A study found that immature beans (Craig, Franca, & Oliveira, 2012) can be differentiated from mature (ripe) beans, using diffuse reflectance infrared spectroscopy. Direct injection electrospray ionisation mass spectrometry has also been used (Amorim et al., 2009) to distinguish between immature, ripe and overripe beans, by measuring methanol extracts of green and roasted beans. The main differences between the beans were in the fatty acid content and the drop in di- and trimeric chlorogenic acids (CGAs) signal intensities. Slight differences have also been found (Jham, Velikova, Muller, Nikolova-Damyanova,

& Cecon, 2001) in lipid content between immature and ripe Enzalutamide coffee beans. It has been found that the chlorogenic acid content in unprocessed coffee beans decreases with maturation of the coffee fruit, and that there is difference between the ripe (pink) and fully ripe fruit (Koshiro et al., 2007). Elemental composition has also been investigated (Valentin & Watling, 2013), but no differences were found with respect to degrees of ripeness. The aim of the presented work was to search for Atezolizumab chemical structure differences in chemical composition between coffee beans of different

degrees of ripeness, using wet-processed green coffee beans from a single origin that are free from defects. The chosen stages of ripeness were all in the range of normal commercial coffee qualities. A range of analytical methods were optimised and developed to analyse selected parameters: chlorogenic acid profile, volatile profile, caffeine, sucrose content and high-molecular weight (HMW) part of the size exclusion chromatogram. Green coffee beans were obtained from the Finca SHANTI

farm of Munaipata Café de Altura S.A., Coroico, Bolivia (16° 13’ 05” S, 67° .43’ 25” W, elevation 1700-1880 m). The coffee plants had been exposed to identical soil and sunshine conditions. Fruits from two varieties of Arabica, Tipica and Catuai, were harvested at three different stages of ripeness, namely unripe, half-ripe Calpain and ripe, as shown in Fig. 1. Unripe fruit were those that had just started to show a red colour on an otherwise mostly green fruit, half-ripe fruit were the opposite and were mostly completely light red in colour with some remaining green spots and ripe fruit were completely deep red in colour. The raw coffee beans were obtained from the fruits by the wet-process post-harvest treatment. All samples were free of defects. Methanol and acetonitrile were obtained from Sigma-Aldrich and were of HPLC gradient grade, sodium phosphate and phosphoric acid were reagent grade from Sigma-Aldrich (Buchs SG, Switzerland) and formic acid was from Fluka eluent additive LC-MS grade. Caffeine and sucrose standards were obtained from Fluka, 3-caffeoyl quinic acid (3-CQA), 4-caffeoyl quinic acid (4-CQA) and 5-feruoyl quinic acid (5-FQA) from Sigma-Aldrich and 5-caffeoyl quinic acid (5-CQA) from Acros Organic (Geel, Belgium).