The aim of this work was to propose a high-performance liquid chromatography with diode array detection (HPLC-DAD) method for determining the three main compounds responsible for determining the quality of saffron (crocetin esters, picrocrocin, and safranal) by preparing an aqueous extract according to the ISO 3632 standard to solve the difficulty that this standard has for aroma and taste determination by ultraviolet-visible spectroscopy. Toward this aim, laboratory-isolated picrocrocin, a safranal standard with a purity of ≥ 88%, trans-crocetin di(β-D-gentiobiosyl) ester (trans-4-GG) and trans-crocetin (β-D-glucosyl)-(β-D-gentiobiosyl) ester (trans-3-Gg) standards, both with a purity of ≥ 99%, and 50 different saffron spice samples from Italy, Iran, Greece, and Spain were used in the intralaboratory validation of the HPLC method. The analytical method proposed was adequate in terms of linearity, selectivity, sensitivity, and accuracy for determining the three foremost parameters that define the quality of saffron using only a saffron solution prepared according to the ISO 3632 standard.

Saffron spice, the most valuable spice worldwide, is the dried stigma that only represents 7.4% of Crocus sativus flowers. Other parts of the flowers different to stigmas are discarded. Flower harvest and all the postharvest steps to produce saffron spice are performed manually. Mechanization of flower collection, stigma separation, and dehydration process is a revolution in saffron spice production, which increases the productive capacity making it possible to extend the uses of C. sativus flowers, beyond the production of saffron spice. Flowers possessed high-phenolic content and excellent antioxidant properties that could contribute to their application as functional ingredients.

A high-performance liquid chromatography with photodiode array detection method (HPLC-DAD) was validated for the analysis of floral bioresidues obtained in saffron spice production by using three different solvent mixtures [water/hydrochloric acid (HCl) (100:1, v/v), water/acetonitrile/trifluoroacetic acid (47:50:3, v/v/v), and water/acetonitrile/HCl (50:50:1, v/v/v)]. Fifteen phenolic compounds were tentatively identified, kaempferol 3-O-sophoroside and delphinidin 3,5-di-O-glucoside being the main ones. The extracts showed very different phenolic profiles obtained by HPLC-DAD coupled with electrospray ionization mass spectrometry (ESI-MS), and several experiments were carried out to explain this. The use of acetonitrile as solvent causes the chromatographic splitting of the peak of the delphinidin 3,5-di-O-glucoside into two peaks. Results obtained in this paper show that the extract prepared with water/HCl (100:1, v/v) would be the best suited for determining phenolic compounds in the quality control of the floral bioresidues from Crocus sativus L.