Liquid Chromatography–Charged Aerosol Detection for Characterization of Natural Toxin Reference Materials

Developing certified reference materials (CRMs) for natural toxins is challenging due to the scarcity of raw materials, their high toxicity, and structural complexity. Typically, purified toxins in microgram to low milligram amounts are quantitated as stock solutions using quantitative nuclear magnetic resonance spectroscopy (qNMR) and then accurately diluted to their final concentrations. With the limited sensitivity of qNMR, additional techniques are required for homogeneity assessment, quantitation, and stability monitoring of less concentrated solutions. Liquid chromatography with charged aerosol detection (LC–CAD) enables nonspecific detection of compounds down to nanogram-on-column levels. A reversed-phase LC–CAD method was developed that also incorporates MS and DAD. Large volume injection programs were implemented to improve detection for samples at low μg mL–1 concentrations. A retention index standard was used to monitor the overall method performance, while selectivity, repeatability, reproducibility, and response uniformity were evaluated using calibration solution CRMs from several toxin classes. Estimated repeatability was 3.9%, while the intermediate precision of normalized relative response factors was 1.6–8.5%. A relative response factor RSD of 13% was observed for 19 toxins from six different classes, but an average relative response factor of 4.8% within the class demonstrated that LC–CAD is highly suitable for within-class quantitation. The utility of LC–CAD is demonstrated at multiple stages of toxin calibration solution CRM development including purity assessment of stock materials, postcertification stability monitoring, and characterization measurements. The work highlights the important and broadly applicable role that LC–CAD can play in the development of vital reference materials for sample-limited natural products.