Journal of Chromatography & Separation Techniques
Open Access

Quantitation of N-terminal formaldehyde adducts to hemoglobin using UPLC-MS/MS

International Summit on Current Trends in Mass Spectrometry

July 13-15, 2015 New Orleans, USA

Min Yang1, Chui Tse2, Maria Ospina2 and Hubert Vesper2

Posters-Accepted Abstracts: J Chromatogr Sep Tech

Abstract :

Introduction: Formaldehyde (FA) is classified as carcinogenic by the International Agency for Research on Cancer. It is an important industrial and environmental chemical. Its occurrence in the environment created the need for data on human exposure to this chemical to assess the extent of exposure and potential health effects associated with the exposure. FA is highly reactive and forms covalent reaction products (adducts) with DNA and proteins. Hemoglobin adducts are successfully used for the assessment of human exposure to a wide range of chemicals. These adducts reflect the exposure over the past 3 month (lifetime of the erythrocyte). A reliable, accurate analytical method for measuring hemoglobin FA adducts was developed to address the need for data about human exposure to FA. Methods: Sample preparation was performed using an automated liquid handler. Total hemoglobin content in the sample was determined using a commercial assay. We used TFE to denature proteins. After removal of TFE, hemoglobin was digested for 48 hours using trypsin at 48�?C. We stopped the digestion reaction with formic acid and analyzed the resulting N-terminal peptide containing the adduct (FA-VHLTPEEK) using UPLC-MS/MS (Thermo Accela UPLC with a Luna C18(2) 100 x 2.1 mm column and Thermo TSQ). Results were normalized to the amount of hemoglobin used for digestion. Quality control (QC) samples were prepared from erythrocytes in house for method validation. Preliminary Data: The method is linear between 21.4 nmol/g - 177 nmol/g FA-VHLTPEEK. The limit of detection is 3.4 nmol FA-VHLTPEEK/g Hb and the limit of quantitation is 11.3 nmol FA-VHLTPEEK/g Hb. The mean recovery is 96.4%±2.0%. The inter-assay and within-assay imprecision, expressed in %CV, determined with three QC samples containing different adduct levels range between 9.8% and 12.2%, and 7.1% and 13.8%, respectively. We measured FA-VHLTPEEK in 192 RBCs specimens. FA-VHLTPEEK was detected in all human samples, with FA-VHLTPEEK concentrations ranging from 41.0 �?? 92.7 nmol/g Hb. This method is suitable for determining FA exposure in the general population.