Direct HPLC Method for Reduced Pyrroloquinoline Quinone in Functional and Biological Matrices

Abstract

Pyrroloquinoline quinone (PQQ) is a redox-active compound with physiological functions, widely used in functional foods and supplements. However, quantifying the reduced form, PQQred, is difficult due to its low solubility and susceptibility to oxidation. This study presents a robust HPLC method for direct quantification of PQQred in complex matrices. By changing to a strongly acidic eluent, the oxidation of PQQred was suppress, and the optimized method successfully separated PQQred from its oxidized counterpart (PQQox)and matrix interferences, enabling accurate quantification. A pretreatment using ascorbic acid and {gamma}-CD effectively reduced and solubilized PQQred, and total PQQ can be analyzed as PQQred. The method achieved excellent linearity (R2 = 1), low detection limits (LOD: 0.20 mg/L, LOQ: 0.50 mg/L), and high precision (RSD < 3%). Application to commercial beverages showed consistent recovery (99-101%) with minimal interference. Moreover, the method suctandem mass spectrometry.cessfully detected biologically generated PQQred in yeast cultures, demonstrating its utility in physiological systems. This redox-specific and practical approach enables routine analysis and quality control of PQQ-enriched products, especially where accurate assessment of redox state is essential.