Reproducibility Relative Standard Deviation Research Articles

A nanobody based ultrasensitive electrochemical biosensor for the detection of soluble CTLA-4 –A candidate biomarker for cancer development and progression

A soluble isoform of cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) has been found in the serum of healthy individuals and alterations in its expression level have been linked with the development and progression of various cancers. Conventionally, soluble CTLA-4 (sCTLA-4) has been quantified by techniques such as ELISA, western blot, and flow cytometry, which however are time-consuming, highly expensive and require large sample volumes. Therefore, rapid, cost-effective and real-time monitoring of soluble CTLA-4 levels is much needed to facilitate timely diagnosis of a worsening disease and help patient selection for immunotherapeutic interventions in cancer. Here, for the first time, we report an ultrasensitive, highly selective electrochemical nanobody (NAb) based biosensor for the quantitative detection of soluble CTLA-4 employing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and gold nanoparticles modified electrode with attomole sensitivity. Incorporating nanomaterials with conductive polymers enhances the sensitivity of the electrochemical biosensor, while the nanobody's stability, specificity and ease of production make it a suitable choice as a bioreceptor. The proposed NAb-based sensor can detect sCTLA-4 from pure recombinant protein in a wide concentration range of 100 ag mL−1– 500 μg mL−1, with a limit of detection of 1.19 ag mL−1 (+3σ of the blank signal). The sensor's relative standard deviation for reproducibility is less than 0.4% and has effective real sample analytics for cell culture supernatant with no significant difference with pure recombinant protein (p < 0.05). Our proposed nanobody based sensor exhibits stability for up to 2 weeks (<3% variation). Moreover, this nanobody-based sensor presents a future opportunity for quantitative, ultrasensitive, and economical biosensor development that can be adapted to monitor the immune landscape of cancer patients to provide a larger therapeutic window.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products.

Osteopontin is a crucial protein ingredient that has been applied in fortified dairy products and infant formula. It has great significance to infant gut health and brain development. However, current techniques including enzyme-linked immunosorbent assay and liquid chromatography coupled with mass spectrometry are still facing the bottleneck of low sensitivity and indirect quantification. Moreover, the unavailable certified commercial OPN standard hinders its accurate quantification. Herein, a novel method of anion-exchange chromatography was established to determine OPN concentration in several dairy matrices. The polarity-reversed capillary isoelectric focusing was utilized to measure the exact isoelectric point (pI) to support method development for OPN separation. Analytical ultracentrifugation was used to calibrate the purity of intact OPN to develop an in-house reference standard. The method showed the merits of limits of detection to 0.04 mg/100 g, relative standard deviation of reproducibility <5% for 13 out of 14 tested matrices, and an average recovery rate of 101.3%. This method has shown the potential to be adopted as an international standard method for the quantification of intact OPN in infant formula and dairy products.

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.

Facile synthesis of MoS2 nanoflower-Ag NPs grown on lignin-derived graphene for Troponin I aptasensing

This article presents the development and application of a green lignin-derived graphene biosensor for Troponin I, a biomarker for Acute Myocardial Infarction (AMI). The graphene was synthesized from oil palm lignin through an optimized laser scribing process. While the three-dimensional nature of the laser-scribed lignin-derived graphene (3D LSG) is advantageous, it suffers from poor electrical conductivity due to the amorphous nature of lignin. Therefore, semi-conductive molybdenum disulphide (MoS2) precursor with conductive green silver nanoparticles (Ag NPs) was added to 0.5, 1.0, 1.5, and 2.0 g of 3D LSG to synthesize 3D LSG_MoS2_Ag NPs hybrids via an aqueous hydrothermal process. Morphological, physical, and structural analyses showed the presence of petal-like MoS2 nanoflower with Ag NPs on the 3D LSG surface. The strong interrelation between 3D LSG, MoS2, and Ag NPs was confirmed by X-ray spectroscopy, Raman spectroscopy and energy dispersive spectroscopy (EDS). Specifically, X-ray spectroscopy revealed the formation of O1s, Ag 3d, C1s, Mo 3d, and S2p in the 3D LSG_MoS2_Ag NPs-2.0 hybrid. Raman spectroscopy revealed an enhancement in the surface area of the 3D LSG_MoS2_Ag NPs-2.0 hybrid, which enhances the detection sensitivity. The 3D LSG_MoS2_Ag NPs hybrid was subsequently chemically modified and immobilised with an aptamer to interact with Troponin I on an impedimetric sensor. The 3D LSG_MoS2_Ag NPs hybrid showed high analytical performance, high specificity, and a ∼ 4-fold increment in selectivity, with a detection limit of 100 attomolar. This biosensor has a sensitivity of 31.45 µA mM−1 cm−2, stability of 87%, with a relative standard deviation for reproducibility of 3.8%.

