In this study, a new miniaturized and integrated analytical system was developed based on the in-line coupling of boronate affinity solid phase extraction with capillary isoelectric focusing separation and UV detection. This original coupling takes advantage of the selective enrichment of cis-diol-containing compounds using a boronate affinity sorbent and the exceptional focusing features of isoelectric focusing process. Such coupling has been used for preconcentration/purification and separation of urinary catecholamines (dopamine, adrenaline and noradrenaline) as proof of concept. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column (8 cm) was chosen as solid phase extraction support due to its good chemical stability and its easy and versatile surface functionalization. Characterization of the miniaturized boronate affinity monolith column (μBAMC) was done by frontal affinity chromatography. An active-site amount of about 0.16 nmol cm-1 (75 μm i. d.) of phenyl boronic acid groups and Kd values ranging from 224 to 106 μM were obtained for catechol and catecholamines. A high loading volume (up to 15 times the affinity column volume) can be introduced with quantitative recovery yields. Optimization of the in-line coupling concerned the adaptation of (i) the μBAMC volume, (i.e. length and inner diameter of the monolithic column) for loading of large sample volumes and (ii) the CIEF experimental conditions. The ampholyte mixture was adapted (i.e. nature and concentration of carrier ampholytes, volume of sacrificial electrolytes) in order to ensure elution and separation of catecholamines and to decrease limit of detection down to 10-20 ng ml-1. The optimized method was applied to analyze urine samples.
Keywords: Boronate affinity chromatography; Capillary isoelectric focusing; Catecholamines; In-line coupling; Poly(GMA-co-EDMA) monolith; Urine.
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