[Inter-laboratory Study on the Modified Methods for Analyzing Bisphenol A Content for Migration Tests from Polycarbonate Food Apparatuses, Containers, and Packaging].

An inter-laboratory study involving 24 laboratories was conducted to validate the modified analytical method for the migration solution of heptane for the determination of bisphenol A migrating from polycarbonate food processing materials. In this study, two concentrations of samples were blindly coded. Each laboratory determined the analyte (bisphenol A, phenol and p-tert-butylphenol) concentration in each sample according to the established protocol. The obtained values were analyzed statistically using internationally accepted guidelines. Horwitz ratios were calculated based on the reproducibility relative standard deviation (RSDR), which was estimated from the inter-laboratory study, and predicted RSDR, which was calculated using the Horwitz/Thompson equation. Horwitz ratios of the two samples ranged from 0.15 to 0.37 for the three compounds, meeting the performance criteria of less than 2 set by the Codex Alimentarius for analytical method approval. These results showed that this modified analytical method shows good performance as an analytical method for the migration solution of heptane.

Correlating Sensory Assessment of Smoke-Tainted Wines with Inter-Laboratory Study Consensus Values for Volatile Phenols.

Vineyard exposure to wildfire smoke can taint grapes and wine. To understand the impact of this taint, it is imperative that the analytical methods used are accurate and precise. This study compared the variance across nine commercial and research laboratories following quantitative analysis of the same set of smoke-tainted wines. In parallel, correlations between the interlaboratory consensus values for smoke-taint markers and sensory analyses of the same smoke-tainted wines were evaluated. For free guaiacol, the mean accuracy was 94 ± 11% in model wine, while the free cresols and 4-methylguaiacol showed a negative bias and/or decreased precision relative to guaiacol. Similar trends were observed in smoke-tainted wines, with the cresols and glycosidically bound markers demonstrating high variance. Collectively, the interlaboratory results show that data from a single laboratory can be used quantitatively to understand smoke-taint. Results from different laboratories, however, should not be directly compared due to the high variance between study participants. Correlations between consensus compositional data and sensory evaluations suggest the risk of perceivable smoke-taint can be predicted from free cresol concentrations, overcoming limitations associated with the occurrence of some volatile phenols, guaiacol in particular, as natural constituents of some grape cultivars and of the oak used for barrel maturation.

[Inter-laboratory Study on the Modified Method Assessment for the Determining Methanol Content in Kitchen Detergents].

We modified a method for determining methanol content in detergents used in kitchens. Furthermore, an inter-laboratory study was conducted in 10 laboratories to validate the modified method. In this study, two concentrations of samples were blindly coded. Each laboratory determined the methanol content in each sample according to a protocol. The determined values were statistically analyzed according to an international harmonized guideline. HorRat values were calculated based on the reproducibility relative standard deviation (RSDR) which was estimated from the interlaboratory study, and predicted RSDR calculated from the Horwitz/Thompson equation. The HorRat values of the two samples were 0.8 and 1.8, meeting the performance criteria of less than 2 set by the Codex Alimentarius for analytical method approval. These results confirm that this modified analytical method shows good performance as an analytical method for determining methanol content in kitchen detergents.

[Validation Study on Migration Test for Plastic Food Utensils, Containers, and Packaging].

Migration test of food utensils, containers, and packaging is an important test method for confirming the safety and their compliance to the standards. However, there is little report on inter-laboratory study which was performed to evaluate the entire migration test, including migration operations and quantification. An interlaboratory study was performed participating 22 laboratories using 8 types of model synthetic resin samples containing 10 substances with a wide range of Log Pow values to evaluate the accuracy of the entire migration test. As a result, most of HorRat (r) values met the target criteria (0.3

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA.

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer's recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes.

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs. Graphical abstract.

[Inter-laboratory Study of an HPLC-UV Method for Free Asparagine in Grains].

In order to validate an HPLC-UV method for the determination of free asparagine, which is a precursor of acrylamide, in grains using dansyl derivatization, an inter-laboratory study was performed in 9 laboratories using 5 kinds of grains (non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour), which naturally contain free asparagine. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDr) were in the ranges of 0.5-2.2 and 2.3-5.9%, respectively. The HorRat values ranged from 0.4 to 0.6. These results were within the range of the procedural manual of the Codex Alimentarius Commission, and therefore the method is effective.

