Exploring the Potential of 87Sr/86Sr Isotope Ratio with Strontium and Rubidium Levels to Assess the Geographic Origin of Saffron

Saffron is regarded as the most expensive spice, mainly because of its laborious harvest. Only a few countries dominate the global saffron market, with Iran producing by far the most saffron, and the saffron production of all other countries thus being much smaller. However, the respective national production (not only of saffron) is usually preferred by local consumers with respect to foreign products and often has a higher price. Cases of saffron with mislabeled geographic origin have repeatedly occurred. Thus, to protect local saffron production, control of the declared geographic origin is required. In the present case, differentiation of the geographic origin by 87Sr/86Sr is performed. The results show the saffron of several countries of origin to vary within the range of marine carbonates; however, saffron samples of Moroccan and Indian origin mainly show elevated 87Sr/86Sr values. Within the Indian saffron samples, one sample from Kishtwar Valley can be differentiated from the Kashmir saffron samples. The results are thus promising, especially when using the combination of Sr and Rb concentrations to differentiate geographic origin whenever the regions are of homogenous bedrock geology within and of different geology between the regions. However, the reported findings need to be checked and confirmed by further and additional saffron samples.


Introduction
Saffron is regarded as the most expensive spice in the world with respect to its weight [1]. This is mainly due to the laborious harvesting by hand, as 1 kg of saffron consists of ca. 250,000 crocus blossom stigmata [1]. Thus, since ancient times, saffron buyers risk being deceived by unscrupulous fraudsters who increase the weight of the saffron by the addition of substances, admix or even replace the saffron with other materials, and, more recently, incorrectly declare the geographic origin of the saffron. About 90% of all saffron produced globally originates from Iran. Iranian saffron is usually also cheaper than the saffron of other geographic origins. In many countries and regions, local and regional products are preferred to "alien" saffron, and substantially higher prices are paid for the local/regional product, especially in, but not restricted to, Europe. Therefore, there exists a relevant difference in price between the cheapest saffron available and the more expensive European (e.g., Greece, Italy, Spain, Austria, Switzerland, UK) saffron, and thus, there is the risk of consumer deception by incorrect declaration of the geographic origin of saffron. For example, there have been repeated claims of (Iranian) saffron being sold under the gneiss usually have ratios beyond the range of the marine carbonates. Moreover, siliciclastic impurities in carbonates can result in values beyond the marine carbonate range. Cellier et al. (2021) [29] demonstrated that the 87 Sr/ 86 Sr values in Champagne have a narrow range (within the marine carbonate range), and thus the 87 Sr/ 86 Sr values of sparkling wines from many other countries, even though they are overlapping with the Champagne range, are outside of this narrow interval. Epova et al. (2018) [34] documented the strong influence of salt in cured ham on its 87 Sr/ 86 Sr values, similar to the study of Tchaikovsky et al. (2019) [35] on salted caviar. A further advantage of the 87 Sr/ 86 Sr proxy is the fact that its isotope ratio is transferred into biogenic tissue without fractionation [36]. Thus, potentially, different types of materials can be used as references, as there is no need to compare the exact same type of sample and reference material (as is the case for the light element SIA).
However, some investigations have demonstrated the limitations (e.g., because of geological heterogeneities, difference in the 87 Sr/ 86 Sr of bedrock and covering sediment, changing of the natural 87 Sr/ 86 Sr owing to anthropogenic activities, etc.) of this method (e.g., [28,33,[37][38][39]), and Horacek et al. (2022) [40] concluded that the value and power of this approach depends on the exact question that needs to be answered and the respective geological setting.
In the present study, the differentiation of saffron samples from various origins (most of them relevant saffron production countries/areas) by 87 Sr/ 86 Sr analysis is explored. To our knowledge, this is the first study authenticating the geographic origin of saffron using 87 Sr/ 86 Sr. We hypothesize that differentiation of the respective origins should be possible by analysis of the 87 Sr/ 86 Sr ratio.

Materials and Methods
In all, 27 saffron samples were collected in different countries and regions, specifically: 9 from Morocco (see Table 1), 13 from India, 3 from Spain (La Mancha region), 1 from Iran, and 1 from Sardinia/Italy ( Figure 1A,B). All but one of the Indian samples originated from Kashmir, whereas the one other sample came from the Kishtwar Valley in Jammu Province ( Figure 1B). The Moroccan samples were collected by one of the authors (M. Lage). The Kashmir saffron samples were bought by M. Horacek in Kashmir from reliable retailers, the Kishtwar saffron sample was collected by J. Vakhlu, and the other samples were provided by colleagues from the respective countries. Each sample consisted of at least one gram of saffron filaments. The dry samples were stored in either small paper bags or small plastic tubes with tightly sealed caps at room temperature.

