# Strategy for integrated GC- and LC-MS/MS analysis of extraterrestrial biomolecules

Ramzi Timoumi, Cedrick Bourseau, Boris Laurent, Laurent Remusat, Marc David, Guillaume Rioland, Brian Grégoire, Pauline Poinot, Claude Geffroy-Rodier

► To cite this version:

Ramzi Timoumi, Cedrick Bourseau, Boris Laurent, Laurent Remusat, Marc David, et al. Strategy for integrated GC- and LC-MS/MS analysis of extraterrestrial biomolecules. Microchemical Journal, 2025, 218, pp.115530. 10.1016/j.microc.2025.115530. hal-05337144

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# Strategy for integrated GC- and LC-MS/MS analysis of extraterrestrial biomolecules

Ramzi Timoumi<sup>a,b</sup>, Cedrick Bourseau<sup>a</sup>, Boris Laurent<sup>c</sup>, Laurent Remusat<sup>c</sup>, Marc David<sup>d</sup>, Guillaume Rioland<sup>b</sup>, Brian Grégoire<sup>a</sup>, Pauline Poinot<sup>a</sup>, Claude Geffroy-Rodier<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Silylation

Integrated gas-liquid tandem mass

spectrometry

Amino acids

Nucleobases

Peptides

Meteorites

## ABSTRACT

The study aims to develop and optimise a derivatisation strategy to combine gas and liquid chromatography analysis of a single sample. Silylation was selected for gas chromatography analysis, as it increases the volatility and detectability of biologically related molecules. Deprotection of *tert*-butyldimethylsilyl derivatives was performed, enabling efficient conversion into the native molecular sample for further analysis using sensitive hydrogenated techniques, thereby broadening the target scope. The deprotection reaction proceeded simply and rapidly using water and methanol at room temperature. The absolute configuration of chiral molecules was preserved throughout the procedure. No significant molecular loss or analytical bias was observed. This new strategy was applied to samples of astrobiological interest to provide both a broad molecular mapping and detailed data on indigenous compounds by decreasing misinterpretation. Application to meteoritic samples highlighted the efficacy and relevance of the proof of concept.

## 1. Introduction

Unravelling the complexity of organics in extraterrestrial environments requires returned samples, especially if preliminary *in situ* analyses yield positive results [1–5]. *In situ* and laboratory analyses face different constraints. The former suffers from low sensitivity owing to miniaturised automated instrumentation operating under extreme conditions, while the latter risks terrestrial contamination. Both types of analyses are, however, mandatory and complementary. Understanding how protocol might affect materials in their native condition becomes important for developing adequate sample-processing strategies [6,7] Until sample return missions like JAXA’s Hayabusa 1 and 2, which returned asteroid samples [8–10], meteorites were the primary representatives of extraterrestrial mineral matrices. Their analysis revealed space molecular diversity and supported the development of analytical protocols for returned samples. Carbonaceous chondrites, a carbon-rich type of meteorite, represent less than 4 % of meteoritic falls on Earth [11,12]. Their diversity in organic molecules, which are of extraterrestrial origin while not entirely free from terrestrial contamination

[13–15], has rendered them a focus of astrobiological studies. Such rare samples must be preserved. New analytical strategies should use a minimal amount of sample for destructive analysis, combine multiple analysis methods and reuse the same sample extract (using “leftovers” as new sample) when possible. Although several integrative strategies have already simplified analytical workflows [16,17], future analyses may benefit from emerging developments, including automation [18].

