Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white

Abstract

This work demonstrates the determination of lysozyme in egg-white samples after enrichment and cleanup by weak cation exchange (WCX) following separation by reversed-phase liquid chromatography (RPLC). The WCX column was prepared from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) and functionalized with iminodiacetate (IDA). Reversed-phase columns were prepared using butyl methacrylate (BMA) and EDMA. Photopolymerization formed the poly(GMA-co-EDMA) column inside vinylized polypropylene tubes whereas poly(BMA-co-EDMA) used thermal polymerization inside functionalized Silcosteel® tubes. The preparation of poly(GMA-co-EDMA) was fast (about 2 h), from preparing the polypropylene tube to washing the formed monolith with acetonitrile (ACN), but functionalization demanded an overnight period of pumping IDA through the column immersed in a water bath thermostated at 80 °C. Preparation of the poly(BMA-co-EDMA) also demanded overnight heating at 60 °C, with subsequent washing of the formed monolith with ACN. Egg-white samples diluted at a 1:10 m v⁻¹ ratio in phosphate buffer (pH 7.0) were injected first through IDA@poly(GMA-co-EDMA) to retain lysozyme (pI 11.4) and remove the proteins with a pI < 7.0. Elution of the lysozyme from the cation exchange column was made with 5% (v v⁻¹) acetonitrile in 0.1% (v v⁻¹) TFA. RPLC then analyzed the eluate with a gradient from 5 to 50% ACN in 0.1% TFA. The limits of detection and quantification were 0.07 and 0.23 mg mL⁻¹, respectively. Egg-white lysozyme concentrations varied between 2.26 ± 0.06 and 4.41 ± 0.08 mg g⁻¹, and spiking/recovery experiments at two concentration levels (0.25 and 0.50 mg mL⁻¹) resulted in recoveries from 94 to 115%, thus demonstrating the columns working with orthogonal selectivity provided enrichment of less abundant lysozyme and accurate results, provided by an efficient cleanup of the sample matrix.