1. Introduction
Crocus sativus L., commonly known as saffron, is the most cultivated plant species belonging to the
Crocus genus [
1]. It grows predominantly on the highlands and mountain areas of Western Asia and Mediterranean Europe thanks to the optimal continental-temperate or continental-Mediterranean climates [
2]. Iran is the main producer with over 160 tons/year, representing around 90% of the total production, whereas other producers include India, Greece, Italy and Spain [
3]. One of saffron’s unique features is represented by the three stigmas growing from the flower, which are responsible for the characteristic red colour of the final product; they are manually or mechanically separated and dried to produce commercially available saffron in filaments or powder [
4].
Saffron is mainly used as a spice added to numerous foods and drinks [
5], as well as a key component of many dyes used in the cosmetic industry [
6]. Saffron is also a strong antioxidant and anti-inflammatory agent with several therapeutic properties [
7]. For centuries, saffron extracts have been used to treat numerous conditions such as genital diseases, eye diseases and menstrual disorders [
8]. More recently, new pharmacological effects of saffron and its metabolites have been discovered, including anti-depressant effects [
9], improvement of sleep quality [
10], effects in aiding depression and anxiety [
11] and many others [
8].
Due to a series of reasons, such as a long and laborious process necessary to achieve the final product [
12] and the need for between 22,000 and 300,000 flowers to produce 1 kg of stigmas [
13], saffron is the highest priced high value agricultural product (HVAP), reaching up to 20,000 EUR/kg [
14]. Therefore, this spice is a major candidate for adulteration, especially the product sold as powder, in which extraneous material can be more easily introduced [
13,
15]. Throughout history, examples of adulteration involved mixing with condensed or older saffron and the addition of artificial substances, organic dyes, other parts of the saffron plant, substances that increase weight such as syrup, honey or glycerine, parts from other plants, or animal substances [
16]. Hence, proper control of the quality of saffron is an important issue concerning the food industry as well as consumers.
Currently, the norm assessing the quality of saffron and specifying the methods to detect adulteration is ISO 3632, which is divided in two parts: ISO 3632-1:2011 (Specifications) and ISO 3632-2:2010 (Test Methods) [
17,
18]. The latter describes a UV-Visible (UV-Vis) spectrophotometric method to classify saffron into three commercial categories (as reported in
Table 1) based on the absorbance of a 1% solution by weight at three different wavelengths (440 nm, 270 nm and 330 nm), which are correlated, respectively, to the content of crocin, picrocrocin and safranal. Crocin is the main carotenoid of saffron and is responsible for the colour of the spice, picrocrocin is the molecule which is responsible for its bitter taste, whereas safranal is one of the many molecules imparting the characteristic aroma [
8]. For these reasons, the absorbances previously mentioned are commonly referred to as: “colouring power”, “bittering power” and “odorous power” of saffron.
In order to be classified as category I, II or III, the sample needs to satisfy the requirements of all three parameters and have a moisture and volatile matter content below 12% for saffron filaments or below 10% for saffron powders [
18].
Despite the fact that the ISO 3632 grading remains the main frame of reference for saffron quality, several studies suggest that the spectrophotometric method is not the best way to assess the overall quality of this spice [
19]. One of the main limitations is the inability to correctly quantify the amount of safranal in the sample [
19,
20,
21] and consequently the failure to assign saffron to the proper commercial category. Despite attempts made to quantify the three molecules responsible for saffron quality through the use of an alternative HPLC-DAD methodology [
20], other studies show that the problems of the ISO method do not lie solely in the detection of safranal [
3,
14,
22,
23]. For example, the method is incapable, in certain cases, of revealing adulterations with other types of plants, and the use of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) needs to be employed as an alternative [
15]. Moreover, the ISO method is also unable to distinguish between synthetic components and natural ingredients; in this case a three-step approach focused on the use of two innovative techniques based on microscopic examinations and DNA barcoding needs to be performed in addition to the ISO methodology [
3].
