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

BACKGROUND: 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. METHOD: 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. RESULTS: 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%. CONCLUSIONS: 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. HIGHLIGHTS: The EnzytecTMLiquid Ethanol was approved as AOAC Method 2017.07 Final Action.

Validation of the RIDASCREEN® Peanut for Determination of Peanut Protein in Cookies, Milk Chocolate, Ice Cream, Trail Mix, Puffed Rice Cereals, and Granola Bar: AOAC Performance Tested MethodSM 112102.

BACKGROUND: Regulations in many countries worldwide prescribe that peanut must be listed on food labels as a cause of food allergies. Re-evaluated voluntary incidental trace allergen labelling (VITAL) values for peanut revealed the eliciting dose (ED01) value-at which 99% of all peanut-allergic individuals will not react-is 0.2 mg peanut protein. OBJECTIVE: Validation of a sandwich ELISA based on monoclonal antibodies to detect peanut proteins. METHODS: Non-processed and processed samples are extracted by an easy procedure at 60°C within 10 min. The measurement range is between 0.75 and 6 mg/kg peanut using a national institute of standards and technology (NIST) reference material as calibrator. RESULTS: The system shows no cross-reactivity against 91 different food commodities. The LOD was 0.15 mg/kg for food matrixes such as cookies, milk chocolate, ice cream, trail mix, puffed rice cereal, and granola bar. LOQ was verified at a level of 0.75 mg/kg. Recovery studies with incurred milk chocolate and ice cream revealed consistent recoveries between 67 and 85%. Mean recoveries for incurred cookies depend on the baking temperature and time and ranged from 60 to 109%. Repeatability was between 5.2 and 12.3%, whereas relative intermediate precision was between 6.4 and 13.0%. The results for incurred cookies and milk chocolate in the independent laboratory study showed mean recoveries between 99 and 104% with RSDs between 3.56 and 19.5% under repeatability conditions. CONCLUSION: The results from the in-house validation study and the independent lab confirmed that the method is accurate and in accordance with requirements laid down in Standard Method Performance Requirement 2017.020. HIGHLIGHTS: RIDASCREEN® Peanut quantifies proteins from peanut in a wide range of food categories.

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

BACKGROUND: According 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. METHOD: The 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. OBJECTIVE: Therefore, 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. RESULTS: Gliadin 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%. CONCLUSIONS: The collaborative study results confirmed that the method is accurate and suitable to measure gliadin in important gluten-free food matrixes. HIGHLIGHTS: The title and applicability statement of Official Method 2012.01 were changed as proposed.

Detection of SARS-CoV-2 Virus on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 022102.

BACKGROUND: The SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs. OBJECTIVE: To validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute's Emergency Response Validation Performance Tested Method(s)SM program. METHOD: The SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method. CONCLUSIONS: The SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces. HIGHLIGHTS: SureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.

Next Generation of LFD Testing - Smartphone-Based Mycotoxin Analysis with RIDA®SMART APP.

BACKGROUND: Grain mills, feed mills, ethanol plants, flour producers, but also mobile laboratory services need simple and reliable possibilities to analyze grain for mycotoxins during harvest in the field. OBJECTIVE: The RIDA®SMART APP is a smartphone application, which was developed together with a Lateral Flow Device as a quantitative and fast on-site/on-field tool for mycotoxin analyses. METHOD: It is a small and light device with its own power supply, very versatile, flexible and easy to use. For quantification, a lot specific calibation curve is uploaded to the RIDA®SMART APP via a QR code from the certificate of analysis. The software application in combination with a compatible smartphone, analyzes the LFD strip via a picture taken with a smartphone and quantifies the mycotoxin contamination within 4 second. Due to the possibility to use mobile networks or WIFI, there are no difficulties in the organization of the collected results. Currently, nine different android-based smartphones from various manufacturers are compatible with the RIDA®SMART APP. RESULTS: In combination with one of the compatible smartphones, the RIDA®SMART APP is able to generate results with a coefficient of variation CV ≤ 13%, even at highly varying lighting conditions. The recovery of certified reference corn samples, previously analyzed with HPLC or LC-MS/MS, shows a mean value between 91% - 119%. CONCLUSION: The RIDA®SMART APP is highly suitable for an inexpensive, quick and accurate quantitative measurement of mycotoxins in agricultural products (e.g. aflatoxins in corn). HIGHLIGHTS: To our best knowledge, the RIDA®SMART APP is the only smartphone-based method for quantitative mycotoxin analysis worldwide combined with a broader measuring range as most usual lateral flow analyzers.

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oat Products by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action 2018.15.

Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples >10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.

Validation of the R-Biopharm AG RIDASCREEN® Histamine (Enzymatic) Kit.

BACKGROUND: RIDASCREEN® Histamine (enzymatic) is an enzymatic test kit for quantification of histamine in fresh fish, canned fish, fish meal, cheese, and wine. METHODS: Fish products are extracted with boiling water, while cheese is extracted with water, and the extract is treated with perchloric acid/potassium hydroxyde. Wine is extracted with the reagents of RIDA® Sample Decolorant kit to eliminate color pigments and interfering compounds. The enzymatic determination is based on histamine dehydrogenase, which catalyzes the oxidative deamidation of histamine in the presence of an electron carrier that converts a dye to a color product. The resulting color intensity is directly proportional to histamine concentration and is measured at 450 nm. The substrates are coated on the microtiter plate. RESULTS: The linear range is from 1 to 20 mg/L in the extract. LOQs are 2 mg/kg for fresh fish, canned fish, and cheese, 1.4 mg/L in wine, and 10 mg/kg for fish meal. The linear range is from 5 to 100 mg/kg for fish products and cheese, 3.6 to 72 mg/L for wine, and 25 to 500 mg/kg for fish meal. Recovery and precision are very good for all matrices, and comparisons with HPLC reference methods revealed that the method also delivers true results for fish products and wine. Agmatine shows a low side activity of around 0.75% at 10 g/kg. Possible interfering substances are ascorbic acid (more than 250 mg/L in wine or 250 mg/kg in fish). A special sample preparation to deplete ascorbic acid from fresh fish is described.

The Validation of the RIDA®QUICK Gliadin for AOAC Research Institute.

RIDA®QUICK Gliadin is an immuno-chromatographic test for the detection of gluten in foods, on surfaces, and in Cleaning-in-Place (CIP) waters. This test kit has been adopted as Final Action AOAC INTERNATIONAL Official Methods of AnalysisSM 2015.16 for gluten in corn products. The assay is based on the monoclonal antibody R5, which recognizes gluten in wheat, barley, and rye. Four different surfaces were contaminated with a gliadin material and analyzed by a direct swabbing of the surface with the dip-stick. The outcome was an LOD95% concentration of the assay between 1.6 and 3.0 µg/100 cm2 gluten. For CIP waters that contain cleansing reagents, 100% positive results were obtained for minimum gluten concentration between 50 and 100 ng/mL. If the CIP water does not contain these reagents, the minimum detectable gluten level is 10 ng/mL. The independent validation study consisted of a method comparison study of recovery from a CIP solution and from a stainless-steel surface. The test kit was evaluated at six different concentration levels for both matrices, with 20 or 30 replicates per concentration level. The probability of detection was calculated for each contamination level. Additionally, the LOD95% concentration was estimated for each matrix analyzed.

Determination of Ethanol in Kombucha, Juices, and Alcohol-Free Beer by EnzytecTMLiquid Ethanol: Single-Laboratory Validation, First Action 2017.07.

EnzytecTM Liquid Ethanol is an enzymatic test for the determination of ethanol in kombucha, juices, and alcohol-free beer. The kit contains two components in a ready-to-use format. Quantification is based on the catalytic activity of alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde and converts NAD+ to NADH. Measurement is performed in 3 mL cuvettes at 340 nm within 20 min. Samples with alcohol contents around 0.5% alcohol by volume need to be diluted 1:20 or 1:50 with water before measurement. Acetaldehyde interferes at concentrations higher than 3000 mg/L, whereas sulfite interferes at concentrations higher than 300 mg/L. The linear measurement range is from 0.03 up to 0.5 g/L ethanol, whereas LOD and LOQ are 1.9 and 3.3 mg/L ethanol, respectively. Kombucha with concentrations between 2.85 and 5.82 g/L showed relative repeatability standard deviation around 1%, whereas juices were below 2%. Results from a reproducibility experiment revealed that at a concentration of 0.1 g/L, the RSDR was at 2.5%, whereas at higher concentrations between 0.2 and 0.3 g/L, coefficients around 1% were obtained. Trueness was checked by using Cerilliant aqueous ethanol solutions and beer with concentration of 0.4 and 4 g/L (BCR-651 and BCR-652). Spiking of kombucha and juice samples resulted in recoveries between 95% and 104%. Acceptable stability was found for the whole test kit under accelerated conditions at 37°C for 2 weeks. The kit is also not susceptible to short freezing-thawing cycles and harsh transport conditions.

Commercial ELISA Measurement of Allergens and Gluten: What We Can Learn from Case Studies.

