Epinephrine (also referred to as adrenaline) is a hormone and neurotransmitter. It is a catecholamine, a sympathomimetic monoamine derived from the amino acids phenylalanine and tyrosine. This method can be used to determine and quantitate epinephrine and epinephrine sulfonate in biological fluids (urine, blood, serum) and drug formulations. Obelisc N columns are used to retain and separate epinephrine and epinephrine sulfonate in mixed-mode hydrophilic interaction chromatography. Epinephrine is retained by the combination of cation-exchange and HILIC mechanisms. Epinephrine sulfonate is retained by HILIC mechanism. Buffer concentration and pH, as well as the amount of acetonitrile, can be used to adjust retention of both compounds. Both compounds can be detected by UV, ELSD and LC/MS. Preparative separation is possible with volatile mobile phases (ammonium formate or ammonium acetate.