Preparation of entrapment-based microcolumns for analysis of drug-humic acid interactions by high-performance affinity chromatography

Reversible interactions between drugs and humic acid in water can be an important factor in determining the bioavailability and effects of these pharmaceuticals as micropollutants in the environment. In this study, microcolumns containing entrapped humic acid were used in high-performance affinity chromatography (HPAC) to examine the binding of this agent with the drugs tetracycline, carbamazepine, ciprofloxacin, and norfloxacin. Parameters that were varied to optimize the entrapment of humic acid within HPLC-grade porous silica included the starting concentration of humic acid, the mass ratio of humic acid vs silica, and the method of mixing the reagents with the support for the entrapment process. The highest retention for the tested drugs was obtained when using supports that were prepared using an initial humic acid concentration of 80 mg mL-1 and a humic acid vs silica mass ratio of 600 mg per g silica, along with preincubation of the humic acid with hydrazide-activated silica before the addition of a capping agent (i.e., oxidized glycogen). Characterization of the humic acid support was also carried out by means of TGA, FTIR, SEM, and energy-dispersive X-ray spectroscopy. The binding constants measured by HPAC for the given drugs with entrapped Aldrich humic acid gave good agreement with values reported in the literature under similar pH and temperature conditions for this and other forms of humic acid. Besides providing valuable data on the binding strength of various drugs with humic acid, this work illustrates how HPAC may be used as an analytical tool for screening and characterizing the interactions of drugs and man-made contaminants with humic acid or related binding agents in water and the environment.

Automated summary

Reversible interactions between drugs and humic acid in water explain the bioavailability and effects of pharmaceuticals as micropollutants in the environment. High-performance affinity chromatography (HPAC) was used to study the binding of tetracycline, carbamazepine, ciprofloxacin, and norfloxacin with entrapped humic acid. Optimal entrapment of humic acid in HPLC-grade porous silica was achieved using an initial humic acid concentration of 80 mg mL-1 and a humic acid vs silica mass ratio of 600 mg per g silica. HPAC measurements showed good agreement with literature values, and characterization of the humic acid support was conducted using TGA, FTIR, SEM, and energy-dispersive X-ray spectroscopy. This study demonstrates the utility of HPAC for screening and characterizing drug interactions with humic acid or related binding agents in water and the environment.