Hence, performing both GC- and LC-MS/MS analyses on a single sample provides a more confirmatory identification of extraterrestrial molecules. While LC-MS is the technique of choice for complex, high molecular weight compounds, gas chromatography-tandem mass spectrometry (GC-MS/MS) is clearly preferred when life building blocks are to be targeted. GC, however, requires a derivatisation step to volatilise main astrobiological targets such as amino acids. Silylation has been selected for *in situ* GC analysis of Martian samples [19–22] owing to their characteristic fragmentation patterns, high-mass molecular ion derivatives, low limits of detection, and compatibility with fully automated procedures [23–25]. The silylation-GC-MS/MS strategy enables the detection of amino acids, carboxylic acids, and nucleobases in a

<footnote><sup>a</sup> University of Poitiers, UMR CNRS 7285, Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP), 4 rue Michel-Brunet, TSA 51106, 86073, France</footnote>

<footnote><sup>b</sup> Centre National d'Etudes Spatiales (CNES), Service Laboratoires & Expertise, 18 Avenue Edouard Belin, CEDEX 9, 61401 Toulouse, France</footnote>

<footnote><sup>c</sup> Muséum National d'Histoire Naturelle (MNHN), Sorbonne University, UMR CNRS 7590, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), 57 Rue Cuvier, Case 52, 75231 Paris Cedex 5, France</footnote>

<footnote><sup>d</sup> University of Paris Est Créteil and University of Paris Cité, CNRS, LISA, F-94010 Créteil, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: claud Geffroy@univ-poitiers.fr (C. Geffroy-Rodier).</footnote>
single run and also allows the separation of numerous isomers, such as α/β/γ-aminobutyric acids, α/β-aminoisobutyric acids, valine/novaline/ isovaline, leucine/isoleucine, and serine/isoserine [26]. Silylation provides an initial molecular fingerprint with minimal sample handling, allowing direct comparison with in situ data.

Despite these advantages, less than 15 % of the silylated sample is typically used in analysis (no more than 6 µL/50 µL for triplicate injections). To circumvent this drawback, we developed a deprotection procedure to enable further analysis of the remaining sample. Accordingly, to detect trace levels of organics—particularly amino acids—this study aimed to establish a novel one-pot, two-step integrated analytical procedure. The developed method was applied to a CM-type chondrite meteorite sample, known for its extensive molecular diversity, to detect amino acids and nucleobases [21].

# 2. Experimental section

## 2.1. Reagents and chemicals

A standard amino acid solution contained 17 amino acids, including l-alanine, glycine, l-valine, l-leucine, l-isoleucine, l-proline, l-methionine, l-serine, l-threonine, l-phenylalanine, l-aspartic acid, l-glutamic acid, l-lysine, l-tyrosine, l-cystine, l-arginine, and l-histidine. All amino acids were present at a concentration of 2.5 × 10⁻³ mol/L in 0.1 N HCl, except l-cystine, which was present at a concentration of 1.25 × 10⁻³ mol/L. Individual pure lyophilised powders of amino acids were also used, including sarcosine, β-alanine, N-ethylglycine, 2-aminoisobutyric acid, γ-aminobutyric acid, 5-aminovaleric acid, l-2-aminobutyric acid, l-3-aminoisobutyric acid, dl-3-aminobutyric acid, l-alloisoleucine, l-isoserine, d-alanine, d-2-aminobutyric acid, d-3-aminoisobutyric acid, d-valine, d-norvaline, d-homoserine, d-isoleucine, d-norleucine, d-alloisoleucine, d-glutamic acid, d-methionine, d-proline, and d-phenylalanine, all with purity ≥97 % (Sigma Aldrich). The purity of l-norvaline, d-leucine, l-norleucine, l-homoserine, and d-aspartic acid was ≥99 % (Alfa Aesar, Ward Hill, USA). The purity of l-isovaline, d-isovaline, serine, and d-threonine was ≥97 % (Acros Organics, Geel, Belgium), and the purity of l-β-leucine and d-β-leucine was ≥97 % (Chem-Impex, Wood Dale USA). Nucleobases such as adenine, guanine, thymine, cytosine, and uracil were also obtained as lyophilised powders (≥ 97 % purity). The purity of dipeptides, including glycylglycine (Gly-Gly), glycylalanine (Gly-Ala), alanylglycine (Ala-Gly), alanylalanine (Ala-Ala), and leucylleucine (Leu-Leu), was ≥98 % (Sigma Aldrich) and that of 2,5-diketopiperazine was ≥98 % (Acros Organics). Leucylleucine was used as LC-HRMS internal standard. Standard stock solutions (0.01 M) were prepared for each compound using Milli-Q water (18.2 MΩ·cm-1).