An exhaustive review on the methods found in the literature that have been used for the determination of saffron quality shows that, aside from the ISO methodology, quality assessment has been performed using chromatographic, spectroscopic and biomimetic-based techniques, along with molecular-biological methods [
22]. One of the main points of agreement between the cited methods is that every technique can give valuable information regarding the quality of the spice; nonetheless, no technique is fully exhaustive in performing this evaluation, suggesting that saffron quality can be determined only by using an array of techniques.
In this study, a new multi-analytical approach for the determination of saffron quality is presented, based on the use of two widespread and consolidated techniques, i.e., UV-Vis spectrophotometry (as described in the ISO 3632 standard) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, along with two novel techniques in the field of saffron quality determination, which are scanning electron microscopy coupled to energy dispersive X-ray spectroscopy (SEM-EDX) and inductively coupled plasma optical emission spectroscopy (ICP-OES).
Despite its several weaknesses, the ISO methodology remains a point of reference when performing analyses on saffron samples, and therefore has been included as part of our study. ATR-FTIR spectroscopy is a rapid screening technique requiring minimal sample preparation that provides a characteristic fingerprint of the spice [
24], which is crucial in sample authentication. Moving on to the new techniques, SEM-EDX is a simple and fast technique which is used to perform morphological investigations and, thanks to the EDX probe, also the determination of the elemental composition of the samples. ICP-OES is employed for the quantification of several metals due to its good sensitivity and large applicability. Thanks to the results obtained with the use of these two techniques, elemental composition and metal content are proposed in this work as two new parameters that can be considered when assessing saffron quality.
2. Materials and Methods
A total of 21 saffron samples were examined; 9 in powder form and 12 in filaments (
Table 2). Both private and commercial samples of various origins were analyzed. The latter definition refers to samples which have been packaged, labelled and were available on the market for purchase, whereas the former refers to samples which have been harvested and processed by private collectors. Prior to analysis, all of the samples were stored at 4 °C in the absence of light.
2.1. Determination of Moisture and Volatile Matter Content
Saffron filament samples were crushed using a pestle in an agate mortar until reaching a fine powder, whereas saffron powder samples required no pretreatment. Around 1.000 g of both saffron powder and saffron filaments was weighed to the nearest 0.001 g. The samples were then oven-dried at 103 2 °C for 4 h and allowed to cool in a desiccator. Once they reached room temperature, they were weighed to the nearest 0.001 g.
Results were expressed as percentage of moisture and volatile matter content according to the following equation:
In this equation, mi is mass of the sample prior to drying and mf is the mass after drying.
2.2. UV-Vis Spectroscopy
The analyses were carried out following the specifications indicated in Paragraph 14 of the ISO 3632-2:2010 norm with a UVIKON 943 double-beam UV-Vis spectrophotometer (Kontron Instruments, Milan, Italy). Around 125 mg of dried saffron was transferred to a 250 mL volumetric flask and approximately 230 mL of Milli-Q water was added. The flask was covered with aluminum foil to avoid direct contact with light sources, placed in a cold-water bath (<22 °C), and the content was stirred for 1 h. The solution was diluted up to the mark with Milli-Q water, then 60 mL was filtered using rapid filtration filter paper (Extra Rapida—Perfecte 2, myCordenons, Milan, Italy). The first 40 mL was discarded and the last 20 mL was kept. Half of this volume was withdrawn and diluted up to the mark in a 100 mL volumetric flask.
The absorption characteristics of this solution were measured by recording a spectrum between 200 nm and 700 nm. Maximum absorbances at 440 nm, 257 nm and 330 nm were recorded, corresponding to the absorption of crocin, picrocrocin and safranal, respectively. The results were expressed as the “coloring power”, “bittering power” and “odorous power” of saffron using the following equations:
where A
λ,max is the maximum absorbance at the desired wavelength, m
w is the mass of the sample in grams, and W
d(%) is the percentage weight of dry sample.
