Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L. Nikolaos Stavros Koulakiotis 1,2 , Ernst Pittenauer 2 , Maria Halabalaki 3 , Anthony Tsarbopoulos 1 and Günter Allmaier 2 * 1 Laboratory of Instrumental Analysis, Department of Pharmacy, University of Patras, Patras, Greece 2 Institute of Chemical Technologies and Analytics, Vienna University of Technologies and Analytics, Vienna, Austria 3 Laboratory of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens, Athens, Greece RATIONALE: The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation. METHODS: Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice. RESULTS: LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument. CONCLUSIONS: The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins ( 0,4 A 2 , 3,5 A 2 product ions indicating a 1!6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd. The fall-blooming taxon Crocus sativus L. (saffron), a member of the plant family called Iridaceae, is nowadays best seen as triploid sterile cultivar of the wild-type Crocus cartwrightianus Herb., [1] a native plant from the eastern Mediterranean area. This genus includes roughly 80 different species originating from central and southern Europe, North Africa, Middle East, and central Asia to China, some of which are cultivated as ornamental plants. Interestingly, only the stigmas of the aforementioned cultivar Crocus sativus L. serve as a spice plant and has been applied in traditional Chinese medicine as well as being used as a food colorant, for roughly 5000 years. The exor- bitant market prices of crocus stigmas samples impose the need for developing analytical strategies to determine the authenti- city of saffron originating from different countries across the world. Those include high-performance liquid chromatography (HPLC) with diode-array detection (DAD) [2–7] or in combination with electrospray ionization (ESI) [8–12] mass spectrometry (MS), and only one research report employing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS. [13] Chemically, saffron contains a wide variety of different constituents including carotenoids (e.g., a- and b-carotene, lycopene and zeaxanthin), mono- and bis-esters of crocetin (one of the major constituents of saffron representing C 20 -degradation products of carotene) with glucose, gentiobiose, gentiotriose or neapolitanose, [2,14] respectively, and a rather complex essential oil (containing up to 70% safranal). Picrocrocin, another major constituent of saffron, represents a glucoside of the monoterpene hydroxyl-b-cyclocitral (the precursor molecule of the volatile safranal). As only a limited number of reports deal with the analysis of polar constituents of Crocus sativus L. by modern mass spectro- metric techniques such as ESI-MS, we have evaluated different mass spectrometric desorption/ionization techniques in combi- nation with tandem mass spectrometry for the structural analysis of two selected crocins (trans-crocin-4 and cis-crocin-4, two geometrical isomers) and picrocrocin by ESI ion trap * Correspondence to: G. Allmaier, Institute of Chemical Technolgies and Analytics, Vienna University of Technol- ogy, Getreidemarkt 9, A-1060 Vienna, Austria. E-mail: guenter.allmaier@tuwien.ac.at Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 670–678 Research Article Received: 5 July 2011 Revised: 12 December 2011 Accepted: 20 December 2011 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2012, 26, 670–678 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6142 670