## 1 Introduction
Chemosensory analysis of scent markings has explained how they are vesicles which contain information that aids in the distinctions between animals of different sexes, ages, and social status and define the time during which a scent marking can be detected in tigers and other great cat species [6; 12; 20; 21; 28]. However, what an animal inhales and how it is processed has not been completely identified or understood [4; 11; 15; 33]. 2-Acetyl-1-pyrroline (2-AP) and phenylethylamine are the only compounds that have ever been associated with the characteristic odor resembling basmati rice, of Bengal tiger MF [4; 28]. The methods for the identification of 2-AP aroma were based on simple yet robust human olfaction, which is limited in its ability to only detect odors at trace levels, e.g., \(10^{-7}\) to \(10^{-11}\) M in humans [34; 35]. This method is also limited in identifying other compounds that may be contributing to the overall odor, so the improved sensory characterization with simultaneous chemical and sensory analyses can still be explored. The age of the sample and presumed loss of compounds over time can make it impossible to detect volatile compounds, specifically 2-AP using GC-MS [4]. The inability to identify 2-AP in Bengal tiger MF and urine was believed to be due to its rapid decay, and therefore limited period of odor identification [5]. Also, 2-AP is thought to be formed by a Maillard reaction during previous solvent-based sample preparation and not necessarily by natural occurrence [4; 5; 36]. Presently, no published research reports characterization of specific odorous chemical markers within scent marks to determine precisely which compounds are responsible for eliciting behaviors in tigers. Thus, there is a need to define characteristic odors by identifying key chemical constituents responsible for odor in a more reliable approach using analytical tools. Simultaneous chemical and sensory analysis is a powerful tool that could present a novel approach to odor characterization of MF of various mammals. The use of mdGC-MS-O could potentially define all odorous compounds and provide an improved library of odorous compounds contributing to eliciting behaviors and tiger identity. Multidimensional-GC-MS-O is a modern system that is utilized for the separation of volatile organic compounds (VOCs) and semi-VOCs. It utilizes multiple columns for the separation of polar and non-polar compounds and accounts for co-elution of compounds and chemical odors [37; 38; 39; 40; 41; 42]. These are common problems associated with single column GC analyses [38; 39]. Application of mdGC-MS interfaced with olfactometry (O) has the potential to accurately measure the influence of odor in scent marking detection in species that use chemical cues as their communication method. Simultaneous chemical-sensory analyses have the potential to be more comprehensive, i.e., yielding valuable information about compound-scent links. In addition, methods based on mdGC-MS-O have very low method detection limits, e.g., 0.020 \(\mathrm{ng\cdot L}^{-1}\) to 0.022 \(\mathrm{ng\cdot L}^{-1}\)[40]. MdGC-MS-O has the capability, through its heart-cut mode, to improve the isolation and separation of complex mixtures, enhance odor characterization, and identify compounds [37; 38]. Simultaneous chemical-sensory analysis has enabled the following findings: compounds responsible for the characteristic odor of live _H. axyridis_[37]; compounds contributing to the characteristic odor of livestock and poultry manure, rumen of beef cattle; linking specific odor with a volatile compound; the role of particulate matter as a carrier of odor; characterization of Kairomones and characteristic odorants released by insects; and quantification of nutraceuticalils in wine [37; 38; 39; 40]. Application of mdGC-MS-O has the potential to measure the influence of odor in scent mark detection in species that use chemical cues as their communication method. Solid phase microextraction (SPME) is particularly suited for characterization of volatiles from biological sources.