Comparison of tetramethylammonium hydroxide (TMAH), trimethylsulfonium hydroxide (TMSH), and trimethylphenylammonium hydroxide (TMPAH) thermochemolysis for in situ space analysis of organic molecules in planetary environments

David Boulesteix, Arnaud Buch, A.J. Williams, Y. He, Caroline Freissinet, M.G. Trainer, J.C. Stern, Cyril Szopa

► To cite this version:

David Boulesteix, Arnaud Buch, A.J. Williams, Y. He, Caroline Freissinet, et al. Comparison of tetramethylammonium hydroxide (TMAH), trimethylsulfonium hydroxide (TMSH), and trimethylphenylammonium hydroxide (TMPAH) thermochemolysis for in situ space analysis of organic molecules in planetary environments. Talanta, 2023, 257 (May), pp.124283. 10.1016/j.talanta.2023.124283. insu-03971903

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Comparison of Tetramethylammonium Hydroxide (TMAH), Trimethylsulfonium

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Hydroxide (TMSH), and Trimethylphenylammonium Hydroxide (TMPAH)

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Thermochemolysis for *in situ* Space Analysis of Organic Molecules in Planetary

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Environments

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D. Boulesteix¹,*, A. Buch¹,*, A. J. Williams², Y. He¹, C. Freissinet³, M. G. Trainer⁴, J. C. Stern⁴, C.

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Szopa³

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¹ Laboratoire Génie des Procédés et Matériaux, CentraleSupélec, University Paris-Saclay, 8-10 rue Joliot-Curie,

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91190, Gif-sur-Yvette, France

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² Department of Geological Sciences, University of Florida, Gainesville, FL 32611, USA

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³ LATMOS/IPSL, UVSQ University Paris-Saclay, Sorbonne University, CNRS, 11 Bd d'Alembert, 78280,

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Guyancourt, France

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⁴ Space Science Exploration Division (Code 690), NASA, Goddard Space Flight Center, Greenbelt, MD, 20771,

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USA

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* Corresponding co-authors. E-mail address: david.boulesteix@centralesupelec.fr (David Boulesteix) and

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arnaud.buch@centralesupelec.fr (Arnaud Buch).

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Abstract

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One of the main objectives of present and future space exploration missions dedicated to astrobiology is the detection of organic molecules of interest for life (e.g. amino and fatty acids). With this aim, a sample preparation and a gas chromatograph (connected to a mass spectrometer) are generally used. To date, tetramethylammonium hydroxide (TMAH) has been the first and only thermochemolysis reagent to be used for *in situ* sample preparation and chemical analysis of planetary environments. Although TMAH is widely used in terrestrial laboratories, numerous applications also leverage other thermochemolysis reagents that may be more relevant than TMAH to meet both scientific and technical objectives of space instrumentation. The present study compares the performance of tetramethylammonium hydroxide (TMAH), trimethylsulfonium hydroxide (TMSH), and trimethylphenylammonium hydroxide (TMPAH) reagents on molecules of interest to astrobiology. The study focuses on the analyses of 13 carboxylic acids ($C_7-C_{30}$), 17 proteinic amino acids, and the 5 nucleobases. Here we report the derivatization yield without stirring or adding solvents, the detection sensitivity with mass spectrometry, and the nature of the degradation products from the reagents produced during pyrolysis. We conclude that TMSH and TMAH are the best reagents for analyzing carboxylic acids and nucleobases. Amino acids are not relevant targets for a thermochemolysis over 300 °C as they are degraded and showed high limits of detection. As TMAH, and probably TMSH, meet the space instrumentation requirements, this study informs sample treatment approaches prior to GC-MS analysis in *in situ* space studies. The thermochemolysis reaction using TMAH or TMSH is also recommended for space return missions to extract organics from a macromolecular matrix, derivatize polar or refractory organic targets, and volatilize with the fewest organic degradations.

Keywords:
Thermochemolysis, GC-MS, space application, TMAH, TMSH, TMPAH.

# I. Introduction

Thermally assisted hydrolysis and methylation (THM), or thermochemolysis, is a sample preparation combining both heating processes and derivatization of acidic functional groups (mainly substituting their labile H with a methyl). This process is generally used to enhance and accelerate the extraction, derivatization (for a better GC separation and peak resolution), and sublimation (or volatilization) of organics in a few seconds from solid (or liquid) samples prior to their analysis with gas chromatography combined with mass spectrometry (1,2).

Thermochemolysis derivatizing reagents are generally low molecular weight molecules with alkaline salt forms preventing high reactivity with water and facilitating the chemical interaction and reaction with the targeted labile functional groups (e.g. -COOH, -NH₂/NH) (3). This analytical technique can be used either *off line* (meaning thermochemolysis is performed with an independent step prior to GC-MS analyses) or *on line* (thermochemolysis products are injected in the GC-MS instrument right after their production, usually achieved in a pyrolyzer coupled to the GC-MS instrument) (4,5). The *on line* method can easily be used in spaceflight instrumentation because of its simplicity and high reaction speed. Studies conducted with tetramethylammonium hydroxide (TMAH) are self-sufficient for *on line* Pyr-GC-MS analysis, obtaining faster and comparably similar results to *off line* and conventional methods of derivatization. For some studies, *on line* thermochemolysis even gave more sensitive results than classical approaches on the complex copolymer extracted from *C. necator* or polyhydroxybutyryates (PHB) from the environmental biofilm composed of *Bacillus species* (6,7). Similarly, studies on TMSH did not demonstrate any benefit of the *off line* method compared to the *on line* (8).

With this purpose, thermochemolysis using tetramethylammonium hydroxide (TMAH) has been tested in laboratories on both analytical standards (9–14) and on environmental samples that are March environmental analogs (e.g. Greenland Glacier, Yilgarn Craton in Western Australia, and Icelandic hot springs) (12,15–17). Given its prevalence in the literature and wide analytical application, TMAH is used in GC-MS spaceflight applications and is implemented on the *March Science Laboratory (MSL, or Curiosity rover)* mission and its Sample Analysis at March (SAM) instrument (10,12,18). TMAH is also slated for flight on future missions, such as *ExoMars* with the March Organic Molecule Analyzer (MOMA) instrument (10,19–21), *Dragonfly* with its Dragonfly mass spectrometer (DraMS) instrument (22,23), and *Europa Lander* with the Europan Molecular Indicators of Life Investigation (EMILI) (24,25).

However, TMAH presents some drawbacks (26), such as isomerization and degradation reactions at high temperatures (27). Moreover, the thermal degradation of TMAH in excess produces numerous degradation compounds that could interfere and coelute with the targeted volatiles, such as fatty acids methyl esters (FAMEs), methylated nucleobases, or methylated amino acids (28). Some other thermochemolysis reagents, which have not been studied for the MSL mission in the 2000s (e.g. trimethylsulfonium hydroxide (TMSH), or trimethylphenylammonium hydroxide (TMPAH)) (see Supplementary material 1 (SM1)), may not have these drawbacks and might give a better derivatization yield or a higher sensitivity under space conditions during *in situ* analysis. These alternative reagents would thus allow better detection of organics and might give a lower limit of detection (LOD) and quantification (LOQ), which would make the detection of organics at fmol levels possible (compared to TMAH, which only enables detection down to pmol of organics) (12).
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In the laboratory, several reactants have been proposed to perform thermochemolysis of organic materials, but there is no study comparing their respective efficiency with respect to the compounds of interest in the search for traces of life in (extraterrestrial) environments, or extraterrestrial samples returned to Earth. Among these reactants, we selected trimethylsulfonium hydroxide (TMSH) (8,29–31) and trimethylphenylammonium hydroxide (TMPAH) (26,32–36) for their easy and quantitative formation of methyl esters/ethers/amines. For these reasons, in the present study, we compared the performance of two strong organic bases able to perform methylation on labile H, TMSH (8,29) and TMPAH (26,30,34,38), with TMAH.

