Paving the way for new and challenging matrix reference materials-particle suspensions at the core of material processing providing RMs for method development and method validation.

Sufficient homogeneity of the certified parameter(s) over the whole fill series of a matrix reference material (RM) is a fundamental quality criterion. In practice, the heterogeneity of the target parameter is evaluated, whereby a relative value can be calculated of how much the target parameter is varying over the RM-batch. A high degree of homogeneity (low heterogeneity) is an inherent quality mark of a good RM. Here, we report how challenging matrix RMs were produced by using particle suspensions at the core of the material processing step. The examples of matrix RMs produced span from whole water reference materials for persistent organic pollutants, PM2.5-like atmospheric dust certified for specific ions to microplastic RMs. Most of these RMs were subsequently used in different phases of analytical method development or for method validation. Common to all these matrices is that they cannot be easily mixed, handled, or dosed to prepare larger sample batches. In all cases, a continuously stirred suspension of particles was used during material processing. In general, relative between-bottle heterogeneities from 1.6 to 6% were achieved for the target parameters in these matrix presentations. Concerning developments of new CRMs in emerging fields, the co-dependence between the availability of validated analytical methods with good repeatability and testing materials with a known and high homogeneity of the target parameter(s) becomes particularly challenging. This situation is an RM/Method causality dilemma. To overcome that hurdle, strategies are proposed for stepwise processes where RM producers and a network of analytical method developers could work hand in hand. In addition, development of a portfolio of inexpensive and well-homogenised common samples coupled with a reporting interface is suggested. This would benefit method developers and RM producers alike. As more and more data is compiled for a specific matrix, it paves the way for new and challenging RMs that can later be used by a wider community.

Feasibility of using quantitative 1H-NMR spectroscopy and ultra-microbalances for investigation of a PET microplastic reference material.

Here, we report on the feasibility of using quantitative NMR and ultra-microbalances for additional measurements of the mass of poly-ethylene terephthalate (PET) particles in a reference material (RM). The microplastic (MP) PET particles were immobilised in solid NaCl following freeze-drying of a 1-ml NaCl suspension. The particles ranged from 30 to about 200 µm (Feretmin). In a 3-day process, more than 500 such units of PET particles in the NaCl carrier were prepared and later used in a large-scale inter-laboratory comparison. The homogeneity of PET in the salt carrier over these 500 units had previously been evaluated with respect to the mass of PET using an ultra-microbalance. In addition to the original results obtained by weighing, two independent results of quantitative 1H-NMR have been obtained for further investigation of this reference material together with one additional set of weighing data. The NMR data were used for confirmation of the weighed amount of PET (as weighing is non-specific for PET). Average masses of 0.293 ± 0.04 mg and 0.286 ± 0.03 mg of PET were obtained using two different ultra-microbalances (14% RSD for n = 14 and 9% RSD for n = 4, respectively). The corresponding 1H-NMR data was 0.300 ± 0.02 mg of PET (6.7% RSD for n = 5) and 0.345 ± 0.04 mg of PET (12.5% RSD for n = 14), respectively. The average mass of PET obtained by 1H-NMR measurements was in agreement with the weighed amounts within their standard deviations. A mean value of 0.306 mg PET with an expanded uncertainty of 0.058 mg (± 19% relative) was calculated, and it is traceable to the SI system of measurements. Measurement of PET by quantitative 1H-NMR spectroscopy is also reported for a water sample. The PET contained in one RM sample was transferred to 1 L of water to mimic a drinking water sample for microplastics.

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept.

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely 'mass of total allergen protein per mass of food'. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors. Graphical abstract.

Derivatization of bisphenol A and its analogues with pyridine-3-sulfonyl chloride: multivariate optimization and fragmentation patterns by liquid chromatography/Orbitrap mass spectrometry.

