The seeds of Oroxylum indicum Vent. play an important role in Traditional Chinese Medicine to treat infectious diseases of the respiratory tract. In this manuscript the first HPLC-MS assay for the separation of all major flavonoids as well as their isolation and quantification is reported. The developed method was fully validated and showed to be accurate (recovery rates≥96.2%), precise (intra-day σ(rel)≤4.83%, inter-day σ(rel)≤5.23%), repeatable (σ(rel)≤2.57), sensitive (LOD: 0.05-0.34 μg/ml) and linear (R(2)≥0.999). Four samples of Oroxyli semen from different origins were separated with this optimized HPLC procedure on a Phenomenex Luna 5 μ C8 (2) 100 Å column using acetonitrile and water/0.1% formic acid as mobile phase. Temperature, flow rate and injection volume were set to 25 °C, 0.5 ml/min and 10 μl, respectively. The most dominant flavonoid showed to be Baicalein-7-O-β-D-gentiobiosid, occurring in the specimens in percentages ranging from 0.92 to 4.08%.
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