Rarely do studies focus on thermochemolysis for space applications, or on the comparison of these three reagents (TMAH, TMSH, and TMPAH presented in Supplementary material 1 (SM1)). The present study aims to compare the performances of these three reagents on a common set of organic molecules of interest for astrobiology. These tests will be performed under sample preparation and operating conditions compatible with the current space instrumental constraints. The final objective is to assess which of the thermochemolysis reagents is the most appropriate for *in situ* chemical analysis to be performed by future space instruments on each environmental sample based on the GC-MS technique in terms of thermal stress, GC resolution, and GC-MS sensitivity. The study and optimization reported in this work may also contribute to preparing the analyses of return samples from extraterrestrial surfaces where the quantity of solvent, the LOD/LOQ, and the number of analyses are less critical parameters compared to *in situ* space analysis.

The first objective is the determination of the optimal temperature of thermochemolysis for each derivatization reagent and each molecular target (amino acids, fatty acids, and nucleobases). The second objective is the comparison of the analytical performances of the three studied reagents using their respective optimal reaction conditions. The third objective is a primary study on the reagent resistance against thermal stress (during the thermochemolysis process). We listed the reagent by-products at the different optimal temperatures of thermochemolysis, the GC-MS sensitivity, separation efficiency, and resolution. Within this objective, we determined the limits of detection and quantification for each reagent and each organic molecule. Finally, a more thorough experiment was conducted for a possible reagent synergy with the combination of two different reagents to derivatize targeted molecules.

This study will provide information to select the reagent(s) most suited for detecting bioindicators (fatty acids, nucleobases, and amino acids), for instance on March, Titan, or Europa.

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## II. Materials and methods

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### 1. Standard solutions and reagents

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Considering the current objectives of *in situ* explorations for astrobiology, this study focused on the analysis of three chemical families of organic molecules recognized for their interest to seek life and prebiotic processes, i.e. amino acids, fatty acids, and nucleobases.

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To test the efficiency of each derivatization reagent towards fatty acids (bearing from 7 to 30 carbon atoms ($C_7$ to $C_{30}$)). Two different solutions (S1 and S2) were prepared in dichloromethane (Prolabo, purity ~ 99,5%) to test two sets of fatty acids. The first solution (S1) contained benzoic acid (Fluka, purity > 99,5%), lauric acid (Alfa Aesar, purity ~ 99,5%), palmitic acid (Sigma, purity > 99%), oleic acid (Fluka, analytical purity), stearic acid (Sigma,
1 purity > 98,5%), arachidic acid (Sigma-Aldrich, purity > 99%), lignoceric acid (Sigma, purity > 2 99%), and melissic acid (Aldrich, purity > 98%), and it was dedicated to comparing the 3 largest range of carboxylic acids.

4 The second fatty acid solution (S2) contained benzoic acid, oleic acid, stearic acid, 5 melissic acid, palmitic acid, arachidic acid, hexadecanedioic acid (Aldrich, purity > 96%), 6 palmitoleic acid (Sigma-Aldrich, purity > 98,5%), eicosapentaenoic acid (Sigma-Aldrich, 7 Analytical purity), 2-methyloctadecanoic acid (Aldrich, purity > 97%), 1,3,5-benzene 8 tricarboxylic acid (Aldrich, purity ~ 98%), and it was dedicated to comparing some of these 9 linear-chain acids with aromatic, branched, unsaturated, sterically crowded, or polyfunctional 10 (called featured) carboxylic acids.

11 The nucleobase solution was made in distilled water with adenine (Alfa Aesar, purity ~ 12 99%), thymine (Alfa Aesar, purity ~ 97%), uracil (Alfa Aesar, purity > 99%), cytosine (Fluka, 13 purity ~ 99%), and guanine (Fluka, purity > 99%). Only guanine was diluted in methanol due 14 to its low solubility in water (Sigma-Aldrich, purity > 99,9%) and then added to the pyrolysis 15 cups. These molecules were selected as they represent a bioindicator, and a diversity of 16 chemical structures/configurations that can be derivatized differently according to the reagent 17 (with C₄/C₅ and N₂/N₅ aromatic ring(s) with primary and/or secondary amine, and/or amide, 18 and/or carboxylic acid functions). 19

20 The amino acids solution (C₂ to C₁₁, nitrogen and sulfur-bearing linear and/or cyclic 21 compounds with carboxylic, amine, and/or thiol functions) was a standard industrial solution 22 containing seventeen proteinic amino acids (Sigma Aldrich, analytical purity). These 23 molecules represent bioindicators and are chosen for their wider type of acidic groups in the 24 same molecule.

25 The concentration of these standards in their respective solutions was chosen to inject 26 low amounts in the GC-MS analyzer similar to the quantity we suspect for extraterrestrial 27 samples to inject in the spaceflight GC-MS instrument (from 330 pmol to 75 nmol, except for 28 LOD and LOQ assessment where we went down to 1pmol) (37). Details and concentration 29 information concerning all those solutions are reported in Supplementary Material 2 (SM2).

30 To prevent potential biases introduced by intraday deviations and injected volume 31 variations in the semi-quantitative analysis, [D₈]naphthalene (diluted at 10⁻³ M in 32 dichloromethane Aldrich, purity > 98% and isotopic purity ~ 99 atom % D) was used as 33 internal standard (1 µL at 10⁻³ M for each sample). [D₈]naphthalene is not derivatized either 34 by TMAH, TMSH, or TMPAH and it is fully vaporized and well-eluted with the set-up and 35 operating conditions used. Moreover, it resists the high temperatures used in this study. To 36 quantify the organic recovery and test the reagents' efficiency to each organic, we measured 37 the peak area of each organic molecule in the chromatograms and divided it by the peak 38 area of the internal standard (IS, or [D₈]naphthalene) using Thermo Scientific Xcalibur 39 software:

$$A_{\text{organic}}/A_{\text{internal standard}} = \frac{\text{Area of the organic peak}}{\text{Area of the internal standard}}.$$

40 To calculate the limits of detection (LOD) and quantification (LOQ), we determined the 41 signal-to-noise (S/N) ratio and relative standard deviation (RSD). A value of S/N=3 or 42 RSD≈17% can be used for LOD, S/N=10 or RSD≈5% can be used for LOQ (38–42). 43 Therefore, S/N=3 and S/N=10 were used to determine the LOD and LOQ of the organics in 44 this study. The calibration curves for each organic molecule were obtained by plotting peak 45 area ratio values (the peak area of organic/internal standard peak area) against the 46 concentration ratio of organic (organic amount/internal standard amount).
1. TMAH, TMSH, and TMPAH were purchased diluted in methanol with a final concentration

2. of 0.25 M, 0.25 M, and 0.5 M in reagent, respectively (Sigma-Aldrich, analytical purity).
## 2. Pyrolyzer and GC-MS

Capsules fill in with sample, reagent of derivatization, and the internal standard were introduced in an EGA/PY-3030D micro-oven pyrolyzer (Frontier Lab). The pyrolyzer was connected to the Split/SplitLess (SSL) injector (GL Sciences) of a Trace GC Ultra gas chromatograph coupled to an ISQ LT quadrupole mass spectrometer (Thermo Scientific). The injector part was set at 300 °C to avoid a cold trap ahead of the GC column.

