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.

Sensitivity Enhancement for Separation-Based Analytical Techniques Utilizing Solid-Phase Enrichment Approaches and Analyte Derivatization for Trace Analysis in Various Matrices

“…Thus, major efforts have been made to design and evaluate innovative formats and efficient sorbent materials in order to improve their selectivity, specificity, and sorption capacity towards target analytes and enhance physicochemical or mechanical stability, among other SPE-related properties. Solventless sample preparation approaches based on analyte extraction and enrichment by online SPE, using phosphate buffers as washing solvents, have been proven to be viable and environmentally friendly alternatives to conventional solvent extraction techniques [20,21]. Fluconazole in serum has been directly quantified by trapping on a pre-column and subsequently separating on an analytical column, utilizing an elution mode by online SPE and HPLC methods (Figure 1) [21].…”

“…Solventless sample preparation approaches based on analyte extraction and enrichment by online SPE, using phosphate buffers as washing solvents, have been proven to be viable and environmentally friendly alternatives to conventional solvent extraction techniques [20,21]. Fluconazole in serum has been directly quantified by trapping on a pre-column and subsequently separating on an analytical column, utilizing an elution mode by online SPE and HPLC methods (Figure 1) [21]. Because of its simplicity of use, flexibility, rapid extraction time, safety, minimal organic solvent consumption, and high enrichment factor, solid-phase microextraction (SPME) is a promising sample pretreatment approach [22].…”