docSpectroscopic Database for authentic saffron (Crocus
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Spectroscopic Database for authentic saffron (Crocus
COST Action FA1101 “SAFFRONOMICS” Omics Technologies for Crop Improvement, Traceability, Determination of Authenticity, Adulteration and Origin in Saffron Spectroscopic Database for authentic saffron (Crocus sativus L.) Contributors AGRICULTURAL UNIVERSITY OF ATHENS ARISTOTLE UNIVERSITY OF THESSALONIKI UNIVERSITY OF CASTILLA LA MANCHA Preface This work was conducted in the frame of “COST Action FA 1101: SAFFRONOMICS - Omics Technologies for Crop Improvement, Traceability, Determination of Authenticity, Adulteration and Origin in Saffron”. The aim of this work was to compile a database of UV-Vis, FT-IR, Raman, NMR and MS spectra (LCMS, GC-MS) of the Crocus sativus stigmas. This spectral database can be used as a guide to identify authentic saffron (dried stigmas of Crocus sativus L.) The database contains different sections according to the spectroscopic technique used for the analysis of Crocus sativus stigmas. Each section covers: the name of the technique used, details for the conditions of analysis and spectra recording, images of spectra and accompanying spectral data e.g. max of peaks etc. After these figures we’re going to present a discussion, and then the experimental part and results will be described and will be compared with literature data. Finally, a conclusion. Editors of this database Dr. Petros A. Tarantilis, Assoc. Professor Dr. Stella Ordoudi, Researcher WG3 Leader WG3 co-Leader Laboratory of Chemistry Department of Food Science & Human Nutrition, School of Food, Biotechnology and Development Agricultural University of Athens Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki DATABASE 1. Technique: UV-Vis spectroscopy National Institute of Agronomic Research Morocco Scientific coordinator: Dr Mounira Lage Code: FA1101-M-D-13` Sample: Stigma, Extract from whole stigma Recording conditions: Spectral area: 200-800 nm, Solvent: water (milliQ) According to ISO, picrocrocin, safranal and crocins are expressed as direct reading of the absorbance of 1% aqueous solution of dried saffron at 257, 330 and 440 respectively. Measurements of E1% of an aqueous saffron extract at 440, 330, and 250 nm, respectively, were done using a 1 cm, pathway quartz cell. Spectrum 1.4 1.2 Absorbance 1 0.8 0.6 0.4 0.2 0 190 Spectral data λmax:257, 330, 440 nm 240 290 340 390 440 490 540 Longueure d'onde 590 640 690 1. Technique: UV-Vis spectroscopy Unipr collection Scientific coordinator: Professor Andrea Mozzarelli (WG2 co-leader) Analysts: Dr. Gianluca Paredi, Dr. Samanta Raboni, Dr. Francesco Marchesani Sample preparation and extraction procedure: Samples in stigma form were gently ground and the powder was sifted with a 0.42 millimeter sieve prior to analysis. Extraction was carried out modifying the procedure described in ISO 3632-2 (2010). Powder was suspended in water at 2% concentration (w/v) and sonicated for 15 minutes in a sonicator bath. Sample solutions were centrifuged at 16100 G for 15 minutes and an aliquot of 5 µL was diluted 400 times. Data recording: The UV-vis spectra were acquired in the 200-700 nm range with CARY 400 spectrophotometer using a 1 cm pathway quartz cuvette. A) fresh Italian saffron B) saffron dessicated in an electric oven at 40°C for 45 minutes, stored for 78 days, and C) stored for 212 days 1. Technique: UV-Vis spectroscopy AUTh collection Scientific coordinator: Professor Maria Z. Tsimidou (Chair) Analysts: Dr. Stella A. Ordoudi (WG3 co-leader), Dr. Anastasia Kyriakoudi (WG2 member, ESR) Sample preparation and extraction procedure: Samples in stigma form were carefully ground with an agate pestle and mortar prior to analysis. Extraction was carried out using a mixture of methanol:water (1:1, v/v) (Kyriakoudi et al., 2012) and following the procedure described in ISO 3632-2 (2010). Working solutions were prepared after dilution of the extracts by 1:10 v/v and filtration (RC55, 13 mm i.d., 0.45m pore size). Data recording: The UV−Vis spectra of working solutions were recorded in the region 200−600 nm using a Shimadzu UV 1601 spectrophotometer (Kyoto, Japan) with a slit width of 2 nm and quartz cells (1 × 1 × 4 cm). Second derivative spectra were calculated using the UVPC 1601 (Personal Spectroscopy Software, v.3.9, Shimadzu) software facilities (delta lamda of 10, scaling x1000). 2nd Derivative spectrum Zero order spectrum Sample Code: FA1101-K-D-11 λmax (nm)a,b 439 ± 0.00 327 ± 1.04 262 ± 0.00 Sample Code: FA1101-K-D-12 λmax (nm)a,b 441 ± 0.29 Sample Code: λmax (nm)a,b 439 ± 0.29 331 ± 0.29 262 ± 0.00 FA1101-K-F-12 331 ± 0.29 263 ± 0.00 Sample Code: FA1101-M-D-11 λmax (nm)a,b 440 ± 0.00 322 ± 0.00 262 ± 0.00 Sample Code: FA1101-M-D-12 λmax (nm)a,b 440 ± 0.00 321 ± 0.00 263 ± 0.00 Sample Code: FA1101-M-D-13 λmax (nm)a,b 439 ± 0.00 331 ± 0.00 262 ± 0.00 Sample Code: λmax (nm)a,b 440 ± 0.00 FA1101-M-L-12 322 ± 0.00 263 ± 0.00 Sample Code: FA1101-M-L-13 λmax (nm)a,b 440 ± 0.00 326 ± 0.29 262 ± 0.29 Sample Code: FA1101-S-F-13 λmax (nm)a,b a 440 ± 0.00 321 ± 0.58 262 ± 0.00 Mean value of three independent measurements ± SD; bfrom the corresponding λmin values in the 2nd derivative spectra Discussion The UV-Vis spectra of aqueous-methanolic extracts present three characteristic bands in the zeroorder spectra. After 2nd-order derivatization of the spectra, the peaks are better resolved. The minima (valleys) that correspond to the three max of the original spectrum oscillate between (a) 438.7 - 441.3 nm, (b) 320.4 – 331.3 nm, (d) 262.3 – 263 nm. The samples originating from the area of Kozani (Greece) and corresponded to the 2012 harvest (FA1101-K-D-2012, FA1101-K-L-2012) as well as one sample originating from La Mancha (Spain) and corresponded to the 2013 harvest (FA1101-MD-2013) contributed to the longest red shifts in the absorbance wavelength within the region of 320340 nm. This finding could indicate a high content in cis-isomers of crocetin esters with sugars (Tarantilis, Tsoupras, & Polissiou, 1995). In all cases, the intensity of absorbance around 440 nm was higher than 1.00 a.u. 2. Technique: HPLC-DAD AUTh Collection Scientific coordinator: Professor Maria Z. Tsimidou (Chair) Analysts: Dr. Stella A. Ordoudi (WG3 co-leader), Dr. Anastasia Kyriakoudi (WG2 member, ESR) Apparatus, elution and recording conditions: Pump P4000 Finnigan MAT (Thermo Separation Products Inc., San Jose, CA, USA), autosampler Midas 830 (Spark, Emmen, Holland), diode array detector UV 6000LP (Thermo Separation Products Inc.) and degasser (Spectra System SCM1000, Thermo Separation Products Inc.). Column: LiChroCART Superspher 100 C18 (125×4 mm i.d.; 4 μm) (Merck, Darmstadt, Germany). Elution system: mixture of water:acetic acid (1 %, v/v) (A) and acetonitrile (B). Linear gradient (20 to 100 % B in 20 min). Flow rate: 0.5 mL/min. Injection volume: 20 µL. Chromatographic data were processed using ChromQuest version 3.0 software (Thermo Separation Products). Monitoring was in the range of 200−550 nm. Chromatograms Sample: FA1101-K-F-12 Spectral Data Identitya,b λmax = 247 picrocrocin λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg m AU λmax = 438 trans-2-gg mAU tR λmax = 435/326 cis-4-GG mAU Peak number λmax = 434/325 cis-3-Gg mAU UV-Vis Spectra and identity of peaks : FA1101-K-F-12 λmax = 435 trans-2-G UV-Vis 4.16 Min Lambda Max 300 200 200 100 100 526 mAU 4.16 min 344 1 mAU 247 300 0 0 250 300 350 400 450 500 550 nm 5.32 Min 1000 442 500 mAU 5.32 min 500 260 2 mAU Lambda Max 462 1000 0 0 200 250 300 350 400 450 500 550 nm 5.98 Min Lambda Max 600 5.98 min 462 400 400 200 200 260 3 mAU 441 600 0 0 250 300 350 400 450 500 550 nm 6.87 Min Lambda Max 40 242 6.87 min 20 20 218 4 m AU 438 40 0 0 250 300 350 400 450 500 550 nm 7.73 Min 150 Lam bda Max 150 100 326 50 50 4 35 7.73 min 243 5 mAU 100 0 250 300 350 400 0 450 500 550 nm 8.51 Min 60 434 60 Lambda Max 20 40 325 8.51 min 245 6 mAU 40 20 0 0 250 300 350 400 450 500 550 nm 435 60 40 40 20 20 246 8.75 min mAU 7 Lambda Max 457 8.75 Min 60 0 0 250 300 350 400 450 500 550 nm a Identification based on in-house isolated crocetin digeniobiosyl ester and picrocrocin standards, spectra matching with literature data; bNomenclature was adopted from Carmona et al., 2006; transor cis-4-GG: isomers of crocetin digentiobiosyl ester; trans- or cis-3-Gg: isomers of crocetin gentiobiosyl-glucosyl monogentiobiosyl ester ester; trans-2-gg : crocetin diglucosyl ester ; trans-2-G: crocetin Sample: FA1101-K-D-11 1400 UV6000-440nm sodeia_2011_rep1 1200 1200 1000 1000 800 600 600 400 400 200 200 mAU 800 0 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 UV6000-250nm sodeia_2011_rep1 500 500 400 400 300 300 200 200 100 100 0 mAU mAU 1400 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 mAU λmax = 247 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU 4.20 Min identity λmax = 437 trans-2-gg mAU UV-Vis 300 Spectral Data λmax = 435/325 cis-4-GG mAU tR λmax = 434/323 cis-3-Gg mAU UV-Vis Spectra and identity of peaks : FA1101-K-D-11 λmax = 435 trans-2-G 300 247 Lambda Max 200 100 100 530 330 mAU 4.20 min 200 0 0 200 250 300 350 400 450 500 550 nm 5.29 Min mAU 500 500 260 5.29 min 1000 461 442 Lambda Max 1000 0 0 200 250 300 350 400 450 500 550 nm 6.02 Min mAU 200 200 259 6.02 min 400 462 441 Lambda Max 400 0 0 250 300 350 400 450 500 550 nm 40 40 6.91 Min 244 20 20 224 6.91 min mAU 437 Lambda Max 0 0 250 300 350 400 450 500 550 nm 7.79 Min Lambda Max 150 435 150 100 325 mAU 100 50 50 247 7.79 min 0 0 250 300 350 400 450 500 550 nm 8.61 Min 434 Lambda Max 40 323 20 248 8.61 min mAU 40 20 0 0 250 300 350 400 450 500 550 nm 457 8.77 Min Lambda Max 150 100 100 50 50 435 251 mAU 8.77 min 150 0 250 300 350 400 0 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-K-D-12 1400 UV6000-440nm sodeia_2012_rep1 1200 1200 1000 1000 800 600 600 400 400 200 200 m AU 800 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 UV6000-250nm sodeia_2012_rep1 500 500 400 400 300 300 200 200 100 100 0 0 mAU 0 2 4 6 8 10 Minutes 12 14 16 18 200 200 175 175 150 150 125 125 100 100 75 75 50 50 25 25 0 0 -25 -25 -50 -50 0 mAU 0 2 4 6 8 10 Minutes 12 14 16 18 20 mAU mAU 1400 mAU λmax = 246 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU 