HPLC separation and determination of dicoumarol and other simple coumarins in sweet clover


  • Katarína Hroboňová
  • Jana Sádecká
  • Jozef Čižmárik




HPLC-DAD, Coumarins, Sweet clover, Synchronous fluorescence


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−1 for dicoumarol, 0.05 – 100 μg.mL−1 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).




How to Cite

Hroboňová, K. ., Sádecká, J. ., & Čižmárik, J. . (2018). HPLC separation and determination of dicoumarol and other simple coumarins in sweet clover. Nova Biotechnologica Et Chimica, 17(1), 95–102. https://doi.org/10.2478/nbec-2018-0010



Research Articles