Alkylating substances – Determination of haemoglobin adducts as N-methylvaline, N-ethylvaline, N-propylvaline, N-butylvaline, N-(2-hydroxypropyl)valine, N-(2-hydroxyethyl)valine, N-(2-cyanoethyl)valine, N-benzylvaline, and N-(2-carbamoylethyl)valine in the erythrocyte fraction of whole blood by GC-MS/MS

Biomonitoring Method – Translation of the German version from 2023

Timo Christ¹ (Method development)
Dietmar Ellrich¹ (Method development)
Gabriele Leng¹ (Method development)
Christoph Schmidtkunz¹ (Method development)
Wolfgang Gries¹ (Method development)
Fabian Pilz² (External verification)
Gerhard Scherer² (External verification)
  Thomas Göen³ (Head of the working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft)
  Andrea Hartwig⁴ (Chair of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft)
  MAK Commission<sup>5

1</sup> Currenta GmbH & Co. OHG, CUR-SIT-SER-GS-BLM – Institute for Biomonitoring, Chempark Building Q 18, 51368 Leverkusen, Germany
² ABF – Analytisch-biologisches Forschungslabor GmbH, Semmelweisstraße 5, 82152 Planegg, Germany
³ Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute and Outpatient Clinic of Occupational, Social, and Environmental Medicine, Henkestraße 9–11, 91054 Erlangen, Germany
⁴ Institute of Applied Biosciences, Department of Food Chemistry and Toxicology, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, Building 50.41, 76131 Karlsruhe, Germany
⁵ Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft, Kennedyallee 40, 53175 Bonn, Germany

Abstract

The working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed and verified the presented biomonitoring method. Alkylating substances play an important role in many industrial processes and include n-alkyl and benzyl halides, α,β-unsaturated carbonyl compounds, epoxides, alkylnitrosamines, chloroalkyl ethers, dialkyl sulfates as well as alkyl alkane sulfonates. As alkylating substances are mainly used in industry, most exposure occurs in the workplace. The general population is primarily exposed through cigarette smoke, which contains a variety of alkylating substances. In addition, certain foods, especially highly heated, starchy foods, are a source of exposure to acrylamide, another alkylating substance. The aim of this work was to develop a selective method to simultaneously determine the adducts of different alkylating substances with the N-terminal valine of haemoglobin, namely N-methylvaline, N-ethylvaline, N-propylvaline, N-butylvaline, N-(2-hydroxypropyl)valine, N-(2-hydroxyethyl)valine, N-(2-cyanoethyl)valine, N-benzylvaline, and N-(2-carbamoylethyl)valine. The method has been comprehensively validated, and the reliability data have been confirmed by replication and verification of the procedure in a second, independent laboratory. For the determination of the adducts at the N-terminal valine of haemoglobin, erythrocytes are isolated from whole blood and subjected to lysis. Globin is precipitated from the haemoglobin solution and, after the addition of N-(2-ethoxyethyl)valine-alanine-anilide as internal standard, the alkylated N-terminal valines are derivatised and cleaved off by a modified Edman degradation using pentafluorophenyl isothiocyanate. The corresponding pentafluorophenyl thiohydantoin derivates are extracted with tert-butyl methyl ether, washed, and analysed by GC-MS/MS. The good precision and accuracy data show that the method is reliable and accurate. The method is both selective and sensitive, and the limits of detection between 10 and 70 pmol/g globin are sufficient to determine occupational exposure and, for some analytes, exposure of the general population to the respective alkylating substances.

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