jats:titleAbstract</jats:title>jats:pMycobacterium acquire iron by producing siderophores called mycobactins, with an extremely high affinity complexation of Fejats:sup3+</jats:sup>. The iron complex shows distinctive electrochemistry predicting 18 orders of magnitude greater affinity of the mycobactin for Fejats:sup3+</jats:sup> than Fejats:sup2+</jats:sup>. A heterogeneous standard rate constant, jats:italickjats:subs</jats:sub></jats:italic> of the order of 10jats:sup−5</jats:sup> cm sjats:sup−1</jats:sup> confirms quasi reversible electrochemistry and based on the equilibrium in the presence of excess solution Fejats:sup3+</jats:sup>/Fejats:sup2+</jats:sup> the oxidation and reduction peaks for the siderophore complex could be calibrated for ferric mycobactin J (FeMJ). FeMJ spiked urine collected from cows showed a matrix effect on the current peak height. For example, 240±15 µM FeMJ was estimated in 600 µM spiked urine. However, in the presence of excess solution Fe(acac)jats:sub3</jats:sub> the same sample yielded an estimated 580±25 µM FeMJ.</jats:p>