Micro-extraction by packed sorbent coupled on-line to a column-switching chromatography system – A case study on the determination of three beta-blockers in human urine

Abstract

A new approach for the automation of Micro-Extraction by Packed Sorbent (MEPS) as a solid phase extraction technique coupled directly to a column-switching chromatography system was presented. A new on-line hyphenation of the modern micro-extraction procedure with liquid chromatography was developed and used for human urine sample clean-up and pre-concentration of three beta-blockers - metoprolol, labetalol and propranolol before separation and determination. A commercial MEPS C-18 cartridge was coupled using Teflon holders into an HPLC system through a high-pressure six-port switching valve. Chromatographic separation was performed on a Triart YMC C-18 (50 mm × 4.6 mm) analytical column with 5 μm multi-layered particles. The multi-layered particle column showed extremely low flow-resistance (<4.1 MPa) at a flow rate of 1.0 mL min−1, and thus direct connection with a MEPS cartridge was possible. A mixture of acetonitrile and an aqueous solution of 0.5% triethylamine with acetic acid, with pH adjusted to 4.5 in the linear gradient elution mode was used. This mobile phase eluted beta-blockers from the MEPS mini-column directly onto the analytical column where the separation was carried out. Metoprolol, labetalol and propranolol were detected by a fluorescence detector at the λex = 220 nm and λem = 315 nm wavelengths. The developed on-line MEPS-HPLC coupling showed satisfactory validation results with recovery ranging from 94.0 to 104.7% and precision in the range of 0.6–9.5% for all beta-blockers.