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.
An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quadrupole 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 baker's 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.
Middle East Technical University, Department of Chemistry, 06531 Ankara, Turkey. bsezgin23@yahoo.com
Sezgin
S
Bakirdere
Emilia
E
Bramanti
Alessandro
A
D'ulivo
O Yavuz
OY
Ataman
Zoltan
Z
Mester
D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000032:analysis; D057230:Limit of Detection; D013058:Mass Spectrometry; D013438:Sulfhydryl Compounds / Q000032:analysis*; D015003:Yeasts / Q000737:chemistry
D013438:Sulfhydryl Compounds; D004229:Dithiothreitol