Abstract:In this paper，a high-performance liquid chromatography（HPLC）method was established to detect 5-hydroxymethylfurfural（5-HMF）in royal jelly，and the content change of 5-HMF in royal jelly during storage was monitored，so as to lay a foundation for the screening and evaluation of freshness markers for royal jelly.The optimized 5-HMF determination method included the precipitation of proteins by trichloroacetic acid，isocratic elution with a mixture of 0.4% phosphoric acid and acetonitrile（volume ratio 90 ∶10），separation with a C18 column，and detection at the wavelength of 285 nm.The method was simple，the 5-HMF chromatographic peak was symmetrical without interference，and the retention time was about 5.296 min.The linearity was good in the range of 0.01 μg/mL-4.00 μg/mL，and the determination coefficient R2was 0.999 8.The limits of detection and quantitation were 0.01 μg/mL and 0.025 μg/mL，respectively.The recoveries were in the range of 91%-105%，and the relative standard deviations were in the range of 1.5%-3.5%.When the royal jelly was stored in the dark at-18℃，4℃，and 25℃ for 150 d，the 5-HMF increased linearly with storage time，and the slope of the fitting equation increased with the temperature increasing.When the royal jelly was stored at room temperature in the light and 37℃ in the dark，5-HMF increased exponentially with storage time，and the rate of 5-HMF increased faster at 37℃.The content of 5-HMF could effectively reflect whether the royal jelly was overheated，stored for too long，and fresh.Combined with functional evaluation of royal jelly，5-HMF was expected to be used as a freshness marker for royal jelly.