A new enzymo-chemical method for simultaneous assay of methanol and formaldehyde

A new enzymo-chemical method for the simultaneous assay of methanol and formaldehyde in mixtures is described which exploits alcohol oxidase (AO) and aldehyde-selective reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme is used for methanol oxidation to formaldehyde and MBTH plays a double role: 1) at the first step of reaction, it forms a colorless azine adduct with pre-existing and enzymatically formed formaldehyde and masks it from oxidation by AO; 2) at the second step of reaction, non-enzymatic oxidation of azine product to cyanine dye occurs in the presence of ferric ions in acid medium. Pre-existing formaldehyde content is assayed by colorimetric reaction with MBTH without treating samples by AO, and methanol content is determined by a gain in a colored product due to methanol-oxidising reaction. Possibility of differential assay of methanol and formaldehyde by the proposed method has been proved for model solutions as well as for real samples of industrial waste and technical formaline. A threshold sensitivity of the assay method for both analytes is near 1 microM that responds to 30-32 ng analyte in 1 ml of reaction mixture and is 3.2-fold higher when compared to the chemical method with the use of permanganate and chromotropic acid. Linearity of the calibration curve is reliable (p < 0.0001) and standard deviation for parallel measurements for real samples does not exceed 7%. The proposed method, in contrast to the standard chemical approach, does not need the use of aggressive chemicals (concentrated sulfuric, phosphoric, chromotropic acids, permanganate), it is more simple in fulfillment and can be used for industrial wastes control and certification of formaline-contained stuffs.