A 0.1 mM or 0.01 mM aqueous solution containing glycyl tri-, tetra, and pentapeptides (Sigma-Aldrich, purity >98 %), alanyl tetra- (Sigma-Aldrich, purity >98 %) and hexapeptides (Bachem, Bubendorf, Suisse, purity >95 %) was prepared. β-lactamase IV (from *Enterobacter cloacae*) and β-glucuronidase (from *Escherichia coli* K12) were purchased from Sigma Aldrich, while sequencing grade modified trypsin (20 µg) was purchased from Promega Corp (Madison, WI, USA). β-Lactamase and β-glucuronidase, respectively at 100 µM and 6 µM in aqueous solution, were subjected to trypsin digestion. For that, protein disulfide bridges were reduced with 15 µL of a 100 mM DL-dithiothreitol (> 99 %, Sigma-Aldrich) solution at 60 °C for 60 min and alkylated with 15 µL of a 240 mM iodoacetamide solution (> 99 %, Sigma-Aldrich) at room temperature for 45 min in the dark. Finally, they were digested for 24 h at 37 °C using a 1:10 (w/w) trypsin-to-protein ratio. The digestion was stopped with formic acid (0.4 % final concentration).

N-Methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA), methanol (MeOH), dimethylformamide (DMF), methyl chloroformate (MCF), pyridine, and trifluoroacetic acid (TFA) required for derivatisation and subsequent analysis were of analytical grade (≥ 98 %, Sigma Aldrich). The purity of chloroform (Acros Organics) was ≥99 %. n-Pentadecane and l-alanine-1-¹³C (≥ 99 %, Sigma Aldrich)

were used as internal standards for GC-MS and LC-MS analysis, respectively.

## 2.2. Derivatisation reactions

Standard solutions of each compound used for silylation were prepared at a concentration of 2.5 mM in Milli-Q water and further diluted in Milli-Q water. A 50 µL aliquot of the solution was evaporated to dryness under a gentle nitrogen stream at 40 °C. Derivatisation of the dry residue was performed using 30 µL of MTBSTFA and 10 µL of DMF. After 30 s of sonication, the mixture was heated at 75 °C for 30 min. An internal standard was added to the mixture (10 µL of 0.1 mM n-pentadecane in chloroform) and 1 µL was used to wash syringe and 1 µL was injected.

For chloroformate derivatisation, 15 µL of MCF was added to a mixture containing 50 µL of amino acid solution, 30 µL of methanol, and 15 µL of pyridine. The derivatives were extracted with 25 µL of chloroform by vigorous shaking, followed by centrifugation. A 1 µL aliquot of the chloroform layer was injected.

## 2.3. Deprotection reaction (desilylation)

First, one-step experiments were conducted. To the silylated sample, 100 µL of H₂O, 100 µL of MeOH, or 100 µL of H₂O:MeOH (1:1, v/v) was added, and the resulting solution was left at room temperature for 5–60 min. Then, a two-step procedure was employed: 100 µL of H₂O was added to the silylated sample and allowed to rest for 1 h, then evaporated to dryness under a gentle stream of dry nitrogen at 40 °C, followed by the addition of 100 µL of MeOH allowed to rest for 1 h. Regardless of the procedure used, the sample was evaporated to dryness and either silylated or solubilised in 50 µL of water for direct LC injection.