To achieve the separation of the analytes, Rxi-5Sil MS Restek columns (30 m × 0.25 mm i.d. × 0.25 µm film thickness) with a 5 m long integrated guard column were used. The temperature programming of the column started at 50 °C, then increased to 250 °C at a rate of 3 °C min⁻¹ (for amino acid tests), 305 °C at a rate of 7 °C min⁻¹ (for nucleobases) optimized after numerous experiments for an efficient GC separation, and a short time of analyses. For carboxylic acids, a non-linear program was set to separate fatty acids close in polarity and molecular weight (comparing linear, unsaturated, and branched) with the first temperature ramp from 50 °C to 100 °C at 7 °C min⁻¹, then up to 250 °C at 5 °C min⁻¹, and finally up to 305 °C at a rate of 7 °C min⁻¹ optimized after a set of experiments to optimally separate and identify all organics. The final temperature for each program temperature was held for 3 minutes.

Helium (99,9999% purity, Air Liquide) was used as the carrier gas, with a constant flow rate in the column set to 1.2 mL min⁻¹. The split flow was 30 mL min⁻¹. The temperature of the SSL injector was set to 270 °C, and the MS ion source was at 350 °C. The ions were scanned from m/z 40 to m/z 500. The temperature of the transfer line from GC to MS was set to 300 °C.

## 3. Sample preparation and gas chromatography analysis

Prior to proceeding to the thermochemolysis reaction, an *in-open capsule* is filled with the reactive mixture. In these capsules, we added either 5 µL (for nucleobases and carboxylic acids) or 30 µL (for amino acids) of the solutions detailed in SM2. Solutions were evaporated to throw the excess water and methanol. Then, 5 µL of derivatizing reagent and 1 µL of the internal standard solution ([D₈]naphthalene) were added to the capsules. Each reagent was added in excess into the pyrolyzer (6 reagent molecules for 1 organic (6:1), according to previous studies (26,29,36,43–45)).

The capsule was then quickly introduced into the pyrolyzer already heated to the studied temperature to proceed to the reaction. Then, volatile molecules produced in the pyrolyzer are quickly carried by the flow of helium to the column for GC-MS analysis.

Molecules were identified by comparing the acquired mass spectra to those from the NIST library (National Institute of Standards and Technology: reference mass spectra library). For each result, we have been careful not to take into account matched molecules with a Reversed Search Index (RSI) and Similarity Index (SI) lower than 700 – corresponding and inverse research corresponding factor.

In SM2 are presented the targeted organics (derivatized/methylated forms observed in GC-MS) with their molecular ion and major fragments.
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### III. Results and discussion

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#### 1. Optimal temperatures for derivatizing organic acids and nucleobases
performed with TMAH, TMSH, and TMPAH

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##### i. TMAH thermochemolysis

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TMAH, TMSH, and TMPAH can derivatize organics between 200 °C and 800 °C, according to previous papers (12,17). However, for future laboratory and resource-limited *in situ* space applications, we must assess the optimal thermochemolysis temperature with the optimal derivatization yield for each organic tested. For an environmental sample (where amino acid, fatty acid, nucleobase, and other organic molecules are mixed in a solid matrix), we have to determine the trade-off in temperature for detecting the widest range of organics.

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To determine the optimal temperature of thermochemolysis with each reagent through *on line* Pyr-GC-MS analysis, the mixture S2 of fatty acids and the mixture of nucleobases were heated separately in the presence of TMAH at different temperatures from 400 °C to 800 °C in the pyrolyzer under He inert atmosphere. 400 °C has been chosen for the minimum temperature of thermochemolysis because the ebullition temperatures of most of the organics are over 350 °C and the derivatization yield is low at lower temperatures, according to previous studies (12,34). For the mixture of amino acids, the range of temperature was limited from 250 °C to 400 °C maximum due to amino acid thermal stability. Figure 1 shows the results obtained for TMAH thermochemolysis on the selected compounds at different temperatures.

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The results and trends associated with increasing temperature in Figs. 1 to 5 can be attributed to different factors: either through structural aspects or operating aspects. Structural aspects concern (i) the ebullition temperature of the organics; (ii) their molecular weight; (iii) their features (e.g. unsaturations, aromatic rings) influencing the kinetic reaction; (iv) the stability of the complex molecules (with an aromatic ring, π-conjugated system, or polyunsaturations); (v) the type of chemical functions to derivatize and the derivatization yield applied to each derivatization reagent; (vi) the ionization and fragmentation in the MS source. The operating aspects concern (i) the MS sensitivity; (ii) the thermal degradation of the organic molecules (as well as for the derivatization reagent); (iii) the potential loss by adsorption on the walls from the pyrolyzer to the MS detector (kept controlled with all transfer lines heated over 300 °C). These listed overall factors are individually detailed for each organic compound and each reagent in this work.
<figure><img src="image_2.png" /></figure>

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<figure><img src="image_3.png" /></figure>

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<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Comparison of the efficiency of the TMAH thermochemolysis reaction at different temperatures (from 400 °C to 800 °C) on (a) carboxylic acids (S2 solution), (b) nucleobases, and from 250 °C to 400 °C for (c) amino acids. The comparison is made by calculating the ratio between the peak area of the target compound over the peak area corresponding to the internal standard ([D₈]naphthalene). Experiments for the other figures are done in triplicates with the statistical analysis done on it. The efficiency is calculated by using the chromatographic peak corresponding to the fully derivatized forms, except for valine, leucine, and phenylalanine where two forms have been observed with one (partially derivatized) or two (fully derivatized) methylations identified. We measured the peak area of each derivatized form and compared them between samples without summing peak areas for a same amino acid.</figcaption></figure>

In Figure 1, over 600 °C, we observed a decrease in the area while the temperature increased. The carboxylic acids are either thermally degraded into the pyrolyzer, or interact less with TMAH, which is also highly degraded (loss of 50% of the reagent at 800 °C compared to 400 °C) despite an initial 6:1 threshold ratio (TMAH:carboxylic function). For the arachidic acid (medium linear-chain acid representative), no differences are observed between 400 °C and 600 °C. However, for a shorter chain than arachidic acid (e.g. oleic acid), results revealed a decrease as 600 °C in the recovery yield of the acid, mostly due to thermal degradations and its lower boiling point. In contrast, for melissic acid (that might be taken as a representative of the long-linear chain acidic group), we observed a statistically significant rise in derivatization and detection of melissic acid at 800 °C (relative to lower temperatures). Indeed, melissic acid possesses a high molecular weight and a high ebullition temperature (441 °C). At 400 °C, the volatilization is not completed compared to 600 °C and 800 °C, where the melissic acid detection increase with the highest temperature which might be due to the heat of the gas flowing and the exceed of the energy barrier necessary to form the methyl melissate ester (derivatized form of the melissic acid).

