Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methodsby Nikola Pluym, Gerhard Gilch, Gerhard Scherer, Max Scherer

Anal Bioanal Chem


Analytical Chemistry / Biochemistry



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

Nikola Pluym1 & Gerhard Gilch1 & Gerhard Scherer1 & Max Scherer1

Received: 26 February 2015 /Revised: 13 April 2015 /Accepted: 16 April 2015 # Springer-Verlag Berlin Heidelberg 2015

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 ofMAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3butadiene, 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 individualMAs.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.

Keywords Mercapturic acid . Biomarkers . Biomonitoring .

LC-MS/MS . Urinary metabolites . Restricted accessmaterial (RAM)


Volatile organic compounds (VOCs) are numerous, varied, and ubiquitous, including both human-made and naturally occurring chemical compounds. Amongst these are several highly toxic and in part carcinogenic chemicals, including acrylamide, benzene, 1,3-butadiene, and crotonaldehyde. The absorption into the human body mainly occurs via the lung or skin. Tobacco smoke has been described as the major nonoccupational source of exposure to many VOCs [1]. Such

VOCs can be determined either by directly measuring mainstream and environmental tobacco smoke (ETS) which, however, does not reflect the actual absorbed dose or by exposure assessment of appropriate biomarkers in different biofluids.

VOCs are mainly determined in blood either directly as parent compounds [2–4] and DNA/protein adducts [5, 6], or suitable metabolites are determined in urine [7–10].

Most VOCs exhibit their toxic potential due to an initial bioactivation step resulting in reactive metabolites which readily form adducts with, inter alia, DNA, hemoglobin, or other proteins. On the other hand, these electrophilic intermediates usually conjugate with glutathione (GSH) in a wellcharacterized detoxification pathway, also known as mercapturic acid (MA) pathway [11]. Mercapturic acids (MAs), N-acetyl-L-cysteine-S-conjugates, represent the * Max Scherer 1 ABF, Analytisch-Biologisches Forschungslabor München,

Goethestrasse 20, 80336 Munich, Germany

Anal Bioanal Chem

DOI 10.1007/s00216-015-8719-x metabolic end products from GSH conjugation and may serve as biomarkers of exposure (BoE) for various VOCs.

MAs are frequently analyzed in exposure assessment of various toxicants for several reasons. Firstly, as MAs are predominantly excreted in urine [12], samples can be obtained non-invasively in sufficient amounts. Furthermore, the short elimination half-lives of MAs allow the determination of recent exposures. Besides, the short half-lives allow repeated sampling and the correlation between the internal dose and a specific environmental impact [13].

Several LC-MS/MS methods have been reported in the past for the analysis of MAs resulting from exposure to toxicants from various sources [8, 11, 14, 15], including tobacco smoke [1, 7]. However, the vast majority of methods comprise only single analytes and small sets of MAs [7–10, 12, 16–26].

Recently, Alwis et al. published a method covering 16 MAs relevant for assessing the exposure to a total of 28 VOCs [27].

However, in order to quantify several MAs resulting from environmental (background) exposure, there is a need for sensitive methods.

Our goal was to develop a robust and quantitative methodology which allows for a complementary, rapid, and sensitive determination of multiple MAs resulting from different major toxicants, for example those identified as harmful or potentially harmful (HPHC) in tobacco smoke by the FDA [28]. The toxicants covered by our methodology are acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,

N-dimethylformamide, ethylene oxide, propylene oxide, styrene, toluene, vinyl chloride, and (m)ethylating agents. It emerged that two multiplex-LC-MS/MS methods for the simultaneous determination of 18 MAs in human urine were required. Both methods were applied to urine samples from a confined, diet-controlled clinical study to evaluate the suitability of the MAs as BoE to environmental and tobacco smoke toxicants.

Materials and methods

Chemicals, standards, stock solutions, and quality controls

N-Acetyl-S-(3-hydroxypropyl)cysteine (3-HPMA), N-acetylS-(3-hydroxypropyl)cysteine-15N13C3 (3-HPMA15N13C3), and N-acetyl-S-(2-hydroxypropyl)cysteine (2-HPMA) were purchased from AptoChem (Montreal, Canada). N-AcetylS-(2-hydroxypropyl)cysteine-D3 dicyclohexylammonium salt (D3-2-HPMA), (R,S)-N-acetyl-S-[1-(hydroxymethyl)-2propenyl-L-cysteine+(R,S)-N-acetyl-S-[2-(hydroxymethyl)3-propenyl-L-cysteine (MHBMA), (R,S)-N-acetyl-S-[1-(hydroxymethyl)-2-propenyl-L-cysteine-D6+(R,S)-N-acetyl-S-[2-(hydroxymethyl)-3-propenyl-L-cysteine-D6 (D6MHBMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine-D7 (D7-DHBMA), N-acetyl-S-(N-methylcarbamoyl)-L-cysteine (AMCC), N-acetyl-D3-S-(N-methylcarbamoyl)-L-cysteine (D3-AMCC), N-acetyl-S-(2-hydroxyethyl)-L-cysteine sodium salt (HEMA), N-acetyl-S-(2-hydroxyethyl-D4)-L-cysteine (D4-HEMA), N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA),