Fluorescence and Mass Spectrometric Detection Schemes for Simultaneous Enzymatic Conversions: Method Development and Comparison

Fluorescence and mass spectrometric detection schemes are developed and compared for the simultaneous activity determination of two enzymes in solution. As model system, the following reactions are used: The alkaline phosphatase catalyzed reaction with 5-fluorosalicyl phosphate yields the fluorescent 5-fluorosalicylic acid, whereas microperoxidase 11 reacts with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole and H2O2 to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole. As the emission spectra of the fluorescent products as well as the molecular masses of substrates and products do not interfere with each other, is it possible to determine both reactions in parallel with both detection schemes. The measurements resulted in the same limits of detection, limits of quantification and linear ranges of the single/simultaneous enzyme determination for fluorescence and MS detection. While the relative standard deviations were significantly lower in case of fluorescence detection (1.4–3.2%) than in mass spectrometry (5.7–10.1%), the latter proved to be the more versatile approach for multianalyte determination.