Thus, depending on the molecules, their stability, and their steric facility to interact with TMAH, we observed either an optimal thermochemolysis at 600 °C (for most fatty acids)
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or 800 °C (for complex and thermally resistant molecules). Indeed, the comparison of organic molecule yields at the same thermochemolysis temperature revealed that short and medium chains (branched, or not) have higher organic peak areas compared to either aromatic or unsaturated acids. This latter difference (by a factor of two) is low compared to long chains, polyfunctional acids, or polyunsaturated acids (where the difference is at least a factor of ten). Finally, trimesic acid was not detectable using TMAH even with Single Ion Extract (SIE), as it is strongly stable with a π-conjugated system and three functions to derivatize, and in parallel as 400 °C, we might have a high decarboxylation rate leading to the formation of benzene and CO2.

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With regards to the nucleobase experiments, adenine and uracil demonstrated an increase in recovery as pyrolysis temperature increased from 400 °C to 600 °C. These nucleobases experienced a yield decrease at 800 °C due to thermal degradation on the acidic functions. Compared to adenine, the recovery yield of the uracil at 400 °C is very low and increases 60% at 500 °C presumably due to its 440.5 °C boiling point. The same trend was observed for cytosine from 400 to 600 °C, but at 800 °C no cytosine was detected due to its thermal degradation. For these three molecules, the optimal temperature of derivatization is within the range of 500 °C to 600 °C due to the energetic barrier reaching over 400 °C and their relative molecular boiling points. No statistically significant differences are noted for thymine and guanine derivatized between 400 °C and 800 °C. Thus, 600 °C is the optimal average temperature for thermochemolysis of nucleobases (despite 500 °C may be also considered an acceptable trade-off). This conclusion agrees with previous work (12).

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Regarding the mechanistic aspects explaining these differences between derivatization temperatures and differences between nucleobase recovery, we observed that thymine and uracil – bearing two secondary amine functions – are detected in higher abundance than nucleobases with two acidic sites and one secondary amine function, such as cytosine, or with multiple acidic functions, such as guanine (difference of a factor of ten). However, a difference is observed between adenine and cytosine presenting both two acidic sites (one primary amine, and one secondary amine), where the first mentioned is not statistically significantly different from uracil or thymine recovery yield. Cytosine has a strong sterical hindrance and a single aromatic with a stable π-conjugated system compared with adenine, which might explain the two orders of magnitude difference with a slower kinetic attack on both sites for cytosine than adenine.

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For amino acids, from 250 °C to 400 °C, we observed a global decrease in the recovery yield. This can be inferred from their high sensitivity to temperatures higher than 200-250 °C, where the carboxylic acid function is easily degraded. Thus, for all amino acids derivatized by TMAH fully or partially (as observed with valine, leucine, and phenylalanine), the optimal temperature is 250 °C.

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In addition to nucleobases and fatty acids, we observed that low sterically hindranced acids, such as valine, leucine, or phenylalanine, and those with a low number of acidic functions to derivatize, such as isoleucine, proline, or methionine, have a higher recovery yield compared to complex amino acids. The latter (multi-functional acidic site, high sterical hindrance, and stable or π-conjugated systems) presents a high ebullition temperature and basic pKa for derivatizable sites, such as threonine, serine, histidine, or aspartic acid (factor of minimum four, and up to twenty) explaining the lower derivatization yield observed.

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However, we observed that different forms of amino acids are detected including valine, leucine, and phenylalanine (full and partial derivatization amino acids) (Figure 1). Indeed, we noted for almost all amino acids, all derivatized forms possible (passing from one acidic site derivatized to a full derivatized molecule that we expect for GC-MS space analysis
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as alkylation helps to decrease the polarity of the molecule, and so to increase its volatility and detection).

TMAH methylates in a matter of seconds over 250 °C and generates higher yields for carboxylic acid functions and secondary amine functions than primary amine functions. This finding is consistent with the lower derivatization yield for polyfunctional molecules compared to monofunctional organics, or bifunctional nucleobases. For a general case, it appears that more than two TMAH molecules are required to interact on the same or close acidic sites. As an example, guanine has three acidic functions that are close to one another and are stabilized by a π-conjugated system leading to a low derivatization yield.

Therefore, we noted the same observations on the derivatization of fatty acids, amino acids, and nucleobases: high organic integrated peak areas measured for (i) molecules easily ionizable by the MS source, (ii) accessible acidic sites for derivatization, (iii) few features and acidic functions, (iv) carboxylic and secondary amine functions compared to π-conjugated stabilized systems, featured and polyfunctional molecules, or primary amine functions, and (v) with low ebullition temperatures. The observations are explained by physio-chemical parameters belonging to the organics (temperature of ebullition, steric conformation, thermal stability), or the TMAH reaction (interaction easier at high temperature to pass the energy barrier, at basic pH imposed by the salts used as a reagent). The optimal temperatures determined here were used here to compare TMAH to TMHS and TMPAH thermochemolysis (600 °C taken for carboxylic acids and nucleobases as an average of all compounds (except guanine at 500 °C), and 250 °C for amino acids, except proline at 300 °C). For carboxylic acids and guanine, 800 °C has not been considered for TMAH as numerous TMAH by-products are observed and detailed in Y. He et al. (2021); they represent drawbacks for space analyses and data interpretations. Indeed, these by-products coelute with most of the carboxylic acids (introduced at trace levels (~20 pmol) and easily indistinguishable from the baseline that raised due to TMAH degradation), making this higher pyrolysis temperature highly undesirable.

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## ii. TMSH thermochemolysis

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We repeated the experiments performed with TMAH to test the thermochemolysis with TMSH, and the obtained results are presented in Fig. 2.

<figure><img src="image_5.png" /></figure>

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<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Comparison of the efficiency of the TMSH thermochemolysis reaction at different temperatures (from 400 °C to 800 °C) on (a) carboxylic acids (second carboxylic acid solution), (b) nucleobases and (c) amino acids. The comparison is made by calculating the ratio between the peak area of the target compound over the peak area corresponding to the internal standard ([D₈]naphthalene). The nucleobases presented are fully derivatized forms.</figcaption></figure>

In Fig. 2 we globally observed a decrease in the integrated peak area as the pyrolysis temperature increased. One exception is the melissic acid, where the MS detection increases between 400 °C and 600 °C. At 800 °C, a decrease of the response is nearly globally noted probably due to the optimal temperature of use of TMSH compared to TMAH at low temperatures (between 200 and 600 °C as observed in previous studies) visible for all fatty acids. Compared to TMAH, the TMSH experiments successfully yielded trimesic and eicosapentaenoic acids at 400 °C and 600 °C, which suggest different chemical interactions with the sulfur of the TMSH compared to the TMAH reactant. It is relevant to note that they
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were detected at very low intensities near the detection limits (specified in Table 2) and decreased in the organic recovery with temperature for eicosapentaenoic (compared to trimesic acid, which is more stable due to its π-conjugated system).

