Enantiomer Separation of Amino Acids in Immunoaffinity Micro LC-MS JESSICA M. ZELEKE, GREGORY B. SMITH, HEIKE HOFSTETTER, AND OLIVER HOFSTETTER * Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois ABSTRACT Chiral immunoaffinity microbore columns were directly interfaced with MS detection, and the effect of column length and temperature on the enantiomer separation of a number of underivatized aromatic and aliphatic amino acids was investi- gated utilizing an antibody chiral stationary phase that had been prepared by immobi- lizing a monoclonal anti-D-amino acid antibody onto silica. The stronger affinity of the antibody towards aromatic and bulky amino acids allowed separation of such analytes in a 0.75 3 150 mm column, while an increase in column length enabled separation of more weakly bound compounds. The strength of interaction between chiral selector and analytes could be modulated conveniently by lowering the temperature. For the first time, simultaneous enantiomer separation of mixtures of amino acids was achieved on antibody-based chiral stationary phases using extracted ion chromatograms. Chirality 18:544–550, 2006. V V C 2006 Wiley-Liss, Inc. KEY WORDS: enantiomer separation; antibody; protein chiral stationary phase; high- performance liquid chromatography; miniaturization; micro LC-MS Since 1992, regulatory agencies such as the U.S. Food and Drug Administration require determination of the stereoisomeric composition of chiral drugs. 1 Although a variety of methodologies have been utilized for enan- tiomer analysis, 2–4 high-performance liquid chromatogra- phy (HPLC) remains the most successful technique in this area. Direct separation of enantiomers in HPLC is based on differential interactions with a chiral selector, which may either be added to the mobile phase or immo- bilized onto a solid support material (e.g., silica and syn- thetic polymers) to produce a chiral stationary phase (CSP). The use of CSPs is generally advantageous since consumption of chiral selector is minimized and interfer- ence with analyte detection, often observed if the selector is added to the mobile phase, is prevented. A large num- ber of chiral selectors have been applied successfully for enantiomer separation, and about 200 CSPs are commer- cially available. 5 However, since these selectors are not tailor-made for a specific separation problem, reliable pre- diction of selectivity and appropriate separation conditions is generally limited. 6,7 In addition to novel screening tech- niques and data banks, which promise to facilitate the search for a suitable selector, 6,8 the utilization of chiral host-molecules of predetermined specificity represents an intriguing alternative approach to enantiomer analysis. It has long been recognized that miniaturized chroma- tographic systems are particularly attractive for the evalu- ation of potentially cost-intensive new selectors, such as antibodies or receptor proteins. 9 The advantages of mini- aturized LC include not only a significant decrease in sta- tionary phase consumption, but also the ability to work with small sample sizes and low flow rates, improved detection as a result of reduced chromatographic dilution of the sample, 10,11 and the possibility to pack longer col- umns with potentially higher resolution. 12 Although UV- spectroscopy is still the most widely employed detection method in LC, miniaturization also facilitates coupling to more sensitive detectors, for example, mass spectrome- ters. Significant advances in the field of ionization techni- ques (especially atmospheric-pressure ionization meth- ods) have resulted in a dramatic increase in the use of hyphenated LC-MS techniques in chemical and biomedi- cal research, 13 and a number of reports have demon- strated the utility of LC-MS for the analysis of chiral phar- maceuticals. 14,15 Various chiral selectors have been applied in standard- size or miniaturized LC-MS for enantiomer separa- tion; 15,16 these include proteins such as a-acid glycopro- tein, 17–19 conalbumin, 20 human serum albumin, 21 and avi- din. 22,23 As protein-based CSPs are typically operated in the reversed-phase mode (i.e., in aqueous buffers that may contain organic additives), transformation of chroma- tographic conditions from UV- to MS-detection can read- ily be achieved. This is in contrast to classical affinity chromatographic systems, which are usually not compati- ble with MS. The generally stronger interactions between, e.g., a receptor protein (such as an enzyme or antibody) *Correspondence to: Oliver Hofstetter, Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115-2862. E-mail: ohofst@niu.edu Contract grant sponsor: National Institutes of Health; Contract grant number: 1 R15 GM066865-01; Contract grant sponsor: National Science Foundation; Contract grant number: CHE-0130635. Received for publication 2 November 2005; Accepted 6 March 2006 DOI: 10.1002/chir.20286 Published online 26 April 2006 in Wiley InterScience (www.interscience.wiley.com). CHIRALITY 18:544–550 (2006) V V C 2006 Wiley-Liss, Inc.