Analytical Determination of Aflatoxin in Ground Corn Check Samples Completed by Multiple Laboratories over Several Years.

Mycotoxins are toxic molecules produced by multiple fungal species, including Aspergillus and Fusarium. Fungal infection of crops can result in mycotoxins entering the animal and human food supply. Enzyme-linked immunosorbent assays and other immunological assays have been developed to detect mycotoxins in foods. To calibrate the response of those methods, reference materials with known amounts of homogeneously dispersed mycotoxins are often utilized, where the mycotoxin concentrations have been determined using high-performance liquid chromatography coupled with absorbance or fluorescence detection methods, or high-performance liquid chromatography coupled with mass spectrometry detection methods. Therefore, it is important that the analytical methods provide accurate and precise quantitation of mycotoxins. The reference materials must also contain homogeneously dispersed known quantities of mycotoxin. To evaluate the accuracy and precision of mycotoxin reference materials and the analytical methods, quantitative results from multiple laboratories were completed each year for several years on ground corn check samples containing known levels of mycotoxins. Results for the quantitation of aflatoxin-containing corn reference samples are presented in this article.

An interlaboratory study on the detection method for Escherichia albertii in food using real time PCR assay and selective agars.

Escherichia albertii is an emerging enteropathogen. Although E. albertii-specific detection and isolation methods have been developed, their efficiency on food samples have not yet been systematically studied. To establish a series of effective methods for detecting E. albertii in food, an interlaboratory study was conducted in 11 laboratories using enrichment with modified E. coli broth supplemented with cefixime and tellurite (CT-mEC), real-time PCR assay, and plating on four kinds of selective agars. This study focused on the detection efficiency of an E. albertii-specific real-time PCR assay (EA-rtPCR) and plating on deoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), DHL supplemented with rhamnose and xylose (RX-DHL), and MAC supplemented with rhamnose and xylose (RX-MAC). Chicken and bean sprout samples were inoculated with E. albertii either at 17.7 CFU/25 g (low inoculation level) or 88.5 CFU/25 g (high inoculation level), and uninoculated samples were used as controls. The sensitivity of EA-rtPCR was 1.000 for chicken and bean sprout samples inoculated with E. albertii at low and high inoculation levels. The Ct values of bean sprout samples were higher than those of the chicken samples. Analysis of microbial distribution by 16S rRNA gene amplicon sequencing in enriched cultures of bean sprout samples showed that approximately >96 % of the population comprised unidentified genus of family Enterobacteriaceae and genus Acinetobacter in samples which E. albertii was not isolated. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a high inoculation level of E. albertii was 1.000 and 0.848-0.970, respectively. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a low inoculation level of E. albertii was 0.939-1.000 and 0.515-0.727, respectively. The E. albertii-positive rate in all colonies isolated in this study was 89-90 % in RX-DHL and RX-MAC, and 64 and 44 % in DHL and MAC, respectively. Therefore, the sensitivity of RX-supplemented agar was higher than that of the agars without these sugars. Using a combination of enrichment in CT-mEC and E. albertii isolation on selective agars supplemented with RX, E. albertii at an inoculation level of over 17.5 CFU/25 g of food was detected with a sensitivity of 1.000 and 0.667-0.727 in chicken and bean sprouts, respectively. Therefore, screening for E. albertii-specific genes using EA-rtPCR followed by isolation with RX-DHL or RX-MAC is an efficient method for E. albertii detection in food.

Erythropoietin N-glycosylation of Therapeutic Formulations Quantified and Characterized: An Interlab Comparability Study of High-Throughput Methods.

Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO N-glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO N-glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique N-glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric N-glycans, i.e., N-acetyllactosamine repeats and O-acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX.

Do GC/MS methods available for furan and alkylfurans in food provide comparable results? - An interlaboratory study and safety assessment of 2-pentylfuran in food.

