HPLC-DAD, HPLC-ESI-MS and HPLC-MS/MS analyses of aqueous preparations of Tiliae flos (Tilia platyphyllos)

Lime flower is used worldwide for its sedative and antispasmodic properties. Traditionally it is used for migraine, hysteria, feverish colds, and for raised arterial pressure associated with arteriosclerosis and nervous tension [1]. Besides flavonols, little is known for the rest of the phenol content. In the present study extensive HPLC-DAD, HPLC-ESI-MS and HPLC-MS/MS analyses were undertaken in the aqueous preparations of Tilia platyphyllos Scop. influorescences. An HPLC-DAD-ESI-MS method was developed and optimised for the quantitative determination of the constituents. Analyses of the ethanol extracts confirmed the predominance of flavonol glycosides and protocatechuic acid. In contrast, both decoction and infusion, which are nevertheless the traditional herbal preparations, were more complex, containing polar simple phenolics and low molecular weight procyanidins. The use of different HPLC columns permitted a good separation of the constituents and enabled their quantitation. The method showed good linearity, (r2 0.9999 for tiliroside and catechin and 0.9986 for quercetin-3-O-glucoside), intra/inter-day variability (%RSD <1.56 and 1.33) and real sample repeatability (%RSD <4.0). Preparative chromatographic investigations (Sephadex LH-20) and NMR analyses revealed the presence of procyanidin B4, while HPLC-MS/MS analyses enabled the identification of procyanidin trimers and tetramers. Overall, 20 constituents were detected and identified, belonging mainly to three classes of compounds: phenolic acid derivatives, condensed tannins and flavonol glycosides. Aqueous extracts contain a higher amount of procyanidins (strong chelating properties) than flavonols and caution should be taken upon frequent use of the drug. This is the first report of detailed analysis of the chemical composition of Tiliae flos.