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On the other hand, we observed for carboxylic acids that short and medium (linear or branched) chains are more efficiently derivatized than featured and long linear-chain acids (by a factor of two to three). For TMSH, the higher derivatization yield is met at 400 °C, except for the melissic acid (C30) at 600 °C. TMSH proves to be a better derivatizing reagent at a lower temperature as noted in previous work (31,43).

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For nucleobases, results revealed an increase of the nucleobase detection for temperatures up to 500 °C, then a decrease at higher temperatures, except for uracil where there is a constant decrease after 400 °C. The bifunctional and non-π-conjugated aromatic systems (adenine, thymine, and uracil) are detected in higher intensity than the stabilized and polyfunctional nucleobases (cytosine and guanine) with a difference by a factor of three to ten. This observation is also explained for TMAH by a lower derivatization yield of primary amines that must be derivatized twice on the same acidic function.

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Thus, 500 °C is the best temperature for TMSH derivatization compared to 600 °C for TMAH, for the low functionalized and sterically hindered nucleobases (adenine, thymine, and uracil). 500 °C and 600 °C are both optimal temperatures for cytosine and guanine. As for TMAH, at these high temperatures, the primary amines are more derivatized than at 400 °C but are not as well derivatized as secondary amines. Indeed, we noticed the presence of the partial derivatized forms for adenine, cytosine, and guanine at 400 °C, but not at 500 °C, 600 °C, and 800 °C.

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For the amino acids, we only detected ten amino acids over the seventeen due to combined factors (thermal degradation of amino acids, low derivatization yield for primary amines, and pH issues with the pKb of 12 while most amino acids have an amine pKa around 10, and likely MS sensitivity) (SM1). As for TMAH, we noticed that valine, leucine, isoleucine, proline, and phenylalanine are detected at a higher intensity than other featured amino acids mostly due to a higher MS sensitivity for these compounds and a higher derivatization yield with TMSH from the kinetic and thermodynamic perspectives.

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### iii. TMPAH thermochemolysis

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We performed the same experiment for TMPAH in Fig. 3 as TMAH seen previously in Fig. 1, and TMSH in Fig. 2 with the same protocols and solutions.
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<figure><img src="image_7.png" /></figure>

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<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Comparison of the efficiency of the TMPAH thermochemolysis reaction at different temperatures (from 400 °C to 800 °C) on (a) carboxylic acids (second carboxylic acid solution), (b) nucleobases and (c) amino acids. The comparison is made by calculating the ratio between the peak area of the target compound over the peak area corresponding to the internal standard ([D₈]naphthalene). The nucleobases presented are fully derivatized forms.</figcaption></figure>

For the case of TMPAH (Fig. 3), we observed no differences between 400 °C and 600 °C for linear and branched carboxylic acid relative areas, except for oleic acid which increased. In contrast, the complex acids: i.e. trimesic acid (polyfunctional and aromatic system), hexadecanedioic acid (polyfunctional), and eicosapentaenoic acid (polyunsaturated) decrease down to their detection limit at 600 °C (Table 2), and not observed over the GC baseline at 800 °C.

Carboxylic acids are optimally derivatized at 400 °C regardless of the fatty acid features. TMPAH is comparable to TMSH in that both are reagents best utilized at lower temperatures and are greatly degraded over 400 °C. In comparison, TMAH is highly degraded over 600 °C. We observed a lower recovery yield for very short and medium chains (e.g. palmitoleic and benzoic acids) than medium chains, such as arachidic or 2-methyloctadecanoic acids (by a factor of two), or long chains, such as melissic acid (by a factor of at least ten) at the optimal temperature.

Concerning nucleobases, we observed a notably different trend depending on the nucleobase and the conjugation of the derivatizable functions on the aromatic ring. For adenine and thymine, we noted an optimal temperature of 400 °C with a constant decrease to 800 °C of the integrated peak area measured. Conversely, for uracil we noted a constant increase from 400 °C to 600 °C, then a decrease due to thermal degradations over 600 °C. Uracil possesses a strong π-conjugated stabilized system but is easily derivatized as it contains only two secondary amines. We did note that higher temperatures allow the kinetic approaches (favoring the reactions despite the π-conjugated system) to pass the energy barrier (thermodynamic constraint). Indeed, the aromatic ring of the TMPAH could interfere with the approach of the methyl group to the amine functions, especially in the cases of uracil and guanine. Finally, for the complex, stabilized, and sterically hindered nucleobases
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cytosine and guanine, we have an optimal detection at 500 °C with the energetic barrier and the ebullition temperature reached, then a decrease as observed with the other nucleobases. However, the nucleobase recoveries are low, which can be explained by the nature of the molecule used (TMPAH and nucleobases) that present aromatic rings which do not favor a full interaction, and thus full derivatization in a matter of seconds due to kinetic aspects (the substitution of a labile H by a methyl group is complex when acidic nucleobase functions are not accessible by high molecular weight and sterically hindered molecule as TMPAH).

Thus, we conclude for TMPAH an optimal pyrolysis temperature is between 400 °C and 600 °C. For accessible labile hydrogens, we observed therefore a better recovery yield for derivatization at 400 °C (adenine and thymine), and a higher optimal temperature at 500 °C for polyfunctional or complex nucleobases (e.g. cytosine and guanine), and 600 °C for uracil.

With regard to amino acids, we observed a constant increase in the integrated peak area measured with temperature increasing from 250 °C to 400 °C where TMPAH is the most efficient as observed for fatty acids and some nucleobases (and is at a minimum in its thermal degradation). However, some amino acids are more sensitive to temperature (e.g. leucine, threonine, or histidine) than others due to their stabilization by an aromatic ring (e.g. phenylalanine, tyrosine), which is denoted by a decrease in the detected derivatized amino acids (decreasing to below the limits of detection for leucine and isoleucine over 300 °C). Compared to fatty acids and nucleobases, the amino acids detected at the beginning (e.g. threonine) and the end (e.g. histidine) of the GC-MS runs in a lower integrated peak area scale (difference by a factor of ten) than the rest of the organics. This loss factor is mostly due to MS sensitivity and to a lesser extent to the derivatization reaction yield of TMPAH with these compounds, except for histidine and serine that have three functional groups to derivatize with one primary amine function (and one alcohol for serine) that are not the main targeted acidic groups for optimal and full derivatization on all groups (likely with TMAH and TMSH).

To conclude, in this first part, we searched for the optimal temperature of individual organics for each thermochemolysis reagent. We noticed similar behaviors for TMAH and TMSH derivatization, but also some similarities with TMPAH for derivatizing short or medium chain and low sterically hindrance systems compared to complex or π-conjugated systems.

However, on linear and branched carboxylic acids with TMSH and TMPAH, we observed the opposite behavior compared to TMAH with a higher yield of derivatization at 400 °C than 600 °C and 800 °C, as expected according to previous studies (26,34,44–46). TMSH can derivatize a wider range of carboxylic acids in high abundance compared to TMAH and TMPAH. Moreover, TMPAH compared to TMSH shows lower derivatization yield on unsaturated, aromatic, polyfunctional, or branched carboxylic acids due to its high molecular weight and its steric attack on labile and hindered functions. For all reagents, the trade-off for in situ spaceflight analyses would be 500-600 °C to detect the widest range of organics.