Quantification of Lactose in Lactose-Free and Low-Lactose Milk and Milk Products by BIOMILK 300/3000 Lac, Collaborative Study: Final Action 2020.09.

In April 2020, the BIOMILK 300 LAC method for the quantification of lactose in lactose-free and low-lactose dairy products was adopted as First Action Official Method of AnalysisSM2020.09 by AOAC INTERNATIONAL. To test the reproducibility of the method, as the last step toward the Final Action status, a collaborative study was organized by BIOLAN Microbiosensores. Fifteen collaborators within the European Union took part in this study, where nine different samples were sent as duplicate blind test portions for lactose determination. The data obtained were used to determine method repeatability and reproducibility and also to validate the new version of the biosensor. The test method is based on the direct enzymatic recognition-electrochemical detection of trace levels of lactose over a wide range of dairy samples by means of the BIOMILK 300 biosensor, in less than 5 min, and without intricate sample pretreatments. All samples resulted in a repeatability relative standard deviation (RSDr) of <8.1% and a reproducibility relative standard deviation (RSDR) of 14% maximum, meeting requirements from Standard Method Performance Requirement (SMPR®) 2018.09. The method has proved to give reproducible results for the quantification of lactose in lactose-free and low-lactose dairy products. On the basis of these results, the enzymatic amperometric biosensor method developed by BIOLAN Microbiosensores was adopted as a Final Action Official Method in July 2022.

Semi-Automated Enzymatic Determination of Ethanol in Beverages: Collaborative Study for RIDA&amp;#174;CUBE Ethanol

Easy and quick methods to quantify ethanol reliably in beverages are always important. In 2022, the EnzytecTM Liquid Ethanol test kit was approved as AOAC Official MethodSM 2017.07 Final Action after a collaborative study was conducted with different beverages such as kombucha, juices, and beer. During set-up of this collaborative test, small sized companies asked to include the RIDA®CUBE Ethanol/RIDA®CUBE SCAN device since it is easy to use, suitable for a few samples only and contains the identical reagents as the EnzytecTM Liquid system. It is applicable to quantify ethanol in diluted kombucha, fruit juices, and alcohol-free beer samples around 0.5% alcohol-by-volume within 12 min. The overall relative reproducibility standard deviation across a wide concentration range for kombucha, was calculated to be 6.29%. Analysis of juices and beer showed an overall higher variation with an estimated overall RSD(R) value by regression of 14.4%. The data obtained by this collaborative study show that the RIDA®CUBE Ethanol in combination with the RIDA®CUBE SCAN device is suitable to quantify ethanol from matrices representing important alcohol-free liquid food categories.

Determination of Ethanol in Food by Enzymatic Method Enzytec™ Liquid Ethanol, Collaborative Study: Final Action 2017.07.

