographic (GC) method for determination of 21 organochlorine pesticides, 6 pyrethroid pesticides, and 7 polychlorobiphenyl (PCB) congeners in milk, beef fat, fish, and eggs. The method was performed at low contamination levels, which represent relevant contents in food, and is an extension of the European standard (method NF-EN-1528, Parts 1-4). It enlarges the applicable scope of the reference EN method to pyrethroid pesticides and proposes the use of solid-phase extraction (SPE) as a cleanup procedure. Cryogenic extraction was made, and SPE cleanup was performed with 2 successive SPE cartridges: C18 and Florisil. After injection of the purified extract onto a GC column, residues were measured by electron capture detection. Food samples (liquid milk, beef fat, mixed fish, and mixed eggs) were prepared, tested for homogeneity, and sent to 17 laboratories in France. Test portions were spiked with 27 pesticides and 7 PCBs at levels from 26 to 45, 4 to 27, 31 to 67, and 19 to 127 ng/g into milk, eggs, fish, and fat, respectively. Based on results for spiked samples, the relative standard deviation for repeatability ranged from 1.5 to 6.8% in milk, 3 to 39% in eggs, 4.5 to 12.2% in fish, and 7 to 13% in fat. The relative standard deviation for reproducibility ranged from 33 to 50% in milk, 29 to 59% in eggs, 31 to 57% in fish, and 30 to 62% in fat. This method showed acceptable intra- and interlaboratory precision data, as corroborated by HORRAT values at low levels of pesticide and PCB contamination. The statistical evaluation of the results was performed according to the International Organization for Standardization (ISO; ISO 3534 standard) and 5725-2 Guideline.

DNA microarray technology used for studying foodborne pathogens and microbial habitats: minireview.

Abstract: Microarray analysis is an emerging technology that has the potential to become a leading trend in bacterial identification in food and feed improvement The technology uses fluorescent-labeled probes amplified from bacterial samples that are then hybridized to thousands of DNA sequences immobilized on chemically modified glass slides The whole gene or open reading frame(s) is represented by a polymerase chain reaction fragment of double-strand DNA, approximately 1000 base pair (bp) or 20-70 bp single-strand oligonucleotides The technology can be used to identify bacteria and to study gene expression in complex microbial populations, such as those found in food and gastrointestinal tracts Data generated by microarray analysis can be potentially used to improve the safety of our food supply as well as ensure the efficiency of animal feed conversion to human food, eg, in meat and milk production by ruminants This minireview addresses the use of microarray technology in bacterial identification and gene expression in different microbial systems and in habitats containing mixed populations of bacteria

State of the Art and Limitations of Quantitative Polymerase Chain Reaction

Abstract: Consequential to the implementation of European Commission (EC) Regulation 1139/98, EC Regulation 49/2000, and EC Regulation 50/2000 has been the need to measure accurately the levels of the genetically modified (GM) species Roundup Ready Soya and Bt 176 Maize that are present in food. Analytical methods to detect and quantitate these transgenic species have received much attention particularly with respect to the deminimus threshold of 1% for their presence in materials derived from non-GM identity-preserved (IP) supplies. The relative advantages and limitations of threshold analysis by double-competitive polymerase chain reaction (PCR) and quantitative real-time PCR are discussed in their application to the quantitative analysis of processed foods. Consideration is also given to other factors involved in the analyses that affect the performance of quantitative procedures, and to the many uncertainties involved in the precision of a reported analytical result.

Determination of Total Dietary Fiber in Selected Foods Containing Resistant Maltodextrin by Enzymatic-Gravimetric Method and Liquid Chromatography: Collaborative Study

