Production of certified reference materials for mycotoxins: IRMM’s view on the assessment of uncertainties

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M₁ (AfM₁) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.     

Evaluation of results derived from the analysis of certified reference materials – a user-friendly approach based on simplicity

Certified reference materials (CRMs) have now been in regular use for several decades. Their production and certification are regulated by international standards. But, even today there are no agreements on procedures for evaluating results obtained by the users. As a consequence, the way CRM results are treated in the literature leaves a lot to be desired. A statistical evaluation is rarely, if ever, described in published reports. The most common approach is to compare the found mean and/or range with the certified range and then state if the mean falls within the certified range, or if the two ranges overlap. If this happens, the analyst is usually satisfied. In addition, usually no regard is paid to the fact that the certified interval is based on a 95% confidence interval (CI) and the found interval on standard deviation and that this evaluation has little, if any, statistical relevance. Long-term evaluation of a CRM often consists in nothing more than producing a control chart, which relates the found results to the certified mean and CI. This paper is an attempt to improve the situation by providing a set of easy-to-use guidelines for evaluating results from CRMs. During the process we have identified different areas in which there is a need for such guidelines: 1.?short-term evaluation of a single, or multiple, determination at one or several specific times; 2.?identification of systematic and random errors; 3.?evaluation of CRMs when used in a collaborative trial of a method; and 4.?long-term evaluation for monitoring an analytical process over extended periods of time. It is important that the guidelines do not require expert competence in statistics from the analyst. Such obstacles would probably render most guidelines unused.     

The role of certified reference materials in ensuring the quality of spectrochemical measurements – the present status in Romania

 The objective of quality assurance programme for spectrochemical measurements is to reduce the measurement errors to accepted limits. Reference materials are being widely used as measurement standards in the fields of industrial production, environmental protection and clinical chemistry, and are playing an important role in ensuring the quality of measurement results. This paper presents some aspects, practices and examples of the activity of the Reference Materials Laboratory of the National Institute of Metrology, Bucharest, in the field of spectrochemical measurements. An attempt to describe the role and use of reliable certified reference materials to ensure the quality of spectrochemical measurements is presented. A short review of the locally available certified reference materials used in spectrochemical measurements is given. The use of reference materials data in estimating the measurement uncertainty is discussed. An interlaboratory comparison, recently organized in Romania, is also presented as a useful response to the need for quality assurance of spectrochemical results. Received: 20 March 1999 / Accepted: 25 February 2000     

Estimation of the homogeneity of reference materials by SS–ETAAS and use of the "tape-sandwich" sample-introduction technique

The so called "tape sandwich", a novel sample-introduction technique developed for SS-ETAAS, has been employed in a study of the microheterogeneity of some biological CRM. Pb, Cd, and Cr were measured by use of a laboratory-assembled instrument with graphite-cup atomizer. Aqueous standards were found satisfactory for calibration. The average mass of sub-samples atomised was in the range 0.250-0.660 mg+/-30%. The relative sampling error found for the CRM investigated varied from 15-34%. Calculated relative homogeneity constants of the order of 4-21 mg(1/2) were obtained, and were reduced considerably by reduction of the particle size. The minimum sample sizes representative of the CRM were, with one exception, much lower (11-75 mg) than those recommended in the documentation accompanying the standards (100-200 mg). In a Chinese hair reference sample Cr data were normally distributed but the presence of nuggets was observed for Cd and Pb; this was associated with severe radial and longitudinal gradients of these elements in hair.     

Matrix reference materials – provision in Australia

 Until quite recently, Australia has not been much involved in the preparation and certification of matrix reference materials for chemical testing. Even today, the vast bulk of chemical reference materials used in Australia are imported from other world producers. Increased international focus on the accuracy, traceability and comparability of chemical measurements has led to the establishment of the National Analytical Reference Laboratory (NARL) within the Australian Government Analytical Laboratories. Part of the work of NARL will be to supply matrix reference materials, not available from existing sources, to meet specific Australian requirements. This need has been addressed in the past by a combination of industry and government initiatives. Examples include a series of certified matrix reference materials for chemical testing of iron ore, coal and mineral sands produced by Standards Australia and a series of three animal fat matrix reference materials certified for a range of pesticide residues produced by the Australian Chemical Standards Laboratory (now part of NARL). To make effective use of limited resources, it will be important for NARL to focus on identified priorities and to maximise the use of available Australian resources and expertise through technical collaboration for reference material production. An important part of this process should be input on needs and priorities from reference material "users" such as government legislators, regulatory authorities, standards setting bodies, industry and the analytical community. The aim will be to produce matrix certified reference materials that are traceable to SI or other international standards at a stated level of measurement uncertainty.     

