Authentication of saffron spice accessions from its common substitutes via a multiplex approach of UV/VIS fingerprints and UPLC/MS using molecular networking and chemometrics
Introduction
Since antiquity, the peculiar and extravagant spices have gained much attention being used in top cuisines worldwide. Among these elite spices, saffron is the most expensive and most revered owing to its unique flavor, health benefits, and traditional production (Farag, Hegazi, Dokhalahy, & Khattab, 2020).
Saffron is derived from the dried stigmas of Crocus sativus, family Iridaceae. It is recognized not only for its unique color and aroma but also for its several health benefits. Folk medicinal uses of saffron include treatment of depression, insomnia, menstrual disturbances, digestive and cardiovascular problems (Ghaffari & Roshanravan, 2019). Additionally, recent in vitro and in vivo studies confirmed its antioxidant, antitumor, cardio-protective, antidepressant, and antibacterial properties (Farag, Hegazi, Dokhalahy, & Khattab, 2020).
The predominant chemical coloring matter in saffron are crocins including the mono- and di- glycosyl esters of crocetin (polyene dicarboxylic acid). While the monoterpene aldehyde; safranal and its glycoside picocrocin are the main contributors to its distinctive aroma and taste. Saffron quality and economic value are highly dependent on its cultivation origin and further processing methods (Carmona, Sánchez, Ferreres, Zalacain, Tomás-Barberán, & Alonso, 2007). It is cultivated and produced in only a few countries including Afghanistan, Azerbaijan, China, Italy, Iran, India, Greece, Morocco, and Spain. Among which Iran is the biggest producer with a market share of 90% of the produced saffron globally (Ghorbani & Koocheki, 2017). On the other side, Spain has been one of the oldest producers of saffron with a reputation for its premium quality (Sanjuán‐López & Resano‐Ezcaray, 2020).
For the sake of its preciousness and limited sources, its market value is estimated to increase globally from USD 645.9 million in 2015 up to USD 730.0 million by 2025 (Khilare et al., 2019). This escalating market pressure led to the increased fraudulence practices through the addition of other inferior plant parts viz. corn stigma, chrysanthemum, calendula, and safflower which warrants the development of quality control tools for its authentication and quality assessment.
Globally, saffron quality is assessed through the normative ISO3632 (ISO, 2010, ISO, 2011) based on spectrophotometric and chromatographic determination of picrocrocin, safranal, and crocins. Nevertheless, this protocol proved insufficient especially when saffron is adulterated with other plant parts with similar color and morphology. The ISO protocol failed to detect saffron adulteration with safflower, marigold, and turmeric at levels as high as 20% (Sabatino, Scordino, Gargano, Belligno, Traulo, & Gagliano, 2011). Consequently, there is an increasing demand for the continuous development of sensitive analytical methods for the assessment of saffron quality.
Two important aspects contribute to saffron genuineness, which are its geographical origin and substitution practices. The commercial value of saffron is directly correlated to its geographical origin, which significantly affects its organoleptic properties. In this context, various analytical tools have been employed for the quality assessment of saffron based on its geographical origin (Bononi, Tateo, Scaglia, & Quaglia, 2020) and for fraudulence and adulteration detection. Commonly employed protocols included spectroscopic analysis like UV–vis, mid-infrared (MID), and nuclear magnetic resonance (NMR), chromatographic analysis mostly coupled to mass spectrometry, i.e. gas chromatography and high-performance liquid chromatography, ultra-high performance liquid chromatography (UPLC) (Koocheki & Milani, 2020).
Owing to the complexity of the data acquired from the aforementioned techniques, multivariate data analysis (i.e. chemometrics) is often recruited, allowing for unbiased sample discrimination. On this basis, untargeted GC–MS analysis coupled to chemometrics was employed for discrimination between Spanish and Iranian saffron specimens, and the detection of its common substitutes (i.e. safflower and calendula). Significant discriminatory volatile markers for the discrimination of saffron from its common substitutes were detected. Nevertheless, the aroma profile failed to distinguish saffron from different geographical origins (Farag, Hegazi, Dokhalahy, & Khattab, 2020).
On that account, the analysis of the nonvolatile constituents of saffron for discriminating its origin and detecting fraudulence was targeted in the present study. For that purpose, UPLC coupled to high-resolution mass spectrometry (UPLC-HRMS/MS) was the analytical platform of choice owing to its robustness, sensitivity, and selectivity. Furthermore, molecular networking via the Global Natural Products Social molecular networking platform (GNPS) was exploited for the visual exploration of the acquired datasets. Molecular networks enable the robust visual sample-to-sample comparison and highlight for the differentiating features (Farag, Hegazi, & Donia, 2020), which could be leveraged for the detection of saffron substitution.
Thus, the present work highlights the application of UPLC-HRMS/MS-based molecular networks for the differentiation of saffron from different geographical origins (Iran vs. Spain) and further from its common substitutes (i.e. safflower and calendula). Additionally, the classification potential of the UV–vis spectroscopy was compared to sophisticated UPLC-MS based on models from each platform for the same dataset and extracted under same conditions. Multivariate data analysis was employed for exposing the discrepancy of metabolites among Iranian and Spanish saffron, and among saffron and its common substitutes based on their UPLC/MS profiles and UV/VIS fingerprints.