A call for data on the occurrence of alkylfurans in food and feed from EFSA triggered the development of new methods to cover next to furan also 2- and 3-methylfuran, 2,5-dimethyl- and 2-ethylfuran as well as 2-pentylfuran. A significant variability was noticed when comparing analysis of 2-pentylfuran and furans in the same matrix performed by different laboratories. To assess the variability an interlaboratory study including eight laboratories was organised. The highest variabilities were observed when analysing cereals, with measurements of 2-pentylfuran indicating concentrations from 8 mg/kg up to more than 1000 mg/kg in the same sample. This study illustrates that the analysis of 2-pentylfuran requires special attention, and that additional method development would be necessary to ensure reliable and reproducible determination of 2-pentylfuran at contamination level. Moreover, a recent evaluation of the EFSA Panel on Food Additives and Flavourings indicates that concerns for genotoxicity, reason why it was grouped with the shorter alkylfurans, are now ruled out. We question the need and justification to include 2-pentylfuran in the analytical method as requested by EFSA, from both the analytical and the safety perspective.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Benchtop nuclear magnetic resonance performance evaluation according to ASTM E691-22 on a population of instruments: Molar substitution determination in hydroxypropyl betadex as a case study for use in quality control environments.

The inclusion of quantitative nuclear magnetic resonance (qNMR) spectroscopy in industry has historically been stifled by a lack of accessibility, caused in-part by the large costs of traditional high-field spectrometers, the maintenance required for these, and the expertise necessary to manage and use them. In recent years, the emergence of benchtop NMR technology, an accessible, affordable, and automatable alternative, has led to a more feasible incorporation of NMR into quality control spaces, an area traditionally reserved for other techniques such as gas chromatography and liquid chromatography, which are routinely combined with detection techniques such as mass spectrometry. While these techniques are commonly used in analyzer-type applications using gold standard methods of analysis, wherein an instrument is dedicated to performing specific assays, this remains uncommon for NMR. Herein, we perform a full method verification using benchtop qNMR on a population of benchtop NMR instruments according to the ASTM designation E691-22, a standard used to determine the precision of a test method. To our knowledge, this is the first published example of this type of study for benchtop NMR spectroscopy. For this work, a total of five analysts performed assays on 23 different benchtop NMR instruments for the analysis of hydroxypropyl betadex according to the USP-NF method, and the results are compared using a variety of statistical methods. The results of this work demonstrate that benchtop NMR technology is effective and robust under repeatability and reproducibility conditions and is a powerful tool for these types of routine quality control analyses.

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens.

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.

Toxic or not toxic? The specifications of the standard ISO 10993-5 are not explicit enough to yield comparable results in the cytotoxicity assessment of an identical medical device.

Background: Medical device manufacturers are obliged to prove the biocompatibility of their products when they come into contact with the human body. The requirements for the biological evaluation of medical devices are specified by the international standard series ISO 10993. Part five of this series describes the performance of in vitro cytotoxicity tests. This test evaluates the effects of medical device use on cell health. The existence of the specific standard suggests that the tests will produce reliable and comparable results. However, the ISO 10993-5 offers wide latitude in the test specifications. In the past, we noticed inconsistencies of the results from different laboratories. Objective: To determine if the specifications of the standard ISO 10993-5 are explicit to ensure the comparability of test results and, if not, identify potential influencing factors. Methods: An interlaboratory comparison was conducted for the in vitro cytotoxicity test according to ISO 10993-5. Fifty-two international laboratories evaluated the cytotoxicity for two unknown samples. One was polyethylene (PE) tubing, which is expected to be non-cytotoxic and the other was polyvinyl chloride (PVC) tubing, for which a cytotoxic potential was presumed. All laboratories were asked to perform an elution test with predefined extraction specifications. The other test parameters were freely chosen by the laboratories according to the guidelines set by the standard. Results: To our surprise only 58 percent of the participating laboratories identified the cytotoxic potential of both materials as expected. Particularly for PVC a considerable variation of the results between the laboratories was observed [mean = 43 ± 30 (SD), min = 0, max = 100]. We showed that ten percent serum supplementation to the extraction medium, as well as longer incubation of the cells with the extract, greatly increased the test sensitivity for PVC. Conclusion: The results clearly show that the specifications set by the ISO 10993-5 are not explicit enough to obtain comparable results for an identical medical device. To set requirements that ensure reliable cytotoxicity assessments, further research will be necessary to identify the best test conditions for specific materials and/or devices and the standard needs to be revised accordingly.

An interlaboratory study to evaluate the utility of gas chromatography-mass spectrometry and gas chromatography-infrared spectroscopy spectral libraries in the forensic analysis of fentanyl-related substances.

Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.