Despite thermal degradations, and low derivatization yield of our reagents on primary amine and alcohol functions, an additional reactional parameter is important to introduce in this discussion to explain these low recovery yields on some amino acids and nucleobases, which might be the trans-esterification reaction. Indeed, TMSH (as well as TMAH and TMPAH) is well-known to promote the methylation of acidic groups, but also the trans-esterification of non-hydrolyzed esters (47). Thus, TMSH reacts more than one time by carboxylic acid functions, and previous studies demonstrate that the mole ratio of TMSH/lipid constitutes a critical parameter for the control of the degree of isomerization of PUFA (48–
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50). The maximum we could get with our device and without damaging the GC column and MS detector was 5 µL for solutions at 0.5 M or 0.25 M, however, a higher concentration could enhance the present derivatization yields.

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Thus, for each targeted molecule and different features, we noted statistically significant changes between reagents with different optimal temperatures for thermochemolysis. For this reason, the goal of the second part of this work is to compare reagents together on the same compounds at the different optimal temperatures estimated above to make recommendations for future space applications.

## 2. Comparison of TMAH, TMSH, and TMPAH for thermochemolysis pre-treatment

### i. Derivatization on fatty acids

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To compare the performances of the three reagents, we used the integrated peak area obtained with each reagent for every chemical species with all three thermochemolysis reagents at their optimal temperature of thermochemolysis (determined in part 1) (Fig. 4).

<figure><img src="image_10.png" /></figure>

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**Fig. 4.** Integrated peak area of carboxylic acids detected by GC-MS (divided by the integrated peak area of the internal standard) after different derivatization reagent interactions with an *on line* thermochemolysis using an optimal Pyr-GC-MS temperature of 400 °C or 600 °C for a minute with 5 µL of reagent. Solutions S1 and S2 of carboxylic acids have been used for this comparison. For TMSH, two optimal temperatures have been found for the range of organics studied differentiated in the graph in full (600 °C) and hatched (400 °C). Experiments were made in triplicates. Trimesic acid was observed, but close to the theoretical detection limit.
In Fig. 4, we observed for short and medium linear carboxylic acids that TMSH at 600 °C gives the same organic recovery as TMAH, except for stearic and palmitic acids, which have a higher yield with TMSH. For long straight-chain, aromatic, or unsaturated carboxylic acids, namely melissic, benzoic, and palmitoleic acids, a lower recovery yield is observed for TMAH compared to TMSH. In comparison, for branched or polyfunctional acids the same or a better detection is observed with TMAH, namely 2-methyloctadecanoic and hexadecanedioic acid. Eicosapentaenoic acid is observed only with TMSH, and trimesic acid with TMSH and TMPAH.

For short and medium linear, aromatic, and polyfunctional carboxylic acids, TMPAH had the same recovery yields as TMAH and TMSH. However, for unsaturated acids such as palmitoleic acid, we observe a higher yield of derivatization with TMPAH than with TMAH and TMSH reagents. We also noted a lower recovery rate for the polyunsaturated eicosapentaenoic acid, and the branched 2-methyloctadecanoic acid, regardless of the thermochemolysis temperature (with the factor of differences summarized in Table 1).

<table><caption>**Table 1.** Averaged factors of differences between reagents for the different organics on the left table, and factors between the type of featured organic molecules on the right table with the solutions S1 and S2.</caption><thead><tr><th></th><th>TMSH<br>TMAH</th><th>TMPAH<br>TMAH</th><th>TMSH<br>TMPAH</th></tr></thead><tbody><tr><td>Benzoic</td><td>1.5</td><td>0.5</td><td>2</td></tr><tr><td>Trimesic</td><td>/</td><td>/</td><td>1</td></tr><tr><td>Palmitoleic</td><td>1.5</td><td>3</td><td>0.5</td></tr><tr><td>Palmitic acid</td><td>1.5</td><td>1</td><td>1.5</td></tr><tr><td>Oleic</td><td>1</td><td>1</td><td>1.5</td></tr><tr><td>Stearic acid</td><td>1</td><td>1</td><td>1.5</td></tr><tr><td>2-methyloctadecanoic</td><td>1</td><td>0.5</td><td>2</td></tr><tr><td>Hexadecanedioic</td><td>0.5</td><td>1</td><td>0.5</td></tr><tr><td>Eicosapentaenoic</td><td>/</td><td>0</td><td>/</td></tr><tr><td>Arachidic</td><td>1</td><td>1.5</td><td>1</td></tr><tr><td>Melissic</td><td>3.5</td><td>4.5</td><td>1</td></tr></tbody></table>

where / corresponds to the detection of the organic with one reagent, but not the second one (generally TMAH, and TMPAH).

<table><thead><tr><th></th><th>Branched<br>Linear</th><th>Unsaturated<br>Linear</th><th>Polysaturated<br>Linear</th><th>Aromatic<br>Linear</th><th>Bifunctional<br>Linear</th><th>Trifunctional<br>Linear</th><th>Long linear chain<br>Short - medium chain</th></tr></thead><tbody><tr><td>TMAH</td><td>2</td><td>2</td><td>0</td><td>8</td><td>100</td><td>0</td><td>60</td></tr><tr><td>TMSH</td><td>2</td><td>2</td><td>25</td><td>8</td><td>100</td><td>400</td><td>100</td></tr><tr><td>TMPAH</td><td>3</td><td>1.5</td><td>0</td><td>8</td><td>70</td><td>400</td><td>30</td></tr></tbody></table>

Results revealed that all three reagents efficiently derivatize carboxylic functions even for compounds injected in trace amounts (e.g. 13-28 pmol injected in the chromatographic column). Overall, despite differences between reagents for each molecule, a factor of two to three in the organic area detected by GC-MS is the maximum observed. However, the higher the molecular weight of the carboxylic acid, the lower the derivatization yield, regardless of the reagent volume and the pyrolysis temperature (SM1). Our experimental conditions spanned a temperature range from 400 °C to 800 °C, and derivatization reagent volumes of 2 µL, 4 µL, and 5 µL and no differences were observed. Therefore we used 5 µL to ensure an excess in reagent compared to derivatized functions with a minimum value of 6:1 (46). The trade-off for TMAH and TMSH space applications might be 500 °C to derivatize with a high recovery of a wide range of fatty acids.

## ii. Derivatization on nucleobases

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Integrated peak area of nucleobases detected by GC-MS (divided by the internal standard integrated peak area) using three different derivatization reagents with an *on line*</figcaption></figure>
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We next compared using nucleobases the reagents based on their optimal pyrolysis temperatures and temperatures used on spaceflight missions (500 and 600 °C) (Fig. 5). We observed that TMSH remains the best performing reagent, except for guanine and uracil. These observations confirm the hypothesis put forward in part 3.1, where results revealed that TMAH and TMPAH allow better derivatization for primary amine functions (even if TMSH derivatizes polyfunctional, sterically hindered, and heavy systems as concluded in the fatty acid analysis). However, the more the system is stabilized, especially by π-conjugated systems, the more the interaction with all reagents is influenced (e.g. cytosine). Indeed, TMSH allowed a five to ten times higher cytosine recovery than TMAH and TMPAH.

