Analysis of potato glycoalkaloids by immunoassay coupled to capillary electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry.

摘要

The use of separation or spectroscopic techniques to analyze the products of an immunoassay can result in faster analysis times, more accurate analyte identification, or increased sensitivity. The objective of this thesis was to develop methods for determining potato glycoalkaloids (GAs) by coupling immunoassays with capillary electrophoresis or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).; A solution-phase immunoassay for the determination of GAs was developed based on quantitation by capillary electrophoresis with laser-induced fluorescence detection. Solanidine coupled to 4′-(aminomethyl)fluorescein and polyclonal antibody solution were used as the immunoreagents. Unbound fluorescent solanidine was detected by capillary electrophoresis. A calibration curve of signal vs log [GA] was linear from 50–400 nM. The relative standard deviation (RSD) for duplicate and day-to-day analyses averaged 5.7% and 12%, respectively. Spike recoveries ranged from 85–97% for spike levels ranging from 43–170 μg/g fresh potato. MALDI-TOF MS was used to determine individual potato GA concentrations in tubers. Samples were extracted with 50% aqueous methanol, deposited on 2′,4′ ,6′-trihydroxyacetophenone crystals and analyzed by MALDI-TOF MS. Analyte ion intensities relative to an internal standard were used to determine α-chaconine and α-solanine concentrations. The RSD of triplicate measurements ranged from 1 to 16%, with an average of 9%. The day-to-day RSD for replicate determinations was 11%. Analyst prepared spike recoveries (50 μg/g) averaged 104% for α-chaconine (RSD 8%) and 98% for α-solanine (RSD 4%). The method limit of detection was estimated to be 2 μg/g. The method was modified to determine GAs in potato pulp, potato protein concentrate, fruit water, and starch.; A sample purification technique was developed for detecting GAs in blood serum by MALDI-TOF MS. GAs were extracted from spiked serum (5 mL) using a C₁₈ solid-phase extraction cartridge. The GAs were then selectively captured on antibody-coated agarose beads. The agarose beads were washed with water and the GAs eluted with 25 μL methanol. MALDI-TOF MS was used to detect the GAs in the methanol eluent. α-Chaconine and α-solanine were detected in serum spiked with 1 ng/mL of each GA. This represents the first known use of immunoaffinity purification with MALDI-TOF MS for food analysis.