Simultaneous Quantitation of Multiple Biological Thiols\nUsing Reactive Ionization and Derivatization with Charged Mass Tags

The biologically important thiols\n(cysteine, homocysteine, N-acetyl\ncysteine, and glutathione) are key species in redox homeostasis, and\nthere is a clinical need to measure them rapidly, accurately, and\nsimultaneously at low levels in complex biofluids. The solution to\nthe challenge presented here is based on a new derivatizing reagent\nthat combines a thiol-selective unit to optimize the chemical transformation\nand a precharged pyridinium unit chosen to maximize sensitivity in\nmass spectrometry. Derivatization is performed simultaneously with\nionization (“reactive ionization”), and mass spectrometry\nis used to record and characterize the thiol reaction products. The\nmethod is applicable over the concentration range from 1 μM\nto 10 mM and is demonstrated for 25 blood serum, 1 plasma, and 3 types\nof tissue samples. The experiment is characterized by limited sample\npreparation (<4 min) and short analysis time (<1 min). High\nprecision and accuracy (both better than 8%) are validated using independent\nHPLC-MS analysis. Cystine–cysteine redox homeostasis can be\nmonitored by introducing an additional reduction step, and the accuracy\nand precision of these results are also validated by HPLC-MS.