Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals

Poorly water-soluble iron-containing compounds are promising iron fortificants. However, ensuring high bioaccessibility and low reactivity of iron is challenging. We present the potential application of ferrous pyrophosphate (Fe(II)PP) and Fe(II)-containing M2(1–x)Fe2xP2O7 salts (0 < x < 1, M = Ca, Zn, or Mn) for delivery of iron and a second essential mineral (M). After preparation by a facile and environment-friendly coprecipitation method, the salts were investigated for their composition, pH-dependent dissolution, iron-mediated discoloration of a black tea solution, and oxidation of vitamin C. Our results suggest that these salts are possible dual-fortificants with tunable composition that compared to Fe(II)PP (i) show lower (<0.5 mM) and enhanced (to 5 mM) iron dissolution in moderate and gastric pH, respectively, (ii) exhibit less discoloration and dissolved iron in tea when x = 0.470 for M = Ca or Zn and x = 0.086 for M = Mn, and (iii) do not increase the oxidation extent of vitamin C over 48 h when x = 0.06, 0.086, or 0.053 for M = Ca, Zn, or Mn, respectively.

Supplementary material for "Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals" by Moslehi, van Eekelen, Velikov, and Kegel

Preparation S1: Pure divalent metal pyrophosphate salts
Pure divalent metal pyrophosphate salts were synthesized as references for comparative purposes.The preparation was done via a well-established co-precipitation method which has been described elsewhere previously [1][2][3]  , respectively.This was done while the NaPP solution was stirring vigorously (~ 400 rpm) with a magnetic stir bar.In the case of the Fe(II)PP a turbid light green, and in other cases turbid white/off-white dispersions were formed within a few seconds after the addition.The samples were then centrifuged at 3273 × g for 30 minutes in 50 ml volume polypropylene conical centrifuge tubes using an Allegra X-12R Centrifuge (Beckman Coulter, Brea, CA, USA).This was followed by washing the precipitate with MQ water twice.Finally, the salts were dried overnight in an oven at 45 °C (Fe(II)PP: 88%, CaPP: 74%, ZnPP: 80%, and MnPP: 72% yield).

Preparation S2: Mixed divalent metal pyrophosphate salts
The mixed divalent metal salts were prepared by the same procedure as the pure salts, by addition of 50 ml of a mixed solution of FeSO  4, where M = Ca, Zn, or Mn).After adding the mixed solution to NaPP, the solutions were stirred vigorously (~ 400 rpm) with a magnetic stir bar (final concentration of NaPP: 4.29 mM).In all ratios, a turbid dispersion was formed a few seconds after the addition.The samples were then centrifuged, washed, and dried in an oven following the same procedure as explained for the pure divalent metal salts.
Consequently, the molar ratios were calculated based on which the x-value was found in the structural formula.The molar ratio of total metal ions ([M] + [Fe], final concentration: 8.573 mM) to pyrophosphate ions was 2:1.The average yields of the prepared mixed salts with M = Ca, Zn, and Mn were 33 ± 3%, 69 ± 10%, and 82 ± 7%, respectively.
Supplementary material for "Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals" by Moslehi, van Eekelen, Velikov, and Kegel Supplementary material for "Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals" by Moslehi, van Eekelen, Velikov, and Kegel

Characterization S2: High-Angle Annular Dark Field Scanning TEM (HAADF-STEM)
High-angle annular dark-field scanning TEM (HAADF-STEM) was performed on a Talos™ F200X (Thermo Fisher Scientific, San Jose, CA, USA) operated at 200 kV.The elemental mapping was recorded by assigning a color to each element.Color indications are as follows: second divalent metal (i.e., M = Ca, Zn, or Mn): green, iron: red, and phosphorus: blue.
Supplementary material for "Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals" by Moslehi, van Eekelen, Velikov, and Kegel

Spectroscopy (ICP-AES)
For the mixed salts in which M = Mn, the chemical composition was obtained by elemental analysis using ICP-AES.After ensuring the identical morphology and homogeneous distribution of Fe and Mn in the salts (i.e., MnMix1-3), their samples were dissolved in 10 ml of a 2% HNO 3 solution to achieve optimal measurement concentration ranges.ICP-AES measurements were performed using an Optima 8300 instrument (PerkinElmer,Waltham, MA, USA) and in triplicate.Finally, the ratios of Mn/Fe and P/Fe were used to obtain the average x-value for each salt based on the general formula.For the salt with heterogeneous morphology (i.e., MnMix4), the approximate x-values were estimated using EDX analysis on the different morphological phases separately, as described in Characterization S1.

