Nutritional Quality and Safety of the Spirulina Dietary Supplements Sold on the Slovenian Market

The microalgae Spirulina may be a popular dietary supplement rich in essential nutrients and vitamins, but oversight of the supplement industry, in general, remains limited, and increasing incidents of adulteration, misbranding, and undeclared ingredients together with misleading claims create potential risks. In response, this study characterized the elemental, amino acid and fatty acid content of commercially available Spirulina supplements in Slovenia using EDXRF, ICP-MS and GC-MS and compared the results with their nutritional declaration. The gathered data confirm that Spirulina supplements are a good source of calcium (0.15 to 29.5% of RDA), phosphorous (3.36–26.7% of RDA), potassium (0.5 to 7.69% of RDA) and selenium (0.01 to 38.6% of RDA) when consumed within recommended amounts. However, although iron contents were relatively high (7.64 to 316% of RDA), the actual bioavailability of iron was much lower since it was mainly present as the ferric cation. This study also confirms that pure Spirulina supplements are a good source of essential and non-essential amino acids, and ω-6 but not ω-3 polyunsaturated fatty acids. The presence of additives resulted in significant variation in nutrient content and, in some instances, lower product quality. Moreover, a high proportion (86.7%) of inappropriate declarations regarding the elemental content was observed. Overall, the study conclusions underline the need for a stricter control system for Spirulina-based supplements.


Introduction
The challenges associated with living sustainability, keeping food production costs down while meeting increasing food demand, has meant that sourcing alternative lipid, protein, pigment and polymer sources has become a global trend. In this context, microalgae rich in functional nutrients that positively affect human health is an excellent example of an alternative nutrient source [1][2][3][4][5]. Spirulina, or correctly Arthrospira spp., is one of the most important microalgal groups currently produced and contains macro-and micronutrients such as high-quality proteins, minerals, vitamins, fatty acids, polysaccharides and other bioactive compounds [6][7][8][9].
Spirulina is a multicellular filamentous cyanobacterium (blue-green microalgae) with nitrogen-fixing symbiotic bacteria. Its multicellular cylindrical trichomes are typically arranged along its entire length in a left-handed open helix, and its surface is without covering and smooth. It is a photosynthetic autotroph with phycocyanin as its primary

Sample Preparation
Fresh samples were freeze-dried and ground to obtain a fine powder, the tablets were also finely ground, and the capsules were opened and the contents used for analysis. The samples were stored in plastic containers and kept refrigerated (4 • C) until analysis. Samples were analyzed in duplicate in small batches. All analyses were performed within one to three months after collection.

Macro-Elemental Analysis by X-ray Fluorescence Spectrometry
The macro-elemental composition (Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Zn, Br, Rb and Sr) was determined non-destructively using Energy Dispersive X-Ray Fluorescence Spectrometry (EDXRF). Pellets (0.5-1.0 g) were prepared using a pellet die and a hydraulic press, and the disc radioisotope excitation sources Fe-55 (25 mCi, Eckert & Ziegler, Berlin, Germany) and Cd-109 (20 mCi, Eckert & Ziegler, Berlin, Germany) used for fluorescence excitation. Fluorescence was measured using the EDXRF spectrometer with an XR-100 SDD detector (Amptek, Bedford, MA, USA), a PX5 digital pulse processor (Amptek, Bedford, MA, USA), and a PC-based, multichannel analyzer software package (DPPMCA). In Fe-55 mode, the spectrometer was equipped with a vacuum chamber to measure light elements (Si, P, S, and Cl), and in Cd-109 mode, in the air for K, Ca, Ti, Mn, Fe, Zn, Br, Rb and Sr. The energy resolution was 125 eV at 5.9 keV. X-ray spectra were analyzed using AXIL Spectral Analysis software. Quantification was performed using the Quantitative Analysis of Environmental Samples (QAES) software developed in-house [26,27]. The estimated uncertainty budget of the EDXRF analysis was 11%. The method was validated by analyzing NIST 1547 (peach leaves) and NIST 1573a (tomato leaves).

Iron Speciation by Fe K-Edge X-ray Absorption Near Edge Structure (XANES)
Powdered Spirulina samples were pressed into pellets (0.1-0.3 g), fixed on Teflon holders with Fe free scotch tape and mounted on an LN 2 cooled stage. X-ray absorption spectra were obtained in fluorescence detection mode using an unfocused CLAESS beamline at the ALBA synchrotron facility (ALBA, Barcelona, Spain). A pair of horizontal and vertical slits allowed the reduction of the beam size on the sample to about 5 mm × 1 mm, illuminating a major part of the pellet. A silicon (Si 111) double crystal monochromator was used with 1 eV resolution at the Fe K-edge (7112 eV). The samples were inserted between the first and the second ionization cell at 45 • relative to the beam. An SDD fluorescence detector, positioned at 90 • to the beam, was used to measure the intensity of the Fe-Kα fluorescence radiation. The fluorescence spectra were recorded as the ratio of the fluorescence detector signal and the signal of the incident photon beam from the first ionization chamber with an integration time of 4 s/step. The absorption spectra were measured within the interval −150 eV to 350 eV relative to the Fe K-edge. In the XANES region, equidistant energy steps of 0.2 eV were used and 1 eV steps elsewhere. Three replicates were measured to check scan reproducibility and improve the signal-to-noise ratio. No evidence of Fe K-edge shifts in consecutive scans of the samples was observed due to the absorbed dose of ionizing radiation. The monochromator's exact energy was calibrated against a 5 µm thick Fe metal foil. The first inflection point in the XANES spectrum of Fe metal was at 7112 eV, while the absolute energy reproducibility was ± 0.03 eV or better. The Fe K-edge XANES spectra were analyzed using the IFEFFIT software package ATHENA [28].
The bioavailability of Fe was estimated by measuring the relative amounts of Fe 2+ (ferrous iron) and Fe 3+ (ferric iron). For this, we used the linear combination fit (LCF) method as described in [28,29]. The relative amounts of each Fe cation (Fe 2+ and Fe 3+ ) in the sample were determined based on a linear combination fit of the XANES spectrum of the sample, that of the Fe reference compound with known valence states of Fe.

