In-depth analysis of crocetin ester glycosides from dried/processed stigmas of Crocus sativus L. by HPLC-ESI-MSn (n = 2, 3).

INTRODUCTION: Saffron stigmas from Crocus sativus L. (Iridaceae) are used as a drug in folk medicine, as a food additive and as a dying agent for at least 3500 years. Despite this long-term use the chemical composition of saffron seems still to be not fully known. OBJECTIVE: An analytical strategy for detailed investigations of aqueous saffron extract is developed based on reverse-phase high-performance liquid chromatography electrospray ionisation (HPLC-ESI) multistage mass spectrometry (MSn ) for crocins. METHODS: Commercially available stigmas are analysed by reverse-phase HPLC in combination with ESI/three-dimensional (3D)-ion trap mass spectrometry (MS) and MSn (n = 2 and 3). Sodium chloride is added to the analyte solution ready for injection to promote abundant [M + Na]+ adduct ions of crocins, being ideal precursor ions for low-energy collision-induced dissociation (CID)-MS2/3 . RESULTS: This strategy allows the detailed structural elucidation of known as well as previously unknown crocin derivatives (molecular mass of the aglycon, oligosaccharide chain length and linkage determination). The two isomeric trisaccharide substituents neapolitanose and gentiotriose are distinguished based on linkage-specific cross-ring cleavage for the first time. Furthermore, crocins containing up to six hexose units are also observed. Five novel crocin ester glycosides shifted by a mass difference of -40 Da indicate the presence of the here newly described C17 -aglycon, termed norcrocetin (crocetin = C20 ). CONCLUSIONS: These findings indicate the action of at least two different carotenoid cleavage dioxygenases (CCD2 and tentatively CCD4) during biosynthesis of this new bis-apocarotenoid aglycon (norcrocetin) and the existence of even higher glycosylated crocin derivatives at trace level.

A laser desorption ionization/matrix-assisted laser desorption ionization target system applicable for three distinct types of instruments (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF) with different performance characteristics from three vendors.

RATIONALE: We have developed a target system which enables the use of only one target (i.e. target preparation set) for three different laser desorption ionization (LDI)/matrix-assisted laser desorption ionization (MALDI) mass spectrometric instruments. The focus was on analysing small biomolecules with LDI for future use of the system for the study of meteorite samples (carbonaceous chondrites) using devices with different mass spectrometric performance characteristics. METHODS: Three compounds were selected due to their potential presence in meteoritic chondrites: tryptophan, 2-deoxy-d-ribose and triphenylene. They were prepared (with and without MALDI matrix, i.e. MALDI and LDI) and analysed with three different mass spectrometers (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF). The ion sources of two of the instruments were run at high vacuum, and one at intermediate pressure. Two devices used a laser wavelength of 355 nm and one a wavelength of 337 nm. RESULTS: The developed target system operated smoothly with all devices. Tryptophan, 2-deoxy-d-ribose and triphenylene showed similar desorption/ionization behaviour for all instruments using the LDI mode. Interestingly, protonated tryptophan could be observed only with the LinTOF/curved field RTOF device in LDI and MALDI mode, while sodiated molecules were observed with all three instruments (in both ion modes). Deprotonated tryptophan was almost completely obscured by matrix ions in the MALDI mode whereas LDI yielded abundant deprotonated molecules. CONCLUSIONS: The presented target system allowed successful analyses of the three compounds using instruments from different vendors with only one preparation showing different analyser performance characteristics. The elemental composition with the QqRTOF analyser and the high-energy 20 keV collision-induced dissociation fragmentation will be important in identifying unknown compounds in chondrites.

Processed stigmas of Crocus sativus L. imaged by MALDI-based MS.

The processed, i.e. dried under certain conditions, stigmas of Crocus sativus L. are one of the most expensive plant parts used commercially. For the color, aroma and biological activity a very complex mixture of glycolipids termed crocins are responsible. Therefore studying structural composition and distribution in the commercial plant material is of great interest. We showed successfully the application of a MALDI-based mass spectrometric imaging (MSI) approach for stigmas towards different crocin species. MSI opens up the investigation of processed plant materials in various fields allowing studying the processing in detail as well as adulteration attempts (which are quite frequent due to the price of the material). Furthermore, we could demonstrate that a similar number of crocins present in stigmas could be detected by MALDI MSI compared to the classical approach of analyzing the solvent-extract of stigmas by MALDI-MS.