Sample Preparation
The samples were homogenized in an agate mortar. A total of 320 mg of saffron was digested with 3 mL of HNO 3 acid (68%, optima) and 1 mL H 2 O 2 31%, after a pre-digestion step (with 3 mL HNO 3 overnight at room temperature, ca. 22 • C), by using a High Pressure Asher as described elsewhere [42]. Samples were digested in triplicate. The obtained extracts were stored at 4 • C until analysis.

Isotopic Analyses
Approximately 3.5 mL of the digested extract was evaporated until dryness in a hot block at 95 • C. After evaporation, the sample was diluted in 4 mL of HNO 3 3M and loaded in a column containing 0.16 g of SR-B50-S resin in order to separate Sr from sample's matrix. The Sr specific isolation procedure from the matrix was based on the method already detailed by Martin et al. (2013) [43], after obtention of the elution profile of Sr in the saffron matrix. In brief, Sr was eluted by the addition of 10 mL of H 2 O, once the matrix was removed with 8 mL HNO 3 3M. The Sr-containing fractions were diluted in HNO 3 2% (v/v) before MC-ICP-MS (Multicollector Inductively Coupled Plasma Mass Spectrometry) analyses.
The analysis was carried out on a "Nu Plasma" MC-ICPMS instrument (Nu Instruments, UK), at Pau University, France, as described in Cellier et al. (2021) [29]. Table S1 shows the instrumental parameters. The isotopically certified standard NIST SRM 987 (pure SrCO 3 , NIST, Gaitherburg, MD, USA, certified for its isotopic composition with a certified value for 87 Sr/ 86 Sr of 0.710255 ± 0.00023) was used as both bracketing standard and quality control. Mass bias and interference correction were carried out as described in Ehrlich et al. (2001) [44]. All samples were measured in triplicate. Standard deviation was usually better than 0.00007.

Multi-Elemental Analyses
Approximately 0.15 mL of the digested sample was diluted in 2% HNO 3 (v/v) and among other element isotopes 85 Rb and 86 Sr were monitored. In addition, an SRM, in duplicate, and a CRM (Certified Reference Material), in triplicate, both of vegetal origin, were processed. These materials were SRM 1570a Spinach Leaves and CRM No. 23 Tea Leaves II, certified on Sr (55.54 ± 0.5 and 3.93 ± 0.25 mg/kg, respectively) and Rb (12.7 ± 1.6 and 17 (the latter with no certified or reference value) mg/kg, respectively), which were also digested, and the measurements used for the validation of this crucial step. The concentration of Sr was determined by ICP MS in the saffron samples, the SRM and CRM. The obtained value for the 28 saffron samples with different geographical origins was between 1.82 and 16.29 mg/kg. The results were validated by the match of the certified and obtained Sr concentrations in the vegetal SRM and CRM, 50.46 and 4.14 mg/kg, respectively, and Rb concentrations of 12.96 and 16.79 mg/kg, respectively. The limit of quantification of Sr and Rb by ICP MS was usually below 0.05 mg/kg.
The instrument used was a NexION 300x (PerkinElmer Inc., Paris, France) equipped with a universal cell to remove potential interferences, which may have an influence on achieving accurate measurements of the elements. The aforementioned cell can be operated in two different modes, depending on the principles of interference removal: collision or reaction mode. The cell can also be simply turned off when no interference removal is intended to be performed, which is called standard mode. In this work, the collision cell technology (CCT) was used in order to achieve an efficient removal of polyatomic interferences by kinetic energy discrimination (KED) by means of helium as the non-reactive gas. The general operating parameters used for the analysis are shown in Table S2.

Statistical Evaluation
For statistical evaluation the software environment R (Vienna) was used to perform a Kruskal-Wallis Test. The coefficient of determination (R 2 ) was calculated using Excel.

Results
A summary of the results is found in Table 2

Rb Concentrations
The Rb concentrations of the saffron samples from Morocco ranged from 2.2 to 16.5 mg/kg with an average of 7.1 mg/kg, the samples from India varied from 8.5 to 16.9 mg/kg with the average of 11.8 mg/kg (Kashmiri samples varied from 8.6 to 16.9 mg/kg with an average of 12.0 mg/kg, and the Kishtwar sample had a Rb content of 8.5 mg/kg), the saffron samples from Spain ranged from 2.6 to 6.1 mg/kg, averaging 3.9 mg/kg, and the Iranian sample and the sample from Sardinia/Italy possessed 5.2 and 4.3 mg/kg, respectively.

