Cocoa flavanols (catechins and procyanidins) can exist in various polymerization states and are commonly classified by their degree of polymerization (DP). There is increasing evidence that flavanols of distinct DP possess different biological activities, but separation and quantification of the higher DP procyanidins is challenging and has thus created the need for new methodologies that utilize advancements in columns and LC-MS/MS systems. An aqueous normal phase (hydrophilic interaction liquid chromatography, HILIC), UPLC method with post-column ESI adjuvant infusion was developed to reduce the total analysis time, increase peak separation, and increase detection specificity (compared to traditional fluorescence methods) by coupling with mass spectrometry detection. The total elution time was reduced from 70 to 90 min (typically used for normal phase and HILIC HPLC separation of procyanidins) down to 9 min by employing UPLC. Results indicate that by using a post-column 0.04 M ammonium formate infusion (5 μL/min), ionization of procyanidins was significantly enhanced. Lower limits of detection ranged from 3.19 × 10-2 to 4.56 pmol-on-column, and lower limits of quantification ranged from 2.79 × 10-2 to 1.17 × 102 pmol-on-column across compounds DP 1-9. This method builds upon the foundation set by existing analytical methods and employs new technologies to dramatically increase sample throughput and enhance detection limits and specificity, facilitating improved analysis for procyanidins.
Keywords: Ammonium formate; Cocoa; Diol; Electrospray ionization; Sensitivity.
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