Use of secondary equilibria in reversed-phase high-performance liquid chromatography for the determination of dissociation constants of polyprotic leukotrienes

Observed retention factors were determined for leukotriene B-4, leukotriene E-4 and N-acetylleukotriene E-4 using mobile phases with different percentages of organic modifier in the pH range from 2 to 12. A two-stage least squares fitting procedure for fitting a general equation to the observed retention factors was used to calculate the dissociation constants at each percentage of organic modifier. The obtained dissociation constants were extrapolated to give pK(a) values in 100% aqueous solutions. The pK(a) values thus calculated for the leukotrienes were of a magnitude to be expected for compounds of this kind. The polyprotic leukotriene E4 has a pK(a1) of 1.54, a pK(a2) of 3.41, and a pK(a3) of 10.16.