Analysis of derivatized and underivatized theanine enantiomers by high‐performance liquid chromatography/atmospheric pressure ionization‐mass spectrometry

Abstract Theanine, a naturally occurring non‐proteinic amino acid found in tea leaves, has demonstrated wide‐ranging physiological activity, from lowering blood pressure to enhancing the anti‐tumor activity of chemotherapeutic drugs. The chiral nature of theanine suggests that enantiospecificity plays a significant role in its various pharmacological functions. Using the Chirobiotic T (teicoplanin) chiral stationary phase, native and derivatized theanine enantiomers were separated and detected via high‐performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization mass spectrometry (API‐MS). With the use of flow rates compatible with each ionization source, native theanine standards achieved excellent sensitivity and detection limits (10 ng/mL) for both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Optimum sensitivity and detection limits for derivatized theanine standards were achieved using ESI‐MS. The enantiomeric composition of six commercially available L ‐theanine samples was evaluated using the high‐flow APCI‐MS method and confirmed with photodiode array detection. Five of the six products contained significant amounts of D ‐theanine. Only one product, SunTheanine®, appeared to contain only the L ‐theanine enantiomer. Copyright © 2004 John Wiley & Sons, Ltd.