RATIONALE: Due to the growing restrictions on the use of bisphenol A (BPA), several other bisphenols are gaining importance as substitutes for BPA in a variety of applications. There is, therefore, a real need for selective and sensitive methods based on mass spectrometry which will allow the human exposure to these new bisphenols to be assessed. METHODS: Derivatization of BPA and its substitutes with pyridine-3-sulfonyl chloride is used to enhance the detection capability of bisphenols by electrospray ionization mass spectrometry. A multivariate experimental design, Box-Behnken response surface, was used to evaluate the influence of the main variables potentially affecting the derivatization yield. Fragmentation patterns for all the derivatized bisphenols were acquired by high-resolution/accurate-mass Orbitrap mass spectrometry. RESULTS: Temperature and pH were identified as the most important factors affecting the derivatization yield of bisphenols. Fragmentation of the protonated molecules produced abundant analyte-specific product ions. Most of the derivatized bisphenols showed significant improvements in their signal-to-noise ratios compared with the underivatized forms. The stability of these derivatives was demonstrated through several freeze/thaw cycles, short-term room temperature and long-term cold storage. CONCLUSIONS: Derivatization of BPA and its structural analogues with pyridine-3-sulfonyl chloride is proposed as a specific, sensitive, high-throughput approach to their analysis by liquid chromatography coupled to electrospray ionization mass spectrometry.

Two-dimensional heart-cut LC-LC improves accuracy of exact-matching double isotope dilution mass spectrometry measurements of aflatoxin B1 in cereal-based baby food, maize, and maize-based feed.

Aflatoxins, mycotoxins of fungi of the Aspergillus sp., pose a risk to consumer health and are, therefore, regulated by more than 100 countries. To facilitate method development and validation as well as assessment of measurement capabilities, availability of certified reference materials and proficiency testing schemes is important. For these purposes, highly accurate determinations of the aflatoxin content in the materials used are necessary. We describe here the use of two-dimensional heart-cut LC-LC in combination with exact-matching double isotope dilution mass spectrometry to determine the content of aflatoxin B1 in three materials used in a proficiency testing scheme. The serious reduction in ionization suppression afforded by the two-dimensional heart-cut LC-LC had a positive effect on the precision of the measured isotope ratios of the exact-matching double isotope dilution mass spectrometry. This is evidenced by the expanded measurement uncertainty (k=2) of 0.017 µg/kg or 8.9 % relative to a mass fraction of aflatoxin B1 in a cereal-based baby food of 0.197 µg/kg. This value is in perfect agreement with the consensus value of this material from a proficiency test (PT) scheme for National Reference Laboratories executed by the European Reference Laboratory for Mycotoxins. The effort necessary to perform the described methodology precludes its frequent use but for specific applications we see it as a valuable tool.

Assignment of a Reference Value of Total Cow's Milk Protein Content in Baked Cookies Used in an Interlaboratory Comparison.

Interlaboratory comparisons (ILC) in the food allergens field mainly rely on the use of consensus values per applied methodology or even per type of an ELISA test kit. Results suggest good reproducibility; however, possible biases may not be recognized since metrological traceability to an independent reference is lacking. The work presented here utilizes isotope dilution mass spectrometry (IDMS) to assign a reference value of the total cow's milk protein (TCMP) content in a baked cookie and its associated uncertainty. TCMP consists of several individual proteins, of which five (representing 92%) served us as markers for TCMP. Per marker, one to four proteotypic peptides were selected for the quantification. These were synthesized, and the mass fractions of respective reference solutions were determined with peptide-impurity-corrected amino acid analysis to establish traceability to SI units. Stable isotope labelled ("heavy") analogues of the proteotypic peptides were also synthesized and blended with extracts of the test material or the reference solutions for IDMS. Through careful measurement design minimizing biases, well-defined model equations were developed, allowing appropriate estimation of the associated uncertainty. The determined reference value of 11.8 ± 1.1 mg TCMP/kg cookie was used for scoring of a novel ILC.

Capabilities of laboratories to determine melamine in food-results of an international proficiency test.

A proficiency test to assess the capabilities of laboratories to determine melamine in a milk powder and a baking mix, representing starch-containing foods like bread and biscuits, was carried out in January 2009. The need for such an interlaboratory comparison arose from a health scare in China about melamine-tainted powdered milk in the second half of 2008. Laboratories in 31 countries, including Australia, China, India, Japan, New Zealand and the USA, and 21 of the 27 Member States of the European Union participated and reported back 114 results for the milk powder and 112 for the baking mix test materials. The reported results were compared to reference values determined by exact-matching double isotope dilution mass spectrometry. The so-determined assigned values were 10.0 +/- 0.6 mg/kg melamine in the milk powder and 3.18 +/- 0.17 mg/kg melamine in the baking mix. A coverage factor k of 2 was applied to calculate the expanded uncertainties. Three quarters of all reported results for both materials had associated z scores which were satisfactory (z

A Greener, Quick and Comprehensive Extraction Approach for LC-MS of Multiple Mycotoxins.