4.24 Min identity λmax = 438 trans-2-gg mAU UV-Vis 400 Spectral Data λmax = 435/326 cis-4-GG mAU tR λmax = 434/324 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in FA1101-K-D-12 λmax = 435 trans-2-G 400 200 200 472 339 4.24 min mAU 246 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm 5.30 Min Lambda Max 462 442 1000 500 500 260 5.30 min mAU 1000 0 200 0 250 300 350 400 450 500 550 nm 6.07 Min 400 441 200 200 239 6.07 min mAU Lambda Max 462 400 0 200 0 250 300 350 400 450 500 550 nm 40 40 6.93 Min 20 20 297 6.93 min mAU 242 438 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm 435 7.83 Min Lambda Max 326 100 50 50 247 7.83 min mAU 100 0 0 250 300 350 400 450 500 550 nm 434 40 20 324 mAU 20 8.70 min 8.70 Min Lambda Max 247 40 0 0 -20 250 300 350 400 450 500 -20 550 nm 8.84 Min 435 100 50 50 246 mAU 8.84 min 457 Lambda Max 100 0 0 250 300 350 400 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-M-L-12 1400 UV6000-440nm spanish_fresh_stigmas_2012_a 1200 1200 1000 1000 800 600 600 400 400 200 200 mAU 800 0 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 UV6000-250nm spanish_fresh_stigmas_2012_a 500 500 400 400 300 300 200 200 100 100 0 mAU mAU 1400 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 Spectral Data identity mAU λmax = 248 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU λmax = 439 trans-2-gg mAU λmax = 435/326 cis-4-GG mAU tR λmax = 433/324 cis-3-Gg mAU UV-Vis Spectra and identity of peaks FA1101-M-L-12 UV-Vis λmax = 435 trans-2-G 4.14 Min Lambda Max 400 200 200 527 345 4.14 min mAU 248 400 0 0 200 250 300 350 400 450 500 550 nm 5.25 Min mAU 500 500 260 5.25 min 1000 462 442 Lambda Max 1000 0 0 200 250 300 350 400 450 500 550 nm 5.98 Min 462 400 400 200 200 259 mAU 5.98 min 441 Lambda Max 0 0 200 250 300 350 400 450 500 550 nm 6.83 Min 40 40 20 20 223 6.83 min mAU 242 439 Lambda Max 0 200 0 250 300 350 400 450 500 550 40 435 nm 7.67 Min 40 20 326 20 244 7.67 min mAU Lambda Max 0 200 0 250 300 350 400 450 500 550 nm 8.49 Min Lambda Max 433 15 15 10 324 5 247 8.49 min mAU 10 5 0 0 -5 -5 250 300 350 400 450 500 550 nm 200 200 8.69 Min 435 100 100 248 mAU 8.69 min 458 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-M-D-11 1400 1400 UV6000-440nm spanish_dry_stigmas_harvest_2011_a 1200 1000 1000 800 800 600 600 400 400 200 200 mAU mAU 1200 0 0 2 mAU 600 4 6 8 10 Minutes 12 14 16 18 20 600 UV6000-250nm spanish_dry_stigmas_harvest_2011_a 500 500 400 400 300 300 200 200 100 100 0 mAU 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 mAU λmax = 248 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU 4.19 Min identity λmax = 439 trans-2-gg mAU UV-Vis 400 Spectral Data λmax = 435/325 cis-4-GG mAU tR λmax = 434/323 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in FA1101-M-D-11 λmax = 435 trans-2-G 400 200 200 419 334 4.19 min mAU 248 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm 463 5.33 Min 1000 500 500 442 260 5.33 min mAU Lambda Max 1000 0 200 0 250 300 350 400 450 500 550 nm 6.02 Min 600 463 400 400 200 200 259 mAU 6.02 min Lambda Max 441 600 0 200 0 250 300 350 400 450 500 550 nm 6.91 Min 462 439 30 20 mAU 20 6.91 min Lambda Max 243 30 10 10 0 0 250 300 350 400 450 500 550 nm 7.71 Min Lambda Max 40 325 244 20 20 435 7.71 min mAU 40 0 250 300 350 400 0 450 500 550 nm 8.52 Min Lambda Max 434 40 20 323 20 245 8.52 min mAU 40 0 0 250 300 350 400 450 500 550 nm 200 8.75 Min 200 458 100 100 246 8.75 min mAU 435 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-M-D-12 1400 UV6000-440nm spanish_dry_stigma_harvest_2012_a 1200 1200 1000 1000 800 600 600 400 400 200 200 mAU 800 0 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 UV6000-250nm spanish_dry_stigma_harvest_2012_a 500 500 400 400 300 300 200 200 100 100 0 mAU mAU 1400 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 identity mAU λmax = 248 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU λmax = 440 trans-2-gg mAU UV-Vis 400 Spectral Data λmax = 435/326 cis-4-GG mAU tR λmax = 434/324 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in FA1101-M-D-12 λmax = 435 trans-2-G 400 4.15 Min 200 200 517 341 4.15 min mAU 248 Lambda Max 0 200 0 250 300 350 400 450 500 550 nm 5.33 Min 1000 462 442 Lambda Max 500 500 260 5.33 min mAU 1000 0 200 0 250 300 350 400 450 500 550 nm 5.99 Min 400 400 200 200 260 mAU 5.99 min 600 462 441 Lambda Max 600 0 200 0 250 300 350 400 450 500 550 nm 6.88 Min 40 242 mAU 6.88 min 461 440 Lambda Max 40 20 20 0 200 0 250 300 350 400 450 500 550 nm 7.70 Min 435 Lambda Max 50 326 50 100 245 7.70 min mAU 100 0 200 0 250 300 350 400 450 500 550 nm 8.51 Min 434 Lambda Max 40 324 20 245 8.51 min mAU 40 20 0 0 -20 250 300 350 400 450 500 -20 550 nm 8.76 Min 100 50 50 248 200 435 0 250 300 350 400 0 458 8.76 min mAU Lambda Max 100 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-M-L-13 1400 Detector 1-440nm spanish 2013 fresh 20ul rep1 1200 1200 1000 1000 800 800 600 600 400 400 200 200 mAU mAU 1400 0 0 2 mAU 600 4 6 8 10 Minutes 12 14 16 18 20 600 Detector 1-250nm spanish 2013 fresh 20ul rep1 500 500 400 400 300 300 200 200 100 100 0 mAU 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 Spectral Data identity mAU λmax = 249 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU λmax = 438 trans-2-gg mAU λmax = 434/326 cis-4-GG mAU tR λmax = 433/325 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in Sample: FA1101-M-L-13 UV-Vis λmax = 434 trans-2-G 3.80 Min Lambda Max 200 100 100 442 320 3.80 min mAU 249 200 0 0 200 250 300 350 400 450 500 550 nm 750 461 Lambda Max 500 500 250 250 260 mAU 5.08 min 442 5.08 Min 750 0 0 200 250 300 350 400 450 500 550 nm 5.82 Min 400 mAU 200 200 239 5.82 min 461 441 Lambda Max 400 0 0 200 250 300 350 400 450 500 550 nm 6.80 Min Lambda Max mAU 20 20 239 332 6.80 min 40 438 40 0 0 250 300 350 400 450 500 550 nm 7.56 Min Lambda Max 434 30 30 258 326 20 mAU 20 7.56 min 10 10 0 0 250 300 350 400 450 500 550 nm 8.41 Min Lambda Max 10 433 20 224 mAU 20 8.41 min 30 247 30 10 0 0 250 300 350 400 450 500 550 nm 100 456 Lambda Max 50 50 256 mAU 8.61 min 434 8.61 Min 100 0 200 0 250 300 350 400 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Sample: FA1101-M-D-13 1400 Detector 1-440nm spanish 2013 dry 20ul rep1 1200 1200 1000 1000 800 600 600 400 400 200 200 mAU 800 0 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 Detector 1-250nm spanish 2013 dry 20ul rep1 500 500 400 400 300 300 200 200 100 100 0 mAU mAU 1400 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 Spectral Data identity mAU λmax = 249 picrocrocin mAU λmax = 441 trans-4-GG mAU λmax = 441 trans-3-Gg mAU λmax = 438 trans-2-gg mAU λmax = 434/325 cis-4-GG mAU tR λmax = 434/325 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in FA1101-M-D-13 UV-Vis λmax = 434 trans-2-G 3.71 Min 300 200 200 100 100 400 326 mAU 3.71 min 244 Lambda Max 300 0 0 250 300 350 400 450 500 550 nm 1000 1000 4.87 Min 461 500 500 238 4.87 min mAU 441 Lambda Max 0 0 200 250 300 350 400 450 500 550 nm 400 200 200 238 mAU 5.53 min 461 441 5.53 Min Lambda Max 400 0 0 200 250 300 350 400 450 500 550 nm 6.63 Min Lambda Max 75 438 75 mAU 50 50 25 25 0 238 331 6.63 min 0 250 300 350 400 450 500 550 434 nm 7.37 Min Lambda Max 100 325 7.37 min mAU 239 100 50 50 0 0 250 300 350 400 450 500 550 nm 8.11 Min 75 Lambda Max 434 240 75 50 325 mAU 50 8.11 min 25 25 0 0 250 300 350 400 450 500 550 nm 8.29 Min 456 434 50 mAU 50 8.29 min 75 Lambda Max 242 75 25 25 0 0 250 300 350 400 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) 1400 1400 1200 1200 1000 1000 800 800 600 600 400 400 200 200 mAU mAU Sample: FA1101-S-F-13 0 0 mAU 600 2 4 6 8 10 Minutes 12 14 16 18 20 600 Detector 1-250nm italikos krokos saffronomics meoh_h2o_50_50 araiwsh 1_10 rep1 500 500 400 400 300 300 200 200 100 100 0 mAU 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 100 100 80 60 60 40 40 20 20 mAU mAU 80 0 0 0 2 4 6 8 10 Minutes 12 14 16 18 20 Spectral Data identity mAU λmax = 247 picrocrocin mAU λmax = 442 trans-4-GG mAU λmax = 441 trans-3-Gg mAU λmax = 437 trans-2-gg mAU λmax = 435/326 cis-4-GG mAU (tR) λmax = 433/325 cis-3-Gg mAU UV-Vis Spectra and identity of peaks in FA1101-S-F-13 UV-Vis λmax = 435 trans-2-G 4.23 Min Lambda Max 400 200 200 416 347 4.23 min mAU 247 400 0 0 200 250 300 350 400 450 500 550 nm 1500 442 5.35 Min Lambda Max 1000 1000 500 500 260 mAU 5.35 min 461 1500 0 0 200 250 300 350 400 450 500 550 nm 6.07 Min 600 400 400 200 200 260 mAU 6.07 min 462 441 Lambda Max 600 0 0 200 250 300 350 400 450 500 550 nm 6.94 Min 40 40 20 20 0 0 mAU 6.94 min 437 242 Lambda Max -20 200 250 300 350 400 450 500 -20 550 nm 7.75 Min Lambda Max 20 246 434 40 229 7.75 min mAU 40 20 0 0 -20 200 250 300 350 400 450 500 -20 550 nm 8.57 Min 227 mAU 8.57 min 20 433 247 Lambda Max 20 0 -20 200 0 250 300 350 400 450 500 -20 550 nm 8.79 Min 457 150 Lambda Max 150 100 50 435 50 248 mAU 100 8.79 min 0 200 250 300 350 400 0 450 500 550 nm (Peak numbers as annotated in the chromatogram of FA1101-K-F-12) Discussion At least seven apocarotenoid metabolites are detected in samples of high quality originating from Kozani, La Mancha and Sardinia regions. Picrocrocin peak with a max value of 246-249 nm was evident in all of the samples. The most abundant crocetin ester, that is the trans isomer of crocetin digentiobiosyl ester (trans-4-GG) absorbs at 260/441-442/461-463 nm and the corresponding cisisomer at 258-260/325-326/434-435 nm. The ester derivative with three sugar moieties, that is the trans-isomer of crocetin gentiobiosyl-glycosyl ester (trans-3Gg), absorbs at 259-260/441/461-463 nm while the cis-isomer at 323-325/433-434. Thus, compounds with lower number of sugars may show a blue shift in the absorbance maxima in the UV region. Moreover the trans-2gg and trans-2G esters of crocetin, each bearing two glucose ester moieties, have characteristic max at 437-440 and 434-435 nm, respectively. On the basis of the peak intensity it could be suggested that FA1101-K-F-12, FA1101-K-D-12, FA1101-K-D-11, FA1101-M-D-13 and FA1101-M-D-12 were the richest in cis-4-GG ester. Literature Kyriakoudi, A., Chrysanthou, A., Mantzouridou, F., Tsimidou, M.Z. Revisiting extraction of bioactive apocarotenoids from Crocus sativus L. dry stigmas (saffron). Anal Chim Acta. 2012, 755, 77-85. Carmona, M., Zalacain, A., Sanchez, A.M., Novella, J.L., Alonso, G.L. Crocetin esters, picrocrocin and its related compounds present in Crocus sativus stigmas and Gardenia jasminoides fruits. Tentative identification of seven new compounds by LC-ESI-MS. 2006, J. Agric. Food Chem. 54, 973-979. ISO 3632-2 (2010) Saffron (Crocus sativus Linneaus) Part 2: Test methods. International Organisation for Standardization, Geneva. Tarantilis, P., Tsoupras, G., & Polissiou, M. Determination of saffron (Crocus sativus L.) components in crude plant extract using high -performance liquid chromatography-UV-visible photodiode-array detection mass spectrometry. Journal of Chromatography A, 1995, 699, 107-118. 