## 2.4. Meteorite sample analysis

A fragment of the Murchison meteorite was subjected to ultrasonic extraction with H₂O:MeOH (1:1, v/v) (see Supporting Information for additional details) [27]. 500 µL of the extract, corresponding to the extraction of 250 mg of meteorite, was evaporated to dryness analysed. The sample was silylated and a total of 3 µL of the silylated sample was used for GC-MS/MS analysis (syringe washes: 2 µL; injection: 1 µL). The remaining 47 µL silylated sample was deprotected using the optimised protocol: 100 µL of H₂O + MeOH (1:1, v/v) for 30 min, followed by evaporation to dryness at 40 °C under a nitrogen stream. Subsequently, 47 µL of water and 5 µL of an aqueous solution of l-alanine-1-¹³C (5 × 10⁻⁵ M) were added as the internal standard, and 5 µL of the sample was injected. Finally, the remaining part of the aliquot was evaporated and used for MCF/MeOH derivatisation. A total of 3 µL was used for MCF/MeOH GC-MS/MS analysis. Calibration curves and blanks were prepared following the same protocols. Procedural blank was analysed by the 4 different methods. Another 500 µL of the extract solubilised in 47 µL of water with 5 µL of an aqueous solution of l-alanine-1-¹³C (5 × 10⁻⁵ M) was injected directly into the LC-MS/MS system.

# 3. Instrumentation

## 3.1. GC-MS/MS analysis

Analysis was carried out on a TSQ 9000 Thermo Scientific GC-triple quadrupole system equipped with an HP5-MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness; Agilent Technologies, Santa Clara, CA, USA). Helium (≥ 99.9999 % purity, Air Liquide, Paris, France) flow was set at 1 mL min⁻¹ with a split ratio of 10:1. The temperature was increased from 90 to 180 °C at 6 °C min⁻¹, held for 5 min, then increased to 300 °C at 10 °C min⁻¹, and held for 8 min. Injector and detector temperatures were set at 280 °C. Multiple reaction monitoring (MRM) mode was used following prior optimisation of transitions and collision
energies on silylated standard solutions (see Table S1 for retention times, MRM transitions, and LODs). Data were processed using the TraceFinder software. One microlitre of the sample was injected.

For the enantiomeric separation of amino acids, chloroformate-
derivatised amino acids were analysed on a TQ7010 Agilent GC-triple
quadrupole system equipped with a CP-Chirasil-Dex (25 m × 0.25
mm, 0.25 µm film thickness; Agilent Technologies) capillary column.
The carrier gas, helium (≥99.9999 % purity, Air Liquide), was flowed at
1 mL min⁻¹, with a 20:1 split ratio. The oven temperature program was
set as follows: 100 °C (5 min) to 128 °C (3 °C min⁻¹), then to 137 °C
(1 °C min⁻¹), and finally to 190 °C (10 min; 3 °C min⁻¹). MRM mode was
also used for MS² analyses after optimisation of transitions and collision
energies on standard solution mixtures (see Table S2). Data were pro-
cessed using the MassHunter software. One microlitre of the sample was
injected.

3.2. UPLC–MS analysis

Desilylated compounds were analysed by liquid chromatography
coupled with mass spectrometry. Five microlitres of the sample was
injected into a Shimadzu Nexera X2 UPLC-8050 triple quadrupole mass
spectrometry system (Shimadzu, Kyoto, Japan) equipped with a
Crownpak CR guard column (10 mm × 4.0 mm, 5 µm film thickness;
Chiral Technologies Europe, Illkirch-Graffenstaden, France) in line with
a Crownpak CR(+) chiral analytical column (150 mm × 4.0 mm, 5 µm
film thickness; Chiral Technologies). The mobile phase was H₂O:MeOH
(95:5, v/v) with 0.5 % TFA. Separation was carried out under isocratic
conditions at a flow rate of 650 µL min⁻¹. MRM transitions and collision
energies were previously optimised using standard solutions [28]. Data
were processed using the LabSolution software. Analysis of peptides
were performed on Vanquish Ultra-Performance Liquid Chromatog-
raphy (UPLC) pumps interfaced with a High-Resolution Mass Spec-
trometer (Exploris 240, Hybrid Quadrupole-Orbitrap Mass
Spectrometer, Thermo Fisher Scientific, Waltham, MA, USA). Com-
pound ionization was performed by ElectroSpray Ionization (ESI). A
stack cooler allows samples storage at 5 °C before analysis. Freestyle
data system (Thermo Fisher Scientific) controls UPLC mobile phases and
MS functions. For accurate mass calibration, a Pierce FlexMix Calibra-
tion Solution is injected every day (Fisher Scientific, Waltham, MA,
USA).