[Development of LC-MS and LC-MS/MS Methods for Free Asparagine in Grains].

New analytical methods for the determination of free asparagine （Asn）, which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5％ （w/v） aqueous trichloroacetic acid solution, appropriately diluted with 0.1％ （v/v） formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl （PFP） column using 0.1％ （v/v） formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 µg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9％, repeatability （RSD） ranged from 0.9 to 6.0％, and within-laboratory reproducibility （RSDwr） ranged from 2.8 to 7.1％. Limits of quantitation （LOQs） were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains （non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour）, which naturally contained free asparagine. The HORRATR values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.

Performance Characteristics of Mass Spectrometry-Based Analytical Procedures for Quantitation of Nitrosamines in Pharmaceuticals: Insights from an Inter-laboratory Study.

With the discovery of carcinogenic nitrosamine impurities in pharmaceuticals in 2018 and subsequent regulatory requirements for risk assessment for nitrosamine formation during pharmaceutical manufacturing processes, storage or from contaminated supply chains, effective testing of nitrosamines has become essential to ensure the quality of drug substances and products. Mass spectrometry has been widely applied to detect and quantify trace amounts of nitrosamines in pharmaceuticals. As part of an effort by regulatory authorities to assess the measurement variation in the determination of nitrosamines, an inter-laboratory study was performed by the laboratories from six regulatory agencies with each of the participants using their own analytical procedures to determine the amounts of nitrosamines in a set of identical samples. The results demonstrated that accurate and precise quantitation of trace level nitrosamines can be achieved across multiple analytical procedures and provided insight into the performance characteristics of mass spectrometry-based analytical procedures in terms of accuracy, repeatability and reproducibility.

Reliability in viscosity measurement of thickening agents for dysphagia management: Are results obtained by cone-and-plate rheometers reproducible between laboratories?

Viscosity measurement using a cone-and-plate rheometer is considered to provide an objective and reliable evaluation of thickening agents for dysphagia management. Here, we showed its measurement uncertainty in the context of an inter-laboratory study. Eight test samples (i.e., four viscosity standard liquids, one xanthan gum reagent powder, and three commercial thickening agent powders) were distributed to 10 laboratories in a blinded manner. According to the same standard operating procedure, each laboratory dissolved the xanthan gum or thickening agents at four concentrations (0.5-4.0 g/100 g) and then measured their viscosity (35-803 mPa∙s). As for the viscosity of the standard liquids, the grand means were 98-100% of the certified values, and the relative standard deviations for repeatability (RSDr ) and reproducibility (RSDR ) were ca. 1% and ca. 5%, respectively, suggesting good accuracy in the measurement process. On the other hand, as for the viscosity of the thickening agents, RSDr and RSDR were ca. 2-6% and ca. 5-8%, respectively; however, heterogeneity in the preparation process comprising a manual dissolving step may increase these to near 60%. Furthermore, RSDr and RSDR of estimated additive concentrations to achieve targeted viscosities (50-500 mPa∙s) based on concentration-viscosity curves were ca. 1-3% and ca. 3-5%, respectively, with a few exceptions. These findings suggest that a strictly standardized procedure provides reliable data on the viscosity measurements for thickening agents.

Statistefix 4.0: A novel probabilistic software tool.

Latest innovations indicate that continuous tools are promising DNA trace assessment methods. In this study, we present the continuous software solution Statistefix 4.0. The software supports DNA experts in deducing DNA profiles for database queries and can help to preselect DNA samples suitable for further processing using advanced probabilistic search engines. The novel tool weights genotype contributions and deduces major contributors from high- and low-quality DNA traces. Peak height, degradation, stutter as well as allelic drop-in/-out events are incorporated in the statistical model. We analyzed reference and casework samples as well as artificially generated mixture samples for software evaluation. The tool offers the completely automated assessment of reference and mixture samples. Deconvolution outcomes of mixtures are compared with EuroForMix, GenoProof Mixture 3 and STRmix™. Data show that Statistefix 4.0 is as successful as analogously tested and implemented software. Deduced DNA profiles from casework samples highlight the potential benefit in routine casework. Statistefix 4.0 is freely available, works with replicates of different autosomal kits and enables bulk sample processing. This inter-laboratory study includes a variety of sample types and indicates a timesaving, robust and easily implemented software that supports DNA analysts in evaluating DNA traces.