2.3. ATR-FTIR Spectroscopy
A small portion of the sample was retrieved and placed under the tip of a Nicolet 380 FTIR spectrometer (Thermo Electron Corporation, Waltham, MA, USA). A spectrum between 400 cm−1 and 4000 cm−1 was recorded in transmittance mode using ATR as a sampling technique and carrying out 64 scans with a resolution of 4 cm−1 and performing smoothing operations (15 points).
2.4. Scanning Electron Microscopy Energy Dispersive X-ray Spectroscopy (SEM-EDX)
An appropriate amount of powder or filaments was placed on a standard circular 19 mm pin stub previously covered with a graphite coating and analyzed using a TM4000PlusII Scanning Electron Microscope (Hitachi, Tokyo, Japan) coupled with an EDX microprobe. Nine different areas were selected (3.8 mm × 2.4 mm) with a 50× magnification and for each of them a microscopic image was obtained using back-scattered electrons in low vacuum conditions. Point and aerial analyses at greater magnifications were performed when necessary. Semi-quantitative elemental analysis of saffron’s main components was carried out with the EDX microprobe on all of the nine areas mentioned and mean average values were then calculated.
2.5. Principal Component Analysis (PCA)
Principal component analysis (PCA), which is an unsupervised method used to evaluate similarities and differences between samples in terms of elemental composition, was carried out on EDX data acquired as described in the previous section. Score plots and loading plots were obtained from the starting dataset and evaluated. All analyses were carried out with the statistical package STATISTICA 7.1 (StatSoft Italia, Vigonza, Italy).
2.6. Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES)
A total of 13 different metals were analyzed using an Optima 8000 Optical Emission Spectrometer (Perkin Elmer, Waltham, MA, USA): Na, Mg, Ca, K, Al, Fe, Pb, Cd, Cr, Mn, Zn, Ni and Cu.
2.6.1. Reagents
The reagents used in the digestion procedures were: nitric acid (67%, NORMATOM®, Ultrapure, VWR Chemicals, Radnor, PA, USA), perchloric acid (65–71%, NORMATOM®, Ultrapure, VWR Chemicals, Radnor, PA, USA) and hydrogen peroxide (30%, NORMATOM®, Ultrapure, VWR Chemicals, Radnor, PA, USA). High-purity Milli-Q water (Merck Millipore Milli-Q, Burlington, MA, USA) was employed in all the steps that required its use.
Calibration curves were constructed using multielement external standards. These were prepared starting from 1000 mg L−1 single-element standard solutions (Perkin Elmer Pure, Atomic Spectroscopy Standard, Shelton, CT, USA).
2.6.2. Sample Preparation
Two different digestion procedures were carried out to perform the ICP-OES analysis on the samples: open vessel (OV) and microwave-assisted (MW) digestion. Both have been used in the determination of the metal content of saffron [
25,
26] and both possess qualities that make them suitable for this type of analysis [
27].
With regards to the first, approximately 50 mg of dried saffron was weighed to the nearest 0.1 mg. A solution containing nitric acid and perchloric acid in a 10:1 ratio was prepared and 10 mL was transferred to a clean and dry Teflon container with the previously weighed saffron. The solution was heated at 120 °C on a heating plate for 2 h.
Moving on to the second method, the same amount of saffron was weighed to perform the MW digestion and transferred in a PFA vessel along with 5 mL of nitric acid and 1 mL of hydrogen peroxide. The vessel underwent the following mineralization program (“W” stands for Watt, unit of measure of power):
1 min—250 W
1 min—0 W
5 min—250 W
4 min—400 W
4 min—600 W
Both solutions were cooled at room temperature and diluted with Milli-Q water to a final volume of 25 mL. They were then filtered using 0.45 μm non-sterile hydrophilic membranes (PTFE Millex-14 LCR, 25 mm, Millex® Syringe Filters, Merck Millipore, Burlington, MA, USA) and centrifuged at 3000 rpm for 3 min.
