High-pressure hydrolysis: A rapid sample preparation strategy for amino acid analysis of complex biological matrices

The extended acid hydrolysis step (typically 20-24 h) is a pervasive bottleneck in amino acid analysis, particularly for complex, protein-rich biological matrices. To overcome this, we developed and rigorously validated a novel high-pressure hydrolysis strategy. Complete protein hydrolysis in a challenging model matrix (Pheretima, a complex animal-derived material) was achieved in only 2 h (120 °C, saturated steam pressure of 0.12 MPa, ∼1.2 atm)-a > 90% reduction in time compared to the conventional approach. Critically, direct comparison with the 24-h gold-standard method confirmed analytical equivalence, with two one-sided t-tests (TOST) confirming equivalence for 15 out of 16 amino acids within the equivalence margin of ±5%. Coupled with a robust phenyl isothiocyanate (PITC) pre-column derivatization HPLC-UV protocol, the method demonstrated excellent performance: specificity, linearity (R² > 0.99), precision (RSD < 2.5%), and accuracy (recoveries: 95.1-104.9%). Its practical utility was proven by the rapid generation of consistent chromatographic fingerprints and the revelation of statistically significant, origin-linked variations in key amino acids (e.g., proline, tyrosine, phenylalanine) from 13 sample batches. This work provides a significant advancement in sample preparation, offering a fast, reliable, and broadly applicable solution that can transform throughput in the amino acid profiling of complex biological materials.