Quantification of Protodioscin and Rutin in Asparagus Shoots by LC/MS and HPLC Methods MINGFU WANG,* YAAKOV TADMOR,QING-LI WU,CHEE-KOK CHIN, STEPHEN A. GARRISON, AND JAMES E. SIMON New Use Agriculture and Natural Plant Products Program and Department of Plant Biology and Pathology, Cook College, Rutgers University, and New Jersey Agricultural Experiment Station, 59 Dudley Road, New Brunswick, New Jersey 08901 A liquid chromatography/mass spectrometry (LC/MS) method with selected ion monitoring was developed and validated to analyze the contents of protodioscin and rutin in asparagus. The distribution of rutin and protodioscin within the shoots was found to vary by location, with the tissue closest to the rhizome found to be a rich source of protodioscin, at an average level of 0.025% tissue fresh weight in the three tested lines, while the upper youngest shoot tissue contained the highest amount of rutin at levels of 0.03-0.06% tissue fresh weight. The lower portions of the asparagus shoots that are discarded during grading and processing should instead be considered a promising source of a new value-added nutraceutical product. KEYWORDS: Asparagus; protodioscin; rutin; LC/MS; quantification INTRODUCTION Asparagus (Asparagus officinalis L., Family Liliaceae) is a popular vegetable that is consumed in many parts of the world for its edible shoots used in salads, vegetable dishes, and soups. Historically, asparagus has been used as a traditional medicine in both European and Asian cultures. In China, asparagus has been used in traditional Chinese medicine as a tonic, antifebrile, antitussive, hair growth stimulator, and diuretic agent (1). Asparagus root is also formally listed by the German Commis- sion E (2) as an approved medicinal product in the treatment of inflammatory diseases of the urinary tract, for prevention of kidney stones, and with a noted diuretic effect. Modern pharmacological studies have found the extracts of asparagus to possess certain biological activities including antifungal (3), diuretic (4), cytotoxic (1, 5-7), antiviral (8), and molluscicide (9) properties. Asparagus is a rich source of phytochemicals. Several types of compounds including flavonoids, sterol saponins, oligosac- charides, carotenoids, sulfur-containing acids, and amino acids have been identified in asparagus (5, 10-12). Among this wide group of compounds, protodioscin (C 51 H 84 O 22 ), the major saponin in asparagus, has attracted more attention. Protodioscin has been associated with bioactivities including cytotoxicity against several human cancer cell lines, especially selectively against one leukemia line (MOLT-4), one NSCLC line (A549/ ATCC), and two colon cancer lines (HCT-116 and SW-620) in a brand new pattern when compared in the NCI’s anticancer drug screen database (13), antiherpes simplex virus type 1 (HSV- 1) activity (14), cytotoxic activity against human leukemia HL- 60 cells (1, 5), cytotoxicity on cultured C6 glioma cells (15), xanthine oxidase inhibitors (16), antineoplastic activity (17, 18), and improving sexual desire and enhancing erections (19). It is of interest to note that protodioscin is the major component in a popular libido-increasing dietary supplement, Tribulus ter- restris (20). Given these activities, this compound became a major focus of our research, but the accurate analysis of protodiosin from botanical sources has limited research and is challenging due to its weak UV absorption. Only a few papers provide reliable procedures for the quantification of protodiosin, and of these, the analytical focus was on the determination of this compound in Tribulus. Those protocols focused on the analysis of protodioscin by a colorimetric method (21) and quantification of the aglycone diosgenin by high-performance liquid chromatography (HPLC) and then converting it back to protodioscin (22) and direct quantification of protodioscin by HPLC using evaporative light scattering detection (20). How- ever, these methods may not directly apply to protodioscin analysis in asparagus. Rutgers University has an active program in asparagus re- search. Among the goals of this program is to increase the levels of bioactive compounds (protodioscin and rutin) in asparagus by breeding. This requires accurate identification and quantifica- tion of these compounds in the parent plants and a large number of the progenies. Here, we report a positive and negative electrospray ionization (ESI)-mass spectrometry (MS) method to characterize the major compounds including saponins and flavonoids in asparagus shoots. We also developed and validated a LC/MS method with selected ion monitoring to quantify the major saponins protodioscin and rutin in asparagus shoots. This method is accurate, reliable, sensitive, and simple and can be used to analyze various asparagus samples with varied amounts of rutin and protodioscin. * To whom correspondence should be addressed. Tel: 732-932-9711 ext. 367. Fax: 732-932-9441. E-mail: mwang@aesop.rutgers.edu. 6132 J. Agric. Food Chem. 2003, 51, 6132-6136 10.1021/jf0344587 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/09/2003