2.6.3. Calibration
A series of standard solutions between 0.005 and 1 mg L
−1 were prepared for each metal and analyzed. Calibration curves were constructed, and regression analysis performed: correlation coefficients were no less than 0.98. The values for the limit of detection (LOD) and limit of quantification (LOQ) for each metal were calculated using the following formulas:
where
Sb is the average blank signal,
sb is the blank standard deviation and m the slope of the calibration curve. LOD values ranged between 0.7 μg L
−1 and 75 μg L
−1, whereas LOQ values ranged between 2 μg L
−1 and 90 μg L
−1.
4. Discussion
As demonstrated in this work, saffron quality can be assessed using a wide array of analytical techniques. Limiting this assessment to the UV-VIS spectrophotometric analysis described in the ISO 3632 technical standard can be misleading in some cases and can give an incomplete characterization of the sample. This work shows that with the aid of ATR-FTIR spectroscopy, along with SEM-EDX and ICP-OES analysis, it is possible to obtain a clearer picture of saffron quality. In this regard, morphological characterization and elemental composition analysis of saffron emerged as two promising and innovative factors for the analysis of this spice.
In fact, most of the samples studied showed little to no signs of adulteration or contamination despite being denied classification in one of the three commercial categories. This was especially the case for saffron filament samples, which all appeared similar in terms of morphological appearance, with no floral contamination, being homogenous in terms of elemental composition and with a metallic content in line with literature values. With the exception of PFL-3, all of these samples were denied classification solely due to the odorous power being outside the specified range, whereas the coloring and bittering power values were at least above the minimum requirements for classification in category III and the moisture and volatile matter content was below the ISO thresholds, indicating no signs of adulteration aimed at increasing saffron mass by addition of volatile substances. This confirms the previously mentioned findings of other studies which have demonstrated the inability of the ISO method in establishing saffron aroma [
1,
19] and the inadequacy of the odorous power as a parameter assessing the quality of the spice [
21].
This was true also for saffron powders: if we do not consider the results obtained for the odorous power, samples characterized by coloring and bittering powers above the minimum requirement of category III and moisture and volatile matter content below 10% also showed a good quality when assessed with the other techniques. However, samples with a coloring and/or bittering power below the minimum value for classification in category III also showed a lower quality according to the other analyses. Specifically, these samples showed a higher content of all terrigenous elements (Al, Si, Na, Mg, Ca and Fe), their morphological appearance differed from the typical features of saffron, and in some cases floral contaminants were identified.
With the aim of trying to highlight some differences between saffron samples, a multivariate data analysis technique i.e., principal components analysis (PCA), was applied to the results of the EDX aerial analyses presented in
Table 2. The loading plot and the score plot are reported in
Figure 6. In spite of the limited number of samples, it seemed interesting to us to apply this approach, which in fact allowed us to draw some useful preliminary indications.
These results show that the majority of the samples are distinguished by the presence of potassium, phosphorous and sulphur, three elements which occur naturally in saffron and are essential for plant growth [
32], and that are not generally related to sources of contamination. The correlation identified between these elements is in our opinion a confirmation of the validity of the approach. However, samples PFL-3, PFL-4 and CPW-1 differed from the others in their high chlorine content, which is the element with the highest loading on the second component. Finally, the samples of lower quality (PPW-2, PPW-3) correlate with terrigenous elements such as Fe, Al, Si, Ca, and Mg.
This was particularly evident for sample PPW-3, which is the one of low quality according to all the analyses performed. This sample was also singled-out by the results of the ICP-OES analysis. In fact, this was the only sample in which the values obtained were consistently above the literature values for almost all of the analysed metals, suggesting that metal content can also be used as a way to discriminate between high- and low-quality samples. In this regard, it is interesting to underline the complementarity of the two novel techniques employed in the determination of saffron quality, in addition to the fact that elemental composition and metal content revealed to be two important parameters in the spice’s evaluation. Indeed, both the absolute amounts (determined via ICP-OES) and the relative percentages (determined via SEM-EDX) can be used to distinguish between the quality of samples.