Green analytical chemistry approach for microwave-assisted digestion of collagen in meat products: A case study

Collagen, the primary structural protein in connective tissue, plays a key role in the texture, water-holding capacity, and shelf life of meat products. This study aimed to validate and adapt the AOAC 990.26/NMKL 127 reference method (AOAC-NMKL) for collagen quantification - based on hydroxyproline (Hyp) determination - by incorporating microwave-assisted digestion (MAD) as a faster and more sustainable alternative. Following hydrolysis, Hyp was oxidized with chloramine-T and reacted with 4-dimethylaminobenzaldehyde for spectrophotometric detection at 558 nm. Firstly, a method verification using various meat matrices (hams, sausages, and hamburgers) was conducted, achieving proper linearity (R² = 0.9990), repeatability and intermediate precision (CV ≤ 8.8 %), accuracy (recoveries of 103-104 %), limit of quantification (LOQ = 0.60 μg 2 mL^-1), and measurement uncertainty (U = 4.6 %). After that, the alternative microwave-assisted acid digestion was optimized and compared with the AOAC-NMKL protocol. The MAD protocol using HCl reduced acid volume by 67 % and digestion time by more than 96 % compared to the reference method. These improvements, combined with the use of less hazardous reagents and reduced waste generation, align with the principles of Green Analytical Chemistry. Overall, the proposed method proved to be efficient and environmentally favorable, representing a valuable alternative for routine collagen determination in meat product analysis.