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

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 (R) 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 degrees C. Preparation of the poly(BMA-co-EDMA) also demanded overnight heating at 60 degrees 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.