Available online at www.sciencedirect.com Journal of Chromatography B, 863 (2008) 206–214 Quantitative chiral analysis of salsolinol in different brain regions of rats genetically predisposed to alcoholism Tatiana Rojkovicova a , Yehia Mechref a,b,∗ , Jason A. Starkey a , Guangxiang Wu a,b , Richard L. Bell c , William J. McBride c , Milos V. Novotny a,b,∗ a Department of Chemistry, Indiana University, Bloomington, IN 47405, United States b METACyt Biochemical Analysis Center, Bloomington, IN 47405, United States c Department of Psychiatry, Indiana University, Indianapolis, IN 46202, United States Received 27 July 2007; accepted 2 January 2008 Available online 17 January 2008 Abstract A method to determine the catecholamine content in putamen (CPU) and midbrain (MB) regions of the brain of alcohol-preferring rats (P) is presented with a focus on the low-level detection of S,R-salsolinol, a metabolite of dopamine and a putative alcoholism marker. The developed strategy allows both quantitative profiling of related catecholamines and the enantiomeric separation and quantification of the S- and R-salsolinol isomers and their ratios. The described LC/MS strategy simplifies the current methodology that typically employs GC–MS by eliminating the need for derivatization. The data also suggest an increase in the non-enzymatic formation of salsolinol as a consequence of ethanol exposure. Published by Elsevier B.V. Keywords: Catecholamines; Salsolinol; Alcoholism; Tandem mass spectrometry; Multiple reaction monitoring (MRM); Chiral separations 1. Introduction The metabolic formation of salsolinol is thought to have a role in alcohol addiction [1–4]. It is assumed that there is a relationship between ethanol intake and the levels of sal- solinol in different brain regions [4–6]. While salsolinol can produce reinforcing effects in the mesolimbic dopamine system, its mechanisms of action are largely unknown [7–10]. Salsolinol is postulated to result from the condensation of acetaldehyde, the main metabolite of ethanol, with dopamine in the brain of mam- mals [3,11,12]. It can be apparently formed in vitro through a non-enzymatic condensation involving the Pictet-Spengler mechanism [4,13–15]. Another proposed pathway for the in vivo formation of salsolinol is through the condensation of dopamine with pyruvic acid, followed by enzymatic decarboxylation and ∗ Corresponding authors at: Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, IN 47405, United States. Tel.: +1 812 855 4532; fax: +1 812 855 8300. E-mail addresses: ymechref@indiana.edu (Y. Mechref), novotny@indiana.edu (M.V. Novotny). reduction [16]. In vivo, however, the enantiospecific occurrence of R-salsolinol in the cerebrospinal fluid [17,18], intraventricu- lar fluid [17,19], and in human brain [17,20–22] suggests that its formation is mediated by an enzyme, R-salsolinol synthase [17,21,22]. The presence of optical isomers in the many regions of the brain, where the synthesis of salsolinol is possible, may allude to the different mechanisms of formation being either enzymatic or non-enzymatic (depending on local metabolic conditions). The formation of salsolinol via the non-enzymatic mechanism, following ethanol consumption, is thus expected to result in the formation of both enantiomers, which would not be the case through the enzymatic route [4,23]. Conversely, if the formation of one enantiomer preferentially occurs over the other, this indicates that the metabolite formation is regulated through an enzyme which is selective for the geometry of substrate and product [21]. Until recently, isoquinolines had been considered to occur as a racemate in humans, being generated through the non-enzymatic condensation of monoamines [21,22]. However, more accu- rate chromatographic methods for the analysis of salsolinol enantiomers seem to indicate the predominant occurrence of 1570-0232/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jchromb.2008.01.016