Glycine and alanine peptides were separated on a Hydrophilic
Interaction Liquid Chromatography (HILIC) column. For that, an Acq-
uity UPLC BEH amide analytical column (150 mm × 2.1 mm, 1.7 µm;
Waters Corporation, Milford, MA) was used. To achieve compound
identification, 10 µL of a standard solution containing glycyl and alanyl
peptides at 1.10⁻⁶ mol.L⁻¹ were injected. To obtain an efficient sepa-
ration, samples had to be diluted in the mobile phase B (i.e. acetonitrile:
water; 85:15).

Elution was performed at a constant flow of 300 µL.min⁻¹ at 30 °C.
Water with 0.15 % formic acid and 10 mmol.L⁻¹ ammonium formate
was used as mobile phase A and acetonitrile:water (85:15) with 0.5 %
formic acid as mobile phase B. Gradient program was used as follows:
0–6 min, 100 % of B; 6–6.8 min, 100–95 % of B; 6.8–7.8 min, 95–80 % of
B; 7.8–11.8 min, 80–70 % of B; 11.8–12.8 min 70–60 % of B. The column
was then reconditioned for 3.2 min with 100 % of B.

Peptides resulting from β-lactamase, β-glucuronidase and trypsin
digestion were separated on a C18 hydrophobic column (Hypersil Gold
Q C18, 150 × 2.1 mm ID, 1.9 µm, 175 Å; Thermo Fisher Scientific,
Waltham, MA, USA). Elution was performed at a constant flow of 500
µL.min⁻¹ at 30 °C. Water with 0.1 % formic acid and 10 mmol.L⁻¹ of
ammonium formate was used as mobile phase A and acetonitrile with
0.1 % formic acid as mobile phase B. Gradient program was used as
follows: 0–0.7 min, 10 % of B; 0.7–1.2 min, 10–15 % of B; 1.2–2.05 min,
15 % of B; 2.05–3.5 min, 15–20 % of B; 3.5–9.5 min 20–55 % of B;
9.5–10 min, 55–60 % of B; 10–11 min 60 % of B; 11–11.5 min 60–95 %
of B; 11.5–12 min 95 % of B. The column was then reconditioned for 3

min with 10 % of B.

Mass spectrometry detection was done in a full scan mode to provide
a non-targeted fingerprint of peptides (Fig. 3, Fig. S4 and Table S1). The
electrospray voltage was set at 3.25 kV, the capillary temperature held
at 400 °C and the heater temperature at 450 °C. Sheath, sweep and
auxiliary gas flow rates were set at 20, 0 and 5, respectively (arbitrary
units). The Exploris 240 worked at 90000 resolution level with an AGC
target of 3.10⁶, a max IT of 200 ms and a mass range of 73–1090 m/z.

Total Ion Current chromatograms (TICs) were plotted with the Origin software (Origin 9.0.0, OriginLab Corporation, Northampton, USA) (Fig. 3).

4. Results and discussion

N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide silylation, selected for past and future Martian missions, is useful for single-run analysis, as it allows the detection of amino acids, nucleobases, and dipeptides in a single run. However, FTICR-MS and LC-MS/MS analyses provide broader coverage for high molecular weight compounds [8,19].

4.1. Optimisation of the deprotection reaction

4.1.1. Amino acids

To perform simple, fast, and effective deprotection without any
racemisation, a room-temperature procedure was optimised. Within this
framework, several experiments were conducted on proteinogenic
amino acids. First, the efficacy of water and methanol as deprotecting
agents was evaluated (Fig. 1). For this, a solution of amino acids was
prepared, silylated, and analysed by GC-MS/MS. The resulting signals
are reported as the silylation reference signals. To evaluate the yields of
deprotection, silylated amino acids were subsequently deprotected
using water, methanol, or a combination of both. To enable GC-MS
analysis, these desilylated amino acids were derivatised again. Peak
areas were finally compared to the silylation reference peak area (set at
100 %).

