Speciation and determination of thiols in biological samples using high performance liquid chromatography–inductively coupled plasma-mass spectrometry and high performance liquid chromatography–Orbitrap MS Original Research Article

An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quaduropole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC–ICP-MS and HPLC–Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103 fmol, respectively, using HPLC–Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC–ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC–ICP-MS instrument, therefore HPLC–Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of bakerʹs yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 μmol g−1 and 0.61 to 1.32 μmol g−1, respectively.