Characterization S4: X-Ray Diffraction (XRD) Spectroscopy
The dried powders of the salts were analyzed at room temperature with an AXS D2 Phaser powder X-ray diffractometer (Bruker®, Billerica, MA, USA), which was equipped with a LYNXEYE® detector in Bragg-Brentano mode.The radiation used was cobalt Kα 1,2 , λ = 1.79026Å, operated at 30 kV, 10 mA for 2θ = 5 to 70 degrees.A silicon holder was used, and the measurements were repeated twice on the salts from independent synthesis batches.

Characterization S5: Fourier Transform Infrared (FT-IR) Spectroscopy
FT-IR measurements were performed on dried powders of the salts using the KBr pellet technique 4 and by an FT-IR spectrometer (PerkinElmer, Waltham, MA, USA).2.5 mg of the powder of each salt was mixed thoroughly with 250 mg of KBr powder (FT-IR grade), and dried in an oven at 60 °C overnight.Pellets were prepared using a press and the measurements were done in independent duplicate.The interferograms were collected over Supplementary material for "Ferrous Pyrophosphate and Mixed Divalent Pyrophosphates as Delivery Systems for Essential Minerals" by Moslehi, van Eekelen, Velikov, and Kegel the spectral range of 1600 -400 cm -1 using a nominal resolution of 4 cm -1 , with a background scan recorded before each measurement.with a magnetic stir bar (final concentration: 10 mg/ml).Then, the pH of the dispersion was adjusted using a pH-stat device (Metrohm, Herisau, Switzerland) by the addition of 0.1 M HCl or 0.1 M NaOH.Subsequently, all dispersions were incubated at 1000 rpm using an Eppendorf ThermomixerR F1.5 (Eppendorf, Hamburg, Germany) at pH values ranging from one to eleven (steps of two pH units), for 2 h at 23 °C.After incubation, the final pH of each sample was measured.Finally, the samples were centrifuged at 15000 × g for 10 min using an Eppendorf Centrifuge 5415R and the supernatants were separated to quantify the dissolved elements concentrations.

4.1.1 Iron concentration measurement by a ferrozine-based colorimetric assay
The concentration of the dissolved iron from Fe(II)PP and the mixed Fe(II)-containing pyrophosphate salts was monitored by a ferrozine-based colorimetric assay 5 .An excess amount of ascorbic acid (50 μl, 100 mM) was added to 50 l sample (supernatant).After 30 minutes incubation of the sample with ascorbic acid, ferrozine (50 l, 10 mM) was added.
The absorbance at 565 nm was measured at room temperature by a SpectraMax M2e (Molecular Devices, Sunnyvale, CA, USA).Quantification of the dissolved iron was performed based on intensity and a calibration curve of FeSO 4 (0.0078 -1 mM, R 2 > 0.99).
Evaluation of the significance of differences in iron concentration was carried out by statistical analysis (significant at p < 0.05).

Figure S1 .
Figure S1.The images and details of color conversion (i.e.,  *  *  * values) of (A) pure Fe(II)PP, the mixed pyrophosphate salts with the general formula M 2(1-x) Fe 2x P 2 O 7 where (B) M = Ca, (C) M = Zn, and (D) M = Mn.The pure CaPP, ZnPP, and MnPP are shown for comparison.The details of color conversions of the salts are obtained by an online color measurement tool (https://imagecolorpicker.com/) and are reported under the images of the salts by  * ,  * ,  * values from left to right, respectively.* The average of the two x-values corresponding to the two morphological phases of the salt MnMix4 is used here (0.440).
Water dispersions of the salts were dried on carbon-coated copper (for M = Ca and Zn) or nickel (for M = Zn) grids and analyzed by transmission electron microscopy and energydispersive X-ray spectroscopy (TEM-EDX).This was performed on a Talos™ F200X (Thermo Fisher Scientific, San Jose, CA, USA) operated at 200 kV.The elemental composition of the mixed salts was obtained from EDX and used for finding the experimental x-value based on the general formula of the mixed salts.The ratios of the atomic percentages (i.e., M/Fe = 2(1-x)/2x, M/P = 2(1-x)/2, Fe/P = 2x/2) were used to find x in the structural formula M 2(1-x) Fe 2x P 2 O 7 .The average x-values were incorporated in the general formula of the mixed divalent metal pyrophosphate salts to obtain the actual chemical formula of the salts.Elemental composition of the mixed salts in which M = Mn could not be obtained accurately by this technique (TEM-EDX) due to the overlapping Mn and Fe lines in the energy spectrum.

4. 1 .
Dissolution S1: pH-dependent dissolution behavior of the pure and mixed divalent metal Fe(II)-containing pyrophosphate salts The dried powders of the salts were re-dispersed in MQ water by stirring (∼ 250 rpm)