Toxic Trace Element and Se Analysis by Inductively Coupled Plasma-Mass Spectrometry
Inductively coupled plasma-mass spectrometry (ICP-MS) was used to measure Se, As, Cd, Hg and Pb levels in commercial Spirulina samples. Powdered samples (0.05-0.1 g) were weighed into Teflon vials, followed by 2 mL of 65% HNO 3 (Suprapur ® , Merck, Darmstadt, Germany). Samples S10, S20, S31, S40, S41, S46 and S47 were prepared in duplicate. The samples were then digested in an UltraWave closed vessel microwave digestion system (Milestone, Sorisole (BG), Italy) at 1500 W and 100 bar maximum pressure. The temperature program was as follows: ramped to 240 • C in 20 min, held for 15 min and then cooled to 40 • C. The digests were then quantitatively transferred to 10 mL polyethylene graduated vials and filled to the mark with MilliQ water. The samples were then filtered through hydrophilic syringe filters (Millipore Millex-HV, Merck, Darmstadt, Germany) (0.45 µm) and diluted in a 1:10 ratio. The reference material BCR-414 (plankton trace elements) and blank samples (HNO 3 ) were prepared similarly.

Fatty Acid Analysis by Gas Chromatography-Mass Spectrometry Method
The analysis of fatty acids in Spirulina samples was determined using Gas Chromatography-Mass Spectrometry (GC-MS).

Fatty Acid Extraction and Esterification
Powdered Spirulina samples (150 mg) were weighed directly into screw-cap vials, and 500 µL of dichloromethane and 3 mL of 0.5 M sodium hydroxide in methanol were added for total lipid extraction. Samples were then purged with nitrogen and heated for 10 min at 90 • C. Once cool, 3 mL of BF 3 -MeOH was added to generate the fatty acids methyl esters (FAMEs) and purged with nitrogen. The samples were heated for 10 min at 90 • C. Once cool, the FAMEs were extracted using 1.5 mL of hexane. The hexane phase was transferred directly into a GC vial and stored at −20 • C. All the samples were prepared in triplicate.

FAME Analysis
Analysis was carried out using a 7890B GC and 5977A Series GC/MSD (Agilent, Santa Clara, CA, USA). Separation was achieved on a 30 m × 0.25 mm × 0.25 µm VF-WAXms capillary column (Agilent J&W, Santa Clara, CA, USA). The injection volume was 1 µL, with a split ratio of 10:1. The carrier gas was helium maintained at a 1.5 mL/min constant flow. The injector temperature was 280 • C and the detector temperature 350 • C. The temperature program was as follows: initial column temperature set at 50 • C for 1 min then programmed to 170 • C at 15 • C/min and held for 5 min, then from 170 • C to 200 • C at 3 • C/min, held 5 min, and from 200 • C to 230 • C at 5 • C/min, and held 17 min.
A standard Supelco 37 component FAME Mix in dichloromethane (Bellefonte, PA, USA) was used for identification and quantification. Compounds were identified based on a comparison of retention times with authentic compounds. With each set of samples, blank samples and the FAME Mix standard were analyzed to verify the stability of the analytical system. The results are expressed as the weight percent of an individual fatty acid to the total fatty acid (TFA) content calculated from the peak area using the appropriate correction factors [30].

Amino Acid Analysis by Gas Chromatography-Mass Spectrometry
The amino acid composition was determined using Gas Chromatography-Mass Spectrometry (GC-MS).

Liquid Phase Hydrolysis
The total amino acid extraction was based on hot protein hydrolysis and simultaneous free amino acid solubilization. Before the analysis, the hydrolysis micro-reaction vessels (5 mL, heavy-wall borosilicate glass, 20 mm × 65 mm, screw top, with a solid phenolic cap) were cleaned by pyrolysis at 500 • C for 6 h and left to cool overnight. The hydrolyzing agent (6N HCl) was prepared fresh from a 30% hydrochloric acid solution. The samples (15 mg) were weighed directly into the reaction vials and the hydrolyzing agent (1 mL) containing 4% thioglycolic acid, which acts as a reducing agent to prevent amino acid oxidation. After, 1% phenol was added to prevent halogenation of the tyrosine. Oxidation was prevented by purging the samples with N 2 (5 min). The vials were then sealed and heated at 110 • C for 24 h.