Section snippets
Plant material
Five saffron accessions (belonging to Spanish “SF_SP” and Iranian “SF_IR” origin), and their common substitutes viz. safflower “Saffl” and calendula “Calend” were selected for this study (Suppl. Table S1). Each specimen was analyzed using three biological replicates.
Chemicals and materials
MilliQ water supplied by a Millipore MR3 purifier system was used for UPLC analysis. Acetonitrile (J. T. Baker, Deventer, The Netherlands, LC-MS grade, purity ≥ 99.5%) and formic acid (J. T. Baker, Deventer, The Netherlands, LC-MS
UPLC-HRMS/MS profiling of Iranian and Spanish saffron, safflower, and calendula metabolites
Metabolites profiling of saffron, safflower, and calendula was executed via UPLC-HRMS/MS in the negative ionization mode. Elution gradient employed using formic acid in water (0.1%): acetonitrile allowed for metabolites elution within 22 min.
Overlaid base peak chromatogram of the studied samples is depicted in (Fig. 1). Metabolite elution was in the following order: chalcones, cinnamic acid esters, flavonoid glycosides, C-glycosyl chalcones, crocetin esters, cinnamoyl amides, and lastly
Conclusion
The present study aimed at determining Spanish saffron genuineness and differentiation from its inferior counterpart (Iranian Saffron) and/or its common substitutes i.e. safflower and calendula. For that purpose, profiling of its secondary metabolites via untargeted UPLC-HRMS/MS was employed and synchronized with molecular networking for the robust discovery of discriminating features among the samples. The metabolic profile of saffron from different geographical origins allowed for sample
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
Dr. M. A. Farag thanks the Alexander von Humboldt-foundation, Germany for financial support. We thank Mr Eric Dokhalahy, Chemistry Department, the American University in Cairo, New Cairo 11835, Egypt for assistance in UV measurement.
References (57)
- et al.
Polyphenolic contents in Citrus fruit juices: Authenticity assessment
European Food Research and Technology
(2014) - et al.
Qualitative and quantitative high performance thin layer chromatography analysis of Calendula officinalis using high resolution plate imaging and artificial neural network data modelling
Analytica chimica acta
(2013) - et al.
Anti-inflammatory and potential cancer chemopreventive constituents of the fruits of Morinda citrifolia (Noni)
Journal of natural products
(2007) - et al.
A review on phytochemistry and ethnopharmacological aspects of genus Calendula
Pharmacognosy reviews
(2013) - et al.
Fragmentation trees reloaded
Journal of Cheminformatics
(2016) - et al.
δ13C data of the total water-soluble fraction and triacylglycerols as related indexes for differentiating the geographical origin of saffron (Crocus sativus L.)
Food Chemistry
(2020) - et al.
Identification of the flavonoid fraction in saffron spice by LC/DAD/MS/MS: Comparative study of samples from different geographical origins
Food Chemistry
(2007) - et al.
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
Journal of Agricultural and Food Chemistry
(2006) - et al.
Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in safflower (Carthamus tinctorius L.) under MeJA treatment
BMC Plant Biology
(2020) - et al.
Two new phenylpropanoid glycosides from the aerial parts of Paederia scandens
Bulletin of the Korean Chemical Society
(2010)
Searching molecular structure databases with tandem mass spectra using CSI: FingerID
Proceedings of the National Academy of Sciences
Metabolome based classification of artichoke leaf: A prospect for phyto-equivalency of its different leaf origins and commercial preparations
Journal of Pharmaceutical and Biomedical Analysis
UPLC-MS metabolome based classification of Lupinus and Lens seeds: A prospect for phyto-equivalency of its different accessions
Food Research International
Chemometrics based GC-MS aroma profiling for revealing freshness, origin and roasting indices in saffron spice and its adulteration
Food Chemistry
Molecular networking based LC/MS reveals novel biotransformation products of green coffee by ex vivo cultures of the human gut microbiome
Metabolomics
Comparative metabolite profiling and fingerprinting of genus Passiflora leaves using a multiplex approach of UPLC-MS and NMR analyzed by chemometric tools
Analytical and bioanalytical chemistry
Unraveling the metabolome composition and its implication for Salvadora persica L. use as dental brush via a multiplex approach of NMR and LC–MS metabolomics
Journal of Pharmaceutical and Biomedical Analysis
Combination of chemical fingerprinting with bioassay, a preferable approach for quality control of Safflower Injection
Analytica chimica acta
Nutrients, phytochemicals and botanical origin of commercial bee pollen from different geographical areas
Journal of Food Composition and Analysis
Saffron; An updated review on biological properties with special focus on cardiovascular effects
Biomedicine & Pharmacotherapy
Sustainable cultivation of saffron in Iran
Identification and quantification of flavonols, anthocyanins and lutein diesters in tepals of Crocus sativus by ultra performance liquid chromatography coupled to diode array and ion trap mass spectrometry detections
Industrial Crops and Products
Metabolomic fingerprinting of saffron by LC/MS: Novel authenticity markers
Analytical and bioanalytical chemistry
Molecular networking aided metabolomic profiling of beet leaves using three extraction solvents and in relation to its anti-obesity effects
Journal of Advanced Research
Chemical profile and nutraceutical features of Salsola soda (agretti): Anti-inflammatory and antidiabetic potential of its flavonoids
Food Bioscience
International Standard ISO 3632–2: Saffron (Crocus sativus L.) Test Methods
International Standard ISO 3632–1: Saffron (Crocus sativus L.) Specification
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