Variation in assessments of suitability and number of contributors for DNA mixtures.

The interpretation of a DNA mixture (a sample that contains DNA from two or more people) depends on a laboratory/analyst's assessment of the suitability of the sample for comparison/analysis, and an assessment of the number of contributors (NoC) present in the sample. In this study, 134 participants from 67 forensic laboratories provided a total of 2272 assessments of 29 DNA mixtures (provided as electropherograms). The laboratories' responses were evaluated in terms of the variability of suitability assessments, and the accuracy and variability of NoC assessments. Policies and procedures related to suitability and NoC varied notably among labs. We observed notable variation in whether labs would assess a given mixture as suitable or not, predominantly due to differences in lab policies: if two labs following their standard operating procedures (SOPs) were given the same mixture, they agreed on whether the mixture was suitable for comparison 66% of the time. Differences in suitability assessments have a direct effect on variability in interpretations among labs, since mixtures assessed as not suitable would not result in reported interpretations. For labs following their SOPs, 79% of assessments of NoC were correct. When two different labs provided NoC responses, 63% of the time both labs were correct, and 7% of the time both labs were incorrect. Incorrect NoC assessments have been shown to affect statistical analyses in some cases, but do not necessarily imply inaccurate interpretations or conclusions. Most incorrect NoC estimates were overestimates, which previous research has shown have less of an effect on likelihood ratios than underestimates.

Combining estimators in interlaboratory studies and meta-analyses.

Many statistical methods (estimators) are available for estimating the consensus value (or average effect) and heterogeneity variance in interlaboratory studies or meta-analyses. These estimators are all valid because they are developed from or supported by certain statistical principles. However, no estimator can be perfect and must have error or uncertainty (known as estimator uncertainty). For a given dataset, the consensus value and heterogeneity variance given by different estimators can often differ significantly. Consequently, the choice of different estimators can affect the conclusion of an interlaboratory study or meta-analysis. However, there is no universally accepted metric for determining which estimator is optimal among a set of candidate estimators. Instead of selecting and using a single estimator, this paper proposes an estimator-averaging approach to combine a set of individual estimators. The final averaged estimator is a linear combination of individual estimators, which accounts for three sources of uncertainties including the estimator uncertainty. Monte Carlo simulations were performed to examine the long-run performance of four individual estimators and the proposed averaged estimators. A case study: the determination of the Newtonian constant of gravitation is presented, where 10 individual estimators (eight frequentist weighted average methods and two Bayesian methods) are combined using the proposed estimator-averaging approach.

Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody.

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.

Validation of PCR methods for confirmation and species identification of thermotolerant Campylobacter as part of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp.

This article describes the outline and organisation of the validation of three multiplex PCR methods for species identification and/or confirmation of thermotolerant Campylobacter spp. The three PCR methods were validated against the reference method described in the EN ISO standard 10272:2017. The results of the PCR methods were compared against the reference method in a method comparison study and an interlaboratory study based on EN ISO 16140-6:2019. The performance, in terms of inclusivity and exclusivity, of each of the eight PCR targets were comparable to the performance of the reference method: close, equal, or better depending on the target. In total, all three PCR methods were concluded to be equally qualified as the reference method for molecular identification and/or confirmation of thermotolerant Campylobacter spp., C. jejuni, C. coli and C. lari isolated from the food chain and have been included in Amendment 1 of ISO 10272:2017.

Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line.

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1-3.4% and interlaboratory CVs of 1-4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics.

Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development.

Global interlaboratory assessments on PCBs, organochlorine pesticides and brominated flame retardants in various environmental matrices 2017/2019.

In support of the United Nations Environment Programme (UNEP) global monitoring plan under the Stockholm Convention contributing laboratories were offered to take part in a series of interlaboratory assessments on persistent organic pollutants (POPs). The results of two rounds of these assessments are reported. The target compounds were polychlorinated biphenyls, organochlorine pesticides, polybrominated diphenylethers, one polybrominated biphenyl and hexabromocyclododecane diastereomers. The matrices distributed were a test solution, fish, sediment, human milk, and air extracts. The number of participants in each round was well over 100, showing the interest of laboratories worldwide. The results showed that many laboratories still struggle to obtain acceptable standard deviations of around 25% for their determinations. In particular for organochlorine pesticides serious improvement in quality is required. Acceptable results were obtained for the air extracts and for the determination of polybrominated diphenylethers in various matrices.

