Determination of ethylene oxide-hemoglobin adduct by silylation and gas chromatography-electron impact-mass spectrometry

A gas chromatography-electron impact ionization-mass spectrometric (GC-EI-MS) assay was developed for the determination of ethylene oxide-hemoglobin adduct (N-2-hydroxyethylvaline, HEVal). HEVal and deuterated HEVal (d(4)-HEVal) were synthesized for identification and quality control. Globin samples were separated from red blood cells (RBCs) by acidic isopropanol and extracted with ethyl acetate. HEVal adduct in globin was transformed to HEVal-pentafluorophenylthiohydantoin derivative by modified Edman-degradation method, which was extracted from globin with diethylether. d(4)-HEVal was used as an internal reference standard. The dried extract was derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)-NH(4)I (1000:4, w/w) containing 0.4 mg of dithioerthritol. The TBDMS derivative of HEVal had very good chromatographic property and offered sensitive response of GC-EI-MS. The recovery of HEVal was about 81.6% and the coefficient of variation was 5.0% at the concentration of 311 pmol/g. Low limit of detection (LOD) of the assay was 1.8 pmol/g in 0.1g hemoglobin. The experiments have demonstrated to detect background level of HEVal adduct in human blood. HEVal adduct in globin was detected between 12 and 6573 pmol/g.