*3.4. Organic Compounds Detection by Gas Chromatography Associated to Mass Spectrometry*

The Mars Organic Molecule Analyzer instrument onboard the ExoMars rover will employ thermal extraction protocols [41]. The characterization of thermal extracts will be carried out by GC-MS detection [42]. This analysis was performed only on a selection of samples, control and samples exposed to simulated space and Mars-like conditions (Top condition).

The GC-MS analysis revealed the presence of several compounds using mass-tocharge ratios (*m/z*): azelaic acid (1,7-epta-didecanoic acid), myristic acid (1-tetradecanoic acid), pyruvic acid (2-Oxopropanoic acid), glucose, fructose, glucitol, glycerol (1,2,3-trihydroxy propane) and ethylene glycol (1,2-dihydroxy ethane). The identified compounds are shown in Table 2; the *m/z* fragmentation spectra of the identified compounds are reported in Table 3. Particularly, the results of pyrolysis analyses showed the presence of low-molecular-weight saturated carboxylic acids. Dicarboxylic acids, such as azelaic acid, were detected in Control and Top samples in OS, P-MRS, and S-MRS analogues (Table 2). In particular, azelaic acid was found in all examined samples, allowing to consider this molecule as a distinctive biomarker for the presence of *C. antarcticus*. Myristic acid was detected only in S-MRS samples exposed to Top conditions and in the relative Control (Table 2). Surprisingly, the amount of azelaic and myristic acids increased after the treatments. A low amount of pyruvic acid was found only in S-MRS control samples; no amount was shown in exposed samples, probably due to the effect of radiation. These results are similar to those reported for ethylene glycol, detected only in the case of S-MRS and P-MRS Top samples.


**Table 2.** Abundance of main compounds detected with Gas Chromatography–Mass Spectrometry analysis after groundbased simulation.

[a] The analyses were performed after silylation with *N*,*N*-bis-trimethylsilyl trifluoroacetamide in pyridine (620 μL) at 60 ◦C for 4 h in the presence of betulinic acid [3b-hydroxy-20(29)-lupaene-oic acid] as the internal standard (0.2 mg). All quantities are expressed in μg. GC-MS analysis of OS, P-MRS, and S-MRS samples. Original substrate (OS), Phyllosilicatic Mars Regolith Simulant (P-MRS) and the Sulfatic Mars Regolith Simulant Phyllosilicate (S-MRS). Top samples exposed on the Top of the payload in comparison with Control samples, not exposed to treatments. nd: not determined.

**Table 3.** Mass-to-charge ratio (*m/z*) value and the abundance of peaks of identified compounds.


[a] Mass spectroscopy was performed by using a GC-MS Varian 410 GC-320 MS. The peak abundance is reported in parenthesis [b] Product analyzed as the monosilyl derivative; [c] Product analyzed as the bis-silyl derivative; [d] Product analyzed as the tris-silyl derivative; [e] Product analysed as the penta-silyl derivative; [f] Product analysed as the hexa-silyl derivative; [g] Ions characteristic for EI/MS sugar degradation: *m/z* 217 [(CH3)3SiOCH=CH-CH=OSi(CH3)3] +, *m/z* 204 [(CH3)3SiOCH=CHOSi(CH3)3] <sup>+</sup>•, *m/z* 191 [(CH3)3SiOCH=OSi(CH3)3] +.
