Stereospecific Radioimmunoassays for d-Pseudoephedrine in Human Plasma and Their Application to Bioequivalency Studies

Abstract

Antiserum to d-pseudoephedrine was raised in New Zealand White rabbits in response to immunization with a conjugate of bovine serum albumin and d-pseudoephedrine-N-3-propionic acid. The hapten was prepared by reaction of methyl acrylate with d-pseudoephedrine, followed by ester hydrolysis. Sodium boro[3H]hydride reduction of dl-ephedrone gave [α-3H]-dl-ephedrine, and a Welsh rearrangement with acetic anhydride followed by deacetylation gave [α-3H]-dl-pseudoephedrine, which was used as a radioligand in radioimmunoassay procedures for direct plasma analyses. Three sensitive radioimmunoassay procedures were developed, two using [3H]pseudoephedrine as the radioligand and either adsorption on coated charcoal or polyethylene glycol precipitation for separation of antibody-bound from free radioligand. The third method used an [125I]tyrosine methyl ester analog of pseudo-ephedrine and charcoal separation, preceded by extraction and derivatization of pseudoephedrine with methyl acrylate. All three assays could detect 2.5ng of pseudoephedrine/ml. The antiserum was stereospecific, showing low cross-reactivities with l-pseudoephedrine and d-and l-ephedrines. d-Norpseudoephedrine and some other related compounds also had low cross-reactivity in these radioimmunoassay procedures. Excellent agreement was found between pseudoephedrine concentrations in human plasma determined by radioimmunoassay and by a standard GLC method. The utility of radioimmunoassay was illustrated by application of one of these procedures to an assessment of the bioequivalence of immediate- and sustained-release pseudoephedrine formulations in normal volunteers. A sustained-release preparation containing 120mg of pseudoephedrine hydrochloride given every 12hr was shown by AUC comparisons to be bioequivalent to an immediate-release tablet (containing 60mg of pseudoephedrine hydrochloride) given every 6hr.