Some recent high-performance liquid chromatography assays of steroid hormones and their conjugates.

because they reported the two isomers Galp1-3GlcNAcfil3GalNAc-01 and Galp 1 -4GlcNAc~l-3GalNAc-01 in pure form from a preparation of human bronchial mucins which had initially been purified in this way rather than by BioGel P4 gel filtration. Several studies have been reported using classical ion-exchange chromatography in monoand oligo-saccharide analysis. In general (Jandera & Churacek, 1974; Goulding, 1975; Ladisch et al., 1978; Scobell & Brobst, 1981), it was found that for cation-exchange resins eluted with water and anion-exchangers eluted with water or borate buffers. but not aqueous ethanol, the retention times for oligosaccharides increases with decreasing number of hydroxyl groups. A separation is therefore achieved mainly by size exclusion. These ion-exchange systems adapted to h.p.1.c. may therefore offer an alternative to classical size exclusion chromatography and, as shown by the study reported above (Van Halbeek et al., 1982), may allow further separation of isomers. Interestingly, Bio-Gel P4 chromatography also gave a partial separation of the two trisaccharide isomers referred to above from human meconium and differing in a 1-3 or 1-4 linkage only, as they were obtained in different ratios in two fractions from human meconium oligosaccharides (Hounsell et a/ . , 1985). Studies using classical ion-exchange columns eluted with aqueous ethanol showed a different type of separation based on the stronger interactions of oligosaccharides having a greater number of hydroxy groups with the waterrich hydration sphere of the resin (Jandera & Churacek, 1974). We have used a Micropak AX-5 anion-exchange column (Varian Associates) with aqueous acetonitrile, as reported by Mellis & Baenziger (1981), for the separation of oligomers of glucose and of N-acetylglucosamine and for high-mannose-type oligosaccharides, and have achieved the separation of the two isomers Galpl-3GlcNAc~1-3GalNAc-ol and Gal01 -4GlcNAc01-3GalNAc-01 obtained from meconium glycoproteins, with the latter having the longer retention time. However, a separation of LNT-ol and LNNT01 could not be achieved using several different acetonitrile/ water eluents. This system is currently under investigation for separation of other oligosaccharide isomers isolated from milk and human meconium glycoproteins.