TECHNICAL NOTE Lactose does not interfere with the analysis of sialic acids as their 1,2-diamino-4,5-methylenedioxybenzene derivatives Véronique Spichtig & Philippe Rohfritsch & Sean Austin Received: 20 August 2010 / Revised: 17 November 2010 / Accepted: 29 November 2010 / Published online: 14 December 2010 # Springer-Verlag 2010 Abstract In 2007, Martin et al. developed a method for the analysis of sialic acids by HPLC following 1,2-diamino- 4,5-methylenedioxybenzene (DMB) derivatisation (Martín et al., Anal Bioanal Chem 387:2943–2949, 2007). Within the article, the authors noted that lactose interfered with the analysis, giving erroneously high results when lactose- containing products were analysed. Such an observation is important when analysing milk-based products, yet was contradictory to the observations of Nakamura et al. (Chem Pharm Bull 35(2):687–692, 1987) who demonstrated that DMB was specific for α-keto acids and did not react with simple sugars such as glucose or lactose. In order to clarify the situation, this phenome- non was investigated and it was confirmed that lactose does not interfere with the analysis. However, it was found that most commercial preparations of lactose do contain small amounts of sialic acids, either as the free monosaccharide or bound to lactose in the form of 3′ - and 6′ -sialyllactose. Keywords Sialic acid . DMB . N-acetylneuraminic acid . HPLC . Fluorimetry . Infant formula Abbreviations DMB 1,2-Diamino-4,5-methylenedioxybenzene HPAEC- PAD High-performance anion exchange chromatography with pulsed amperometric detection HPLC High-performance liquid chromatography Neu5Ac N-acetylneuraminic acid Neu5Gc N-glycolylneuraminic acid 3′SL 3′-Sialyllactose 6′SL 6′-Sialyllactose Introduction Sialic acids are becoming recognised as important components of milk-based products, and many companies now label the sialic acid content of such products. Methods suitable for controlling the content claims may be based on the derivatisation of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by reversed phase high-performance liquid chromatog- raphy (HPLC) [ 1, 2]. Recently, Martín et al. published an article on the analysis of sialic acid in infant formula using such a method [3]. In this publication, it was postulated that neutral sugars such as lactose may interfere with the analysis and should be removed prior to analysis. To this end, they introduced a clean-up procedure using a Dowex ion exchange resin and observed that the apparent sialic acid content of their samples was reduced after passing over the resin (1.51 mg/g compared with 1.79 mg/g). At the same time, our lab was working on a similar method [4]. However, lactose interference had not been investigated since Nakamura et al. [5] already carried out a thorough investigation of possible interfering compounds including a selection of neutral sugars (including lactose) and amino acids. They concluded that DMB was a specific label for α-keto acids, and the only non- α-keto acids V. Spichtig : S. Austin (*) Quality and Safety Department, Nestlé Research Centre, Vers-Chez-Les-Blanc, 1000 Lausanne, Switzerland e-mail: sean.austin@rdls.nestle.com P. Rohfritsch Chemistry Section, Nestlé UK Quality Assurance Laboratory, Haxby Road, York YO91 1XY, UK Anal Bioanal Chem (2011) 399:1917–1922 DOI 10.1007/s00216-010-4564-0