Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods

Abstract

Mercapturic acids (MAs) are metabolic end products formed from conjugates between glutathione and electrophilic compounds. MAs are, therefore, suitable biomarkers of exposure to toxicants, which are either electrophiles by themselves or metabolized to electrophilic intermediates. We developed and validated two LC-MS/MS methods which allow the complementary, rapid, and sensitive determination of MAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,N-dimethylformamide, ethylene, ethylene oxide, vinyl chloride, propylene oxide, styrene, toluene as well as methylating and ethylating agents. Since separate determinations of single or small groups of MAs are time-consuming and expensive, we multiplexed several individual methods into two LC-MS/MS methods covering 18 individual mercapturic acids. Method validation according to FDA guidelines showed excellent results in terms of robustness, accuracy, and sensitivity of the methods. Moreover, the use of a minimal, simple, and straightforward sample cleanup procedure further accelerated the analytical workflow, which allows a time- and cost-efficient analysis of up to 18 MAs derived from various toxicants in environmental levels. The methods were applied to urine samples derived from a strictly diet-controlled clinical study, including 25 smoking and 25 non-smoking subjects. Significant increase in the urine concentrations in smokers as compared to non-smokers (p < 0.01; Student t test) was observed for 13 individual MAs. Moreover, a dose dependence was obtained for the majority of the analytes. In conclusion, the newly developed assays represent a powerful tool for the fast and reliable quantification of 18 MAs in clinical studies. A first method application suggests several suitable biomarkers for nine relevant toxicants in tobacco smoke.

Urinary Mercapturic Acids to Assess Exposure to Benzene and Other Volatile Organic Compounds in Coke Oven Workers

“…The levels of mercapturic acids found in the present study were largely comparable with those previously reported for non-smokers of the general population; differences were noted only for HMPMA and SBMA, with control subjects of this study showing higher levels for HMPMA and lower levels for SBMA than in previous works [44][45][46].…”

Mercapturic acids: recent advances in their determination by liquid chromatography/mass spectrometry and their use in toxicant metabolism studies and in occupational and environmental exposure studies

“…Application of UHPLC specifically to bioanalysis has been reviewed (Varma et al, 2010). UHPLC analysis of structurally similar mercapturates found in the urine of smokers (Wu et al, 2012, Pluym et al, 2015) and multiple mercapturates in workers exposed to multiple volatile organic chemicals (Alwis et al, 2012) will be considered in later sections of this review.…”

“…Simultaneous determination analysis is well suited to investigate complex exposures to volatile organic compounds (VOC) in occupational settings, in exposure of urban populations to air pollutants, and in cigarette smoke (Schettgen et al, 2008b; Ding et al, 2009; Scherer et al, 2010; Eckert & Goen, 2014; Zhang et al, 2014; Chiang et al, 2015; Li et al, 2015b; Pluym et al, 2015). Sabatini developed a simultaneous determination for the mercapturates of benzene, toluene and xylene (BTX) using FDA validation guidelines (US Department of Health and Human Services Food and Drug Administration, May 2001) to measure BTX co-exposure in traffic warden exposures to automobile exhaust and urban air pollutants examining the urine of men and women, including smokers and non-smokers (Sabatini et al, 2008, Table 3).…”

Editors’ Note

“…Die CVR für LU-und PU-Test-Proben lagen bei 9,1-31,9% (Mittelwert: 18,8%) bzw. 13,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27],0% (Mittelwert: 18,5%) und wiesen auf eine begrenzte Reproduzierbarkeit in der Analyse von 3-HPMA bei beiden Probentypen hin. Die Neuberechnung der PU-Ergebnisse unter Anwendung einer eingebettenen Kalibrierung, die aus den berichteten Peak-Bereichen für die LU-Testproben abgeleitet war, führte zu einer gewissen Verbesserung der CV R -Werte (Bereich: 9,6-28,8%, Mittelwert: 16,7% The measurement of suitable biomarkers of exposure and effect is central to the evaluation of these products in humans.…”

“…The mercapturic acid is formed by reaction of acrolein with glutathione (either spontaneously or enzymatically catalyzed by a glutathione transferase) and subsequent degradation of the tripeptide, followed by N-acetylation of the cysteine residue and reduction of the aldehyde group ( Figure 1). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is the most frequently used methodology for 3-HPMA determination in urine (11)(12)(13)(14)(15)(16)(17). In a series of studies, urinary 3-HPMA levels were analyzed in smokers and non-smokers, showing, on average, about 5-fold higher levels in smokers (12,13,16,(18)(19)(20)(21)(22)(23)(24).…”