In food/feed control, mycotoxin analysis is often still performed "one analyte at a time". Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, forgoing sample extract clean-up, and minimizing solvent consumption. For this, 2 g of test material were suspended in 8 mL water and 16 mL ethyl acetate were added. Extraction was accelerated through sonication for 30 min and subsequent addition of 8 g sodium sulfate. After centrifugation, 500 µL supernatant were spiked with isotopologues, dried down, reconstituted in mobile phase, and measured with LC-MS. The method was validated in-house and through a collaborative study and the performance was fit-for-purpose. Repeatability relative standard deviation (RSDs) between 16% at low and 4% at higher contaminations were obtained. The reproducibility RSDs were mostly between 12% and 32%. The trueness of results for T-2 toxin and Zearalenone were not different from 100%, for Deoxynivalenol and HT-2 toxin they were larger than 89%. The extraction was also adapted to a quick screening of Aflatoxin B1 in maize by flow-injection-mass spectrometry. Semi-quantitative results were obtained through standard addition and scan-based ion ratio calculations. The method proved to be a viable greener and quicker alternative to existing methods.

Comparative evaluation of methods for the detection of 2-alkylcyclobutanones as indicators for irradiation treatment of cashew nuts and nutmeg.

Irradiation of food products and ingredients must be indicated by proper labeling. This study evaluated the appropriateness of the European Standard EN 1785:2003 for the detection of 2-alkylcyclobutanones, which are radiolysis products of fatty acids, in cashew nuts and nutmeg and confirmed its suitability to detect irradiation of cashew nut samples at average absorbed doses of 1 kGy and above. An alternative method was developed, which is based on matrix solid phase dispersion and subsequent separation and detection of oxime derivatives of 2-alkylcyclobutanones by high performance-high resolution mass spectrometry. It is more rapid, less resource consuming, and more sensitive than EN 1785:2003. This method allowed detection of 2-alkylcyclobutanones in cashew nuts irradiated at 100 Gray and in nutmeg irradiated at 400 Gray. None of the 26 cashew nut and 14 nutmeg samples purchased in different EU Member States contained traces of 2-alkylcyclobutanones.

Detection of recombinant human erythropoietin in urine by isoelectric focusing.

BACKGROUND: Doping with erythropoietic proteins such as recombinant human erythropoietin (rHuEPO) and darbepoetin alfa is a serious issue in sport. There is little information on the time course of detection of rHuEPO in urine and on methods to evaluate electrophoresis-based data. METHODS: We used a recently described isoelectric focusing method for detecting rHuEPO and endogenous EPO in urine obtained from individuals treated with placebo or epoetin alfa. The latter was administered subcutaneously at 50 IU/kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. Blood and urine samples were collected during the morning of study days -3, 0, 2, 4, 7, 9, 11, 14, 16, and 18 and on days 2, 3, 4, and 7 postadministration. We developed visual and numerical (two-band ratio) techniques to evaluate the electropherograms for the presence of rHuEPO. RESULTS: Compared with the placebo group, the epoetin alfa-treated group responded with increases in hematocrit, reticulocytes, macrocytes, serum EPO, and serum soluble transferrin receptor. The electropherograms showed that the pattern of bands arising from urinary rHuEPO is different from that of endogenous urinary EPO. Both the two-band ratio and the visual technique detected rHuEPO in all 14 epoetin alfa-treated individuals 3 days after the last dose. On the 7th day after the last dose, both techniques detected rHuEPO in approximately one-half of the participants. rHuEPO was not detected in the placebo-treated individuals. CONCLUSIONS: The isoelectric focusing method detects rHuEPO in most urine samples collected 3 days after nine doses of epoetin alfa. The numerical two-band ratio was equivalent to a visual method for detecting rHuEPO in urine.