Amino Acid Derivatization
For derivatization, a commercial EZ:faast Amino Acid Hydrolysate kit (Phenomenex, Torrance, CA, USA) was used. The procedure was as follows: 355 µL of the Na 2 CO 3 solution was added to the hydrolysate sample (100 µL) to obtain a pH of 2-2.5. To this was added 20 µL of the norvaline internal standard solution (0.2 mM) and 100 µL 10% n-propanol. The samples were then extracted using solid-phase extraction and the amino acids eluted with 200 µL freshly prepared eluting medium (sodium hydroxide:n-propanol in 3-picoline, 3:2, v/v). Further, the amino acids were derivatized in a mixture of chloroform and propyl chloroformate (50 µL). The amino acids were then extracted into the organic chloroform layer by repeated emulsification and allowing the reactions to proceed for 1 min in between vortexing. Iso-octane (100 µL) was then added, and the mixture was emulsified for an additional 5 s and allowed to react for 1 min. The organic layer was then transferred into a GC vial and reduced to dryness (N 2 ). The amino acid derivatives were then reconstituted in a solution (100 µL) of iso-octane:chloroform (80:20, v/v). All samples were prepared in duplicate.

Gas Chromatography-Mass Spectrometry Method for Amino Acid Analysis
Amino acid analysis was performed using a 7890B GC and 5977A Series GC/MSD (Agilent, Santa Clara, CA, USA). Separation was achieved on a 10 m × 0.25 mm × 0.15 mm ZB-AAA GC column provided in the EZ:faast kit together with a FocusLiner ® . The split ratio was 15:1, and the injector temperature was 250 • C. The injection volume was 1.5 µL. Helium was used as the carrier gas at a 1.5 mL/min flow rate. The temperature program was 110 • C to 320 • C at 30 • C/min. The detector temperature was set to 310 • C. Calibration curves were prepared for individual amino acids at concentrations of 50, 100 and 200 nmol/mL using the standard mixture (SD) provided. The amino acid standard mixture consisted of 200 nmoles/mL of each amino acid: alanine (ALA), glutamic acid (GLU), hydroxylysine (HLY), leucine (LEU), phenylalanine (PHE), threonine (THR), valine (VAL), aspartic acid (ASP), glycine (GLY), hydroxyproline (HYP), lysine (LYS), proline (PRO), tryptophan (TRP), cystine (C-C), histidine (HIS), isoleucine (ILE), methionine (Met), serine (SER) and tyrosine (TYR). Each calibrant was prepared in triplicate. From then on, the SD solutions were treated following the same procedure as the samples. Individual amino acids were identified by comparing peak retention times with known amino acids in the standard. The amino acid content results are expressed in mg/g of sample dry weight (dwt).

Statistical Analysis
Statistical analysis was performed using XLSTAT software (Addinsoft, Long Island, NY, USA, 2019). First, basic statistical methods were used for data analysis (median and quartiles, minimum, maximum, average). Principal component analysis (PCA) was applied further to identify characteristic parameters to discriminate samples based on their macro and trace-elemental composition, amino acid and fatty acid composition. The results are presented as biplots, simultaneous variables and as PCA plots.

