This study aimed to establish a high performance liquid chromatography coupled with refractive index detector (HPLC-RID) method for the determination of trehalulose in stingless bee honey, and apply it to actual samples from different stingless bee species, geographic origins, and harvest dates.

We collected stingless bee honey samples produced by different bee species in Yunnan and Hainan as well as Malaysia in 2019–2021 for analysis by HPLC-RID.

The samples were dissolved in pure water and filtered through a 0.22 μm nylon membrane. Baseline chromatographic separation of trehalulose from other oligosaccharides in honey was achieved on a Hi-Plex Pb column (300 mm × 7.7 mm, 8 μm) using pure water as the mobile phase. The method had high recoveries (ranging from 93.50%–95.71%, with relative standard deviation (RSD) not larger than 0.61%) and good repeatability and accuracy. The content of trehalulose in stingless bee honey ranged from 7.0% to 30.8%, and varied significantly across harvest years and bee species (P < 0.05).

The proposed method is simple, rapid and accurate and can be used for the determination of trehalulose in stingless honey for quality control purposes. Stingless bee honey is rich in trehalulose, which can be a potential quality index for the identification and evaluation of stingless bee honey.