Reversed-Phase Liquid Chromatography Methods Based on C18 Polymer Monoliths for the Determination of Urinary Myoglobin

Abstract

This study describes reversed-phase liquid chromatography (RPLC) methods to quantify urinary myoglobin using polymer monolithic columns produced by copolymerization of stearyl methacrylate (SMA) and ethylene glycol dimethacrylate (EDMA). The columns were prepared in the coffins of 1.5 mm internal diameter (i.d.) ethylene tetrafluoroethylene (ETFE) tubing for use in sequential injection chromatography (SIC) and solid phase extraction (SPE), and inside 1.0 mm i.d. Silcosteel® tube for use in narrow-bore liquid chromatography. The monoliths inside the ETFE were produced via UV, whereas thermal polymerization formed the monoliths inside the Silcosteel® tube. The separation of carbonic anhydrase, lysozyme, and myoglobin was demonstrated because they may occur simultaneously in urine samples. Quantification was undertaken by external calibration, and the accuracy was evaluated by the spiking/recovery strategy. The methods exhibited linearity from 5.0 to 60 µg mL-1 (SIC), 2.5 to 50 µg mL-1 (high-performance liquid chromatography (HPLC)), and 1.0 to 7.5 µg mL-1 for an SPE-HPLC method. The lowest limits of detection and quantifications were 0.13 and 0.43 µg L-1, respectively, obtained after concentrating myoglobin by SPE. Recoveries ranged from 98 to 105%. The low cost, simplicity, reusability, and analytical features provided by these polymeric stationary phases make them affordable alternatives to routine analyses of urinary myoglobin.