Elemental Composition
The results are presented in Table 2. The elemental content in the supplements was as follows: Se < Rb < Br < Ti < Zn < Sr < Mn < Fe < Ca < Cl < Si < S < P < K. We specifically focused on elements Fe, Ca, K, Se and P since Spirulina is promoted as a rich source of these elements [13,15,31]. Although the recommended daily intake (RDI) varies, the majority of the producers recommend 3-10 g of Spirulina supplement intake per day, regardless of the product form (powder, capsule or tablet). As well as the RDI, the data are further evaluated using Dietary Reference Values (DRV) such as Population Reference Intake (PRI), which represents the level of nutrient intake adequate for all people in a population group, or Adequate Intake (AI), which is used when a PRI cannot be determined. Where possible, the elemental composition was also evaluated in terms of Tolerable Upper Intake Level (UL), i.e., the maximum daily intake of a nutrient from all sources unlikely to pose adverse health effects on humans [32]. The data are presented as the median value and interquartile range (IR, in parentheses).
Given the recommended daily dose, the minimal intake of Ca is 1.38 to 4.60 mg Ca/day, the maximal Ca intake is 84.0 to 280 mg Ca/day, and a median value of 4.38 (2.92-7.8)-14.6 (9.72-26.0) mg Ca/day. The PRI for Ca is 950 mg/day [33]. Therefore, the values are within the recommended PRI values of 0.15% to 29.5%. This variability can be attributed to the differences in supplement formulation and the presence of additives. For example, values were found in S19, which contains calcium carbonate, and S1, which contains calcium carbonate in the form of edible scallop shell powder [34]. The lowest values were in S41, S42 and S46. The highest amount of Ca was in S18, a mixed sample containing Spirulina, Chlorella and Lithothamnium algae (191-635 mg/day for 3 g to 10 g of supplement/day, respectively). This value is likely due to Lithothamnium calcareum (L. calcareum), a seaweed that crystallizes calcium carbonate in its cell walls [35]. However, the UL of 2500 mg/day is unlikely to be exceeded by including Spirulina supplements in the diet [36].
The minimal and maximal P intake based on RDI was 18.5-61.6 and 44.1-147 mg/day, respectively. The median value was 32.7 (30.3-35.9)-109 (101-120) mg P/day. The P content represents 3.36-26.7% of the AI (550 mg/day) [33]. The lowest value among all samples was in S9, a mixed sample containing wheatgrass, barley grass, Chlorella and Spirulina, i.e., 15.2 (for 3 g of supplement/day)-50.6 mg/day (for 10 g of supplement/day). This most likely results from the lower amount of Spirulina and Chlorella algae in the supplement, especially since Spirulina and Chlorella typically contain much higher phosphorous content than cereal grasses [13,15,37]. Like Ca, if the Spirulina supplement consumption remains within the RDI, the UL determined for P (3000 mg/day) [36] is unlikely to be exceeded.
The minimal intake of K varies from 17.5 to 58.3 mg/day and maximal from 80.7 to 269 mg/day, with a medium intake of 45.6 (42.8-50.0)-152 (143-167) mg/day. This amount would account for 0.5-7.69% of the AI value (3500 mg/day) for an adult [33]. Three of the highest K values were in S5, S44 and S46, where S44 and S46 originate from Italy. The lowest values were measured in S37 and S22, which contained additives and did not declare an origin. No UL was set for K consumption.
The daily intake of Fe (mg Fe/day) was from 0.84-2.81 (minimal value) and 10.4-34.8 (maximal value) with a median of 2.07 (1.47-3.40)-6.89 (4.90-11.3). Such amounts account for between 5.25 to 218% of the PRI for females (16 mg/day) and 7.64 to 316% for males (11 mg/day) [33]. The high deviation observed among the samples is due to the high amount of Fe in S4 and S26 from Hawaii and S2 with no declared origin. High Fe values likely result from a high concentration of Fe in the growth medium. The Fe content in the Spirulina microalgae has been proven to reflect that in the growth medium [38,39].
Iron bioavailability in Spirulina was determined by analyzing the relative ferrous and ferric iron amounts using XANES analysis. The results are presented in Table 3. The results show that most iron (82-92%) is present as Fe 3+ , which means that the bioavailability of Fe from the supplements is low, as only a small amount of iron is available in a more bioavailable ferrous form, Fe 2+ [40][41][42]. This finding also means that promoting Spirulina as a rich source of dietary iron should be reconsidered.
The minimal and maximal Se intake was 0.01 to 0.04 and 8.10 to 27.0 µg/day, respectively, with the median being 0.30 (0.20-0.80)-1.01 (0.66-2.67) µg Se/day. The amounts of Se also varied significantly, but S14, S15 and S32 stand out due to their high Se value. The former two are from Taiwan, and the latter is from China. High Se values in these samples are believed to be due to higher amounts of Se in the growth medium. However, Se from these types of samples has been shown to have a lower bioavailability than classical sources such as selenomethionine and inorganic Se salts. In addition, Se from Spirulina is metabolized differently due to its chemical form [43]. The lowest Se values were determined in pure Spirulina samples from Italy (S44 and S46). The Se accounts for 0.01% to 38.6% of the recommended AI of 70 µg/day [33]. The UL for Se is 300 µg/day [36] but is unlikely to be exceeded by adding daily Spirulina supplements to a regular diet.
Determined values of all elements are similar to those previously reported in the literature [13,15,20,44]. However, the significant variability observed in the amounts of certain elements is likely related to the growth medium used and intentional enrichment. It has been shown that micronutrients in the growth medium significantly improve uptake and accumulation of the macro-and micronutrients [39,43,45]. The growth medium pH can also affect Spirulina mineral assimilation, i.e., metal ion assimilation increases at higher pH [46]. According to the data, Spirulina food supplements are a good source of iron, calcium and phosphorous and can provide substantial amounts of potassium and selenium. However, the intake of specific nutrients is product dependent and depends on the amount of supplement consumed, since the recommended daily consumption values differ among producers.
The high iron content in Spirulina supplements is significant for those who consume, for example, fewer foods of animal origin and therefore have a lower iron intake in their diet. In addition to containing high amounts of iron, Spirulina also does not contain phytates or oxalates that would cause iron chelation, making Spirulina iron highly available for absorption in the human intestine [15,47]. However, as this study has shown, more research is needed to assess the actual iron bioavailability from Spirulina due to the predominance of the ferric (Fe 3+ ) iron form.

Compliance with Their Nutrient Declaration
Measured elemental values were compared to the values declared on the products. Table 4 lists the 15 products that provided information on the content of Fe, Mn, Ca, Zn and P together with the degree of deviation (%) from the declared values. Iron had the most declarations (32.6% of the products), followed by Mn (13.0%), Ca and Zn (8.70%), P (4.3%) and finally K and Se (2.17%). The maximum permissible deviation of mineral content in food supplements is from −20% to 45% [48]. An excessive negative deviation from the declared Fe content was found in S5, S24 and S28 and a positive deviation in S4, S8, S10, S13, S14, S15, S17 and S36. The Mn content deviated positively in S4 and negatively in S12, S13 and S17, while S28 had an insufficient Ca content. The Zn content was low in S17, S23 and S24, while the P content was high in S12. In the case of K, the measured content in S17 was within the declared limits (+10.7%), while Se was much lower (−95.8%) than the declared value. However, several producers state that mineral levels can deviate due to seasonal fluctuations. In addition, even though some values are high, they remain below the UL and are unlikely to pose a risk to human health. However, the proportion of inappropriate declarations (86.7%) is a cause for concern and could undermine consumer confidence, and supports the need for regular monitoring and improved quality control.

