Abstract

Urinary urea is a key biomarker for tracking protein oxidation and nitrogen balance. A persistent analytical limitation is that conventional total-nitrogen assays do not deconvolute urinary urea from urinary ammonia, and direct measurement still requires capital-intensive clinical analyzers. Here, we implement and independently validate two cost-effective methods that resolve urinary urea by pairing the presence and absence of urease: Method 1 is a urease-coupled potentiometric assay using an ammonium ion-selective electrode (ISE method); Method 2 is a microplate Berthelot–salicylate colorimetric assay employing a repurposed consumer-grade aquarium ammonia test kit (API®) (API method). Each method was validated against the Roche Cobas C311 clinical analyzer (ISE for urinary ammonia and API for urinary urea) in healthy adult volunteers (n = 10 each), and API–ISE were then compared head-to-head for urinary urea. ISE vs. Cobas yielded R2 = 0.967, slope 95% CI [0.867, 1.173], and Bland–Altman bias of −1.02 mM (95% LoA [−7.40, +5.36] mM). API vs. Cobas yielded R2 = 0.973, slope 95% CI [0.790, 1.039], and bias of −1.55 mM (95% LoA [−11.56, +8.46] mM). API–ISE urinary urea comparison confirmed mutual agreement (R2 = 0.9976, slope 95% CI [0.946, 1.024]; bias of +3.47 mM; 95% LoA [−14.60, +21.53] mM), with mean recoveries near 100% (API: 100.7 ± 2.9%; ISE: 100.6 ± 5.4%; 20–350 mM). Both methods agreed with the clinical analyzer, supporting their potential for decentralized, resource-limited testing; clinical and point-of-care validations are needed.

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