Novel analytical strategy integrating enzymatic hydrolysis with UPLC-MS/MS for precise quantification of free and...

The discovery of bound heterocyclic amines (HAAs) has led to the realization that the HAAs content in meat may have been underestimated before, so the determination of bound HAAs content in meat products has become important. In this method, methanol-hydrochloric acid solution was selected as the extraction solution of free HAAs in crispy fried pork, and then the bound HAAs in meat was extracted by innovative enzymatic hydrolysis method. The optimal extraction process parameters of bound HAAs were determined by single-factor experiment combined with response surface method. Under the optimal extraction conditions, with a neutral protease to bromelain ratio of 75:25, enzyme concentration of 5 %, and pH of 6, the HAAs concentration reached 127.63 ng/g. SelectPrep MCX solid-phase extraction column (3 cc / 60 mg) and 10 % ammonia-methanol solution (6 mL) were selected as SPE conditions. The established method showed good linearity (R² ≥ 0.9920), standards-compliant recoveries (recovery rates for free HAAs matrix peaks ranged from 74.48 % to 116.12 %, recovery rates for bound HAAs matrix peaks ranged from 68.34 % to 110.25 %), and high precision (intra-day and inter-day RSD < 14.95 %). The limit of detection (LOD) ranged from 0.030 to 0.122 ng/g, and the limit of quantification (LOQ) ranged from 0.122 to 0.488 ng/g. This method has been successfully applied to the analysis of both free and bound HAAs in crispy fried pork, with satisfactory recovery rates. However, it is still necessary to explore more diversified enzymatic hydrolysis conditions in future studies to improve the extraction efficiency of HAAs bound in different substrates.