Heparan sulphates (HSs), a specific class of glycosaminoglycans (GAGs), are important
participants of cellular signalling. Analytical characterization of GAGs requires
a complex sample preparation workflow. Although a detailed stability and recovery
study is available for the chondroitin sulphate GAG class, the literature concerning
HS is incomplete in this regard. Therefore, our aim was to systematically investigate
various parameters that could potentially influence the stability and recovery of
HS samples when performing disaccharide analysis using high‐performance liquid chromatography–mass
spectrometry. First, effects concerning vacuum evaporation and freezing were investigated.
Next, the storage stability of the HS disaccharides was analysed under several conditions
such as temperature, pH, digestion buffers, injection solvents and storage vessels.
We have identified several critical parameters influencing the stability and recovery
of HS disaccharides. We concluded that major sample loss is expected when Tris–HCl
is used as digestion buffer, followed by vacuum evaporation at elevated temperatures,
or samples are stored under alkaline conditions. Following the practical considerations
of this paper can contribute to increasing the reliability of future analytical measurements.
