# Spirulina Supplements as a Source of Mineral Nutrients in the Daily Diet
## 2 Materials and Methods
### Sample Determination  
Briefly, samples were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP 7400 Duo, Thermo Scientific, Waltham, MA USA), which allows measurement of various elements, including in plant samples [27; 28]. An ICP-OES with a concentric nebulizer and cyclonic spray chamber was used. The analysis was performed in radial and axial modes. The wavelengths used in the analysis were Zn 206,200, Cr 205,560, Mn 257,610, Cu 224,700, Fe 259,940. Validation was performed by evaluating reference material NIST SRM 8414 (National Institute of Standards and Technology, Gaithersburg, MA, USA), limits of detection (LOD) and recovery of internal standard (yttrium) (Table 2). To eliminate potential interferences, emission lines were empirically selected in a pilot measurement. Such a validation model is often used in ICP-OES studies [28]. Y recovery ranged from 90-106%. R2 values for all standard curves ranged from 0.998 to 1.000. Briefly, selenium (Se) concentration was determined by spectrofluorimetry using a SHIMADZU RF-5001 PC analyzer. Samples were wet-digested in concentrated HNO\({}_{3}\) (230 \({}^{\circ}\)C/180 min) and HClO\({}_{4}\) (310 \({}^{\circ}\)C/20 min). Then, 9% HCl was added to reduce selenate VI to selenate IV. Subsequently, selenate IV was complexed with 2,3-diaminonaphthalene (Sigma), and the resulting complex was extracted with cyclohexane (Chempur, Piekary Slaskie, Poland). Fluorescence was measured at an emission wavelength of 518 nm and an excitation wavelength of 378 nm. The accuracy of the method was based on NCS-ZC 71001 reference material (certified reference material--BCR-185R bovine liver) from China NatiAnalysis Center for Iron and Steel (Beijing, China). The determined Se concentration was 90.9% of the standard value. Two replicates were performed for each sample, and the average of the data, expressed in milligrams per kilogram wet weight, was used in the statistical analysis. The certified selenium concentration was 1.68 \(\mu\)g/g, the labeled concentration was 1.56 \(\mu\)g/g, and the recovery was 93%. \begin{table}
\begin{tabular}{c c c c c} \hline
**Element** & **Certified [mg/L]** & **Measured [mg/L] (\(n=3\))** & **LOD [mg/L]** & **\% RSD Range** \\ \hline Ca & \(145\pm 20\) & 141 & 0.00676 & 0.4–2.5 \\ Mn & 0.37 \(\pm\) 0.09 & 0.43 & 0.00026 & 1.0–6.2 \\ K & 15,170 \(\pm\) 370 & 15290 & 0.08426 & 0.3–1.4 \\ Zn & 142 \(\pm\) 14 & 138 & 0.00065 & 1.5–5.7 \\ Cu & 2.84 \(\pm\) 0.45 & 3.06 & 0.00186 & 2.4–8.2 \\ Fe & 71.2 \(\pm\) 9.2 & 76.1 & 0.00022 & 1.8–6.7 \\ Na & 2100 \(\pm\) 80 & 2146 & 0.08137 & 0.8–4.1 \\ Pb & 0.38 \(\pm\) 0.24 & 0.48 & 0.00178 & 5.3–11.2 \\ Cr & 0.071 \(\pm\) 0.038 & 0.080 & 0.00044 & 3.9–9.2 \\ P & 8360 \(\pm\) 450 & 8874 & 0.00532 & 0.8–3.2 \\ Mg & 960 \(\pm\) 95 & 923 & 0.00159 & 0.5–1.9 \\ \hline \end{tabular}
\end{table} 
Table 2: Analysis of reference material Bovine Muscle NIST-SRM 8414, limits of detection (LOD), and relative sample deviation (% RSD) range [29]. Selenium (Se) concentration was determined by spectrofluorimetry with a SHIMADZU RF-5001 PC analyzer. Samples of spiruline were wet-digested in concentrated HNO\({}_{3}\) (230 \({}^{\circ}\)C/180 min) and HClO\({}_{4}\) (310 \({}^{\circ}\)C/20 min). An amount of 9% HCl was added to the digested samples to reduce selenate VI to selenate IV. Subsequently, selenate IV was complexed with 2,3-diaminonaftalene (Sigma) and the resulting complex was extracted with cyclohexane (Chempur). Fluorescence was measured from the organic layer (cyclohexane) at 518 nm emission wavelength and 378 nm excitation wavelength. The accuracy of the analytical method was based on NCS-ZC 71001 (beef liver) reference material from China NatiAnalysis Center for Iron and Steel (Beijing, China). The determined Se concentration was 90.9% of the standard value. Two replicates were performed for each sample, and statistical analysis used the average of the data, expressed in milligrams per kilogram wet weight.