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

Pyrroloquinoline quinone (PQQ) is a redox-active compound with physiological functions, widely used in functional foods and supplements. However, quantifying the reduced form, PQQ_red, is difficult due to its low solubility and susceptibility to oxidation. This study presents a robust HPLC method for direct quantification of PQQ_redin 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 (PQQ_ox)and matrix interferences, enabling accurate quantification. A pretreatment using ascorbic acid and γ-CD effectively reduced and solubilized PQQ_red, and total PQQ can be analyzed as PQQ_red.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 PQQ_redin 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.