Toxic Trace Element Content
The content of Cd, Hg and Pb was evaluated according to the maximum allowed European Commission levels [49]. In contrast, the As content was evaluated according to benchmark dose lower confidence limit (BMDL 01 ) for cancers of the lung, skin and bladder, and skin lesions determined by The Scientific Panel on Contaminants in the Food Chain (CONTAM) [50]. Toxic trace element content and their maximum allowed values are presented in Table 5.
The maximum measured values of Cd and Pb were 226 µg/kg dwt and 1320 µg/kg dwt, respectively, and did not exceed the maximum allowed value in food supplements. The daily BMDL 01 values were calculated based on the average male (87 kg) and female (68 kg) body weight in the Slovenian population [51]. The BMDL 01 values for As range between 0.3 and 8 µg/kg b.w. per day. Based on an RDI of 3-10 g/day, the maximum daily intake from consuming Spirulina was 8.11-27.0 µg/day and did not exceed the upper BMDL 01 value for all of the supplements tested. In the case of Hg, S39 exceeded the maximum value of 100 µg/kg dwt by 1% (101 µg/kg dwt). All others were below the maximum allowed Hg value in food supplements. The samples' median value (interquartile range) was 5.57 (3.57-7.63) µg/kg dwt. The lowest values of As, Cd and Pb were found in S46 produced in Italy, while the lowest Hg value was measured in S18, a mixed sample containing Spirulina, Chlorella and Lithothamnium algae. The measured values of elements Cd, Hg and As are comparable to literature values [6,21,52,53]. Pb values are also consistent with those determined by Al-Homaidan [53] but are lower than in commercial samples tested by Hsu et al. and Campanella et al., which ranged between 5600-15,200 µg/kg dwt, and 30.8% of the Spirulina samples tested by Rzymski et al., which ranged from 3500 µg/kg dwt to 5000 µg/kg dwt. The authors attributed the high values to local contamination, greater propensity toward Pb assimilation by microalgae and natural background levels [6,52,54,55].   Based on the data from the present study, Spirulina supplements do not contribute significantly to the intake of toxic trace elements and do not pose a serious risk. Nevertheless, Spirulina is an effective accumulator of trace elements which is an advantage when it accumulates elements essential for human health but a disadvantage for toxic elements [21]. Spirulina is also a potential source of Pb, Hg, Cd, and As in open production systems, where pedoclimatic conditions and agricultural practices could contribute to their presence in higher values and again supports the need for regular monitoring [56][57][58]. In contrast, this is not an issue when grown in controlled (closed) environments [21].

Amino Acid Content
Amino acids asparagine (ASN) and glutamine (GLN) were quantitatively converted to aspartate (ASP) and glutamate (GLU) during acid hydrolysis. Tryptophan (TRP) was lost during acid hydrolysis, and arginine (ARG) and cysteine (CYS) were not included in the GC-MS EZ:faast kit due to their thermal instability [59,60]. Again, the results (Table 6) are presented according to the RDI of 3-10 g. The measured values are compared to the daily recommended amounts [61] for the average male (87 kg) and female (68 kg) in Slovenia [51]. Table 6. Total amino acid content (mg/day) in Spirulina supplements, presented for minimal (3 g) and maximal (10 g) recommended daily intake. The amino acid values determined in this study are presented in Table S1 and are within the literature values, except for VAL and HIS, where the measured values were higher [6,62,63]. The VAL content ranged from 50.8-123 mg/g dwt in this study, while the previously reported values range from 35.8-60 mg/g dwt. The HIS content varied from 16.3-22.2 mg/g dwt, compared to 6.00-11.9 mg/g dwt reported in the literature (Figure 1; Table S1). The lowest content of all amino acids except MET was determined in S22, closely followed by S19 and S37. These samples contain Spirulina and excipients, especially S22 and S19, which have the most declared excipients among all the samples. It is known that a high excipient content affects the amino acid content due to the reduced amount of Spirulina [6]. Valine, ILE and HIS were present in the lowest amounts in all samples. The sample with the second-lowest content of all other analyzed amino acids was S9-a mixed sample of wheatgrass, barley grass, Spirulina and Chlorella. This result could also be due to the low microalgae content in the supplement, as cereal grasses are not as rich in protein as Spirulina and Chlorella [64,65]. These results suggest that, combined with elemental composition, the amino acid content could distinguish authentic samples from adulterated ones. The highest values of ALA, GLY, VAL, LEU, ILE, THR, SER, ASP and TYR were found in S36 and S40, PRO and LYS were highest in S6, PHE in S8, HIS in S28, MET in S39 and GLU in S46-all were declared as pure Spirulina products. In addition to product composition, other factors affecting the amino acid content have been reported. For example, cells grown under stress conditions, including salinity stress, have a lower capacity for protein synthesis, which is seen in the lower protein content found in biomass grown in salinated water [66,67]. Differences also occur due to different cultivation times, light intensities, temperatures, and the growth medium's nutrient composition. For example, Spirulina grown in a urea growth medium has a higher amino acid content than others [62]. The drying processes used can also affect the amount of protein and, consequently, amino acid content. The highest protein losses are connected to convective and infrared drying in spreading cylinders, while freeze-drying gives the highest protein recoveries. In addition, thin layer drying results in higher protein recoveries than cylinder drying [68]. Drying at low temperatures (40-50 °C) does not affect the products' nutritional quality compared to the fresh Spirulina [69].
This study shows that the Spirulina supplements contained all the essential and nonessential amino acids measured. Coverage of daily requirements for adults [61]  According to these results, the tested Spirulina supplements are a good source of essential and nonessential amino acids, as shown by other authors analyzing commercial Spirulina products [9,63,66]. However, despite that S22, S19 and S37 stand out because of their low amino acid content, overall, Spirulina food supplements would be reasonable when choosing high amino acid content products.