Abstract: A method was developed for determination of total dietary fiber (TDF) in foods containing resistant maltodextrin (RMD) which includes nondigestible carbohydrates that are not fully recovered as dietary fiber by conventional TDF methods such as AOAC 985.29 or 991.43. Because the average molecular weight (MW) of RMD is 2000 daltons, lower MW soluble dietary fiber components do not precipitate in 78% ethanol; therefore, RMD is not completely quantitated as dietary fiber by current AOAC methods. The accuracy and precision of the method was evaluated through an AOAC collaborative study. Ten laboratories participated and assayed 12 test portions (6 blind duplicates) containing RMD. The 6 test pairs ranged from 1.5 to 100% RMD. The method consisted of the following steps: (1) The insoluble dietary fiber (IDF) and high MW soluble dietary fiber (HMWSDF) were determined by AOAC 985.29. (2) Ion exchange resins were used to remove salts and proteins contained in the AOAC 985.29 filtrates (including ethanol and acetone). (3) The amount of low MWRMD (LMWRMD) in the filtrates were determined by liquid chromatography. (4) The TDF was calculated by summation of the IDF, HMWSDF, and LMWRMD fractions having nondigestible carbohydrates with a degree of polymerization of 3 and higher. Repeatability standard deviations (RSDr) were 1.33-7.46%, calculated by including outliers, and 1.33-6.10%, calculated by not including outliers. Reproducibility standard deviations (RSDR) were 2.48-9.39%, calculated by including outliers, and 1.79-9.39%, calculated by not including outliers. This method is recommended for adoption as Official First Action.

Comparison of two post-column derivatization systems, ultraviolet irradiation and electrochemical determination, for the liquid chromatographic determination of aflatoxins in food

Abstract: This study compared 2 post-column derivatization (PCD) techniques for the determination of aflatoxins B1, B2, G1, and G2 (AFB1, AFB2, AFG1, and AFG2) by fluorescence detection after liquid chromatographic separation: ultraviolet (UV) irradiation (PCD(UV)) and electrochemical bromination (PCD(EC)). Photochemical fluorescence enhancement was obtained with 2 different commercially available systems (PCD(UV1) and PCD(UV2)). An electrochemical bromination apparatus was used for bromination. Analyses of naturally contaminated or spiked samples of corn, pistachio paste, peanut butter, fig paste, and animal feed showed that neither of the techniques resulted in derivatization-specific matrix interferences for any of the matrixes under study, even when extracts were not completely purified. The response ratios PCD(UV)/PCD(EC) for AFB1, AFB2, AFG1, and AFG2 were 0.86, 0.96, 0.70, and 0.96, respectively, for PCD(UV1) and 0.82, 0.95, 0.60, and 0.90, respectively, for PCD(UV2). The long-term use of the UV lamps (300 h for PCD(UV1) and 343 h for PCD(UV2)) in the photochemical detectors showed that these ratios remained stable throughout the time frame investigated. The relative standard deviation obtained for each of the devices during the in-house validation study ranged from 0.3 to 1.8% for PCD(UV1), from 0.8 to 1.3% for PCD(UV2), and from 0.9 to 2.0% for PCD(EC).

Rapid determination of thiamine, riboflavin, pyridoxine, and niacinamide in infant formulas by liquid chromatography.

Abstract: A simplified, simultaneous determination of vitamins B 1 , B 2 , B 3 , and B 6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified methanol-water mobile phase. The dioctylsulfosuccinate counter-ion facilitates unique retention of the pyridine-based vitamins (niacinamide and pyridoxine) and allows for concurrent measurement of both the pyridoxal and riboflavin 5'-phosphate endogenous components of milk. Other naturally occurring undetected vitamin congeners have minimal analytical significance. UV detection is used for niacinamide, and programmed fluorescence detection is used for riboflavin and the B 6 vitamins. Thiamine is routinely determined sequentially under modified elution conditions.

Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan, Scorpion, and SYBR Green I chemistries.

Abstract: A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.

Predictions for rapid methods and automation in food microbiology.

Abstract: A discussion is presented on the present status of rapid methods and automation in microbiology. Predictions are also presented for development in the following areas: viable cell counts; real-time monitoring of hygiene; polymerase chain reaction, ribotyping, and genetic tests in food laboratories; automated enzyme-linked immunosorbent assay and immunotests; rapid dipstick technology; biosensors for Hazard Analysis Critical Control Point programs; instant detection of target pathogens by computer-generated matrix; effective separation and concentration for rapid identification of target cells; microbiological alert systems in food packages; and rapid alert kits for detecting pathogens at home.

Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column.

Abstract: Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.