Enzytec™ Liquid Ethanol was approved as AOAC Official MethodSM2017.07 First Action in September 2017 and is now further characterized by a collaborative study using the manual and automated version of the test. It is applicable to quantify ethanol in diluted or undiluted kombucha, fruit juices, vegetable juices, and alcohol-free beer samples around 0.5% ABV within 20 min using two ready-to-use reagents and measurement of absorbance at 340 nm. The overall relative reproducibility standard deviation across a wide concentration range for kombucha was calculated to be 6.99% by modeling the reproducibility standard deviation by the mean concentration for each of the six kombucha pairs by a linear regression. Analysis of juices and beer showed an overall higher variation with an estimated overall RSD(R) value by regression of 10.1%. Mean recovery of aqueous ethanol reference solutions tested by each participant was between 100 and 103%. The data obtained by this collaborative study show that the EnzytecTMLiquid Ethanol is suitable to quantify ethanol from matrices representing important alcohol-free liquid food categories. The EnzytecTMLiquid Ethanol was approved as AOAC Method 2017.07 Final Action.

Validation of the Test Method-Determination of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Related Food Products and Animal Feeds: Collaborative Study, Final Action 2020.07.

A simple, accurate, and reliable method to measure available carbohydrate components of food products, including cereal and dairy products, fruits, vegetables, processed food, food ingredients, and animal foods, was developed by Megazyme (product K-AVCHO, Bray, Ireland). A single-laboratory validation of the enzymatic method resulted in First Action status as Official Method of AnalysisSM2020.07. A collaborative study was conducted to evaluate the repeatability and reproducibility of Official Method 2020.07 for the measurement of available carbohydrates, including digestible starch, lactose, sucrose, isomaltose, maltose, glucose, fructose, and galactose in a broad range of food and feed products. Samples are defatted if containing >10% fat content, and incubated with pancreatic α-amylase and amyloglucosidase under conditions that simulate those in the small intestine (pH 6, 37°C, 4 h). The reaction solution is clarified and diluted, and an aliquot is incubated with sucrase, maltase, oligo-1,6-α-glucosidase, and β-galactosidase to hydrolyze sucrose, maltose, isomaltose, and lactose to glucose, fructose, and galactose, which are then measured enzymatically. The multi-laboratory validation (MLV) matrixes included cereal, animal feeds, fruit, vegetables, infant formula, powdered milk drink, a dessert product, and mushrooms. Additional materials were analyzed by collaborators as "practice samples." All MLV matrixes resulted in repeatability relative standard deviations (RSDr) <3.91% and reproducibility relative standard deviations (RSDR) ranging from 3.51 to 11.58% with 9 of the 10 matrixes having RSDR of <6.19%. For the practice samples, the RSDR ranged from 2.7 to 11.4% with 7 of the 8 samples having RSDR of <4.4%. Official Method 2020.07 meets the AOAC requirements for repeatability and reproducibility, and the data support Final Action status. Official Method 2020.07 is a robust, simple to use, and reproducible method for the analysis of available carbohydrates in a wide range of matrixes.

Reproducibility of methods required to identify and characterize nanoforms of substances

Nanoforms (NFs) of a substance may be distinguished from one another through differences in their physicochemical properties. When registering nanoforms of a substance for assessment under the EU REACH framework, five basic descriptors are required for their identification: composition, surface chemistry, size, specific surface area and shape. To make the risk assessment of similar NFs efficient, a number of grouping frameworks have been proposed, which often require assessment of similarity on individual physicochemical properties as part of the group justification. Similarity assessment requires an understanding of the achievable accuracy of the available methods. It must be demonstrated that measured differences between NFs are greater than the achievable accuracy of the method, to have confidence that the measured differences are indeed real. To estimate the achievable accuracy of a method, we assess the reproducibility of six analytical techniques routinely used to measure these five basic descriptors of nanoforms: inductively coupled plasma mass spectrometry (ICP-MS), Thermogravimetric analysis (TGA), Electrophoretic light scattering (ELS), Brunauer–Emmett–Teller (BET) specific surface area and transmission and scanning electron microscopy (TEM and SEM). Assessment was performed on representative test materials to evaluate the reproducibility of methods on single NFs of substances. The achievable accuracy was defined as the relative standard deviation of reproducibility (RSDR) for each method.Well established methods such as ICP-MS quantification of metal impurities, BET measurements of specific surface area, TEM and SEM for size and shape and ELS for surface potential and isoelectric point, all performed well, with low RSDR, generally between 5 and 20%, with maximal fold differences usually <1.5 fold between laboratories. Applications of technologies such as TGA for measuring water content and putative organic impurities, additives or surface treatments (through loss on ignition), which have a lower technology readiness level, demonstrated poorer reproducibility, but still within 5-fold differences. The expected achievable accuracy of ICP-MS may be estimated for untested analytes using established relationships between concentration and reproducibility, but this is not yet the case for TGA measurements of loss on ignition or water content. The results here demonstrate an approach to estimate the achievable accuracy of a method that should be employed when interpreting differences between NFs on individual physicochemical properties.

Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients by High Sensitivity Enzymatic Method (K-LOLAC), Collaborative Study: Final Action 2020.08.

The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods in 2018 for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPR®) 2018.009. Megazyme's Lactose Assay Kit (K-LOLAC) was reviewed and accepted as a First Action Official MethodSM in 2020 (2020.08). A collaborative study was conducted to evaluate the to evaluate the reproducibility of AOAC Official MethodSM2020.08 for the measurement of lactose concentration in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. Samples are deproteinated and clarified by treatment with Carrez reagents, and then free glucose is removed using a glucose oxidase and catalase treatment system. Quantification of lactose is based on the hydrolytic activity of β-galactosidase, which hydrolyses lactose to glucose and galactose. Any remaining free D-glucose is first measured using a hexokinase (HK)/glucose 6-phosphate dehydrogenase (G-6PDH)/6-phosphogluconate dehydrogenase (6-PGDH) based assay procedure, and then β-galactosidase is added to hydrolyze the lactose in the same reaction vessel with concurrent measurement of the released D-glucose. The samples analyzed included a number of lactose-free and low-lactose milk samples, lactose-free infant formula, lactose-free milkshake, lactose-free adult nutritional drink, lactose-free cream, and lactose-free cheese. All materials had repeatability relative standard deviations (RSDr) <7%. The reproducibility relative standard deviation (RSDR) varied from 3.8 to 14.9% with seven of the 10 test samples having an RSDR of <10%. The Lactose Assay Kit (K-LOLAC) meets the requirements for reproducibility set out under SMPR 2018.009. The Lactose Assay (K-LOLAC) is a robust, simple, and reproducible method for analysis of lactose in foodstuffs and beverages.

Detection and Stability of Cyanogen Bromide and Cyanogen Iodide in Drinking Water

This study systematically summarized the factors affecting the stability of CNXs, providing a reference for better control and elimination of CNXs. A method for the detection of CNBr and CNI in solution was established using a liquid–liquid extraction/gas chromatography/electron capture detector. Specifically, the method was used to investigate the stability of CNBr and CNI in drinking water, especially in the presence of chlorine and sulfite, and it showed good reproducibility (relative standard deviation &lt;3.05%), high sensitivity (method detection limit &lt;100 ng/L), and good recovery (91.49–107.24%). Degradation kinetic studies of cyanogen halides were conducted, and their degradation rate constants were detected for their hydrolysis, chlorination, and sulfite reduction. For hydrolysis, upon increasing pH from 9.0 to 11.0, the rate constants of CNCl, CNBr, and CNI changed from 8 to 155 × 10−5 s−1, 1.1 to 34.2 × 10−5 s−1, and 1.5 to 6.2 × 10−5 s−1, respectively. In the presence of 1.0 mg/L chlorine, upon increasing pH from 7.0 to 10.0, the rate constants of CNCl, CNBr, and CNI changed from 36 to 105 × 10−5 s−1, 15.8 to 49.0 × 10−5 s−1, and 1.2 to 24.2 × 10−5 s−1, respectively. In the presence of 3 μmol/L sulfite, CNBr and CNI degraded in two phases. In the first phase, they degraded very quickly after the addition of sulfite, whereas, in the second phase, they degraded slowly with rate constants similar to those for hydrolysis. Owing to the electron-withdrawing ability of halogen atoms and the nucleophilic ability of reactive groups such as OH− and ClO−, the rate constants of cyanogen halides increased with increasing pH, and they decreased in the order of CNCl &gt; CNBr &gt; CNI during hydrolysis and chlorination. The hydrolysis and chlorination results could be used to assess the stability of cyanogen halides in water storage and distribution systems. The sulfite reduction results indicate that quenching residual oxidants with excess sulfite could underestimate the levels of cyanogen halides, especially for CNBr and CNI.