Fatty Acid Content
The amounts of individual fatty acids (FA) are presented in Table 7 and Table S2. The results in Table 7 are presented as the median value (interquartile range in parentheses) of weight percent of individual fatty acids of total fatty acids (TFA). The most abundant FA was palmitic acid (16:0) followed by linoleic (C18:2 ω-6c), γ-linolenic (C18:3 ω-6), palmitoleic (C16:1 ω-7), stearic (C18:0) and oleic/elaidic acid (C18:1 ω-9c/9t). The amounts of fatty acids agree with published data [6,70], although levels of γ-linolenic, linoleic, palmitoleic and stearic acid vary [71][72][73][74]. These differences could be due to higher or lower content of saturated/unsaturated FA in the algal cells, which depends on metabolic needs, such as membrane fluidity, which depends on the growth conditions [75]. It is known that In addition to product composition, other factors affecting the amino acid content have been reported. For example, cells grown under stress conditions, including salinity stress, have a lower capacity for protein synthesis, which is seen in the lower protein content found in biomass grown in salinated water [66,67]. Differences also occur due to different cultivation times, light intensities, temperatures, and the growth medium's nutrient composition. For example, Spirulina grown in a urea growth medium has a higher amino acid content than others [62]. The drying processes used can also affect the amount of protein and, consequently, amino acid content. The highest protein losses are connected to convective and infrared drying in spreading cylinders, while freeze-drying gives the highest protein recoveries. In addition, thin layer drying results in higher protein recoveries than cylinder drying [68]. Drying at low temperatures (40-50 • C) does not affect the products' nutritional quality compared to the fresh Spirulina [69].
This study shows that the Spirulina supplements contained all the essential and non-essential amino acids measured. Coverage of daily requirements for adults [61]  According to these results, the tested Spirulina supplements are a good source of essential and non-essential amino acids, as shown by other authors analyzing commercial Spirulina products [9,63,66]. However, despite that S22, S19 and S37 stand out because of their low amino acid content, overall, Spirulina food supplements would be reasonable when choosing high amino acid content products.

Fatty Acid Content
The amounts of individual fatty acids (FA) are presented in Tables 7 and S2. The results in Table 7 are presented as the median value (interquartile range in parentheses) of weight percent of individual fatty acids of total fatty acids (TFA). The most abundant FA was palmitic acid (16:0) followed by linoleic (C18:2 ω-6c), γ-linolenic (C18:3 ω-6), palmitoleic (C16:1 ω-7), stearic (C18:0) and oleic/elaidic acid (C18:1 ω-9c/9t). The amounts of fatty acids agree with published data [6,70], although levels of γ-linolenic, linoleic, palmitoleic and stearic acid vary [71][72][73][74]. These differences could be due to higher or lower content of saturated/unsaturated FA in the algal cells, which depends on metabolic needs, such as membrane fluidity, which depends on the growth conditions [75]. It is known that certain microalgae, including Spirulina, regulate their lipid composition at low temperatures to achieve better membrane fluidity, which results in increased levels of unsaturated FA.  High temperatures during growth also favor the formation of saturated FA [70,76,77], and the formation of certain fatty acids is more intensive at specific temperatures for certain strains, i.e., the highest γ-linolenic FA production in Spirulina maxima is between 35 and 40 • C. In contrast, in Spirulina platensis, the production of γ-linolenic is higher at 30 • C [78]. Culture age and growth medium salinity are also important, and prolonged exposure to high salinity stress results in higher values of γ-linolenic acid in different Spirulina strains [72]. As with amino acids, the drying process can affect lipid yield and total fatty acid content. Namely, the maximum monounsaturated and polyunsaturated FA levels are obtained in the freeze-dried samples, while the sun-dried samples contain higher amounts of saturated acids. In addition, freeze drying provides better nutrient preservation than other dehydration methods such as sun drying, oven drying or spray drying [79].
In most samples, only small amounts or no ω-3 fatty acids were found, making commercial Spirulina samples a good source of ω-6 (especially LA and GLA), but not ω-3 fatty acids, which was also confirmed by other studies [6,70,74,80].
In addition, S18 differed from supplements containing only Spirulina. This sample contains high amounts of C16:2 ω-6 (14.6% of TFA), C18:2 ω-6c (34.4% of TFA) and C18:3 ω-3 (2.52% of TFA). In contrast, the amounts of C16:0 (28.3% of TFA), C18:0 (0.88% of TFA) and C18:3 ω-6 (8.84% of TFA) were exceptionally low. Higher levels of C18:3 ω-3 (ALA, α-linolenic acid) in mixed samples likely result from the presence of Chlorella, which is known to contain large amounts of this FA. Alternatively, low GLA and palmitic acid levels point to a low Spirulina content in S9 and S18, as their high content is typical for pure Spirulina products [74]. The fatty acid distribution (% of TFA) in the analyzed samples is presented in Figure 2. The data make it possible to distinguish between mixed samples and samples containing pure Spirulina based on the fatty acid composition. Such knowledge could help separate authentic from adulterated samples. In addition, the Spirulina supplements tested proved to be a good source of ω-6, but not ω-3 polyunsaturated fatty acids. However, since the FA content varied in samples, it would be sensible to choose pure Spirulina products for consumption with no added excipients from a trusted producer to guarantee optimal FA composition.

