# A Colorimetric Multimetabolite Assay for Quantitative Measurement of Keto Acids in Urine for At‐Home Monitoring of Metabolic Disorders

## TL;DR

A new at-home test was developed to quickly and accurately measure harmful keto acids in urine for monitoring metabolic disorders like PKU and MSUD.

## Contribution

A low-cost, quantitative, colorimetric assay for at-home detection of multiple keto acids in urine is developed.

## Key findings

- The DNPH-based assay quantifies keto acids in urine within 10 minutes with high accuracy.
- The assay showed strong correlation with NMR-based metabolomics for PKU and MSUD samples.
- The prototype at-home kit uses caprolactam-immobilized NaOH for stability and ease of use.

## Abstract

Inborn errors of metabolism such as phenylketonuria (PKU) and maple syrup urine disease (MSUD) can cause severe developmental problems. Both conditions can lead to harmful levels of keto acids in biofluids—phenylpyruvic acid (PPA) in PKU and branched‐chain α‐keto acids in MSUD. Monitoring urinary keto acids helps track dietary adherence and reduces the risk of metabolic crisis. However, current at‐home tests are qualitative and difficult to interpret, while existing metabolomic assays require expensive equipment and must be conducted in a lab. This study aimed to develop a simple, quantitative, rapid, at‐home assay for detecting multiple urinary keto acids associated with PKU and MSUD. A modified two‐step 2,4‐dinitrophenylhydrazine (DNPH)–based multimetabolite assay was developed, where sodium hydroxide (NaOH) converts the yellow hydrazone precipitate to a stable amber solution, enabling quantification of multiple keto acids (700–7200 μM) within 10 min. The assay was validated using spiked urine samples and adapted into a prototype at‐home kit using caprolactam‐immobilized NaOH. Nuclear magnetic resonance (NMR)–based metabolomics was used as a reference method to authenticate readings from a PKU patient. Correlation studies demonstrated strong linearity for MSUD (R
2 = 0.91–0.96)‐ and PKU (R
2 = 0.95–0.99)‐spiked samples. Quantification of keto acids in authentic PKU urine samples showed excellent agreement with the results of quantitative NMR‐based metabolomics assays (R
2 = 0.99). Low‐cost, at‐home colorimetric tests for urinary keto acids could enable screening, detection, and monitoring of PKU and MSUD in the 90% of the world without access to advanced metabolic clinics.