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Updated: Mar 19, 2026

Extraction and Analysis of Taiwanese Green Propolis
Published on: January 7, 2019
Determination of Proline Enantiomers in Honey by FMOC-LC-FL and Storage-Associated Changes in Enantiomeric
Koichi Saito1, Ayano Nakazato1, Rie Ito1
1Hoshi University, Department of Analytical Chemistry, School of Pharmacy and Pharmaceutical Sciences, 2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
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.
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