Tandem quadrupole mass spectrometry HPLC-ESI-MS/MS CID-MS/MS Multiple reaction monitory (MRM) Quercetin Apigenin Kaempferol a b s t r a c t The presence of three flavonols (quercetin, kaempferol, myricetin) and two flavones (apigenin, luteolin) were investigated in the extract of fresh spinach leaves. Aqueous spinach extracts were prepared with a leaf/water ratio of 1:2 at 80 °C for 30 min stirring. Ferric ammonium sulphate method was used for measuring total polyphenols in the extracts and expressed as catechins and tannic acid equivalents. The flavo-noids glycosides in the extract were hydrolysed to their aglycons with 1.2 M HCl in boiling 50% water methanol solution. The resulting aglycons were identified and quantified by a C18 reverse phase high-performance liquid chromatography (RP-HPLC). Furthermore, the results were confirmed by HPLC coupled to an electrospray ionisation tandem triple-quadrupole mass spectrometer ESI-MS performing low-energy collision induced dissociation (CID-MS/MS) in the collision cell (HPLC-ESI-MS/MS). Analyses were made in the multiple reaction monitory (MRM) mode. Results showed that total polyphenols contents in fresh spinach leaves were 270 mg kg À1 and 390 mg kg À1 as tannic acid and catechin equivalents, respectively, in which, major flavonoids aglycons were apigenin (170 mg kg À1), quercetin (50 mg kg À1) and kaempferol (30 mg kg À1).