@article{Hroboňová_Sádecká_Čižmárik_2018, title={HPLC separation and determination of dicoumarol and other simple coumarins in sweet clover}, volume={17}, url={https://journals.scicell.org/index.php/NBC/article/view/493}, DOI={10.2478/nbec-2018-0010}, abstractNote={<p>Dicoumarol is a mycotoxin, that acts as a blood anticoagulant, is formed during the microbial action of molds and fungi in spoiled hay or silage containing high-coumarin plant. A HPLC-DAD method for determination of coumarins, including dicoumarol, coumarin, and 4-hydroxycoumarin was developed. Methanol and acetic acid were used as mobile phase with gradient elution. The simultaneous separation was performed using C18 type of stationary phase. The recoveries were 88.6 – 92.6 %, 91.8 – 95.0 %, and 89.7 – 94.1 % (evaluated for three concentration levels) for dicoumarol, coumarin, and 4-hydroxycoumarin respectively. The parameters of system suitability (repeatability of retention times and peak areas) were determined for evaluation of the method. The method showed a good linearity in the concentration range 0.7 – 100 μg.mL<sup>−1</sup> for dicoumarol, 0.05 – 100 μg.mL<sup>−1</sup> for coumarin and 4-hydroxycoumarin with correlation coefficients higher than 0.9885. Extracts of sweet clover herb, hay, and spoiled hay were subjected to HPLC-DAD analysis. The most abundant compound in sweet clover herb and hay extracts was coumarin. In spoiled sweet clover hay extract the 4-hydroxycoumarin was detected in addition. The formation of 4-hydroxycoumarin was also observed in the synchronous fluorescence spectra recorded at the wavelength difference of 90 nm (difference between emission and excitation wavelength).</p>}, number={1}, journal={Nova Biotechnologica et Chimica}, author={Hroboňová, Katarína and Sádecká, Jana and Čižmárik, Jozef}, year={2018}, month={Sep.}, pages={95–102} }