Development of a method for the quantitative metabolite profiling of pharmaceutical drugs using HPLC-ICP-MS following pre-column derivatization of their amino and hydroxyl groups using 4-aminopyridine as a model compound

Chemical derivatization allows the introduction of an ICP-MS accessible element into pharmaceuticals and their metabolites, thus extending the application range of HPLC-ICP-MS in pharmaceutical applications. In this study, such a pre-column derivatization approach was developed with the aim of using HPLC-ICP-MS for the quantitative metabolite profiling of pharmaceutical drugs in human plasma samples. We targeted the amino and hydroxyl groups, two of the most abundant functional groups present in drugs, and used 4-aminopyridine and 3-hydroxy-4-aminopyridine as model compounds. 4Iodobenzoyl chloride was used as a commercially available and multi-functional derivatization reagent to introduce iodine (I) as an element that can be quantified using ICP-MS with high sensitivity. This reagent reacts readily with the amino group present in 4-aminopyridine and the amino and hydroxyl groups present in its metabolite 3-hydroxy-4-aminopyridine. The reaction conditions were systematically optimized to guarantee quantitative formation of the target derivatives. Baseline separation of the target derivatives from one another and from also derivatized endogenous compounds was achieved using reversed phase UHPLC and gradient elution. Accuracy (recovery between 95 and 107%) and precision (repeatability # 6.9% RSD) were fit-for-purpose for both 4-aminopyridine and 3-hydroxy-4aminopyridine. The limit of quantification (LOQ) is 10 mu g L-1 for I, corresponding to 7.5 mu g L-1 of 4-aminopyridine and 5.8 mu g L-1 of 3-hydroxy-4-aminopyridine, respectively.