Homostachydrine (pipecolic acid betaine) as authentication marker of roasted blends of Coffea arabica and Coffea canephora (Robusta) beans Luigi Servillo a,⇑ , Alfonso Giovane a , Rosario Casale a , Domenico Cautela b , Nunzia D’Onofrio a , Maria Luisa Balestrieri a , Domenico Castaldo b,c,d a Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy b Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi (SSEA), Azienda Speciale – Camera di Commercio di Reggio Calabria, Via G. Tommasini 2, 89127 Reggio Calabria, Italy c Ministero dello Sviluppo Economico, MiSE – Via Molise 2, Roma, Italy d Dipartimento di Ingegneria Industriale e ProdAL scarl, Università degli studi di Salerno, via Ponte Don Melillo 1, 84084 Fisciano, Salerno, Italy article info Article history: Received 23 November 2015 Received in revised form 11 February 2016 Accepted 27 February 2016 Available online 3 March 2016 Keywords: Pipecolic acid betaine Homostachydrine N-methylpipecolic acid Betaines Roasted coffee Arabica coffee Robusta coffee LC-ESI-MS/MS abstract The occurrence of pipecolic acid betaine (homostachydrine) and its biosynthetic precursor N-methylpi- pecolic acid was detected for the first time in green coffee beans of Robusta and Arabica species. The anal- yses were conducted by HPLC–ESI tandem mass spectrometry and the metabolites identified by product ion spectra and comparison with authentic standards. N-methylpipecolic acid was found at similar levels in green coffee beans of Robusta and Arabica, whereas a noticeable difference of homostachydrine con- tent was observed between the two green coffee bean species. Interestingly, homostachydrine content was found to be unaffected by coffee bean roasting treatment because of a noticeable heat stability, a fea- ture that makes this compound a candidate marker to determine the content of Robusta and Arabica spe- cies in roasted coffee blends. To this end, a number of certified pure Arabica and Robusta green beans were analyzed for their homostachydrine content. Results showed that homostachydrine content was 1.5 ± 0.5 mg/kg in Arabica beans and 31.0 ± 10.0 mg/kg in Robusta beans. Finally, to further support the suitability of homostachydrine as quality marker of roasted blends of Arabica and Robusta coffee beans, commercial samples of roasted ground coffee blends were analyzed and the correspondence between the derived percentages of Arabica and Robusta beans with those declared on packages by man- ufacturers was verified. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction The genus Coffea L. comprises more than one hundred species but only about 25 produce fruits with commercial value. Among these, 4 are used by industry to produce the coffee used for the preparation of the most popular beverage in the world after tea. Coffea arabica L. and Coffea canephora P. (known as Robusta) are the species most used in the preparation of commercial blends of roasted coffee. C. arabica L. represents about 70% of the world pro- duction and is the most appreciated species by the consumer for superior flavor and lower caffeine content than Robusta. However, the latter is more productive and resistant to diseases and also suitable for the cultivation in lowlands. For all these reasons, the Arabica species has commercial values consistently higher than Robusta. The other two coffee species used by industry, although in much less amount, are the Liberica, grown mainly in Liberia and Côte d’Ivoire, and the Excelsa. These species are grown mainly to meet local consumption (Davis, Govaerts, Bridson, & Stoffelen, 2006; Tafani, 2013). Food and beverage adulteration, which become a major issue in the era of market globalization, involves many different edible products, including coffee (Toci, Farah, Pezza, & Pezza, 2015), and the assessment of their authenticity is of paramount importance for quality and economical reasons. Therefore, various chemical components, such as sterols, volatile components, and metals (Frega, Bocci, & Lercker, 1994; Krivan, Barth, & Morales, 1993; Lercker, Frega, Bocci, & Rodriguez- Estrada, 1994; Murota, 1993), devised to discriminate among dif- ferent coffee species, are mainly utilized as indicators of beans of Robusta species in Arabica coffee lots. Presently, the occurrence of Robusta beans into roasted coffee is mainly detected by measur- ing the concentration of 16-O-methylcafestol (16-OMC), a specific pentacyclic diterpene alcohols derived from cafestol. This metabolite, identified and isolated from Robusta coffee beans http://dx.doi.org/10.1016/j.foodchem.2016.02.154 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail address: luigi.servillo@unina2.it (L. Servillo). Food Chemistry 205 (2016) 52–57 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem