Identification and characterization of selenium species in enriched green onion (Allium fistulosum) by HPLC-ICP-MS and ESI-ITMS Monika Shah, a Sasi S. Kannamkumarath, a Jorgelina C. A. Wuilloud, a,b Rodolfo G. Wuilloud a and Joseph A. Caruso* a a Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA. E-mail: joseph.caruso@uc.edu b Food and Drug Administration (FDA), Forensic Chemistry Center, Cincinnati, Ohio 45237-3097, USA Received 3rd October 2003, Accepted 27th January 2004 First published as an Advance Article on the web 17th February 2004 In this study speciation of selenium in selenium-enriched green onions (Allium fistulosum) was done with reversed-phase ion-pairing high performance liquid chromatography (RP-IP-HPLC) and size-exclusion chromatography (SEC) coupled on-line to ICP-MS for selenium specific detection. The plant extract obtained using sodium hydroxide (0.1 mol l 21 ) analyzed by SEC (CAPS 10 mmol l 21 , pH 10.0) with ICP-MS detection showed the incorporation of selenium in both high (y12 kDa) and low molecular weight (0.36–2 kDa) fractions. Presumably protein bound selenoamino acids were released using enzymes (Proteinase K and Protease XIV) and selenoamino acids found in cytosol in their free form were extracted using 0.1 M HCl. The extracts were analyzed for speciation studies by RP-IP-HPLC [0.1% (v/v) heptafluorobutyric acid, 5% (v/v) methanol, pH 2.5]. Matching the chromatographic retention times with commercially available standards provided evidence for the presence of Se-cystine, methylselenocysteine, Se-methionine, and inorganic selenium. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) confirmed the presence of c-glutamyl-Se- methylselenocysteine in green onions as has been reported in other onion types. Introduction Selenium is a key trace element required in small amounts in humans and animals. Anti-carcinogenic properties of selenium have been suggested by various studies. 1–4 Selenium enriched yeast and different species of Allium plants have been proposed as dietary supplements due to their several medicinal and especially, anti-carcinogenic properties. 5–7 Clark et al. 1 showed that human dietary supplementation with selenium-enriched yeast decreased cancer incidence and mortality rate by nearly 50% for certain tumors. The major species in enzymatic extracts of yeast and garlic have been found to be seleno- methionine and c-glutamyl-Se-methylselenocysteine, respec- tively. 8,9 With the Allium family vegetables, research has been selectively focused on bulbs and the information available on the speciation of selenium in leaves of Allium vegetables is scarce. In fact, the presence of selenium species in leaves of bulb onions (Allium cepa) has recently been demonstrated. 10 However, speciation studies of selenium in green onions (Allium fistulosum), a major portion of which corresponds to leaves, have not been reported and, contrary to most other onions, these leaves are normally eaten. Therefore, the identification of selenium species in green onion is of interest. Size-exclusion chromatography (SEC) was coupled to induc- tively coupled plasma mass spectrometry (ICP-MS) to study the distribution of selenium in covalently bound high molecular weight (HMW) (w12 kDa, likely to be proteins) and low molecular weight (LMW, 0.36–2 kDa likely to be small peptides or free amino acids) fractions. Reversed phase ion- pairing chromatography (RP-IP-HPLC) coupled to ICP-MS for separation of the selenium species was also employed after different extraction procedures. Identification of selenium compounds was performed by matching the retention time with chromatographic peaks of a standard mixture containing different selenium species. Electrospray ionization-ion trap mass spectrometry (ESI-ITMS) was used for identification of an unknown species. Molecular mass was identified in the MS mode and structural information was obtained from MS-MS spectra. Experimental Instrumentation High performance liquid chromatography (HPLC) utilized a Shimadzu (Shimadzu Scientific Instrument Inc, Columbia, MD, USA) LC-6A pump with a 100 ml loop (Rheodyne 7725 injection valve, Rheodyne, Cotati, USA). RP chromatography was performed using a C8 Alltima (Alltech, Deerfield, IL, USA) column (150 mm 6 4.6 mm, 5 mm particle size); while a Superdex Peptide HR 10/30 (Pharmacia Biotech) was used for SEC separations. An Elan 6000 inductively coupled plasma mass spectrometer (PerkinElmer Sciex, Canada) equipped with a cross-flow nebulizer and a Scott-type double-pass spray chamber made from Ryton was used for the determination of total selenium as well as for selenium-specific detection. The outlet of the UV detector was connected online to the liquid sample inlet of the ICP-MS nebulizer using 300 mm long by 0.25 mm id PEEK tubing. It has to be pointed out that in case of size exclusion chromatography, the high flow rate of the mobile phase (0.6 ml min 21 ) and the small dead volume of the PEEK tube results in insignificant difference in chromato- graphic retention times between the two detection systems. The instrumental operating conditions are given in Table 1. The MS experiments were performed using an Agilent 1100 Series LC/ MSD Ion Trap Mass Spectrometer (Agilent Technologies, Tokyo, Japan). The mass spectrometer was equipped with an electrospray interface as the ionization source, which was operated under the conditions indicated in Table 1. Full-scan mass spectra (100–500 amu) were recorded every 39.227 ms in DOI: 10.1039/b312320k J. Anal. At. Spectrom. , 2004, 19 , 381–386 381 This journal is ß The Royal Society of Chemistry 2004