Single and multi-laboratory validation of a droplet digital PCR method

The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control.Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range.In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters.

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.

The Effect of Monitoring System on Risk Assessment of Aflatoxins in Iran's Pistachio Nuts Exported to the E.U. During 2012 - 2018.

Pistachio has high nutritional value and popularity. The susceptibility of pistachio to aflatoxin contamination caused establishing a monitoring system introduced and implemented by the Ministry of Health in Iran to ensure consumers' access to safe and hygienic pistachios. In this research, aflatoxin contamination level in all consignments (7298) exporting to E.U. was examined using HPLC with fluorescence detection after immunoaffinity column clean up from Nov 2012 to Oct 2018. The average recoveries ranged 78.6% - 97.6%, with a relative standard deviation for reproducibility below 8.5% and expanded uncertainty of aflatoxin B1 (AFB1) at spiked levels 1, 4, and 8 ng/g were 0.17, 0.57, 0.89 ng/g, respectively. The results showed that aflatoxin B1 and total (AFT) were detected in 1921 (23.4%) and 1927 cases (23.5%), with the mean values ranging from 2.18 - 4.6 ng/g and 2.8 - 5.1 ng/g during six consecutive years, respectively. Implementing an effective monitoring system for pistachio nuts could determine consignments contaminated with aflatoxins. Concerning AFB1, risk assessments recorded for dietary exposure dose, margin of exposure (MOE), Hazard Index (HI), estimated liver cancer risk, and cancer incidence attributable to dietary ranged 0.0132 - 0.1180 ng/kg.bw/day, 1441 - 12843, 0.21 - 1.84, 0.00071 - 0.00633 cases/105 population/year, 0.02 - 0.2%, respectively. Identification and rejection of contaminated cargos lead to an increase in MOE (> 10000), and it also guarantees that pistachio consumption is safe from a toxicology point of view. Due to the monitoring system, the estimation of liver cancer incidence attributable to dietary AFB1 was reduced (≤ 0.02%). It indicates that the consumption of pistachio poses no health risk for Europeans and Iranians.

Cockle shell-derived nanoparticles for optical urea biosensor development based on reflectance transduction

An optical biosensor for urea based on urease enzyme immobilised on functionalised calcium carbonate nanoparticles (CaCO 3 -NPs) was successfully developed in this study. CaCO 3 -NPs were synthesised from discarded cockle shells via a simple and eco-friendly approach, followed by surface functionalisation with succinimide ester groups. The fabricated biosensor is comprised of two layers. The first (bottom layer) contained functionalised NPs covalently immobilised to urease, and the second (uppermost layer) was alginate hydrogel physically immobilised to the pH indicator phenolphthalein. The biosensor provided a colorimetric indication of increasing urea concentrations by changing from colourless to pink. Quantitative urea analysis was performed by measuring the reflectance intensity of the colour change at a wavelength of 633.16 nm. The determination of urea concentration using this biosensor yielded a linear response range of 30–1000 mM (R 2 = 0.9901) with a detection limit of 17.74 mM at pH 7.5. The relative standard deviation of reproducibility was 1.14%, with no signs of interference by major cations, such as K + , Na + , NH₄ + , and Mg 2+ . The fabricated biosensor showed no significant difference with the standard method for the determination of urea in urine samples.

