Green and facile short path smartphone-based assay for L-proline in dietary supplements using a mild chromogenic reaction combined with simple equipment

A novel method for determining L-proline content in dietary supplements was developed, relying on a chromogenic reaction between L-proline and isatin. Because of the resulting intense blue color, solutions were transferred to short pathlength cuvettes (0.2 cm) in order to lower the color intensity, eliminating the need for dilution, before being subjected to photography using a smartphone and subsequent Red-Green-Blue (RGB) analysis. The intensity of the red color (R), expressed as log (Rblank/R), was used as an analytical signal, providing good linearity (r2 = 0.9994) over a concentration range of 0.8 − 8.0 mM. The assay exhibited satisfactory accuracy, precision, and sensitivity (with a limit of detection of 0.24 mM and a limit of quantification of 0.72 mM), without interference from excipients and other 19 essential amino acids. Additionally, the results were consistent with those obtained through the currently used methods, i.e., nonaqueous titration and spectrophotometry using ninhydrin as a chromogenic reagent. Therefore, a green and simple by design analytical method development, marked by the utilization of a mild reaction in a micro-scale (300 μL), eco-friendlier reagents, reduced solvent usage, and simplified procedures using basic and inexpensive equipment while also minimizing chemical exposure, was effective in overcoming limitations in existing assays for L-proline. Moreover, the use of short pathlength cuvettes for RGB acquisition in smartphone-based digital image colorimetry, introduced for the first time in this work, demonstrates potential for analyzing other substances. [Display omitted] •First report on an innovative short-path smartphone assay using simple equipment.•It was successfully applied for quantification of proline in dietary supplements.•A mild and eco-friendly isatin reaction was utilized with a small-scale setup.•0.2 cm cuvettes reduced image color intensity, eliminating the need for dilution.•Solvent usage and chemical exposure were reduced during signal measurement.