Abstract: Objective: This study explored experimental methods for selenium speciation analysis using high performance liquid chromatography coupled to atomic fluorescence spectrometry (HPLC-AFS) under different conditions, with the aim of identifying optimal conditions for improved efficiency, peak shape and resolution, and establishing a method for determining selenium species in tea. Methods: After passing the sample through a 0.22 μm filter membrane, five selenium species, selenocystine (SeCys2), methyl-selenocysteine (MeSeCys), selenite (Se(IV)), selenomethionine (SeMet), and selenate (Se(VI)), were separated by HPLC using a PRP-X100 ion-exchange column. The five selenium species were completely separated within 9 min via gradient elution using two mobile phases: (A) 40 mmol/L diammonium hydrogen phosphate + 5 mmol/L citric acid, pH 4.3, and (B) 60 mmol/L diammonium hydrogen phosphate + 5 mmol/L citric acid, pH 4.3. They were subsequently detected by AFS. Results: Within the concentration range of 2–100 μg/L, the standard curves for the five selenium species exhibited good linearity, with correlation coefficients not less than 0.999 4. The method detection limits ranged from 0.004 to 0.014 mg/kg. Under optimized conditions, recoveries from spiked sampled ranged from 80.2% to 108.0% with relative standard deviations (RSDs) of 1.1%–8.2%. Conclusion: This method effectively improves the detection efficiency of selenium species using HPLC-AFS and shortens the retention time, and can be widely applied for the determination of selenium species in tea.

Key words: selenium species; high performance liquid chromatography; atomic fluorescence spectrometry; tea; influencing factors