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For amino acids, results are presented in SM3 with a better derivatization yield for TMAH at the optimal temperature of each reagent. These reagents do not derivatize amino acids in high abundance, which reveal that thermochemolysis is not suited to target these organics compared to other derivatizing reagents at lower temperatures (51,52).

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This optimizing work on the thermochemolysis process for space applications leads to two conclusions: (i) thermochemolysis using TMAH or TMSH is suited for laboratory experiments on planetary simulations or return sample analyses as for in situ analyses where the optimal temperature will be chosen according to the sample to analyze. In parallel into the pyrolyzer, a three ramped temperature with isotherms at 300 °C, 500 °C, and 600 °C is possible with a release of volatile organics in a cryotrap in the capillary column before GC runs; (ii) for *in situ* analyses on extraterrestrial planetary systems, the temperature of 500-600 °C is a trade-off with no or few influences on matrix extraction, derivatization, or volatilization of light and heavy organics of astrobiology interests (for TMAH and TMSH with higher analytical interests than TMPAH, according to the work conducted).

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### 3. TMSH and TMPAH Thermochemolysis additional results for future space application

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#### i. TMSH and TMPAH degradation products

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To complete the work and select the best-suited reagent(s) for spaceflight analyses, we conducted thermal tests on the three reagents to document the by-products generated, then analyzed their interaction with our targeted organics during the GC-MS analyses (comparing TMSH and TMPAH with TMAH already studied in previous work (53)).

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We reported in SM4 and SM5 the by-product list and areas measured on the chromatogram observed after pyrolysis at 600 °C and 800 °C for TMSH (plus 400 °C for TMPAH because TMPAH presented high organic recovery at a lower temperature than TMAH and TMSH) (SM4 and SM5). We noted the production of most of the by-products at 600 °C for TMSH (e.g. dimethyl and trimethyl sulfide), and TMPAH (e.g. N-methylbenzaldimine, N-phenylmethylene-benzenamine, and N-methyl-Aniline) – with only seven by-products reported for TMPAH at 400 °C. At 800 °C, the production yield of these by-products increases compared to 600 °C, and this increase is accompanied by the detection of new by-products that are produced in higher quantities than the LOD. Thus, both the diversity and the quantity of by-products increase with the increase in temperature, especially for TMAH (53), and TMPAH. Unique to TMSH, the trend tends to be fixed, although 14 by-products were detected (over the LOD) at 800 °C compared to 600 °C. We can conclude that TMSH possesses interesting properties, such as the low sensitivity relative to the temperature with the fewest number of by-products in diversity and quantity among the three tested thermochemolysis reagents. Moreover, we noticed the lower number of by-
products from TMSH does not overlap with derivatized organic molecules (after the comparison with the TMSH by-products' retention times and the targeted organic molecules, but also with the non-coelutions observed directly during the tests conducted in 3.1) compared to TMAH and TMPAH with numerous degradation products as 600 °C.

## ii. Limits of detection and quantification

In space applications, the limits of detection of organics, and especially the targeted bioindicators studied in this work – that are necessary for the formation of lipid membranes, proteins, DNA, and RNA – would be present in fmol to nmol concentrations in extraterrestrial environments if they are produced by prebiotic chemistry, or through exogenous delivery of macromolecular material (from meteorites or IDPs, for instance) (12,37). The following tests help for determining the limits of detection and quantification we might propose for future space missions to detect the three molecular families targeted here.

**Table 2.** Experimental limits of detection and quantification. The limits have been calculated from previous methods applied to nucleobases, and ANSES and UE guidelines (38,39,54). For experimental limits, the methods of the 3σ for LOD and 5σ for LOQ (where σ referred to the baseline) that we compare with our calibration curves and organic areas.

<table><thead><tr><th></th><th>TMAH</th><th>TMSH</th><th>TMPAH</th></tr><tr><th>pmol</th><th>LOD</th><th>LOD</th><th>LOD</th></tr></thead><tbody><tr><td>Benzoic acid</td><td>2</td><td>2</td><td>10</td></tr><tr><td>Palmitoleic acid</td><td>10</td><td>2</td><td>1</td></tr><tr><td>Palmitic acid</td><td>1</td><td>1</td><td>5</td></tr><tr><td>Oleic acid</td><td>2</td><td>1</td><td>5</td></tr><tr><td>Stearic acid</td><td>2</td><td>1</td><td>5</td></tr><tr><td>2-methyloctadecar</td><td>5</td><td>1</td><td>5</td></tr><tr><td>Hexadecanedioic a</td><td>10</td><td>8</td><td></td></tr><tr><td>Eicosapentaenoic acid</td><td></td><td>10</td><td></td></tr><tr><td>Arachidic acid</td><td>2</td><td>8</td><td>5</td></tr><tr><td>Melissic acid</td><td>4</td><td>8</td><td>10</td></tr><tr><th></th><th>TMAH</th><th>TMSH</th><th>TMPAH</th></tr><tr><th>pmol</th><th>LOD</th><th>LOD</th><th>LOD</th></tr><tr><td>Adenine</td><td>4</td><td>2</td><td>4</td></tr><tr><td>Thymine</td><td>4</td><td>2</td><td>4</td></tr><tr><td>Uracile</td><td>4</td><td>2</td><td>8</td></tr><tr><td>Cytosine</td><td>16</td><td>4</td><td>20</td></tr><tr><td>Guanine</td><td>16</td><td>16</td><td>20</td></tr><tr><th></th><th>TMAH</th><th>TMSH</th><th>TMPAH</th></tr><tr><th>pmol</th><th>LOD</th><th>LOD</th><th>LOD</th></tr><tr><td>Threonine</td><td>500</td><td>1000</td><td>500</td></tr><tr><td>Alanine</td><td>200</td><td>200</td><td>100</td></tr><tr><td>Glycine</td><td>200</td><td>500</td><td>100</td></tr><tr><td>Partially derivatized</td><td>200</td><td>500</td><td>200</td></tr><tr><td>Valine</td><td>100</td><td>500</td><td>100</td></tr><tr><td>Partially derivatized</td><td>100</td><td>200</td><td>100</td></tr><tr><td>Leucine</td><td>100</td><td>200</td><td>50</td></tr><tr><td>Isoleucine</td><td>100</td><td>200</td><td>100</td></tr><tr><td>Aspartic acid</td><td>200</td><td>1000</td><td>500</td></tr><tr><td>Cysteine</td><td>250</td><td>500</td><td>500</td></tr><tr><td>Methionine</td><td>100</td><td>500</td><td>200</td></tr><tr><td>Partially derivatized</td><td>100</td><td>200</td><td>100</td></tr><tr><td>Phenylalanine</td><td>100</td><td>200</td><td>80</td></tr></tbody></table>

