Ultra-high pressure liquid chromatography coupled with mass spectrometry (MS) was employed for the determination of coumestrol in lucerne species (Medicago sativa and Medicago lupulina) growing in Lithuania. Coumestrol was extracted with an acidified (2 mol/L HCl) methanol/water (8:2, v/v) solution. Optimized separations were carried out on the Acquity BEH C18 column in the gradient elution mode using the mobile phase composed of water and methanol containing 0.25% (v/v) acetic acid. MS detection was performed in the selected ion monitoring mode using a negative ion electrospray ionization source. The calibration curve was linear over the concentration range 0.05–5.00 mg/L. The limit of detection and the limit of quantification were 0.015 and 0.05 mg/L, respectively. The measured recoveries of coumestrol were in the range 88.5–96.1% with RSD values ranged from 4.5 to 7.2%.

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