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Robustness testing of the high throughput HPLC-based analysis of plasma N-glycans

Plasma glycan analysis using high throughput HPLC- based 96 well platform became a standard procedure for analyzing large pool of samples for a studies compromising thousands of observed individuals. Analytical method which is used to obtain such a huge amount of data should be well characterized and all potentially critical steps should be known. Robustness of the high throughput method was tested by Plackett Burman two level, 11- factor, 12 experiment screening design. It provides the valuable information about few most important factors on which the further optimization should be focused. For initial screening eleven potentially, but still as critical unproven factors, were chosen according to a long-term laboratory experience. Response variable was calculated as coefficient of variance between area % of each peak in each reaction and the area % obtained after performing the procedure according to the laboratory Standard operating procedure, with standardized plasma samples. Thirteen out of 16 by HPLC separated glycan groups revealed statistically significant changes according to changes in factor levels. Glycan groups did not displayed uniform response to 11 factors, which was expected due to their structural and chemical difference, but definitely showed clear pattern which directs method setup towards optimal standard procedure.

screening design; Plackett-Burman; N-glycosylation; glycan analysis; high throughput HPLC; plasma N-glycans

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