The potential of handheld near infrared spectroscopy to detect food adulteration: Results of a global, multi-instrument inter-laboratory study.

Fraud in the food supply system will be exacerbated by shortages caused by climate change and COVID-19's impact. The dried herbs market exemplifies complex supply chains attractive to criminals seeking financial gain. Real-time remote testing is achievable through development of globally accessible chemometric models for portable near infrared devices, deployed throughout supply chains. This study describes building of models for detection of oregano adulteration, on portable near infrared devices, and comparison to a laboratory-based Fourier-Transform Infrared spectroscopy method. 33/34 portable devices were able to correctly classify 5 out of 6 samples successfully with all adulterated samples being correctly classified following the use of appropriate transferability pre-processing routines. The devices native setup shows limited ability to perform a true screening of oregano using the setup offered. However modifications to the setup could in the future offer a solution that facilitates fit-for-purpose real time detection of adulterated samples within the supply chain.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Inter-laboratory comparison of 2 ELISA kits used for foot-and-mouth disease virus nonstructural protein serology.

Serologic assays used to detect antibodies to nonstructural proteins (NSPs) of foot-and-mouth disease virus (FMDV) are used for disease surveillance in endemic countries, and are essential to providing evidence for freedom of the disease with or without vaccination and to recover the free status of a country after an outbreak. In a 5-site inter-laboratory study, we compared the performance of 2 commercial NSP ELISA kits (ID Screen FMD NSP ELISA single day [short] and overnight protocols, ID.Vet; PrioCHECK FMDV NS antibody ELISA, Thermo Fisher Scientific). The overall concordance between the PrioCHECK and ID Screen test was 93.8% (95% CI: 92.0-95.2%) and 94.8% (95% CI: 93.1-96.1%) for the overnight and short ID Screen incubation protocols, respectively. Our results indicate that the assays (including the 2 different formats of the ID Screen test) can be used interchangeably in post-outbreak serosurveillance.

Using hydrochloric acid and bile resistance for optimized detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from sprouts.

Shiga toxin-producing Escherichia coli (STEC) in sprouts have caused large scale outbreaks in the past involving severe illness. The combination of this very diverse pathogen and a food matrix with high numbers of background microbiota poses a particular challenge for detection and isolation. An acid treatment of the enrichment before plating on agar has been shown to improve the recovery of STEC from sprouts. After enrichment in buffered peptone water (BPW) at 37 °C we applied an acid treatment, followed by plating on tryptone bile x-glucuronide (TBX) agar (acid bile method). An inter-laboratory study was organized with 21 laboratories taking part to evaluate the performance parameters and applicability of the acid bile method. A sample set of six sprout samples was prepared consisting of two uninoculated samples and four spiked samples, each containing one of two STEC strains at one of two concentrations (low and high). Analyzing a set of six samples at the National Reference Laboratory (NRL E. coli), we determined the relative abundance of STEC without, after acid-, after bile- and after acid-bile treatment using real-time PCR. The participating laboratories successfully applied the acid bile method and were better able to detect (sensitivity 92.9% vs. 70.0%) and isolate (sensitivity 87.5% vs. 31.3%) STEC from positive samples using the acid bile method compared to non-acid methods. The relative limit of detection (RLOD) after isolation using the acid bile method (vs. non-acid method) was <1 for both STEC strains used, BfR-EC-14434 O133:H25 (0.146) and BfR-EC-16015 O26:H11 (0.073). A collection of STEC (n = 71) of diverse type and characteristics was assessed for their resistance towards the acid bile treatment selection. The majority (n = 65) of STEC strains could be recovered after acid treatment on TBX plates. However, a few strains (n = 6), among them clinical isolates were (partly) sensitive. These results suggest that an acid bile method is a rapid and reasonable approach to improve the recovery of STEC from sprouts when used in combination with methods targeting other selection markers.

Collaborative study: Quantification of total folate in food using an efficient single-enzyme extraction combined with LC-MS/MS.