High resolution mass spectrometry method for the determination of 13 antibiotic groups in bovine, swine, poultry and fish meat: An effective screening and confirmation analysis approach for routine laboratories

A screening and confirmatory method for the determination of 13 antibiotic groups including 91 substances was developed for muscle samples and validated using ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry. Optimisation of the parameters was performed for every analyte and personal compound database library was created, comprising the mass spectra for following collision energies; 0, 10, 20 and 40 eV. Validation plan scheming and calculation of the parameters was performed using validation software, which included following performance characteristics; selectivity, trueness, precision, decision limit (CCα), detection capability (CCβ) and relative matrix effect. High resolution mass spectrometry operated in a narrow mass window and resolving power of 50,000 FWHM (2 GHz). Accurate mass measurements with mass accuracy below 10 ppm for all analytes were obtained in spiked muscle samples. The validation experiments showed good selectivity and specificity. The method was validated in the measurement range from 0.5 to 1000 μg/kg, where a specific analyte mixed standard solution was prepared depending on the defined maximum residue limit (MRL) or lowest calibration level (LCL). For screening analysis, detection capabilities (CCβ) below the set MRL values or at LCL concentration levels were defined. CCβ, as the concentration above which the sample can be considered suspect, ranged from 0.63 to 207.8 μg/kg. For confirmation of the analyte, CCα was calculated at LCL or at the MRL, where it ranged from 0.38 to 14.3 μg/kg for unauthorised substances and from 13.8 to 1492.6 μg/kg for authorised substances. Relative within-laboratory reproducibility standard deviation ranged from 5.1 to 34.2%. Satisfactory z-score values in the range from −0.87 to 1.88 were obtained as a result of participating in four proficiency tests organized by European Union Reference Laboratories. • Screening and confirmatory HRMS method for 91 antibiotics. • Wide validation range around the established residue limits. • CCβ and CCα as parameters for screening and confirmatory analysis. • Satisfactory proficiency results as confirmation of good method performance.

Determination of Gliadin as a Measure of Gluten in Food by R5 Sandwich ELISA RIDASCREEN® Gliadin Matrix Extension: Collaborative Study 2012.01.

BackgroundAccording to Codex Alimentarius, food products containing less than 20 mg/kg gluten can be labeled as “gluten-free.” Since 2002, the R5 antibody method allowed determination of gluten levels and led to a huge improvement of products available to celiac disease (CD) patients.MethodThe R5-containing test kit RIDASCREEN® Gliadin in combination with the cocktail solution was endorsed as Codex Type 1 Method in 2006 based on a collaborative study with corn-based bread, rice-based dough, wheat starches, rice, and corn flour. In 2012, the method was approved as First Action Official MethodSM2012.01 with an “in foods” claim. For Final Action in 2016, the matrix claim was reduced to rice- and corn-based matrixes.ObjectiveTherefore, R-Biopharm decided to start a collaborative study to demonstrate the wide applicability of Official Method 2012.01 for the quantitative analysis of gliadin in soy, starches, pseudo cereals, legumes, spices, juice, nut nougat crème, cream cheese, pesto, meat, vegetarian meat alternative, cookies, dessert, cake, fish, bread, candies, and potatoes. Materials for incurring were the MoniQA wheat flour and the PWG gliadin preparation. ResultsGliadin levels ranged from 3.4 up to 27.4 mg gliadin per kg. The results of the collaborative study with 14 participating laboratories showed recoveries ranging from 80 to 130%. Relative reproducibility standard deviations for contaminated samples were between 9.8 and 27.7%.ConclusionsThe collaborative study results confirmed that the method is accurate and suitable to measure gliadin in important gluten-free food matrixes.HighlightsThe title and applicability statement of Official Method 2012.01 were changed as proposed.

