An isotope-dilution UPLC-MS/MS technique for the human biomonitoring of the internal exposure to glycidol via a valine adduct at the N-terminus of hemoglobin

- The adduct N-(diHOPr)-valine in hemoglobin is a biomarker of glycidol exposure.
- The fluorescein conjugate of N-(diHOPr)-valine is characterized by NMR spectroscopy.
- The adduct is quantified by a novel isotope-dilution UPLC-MS/MS method.
- The validated method excels in terms of sensitivity, specificity and convenience.
- The glycidol adduct levels in 12 human blood samples were determined.

Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and DHP-Val-d7-FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7 pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2-4.9 pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC-MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC-MS methods.