During the last decade, results from ELISA test kits have often been criticized as being flawed. Therefore, it may appear counterintuitive that ELISAs are used for most food allergen and gluten analytical needs. One reason, in addition to the nonavailability of comparable alternative methods, is the fact that the methods used underwent a long validation and learning period in the market. This publication presents several case studies on interference, cross-reactivity issues, calibrators, fragmented allergens and gluten, matrix influences, and misunderstood intended-use statements. Afterward, the relevant validation parameter LOD, LOQ, selectivity, and precision are discussed. Finally, a comprehensive list of practical recommendations for ELISA test kit users is presented.

Stakeholders' Guidance Document for Consumer Analytical Devices with a Focus on Gluten and Food Allergens.

Until recently, analytical tests for food were performed primarily in laboratories, but technical developments now enable consumers to use devices to test their food at home or when dining out. Current consumer devices for food can determine nutritional values, freshness, and, most recently, the presence of food allergens and substances that cause food intolerances. The demand for such products is driven by an increase in the incidence of food allergies, as well as consumer desire for more information about what is in their food. The number and complexity of food matrixes creates an important need for properly validated testing devices with comprehensive user instructions (definitions of technical terms can be found in ISO 5725-1:1994 and the International Vocabulary of Metrology). This is especially important with food allergen determinations that can have life-threatening consequences. Stakeholders-including food regulators, food producers, and food testing kit and equipment manufacturers, as well as representatives from consumer advocacy groups-have worked to outline voluntary guidelines for consumer food allergen- and gluten-testing devices. These guidelines cover areas such as kit validation, user sampling instructions, kit performance, and interpretation of results. The recommendations are based on (1) current known technologies, (2) analytical expertise, and (3) standardized AOAC INTERNATIONAL allergen community guidance and best practices on the analysis of food allergens and gluten. The present guidance document is the first in a series of papers intended to provide general guidelines applicable to consumer devices for all food analytes. Future publications will give specific guidance and validation protocols for devices designed to detect individual allergens and gluten, as statistical analysis and review of any validation data, preferably from an independent third party, are necessary to establish a device's fitness-for-purpose. Following the recommendations of these guidance documents will help ensure that consumers are equipped with sufficient information to make an informed decision based on an analytical result from a consumer device. However, the present guidance document emphasizes that consumer devices should not be used in isolation to make a determination as to whether a food is safe to eat. As advances are made in science and technology, these recommendations will be reevaluated and revised as appropriate.

Determination of Gluten in Processed and Nonprocessed Corn Products by Qualitative R5 Immunochromatographic Dipstick: Collaborative Study, First Action 2015.16.

In September 2013, the AACC International (AACI) Protein Technical Committee (now Protein and Enzymes Technical Committee) initiated a collaborative study of a method for the qualitative analysis of intact gluten in processed and nonprocessed corn products, using an R5 immunochromatographic dipstick system. It was validated to demonstrate that potential gluten-free products contain gluten lower than the Codex threshold of 20 mg/kg gluten. The results of the collaborative test with 18 participants confirmed that the method is suitable to detect gluten contaminations that are clearly lower than the threshold. It is recommended that the method be accepted by AOAC as Official First Action.

Validation of the RIDASCREEN(®)FAST Milk Kit.

The RIDASCREEN(®)FAST Milk test is a sandwich ELISA for the rapid quantification of milk proteins in various foods. The specific antibodies target casein and ß-lactoglobulin. Samples are extracted and can then be analyzed in less than 40 min. The calibration curve covers a range from 2.5 to 67.5 mg/kg milk protein. The assay was validated with cookies, infant formula, chocolate dessert, ice cream, and sausages. All negative samples were found well below the LOQ of 2.5 mg/kg. Recoveries of the spiked samples were mostly in the range of 80-120%. The LOD of the ELISA was found below 1 mg/kg. The analysis of 39 different substances of interest revealed that no cross-reactivity above the LOQ occurred. Ruggedness testing proved that variations in incubation temperature, reagent volume, incubation time, extraction temperature, and extraction time had no significant influence. The stability at 4-8°C of three independent lots was investigated and found to exceed 18 months. Very good lot-to-lot consistency and no significant loss of the analytical capacity over the shelf life were observed. Incurred cookies and chocolate dessert samples were prepared and analyzed by an independent laboratory; mean recoveries of 94.4 and 102.2% and mean SDs of 10.9 and 6.3%, respectively, were found. For the 0 mg/kg level for both materials, all samples tested returned values of <2.5 mg/kg. Therefore, the analytical performance claims of the manufacturer were confirmed.