Optimisation of preparative HPLC separation of four isomeric kaempferol diglycosides from Prunus spinosa L. by application of the response surface methodology

- Response surface methodology was used for optimisation of preparative HPLC separation.
- Four isomeric kaempferol diglycosides were isolated in high yield and purity.
- Structures were identified by extensive NMR studies and hydrolytic experiments.
- The isolates may be analytical and chemotaxonomic markers of P. spinosa leaves.

A fast and efficient preparative HPLC-PDA method was developed for the separation and isolation of four rare isomeric kaempferol diglycosides from leaves of Prunus spinosa L. The separation procedure of the enriched diglycoside fraction of the 70% (v/v) aqueous methanolic leaf extract was first optimised on analytical XBridge C18 column (100 mm × 4.6 mm i.d., 5 μm) and central composite design combined with response surface methodology was utilized to establish the optimal separation conditions. The developed method was directly transferred to preparative XBridge Prep C18 column (100 mm × 19 mm i.d., 5 μm) and the final separation was accomplished by isocratic elution with 0.5% acetic acid-methanol-tetrahydrofuran (75.2:16.6:8.2, v/v/v) as the mobile phase, at a flow rate of 13.6 mL/min, in less than 12 min for a single run. Under these conditions, four flavonoid diglycosides: kaempferol 3-O-α-l-arabinofuranoside-7-O-α-l-rhamnopyranoside, kaempferol 3,7-di-O-α-l-rhamnopyranoside (kaempferitrin), and reported for the first time for P. spinosa kaempferol 3-O-β-d-xylopyranoside-7-O-α-l-rhamnopyranoside (lepidoside) and kaempferol 3-O-α-l-arabinopyranoside-7-O-α-l-rhamnopyranoside, were isolated in high separation yield (84.8-94.5%) and purity (92.45-99.79%). Their structures were confirmed by extensive 1D and 2D NMR studies. Additionally, the UHPLC-PDA-ESI-MS3 qualitative profiling led to the identification of twenty-one phenolic compounds and confirmed that the isolates were the major components of the leaf material.

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