Simultaneous determination of α-ketoglutarate, ergothioneine, pyrroloquinoline quinone and ascorbic acid in dietary supplements by HPLC-UV: Method validation and quality control application

α-Ketoglutarate (AKG), ergothioneine (EGT), ascorbic acid (VC), and pyrroloquinoline quinone (PQQ) are increasingly co-formulated to combat age-related mitochondrial dysfunction and oxidative stress, driving an urgent need for reliable quality control. However, existing analytical approaches remain fragmented, targeting only one or two compounds, relying on time-consuming derivatization, or employing costly mass spectrometry, and critically failing to address chemical incompatibilities among analytes. This study presents the first validated HPLC-UV method for simultaneous quantification of all four bioactives. A segmented extraction strategy was developed to reconcile their physicochemical conflicts: VC, AKG, and EGT were extracted with 0.1% metaphosphoric acid, while acid-labile PQQ was separately extracted with neutral phosphate buffer. Chromatographic separation was achieved on an AQ-C18 column with a gradient of 50 mM Na₂HPO₄ and acetonitrile. The method demonstrated excellent linearity (R² > 0.99), high sensitivity, good precision (RSD < 2%), and satisfactory accuracy (recoveries: 97.2–103.6%). The Mosaic-WAC score of 82.6 further validates the proposed method. Analysis of commercial products indicated that only VC exhibited degradation, with more pronounced losses in liquid matrices, attributable to its inherent oxidizability. This underscores the need for comprehensive multi-component quality control. The proposed method provides a practical platform for routine analysis and regulatory evaluation of complex nutraceutical products.