Sr Concentrations
The Sr concentrations of the saffron samples from Morocco ranged from 1.9 to 11.6 mg/kg with an average of 4.4 mg/kg, the samples from India varied from 1.5 to 6.7 mg/kg with the average of 4.8 mg/kg (Kashmiri samples varied from 2.6 to 6.7 mg/kg with an average of 5.1 mg/kg, and the Kishtwar sample had a Sr content of 1.5 mg/kg). The saffron samples from Spain ranged from 7.6 to 13.1 mg/kg, averaging 9.7 mg/kg, and the Iranian sample and the sample from Sardinia/Italy possessed 12.8 and 1.2 mg/kg, respectively.

Statistical Evaluations
Statistical evaluation identified one Moroccan saffron sample as an outlier for all three parameters investigated (Figure 2). Furthermore, it also identified the Indian Kishtwar saffron sample as an outlier with respect to 87 Sr/ 86 Sr. Significant differences were identified by the Kruskal-Wallis Test for 87 Sr/ 86 Sr, Sr and Rb concentrations (Table 3A-

Discussion
For land plants (including all materials and tissues from plants) the relevant parameter is the soil and the underlying geology, especially the type of bedrock (Horacek (2022) and references therein). Marine carbonates vary in their 87 Sr/ 86 Sr values within a small and well-defined range (within 0.7068-0.7092, e.g., [33], and references therein), and the results of the samples from Spain, Iran, Sardinia/Italy, and one Moroccan saffron sample lay within this interval, with variable Sr concentrations within the trend of marine carbonates (varying Sr content, narrow range of 87 Sr/ 86 Sr). The Kashmir/India samples were beyond the range of marine carbonates, evidencing the significant/dominant influence of siliciclastics. In

Discussion
For land plants (including all materials and tissues from plants) the relevant parameter is the soil and the underlying geology, especially the type of bedrock (Horacek (2022) and references therein). Marine carbonates vary in their 87 Sr/ 86 Sr values within a small and well-defined range (within 0.7068-0.7092, e.g., [33], and references therein), and the results of the samples from Spain, Iran, Sardinia/Italy, and one Moroccan saffron sample lay within this interval, with variable Sr concentrations within the trend of marine carbonates (varying Sr content, narrow range of 87 Sr/ 86 Sr). The Kashmir/India samples were beyond the range of marine carbonates, evidencing the significant/dominant influence of siliciclastics. In         The Rb and Sr contents of the Moroccan samples showed a positive correlation (R 2 = 0.8115); however, we currently do not have an explanation for that. As saffron of no other origin followed this trend, and as Morocco has a diverse bedrock geology, it might be that there is no single cause for this pattern. The Kashmir/India samples possessed a distinct pattern and only slightly overlapped with a few Moroccan samples. In addition, the Kishtwar sample had a distinctive signal (with respect to 87 Sr/ 86 Sr and Rb and Sr concentrations) and was separated from other saffron samples. The Spanish and Iranian samples showed similar rubidium values (ca. 1.5 to 6.5 ppm) and a stronger variation in Sr concentration between 1 and 13 ppm. Our Spanish and Iranian results were in good agreement with Wakefield et al. (2019) [16] (even though these data might need to be regarded cautiously (see above)). Wakefield et al. [16] reported a slightly smaller range in Rb and Sr for the Spanish saffron than our data suggested, and a much broader range toward higher Sr values. Potentially, elevated Sr concentrations might be a useful indicator for Iranian saffron, even though not all of the Iranian saffron will be identified in this way.
The Moroccan samples showed a large variation in 87 Sr/ 86 Sr isotopes, ranging from below the marine strontium isotope interval to values above 0.715 and thus within the massively siliciclastically influenced range. The sample with the lowest value (0.7063, below the marine interval) evidenced the origin from a basalt bedrock ( [33] and references therein), and the sample within the marine strontium isotope range might evidence the origin from a region with carbonate bedrock; however, an origin from a basalt bedrock with a very minor siliciclastic influence might also be possible. The other Moroccan samples possessing 87 Sr/ 86 Sr values beyond the marine range evidenced a dominant siliciclastic influence exceeding the one on the Kashmiri samples, with a small overlap of these two groups between 0.71228 and 0.71338. The highest values also exceeded the range of "continental volcanics", as shown in Horacek (2022) [33] and references therein. A positive correlation of Sr and Rb content was noted for the Moroccan samples, which is probably founded on the bedrock mineralogy and petrology (with the highest element concentrations in the basaltic bedrock sample). As the 87 Sr/ 86 Sr values also varied strongly within one locality (Taliouine), at least there a very heterogenous geological situation can be assumed also at small scale. The Kishtwar saffron sample showed a very elevated 87 Sr/ 86 Sr value of almost 0.720, indicating a bedrock of (old) granite or gneiss [33], and references therein, as it was present in the Kishtwar Valley area ( Figure 1B). Differentiation of geographic origin by 87 Sr/ 86 Sr ratio of the investigated samples is possible for the Moroccan and Kashmiri saffron samples and the Spanish and the Iranian samples. However, as Spain and Iran are both large countries, each possessing a very diverse and heterogenous geology, it needs to be verified if all saffron produced in these countries stems from areas with carbonate bedrock. Still, the 87 Sr/ 86 Sr value reported for Spanish apricots was also within the range observed for the Spanish saffron [40]. No differentiation between the Spanish and the Iranian samples or the Kashmiri and Moroccan saffron was possible judging solely by 87 Sr/ 86 Sr. However, with respect to the latter two countries of origin, a differentiation between Kashmir and Moroccan saffron might be possible using 87 Sr/ 86 Sr, Sr and Rb concentrations. An excellent separation was achieved between saffron from the Kashmir and Kishtwar regions, as the respective differences in bedrock geology resulted in distinctively different 87 Sr/ 86 Sr signals for saffron from these two regions-even though, up to now we only have one result from the Kishtwar Valley. As both regions also seem to be well confined (geographically, with respect to environmental conditions), and the underlying bedrock geology seems to be homogenous at and differing between these sites ( Figure 1B), no overlap is to be expected. However, this assumption needs to be tested and verified.
As the results of the Kashmir and Kishtwar saffron samples demonstrated, the analysis of the 87 Sr/ 86 Sr value can be an excellent tool for differentiation of commodities from different geographic regions, given that they possess respective differences and homogeneity in their bedrock geology. Furthermore, a differentiation of geographic origin by 87 Sr/ 86 Sr might be achieved for certain regions by combining the 87 Sr/ 86 Sr ratio with Sr and Rb elemental concentrations, as was shown for the Kashmir and Moroccan saffron samples. However, one has to consider that the bedrock geology in Morocco is obviously very heterogenous; thus, we cannot be certain that we obtained a full spectrum of the 87 Sr/ 86 Sr values possible. Potentially, further proxies need to be applied for a complete differentiation of saffron samples from these two areas. Thus, as already mentioned, the power and potential of the 87 Sr/ 86 Sr analysis depends on the exact question one wants to address-and the respective homogenous conditions of the bedrock of the regions to be differentiated.