Fiber Nanoarchitectonics for Pre-Treatments in Facile Detection of Short-Chain Fatty Acids in Waste Water and Faecal Samples.

Short-chain fatty acids (SCFAs) are among the active metabolites in biological process both in the intestinal tract and the bioconversion of organic wastes, which has resulted in various human diseases and environmental problems. In order to accurately detect SCFAs, we introduced a novel extraction sorbent. Electrospun polyacrylonitrile (PAN) nanofiber membrane was synthesized, then poly (3, 4-Ethylenedioxythiophene) (PEDOT) was deposited onto the surface of electrospun PAN nanofibers by in situ polymerization. The morphology of the composite PAN/PEDOT nanofiber was characterized by scanning electronic microscopy (SEM) and FTIR spectrum. PAN/PEDOT was used to isolate and concentrate the SCFAs in waste water and fecal samples before gas chromatography mass spectrometry (GC-MS) analysis. The analytical method was evaluated systematically, and low limits of detection (LODs) of 0.34–0.87 μg/L and good linearity (R2 ≥ 0.9953) were obtained. The method was applied successfully for the determination of SCFAs in waste water and fecal samples, with good recovery (87.5–104.6%) and satisfactory reproducibility (relative standard deviation: 6.5–14.1%). The results indicated that the proposed method can be used as a potential approach for the determination of SCFAs with high sensitivity in waste water and biological samples.

Real-Time PCR Assay for the Detection and Quantification of Roe Deer to Detect Food Adulteration-Interlaboratory Validation Involving Laboratories in Austria, Germany, and Switzerland.

Game meat products are particularly prone to be adulterated by replacing game meat with cheaper meat species. Recently, we have presented a real-time polymerase chain reaction (PCR) assay for the identification and quantification of roe deer in food. Quantification of the roe deer content in % (w/w) was achieved relatively by subjecting the DNA isolates to a reference real-time PCR assay in addition to the real-time PCR assay for roe deer. Aiming at harmonizing analytical methods for food authentication across EU Member States, the real-time PCR assay for roe deer has been tested in an interlaboratory ring trial including 14 laboratories from Austria, Germany, and Switzerland. Participating laboratories obtained aliquots of DNA isolates from a meat mixture containing 24.8% (w/w) roe deer in pork, roe deer meat, and 12 meat samples whose roe deer content was not disclosed. Performance characteristics included amplification efficiency, level of detection (LOD95%), repeatability, reproducibility, and accuracy of quantitative results. With a relative reproducibility standard deviation ranging from 13.35 to 25.08% (after outlier removal) and recoveries ranging from 84.4 to 114.3%, the real-time PCR assay was found to be applicable for the detection and quantification of roe deer in raw meat samples to detect food adulteration.

Development, optimization, and validation of a method for detection of cartap, thiocyclam, thiosultap-monosodium, and thiosultap-disodium residues in plant foods by GC-ECD

A method was developed for the simultaneous determination of four nereistoxin-related pesticides, viz. cartap, thiocyclam, thiosultap-monosodium, and thiosultap-disodium, in 20 plant foods. The samples were extracted using a hydrochloric acid solution containing cysteine hydrochloride, derivatized to nereistoxin under alkaline conditions, and analyzed by gas chromatography with electron capture detector. The average recoveries of the method were 72–108%, with relative standard deviations (RSDs) of 0.3–14.7% (n = 1200, p < 0.05). The intermediate precision and reproducibility experiments using established methods were also carried out. All the results passed the Cochrane and Grubbs tests (n = 2400, p < 0.05). The RSDs of intermediate precision and RSDs of reproducibility among laboratories were in the ranges 1.7–10.9% and 2.4–15.3% (n = 2400, p < 0.05), respectively, indicating that the accuracy and precision of the method are satisfactory. This method can be used to detect nereistoxin-related pesticides in plant foods.