Table 2 presents the experimental limits of detection (and limits of quantification multiplying the LOD by 3.3) that we compared in the laboratory with theoretical limits. The value, for TMSH especially, was higher with theoretical values due to high standard deviations in the calibration curves when closer to the LOD. The organic peak shape and area, in addition to the S/N ratio, is related to a lower quantity injected for the calibration curves (2 and 10 pmol according to the fatty acids, 2 or 4 pmol according to the nucleobases, and 100 pmol for the amino acids) was three to five times higher than the baseline (according to the 3σ for LOD and 5σ for LOQ). This method is currently applied in space mission analysis as we expect to find only trace concentrations of the bioindicators on
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There were a few interesting observations between reagents with differences lower than five sigmas. TMAH and TMSH have the same limits of detection which are both lower than TMPAH. Thus, TMAH and TMSH are more relevant than TMPAH to the detection of trace organics. Furthermore, for fatty acids and nucleobases, the limits of detection are comparable and are a factor of 100 lower than for amino acids. This finding confirms the observed results in Fig. 1 to 5 and SM3 with amino acid detection close to the baseline, and with lower integrated peak areas than the other organics tested even though the amino acid injection volume was greater than the other organics in this study. Moreover, it confirms that all reagents are not suited to derivatize primary and secondary amines with a difference factor of a minimum of two comparing detection of fatty acids by the different reactants (with only one carboxylic acid function – e.g. palmitic acid) compared to nucleobases (with multiple amine functions – primary and secondary amines – e.g. guanine), and factor of ten to a hundred with amino acids (with one carboxylic acid and one amine function – e.g. threonine).

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An important characteristic to consider – that is not visible in the results but observed on chromatograms– is the higher peak resolution and MS sensitivity with TMSH than other reagents to detect and separate properly derivatized peaks from other derivatized peaks, from the baseline, or the few reagent by-products. Indeed, the chromatographic peaks obtained after a TMSH derivatization gave always gaussian peaks, well separated, with a lower baseline allowing the detection of trace elements injected at 4 to 8 pmol and an S/N ratio over three, and finally with high reproducibility and repeatability (SM6). Furthermore, K. Vosmann et al. tested its efficiency for derivatizing thiol groups at 280 °C on numerous organics. They observed a full alkylation for long-chain alkanethiols and bifunctional, thiols only for an excess of TMSH of 2:1 (TMSH:thiol equivalent) (44), in addition to the derivatization of other acidic functions (e.g. fatty acids, alcohols).

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For future space applications on the ocean worlds, such as Titan, Europa, or Enceladus, it would be important to look at the impact of salts also on the thermochemolysis process. Below 2 mole equivalents of NaCl, no influence on the recovery yield has been observed (55). However, over 4 mole equivalents, they noted a decrease in the yield of derivatization by about ten percent (55). It is important to plan additional experiments under space conditions to estimate the loss of organics in the presence of NaCl and other salts, but also to design a salt processing step ahead of the derivatization step to eliminate these pollutants/contaminants. It would be interesting to look for the potential influence of irradiations on the aging of TMSH during space flights and on planetary surfaces, as done on TMAH (56).

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## IV. Conclusions

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To determine the possible future use of TMAH, TMSH, and TMPAH thermochemolysis reagents for *in situ* sample pre-treatment allowing gas chromatography – mass spectrometry analysis on refractory or polar solid organic matter, we compared these three reagents under space conditions looking at the completion of some exobiological space requirements, such as limit of detection, optimal temperature of thermochemolysis, by-product challenges coupled to GC characteristics for each reagent, and universality to derivatize organic compounds with a high recovery yield.
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To conclude on these results, we thus remarked that all three thermochemolysis reagents present globally similar characteristics and recovery yield for carboxylic acids and nucleobases that are the main targets of this sample pre-treatment for future exobiological missions. TMSH seems the most appropriate reagent to be used for exobiological researches (and comparable to TMAH, but with fewer drawbacks with regard to the number of by-products). Moreover, the compounds produced by the degradation of TMSH are only sulfur-bearing molecules that will probably not be found in extraterrestrial surfaces compared to amine by-products from TMAH (and TMPAH) degradation (or reaction with the *in situ* samples), except from some locations on March (37,57,58).

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However, TMSH differed from TMAH and TMPAH in its incapacity to well-derivatized primary amines, and so amino acids, and only some nucleobases (namely, guanine and cytosine) (as observed in (59)). Hence, other derivatization reagents are selected for space applications to detect pmol of amino acids, such as DMF-DMA (and MTBSTFA) in the Dragonfly (and ExoMars) mission (51,52,60). TMSH stands out despite its low thermal degradation in few by-products as in their respective abundances, and also in the spectral chromatographic properties that benefit from a good recognition of the targeted derivatized compounds. Moreover, their properties as salts allow the non-reactivity with salts or water, but also a high resistance to temperature and other external condition variations that represent adapted reagents with space constraints. Further thermal and radiation studies on TMPAH and TMSH should be considered to ensure the theory and first applicated studies under space conditions. This study will serve as a base for future studies and future data comparison once *in situ* GC-MS extraterrestrial analysis will be conducted (comparing the structures of the molecules found, the time of retention, the area in compounds measured on the GC chromatograms, the mechanistic possible interpretations for their detection (and production) on these substrates), and their quantity. A heating process at 500-600 °C with TMAH and TMSH for less than five seconds gave us promising results on the reagents' degradation as on the recovery yield of organics and their limits of detection searching for trace elements in complex matrices on March, Titan, or Europa, for instance, with the future missions ExoMars, Dragonfly, and EuropaLander, respectively. Furthermore, K. Vosmann et al. tested its efficiency for derivatizing numerous featured organics and confirmed our observations showing full derivatization of all kinds of acidic functions (44), which enhance the range of molecules that we can target with this promising reagent. Finally, concerning the EuropaLander mission, which is not likely to fly in the coming decades, and the delayed ExoMars mission, we confirm in the results of the current article that reagents have proven to be useful and are robust for future use with GC-MS analyses (for return sample or *in situ* space missions).

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## Acknowledgments

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I would like to thank the first place all LGPM team for their help in technical issue resolutions, especially Corinne Roussel, Sébastien Gauthier, Joel Casalinho, and Jamila El Bekri. Then, I would like to acknowledge all the work of the LATMOS team, working in collaboration with us.

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Finally, I would like to acknowledge the CNES and all the American laboratories working in collaboration with us and making feedback on all experiments and presentations (UF, NASA-GSFC, APL).
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Thermochemolysis
reagents

$$
\mathrm{H_3C-S^{+}-\underset{\underset{\mathrm{CH_3}}{|}{\mathrm{CH_3}}}{\mathrm{OH}}}^{-}
$$

TMSH

$$
\mathrm{H_3C-N^{+}-\underset{\underset{\mathrm{CH_3}}{|}}{\mathrm{CH_3}},OH}^{-}
$$

TMAH

$$
\[
\begin{array}{c}
\mathrm{CH_3} \\
\mathrm{N-CH_3} \\
\mathrm{CH_3}
\end{array}
\underset{-\mathrm{OH}}{\overset{+\mathrm{CH_3}}{|}}
\]
$$

TMPAH

Planetary targets

Ocean worlds

<figure><img src="image_12.png" /></figure>

Titan

<figure><img src="image_13.png" /></figure>

Europa

Rocky worlds

<figure><img src="image_14.png" /></figure>

Mars

<figure><img src="image_15.png" /></figure>

Bennu

Earth

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

Molecular targets

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>