ORIGINAL PAPER Absolute quantification of free glutathione and cysteine in aquatic insects using isotope dilution and selected reaction monitoring Christopher M. Shuford & Monica D. Poteat & David B. Buchwalter & David C. Muddiman Received: 20 July 2011 /Revised: 8 September 2011 /Accepted: 12 September 2011 /Published online: 29 September 2011 # Springer-Verlag 2011 Abstract A simple and robust isotope dilution mass spectrometry-based assay was developed for the determina- tion of free cysteine and glutathione (GSH) in aquatic insects. Several experimental parameters were evaluated and opti- mized to provide specific and sensitive detection of both compounds by in situ derivatization with N-ethylmaleimide followed by acid alkylation quenching and reverse- phased liquid chromatography coupled with selected reaction monitoring. For both targets, the assay was evaluated over a concentration range of 0.313 to 320 μM and was demonstrated to have a quantitative dynamic range spanning nearly three orders of magnitude, with lower limits of quantification being 0.330 μM for GSH and 0.370 μM for cysteine. Additionally, measurements were observed to be highly reproducible over the course of several days. When applied to the analysis of four different species of insects, large biological variation between and within species was observed. Different feeding regimens were also tested within two species of insects but statistical comparisons revealed no significant difference in the levels of either compound. Keywords Glutathione . Cysteine . Selected reaction monitoring . Absolute quantification . Isotope dilution Introduction Insects are a vital and abundant part of nearly all ecosystems and, as such, the numerous species found therein have the potential to serve as indicators for environmental stress [1]. A major challenge for all organisms that rely upon oxygen for metabolic purposes is the amelioration of reactive oxygen species (ROS) [2]. In addition to ROS generation associated with normal aerobic metabolism, several environmental agents are capable of generating ROS, including ultraviolet radiation [3], trace metals [4, 5], crude oil [6], and organo- phosphates [7, 8]. In addition, recent work has shown that diatoms (a common food source for aquatic insects) can produce polyunsaturated aldehydes when they senesce [9– 11], which are currently receiving attention due to their roles in oxidative stress, carcinogenesis, and aging. Thus, antiox- idant systems are important in determining performance of species in changing environments. Free glutathione (GSH) and free cysteine are thought to be particularly important antioxidants in insects [12] given their vital roles in maintaining appropriate redox conditions in the cell and controlling free concentrations of potentially toxic metal ions. Consequently, these compounds may be critical in determining pollution sensitivity differences among species [13, 14]. This is particularly true in metal contaminated environments because heavy metal exposure results in oxidative stress through generation of reactive oxygen species [15, 16] and thiol scavenging [17]. Though studies of free GSH and cysteine in aquatic insects are limited [18], there appears to be potentially significant differences in the relative roles of antioxidant enzymes and non-enzymatic antioxidants in this diverse group of insects. It is clear that additional Published in the 10th Anniversary Issue. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5416-2) contains supplementary material, which is available to authorized users. C. M. Shuford : D. C. Muddiman (*) W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA e-mail: david_muddiman@ncsu.edu M. D. Poteat : D. B. Buchwalter Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695, USA Anal Bioanal Chem (2012) 402:357–366 DOI 10.1007/s00216-011-5416-2