3. Technique: HPLC–ESI–MS ENEA collection Scientific Coordinator: Giovanni Giuliano, ENEA Sample preparation 3 mg of stigma powder were extracted in 300 µl of cold 50% methanol, homogenized for 40’ in MM 300 at 18Hz, and centrifuged 20’ at 20000 g at 4°C. The supernatant was recovered and analyzed by HPLC-ESI-MS. Apparatus, elution and recording conditions: Twenty microliters of diluted extracts were analyzed by a Discovery LTQ-Orbitrap mass spectrometry system using ElectroSpray Ionization (ESI) (operating in positive ion mode), coupled to an Accela UHPLC system equipped with a photodiode array detector (ThermoFisher Scientific). LC separations were performed using a C18 Luna reverse-phase column (100×2.1 mm, 2.6 m particle size; Phenomenex). The mobile phases used were water + 0.1 % formic acid (A), and acetonitrile +0.1 % formic acid (B). The chromatographic separation was developed using the following gradient: HPLC GRADIENT Time (min) % A % B 0 95 5 0.5 95 5 24 25 75 26 25 75 27 95 5 32 95 5 UV-VIS detection was continuously acquired from 220 to 700 nm. All solvents used were LC-MS grade quality (CHROMASOLV; Sigma-Aldrich). For ESI-MS ionization nitrogen was used as sheath and auxiliary gas, set to 35 and 15 units, respectively. The capillary temperature was 290 °C, the spray voltage, the capillary voltage and tube lens settings were 3 andand 165 V, respectively. Identification was achieved on the basis of accurate masses and, when available, by comparison with authentic reference substances. Metabolites were identified by their order of elution and online absorption spectra co-migration with authentic standards (crocetin, crocins) and on the basis of the m z-1 accurate masses obtained from the Pubchem database (http://pubchem.ncbi.nlm.nih.gov/) for native compounds, or from the Metabolomics Fiehn Lab Mass Spectrometry Adduct Calculator (http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/MSAdduct-Calculator/) for adducts. Metabolite peaks were integrated at their individual λmax and formononetin at 260 nm. For PDA normalization and quantification, all peaks were normalized relative to the internal standard (formononetin) to correct for extraction and injection variability. An external calibration curve of formononetin, run separately, was used to calculate absolute amounts. All peaks underwent a second normalization to correct for their individual molar extinction coefficients. Metabolites were also validated on the base of the accurate masses, or MS/MS validation. MS Quantification was performed in a relative way, as fold level with respect to the formononetin content. In the ppt we reported the PDA and mass spectra of the principal metabolites identified, showing one of the different adducts observed (M+H; M+NH4; M+Na; M+K+Na; M+Na+NH4). SAFFRON POLAR EXTRACT (Castilla‐La Mancha DRIED 2013) 4 RT: 0.00 - 32.04 SM: 9G PDA 10 31 10.31 5 600000 11.22 uAU 500000 400000 7 300000 1 2 8.32 8 52 8.52 100000 5 98 5.98 1.56 1.72 0 6 12.29 7.17 8.43 40000000 3 35000000 8 9 13.70 3 200000 4 13.92 14 72 14.72 15.44 16.93 19.27 24.72 25.84 28.39 29.53 12.98 13.88 NL: 4.24E7 TIC F: FTMS + p ESI Full ms [110.001800.00] MS A TIC 9 10 5 25000000 10 7‐8 10.47 30000000 Intensity NL: 6.89E5 Total Scan PDA A 11.35 20000000 15000000 1 1 27 1.27 10000000 5.42 4.49 5000000 14.81 15 57 15.57 7.65 28.57 18.33 19.13 6.09 23.33 24.14 24 26 29.67 0 0 2 4 6 8 10 12 14 16 18 Time (min) 1: kaempferol‐3‐O‐sophoroside‐7‐glucoside 2: kaempferol‐3,7,4’‐triglucoside 3: picrocrocin 3: picrocrocin 4: trans‐crocin 4 5: trans‐crocin 3 20 22 6: trans –crocin 2’ 7: cis‐crocin 4 8: trans‐crocin 8: trans crocin 2 2 9: cis crocin 3 10: trans‐crocin 1 28 30 32 Kaempferol‐3‐O‐sophoroside‐7‐glucoside RT: 0.00 - 32.04 SM: 9G NL: 4.24E7 TIC F: FTMS + p ESI Full ms [110 00-1800 [110.00 1800.00] 00] MS A 8.43 40000000 10.47 12.98 13.88 a 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1.27 14.81 15.57 7.65 10000000 4 49 4.49 5000000 5 42 5.42 0 28.57 18.33 19.13 6.09 23.33 24.14 29 67 29.67 NL: 5.14E5 m/z= 773.21091773.21401 F: FTMS + p ESI Full ms [110 00 1800 00] [110.00-1800.00] MS A 6.09 500000 kaempferol‐3‐O‐sophoroside‐7‐glucoside Intensityy 400000 b 300000 m/z= 773.2124 / 773 2124 200000 7.32 100000 8.38 0 0 2 4 6 8 10 12 14 16 18 Time (min) 20 22 24 26 28 Mass spectrum at RT 6.09 p A #329 RT: 6.09 AV: 1 SM: 9G NL: 1.09E6 F: FTMS + p ESI Full ms [110.00-1800.00] 200000 237.0000 347 0000 347.0000 180000 449.1077 700000 d 220000 kaempferol‐3‐O‐sophoroside‐7‐glucoside (M+H) (M H) 800000 32 A #1796 RT: 5.98 AV: 1 SM: 9G NL: 2.44E5 microAU F: FTMS + p ESI Full ms [110.00-1800.00] 266.0000 240000 c 773.2125 900000 30 PDA spectrum at RT 5.98 PDA spectrum at RT 5.98 1000000 160000 140000 600000 uAU Intensity TIC 500000 120000 100000 400000 141.9585 80000 300000 200000 100000 60000 182.9852 496.2020 287.0547 414.0697 20000 863.1065 0 200 400 40000 827.1305 611.1600 665.0790 600 800 1000 m/z 1117.6383 1200 1305.2982 1481.9493 1599.3378 1400 1600 0 1800 250 300 350 400 450 wavelength (nm) 500 550 600 650 700 PICROCROCIN 0.00 - 32.04 SM: 9G NL: 4.24E7 TIC F: FTMS + p ESI Full ms [110.00-1800.00] MS A 8.43 40000000 10.47 a 12 98 13.88 12.98 13 88 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1 27 1.27 14.81 15.57 7.65 10000000 0 28.57 18.33 19.13 6.09 5.42 4.49 5000000 23.33 24.14 29.67 NL: 6.60E6 m/z= 348.20100348 20240 F: 348.20240 FTMS + p ESI Full ms [110.00-1800.00] MS A 8.43 6000000 b picrocrocin 5000000 Intensity TIC 4000000 3000000 m/z= 348.2017 m/z 348.2017 2000000 1000000 9.42 10.94 0 0 2 4 6 8 10 30.59 12 14 16 18 Time (min) 20 Mass spectrum at RT 8.43 26 28 30 32 PDA spectrum at RT 8.32 1500000 picrocrocin (M+NH4) 151.1116 4500000 d 1400000 c 5500000 1300000 1200000 1100000 1000000 900000 uAU 4000000 Intensity 24 A #2498 RT: 8.32 AV: 1 SM: 9G NL: 1.59E6 microAU F: FTMS + p ESI Full ms [110.00-1800.00] 250.0000 A #473 RT: 8.43 AV: 1 SM: 9G NL: 6.13E6 F: FTMS + p ESI Full ms [110.00-1800.00] 348.2017 6000000 5000000 22 3500000 800000 700000 523.2189 3000000 600000 2500000 500000 2000000 400000 300000 1500000 100000 687.3051 500000 0 200000 714.2542 1000000 256.9979 200 385.0942 400 604.2447 600 761.2670 1028.4180 1144.3446 800 1000 / 1200 1329.1479 1400 1537.2561 1600 1740.5668 1800 333.0000 0 250 300 350 395.0000 400 450 wavelength (nm) 500 550 600 650 700 CROCIN 4 (crocetin di‐gentiobioside) RT: 0.00 - 32.04 SM: 9G 8.43 40000000 10.47 NL: 4.24E7 TIC F: FTMS + p ESI Full ms [[110.00-1800.00]] MS A a 12.98 13.88 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1.27 14.81 15.57 7.65 10000000 28.57 18.33 19.13 6.09 5.42 4.49 5000000 0 23 33 23.33 24 14 24.14 29.67 NL: 1.67E6 m/z= 994.41058994.41456 F: FTMS + p ESI Full ms [110.00-1800.00] MS A 10.43 1600000 trans crocin 4 1400000 b 1200000 Intensity TIC 1000000 13.81 800000 m/z= 994.4125 / cis crocin 4 600000 400000 200000 0 2 A #593 RT: 10.43 AV: 1 SM: 9G NL: 3.37E6 F: FTMS + p ESI Full ms [110.00-1800.00] 329.1747 4 6 8 10 12 16.37 14 16 18 Time ((min) Ti i ) 20 22 24 26 30 32 p ESI Full ms [110.00 1800.00] PDA spectrum at RT 10.31 442.0000 500000 Mass spectrum at RT 10.43 3000000 465.0000 450000 400000 293.1537 2800000 28 F: FTMS 3200000 trans crocin 4 (M+NH4) 2600000 994.4119 2400000 2000000 350000 300000 uAU 2200000 In ten sity 11.30 9.55 0 670 3067 670.3067 1800000 1600000 250000 200000 1400000 150000 1200000 1000000 100000 506.2232 800000 961.3612 360.1499 635.2697 600000 1031.3041 229.1223 400000 707.1987 473.2166 200000 1135.3776 869.9214 0 200 400 600 800 A #785 RT: 13.81 AV: 1 SM: 9G NL: 1.39E6 F: FTMS + p ESI Full ms [110.00-1800.00] 1000 m/z 994.4112 1300000 1200 1322.5812 1483.1049 1376.0142 1621.5370 1772.0795 1400 1600 262.0000 50000 c 1800 235.0000 d 0 250 F: FTMS 300 350 400 450 wavelength (nm) p ESI Full ms [[110.00 1800.00]] 500 550 600 650 700 435.0000 550000 Mass spectrum at RT 13.81 PDA spectrum at RT 13.70 500000 1200000 450000 1100000 400000 1000000 350000 800000 cis crocin 4 (M+NH4) 700000 600000 500000 516.1559 400000 200000 100000 186.9562 250000 200000 100000 311.1639 150.1124 300000 649.2183 707.1990 487.1655 617.2588 811.2704 942.3162 1091.9570 327.0000 236.0000 150000 1031.3027 300000 uAU u In te n sityy 900000 1271.8606 1444.9579 1551.9673 262.0000 50000 1731.4205 0 0 200 400 600 800 1000 m/z 1200 1400 1600 1800 250 300 350 400 450 wavelength (nm) 500 550 600 650 700 CROCIN 3 (crocetin gentiobioside‐glucoside) RT: 0.00 - 32.04 SM: 9G 8.43 40000000 10.47 NL: 4.24E7 TIC F: FTMS + p ESI Full ms [[110.00-1800.00]] MS A a 12.98 13.88 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1.27 14.81 15.57 7.65 10000000 5.42 4.49 5000000 28.57 18.33 19.13 6.09 0 23.33 24 14 24.14 29.67 11.35 1200000 trans crocin 3 1000000 b 800000 Inten nsity TIC 600000 14.81 400000 NL: 1.35E6 m/z= 832.35808832.36140 F: FTMS + p ESI Full ms [110.00-1800.00] MS A m/z= 832.3597 / cis crocin 3 200000 0 0 2 4 A #649 RT: 11.35 AV: 1 SM: 9G NL: 1.98E6 F: FTMS + p ESI Full ms [110.00-1800.00] 329.1747 9.49 10.43 8 10 6 13.58 12 14 15.85 16 18 Time ((min)) 20 Mass spectrum at RT 11.35 1800000 293.1537 22 24 26 28 30 32 F: FTMS + p ESI Full ms [110.00-1800.00] 441.0000 PDA spectrum at RT 11.22 465.0000 350000 832.3593 1600000 300000 1400000 trans crocin 3 (M+NH4) 1000000 250000 uA AU Inte nsity 1200000 800000 200000 00000 150000 670.3066 236.0000 600000 353.1055 100000 400000 200000 229.1223 435.1298 567.5399 707.1991 473.2168 0 200 400 600 800 1000 m/z 1200 1400 1600 c 1800 250 350 400 450 wavelength (nm) 500 550 600 650 700 240000 Mass spectrum at RT 14.81 550000 500000 PDA spectrum at RT 14.72 220000 200000 236.0000 180000 