Validation of a high‑performance thin‑layer chromatography method for the quantitative determination of trehalulose

A novel, simple and reliable high-performance thin-layer chromatography (HPTLC) method was developed and validated for the quantification of trehalulose in stingless bee honey. The chromatographic separation was performed using silica gel 60 F 254 HPTLC plates and 1-butanol‒2-propanol‒aqueous boric acid solution (5 mg/mL) (30:50:10, V/V) as the mobile phase. The retardation factor (RF ) for trehalulose was found to be 0.045. The method showed linearity over the concentration range of 100–800 ng per band with a coefficient of correlation (R) of 0.9996. The limit of detection and limit of quantification for trehalulose were found to be 20.04 ng per band and 60.72 ng per band, respectively and the mean per cent recovery of trehalulose was 101.8%. The method has been validated for its specificity, linearity, sensitivity, precision, accuracy, repeatability and robustness following the International Council for Harmonisation Q2 (R1), and it has been successfully applied in the determination of trehalulose in stingless bee honey.