An interlaboratory study evaluating the interpretation of forensic glass evidence using refractive index measurements and elemental composition.

Seventeen laboratories participated in three interlaboratory exercises to assess the performance of refractive index, micro X-ray Fluorescence Spectroscopy (µXRF), and Laser Induced Breakdown Spectroscopy (LIBS) data for the forensic comparison of glass samples. Glass fragments from automotive windshields were distributed to the participating labs as blind samples and participants were asked to compare the glass samples (known vs. questioned) and report their findings as they would in casework. For samples that originated from the same source, the overall correct association rate was greater than 92% for each of the three techniques (refractive index, µXRF, and LIBS). For samples that originated from different vehicles, an overall correct exclusion rate of 82%, 96%, and 87% was observed for refractive index, µXRF, and LIBS, respectively. Special attention was given to the reporting language used by practitioners as well as the use of verbal scales and/or databases to assign a significance to the evidence. Wide variations in the reported conclusions exist between different laboratories, demonstrating a need for the standardization of the reporting language used by practitioners. Moreover, few labs used a verbal scale and/or a database to provide a weight to the evidence. It is recommended that forensic practitioners strive to incorporate the use of a verbal scale and/or a background database, if available, to provide a measure of significance to glass forensic evidence (i.e., the strength of an association or exclusion).

Effect of the manufacturing process on Fiore Sardo PDO cheese microstructure by multi-frequency NMR relaxometry.

The quality of Fiore Sardo cheese, a traditional Italian dairy product, was analyzed by means of Multi-frequency Nuclear Magnetic (NMR) relaxometry. Specifically, ten cheese wheels were purchased from different production chains, either industrial (N = 5) or artisanal (N = 5) samples. The former came from large scale productions and the latter were produced by shepherds in small quantities and in very small dairy factories. A preliminary interlaboratory proficiency testing of Time Domain - NMR (TD-NMR, 20 MHz) relaxometry by five laboratories, consistently showed that product quality is significantly different in terms of molecular mobility according to their production chain (i.e. industrial or artisanal). More detailed information about cheese microstructure was obtained by Multi-frequency Fast Field Cycling NMR (FFC-NMR) at lower magnetic fields (0.01-10 MHz). According to the interpretative model adopted to describe FFC-NMR data, industrially processed cheeses showed a higher para-casein hydration, higher protein protons to water protons ratio and a higher disorder (lower fractal dimension df) than artisanal products. It is suggested that differences between artisanal and industrial cheeses generate from the processing steps preceding cheese maturation, and are clearly reflected in the visual appearance of cheeses. This study shows that NMR relaxometry techniques can successfully discriminate Fiore Sardo cheese from different production chains, and paves the way for their implementation in quality control practices of dairy products.

Validation of a multi-analyte HPLC method for the determination of carotenoids used as feed additives in fish and poultry feed: results of an interlaboratory study.

The performance characteristics of a multi-analyte method for the determination of all 10 carotenoids authorised as feed additives within the EU were assessed via an interlaboratory study. The analytical method is based on reversed phase high performance liquid chromatography (RP-HPLC) coupled to an optical detector set at 410 nm. The analysis is particularly challenging due to the presence of various stereoisomers of each carotenoid, and the use of these compounds via natural or synthetic formulations, requiring a special sample preparation. EU regulations specifying the conditions of use set legal limits for these substances in compound feedingstuffs ranging from 6 mg kg-1 to 138 mg kg-1, depending on the individual carotenoid and the target animal for which the feed is supplemented with this carotenoid. The purpose of the multi-analyte method validated in this paper is to facilitate the monitoring of carotenoids at relevant levels when used as feed additives in compound feedingstuffs and pre-mixtures. The interlaboratory study delivered precision data for 43 different analyte/mass fraction/matrix combinations, covering a mass fraction range of the target analytes from 2.6 mg kg-1 to 3861 mg kg-1. The relative standard deviations for repeatability (RSDr) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSDR) varied from 6.8 to 39 % with a mean value of 21 %. Given the broad scope of the method covering 10 carotenoids added to compound feedingstuffs and pre-mixtures via different formulations, this multi-analyte method is considered fit for the intended purpose.

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup.

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.