Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC)

Abstract

N-Hydroxysuccinimide (NHS) esters are the most important activated esters used in many different bioconjugation techniques, such as protein labelling by fluorescent dyes and enzymes, surface activation of chromatographic supports, microbeads, nanoparticles, and microarray slides, and also in the chemical synthesis of peptides. Usually, reactions with NHS esters are very reliable and of high yield, however, the compounds are sensitive to air moisture and water traces in solvents. Therefore, the quantification of NHS would be a very helpful approach to identify reagent impurities or degradation of stored NHS esters. No robust and sensitive method for the detection of NHS (or the more hydrophilic sulfo-NHS) has been reported yet. Here, a chromatographic method based on HILIC conditions and UV detection is presented, reaching a detection limit of about 1 mg L⁻¹, which should be sensitive enough for most of the applications mentioned above. Exemplarily, the hydrolytic degradation of a biotin-NHS ester and a purity check of a fluorescent dye NHS ester are shown. An important advantage of this approach is its universality, since not the structurally variable ester compound is monitored, but the constant degradation product NHS or sulfo-NHS, which avoids the necessity to optimize the separation conditions and facilitates calibration considerably.