Anal. Chem. 1999, 71, 102-108
A Dual-Detection Strategy in the Chromatographic
Analysis of 2-Aminoacridone-Derivatized
Oligosaccharides
Helen Birrell, Joanne Charlwood, Ian Lynch, Simon North, and Patrick Camilleri*
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K.
However, its physicochemical properties permit a wider scope in
the use of this derivatizing agent. In our studies, we have found
that 2-AMAC carbohydrate derivatives can be analyzed by a variety
of separation (reversed and normal-phase chromatography8 and
micellar electrokinetic capillary chromatography9-12) and detection
(UV, fluorescence,8-12 electrospray, and matrix-assisted laser
desorption/ ionization mass spectrometry8,13) techniques.
We now report on the application of 2-AMAC for the prelimi-
nary characterization of glycan mixtures on the basis of their
retention properties on a normal-phase chromatographic stationary
phase, and using an aqueous mobile phase. To enhance the
accuracy of our measurements, we have mixed the 2-AMAC
derivatized mixtures with a dextran ladder derivatized with methyl
4-aminobenzoate (M-4AB) prior to chromatographic separation.
The fluorescence and UV properties of 2-AMAC are complemen-
tary to those of M-4AB, allowing the simultaneous and distinctive
monitoring of the two sets of carbohydrate derivatives by
fluorescence and UV absorbance, respectively. This internal
standardization procedure over the whole length of the chromato-
gram removes the necessity to run dextran ladder standards
before and after the chromatographic analysis of a glycan mixture
of interest. The methodology we describe thus saves time and
ensures that peak movements between one chromatogram and
another can be accounted for. It also allows meaningful interpreta-
tion of chromatographic profiles before and after enzymic diges-
tion.
A protocol has been developed involving the derivatization
of glycan mixtures with 2 -aminoacridone and co-injection
with a dextran ladder derivatized with methyl 4 -ami-
nobenzoate (M-4 AB). These two derivatizing agents have
very different ultraviolet absorbance and fluorescence
characteristics. A chromatographic separation using a
normal-phase column support followed by in-series UV
and fluorescence detection allowed simultaneous analysis
of the two mixtures of the separately derivatized carbo-
hydrates without any interference. This new approach
uses the M-4 AB dextran ladder derivatives as internal
standards spanning the whole chromatogram, allowing an
accurate and detailed comparison of glycosylation profiles.
It also saves much time by avoiding the necessity of
“sandwiching” an unknown glycan mixture between two
chromatographic runs of a dextran ladder. The use of this
technique has been demonstrated in the case of glycans
released from ribonuclease B and human IgG.
The role played by oligosaccharides in modulating the phys-
icochemical and structural properties of glycoproteins is now
widely accepted.1,2 This increased understanding in the biological
function of these molecules largely stems from the fact that, over
the last 10 years, major developments have occurred in the
structural analysis of picomole levels of carbohydrate. Although
direct analysis of oligosaccharides using ion-exchange chroma-
tography at alkaline pH’s and amperometric detection has been
used extensively,3,4 derivatization prior to analysis facilitates
detection due to the formation of highly volatile5 or fluorescent6
molecules which can be analyzed by gas or high-performance
liquid chromatography, respectively.
We have found that 2-aminoacridone (2-AMAC), covalently
linked to carbohydrates via reductive amination, is an excellent
reagent for the analysis of complex glycan mixtures. Besides being
an intense fluorophore, 2-AMAC is highly hydrophobic; it is also
neutral over a wide pH range. 2-AMAC had been used originally
for the analysis of carbohydrates by slab-gel electrophoresis.7
EXPERIMENTAL SECTION
Materials. Sodium cyanoborohydride, ammonium formate,
dimethyl sulfoxide, trifluoroacteic acid, and methyl 4-aminoben-
zoate were supplied as >95% pure from Aldrich (Poole, U.K.).
Glacial acetic acid was purchased as ANALAR grade from Fisher
Scientific (Gillingham, U.K.). Acetonitrile was purchased from
Romil (Cambridge,U.K.). Aqueous solutions were prepared using
(8) Okafo, G.; Burrow, L.; Carr, S. A.; Roberts, G. D.; Johnson, W.; Camilleri,
P. Anal. Chem. 1 9 9 6 , 68, 4424-4430.
(9) Greenaway, M.; Okafo, G. N.; Camilleri, P.; Dhanak, D. J. Chem. Soc., Chem.
Commun. 1 9 9 4 , 1691-1692.
(1) Varki, A. Glycobiology 1 9 9 3 , 3, 97-130.
(2) Dwek, R. A. Chem. Rev. 1 9 9 6 , 96, 683-720.
(10) Camilleri, P.; Harland, G. B.; Okafo, G. Anal. Biochem. 1 9 9 5 , 230, 115-
122.
(3) Kumarasamy, R. J. Chromatogr. 1 9 9 0 , 512, 149-155.
(4) Anumula, K.; Taylor, B. P. Eur. J. Biochem. 1 9 9 1 , 195, 269-280.
(5) Dell, A. Methods Enzymol. 1 9 9 0 , 193, 647-660.
(6) Rudd, P. M.; Guile, G. R.; Kuster, B.; Harvey, D. J.; Opdenakker, G.; Dwek,
R. A. Nature 1 9 9 7 , 388, 205-207.
(11) Harland, G. B.; Okafo, G.; Matejtschuk, P.; Sellick, I. C.; Chapman, G. E.;
Camilleri, P. Electrochemistry 1 9 9 6 , 17, 406-410.
(12) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.;
Camilleri, P. Anal. Biochem. 1 9 9 6 , 240, 68-74.
(13) North, S.; Okafo, G.; Birrell, H.; Haskins, N.; Camilleri, P. Rapid Commun.
Mass Spectrom. 1 9 9 7 , 11, 1635-1642.
(7) Jackson, P. Anal. Biochem. 1 9 9 1 , 196, 238-244.
102 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
10.1021/ac9806759 CCC: $18.00 © 1998 American Chemical Society
Published on Web 11/26/1998