High-Performance Cation-Exchange Chromatography and Pulsed Amperometric Detection for the Separation, Detection, and Quantitation of N-Alkylated Imino Sugars in Biological Samples H. R. Mellor, A. Adam,* F. M. Platt, R. A. Dwek, and T. D. Butters 1 Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; and *Dionex (UK) Ltd., Camberley, Surrey, United Kingdom Received May 2, 2000 The use of imino sugars for the potential treat- ment of lysosomal glycolipid storage diseases and hepatitis virus infections requires accurate, quantitative measurement of these compounds in biological samples. We demonstrate here the versa- tility of cation-exchange chromatography and pulsed amperometric detection of a range of com- pounds that differ in both isometric structure and N-alkyl chain length. Although column retention ap- pears dependent upon residual charge on the imine function, successful isocratic separation can be achieved by secondary hydrophobic interactions. A series of N-alkylated deoxynojirimycin compounds containing C 1–10 alkyl chains are readily separated and detected by pulsed amperometry after cation suppression. Using experimentally derived response factors for imino sugars and measurement of peak areas we have developed a reliable method for quan- titatively determining concentrations in solution. A rapid protocol for the removal of protein and con- taminants in biological samples is described. This has allowed the successful measurement of imino sugars in animal tissues and will be useful for un- derstanding the factors involved in compound bio- availability and in the design of novel therapeutics. © 2000 Academic Press Key Words: imino sugar inhibitors; pulsed amperom- etry; lysosomal glycolipid storage diseases. The glycosphingolipid (GSL) 2 lysosomal storage dis- eases are human metabolic diseases where the sub- strate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunc- tion and pathology. The therapeutic options for treat- ing these diseases are relatively limited, and for the majority of these disorders there are currently no available therapies. The strategy that we have been exploring is substrate deprivation (1, 2). This approach uses a GSL biosynthesis inhibitor, N-alkylated deriva- tives of deoxynojirimycin (DNJ) and deoxygalac- tonojirimycin (DGJ) to balance the rate of GSL synthe- sis with the impaired rate of GSL breakdown. We have treated mouse models of Tay-Sachs and Sandhoff dis- ease with N-butyl-DNJ (NB-DNJ) and showed that substrate deprivation prevented GSL storage in the CNS (3), delayed symptom onset, and increased life expectancy by 40% in the Sandhoff mouse model (4). A clinical trial in type I Gaucher patients has been initi- ated by Oxford GlycoSciences in the United Kingdom, Israel, Czechoslovakia, and Europe. N-alkylated deoxynojirimycin is also a potent -glu- cosidase I inhibitor and nine carbon alkyl chain ana- logues have shown efficacy as antivirals in animal models for hepatitis B (5) and a surrogate tissue cul- ture model of hepatitis C, bovine viral diarrhea virus (BVDV) (6). We have recently determined the mechanism of ac- tion of N-alkylated imino sugar inhibitors of ceramide- 1 To whom correspondence should be addressed. Fax: 01865 275216. E-mail: terry@glycob.ox.ac.uk. 2 Abbreviations used: GSL, glycosphingolipid; DNJ, deoxynojiri- mycin; DGJ, deoxygalactonojirimycin; NB-DNJ, N-butyl-DNJ; CNS, central nervous system; BVDV, bovine viral diarrhea virus; SCX, strong cation-exchange. 136 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 284, 136 –142 (2000) doi:10.1006/abio.2000.4678, available online at http://www.idealibrary.com on