Quantification of the specific folate vitamers to estimate total folate in foods is not standardized. A collaborative study, including eight European laboratories, was conducted in order to determine the repeatability and reproducibility of the method for folate quantification in foods using the plant-origin γ-glutamyl hydrolase as part of the extraction procedure. The seven food samples analyzed represent the food groups; fruits, vegetables, dairy products, legumes, offal, fish, and fortified infant formula. The homogenization step was included, and six folate vitamers were analyzed using LC-MS/MS. Total folate content, expressed as folic acid equivalent, was 17-490 µg/100 g in all samples. Horwitz ratio values were within the acceptable range (0.60-1.94), except for fish. The results for fortified infant formula, a certified reference material (NIST 1869), confirmed the trueness of the method. The collaborative study is part of a standardization project within the Nordic Committee on Food Analysis (NMKL).

Assessment of the transfer, persistence, prevalence and recovery of DNA traces from clothing: An inter-laboratory study on worn upper garments.

Among the various items recovered from crime scenes or persons involved in a crime event, clothing items are commonly encountered and submitted for forensic DNA sampling. Depending on the case circumstances and the activity-of-interest, sampling of the garment may concentrate on collecting DNA from the wearer, or from one or more offenders who have allegedly contacted the item and/or wearer. Relative to the targeted DNA, background DNA already residing on the item from previous contacts, or transferred during or after the crime event, may also be collected during sampling and observed in the resultant DNA profile. Given our limited understanding of how, and from where, background DNA is derived on clothing, research on the transfer, persistence, prevalence, and recovery (TPPR) of DNA traces from upper garments was conducted by four laboratories. Samples were collected from several areas of two garments, each worn on separate working or non-working days and individually owned by four individuals from each of the four laboratories, and processed from DNA extraction through to profiling. Questionnaires documented activities relating to the garment prior to and during wearing, and reference profiles were obtained from the wearer and their close associates identified in the questionnaire. Among the 448 profiles generated, variation in the DNA quantity, composition of the profiles, and inclusion/exclusion of the wearer and their close associates was observed among the collaborating laboratories, participants, garments worn on different occasions, and garment areas sampled.

STRmix™ collaborative exercise on DNA mixture interpretation.

An intra and inter-laboratory study using the probabilistic genotyping (PG) software STRmix™ is reported. Two complex mixtures from the PROVEDIt set, analysed on an Applied Biosystems™ 3500 Series Genetic Analyzer, were selected. 174 participants responded. For Sample 1 (low template, in the order of 200 rfu for major contributors) five participants described the comparison as inconclusive with respect to the POI or excluded him. Where LRs were assigned, the point estimates ranging from 2 × 104 to 8 × 106. For Sample 2 (in the order of 2000 rfu for major contributors), LRs ranged from 2 × 1028 to 2 × 1029. Where LRs were calculated, the differences between participants can be attributed to (from largest to smallest impact): This study demonstrates a high level of repeatability and reproducibility among the participants. For those results that differed from the mode, the differences in LR were almost always minor or conservative.

Elaboration and validation of the method for the quantification of the emetic toxin of Bacillus cereus as described in EN-ISO 18465 - Microbiology of the food chain - Quantitative determination of emetic toxin (cereulide) using LC-MS/MS.

A method for the quantification of the Bacillus cereus emetic toxin (cereulide) was developed and validated. The method principle is based on LC-MS as this is the most sensitive and specific method for cereulide. Therefore the study design is different from the microbiological methods validated under this mandate. As the method had to be developed a two stage validation study approach was used. The first stage (pre-study) focussed on the method applicability and the experience of the laboratories with the method. Based on the outcome of the pre-study and comments received during voting at CEN and ISO level a final method was agreed to be used for the second stage the (final) validation of the method. In the final (validation) study samples of cooked rice (both artificially contaminated with cereulide or contaminated with B. cereus for production of cereulide in the rice) and 6 other food matrices (fried rice dish, cream pastry with chocolate, hotdog sausage, mini pancakes, vanilla custard and infant formula) were used. All these samples were spiked by the participating laboratories using standard solutions of cereulide supplied by the organising laboratory. The results of the study indicate that the method is fit for purpose. Repeatability values were obtained of 0.6 µg/kg at low level spike (ca. 5 µg/kg) and 7 to 9.6 µg/kg at high level spike (ca. 75 µg/kg). Reproducibility at low spike level ranged from 0.6 to 0.9 µg/kg and from 8.7 to 14.5 µg/kg at high spike level. Recovery from the spiked samples ranged between 96.5% for mini-pancakes to 99.3% for fries rice dish.