Flow injection determination of metrifonate by immobilised acetylcholinesterase inhibition

A flow injection system for determination of metrifonate by inhibition of immobilised acetylcholinesterase (AChE) is described. Acetylthiocholine is hydrolysed by AChE, and the thiocholine produced is mixed with the chromogen 5,5-dithiobis(2-nitrobenzoic acid) and the absorbance is measured at 405 nm. The decrease in absorbance due to decrease in enzymatic activity after injecting metrifonate as an inhibitor is measured. On-line re-activation of the inhibited enzyme by buffer, substrate and pyridine-2-aldoxime methiodide is also studied so that system can be reused continuously. The calibration graph is linear in the range of 1 × 10−6 to 1 × 10−5 M metrifonate (r = 0.998, n = 5), the 3σ limit of detection is 8 × 10−7 M and the relative standard deviation for 5 determinations of 6 × 10−6 M metrifonate is 2.0%. Common blood components did not interfere.