Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of L-Mtx and three biosensors for the assay of D-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or L-amino acid oxidase (L-AAOD) and/or horseradish peroxidase (HRP) for the assay of L-methotherexate, and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l(-1) to pmol l(-1), magnitude order with limits of detection in the fmol l(-1) to nmol l(-1) concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).