Assessment of commutability for candidate certified reference material ERM-BB130 “chloramphenicol in pork”

Chloramphenicol (CAP), an effective antibiotic against many microorganisms, is meanwhile banned in the EU for treatment of food-producing animals due to adverse health effects. The Institute for Reference Materials and Measurements (IRMM) is currently developing a certified reference material (CRM) for CAP in pork, intended for validation and method performance verifications of analytical methods. The material will be certified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methods and has a target CAP level around the minimum required performance limit (MRPL) of 0.3 μg/kg. To prove that the material can be applied as a quality control tool for screening methods, a commutability study was conducted, involving five commercially available enzyme-linked immunosorbent assay kits and one biosensor assay (BiaCore kit). Meat homogenates (cryo-milled wet tissue) with CAP concentrations around the MRPL and the candidate CRM (lyophilised powder) were measured by LC–MS/MS and GC–MS as well as the six screening methods. Pairwise method comparisons of results obtained for the two sample types showed that the CRM can successfully be applied as quality control (QC) sample to all six screening methods. The study suggests that ERM-BB130 is sufficiently commutable with the investigated assays and that laboratories applying one of the investigated kits therefore benefit from using ERM-BB130 to demonstrate the correctness of their results. However, differences among the assays were observed, either in the abundance of bias between screening and confirmatory LC and GC methods, the repeatability of test results, or goodness of fit between the methods.     

What’s new with the NRC certified reference material program

The environmental CRMs currently available from NRC Ottawa comprise four natural waters for trace elements, four biological tissues for trace elements and methylmercury, three marine sediments for major and trace elements (one sediment has certified values for butyltins) and one fish tissue for organochlorines (PCBs, dioxins). Although significant effort has been expended to maintain this suite of materials, several new initiatives have also recently been completed or are in progress. The certified value for arsenobetaine in Dogfish Muscle CRM DORM-2 has been established. The certification approaches, as well as the studies to determine extraction efficiency of this organoarsenic compound, are presented. A stability study has been completed for Hg in a natural water sample and plans are underway for production of a CRM. Assessment of micronutrient stability in seawater for the purposes of CRM production is continuing and progress on this initiative is discussed.     

Certified reference materials in analytical chemistry – A century of NIST contribution

Over the course of its first 100 years, the National Institute of Standards and Technology (NIST) has made numerous contributions to advancing the science and practice of analytical chemistry. Contributions to fundamental constants and reference data, such as determination of the Faraday, Avagadro’s number, and atomic masses, began at almost the beginning of the new institution when it was formed in 1901. Instrumentation development, improvement, and reproducible methods for its use have also been an important part of the NIST effort. This paper will describe what may be the organization’s most important and certainly its most unique contribution; namely, certified reference materials. Ultimately these certified reference materials would become known at NIST as standard reference materials (SRMs). It is a contribution that now has been mirrored around the world with reference materials being certified in at least 25 countries and routinely applied in more than twice that number. The result has been more accurate analyses of materials that impact our safety, health, and well-being. Received: 22 November 2000 Accepted: 22 November 2000     

Updated estimates of the selenomethionine content of NIST wheat reference materials by GC–IDMS

Updated estimates of the selenomethionine content of four NIST wheat reference materials have been obtained by use of a revised gas chromatography–stable-isotope dilution mass spectrometric method. The revised method makes use of digestion with methanesulfonic acid, which enables more complete recovery of endogenous selenomethionine than was previously achieved by overnight denaturing treatment in 0.1 mol L⁻¹ HCl. The NIST wheat reference materials each contain approximately 55% of their total Se content as selenomethionine. Information about forms of Se in reference materials adds value to these materials in Se speciation studies. Estimates of selenomethionine content are also provided for other wheat samples, including several grown under conditions of exposure to high Se levels. These samples also contain approximately 55% of their total Se content as selenomethionine. The consistent level of 55% of total selenium occurring in the form of selenomethionine when the total selenium content varies by a factor of 500 is suggestive of an active mechanism of incorporation of selenium into wheat grain. Figure Selenomethionine content of wheat samples     

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods

The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg⁻¹ are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.     

