Pyridinylboronic acid-functionalized organic–silica hybrid monolithic capillary for the selective enrichment and separation of cis-diol-containing biomolecules at acidic pH

Boronate affinity chromatography (BAC) is a unique means for the selective separation and enrichment of 1,2 and 1,3 cis-diol-containing compounds. However, conventional boronate affinity materials require a basic binding pH (usually ≥ 8.5), which gives rise to not only inconvenience in operation but also the risk of degradation of labile compounds. Although the applicable pH has been expanded to 5.0 in recent years, the current boronate affinity materials still fail to meet the acidic pH end of frequently used biosamples, particularly urine (pH 4.5). In this study, we report a 3-pyridylboronic acid-functionalized organic–silica hybrid monolithic capillary that exhibited a binding pH of 4.5, the lowest so far in BAC. Such a binding pH enabled direct extraction of cis-diol-containing biomolecules such as nucleosides from urine samples without pH adjustment. The boronate affinity monolithic capillary showed enhanced affinity toward negatively charged cis-diol-containing analytes such as ribonucleotides. Moreover, it could function as an anion exchanger at acidic pH (∼2). The column was found to retain multiple compounds from urine, which can be assumed to be at least mostly if not entirely cis-diol-containing compounds.