Principal Component Analysis (PCA) of Spirulina Samples from Slovenian Market Analysis Results
The data set of 46 Spirulina samples and 43 analyzed parameters (macro-and traceelemental, amino acid and fatty acid composition data) was analyzed by PCA to identify the trends and examine the distribution of variables in the investigated samples ( Figures  3 and 4). PC1 explained 33.0% and PC2 13.5% of the total variance. Four groups of samples can be identified (Figure 3  In addition, S18 differed from supplements containing only Spirulina. This sample contains high amounts of C16:2 ω-6 (14.6% of TFA), C18:2 ω-6c (34.4% of TFA) and C18:3 ω-3 (2.52% of TFA). In contrast, the amounts of C16:0 (28.3% of TFA), C18:0 (0.88% of TFA) and C18:3 ω-6 (8.84% of TFA) were exceptionally low. Higher levels of C18:3 ω-3 (ALA, α-linolenic acid) in mixed samples likely result from the presence of Chlorella, which is known to contain large amounts of this FA. Alternatively, low GLA and palmitic acid levels point to a low Spirulina content in S9 and S18, as their high content is typical for pure Spirulina products [74]. The fatty acid distribution (% of TFA) in the analyzed samples is presented in Figure 2.
The data make it possible to distinguish between mixed samples and samples containing pure Spirulina based on the fatty acid composition. Such knowledge could help separate authentic from adulterated samples. In addition, the Spirulina supplements tested proved to be a good source of ω-6, but not ω-3 polyunsaturated fatty acids. However, since the FA content varied in samples, it would be sensible to choose pure Spirulina products for consumption with no added excipients from a trusted producer to guarantee optimal FA composition.

Principal Component Analysis (PCA) of Spirulina Samples from Slovenian Market Analysis Results
The data set of 46 Spirulina samples and 43 analyzed parameters (macro-and traceelemental, amino acid and fatty acid composition data) was analyzed by PCA to identify the trends and examine the distribution of variables in the investigated samples (Figures 3 and 4). PC1 explained 33.0% and PC2 13.5% of the total variance. Four groups of samples can be identified (Figure 3), each represented by different variables. Most parameter vectors are directed towards the upper right quadrant of Figure 4. The positive trend is due to an increased amino acid composition as well as C16:0, C16:1 ω-7, C18:2 ω-6c and C18:3 ω-6 fatty acids. The blue group (Figure 3) has the highest content of these compounds, followed by the yellow group, while the red group has the lowest amount. Finally, ungrouped samples (S6, S9, S18, S19, S22 and S37), which have the lowest content of selected amino acids and fatty acids, are located on the left-hand side of the graph. A similar positive trend for elements Cl, Ti, Fe, Zn, Mn, Rb and Br and fatty acids C14:1 and C18:1 ω-9c/C18:1 ω-9t is observed in the upper part of the second PCA graph (Figure 4). The samples from Hawaii (orange group) contained the highest amounts of these elements and fatty acids. Interestingly, the undeclared sample S2 falls in the same group, indicating that this Spirulina supplement might originate from Hawaii. Alternatively, ungrouped samples to the left of the vertical line of Figure 3 (S6, S9, S18, S19, S22 and S37) show a positive Ca, Sr and Cd and C14:0, C18:0 and C16:2 ω-4 trend (Figure 4).
Samples S9 and S18 were expected to stand out since they contain other plant or algae material, and their parameters were expected to differ from the samples containing pure Spirulina. In addition, S19 and S22 contain various excipients, affecting the nutrient composition. In contrast, close inspection of the data for S6 and S37 suggests possible adulteration since S6 is declared as pure Spirulina and S37 contains excipients, which did not affect our results. Overall statistical evaluation of the results supports our previous observations. to an increased amino acid composition as well as C16:0, C16:1 ω−7, C18:2 ω−6c and C18:3 ω−6 fatty acids. The blue group (Figure 3) has the highest content of these compounds, followed by the yellow group, while the red group has the lowest amount. Finally, ungrouped samples (S6, S9, S18, S19, S22 and S37), which have the lowest content of selected amino acids and fatty acids, are located on the left-hand side of the graph. A similar positive trend for elements Cl, Ti, Fe, Zn, Mn, Rb and Br and fatty acids C14:1 and C18:1 ω-9c/C18:1 ω-9t is observed in the upper part of the second PCA graph (Figure 4). The samples from Hawaii (orange group) contained the highest amounts of these elements and fatty acids. Interestingly, the undeclared sample S2 falls in the same group, indicating that this Spirulina supplement might originate from Hawaii. Alternatively, ungrouped samples to the left of the vertical line of Figure 3 (S6, S9, S18, S19, S22 and S37) show a positive Ca, Sr and Cd and C14:0, C18:0 and C16:2 ω-4 trend (Figure 4).
Samples S9 and S18 were expected to stand out since they contain other plant or algae material, and their parameters were expected to differ from the samples containing pure Spirulina. In addition, S19 and S22 contain various excipients, affecting the nutrient composition. In contrast, close inspection of the data for S6 and S37 suggests possible adulteration since S6 is declared as pure Spirulina and S37 contains excipients, which did not affect our results. Overall statistical evaluation of the results supports our previous observations.

Conclusions
This study finds that when consumed in recommended amounts, the analyzed Spirulina supplements are a good source of calcium, phosphorous, potassium, and selenium, while toxic trace elements do not represent a serious health risk. They are also a good source of essential and non-essential amino acids as well as ω-6 polyunsaturated fatty acids. The study data also show that Spirulina contains low amounts of ω-3 polyunsaturated fatty acids, and although they contain high amounts of Fe, since it is mainly present as Fe 3+ , the Fe is less bioavailable.
Therefore, a well-thought-out selection of Spirulina supplements would be advised and choosing pure Spirulina supplements is advisable, especially since pure Spirulina samples have a higher amino acid, γ-linolenic and linoleic fatty acid, P and Se content. Additionally, as different Spirulina products might have a different nutrient composition, the products should be chosen according to the specific nutrient needs of the individual. Supplements from Hawaii are rich in Fe, Zn, Mn, Cl, Ti, Rb and Br.
Notably, a high proportion (86.7%) of inappropriate declarations was found among the analyzed samples regarding the content of Fe, Mn, Ca, Zn, P, K and Se, which is a cause for concern. Deviations of more than 45% over the declared value could pose a risk to human health through excessive elemental intake. Such deviations can also undermine consumer confidence. Fortunately, in this case, UL levels were not exceeded.
This study also showed how adding algal or plant material supplements alters the elemental, amino acid and fatty acid composition. Such data could distinguish mixed products from those containing only Spirulina and therefore be valuable in authenticity studies. Multivariate analysis was able to discern these products from those containing only Spirulina. In addition, the amino acid, fatty acid and mineral composition data suggest that at least two samples were adulterated, since such differences in composition compared to pure Spirulina could not be explained by the addition of excipients.
Overall, Spirulina is a good source of nutrients. However, in the case of dietary supplements, regular monitoring and inspection are advised to identify adulterations and

Conclusions
This study finds that when consumed in recommended amounts, the analyzed Spirulina supplements are a good source of calcium, phosphorous, potassium, and selenium, while toxic trace elements do not represent a serious health risk. They are also a good source of essential and non-essential amino acids as well as ω-6 polyunsaturated fatty acids. The study data also show that Spirulina contains low amounts of ω-3 polyunsaturated fatty acids, and although they contain high amounts of Fe, since it is mainly present as Fe 3+ , the Fe is less bioavailable.
Therefore, a well-thought-out selection of Spirulina supplements would be advised and choosing pure Spirulina supplements is advisable, especially since pure Spirulina samples have a higher amino acid, γ-linolenic and linoleic fatty acid, P and Se content. Additionally, as different Spirulina products might have a different nutrient composition, the products should be chosen according to the specific nutrient needs of the individual. Supplements from Hawaii are rich in Fe, Zn, Mn, Cl, Ti, Rb and Br.
Notably, a high proportion (86.7%) of inappropriate declarations was found among the analyzed samples regarding the content of Fe, Mn, Ca, Zn, P, K and Se, which is a cause for concern. Deviations of more than 45% over the declared value could pose a risk to human health through excessive elemental intake. Such deviations can also undermine consumer confidence. Fortunately, in this case, UL levels were not exceeded.
This study also showed how adding algal or plant material supplements alters the elemental, amino acid and fatty acid composition. Such data could distinguish mixed products from those containing only Spirulina and therefore be valuable in authenticity studies. Multivariate analysis was able to discern these products from those containing only Spirulina. In addition, the amino acid, fatty acid and mineral composition data suggest that at least two samples were adulterated, since such differences in composition compared to pure Spirulina could not be explained by the addition of excipients.
Overall, Spirulina is a good source of nutrients. However, in the case of dietary supplements, regular monitoring and inspection are advised to identify adulterations and deviations from the declared content. In this way, potential hazards for consumer health can be avoided.