Application of a Small Protein-Coated Column to Trap, Extract and Enrich Carbamazepine Directly from Human Serum for Direct Chromatographic Analysis

Abstract

An automated solid phase extraction (SPE) protocol to determine carbamazepine in human serum has been developed and validated using a simple, rabid and sensitive liquid chromatography-based bio-analytical method. Extraction of carbamazepine was carried out using an on-line SPE tool of a short protein-coated (PC) ODS silica pre-column (PC-ODS-pre-column) and phosphate buffer saline (PBS) with a pH of 7.4 as an extraction solvent. There are two distinct chromatographic modes used by PC-ODS-pre-column. While carbamazepine trapping required reversed-phase liquid chromatography, proteins were extracted from serum samples using PBS by size-exclusion liquid chromatography. Then, carbamazepine was eluted from the PC-ODS-pre-column onto the quantification position using a mixture of methanol-distilled deionized water (50:50, v/v) as an eluent and ODS analytical column. At room temperature (22 ± 1 °C), carbamazepine was completely separated from the co-eluted matrix components and detected at 230 nm. Carbamazepine’s linearity was obtained at concentrations ranging from 50 to 10,000 ng/mL. With good accuracy and precision, carbamazepine recoveries in serum samples ranged from 86.14 to 97.82%. The extraction step was conducted using PBS as a safe and green extraction solvent, making this protocol both cost-effective and ecologically safe.