450000 160000 400000 350000 300000 140000 cis crocin 3 (M+NH4) 329.1747 250000 200000 uAU Intensity 300 434 0000 434.0000 832.3593 600000 869.2510 473.2165 1306.8026 1467.8925 1042.2054 40000 1673.8409 0 200 400 600 326.0000 80000 60000 707.1984 670.3069 182.9852 120000 100000 797.3216 435.1297 150.1124 100000 50000 d 0 F: FTMS + p ESI Full ms [110.00-1800.00] A #841 RT: 14.81 AV: 1 SM: 9G NL: 6.30E5 F: FTMS + p ESI Full ms [110.00-1800.00] 150000 262.0000 50000 868.2442 1240.0247 971.8296 1111.0918 1353.9122 1472.0244 1647.6897 800 1000 m/z 1200 1400 1600 1800 20000 0 250 300 350 400 450 wavelength (nm) 500 550 600 650 700 CROCIN 2 (crocetin gentiobioside) RT: 0.00 - 32.04 SM: 9G 8.43 40000000 10.47 NL: 4.24E7 TIC F: FTMS + p ESI Full ms [110 00 1800 00] [110.00-1800.00] MS A a 12.98 13.88 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1.27 14.81 15.57 7.65 10000000 4.49 5000000 5.42 28 57 28.57 18.33 19.13 6.09 0 23.33 24.14 29.67 NL: 1.45E6 m/z= 670.30558670.30826 F: FTMS + p ESI Full ms [110.00-1800.00] MS A 10.47 1400000 b 1200000 Intensity 1000000 trans crocin 2 800000 14.02 400000 200000 16.92 9.49 0 0 2 4 6 8 10 12 14 17.93 16 18 Time (min) 20 22 24 26 28 30 32 Mass spectrum at RT 14.02 PDA spectrum at RT 13.88 F: FTMS + p ESI Full ms [110.00 1800.00] A #801 RT: 14.08 AV: 1 SM: 9G NL: 3.38E5 F: FTMS + p ESI Full ms [110.00-1800.00] 329.1746 435.0000 200000 670.3067 320000 180000 293.1535 236.0000 280000 160000 c 260000 240000 trans crocin 2 (M+NH4) 200000 d 140000 120000 uAU 220000 Intensity m/z= 670.3069 / 11.35 600000 300000 TIC 180000 100000 160000 80000 140000 120000 158.9613 100000 706.1913 80000 60000 994.4125 186.9562 379.0319 516.1553 40000 20000 327.0000 60000 635.2712 753.2054 955.3600 40000 1092.0587 1302.9343 1427.5153 1174.0093 1621.9093 1734.6949 20000 0 0 200 400 600 800 1000 m/z 1200 1400 1600 1800 250 300 350 400 450 wavelength (nm) 500 550 600 650 700 CROCIN 1 (crocetin glucoside) RT: 0.00 - 32.04 SM: 9G NL: 4.24E7 TIC F: FTMS + p ESI Full ms [110 00-1800 [110.00 1800.00] 00] MS A 8.43 40000000 10.47 a 12.98 13.88 35000000 Intensity 30000000 25000000 11.35 20000000 15000000 1.27 14.81 15.57 7.65 10000000 4.49 5000000 5.42 28.57 18.33 19.13 6.09 0 23.33 24.14 29.67 NL: 4.60E3 m/z= 513.20837513.21043 F: FTMS + p ESI Full ms [110.00-1800.00] MS A 11.35 4000 15.57 b trans crocin 1 m/z= 513.2094 / 3000 Intensity TIC 2000 1000 0 0 2 4 6 8 10 12 14 16 18 Time (min) M Mass spectrum at RT 15.57 t t RT 15 57 A #881 RT: 15.57 AV: 1 SM: 9G NL: 5.06E4 F: FTMS + p ESI Full ms [110.00-1800.00] 20 22 24 26 28 30 32 PDA spectrum at RT 15.44 p A #4633 RT: 15.44 15 44 AV: 1 SM: 9G NL: 1.86E5 1 86E5 microAU F: FTMS + p ESI Full ms [110.00-1800.00] 236.0000 180000 536.3058 50000 160000 45000 Intensity 30000 100000 25000 80000 20000 60000 15000 537.3094 d 120000 uAU trans crocin 1 (M+Na) 35000 140000 c 40000 435.0000 40000 10000 20000 513 2103 513.2103 5000 538.3121 511.2561 523.2175 534.2687 527.2503 0 505 510 515 520 525 m/z 530 535 0 250 300 350 400 450 wavelength (nm) 500 550 600 650 700 Table 1: Principal metabolites identified in saffron polar extract (Castilla‐La Mancha dried 2013) Metabolite Monoisotopic mass trans crocin 4 trans crocin 4 cis crocin 4 trans crocin 3 cis crocin 3 trans crocin 2 trans crocin 2 trans crocin 2' trans crocin 1 crocetin picrocrocin i i safranal 3‐OH‐beta‐cyclocitral 3‐Hydroxy‐beta‐ionone Kaempferol 3‐O‐sophoroside‐7 glucoside Kaempferol 3,7,4'‐triglucoside Kaempferol 7‐sophoroside or 3‐sophoroside or rutin 976.37875 976.37875 814.32592 814.32592 652 27310 652.27310 652.27310 490.22028 328.16740 330 16780 330.16780 150.10446 168.11500 208.14642 772.20620 772.20620 610.15338 Kaempferol p 3‐β‐D‐glucopyranoside β g py or 7‐β‐D‐glucopyranoside β g py Kaempferol 3‐rutinoside‐7‐glucoside Kaempferol ‐acetylglucoside Kaempferol 3‐O‐rutinoside or Apigenin_6,8‐digalactoside Dihydrokaempferol‐7‐O‐glucoside Dihydrokaempferol 7 O glucoside Kaempferol isorhamnethin‐3‐4'‐diglucoside Rhamnetin 3‐rutinoside Naringenin 7 O glucoside (isoform 1 2and 3) Naringenin‐7‐O‐glucoside (isoform 1, 2and 3) Quercetin‐3‐diglucoside Quercetin/Delphidin Luteolin 448.10050 756.21120 490.41360 594.15840 450 11620 450.11620 286.04770 640.16390 624.16900 434 12130 434.12130 626.14830 302.04260 286.04770 4. Technique: UHPLC–QTOF University of Chemistry and Technology (UCT), Prague Faculty of Food and Biochemical Technology Department of Food Analysis and Nutrition Scientific Coordinator: Rubert Bassedas Josep Vicent Solid Liquid Extraction A portion of each saffron sample was left in a filter paper, folding the paper, saffron was homogenized crushing by pressure rolling a metallic cylinder, consecutively 50 mg were weight in a 15 mL cuvette and 5 mL of ethanol/water (70/30, v/v) were added. For increasing the effectiveness of this extraction procedure the samples were extracted assisted by sonication during 1h. After that, samples were centrifuged during 5 min at 10,000 rpm. Then, an aliquot (1mL) was transferred into the vial for the UHPLC-HRMS analysis. Ultra high Performance Liquid Chromatography High Resolution Mass Spectrometry The chromatographic separation was performed by Dionex UltiMate 3000 RS UHPLC system (Thermo Fisher Scientific, Waltham, USA), equipped with a Kinetex C18 (Phenomenex, Torrance, USA) 100 mm × 2.1 mm i.d., 1.7 μm particle size maintained at 40°C. UHPLC safe SecurityGuard ULTRA Guard (Phenomenex, Torrance, USA) precolumn of the same size and stationary phase was used. The mobile phases consisted of (A) 5 mM ammonium formate in Milli-Q water for ESI+ mode and 5 mM ammonium acetate in Milli-Q water for ESI- analyses, and (B) in both cases methanol. A multi-step elution gradient was optimized as follow: 0.0 min (95% A; 0.40 mL min-1) a gradient begun up to 11.0 min (0% A; 0.55 mL min-1), subsequently an isocratic step was executed during two minutes, 13.0 min (0% A; 0.60 mL min-1), 13.1 min (95% A; 0.40 mL min-1) a reconditioning period up to 15.0 min (95% A; 0.40 mL min-1) was used (Table III). The sample injection volume was 2 μL for both positive and negative ionization modes and the autosampler temperature was kept at 5°C. Table I. UHPLC Conditions UHPLC system Dionex UltiMate 3000 Column Kinetex C 18 (100 × 2,1 mm; 1,7 µm) A: 5mM CH3COONH4 in water B: methanol Flow Time A B rate (min) (ml/min) % % 0 0.3 95 5 11 0.4 0 100 13 0.5 0 100 13.1 0.4 95 5 15 0.4 95 5 2 µl 40 °C Mobile phase Gradient Injection Column temperature Autosampler temperature 5 °C As MS detector, AB SCIEX TripleTOF® 5600 quadrupole–time-of-flight mass spectrometer (AB SCIEX, Concord, ON, Canada) was used for saffron metabolic fingerprinting. The ion source was a Duo Spray™ with separated ESI ion source and atmospheric-pressure chemical ionization (APCI). ESI was used for the measurement of saffron metabolic fingerprinting, while APCI probe worked as the second gas heater, as well as APCI was also used for exact mass calibration of the TripleTOF instrument. In the positive ESI mode the source parameters, metabolic fingerprinting, were as follows: capillary voltage: +4500 V; nebulizing gas pressure: 60 psi; drying gas pressure: 50 psi; temperature: 550°C; and declustering potential: 80 V. The capillary voltage in negative ESI was -4000 V, other source settings were the same as for ESI-. A TOF MS acquisition method was based on Information Dependent Acquisition (IDA) approach recording both MS and MS/MS spectra. The method consisted of a survey TOF MS experiment ranged from m/z 100 to 1200, and in parallel, Product Ion (PI) spectra for the eight most intense ions of the survey spectra throughout the chromatographic run. Dynamic Background Subtraction was activated to minimize selection of background ions in the IDA experiments. PI spectra were collected for ions ranged from m/z 80 to 1200 with the quadrupole extraction window of 1 Da. The former selected precursor ion was excluded for 3 s (mass tolerance of 30 mDa) and totally excluded after three occurrences. The PI spectra were recorded with collision energy of 35 V and collision energy spread of ±15 V. In this way, both low and high energy fragment ions were present in a single spectrum. The total cycle time took 0.55 s. Table II. MS conditions Mass analyzer TripleTOF 5600 (AB SCIEX) Ionization ESI(+)/ESI(-) Capillary voltage 4.5 kV(+)/4 kV (-) Curtain Gas N2; 35 psi Nebulizing gas pressure N2; 60 psi Drying gas pressure N2; 60 psi Source temperature 550 °C m/z range: 100 - 1200 Spectrum without fragmentation Acquisition time: 100 ms m/z range: 50 - 1000 Product spectra (8×) Acquisition time: 40 ms Collision energy: 35 ± 15 V Resolving power 30,000 FWHM An automatic m/z calibration was performed by the calibration delivery system (CDS) every 15 samples using positive or negative APCI calibration solution (AB SCIEX, Concord, ON, Canada) according to the batch polarity. Each set of samples in each polarity was preceded by 3 blank controls: Milli-Q water, methanol and blank (extraction procedure without sample). At the end, the same MS approach was carried out by ESI- mode. The achieved resolving power was >31,000 (m/z 321.0192) full width at half maximum (FWHM) in both polarities. The PI spectra were measured in high sensitivity mode, which provides half resolving power. Instrument control and data acquisition were carried out with the Analyst 1.6 TF software (AB Sciex, Concord, ON, Canada) and the qualitative analysis was performed using PeakView 2.0 (AB Sciex, Concord, ON, Canada) equipped with the MasterView, Formula Finder and directly linked to ChemSpider database. It should be noted that the in-batch sequence of the samples was random (established based on random number generation) to avoid any possible time dependent changes during UHPLC–HRMS analysis, which would result in false clustering. To address overall process variability, metabolomics studies were augmented to include a set of six samples technical replicates. Reproducibility analysis for compounds detected in theses replicates provided a measure of variation for extraction, injection, retention time (RT) and mass accuracy. 