pubs.acs.org/JAFC Published on Web 05/03/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 6011–6017 6011 DOI:10.1021/jf100143n Quantification of Crocetin Esters in Saffron (Crocus sativus L.) Using Raman Spectroscopy and Chemometrics EIRINI G. ANASTASAKI, † CHARALABOS D. KANAKIS, † CHRISTOS PAPPAS, † LUANA MAGGI, ‡ AMAYA ZALACAIN, ‡ MANUEL CARMONA, ‡ GONZALO L. ALONSO, ‡ AND MOSCHOS G. POLISSIOU* ,† † Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Greece, and ‡ Ca´ tedra de Quı´mica Agrı´cola, ETSI Agro´ nomos, Universidad Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain The feasibility of Raman spectroscopy for predicting the content of crocetin esters (crocins), and coloring strength was assessed. 114 samples from Greece, Iran, Italy and Spain were divided into two sets: a calibration set with 49 samples and a validation one with 65 samples. Calibration models for crocetin esters (r 0.97, RMSEC 0.92, RMSEP 0.97, RPD 3.46) and coloring strength (r 0.95, RMSEC 12.2, RMSEP 11.3, RPD 2.59) were built in the spectral region 1700-955 cm -1 using partial least-squares (PLS) regression. The calibration models were validated using cross-validation, leaving one sample out (r 0.97, RMSECV 1.09 for crocetin esters and r 0.93, RMSECV 14.5 for coloring strength). The crocetin esters content as determined by liquid chromatography fluctuated between 18.8 and 31.7 mg/100 g saffron. The corresponding values, as calculated using the Raman method, fluctuated between 19.2 and 32.0 mg/100 g saffron. The coloring strength determined by the reference method ranged from 177.0 to 296.7 units, while with the Raman method the values were between 186.8 and 297.6 units. The results, as compared to the reference methods (liquid chromatography and UV-vis spectrophotometry), show that the proposed methodology gives data with acceptable accuracy. The proposed models can be used as a tool for rapid screening of quality in saffron samples. KEYWORDS: Saffron; Raman spectroscopy; HPLC chromatography; crocins; coloring strength; partial least-squares PLS INTRODUCTION Saffron’s quality is determined by its taste, aroma and color. Picrocrocin, safranal and crocins are the secondary metabolites that contribute to saffron quality parameters respectively. Picro- crocin, the safranal’s glycoside precursor, contributes to the bitterness and is responsible for its taste. Safranal, a monoterpene aldehyde, is the main compound of the essential oil of saffron and gives its distinctive aroma. Crocetin esters with glucose, gentio- biose, neapolitanose or triglucose sugar moieties are water- soluble carotenoids and responsible for saffron’s yellowish color. The determination of these compounds is important for establish- ing the commercial quality criteria and consequently its price. For world trade, color is the major parameter for saffron’s quality. Under the Technical Specifications of the ISO standard ISO/TS 3632:2003 ( 1 ) this parameter is expressed as coloring strength, which is defined as the absorbance at 440 nm of a 1% aqueous solution in a 1 cm quartz cell (E 1cm 1% 440 nm). According to this value, saffron is classified into three categories. Because this procedure does not give a detailed composition of crocetin esters, thin layer chromatography and liquid chromatography have been applied, instead ( 2 -6 ). Although these techniques are very sensitive and accurate, they are expensive and time-consuming and must be supported by specialized staff. In the case that the saffron sector is made up mostly from medium and small size enter- prises, affordable and fast techniques are needed. The application of spectroscopy techniques is enforced in order to save time, cost and reagents. In this context, spectroscopy techniques such Raman and infrared spectroscopy (IR) in combination with suitable chemo- metric algorithms have been shown to be advantageous for routine basis quality control of foods ( 7 -14 ). Raman and infrared spectroscopy are complementary methods that can be applied for a routine control process. They are fast and solvent free techni- ques. Recently, near-infrared spectroscopy (NIRS) in combina- tion with chemometrics has been shown to be usable for quanti- fying the main saffron compounds ( 15 ). However, an NIR spectrum is difficult to be interpreted due to the fact that NIR consists of overtones and combination bands of fundamental transitions. In a Raman spectrum, there are well-resolved bands of fundamental vibrational transitions thus providing more clear structural information. The polyene structure of carotenoids enhances strong signals, so they can easily be detected in complex biological matrices ( 12 , 16 ). Raman spectroscopy has already *Corresponding author. Tel: þ30 210 529 4241. Fax: þ30 210 529 4265. E-mail: mopol@aua.gr.