Critical Evaluation of Analytical Strategies for the Quantification of Procaine and Its Metabolites Across Diverse Matrices

Procaine (PC) is an ester-type local anesthetic whose rapid enzymatic and chemical hydrolysis to p-aminobenzoic acid (PABA) makes intact-drug quantification exceptionally difficult across plasma, serum, urine, pharmaceutical formulations, and environmental waters, where PC exists only transiently and at ultra-trace levels; this review therefore aims to critically compare chromatographic, electrochemical, and spectroscopic strategies for the selective and sensitive measurement of PC and its metabolites. The evidence shows that physical separation by HPLC is indispensable for resolving PC from PABA and excipients, with fluorescence-enhanced HPLC achieving ng/mL sensitivity, while LC–MS/MS offers demonstrated analytical feasibility molecular selectivity and sub-ng to pg/mL limits enabling simultaneous PC–PABA profiling in ≤5 min; nanostructured and molecularly imprinted electrochemical sensors exploit the electroactive para-aminobenzoate moiety to provide rapid, matrix-tolerant nanomolar to sub-nanomolar detection that may reduce the extent of ex vivo hydrolysis due to rapid analysis and minimal sample handling time. Spectroscopic assays are suitable for high-throughput pharmaceutical and environmental screening, but their applicability is limited by drug instability and cross-reactivity. Given the relative scarcity of chromatographic studies on PC despite its instability-driven analytical complexity, future work should prioritize stabilization-coupled LC–MS/MS and next-generation electrochemical platforms, together with green, miniaturized workflows and AI-assisted optimization, to enable robust, high-confidence quantification of PC in real-world matrices.