Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke.
Alkylating agents occur in the environment and are formed endogenously. Tobacco smoke contains a variety of alkylating agents or precursors including, among others, N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrylonitrile and ethylene oxide. We developed and validated a method for the simultaneous determination of methylmercapturic acid (MMA, biomarker for methylating agents such as NDMA and NNK), 2-hydroxyethylmercapturic acid (HEMA, biomarker for ethylene oxide) and 2-cyanoethylmercapturic acid (CEMA, biomarker for acrylonitrile) in human urine using deuterated internal standards of each compound. The method involves liquid/liquid extraction of the urine sample, solid phase extraction on anion exchange cartridges, derivatization with pentafluorobenzyl bromide (PFBBr), liquid/liquid extraction of the reaction mixture and LC-MS/MS analysis with positive electrospray ionization. The method was linear in the ranges of 5.00-600, 1.00-50.0 and 1.50-900 ng/ml for MMA, HEMA and CEMA, respectively. The method was applied to two clinical studies in adult smokers of conventional cigarettes who either continued smoking conventional cigarettes, were switched to test cigarettes consisting of either an electrically heated cigarette smoking system (EHCSS) or having a highly activated carbon granule filter that were shown to have reduced exposure to specific smoke constituents, or stopped smoking. Urinary excretion of MMA was found to be unaffected by switching to the test cigarettes or stop smoking. Urinary HEMA excretion decreased by 46 to 54% after switching to test cigarettes and by approximately 74% when stopping smoking. Urinary CEMA excretion decreased by 74-77% when switching to test cigarettes and by approximately 90% when stopping smoking. This validated method for urinary alkylmercapturic acids is suitable to distinguish differences in exposure not only between smokers and nonsmokers but also between smoking of conventional and the two test cigarettes investigated in this study.
ABF Analytisch-Biologisches Forschungslabor München GmbH, Goethestrasse 20, 80336 Munich, Germany. gerhard.scherer@abf-lab.com
Gerhard
G
Scherer
Michael
M
Urban
Heinz-Werner
HW
Hagedorn
Richard
R
Serafin
Shixia
S
Feng
Sunil
S
Kapur
Raheema
R
Muhammad
Yan
Y
Jin
Mohamadi
M
Sarkar
Hans-Juergen
HJ
Roethig
D000111:Acetylcysteine / Q000652:urine*; D000477:Alkylating Agents / Q000633:toxicity*; D000837:Anion Exchange Resins; D015415:Biomarkers / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D012015:Reference Standards; D012906:Smoke / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014026:Nicotiana
D000477:Alkylating Agents; D000837:Anion Exchange Resins; D015415:Biomarkers; D012906:Smoke; D000111:Acetylcysteine