A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine

Anal Biochem. 2019 Sep 1:580:1-13. doi: 10.1016/j.ab.2019.05.015. Epub 2019 May 31.


The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2 μM. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.

Keywords: Ascorbate; Colorectal cancer; Colorimetric assay; Dipstick; Urine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ascorbic Acid / urine*
  • Colorimetry / methods*
  • Healthy Volunteers
  • Humans
  • Methylphenazonium Methosulfate / chemistry
  • Tetrazolium Salts / chemistry


  • Tetrazolium Salts
  • Methylphenazonium Methosulfate
  • Ascorbic Acid

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