Optimization, validation, and greenness assessment of a capillary zone electrophoresis method for analysis of inorganic and organic anions in human milk oligosaccharides.

The development and validation of an accurate, selective, and eco-friendly capillary zone electrophoretic detection (CZE) method has been presented for concurrent measurement of inorganic and organic anions including chloride, sulfate, formic acid, citric acid, acetic acid, phosphate, and glutamic acid in Human Milk Oligosaccharides (HMOs) for the first time. An electrolyte composed of an aqueous solution of benzoic acid, 16.38 mM; l-histidine, 24.49 mM; and polyacrylamide 0.0025 % provided a satisfactory separation of all analytes of interest. The electroosmotic flow was reversed using polyacrylamide in the background buffer. UV detection was carried out at a wavelength of 230 nm using benzoic acid in running buffer. Analysis was conducted on a 60 cm (53 cm to the detector) x 0.75 μm i.d. fused-silica capillary at a potential of -30 kV and the temperature of 20 °C. The procedure had a linear response in the concentration range of 2-200 mg/L for all analytes with correlation coefficients ≥0.9997. Validation was performed based on the International Council for Harmonization (ICH). The National Environmental Methods Index and the analytical GREEness metric were the tools applied for greenness assessment. System suitability parameter was determined using the expanded uncertainty of the method estimated from method validation data. The applicability of the proposed procedure was shown in diverse real samples.