1.3 Tentative identification of markers The identification of markers represents the last step and it is crucial in order to understand the metabolite pathway, since these metabolites can be metabolic intermediates, secondary metabolites and/or other components, which were originated during food processing. The use of HRMS instrumentation allowed both MS and MS/MS accurate mass spectra to be acquired, this is vital for a tentative identification of markers, which represents the most laborious and difficult step. In parallel, powerful informatics tools and databases were simultaneously used in order to estimate the elemental formula and MS/MS elucidation of each marker. Table III summarizes m/z, RT and tentative molecular formula and identification for the selected markers. Tentative m/z RT Molecular Formula identification Oxidized PC 36:4 798.5674 11.7 C 4 H NO P 4 80 9 PC 18:2/18:2 PC 36:4 782.5694 12.1 C44H80NO8P 812.6164 12.6 C 4 H NO P 6 86 8 PC 18:2/18:2 PC 38:3 PC 38:4 810.6057 12.4 C 4 H NO P 6 84 8 838.6363 12.7 C 820.5855 12.4 796.5504 11.6 O NP 8 O NP 8 NO P 9 353.2311 7.8 4H 8 88 C 4H 7 82 C 4H 4 78 Unknown PC 18:1/20:3 PC 40:4 PC 39:6 Oxidized PC 36:5 Unknown As an example, OPLS-DA loading plot Spain-PDO, as well as VIP showed that m/z 798.5674 and RT 11.7 min (peak) was the most significant marker PDO (La Mancha-Aragón). Molecular formula estimation, structural elucidation and subsequent tentative identification were performed based on MS and MS/MS accurate mass spectra using PeakView 2.0. Based on the isotope pattern of unknown compounds, for calculation C (n ≤ 50), H (n ≤ 100), N (n ≤ 10), O (n ≤ 20) P (n ≤ 15), S (n ≤ 5) atoms were considered. Variable trend plot m/z 798.5719 RT 11.7 Marker PDO Average Spain Figure I. Variable trend plot highlights variable behavior. PDO sample intensity of m/z 798.5719 and RT 11.7 min shows higher than average value. FormulaFinder software, which was used in this study, was able to predict proposed formulas according to MS and MS/MS ranking, reflecting differences between calculated and measured m/z values for both parent and fragments ions, and the match of experimental and theoretical isotope pattern in terms of spacing and relative intensities. In this example, 47 candidates were obtained using FormulaFinder (mass error < 5 ppm), but only candidates, presence of which were probable in saffron and the highest scores (MS and MS/MS range) were examined. For the molecular feature m/z 798.5674 and RT 11.7 min, the highest scoring generated formula was C44H80O9NP. The MS/MS spectra acquired by IDA method shows the most abundant product ion m/z 184.0740, which is a characteristic fragment of phosphatidylcholines (PC) using ESI+. At the same time, PeakView was directly linked to ChemSpider (www.chemspider.com), where two matches were obtained. In both cases, suggested candidates were oxidized glycerophospolipids, however it was not possible to identify the hydrophobic chains based on products ions, nor PC. Two strategies were therefore applied. The first one, it was to identify non-oxidized PC, C44H80O8NP, based on PeakView and MS and MS/MS spectra. On the other side, LipidView (AB SCIEX, Concord, ON, Canada) catalog (library) was used in order to confirm this PC. The Figure II shows the extracted ion chromatograms of C44H80O8NP (non-oxidized PC) Figure IIa, C44H80O9NP (oxidized PC) Figure IIb and C44H80O10NP (oxidized PC) Figure IIc. Non-oxidize PC, m/z 782.5714 and RT 12.2 min, molecular formula estimation, structural elucidation and subsequent tentative identification were performed based on MS and MS/MS accurate mass spectra using PeakView, even though it was not a marker in the OPLSDA loading plot, but it could be useful for the identification of the most significant marker. Based on the isotope pattern of unknown compounds, 21 candidates were obtained using FormulaFinder (mass error < 5 ppm), but only candidates, presence of which were probable in saffron and the highest scores (MS and MS/MS range) were examined. The peak m/z 782.5714 and RT 12.2 generated the highest scoring formula C44H80O8NP. Again, the most abundant product ion (m/z 184.0738), which is characteristic fragment of phosphatidylcholines. Although the rest of fragments are significantly less abundant, they could be used for a structural elucidation. Other minor product ions (m/z 520.3414) included the polar head and one hydrophobic chain. In addition, loss of water was also detected for this fragment (m/z 502.3307). Tentatively, both hydrophobic chains are identical and they are fatty acid (FA) 18:2. The theoretical and observed pathways were compared and 86% of matches were found. The PC 36:4, concretely PC 18:2/18:2, was tentatively identified (Figure III). a C44H80NO8P PC 36:4 (PC 18:2/18:2) b c C44H80NO9P (Marker) PC 36:4 Oxidized glicerophospolipid + [GP+NH ] 4 C H NO P 44 80 10 PC 36:4 Oxidized glicerophospolipid Figure II. Extracted ion chromatograms of m/z 782.5694 (a), PC 36:4, and their oxidized lipids, extracted ion chromatograms, m/z 798.5643 (b) and m/z 814.5593 (c) of saffron from La Mancha, PDO. C44H81NO8P+ Phosphatidylcholine PC 36:4 (18:2/18:2) C26H49NO6P+ C26H51NO7P+ - H2O C39H67O4+ Figure III. Glycerophospholipid pathway. Secondly, LipidView, specialized software for lipid research and identification was used. This database did not allow to confirm 782.5714 (glycerophospholipid) using the ESI+ library, since the polar head group m/z 184.0738 is the most abundant fragment ion, PC 36:4 was only identified. Nevertheless, taking advantage of negative ionization data, which was previously acquired, this marker was confirmed again. Negative ESI spectrum of m/z 780.5580 RT 12.2 min, showed as the most abundant fragment ion, m/z 279.2337, FA 18:2. They, MS and MS/MS accurate mass, were found LipidView database, this glycerophospholipid corresponded to phospatidylcholine PC 36:4 (PC 18:2/18:2). Figure IV summaries LipidView performance. ESI+ ESI- ESI+ ESIPC 36:4 PC 18:2/18:2 Figure IV. LipidView Catalog shows pseudomolecular ions and fragments for identification of lipids. Obviously, oxidized lipids were the most significant markers of PDO La Mancha-Aragón saffron. This could be as a consequence of the drying process used by saffron producers from La Mancha and Aragón area. The temperatures about 70ºC activates lipid oxidation, which is not activated by procedures used elsewhere. In order to demonstrate this food processing difference, PDO Greece saffron and labeled Spanish saffron were also compared for oxidized lipids. Because of the fact that in western of Macedonia (Greece), fresh stigmas are fully extended in thin layers and stored 12-24h in a room controlling the temperature at 25-30ºC, the lipid oxidation was not activated and therefore oxidized lipids are not present, as documented in the Figure V. Similarly, labeled Spanish saffron from unknown origin highlighted similar behavior for oxidized lipids. In both cases, PCs 36:4 of PDO Greek saffron (Figure Va) and labeled Spanish saffron (Figure Vb) were identified, however, different ratio was observed between them. It should be noted that PC 36:4 was an important marker PCA loading plot, since PC 36:4 accentuated a different behavior according to saffron origin, which differentiated saffron samples. In addition, oxidized PCs for PDO Greek saffron (Figure Vc) and labeled Spanish saffron (Figure Vd) were not detected, or these peaks showed lower responses than PDO La ManchaAragón, therefore, reflected a different food processing such PDO Greek saffron declares. As it was already explained, OPLS-DA statistical model allowed PDO La Mancha-Aragón and labeled Spanish saffron to be distinguished, at the same time metabolite pathway was related to food processing according to PDOs. Nevertheless, labeled Spanish saffron highlighted unknown origin and the food processing remains unknown. a b C H NO P 44 80 C 8 H 44 PC 36:4 (PC 18:2/18:2) NO P 80 8 PC 36:4 (PC 18:2/18:2) c d C44H80NO9P PC 36:4 Oxidized glicerophospolipid C 44 H NO P 80 9 PC 36:4 Oxidized glicerophospolipid Figure V. Extracted ion chromatograms of PDO Greek saffron and labeled Spanish saffron. Extracted ion chromatograms of m/z 782.5694 PC 36:4 for PDO Greek saffron (a) and labeled Spanish saffron (b), and their oxidized lipids, extracted ion chromatograms, m/z 798.5643 for PDO Greek saffron (c) and labeled Spanish saffron (d). Figure VI shows (A) Base Peak Chromatogram (BPC) of fresh saffron sample (FA1101-M-L-13), (B) Crocetin [C22H28O4+H]+ extracted ion chromatogram (XIC), (C) picrocrocin [C16H26O7+H]+ XIC. At the end of the chromatogram, from 10 to 13 min, glicerophospolidids can be also observed. A B C On the other hand, sample FA1101-M-D-13 is summarized in Figure VII. BPC of dried saffron sample (FA1101-M-D13), (B) Crocetin [C22H28O4+H]+ extracted ion chromatogram (XIC), (C) picrocrocin [C16H26O7+H]+ XIC. At the end of the chromatogram, from 10 to 13 min, glicerophospolidids can be also observed. A B C Conclusions Untargeted metabolic fingerprinting, UHPLC-HRMS merged with chemometrics, demonstrated to be a powerful tool for saffron authentication in terms of origin. The analytical method optimized provided sufficient discrimination power and information to discriminate saffron origin using positive data, and promising results for harvest using negative data. The food processing plays a key role, concretely the drying process, which produces metabolites according to the drying temperature, linked to the origin and PDOs. Based on orthogonal partial least square discriminant analysis statistical model built using positive data, authentic discrimination between Spanish saffron was carried out, demonstrating fraudulent behavior in more of 50% of samples. La Mancha-Aragón markers may be identified and confirmed unequivocally using analytical standards and LC-MS; subsequently it could be set up in order to provide the control authorities with applicable routine methodologies based on LC-MS, such as LC-QqQ systems. 5. Technique: GC-MS Scientific Coordinator: Lourdes Gómez-Gómez Contributors Ángela Rubio Moragaa, José Luis Ramblaa,b, Oussama Ahrazema, Antonio Granellb, Lourdes Gómez-Gómeza a Instituto Botánico. Departamento de Ciencia y Tecnología Agroforestal y Genética. Facultad de Farmacia. Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain. b Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain. 1. Introduction The majority of the studies performed with Crocus sativus concentrate on the spice, and little is known about the synthesis and accumulation of metabolites during stigma development, including volatile compounds. In the present study, we have used headspace solid phase microextraction coupled to gas chromatographymass spectrometry to identify the evolution of volatile compounds in the stigma during the different stages of development of C. sativus. We also discuss briefly whether specific volatile production could be paralleled by changes in the level of expression of candidate genes in the metabolic pathways involved. Samples Excised fresh Crocus sativus stigmata at different developmental stages cultivated in Minaya (Albacete, Spain): Yellow: closed bud inside the perianth tubes (around 0.3 cm in length); Orange: closed bud inside the perianth tubes (around 0.4 cm in length); Red: closed bud inside the perianth tubes (0.8 cm in length); -3DPA: three days before anthesis, dark red stigma in closed bud outside the perianth tubes (3 cm); Anthesis: day of anthesis, dark red stigma (3 cm); +2DPA: two days post anthesis, dark red stigma. Volatile compounds capture and analyses Volatiles compounds were captured by headspace solid phase microextraction (HS-SPME) by means of a PDMS/DVB fibre. Compounds were analysed by gas chromatography coupled to mass spectrometry (GC-MS). HS-SPME extraction protocol: 10 mg of fresh C. sativus stigma from different stages were introduced in a 22 ml crimp cap vial, together with 10 µl of a 1 ppm methylsalicylate solution as an internal standard. Then a 65 μm PDMS/DVB fibre (polydimethylsiloxane/divinylbenzene) (Supelco, Pennsylvania, USA) was exposed to the headspace for 1 h at 30 ºC, while being shaken at 300 rpm. After sampling, the fibre was immediately desorbed in the GC injector. A desorption time of 1 min at 250 ºC was used in the splitless mode. Before sampling, each fibre was reconditioned for 5 min in a GC injector port at 250 ºC. Triplicate analyses were performed at each stage. GC-MS analysis: Volatile organic compounds were analysed by GC-MS using a Clarus 600 gas chromatograph from Perkin Elmer (Shelton, USA), equipped with a ZB-5ms capillary column (30 m, 0.25 mm, 0.25 µm) (Phenomenex, Torrance, CA, USA). Oven programming conditions were 40 ºC for 2 min, 5 ºC/min ramp until 180 ºC; 15 ºC/min ramp until 250 ºC, then 5 min at 250 ºC. Helium was used as the carrier gas at a constant pressure of 19.5 psi. Detection was performed by a Perkin Elmer Clarus 600T mass spectrometer in the EI mode (ionization energy, 70 eV; source temperature, 150 ºC). Acquisition was performed in scan mode (m/z range 35-250). Chromatogram Volatile compounds emitted by fresh stigma at anthesis Peak 93 100 % 1 Evolution of emission levels during development 91 92 77 79 39 41 0 35 40 43 42 45 40 45 65 51 55 49 50 52 54 50 94 80 53 36 38 58 55 60 105 121 136 67 78 68 62 63 66 65 70 75 107 81 73 74 75 8283 80 86 89 85 90 95 96 102 95 100 103 119 108109 115 110 115 105 122 126 128 132 133134 137 139 141 120 125 130 135 m/z 140 MS Data m/z: 39, 41, 77, 79, 91(43%), 92(38%), 93(100%), 105, 121, 136. 2 57 % 100 56 43 41 71 85 55 39 37 0 35 38 40 40 98 99 58 42 53 44 46 50 51 45 50 69 70 59 55 60 63 65 67 65 72 68 70 75 77 79 81 75 80 83 86 89 91 92 95 97 85 90 95 100 100 105 112 113 105 110 117 120 123 115 120 125 128 131 130 135 140 135 141 140 142 149 152 145 150 154 156 157 155 161 m/z 160 MS Data m/z: 41, 43(28%), 56(50%), 57(100%), 71, 85, 98, 99, 141. 3 43 100 % 41 57 55 108 69 39 56 58 71 67 68 53 40 36 38 45 47 0 35 40 51 49 50 45 59 54 50 55 61 63 60 70 72 65 85 79 77 73 74 65 70 75 81 84 80 111 93 83 42 91 88 89 85 92 90 99 109 95 97 98 100 101 105 107 95 100 105 126 112 110 113 116 115 120 122 124 127 m/z 120 125 130 MS Data m/z: 39, 41(60%), 43(100%), 55, 57(48%), 69, 71, 108, 111, 126 4 57 % 100 56 41 43 71 85 55 39 37 0 35 38 40 40 58 42 44 45 50 51 53 45 50 69 59 61 63 65 67 55 60 65 72 75 77 79 81 70 75 80 83 99 86 87 88 90 95 97 85 90 95 100 100 102109 105 112 113 114 111 110 115 121 122 125128 120 125 130 136 130 137 140 135 140 149 154 145 150 155 155 156 163 160 167 168 165 170 m/z 170 MS Data m/z: 41 (43%), 43, 55, 56(45%), 57(100%), 71, 85, 99, 112, 113 5 68 100 67 93 % 41 79 39 55 77 70 91 83 94 107 92 53 42 43 80 69 65 52 45 36 38 50 46 0 35 108 51 40 40 54 47 45 50 72 78 66 59 61 55 63 60 70 75 81 105 88 80 98 95 74 76 65 136 121 84 97 89 90 85 109 110 100 103 90 95 100 105 110 115 117 119 122 115 120 137 132 126 128 134 125 130 135 141 m/z 140 MS Data m/z: 39, 41, 67(73%), 68(100%), 79, 93(70%), 107, 121, 136 6 57 % 100 56 43 71 41 70 55 113 85 39 36 38 0 35 40 40 69 58 42 44 45 50 51 45 50 53 55 60 65 112 72 73 59 62 63 65 67 70 75 83 77 79 81 80 86 87 91 94 97 85 90 95 99 100 100 109 105 111 114 110 115 116 120 126 127 128 125 130 137 138 139142 135 140 145 151 154 155 156 159 150 155 160 166 168 171 170 m/z 165 170 MS Data m/z: 41, 43(33%), 55, 56(49%), 57(100%), 70, 71, 85, 112, 113. 7 105 100 % 77 120 51 43 50 78 39 38 41 42 37 0 35 44 40 52 53 45 48 49 45 59 61 55 50 55 62 63 65 66 60 65 74 69 70 106 76 72 73 79 81 83 84 85 86 80 85 75 89 91 92 90 95 97 95 98 99 102 104 100 107 109 111 105 110 121 125 127 122 126 116 118 115 120 m/z 125 MS Data m/z: 39, 43, 50, 51, 52, 77(100%), 78, 105(99%), 106, 120(64%) 8 93 100 71 41 43 55 % 69 80 39 67 68 83 77 121 91 79 53 94 81 56 40 0 35 51 54 50 52 44 45 37 38 48 40 136 42 45 50 55 65 57 59 62 63 60 66 65 96 72 82 70 73 75 70 78 75 84 95 86 80 85 89 90 95 97 98 105 107 109 111 99 103 100 105 110 122 115 115 119 120 123 127 128 133 125 130 134 135 139 140 144 m/z 140 MS Data m/z: 39(87%), 41(92%), 43, 55, 67, 68, 69, 71(89%), 79, 80, 93(100%), 121, 136. 9 43 100 41 % 69 39 112 56 97 70 98 42 72 71 55 57 83 85 40 53 44 45 37 38 0 35 40 45 50 51 54 58 59 49 50 96 67 55 63 65 60 111 81 77 79 82 84 86 87 73 74 65 70 75 80 85 93 95 99 100 91 90 95 100 105 139 113 108 109 110 114 121 115 120 123 138 125 127 125 136 130 135 140 154 155 151 152 142 140 145 150 159 155 161 160 m/z MS Data m/z: 39, 41(69%), 43(100%), 56, 69(60%), 70, 72, 97, 98, 112. 10 56 100 43 85 % 41 125 69 39 55 83 57 153 168 40 42 53 38 37 0 35 68 44 50 51 54 45 49 40 45 50 55 58 59 63 65 60 67 65 70 79 71 73 74 77 80 70 75 80 96 97 82 84 86 94 8791 93 95 98 99 100 85 90 95 100 126 111 107 105 113 110 115 121 122 120 127 135 137 139 140 125 130 135 140 152 154 150 155 150 145 169 158 160 163 165 171 170 175 179 180 m/z 180 MS Data m/z: 39, 41, 43(82%), 55, 56(100%), 69, 85(73%), 125, 153, 168. 11 68 100 % 96 39 40 152 41 109 69 38 42 43 37 0 35 4449 40 50 51 45 53 55 56 54 50 57 55 62 67 63 65 66 70 60 65 81 79 73 74 77 70 75 80 83 84 97 91 94 95 98 99 85 89 85 90 95 137 110 105 107 100 105 112 110 124 119 123 115 125 130 153 138 141 125 128 133 120 135 140 146 145 151 154156 160 m/z 150 155 160 MS Data m/z: 39, 40, 41, 53, 55, 68(100%), 69, 96(73%), 109, 152(29%). 12 107 100 91 % 121 105 79 150 77 39 65 51 92 108 53 52 42 45 37 0 35 40 45 62 64 48 50 78 63 66 56 58 55 119 103 60 41 60 65 68 70 70 115 117 74 76 75 135 122 73 88 89 80 85 90 94 95 110 101 100 105 110 125 130 115 120 125 133 132 130 135 151 139 142 144 149 140 145 150 MS Data m/z: 39, 51, 65, 77, 79, 91(73%), 105, 107(100%), 121(52%), 150. 157 155 159 160 m/z 160 13 137 100 109 152 67 81 123 41 % 39 43 91 79 55 95 77 53 93 107 65 119 51 40 0 35 57 52 42 37 38 69 40 45 46 47 45 50 54 56 58 50 55 60 61 60 63 78 80 68 66 82 65 70 92 83 84 71 74 75 64 96 80 85 153 138 124 121 97 98 90 110 94 85 89 75 105 95 103 111 117 99 100 105 110 115 125 120 131 125 134 135 130 135 139 140 144 148 151 145 150 154 160 155 160 m/z MS Data m/z: 39, 41, 43, 67, 81, 91, 109(86%), 123, 137(100%), 152(87%). 14 83 100 43 41 55 112 98 % 39 69 56 97 111 53 67 70 125 7981 57 40 44 38 45 46 37 0 35 40 45 51 54 50 50 58 60 85 91 65 68 59 63 55 77 71 72 65 70 73 75 78 80 80 87 85 93 95 94 113 99 107 109 100 105 89 90 95 100 105 110 114 115 121 123 126 120 125 127 134138 130 135 139 140 141149 153 154 155 140 145 150 166 167 168 155 160 MS Data m/z: 39, 41(92%), 43(96%), 55, 56, 69, 83(100%), 97, 98, 112. 165 170 15 153 100 43 % 125 41 39 55 111 69 53 65 70 51 57 45 50 5254 63 66 68 71 74 5961 73 46 40 40 45 50 55 60 65 70 107 95 97 98 93 75 80 85 80 85 89 92 90 96 105 99 95 112 135 127 121 120 100 105 110 154 126 109 81 44 38 0 35 77 91 83 79 67 134 115 120 125 130 155 138 139 146149152 157 135 140 145 150 155 165 167 168 160 165 170 MS Data m/z: 39, 41, 43(75%), 55, 69, 91, 107, 111, 125(67%), 153(100%). 16 71 100 % 43 83 56 41 55 98 89 57 73 69 39 97 85 44 38 47 0 40 51 53 50 59 60 65 67 74 79 81 70 80 86 90 95 99 103 90 100 110 111 113 115 121 110 120 143 145 127 128 130 139 142 140 173 146 150 155 161 165 160 171 170 MS Data m/z: 41, 43(95%), 55, 56, 57, 71(100%), 73, 83(38%), 89, 174180 180 98. 71 100 43 56 % 89 41 55 57 73 85 39 45 69 59 60 65 67 46 51 53 38 0 40 50 60 173 83 88 74 77 81 70 80 90 95 97 143 98 91 103 109 90 100 113 110 115 121 127 128 129 110 120 137 130 144 148 142 140 155 150 174 161 162 160 184 170 182 170 180 187 193 190 198 201 m/z 200 MS Data m/z: 41, 43(89%), 55, 56(85%), 57, 71(100%), 73, 85, 89, 173. 18 109 100 39 137 152 180 79 123 4143 % 91 77 138 53 81 55 124 6567 107 69 63 40 50 38 105 66 52 54 57 44 45 37 7880 179 83 62 64 60 61 46 165 110 93 50 60 71 70 89 7375 76 84 80 134 119 85 96 97 98 103 111 121 118 90 100 110 133 125 126 0 40 151 95 51 120 131 130 153 181 139 140 140 166 154 162 163 147 150 160 182 168 175 170 180 187 191 190 MS Data m/z: 39, 41, 43, 79, 91, 109(100%), 123, 137(79%), 152(63%), 180. m/z 19 105 100 91 161 119 41 121 43 % 107 79 133 39 77 55 93 176 81 53 67 95 69 135 65 109 115 123 51 45 82 83 58 0 40 50 60 70 80 90 162 128 102 87 48 136 129 103 96 89 72 74 62 38 131 71 63 57 50 111 100 141 110 120 130 147 150 153 145 140 155 150 159 168 163 177 169 160 179 181 182 170 180 192 194 195 200 m/z 200 190 MS Data m/z: 41, 43, 79, 91(96%), 105(100%), 107, 119, 121, 133, 161(76%). 20 121 100 % 43 93 161 41 123 79 95 81 105 55 65 51 44 38 45 57 50 179 71 82 58 0 40 109 67 69 53 60 63 176 107 77 39 72 70 136 119 91 83 89 76 80 124 96 90 97 103 100 110 110 125 115 120 133 138 131 130 162 137 140 145 147 151 152 159 150 160 194 163 166 170 175 180 195 182 191 192 180 190 197m/z 200 MS Data m/z: 41, 43(84%), 79, 81, 93(48%), 95, 121(100%), 123, 136, 161. 