## 2 Results and Discussion  
### Selection of Marking Fluid Extraction Parameters  
Extraction efficiencies using five fiber types (50/30 \(\upmu\)m divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 85 \(\upmu\)m Carboxen/PDMS (CAR/PDMS), 75 \(\upmu\)m CAR/PDMS, 100 \(\upmu\)m PDMS, and 65 \(\upmu\)m PDMS/DVB, two temperatures (25 \({}^{\circ}\)C and 37 \({}^{\circ}\)C), two sample quantities (0.25 mL and 50 mL), and two extraction times (1 h and 24 h) were compared (Figure 1 and Figure S1-S3). Extraction parameters for the MF were based on the number of total and characteristic compounds detected, and peak area count comparisons of key compounds (Figures S1-S4). Based on these results, the 75 \(\upmu\)m CAR/PDMS fiber with a 0.25 mL sample quantity, 24 h extraction at 37 \({}^{\circ}\)C was selected as the most efficient to characterize the VOCs within tiger MF. The 75 \(\upmu\)m CAR/PDMS fiber was the only fiber coating that extracted enough mass for detecting the matching signature molecular ions and characteristic odors of all the \"nutty\" and \"urinous\" compounds emitted from tiger MF. Although the 65 \(\upmu\)m PDMS/DVB SPME fiber was efficient at extracting enough mass for the detection and chromatographic identification of 2-AP, it was inefficient at the extraction of mass necessary for the detection of all 14 confirmed odorous compounds with a total of 32 odorous events detected with the 75 \(\upmu\)m CAR/PDMS SPME fiber (Table S1, Figure S4, Table 2). Compared with the 75 \(\upmu\)m CAR/PDMS SPME fiber, the 65 \(\upmu\)m PDMS/DVB SPME fiber was only able to extract about half the number of compounds resulting in odorous events (18). In addition to 2-AP, the use of the 75 \(\upmu\)m CAR/PDMS SPME fiber resulted in the identification of two (confirmed with chemical standards) compounds (urea, furfural), and four compounds tentatively identified as ((\(R\))-3-methylbutanamine, 3-hydroxybutanal, propanedionic acid, and (\(R\))-3-methylcyclopentanone)) responsible for characteristic odor in tiger MF (Figure S4, Table 2).  
Figure 1: Effects of fiber coating type on SPME adsorption of 2-acetyl-1-pyrroline, the characteristic odorant compound released from marking fluid of _P. tigris allaia_ with 85 \(\upmu\)m CAR/PDMS, 50/30 \(\upmu\)m DVB/CAR/PDMS, 100 \(\upmu\)m PDMS, 65 \(\upmu\)m PDMS/DVB, and 75 \(\upmu\)m CAR/PDMS SPME fibers. Marking fluid (0.25 mL) and a stir bar were inserted into a 2 mL glass vial with a PTFE coated septa for a period of 30 min for equilibration. Samples (\(n=3\)) were extracted at a temperature of 37 \({}^{\circ}\)C for 1 h. MS scan mode was total ion scan. Two min of the 40 min total scan is shown. Identification of 2-AP was accomplished with two fibers, the 75 \(\upmu\)m CAR/PDMS and 65 \(\upmu\)m PDMS/DVB SPME fibers. The 75 \(\upmu\)m CAR/PDMS fiber had a peak area of 3.5 \(\times\) 10\({}^{5}\) counts and the 65 \(\upmu\)m PDMS/DVB SPME fiber had a peak area of 8.9 \(\times\) 10\({}^{5}\) counts.  
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\begin{table}
\begin{tabular}{l l l l l l l l l l l} \hline \hline No. & \multicolumn{2}{c}{**Compound**} & \multicolumn{2}{c}{**Classification**} & \multicolumn{2}{c}{**KT fusion**} & \multicolumn{2}{c}{**CAS**} & \multicolumn{2}{c}{**Top 33 lines and Relative**} & \multicolumn{2}{c}{**\& **  
2-AP was used as a reference compound to measure changes in peak area counts between different sample volumes and SPME extraction times. There were no statistical differences in concentrations of 2-AP between the 0.25 mL and the 0.50 mL sample size and extraction times using the 75 \(\upmu\)m CAR/PDMS (Figure S3). Due to the limited number of samples available, the 0.25 mL quantity was selected as the sample size for this study. The 24 h extraction time was selected because the number of detectable odorous compounds increased two-fold with the 23 h increase in extraction time (Figure S4).