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Food fortification in low-income settings is limited due to the lack of simple quality control sensing tools. In this study, we field validated a paper-based, smartphone-assisted colorimetric assay (Nu3Px) for the determination of iron in fortified flours against the gold standard method, atomic emission spectrometry (AES). Samples from commercial brands (n = 6) were collected from supermarkets, convenience stores, and directly from companies in Mexico and characterized using both Nu3Px and AES. Nu3Px’s final error parameters were quantified (n = 45) via method validation final experiments (replication and comparison of methods experiment). Qualitative pilot testing was conducted, assessing Nu3Px’s accept/reject batch decision making (accept ≥ 40 μg Fe/g flour; reject &lt; 40 μg Fe/g flour) against Mexico’s fortification policy. A modified user-centered design process was followed to develop and evaluate an alternative sampling procedure using affordable tools. Variation of iron content in Mexican corn flours ranged from 23% to 39%. Nu3Px’s random error was 12%, and its bias was 1.79 ± 9.99 μg Fe/g flour. Nu3Px had a true mean difference from AES equal to 0 and similar variances. AES and Nu3Px made similar classifications based on Mexico’s policy. Using simple, affordable tools for sampling resulted in similar output to the traditional sampling preparation (r = 0.952, p = 0.01). The affordable sample preparation kit has similar precision to using analytical tools. The sample preparation kit coupled with the smartphone app and paper-based assay measure iron within the performance parameters required for the application to corn flour fortification programs, such as in the case of Mexico.

In-Country Method Validation of a Paper-Based, Smartphone-Assisted Iron Sensor for Corn Flour Fortification Programs