21 43 % 100 41 69 39 53 55 0 40 6365 50 60 71 72 77 70 84 80 91 85 90 109 95 96 103 105 111 90 100 151 136 93 83 79 81 58 45 48 51 37 107 67 44 125 121 116 119 110 126 120 137 133 144 130 140 150 152 150 170 173 176 157 161 162 160 170 183 180 MS Data m/z: 39, 41(37%), 43(100%), 67, 69(34%), 93, 107, 125, 136, 151. 22 177 100 % 43 41 91 39 38 53 51 65 57 58 45 63 71 82 83 89 72 50 60 70 80 90 96 119 115 99 103 100 117 122 133 120 149 159 162 136 123 131 129 110 178 121 109 81 69 0 40 105 107 95 55 44 135 93 77 79 130 137 144 140 147 150 150 MS Data m/z: 39, 41(25%), 43(41%), 77, 79, 91, 93, 135, 163 157 160 179 165 174 175 170 180 177(100%), 178. 23 191 100 43 % 119 121 41 109 95 105 91 69 55 135 79 77 57 39 50 60 99 85 71 63 50 40 123 133 59 51 36 0 192 83 67 65 53 44 93 72 70 80 115 103 86 90 100 110 128 131 137 120 130 159161 145 149 157 140 150 160 175 165 173 170 177 189 178 188 180 190 219 193 201 213 217 220 203 200 210 220 MS Data m/z: 41, 43(76%), 69, 91, 95, 105, 109, 119(57%), 121, 191(100%). 24 43 100 41 57 82 % 55 68 69 96 67 83 81 44 71 95 85 54 39 45 36 40 50 50 58 80 59 65 86 91 77 70 124 98 72 60 110 111 109 66 53 0 97 80 90 99 100 112 108 110 123 125 137 113 120 128 130 138 141 151 140 152 154 150 180 182 166 168 169 186 196 197 198 160 170 180 190 200 208 210 210 219 220 226 MS Data m/z: 41(91%), 43(100%), 55, 57(88%), 67, 68, 69, 82, 83, 96. 25 74 100 % 87 43 75 41 55 143 59 199 69 57 39 0 40 45 46 54 50 62 60 71 64 70 88 76 80 97 83 101 90 129 130 103 111 115 121 128 93 100 110 120 130 141 140 144 155157 150 160 165 171 172 180 170 180 197 185 190 195 190 200 200 211 206 210 242 213 223 220 230 235 240 230 MS Data m/z: 41, 43(30%), 55, 59, 69, 74(100%), 75, 87(65%), 143, 199. 240 243 250 3. Discussion The volatile profile was characteristic for each developmental stage from the immature yellow to the fully developed and mature stigmata. Many different volatile compounds emitted by the stigmata were identified (either tentatively or unequivocally) from different metabolic pathways, and a significant number of them revealed to be carotenoid-derived volatile compounds. The emission of each volatile showed a particular evolution during the development of the stigma. Some compounds (such as α-pinene, limonene, acetophenone or peaks 2, 4 and 6) were emitted at the highest levels in the yellow stage and decreased thereafter. Other compounds peaked at the red stage (such as the apocarotenoid β-cyclocitral) or at 3 days before anthesis (peaks 16, 17 and 23). Many volatile compounds showed a sharp increase at the anthesis stage. These include linalool, β-ionone, dihydro-βionone, two compounds tentatively identified as 4-oxoisophorone and megastigma-4,6,8-triene, and peak 14, a putative apocarotenoid. Linalool is a monoterpenic alcohol, and its emission pattern followed that of the expression of the putative terpene synthase CsTS2, whilst monoterpenes α-pinene and limonene followed that of the CsTS1 gene, another putative terpene synthase (Moraga et al., 2009), suggesting that they might be involved in their biosynthesis. Interestingly, all the other identified compounds peaking at anthesis revealed to be apocarotenoids. The emission of these volatiles did not parallel the expression of carotenoid biosynthesis genes of these volatiles, but the expression levels of some carotenoid cleavage dioxygenases (CsCCDs), confirming previous observations (Rubio et al., 2008). Safranal, which is considered to be the major aroma component in saffron, representing as much as 60% to 70% of the essential oil content (Alonso et al., 1995; Tarantilis and Polissiou, 1997) was emitted at low levels from fresh stigmas, in contrast to the high levels observed for picrocrocin, what suggests that safranal is most likely generated by picrocrocin degradation during the dehydration process of the stigma (Raina et al., 1996). 4. Conclusion The volatile composition of C. sativus stigmas changed notably as stigmata developed with each developmental stage being characterized by a different volatile combination. Several apocarotenoids, together with the monoterpenoid linalool, showed a sharp increase at anthesis. In addition to the accumulation of coloured non-volatile apocarotenoids which may act as visual cues, the apocarotenoid volatiles emitted by the stigma together with the monoterpenoids released at the time of anthesis may act synergistically in attracting pollinators to the flower of Crocus in the right moment. References Alonso, G.L., Salinas, M.R., Esteban-Infantes, F.J., Sanchez-Fernandez, M.A., 1995. Determination of safranal from saffron (Crocus sativus L.) by thermal desorption-gas chromatography. Agric. Food Chem. 44, 185188. Moraga, A.R., Rambla, J.L., Ahrazem, O., Granell, A., Gomez-Gomez, L., 2009. Metabolite and target transcript analyses during Crocus sativus stigma development. Phytochemistry 70, 1009-1016. Raina, B., Agarwal, S., Bhatia, A., Gaur, G., 1996. Changes in pigments and volatiles of saffron (Crocus sativus L) during processing and storage. J. Sci. Food Agric. Sci. 71, 27-32. Rubio, A., Rambla, J.L., Santaella, M., Gomez, M.D., Orzaez, D., Granell, A., Gómez-Gómez, L., 2008. Cytosolic and plastoglobule targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-ionone-release. J. Biol. Chem. 283, 24816-24825. Tarantilis, P.A., Tsoupras, G., Polissiou, M.G., 1995. Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV–visible photodiode-array detection-mass spectrometry. J. Chromatogr. 699, 107-118. 5. Technique: GC-MS National Institute of Agronomic Research, Morocco Scientific coordinator: Dr Mounira Lage Code: FA1101-M-L-13 Sample Stigma – Thermal desorption (TD) Recording conditions A joined system made up of Perkin-Elmer (Norwalk, CT, USA) ATD-400 thermal desorption equipment, a model HP-6890 gas chromatograph, and a model HP- 5973 mass spectrometer provided with a NIST library (Hewlett-Packard, Palo Alto, CA) were used. A fused silica capillary column with stationary phase BP21 50 m in length, with an inside diameter of 0.22 mm, and 0.25 m of film was employed (SGE). The carrier gas was helium of chromatographic purity (220 kPa). Fifty milligrams of sample was introduced into the desorption tube and desorbed at 3 min. Other conditions for the thermal desorption equipment were as follows: oven temperature of 250 °C, cold trap temperature of -30 °C, and transfer line temperature of 200 °C. Conditions for gas chromatography were as follows: 100 °C (5 min) increased at a rate of 18 °C/min to 210 °C (15 min). In the mass spectrometer, the electron impact mode (EI) was set up at 70 eV. The mass range varied from 35 to 500 units, and the detector temperature was 150 °C. (11) All compound identification was carried out using the NIST library and by comparison with those reported previously Chromatogram Abundance TIC: 51A,28,11.D\ data.ms 400000 350000 300000 250000 200000 150000 100000 50000 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 Time--> Peak 1. Spectrum Abundanc e Sc an 319 (3.786 min): 51A,28,11.D\ data.ms 121.1 2800 2600 2400 2200 2000 1800 91.1 1600 1400 43.1 1200 1000 800 600 400 150.0 200 278.1 192.9 353.8 230.9 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 121(100), 91(60), 107(50), 79(40), 135(20), 150 (10) Peak 2. spectum Abundanc e Sc an 394 (4.103 min): 51A,28,11.D\ data.ms 107.1 30000 25000 20000 15000 10000 81.1 5000 55.1 140.1 189.1214.4 0 40 60 80 277.9 352.2 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 107(100), 125 (35), 81(33), 55(30), 140 (28) Peak 3. spectum Abundanc e Sc an 445 (4.318 min): 51A,28,11.D\ data.ms 82.1 24000 22000 20000 18000 16000 14000 12000 10000 8000 138.1 6000 4000 54.1 2000 111.0 182.4 0 40 60 80 278.1 354.1 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 82(100), 138(30), 54(10), 95(5) Peak 4. Spectrum Abundanc e Sc an 494 (4.525 min): 51A,28,11.D\ data.ms 107.1 40000 35000 30000 25000 150.1 20000 15000 79.1 10000 5000 41.1 194.0 0 40 60 80 278.1 354.0 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 107(100), 91(80), 121(50), 79(25), 135(10) Peak 5. Spectrum Abundanc e Sc an 543 (4.732 min): 51A,28,11.D\ data.ms 68.1 9000 96.1 8000 7000 6000 5000 152.1 4000 3000 2000 41.0 121.1 1000 202.1 278.1 352.0 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 68(100),96(95),152(46),109(12),41(10) 137(4) Peak 6. Spectrum Abundance Scan 651 (5.188 min): 51A,28,11.D\ data.ms 139.1 7000 56.1 6000 5000 4000 3000 2000 83.2 1000 111.1 278.0 202.0 354.0 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 139 (100), 56(95), 42 (75), 154(60), 69(52),83(24) Peak 7. Spectrum Abundanc e 2000 Sc an 1283 (7.857 min): 51A,28,11.D\ data.ms 109.1 153.0 180.0 45.1 1800 1600 1400 77.0 1200 1000 800 278.1 600 354.1 400 305.1 202.4 200 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 109(100), 137(95), 180(80), 152(78), 123(65), 91(45), 135(35) Peak 8. Spectrum Abundance Sc an 1631 (9.326 min): 51A,28,11.D\ data.ms 2000 45.1 1800 1600 107.0 135.0 1400 1200 1000 800 77.1 168.0 278.1 600 354.1 400 205.1 200 305.1 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/ z--> Spectral Data 107(100), 135(95), 91(70), 121(68), 168(30), 150(20), 153(18) 6. Technique: FT-IR AUA collection Scientific Coordinators: Prof. Moschos G. Polissiou (WG2 leader), Assoc. Prof. Petros A. Tarantilis (WG3 leader) Analyst: Eleftherios A. Petrakis (WG2 member) Sample preparation: All samples of C. sativus dried stigmas were finely ground using an agate pestle and mortar. Subsequently, each saffron sample was mixed with KBr at a 2/148 ratio (w/w) and a KBr disc was prepared after homogenization of the mixture (0.150 g). Data acquisition and processing: FT-IR measurements were performed using a Nicolet 6700 FT-IR (Thermo Scientific, Madison, WI USA) spectrometer operating in the 4000-400 cm-1 wavenumber range (mid-infrared), equipped with a deuterated triglycine sulfate (DTGS) detector, a Nichrome source and a KBr beamsplitter. All spectra were recorded in transmission mode against a KBr background spectrum at constant temperature (25 °C). A total of 100 scans were accumulated for each spectrum, with 4 cm-1 resolution. Spectra were collected in triplicate at least for each sample and averaged