Development of a certified reference material for cachaça: an effective material for quality assurance

Cachaça is the typical and genuine denomination for the sugar cane beverage produced in Brazil. It has an alcoholic content between 38 and 48 % by volume, at 20 °C, and is obtained from the distillation of fermented sugar cane juice with the possible addition of up to 6 g/L of sugar, expressed by sucrose. This paper aims to outline the approach taken for the certification of the mass fraction of individual alcohols and their associated uncertainties in a cachaça certified reference material (CRM) produced by the Organic Analysis Laboratory of the Chemical Metrology Division of Inmetro. The requirements of ISO Guide 31, ISO Guide 34 and ISO Guide 35 were followed for the certification. This included the assessment of the degree of homogeneity of the material, the short-term stability study to determine the transport conditions, the long-term stability study to establish the shelf life and storage conditions of the CRM and the characterization process. The certified values were determined by two analytical techniques: gas chromatography coupled to mass spectrometry and gas chromatography with flame ionization detection. In order to support the routine testing of cachaça, in helping analytical laboratories validate and standardize their methods, the certified reference material (CRM 02.1/10.003b) was developed and certified for the mass fractions of methanol (9.18 mg/100 g ± 0.82 mg/100 g), 2-butanol (4.15 mg/100 g ± 0.40 mg/100 g), 1-butanol (1.30 mg/100 g ± 0.18 mg/100 g), isobutanol (24.0 mg/100 g ± 1.0 mg/100 g) and 1-propanol (29.4 mg/100 g ± 1.3 mg/100 g) in a spiked cachaça.     

Harmonized protocol and certified reference material for the determination of extractable contents of phosphorus in freshwater sediments – A synthesis of recent works

An analytical protocol for the determination of the extractable phosphorus contents in freshwater sediments has been harmonized through interlaboratory studies in the frame of the Standards Measurements and Testing Program of the European Commission. A homogeneous and stable sediment reference material has been prepared and certified on the basis of this protocol named SMT protocol, and will be available in spring of 2001. The SMT protocol, together with the reference material, are useful tools in the field of water management, especially at a time when quality assurance and data comparability are of paramount importance in laboratory analysis. The knowledge of the bioavailable forms of phosphorus is important not only for sediments but also for sludge and soils. Therefore, the SMT protocol could be extended to these materials and new CRMs could be prepared. The SMT protocol was used in a study of a reservoir, which allowed to calculate the P stock, therefore helping to predict the restoration delay of the lake. The paper describes the protocol and the CRM, and gives a brief outline of the case study. Received: 14 November 2000 / Revised: 25 January 2001 / Accepted: 31 January 2001     

Recent developments in Australia — reference materials and proficiency testing

The Australian Government Analytical Laboratories (AGAL) has historically been the major provider of chemical and micro biological analytical services to the Australian Government. An equally important role has been the provision of specialist functions associated with:

?development and implementation of new and improved testing methods;

?evaluation and commissioning of new instrumentation;

?advice and assistance to government with the design, implementation and conduct of proficiency testing programs, particularly in areas of chemical testing related to international trade.

In July 1993, in response to increasing international interest in chemical metrology, AGAL established a Valid Analytical Measurement program focused on improving the acceptability of laboratory data and supporting the development of appropriate standards through the National Standards Commission. AGAL has recently surveyed Australian residue testing laboratories to determine their priority requirements for reference materials and inter laboratory check sample programs. Survey results have clearly demonstrated a need for reasonably priced, certified reference materials for pesticide and antibiotic residue testing in raw agricultural produce. There are few reference materials available in this field of testing, particularly for residues of concern in Australia. AGAL has therefore initiated action as follows:

?Preparation and certification of two priority reference materials

1. organochlorines (dieldrin and heptachlor epoxide) in animal fat,
2. organophosphates (diazinon, chlorpyrifos and ethion) in animal fat.

?Investigation of storage stability of animal liver samples containing avermectin, sulphonamide and other antibiotic residues to ascertain suitability for reference material production.

In the area of proficiency testing, AGAL provides ongoing services (in close consultation with NATA, the National Association of Testing Authorities) to the Australian export meat industry. AGAL is seeking to expand this role to involve a wider range of laboratories engaged in residue testing of both export and domestic produce. The Australian Government believes it has an important role to play, particularly in the South East Asian and Pacific Rim regions, in co-operation, collaboration, assistance with training, and provision of specialist technical services.