Determination of Acrylamide in Coffee, Cereals, Baby Food, Cocoa, Dry Pet Food, Potato Products, Vegetable Crisps, Biscuits, Tea, Nuts, and Spices by LC-MS/MS in a Single-Laboratory Validation: First Action 2023.01.

BACKGROUND: Acrylamide (AA) is a process contaminant naturally formed during the cooking of starchy food at high temperatures. Considering existing risks of misquantification inherent to the analysis of AA, an AOAC initiative raised the need for a consensus standard to determine AA in a broad variety of food. OBJECTIVE: A quantitative LC-MS/MS method for AA determination in food was validated in a single-laboratory study. Targeted performance requirements in terms of target matrixes, limit of quantification, recovery, and precision were as defined per Standard Method Performance Requirement (SMPR®) 2022.006. METHOD: The proposed method derives from EN 16618:2015 standard pending modifications brought to the (1) sample preparation (simplified, potentially automated); (2) scope of application (significantly extended); and (3) LC conditions (improved selectivity). Confirmatory detection of AA is conducted by LC-MS/MS in the Selected Reaction Monitoring mode (SRM), and isotopic dilution was applied for quantification approach using either 2,3,3-d3-acrylamide (d3-AA), or 13C3-2,3,3-d3-acrylamide (13C3-d3-AA) as labeled internal standard. RESULTS: A total of 16 laboratory samples from nine matrix categories were included in the validation process. A full validation was conducted on coffee (instant, roast), infant cereal, cocoa powder, pet food (croquettes), tea (green tea), spices (black pepper), and nuts (roasted almonds) with satisfactory performances both in terms of recovery (97-108%) and precision (RSDr and RSDiR <12%). The method applicability was further demonstrated through the analysis of quality control materials and reference materials including French fries, potato crisps, vegetable crisps, instant coffee, infant food, and biscuits (cookies), with accuracy values determined within a 94-107% range. CONCLUSIONS: The performances of the presented method are in agreement with the acceptance criteria stipulated in SMPR 2022.006. HIGHLIGHTS: The Expert Review Panel for acrylamide approved the present method as AOAC Official First Action 2023.01.

Towards a consensus LC-MS/MS method for the determination of acrylamide in food that prevents overestimation due to interferences.

Acrylamide is prone to misquantification, and critical steps in the analytical procedures need to be identified and controlled to ensure a reliable determination. Four methods were considered to illustrate misquantification issues with acrylamide. For two methods varying by the extent of their sample preparations, cases of overestimation in cocoa samples reaching up to a 20-fold factor are shown. A second example, applied to a variety of food products, includes two other methods varying by their chromatographic conditions. As a follow up of a study conducted in 2020 about the identification of N-acetyl-ß-alanine as an interference of acrylamide in coffee, the extent of this interference was evaluated in a selection of coffee samples, cereal-based products and baby foods. The ultimate objective of this manuscript was to resolve such cases of misquantification and validate a wide scope and robust method allowing an interference free acrylamide analysis. To do so, an extraction procedure based on the EN 16618:2015 standard with water extraction and two consecutive solid phase extraction (SPE) steps was applied with modified liquid chromatographic conditions. The method was validated in coffee, cereals, baby foods, cocoa and pet foods with excellent performance in terms of recovery (97-108%) and precision (RSDr and RSDiR <12 %). The breath of scope was further proved through trueness determination in quality control materials and reference materials including French fries, potato crisps, vegetable crisps, instant coffee, infant food and biscuit (cookie), with trueness values found within a 94-107% range.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.