2104 J. Agric. Food Chem. 1995, 43, 2104-2109 HPLCLMS Application to Anthocyanins of Vitis vinifera L. Alessandro Baldi,+Annalisa Romani,t Nadia Mulinacci,t Franco F. Vincieri,*lt and Bruno Casetta$ Dipartimento di Scienze Farmaceutiche, Universitti degli Studi di Firenze, via Gin0 Capponi 9, 50121 Firenze, Italy, and Perkin-ElmerHolding GmbH, Langen, Frankfurt, Germany An HPLC/MS application to anthocyanic compounds of Vitis vinifera L. is described. A series of liquid-liquid and liquid-solid extractions yielded an extract containing anthocyanic compounds only. This extract was analyzed by HPLCiDAD and HPLC/MS, and W-vis and MS spectra of each compound were obtained. An API ion-spray interface allowed coupling between the chromatographic system and a mass spectrometer. This interface allowed application of the chromatographic conditions normally used in the HPLC analysis of anthocyanic compounds,which led to the identification of the 3-glucosides,the 3-acetylglucosides, and the 3-p-coumaroylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin, already known in the literature. Two 3-caffeoylglucoside derivatives were identified too, and it has been possible to identify, for the first time, some 3,5-diglucosides that are known to be present in Vitis sp. but not in Vitis vinifera L. The investigated cultivars showed the same anthocyanic profile but dramatic quantitative differences. Keywords: Anthocyanin; Vitis vinifera; HPLCIMS INTRODUCTION Mass spectrometry has proved to be effective and of considerable diagnostic value when used as a supporting technique in the characterization of flavonoids. Among the different techniques, FAB is the most commonly used for the mass spectrometric study of anthocyanins (Strack and Wray, 1994). Usually it allows one to obtain the signal corresponding to the molecular peak and excellent fragmentation, thus providing relevant infor- mation on the aglycon and sugars, as well as useful structural information about the examined compound. If the FAB analytical procedure is to yield such infor- mation, it must necessarily be preceded by the purifica- tion of the compound and its solubilization in a polar matrix. Direct analysis of an anthocyanic mixture is also possible (Laing and Cormier, 1991); in this case a series of molecular peaks is obtained, and using MSI MS techniques it is possible to obtain information on the substituents of some compounds. Nevertheless, prior chromatographic separation is a useful prerequi- site for any mass spectrometry analytical method used to test anthocyanic mixtures. When mass spectrometry is coupled to a chromatographicseparation method, both the retention characteristics and the mass spectra provide structural information without the necessity of isolating the individual compounds (Glapgen et al., 1992a). The first applications of this coupling to an- thocyanic compounds were GC/MS techniques. Gas chromatographic analysis of anthocyanins necessarily requires the derivatization of the compounds either by TMCS or HMDS to increase their volatility. This implies a considerable increase in their molecular weight and makes the interpretation of their mass spectra more difficult. Bombardelli et al. (1977) devel- oped a GCiMS analytical technique applied to an anthocyanic extract from blueberry (Vaccinium mirtillus * Author to whom correspondence should be ad- dressed [telephone (39) 55-2757-288; fax (39) 55-240776; e-mail VINCIERI@farmfi.chim.unifi,it] . Universitfi degli Studi di Firenze. Perkin-Elmer. L.). However, HPLC is a better separation method for anthocyanins. The interfacing of a HPLC apparatus with a mass spectrometer (HPLC/MS)has been opti- mized in several applications (Thompson et al., 1982; Whitehouse et al., 1985; Bruins et al., 1987). Such a technique has been little used to analyze anthocyanins (Glapgen et al., 1992b) because of their low volatility and high polarity, which requires the use of RP columns and acid eluents having a high proportion of water. So, the low volatility of the eluents, their high polarity, and the chemical characteristics of the compounds for analy- sis make the choice of the interface between the chromatographic system and the mass spectrometer absolutely crucial for this procedure. This application and the characteristics of the involved molecules require an interface that operates at low temperatures, to avoid degradation of analytes, and permits the use of highly polar eluents under acidic pH conditions, to keep molecules unfragmented until they enter the mass spectrometer. An interface fitting these requirements is the API ion spray interface, which allows ambient pressure ionization and the use of any aqueous eluent. The pool of anthocyans contained in the berry skins of Vitis vinifera L. was taken as a research model to investigate the possible application of HPLC/MS to anthocyanins. The anthocyanic content of the fruits of V. vinifera L. was determined (Riberau-Gayon, 1968) long ago for the presence of the 3-0-glucosides of delphinidin, cyanidin, petunidin, peonidin, and malvi- din, as well as their relative 3-acetylglucosides and 3-p- coumaroylglucosides. This paper aims at providing both a scientific contri- bution to the knowledge of the anthocyanins contained in the grapes of V. vinifera L. and a practical-analytical contribution in the development of a HPLC/MS method. Results have been obtained using a HPLC apparatus coupled with a mass spectrometer equipped with an API (atmospheric pressure ionization) ion spray interface. The use of this technique made it possible to obtain the mass spectra of all the anthocyanic compounds present in the extracts under investigation, even those occurring in traces or some coeluted ones. 0021 -8561/95/1443-2104$09.00/0 0 1995 American Chemical Society