[Determination of acrylamide and ethylene oxide hemoglobin adducts by isotope dilution-ultra-high performance liquid chromatography-triple quadrupole mass spectrometry].

Abstract

Objective: To develop a precise quantitative method for detecting N-alkylated hemoglobin adducts of acrylamide and ethylene oxide.

Methods: Hemoglobin was liberated from anticoagulated whole blood through freeze-thaw lysis of erythrocytes. A reaction mixture containing 250 μL of hemolysate, stable isotope-labelled internal standards, and the Edman degradation reagent fluorescein isothiocyanate was prepared. The mixture underwent vortex mixing and reacted for 16 hours at 37℃ and 800 r/min, facilitating the simultaneous derivatization and cleavage of large molecular weight hemoglobin N-terminal alkylation adducts to yield smaller valine adducts with fluorescein labels. Post-derivatization, the reaction mixture was treated with 1.6 mL of acetonitrile to precipitate proteins. Subsequent centrifugation allowed for the extraction and purification of the target adducts via mixed anionic solid-phase extraction, employing both ion-exchange and reversed-phase retention mechanisms. The purified eluates were then dried under a stream of nitrogen at 55℃, reconstituted in a 1∶1(V/V) acetonitrile-water solution, and subjected to membrane filtration before analysis. Acrylamide and ethylene oxide hemoglobin adducts in human whole blood were detected by an ultra high performance liquid chromatography-electrospray triple quadrupole mass spectrometry using a Waters ACQUITY HSS T3 column(100 mm×2.1 mm, 1.8 μm), and quantified using an isotope dilution internal standard quantification method with simultaneous derivatization of standards.

Results: The detection limits for acrylamide and ethylene oxide adducts were established at 0.1 and 0.2 pmol/g Hb, respectively, with quantification lower limits down to 0.4 and 0.8 pmol/g Hb. The standard curves exhibited broad linearity with correlation coefficients(r) exceeding 0.999. Intra-and inter-day variance remained below 7.2% for both adducts. Quantitative assessment revealed deviations of +15% for acrylamide and-5.8% for ethylene oxide adducts, compared to the standard addition method.

Conclusion: The method demonstrated herein is characterized by its sensitivity, stability, precision, and suitability for biomonitoring hemoglobin adducts of acrylamide and ethylene oxide within the general population. It also proves effective for assessing occupational exposure and acute poisoning cases, as well as for the detection of a variety of N-valine alkylated hemoglobin adducts.