using OMNIC 8.0 (Thermo Fisher Scientific Inc.). All spectra were smoothed (15point Savitzky-Golay smooth), while baseline correction was carried out using the “automatic baseline correct” function (polynomial order 2) of the software. FT-IR Spectra Sample Code: FA1101-K-D-11 Sample Code: FA1101-K-D-12 Sample Code: FA1101-K-F-12 Sample Code: FA1101-M-D-11 Sample Code: FA1101-M-D-12 Sample Code: FA1101-M-D-13 Sample Code: FA1101-M-L-12 Sample Code: FA1101-M-L-13 Sample Code: FA1101-S-F-13 Discussion FT-IR spectroscopy provides valuable information associated with saffron compounds such as crocetin esters (crocins), picrocrocin and sugars, and can be used as a fingerprinting technique. The spectral region from ~1800 to 800 cm-1 is particularly characteristic of saffron fingerprint. According to the FT-IR spectra recorded, most saffron samples presented similar profiles. However, the fresh sample (FA1101-K-F-12) originating from Kozani (Greece) and the lyophilized sample FA1101-M-L-13 from La Mancha (Spain) presented higher peak intensities probably due to less moisture content. The broad band centered at about 3380 cm-1 corresponds to hydroxyl (-OH) group of sugars. The region 3000-2830 cm-1 presents two peaks due to C-H stretching. The spectral area of 1800-1500 cm-1 is the region of characteristic groups. The carbonyl (-C=O) group (esters, ketones, aldehydes) and the C=C bonds absorb in this region. The peak at ~1701 cm-1 is assigned to crocetin esters -C=O vibration. The bands in the region 1500-800 cm-1 are associated with the skeletal vibrations of the components and have been attributed to -CH2-, CH3-, -OH, C-C, C-O, C-OC groups. Specifically, the C-O-C group of crocetin esters is related to the peak at ~1227 cm-1, while the 1200-800 cm-1 spectral region has been correlated with the presence of sugars. The signal observed at ~1071 cm-1 is assigned to C-O vibration of sugars. Furthermore, the signals shown between 970-920 and 780-700 cm-1 result from C-H (trans-) and C-H (cis-) out-of-plane vibrations, respectively. Fig. 1. Chemical structures of major esters of (a) trans- and (b) cis-crocetin Fig. 2. Chemical structure of picrocrocin Literature Tarantilis, P.A., Beljebbar, A., Manfait, M., Polissiou, M. FT-IR, FT-Raman spectroscopic study of carotenoids from saffron (Crocus sativus L.) and some derivatives. Spectrochim. Acta A 1998, 54, 651-657. Anastasaki, E., Kanakis, C., Pappas, C., Maggi, L., del Campo, C.P., Carmona, M., Alonso, G.L., Polissiou, M.G. Differentiation of saffron from four countries by mid-infrared spectroscopy and multivariate analysis. Eur. Food Res. Technol. 2010, 230, 571-577. Ordoudi, S.A., De Los Mozos Pascual M., Tsimidou, M.Z. On the quality control of traded saffron by means of transmission Fourier-transform mid-infrared (FT-MIR) spectroscopy and chemometrics. Food Chem. 2014, 150, 414-421. 7. Technique: Raman AUA collection Scientific Coordinators: Prof. Moschos G. Polissiou (WG2 leader), Assoc. Prof. Petros A. Tarantilis (WG3 leader) Analyst: Eleftherios A. Petrakis (WG2 member) Sample preparation: Saffron samples in stigmas were finely ground using an agate pestle and mortar prior to Raman measurements. Data acquisition and processing: Raman spectra of powdered samples were recorded using an Advantage 785 near-infrared dispersive Raman spectrometer (DeltaNu Inc., Laramie, WY USA) equipped with a 785 nm diode laser for excitation, having a maximum output power of 71.6 mW. The spectral resolution of the instrument is 8 cm-1 and the spectral range 2000-200 cm-1. At least 10 mg of each sample were placed into a 1 mL clear shell vial (VWR International, USA), which was subsequently placed into a prefixed vial sample holder. The spectra were collected with the NuSpec software (DeltaNu Inc., Laramie, WY USA) and referenced with the laser off to subtract ambient light. Each spectrum acquired was an average of three 1 s acquisitions, while four replicates were conducted for each sample. OMNIC software (ver. 8.0, Thermo Fisher Scientific Inc.) was used for baseline correction (polynomial order 2) and averaging multiple spectra. Raman Spectra Sample Code: FA1101-K-D-11 Sample Code: FA1101-K-D-12 Sample Code: FA1101-K-F-12 Sample Code: FA1101-M-D-11 Sample Code: FA1101-M-D-12 Sample Code: FA1101-M-D-13 Sample Code: FA1101-M-L-12 Sample Code: FA1101-M-L-13 Sample Code: FA1101-S-F-13 Discussion Raman spectra provide valuable information about saffron carotenoids. In particular, Raman signal intensities of saffron samples are correlated to crocins content and thus a powerful quantitative tool can be obtained through Raman spectroscopic analysis. Although similar Raman spectra were observed for the different C. sativus samples, the intensity of major Raman peaks was varied. The dry saffron samples originating from Kozani, La Mancha, and Sardinia presented strong Raman signal intensities, directly proportional to the respective ISO values for colouring strength as obtained by UV-Vis spectrophotometry. Also, the bands observed in the Raman spectra acquired from the lyophilized sample FA1101-M-L-13, originating from La Mancha (Spain), as well as from the fresh sample (FA1101-K-F-12) from Kozani (Greece) are weaker due to lower crocins content. The very strong peaks at ~1536 cm-1 and 1165 cm-1 are assigned to C=C and C-C stretching, respectively. The region 1300-1100 cm-1 is assigned to C-C stretching and C-H in-plane bending modes. The weak peak occurring at 1210 cm-1 is mainly due to C-H in-plane bending, while the peaks at 1283 cm-1 and ~1020 cm-1 are related to CH deformation and -CH3 in-plane rocking, respectively. The band at 965 cm-1 is attributed to C-C ring vibration. Fig. 1. Chemical structures of crocetin esters (crocins) Literature Assimiadis, M.K., Tarantilis, P.A., Polissiou, M.G. UV-Vis, FT-Raman, and 1H NMR spectroscopies of cis-trans carotenoids from saffron (Crocus sativus L.). Appl. Spectrosc., 1998, 52, 519-522. Tarantilis, P.A., Beljebbar, A., Manfait, M., Polissiou, M. FT-IR, FT-Raman spectroscopic study of carotenoids from saffron (Crocus sativus L.) and some derivatives. Spectrochim. Acta A 1998, 54, 651-657. Anastasaki, E.G., Kanakis, C.D., Pappas, C., Maggi, L., Zalacain, A., Carmona, M., Alonso, G.L., Polissiou, M.G. Quantification of crocetin esters in saffron (Crocus sativus L.) using Raman spectroscopy and chemometrics. J. Agric. Food Chem. 2010, 58, 6011-6017. 7. Technique: NMR ISMAC Scientific coordinator: Dr. Roberto Consonni Analysts: Dr. Roberto Consonni (STSM coordinator, WG2 member), Laura R. Cagliani (WG2 member) Sample preparation and extraction procedure: Samples in stigma form were ground before the analysis. Extraction was carried out using deuterated dimethylsulfoxyde (DMSO), stirred for 3 minutes at room temperature and after 10 minutes the sample was centrifuged at12,100 g for 10 min. 500 μL of the supernatant was used for the NMR analysis. Data recording: All 1H-NMR spectra have been recorded on a Bruker DMX 600 spectrometer (Bruker Biospin GmbH Rheinstetten, Karlsruhe, Germany) operating at 14.1 T and equipped with a 5-mm reverse probe with z-gradient. Spectra were recorded at 300 K, with a spectral width of 8012 Hz and 32 K data points. Residual water suppression was achieved by applying a presaturation scheme with low power radiofrequency irradiation for 1.2 s. FA1101-S-F-13 FA1101-M-L-12 FA1101-M-D-12 FA1101-M-D-11 FA1101-K-D-12 FA1101-K-F-12 FA1101-K-D-11 Discussion The NMR spectrum of saffron provide useful information concerning the main metabolites present, like crocetin esters, picrocrocin, saccharides (in both free and bound form), fatty acids and flavonoids. Other NMR signals of isomers and other small molecules are detected as well. By the NMR spectra comparison of the investigated samples, kaempherol content resulted enriched in all Kozani samples (Greece) namely K-D-11, K-D-12, and K-F-12, while cis-crocins resulted enriched in La Mancha samples (Spain) namely M-D-11, M-D-12, M-L-12 and in Greek sample K-F12. Finally, samples K-D-11, K-D-12 and S-F-13 resulted enriched in safranal content. Literature Cagliani, L.R., Culeddu, N., Chessa, M., Consonni, R. NMR investigations for a quality assessment of Italian PDO saffron (Crocus sativus L.). Food Control 2015, 50, 342-348. Petrakis, E.A., Cagliani, L.R., Polissiou, M.G., Consonni, R. Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1H NMR metabolite fingerprinting. Food Chem. 2015, 173, 890-896. Ordoudi, S.A., Cagliani, L.R., Lalou, S., Naziri, E., Tsimidou, M.Z., Consonni, R. 1H NMR-based metabolomics of saffron reveals markers for its quality deterioration. Food Res. Intern. 2015, 70, 1-6. Reference samples Code FA1101-K-D-11 FA1101-K-D-12 FA1101-K-F-12 FA1101-M-D-11 FA1101-M-D-12 FA1101-M-D-13 FA1101-M-L-12 FA1101-M-L-13 FA1101-S-F-13 Description Kozani 2011 Kozani 2012 Dry Kozani 2012 fresh La Mancha 2011 Dry La Mancha 2012 Dry La Mancha 2013 Dry La Mancha 2012 Lyophilized La Mancha 2013 Lyophilized Italian Saffron 2013 fresh Sardinia About COST Action FA1101 “SAFFRONOMICS” Omics Technologies for Crop Improvement, Traceability, Determination of Authenticity, Adulteration and Origin in Saffron The main challenge in the scope of the Common Agricultural Policy (CAP) is the development of a sustainable economy based on High Value Agricultural Products (HVAPs). Saffron is the highest priced HVAP and a good example of profitability, sustainability, cultural and social values, and high labour demand within Europe and worldwide. COST FA1101 (SAFFRONOMICs) Action has provided the adequate framework for collaborative research and innovations on saffron crop improvement, traceability, determination of authenticity, adulteration and origin by offering interested players the possibility of joining different inter-disciplinary and multi-sectorial efforts. COST is supported by the EU Framework Programme Horizon 2020 If you wish to learn more about COST Action FA1101, please contact: Prof. Maria Tsimidou, Chair of COST Action FA1101 Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki (AUTh), Thessaloniki, Greece [email protected] Dr. Ioanna Stavridou, Science Officer Food and Agriculture, COST Office, Brussels, [email protected]
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