Tang_2026_Drug.Metab.Pharmacokinet_68_101541

Plasma stability assays are routinely applied in early drug discovery to evaluate enzymatic liability and to support lead optimization efforts. Although these assays are generally regarded as robust, the extent to which pre-analytical variables influence specific plasma hydrolase pathways has not been systematically examined. In this study, the impact of plasma anticoagulant selection on hydrolysis was investigated using procaine and pilocarpine as prototypical substrates of the serine hydrolase butyrylcholinesterase (BChE) and the calcium-dependent hydrolase paraoxonase 1 (PON1), respectively. Plasma stability was assessed in dipotassium ethylenediaminetetraacetic acid (K(2)-EDTA) and lithium heparin plasma collected from the same donors. Procaine exhibited comparable stability across both plasma matrices, consistent with BChE-mediated hydrolysis. In contrast, pilocarpine underwent substantial hydrolysis in lithium heparin plasma but appeared stable in K(2)-EDTA plasma. This effect is most consistent with calcium chelation by EDTA, as evidenced by complete suppression of PON1-mediated pilocarpine hydrolysis in lithium heparin plasma following exogenous EDTA supplementation. Collectively, these findings demonstrate that K(2)-EDTA anticoagulation can selectively mask calcium-dependent PON1-mediated plasma hydrolytic pathways, leading to underestimation of plasma lability for susceptible compounds. Anticoagulant choice therefore represents an underappreciated determinant in plasma stability assessment, with direct implications for mechanistic interpretation of plasma-mediated clearance pathways during early DMPK evaluation.