Determination of Proline Enantiomers in Honey by FMOC-LC-FL and Storage-Associated Changes in Enantiomeric Composition.

Abstract

Background: Proline is one of the most abundant free amino acids in honey and is widely used as a quality indicator. However, its enantiomeric composition has not been systematically investigated in relation to long-term storage.

Objective: This study developed a sensitive and selective method for the determination of D-proline (D-Pro) and L-proline (L-Pro) in honey using 9-fluorenylmethyl chloroformate (FMOC) derivatization followed by LC with fluorescence detection (LC-FL) and evaluated storage-associated changes in enantiomeric composition.

Methods: Fresh honeys and honeys stored at ambient temperature for approximately 20 years were analyzed. After solid-phase extraction cleanup and selective masking of primary amino acids, proline enantiomers were derivatized with FMOC and quantified by LC-FL.

Results: The method provided acceptable recoveries (90-98%) with a method detection limit of 0.03 μg/g, representing markedly improved sensitivity compared with a previous LC with ultraviolet detection (LC-UV) method. Both enantiomers were detected in all honeys. Long-term storage was associated with a marked decrease in L-Pro and a moderate but statistically significant increase in D-Pro (P < 0.05). Notably, the D-Pro/L-Pro ratio clearly differentiated fresh and long-term storage honeys.

Conclusion: These findings indicate that long-term storage is associated with significant changes in proline enantiomer composition, likely related to non-enzymatic processes such as Maillard-type reactions. The present study introduces stereochemically resolved proline analysis as a complementary perspective for honey quality evaluation.

Highlights: A sensitive FMOC-LC-FL method enabled enantiomer-resolved analysis of proline in honey, revealing storage-associated shifts in enantiomeric composition and demonstrating the potential of the D-Pro/L-Pro ratio as an additional quality indicator.