Conclusions
The 87 Sr/ 86 Sr ratio of saffron samples from different regions and countries of origin was analyzed and used for differentiation. An excellent separation was achieved between the saffron samples from the Kashmir and Kishtwar regions, owing to the respective differences in bedrock geology, even though at present this separation was documented for only one sample coming from the Kishtwar Valley/India. A good differentiation between the Kashmir and Moroccan saffron samples investigated was possible by combining the 87 Sr/ 86 Sr value and Sr and Rb concentrations. The investigated saffron samples from Morocco and Kashmir were completely differentiated from the investigated (few) samples from Spain and Iran, as the samples from the latter two countries exclusively showed 87 Sr/ 86 Sr values within the range of marine carbonate bedrock. The Kashmir samples all lay beyond the marine 87 Sr/ 86 Sr range, whereas the Moroccan samples investigated lay beyond or below and one Moroccan sample was within the range of marine carbonate 87 Sr/ 86 Sr, but this sample could be differentiated by the Sr and Rb contents. However, both Spain and Iran are big countries with a very heterogenous geology; thus, it might be expected that saffron samples from these two countries would possess 87 Sr/ 86 Sr values exceeding the ones measured in our study, even though the production of saffron in Spain is restricted to a small region. Therefore, the good differentiation observed in this study might disappear when analyzing further samples from these two countries. Our study confirmed the excellent differentiation of saffron from Kashmir and Kishtwar (even though based on only one sample from Kishtwar so far) because of the seemingly homogeneous geological situation in the saffron-growing area in Kashmir (Pampore region) and the distinctively differing geology in Kishtwar. Furthermore, the Moroccan and Kashmir samples could be differentiated from each other and from the investigated samples of other geographic origins applying 87 Sr/ 86 Sr analysis and Sr and Rb content investigations. However, this claim needs to be checked and confirmed by analysis of further saffron samples.

Data Availability Statement:
The datasets generated for this study are included in this article.

Acknowledgments:
We thank Sylvain Berail from Advanced Isotope Analysis (Pau, France) and three colleagues from Pau University (from whom we have not been able to obtain permission in writing to name them) for the preparation and measurement of the samples. Sabrina Kuchling is thanked for statistical evaluation. We thank two anonymous reviewers for constructive reviews.

Conflicts of Interest:
The authors declare no conflict of interest.