There was no major loss of amino acids resulting from the depro-
tection procedure, since all amino acid recoveries were almost quanti-
tative (Fig. 1). Treatments with both methanol and water gave slightly
better results than either solvent alone. The two-step procedure (H₂O
addition, followed by MeOH addition) could facilitate the evaporation of
residual water. However, this procedure was more time-consuming (2 h)
than the one-step deprotection process with water and methanol (H₂O
+ MeOH, 1:1, v/v). As the number of steps is a critical factor for auto-
mation, the one-step approach was preferred for further experimenta-
tion. Hence, a comparative study was performed to test different
durations ranging from 5 to 60 min. Results demonstrated that the
deprotection process was not significantly affected beyond 30 min
(Fig. S1). Deprotection for 30 min with H₂O + MeOH was considered a
suitable compromise between yield and duration—the time was set at
30 min.

To shed light on the chirality of extraterrestrial samples, race-
misation of chiral molecules such as amino acids must be avoided. Once
the deprotection conditions were optimised, experiments were con-
ducted on several enantiomerically pure amino acids. They were first
silylated and then deprotected using the optimal conditions as defined
above (i.e., H₂O + MeOH (1:1, v/v) for 30 min). The deprotected amino
acids were then directly analysed by LC-MS/MS using a chiral column.
No effect of silylation and deprotection by-products was observed on
chromatographic elution or ESI ionization, except for peak broadening
in *D*-norvaline (Fig. 2). The standard deviation was lower for *D*-amino
acids, which eluted first. No trace of *D*-amino acids was detected for the
deprotection of the *L*-amino acid mixture. Similarly, no *L*-amino acids
were detected during the deprotection of the *D*-amino acids. Therefore,
we assumed that no enantiomeric conversion occurred during the entire
procedure (Fig. S2).
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Yields of recovery of 17 deprotected amino acids. Deprotection was performed at room temperature for 60 min in one step with H₂O, MeOH, H₂O + MeOH (1:1, v/v), or in two steps with (1) H₂O and (2) MeOH (60 min each). Second silylation was performed after evaporation to dryness (n = 6; error bars correspond to standard deviations). Reference was a single silylation experiment; yield was set at 100 %.</figcaption></figure>

### 4.1.2. Nucleobases and peptides

Following the optimisation of the deprotection protocol for amino acids, the same protocol was tested on potential targets such as nucleobases and small peptides [13,21,27]. Solutions of 5 nucleobases and 6 peptides were prepared, silylated, and deprotected following the optimised protocol defined previously (i.e., H₂O + MeOH for 30 min). To allow their GC-MS/MS analysis, the deprotected samples were derivatised. Peak areas were compared to the silylation reference. There was no significant difference in nucleobase recoveries, but higher standard deviations were observed (Fig. S3). Variability reached up to 28 %, even for the reference. Such variability suggested a lack of reproducibility in nucleobase silylation rather than a bias introduced by the deprotection step. No significant difference between the deprotected peptides and the reference sample was observed either. This procedure could therefore be relevant for such biomolecules.

Furthermore, we evaluated the effect of the silylation/desilylation protocol on background molecules that are typically present when analysing natural samples. We specifically focused on compounds that could be partially silylated but remain non-volatile, making them undetectable by GC-MS. For this purpose, we applied the silylation/desilylation protocol to a complex mixture consisting of oligopeptides and a protein digest that are not targeted but could induce interferences. As illustrated in Fig. S4, the chromatographic profile remained largely unchanged before and after the procedure. Additionally, no shift in peptide retention times was observed (Fig. 3). Overall, the protocol did not significantly affect peptide signal intensities (area variation <15 %). However, we observed a notable decrease in the signal of one long β-glucuronidase peptide (IAVPGSFNDQFADADIR) and both trypsin peptides (VATVSLPR and IQVR) following desilylation. Given that peptides contain multiple silylation sites with arginine as C terminal amino acid, it is likely that desilylation was incomplete, resulting in a shift in their exact masses. If the procedure is to be used for biological complex mixture deprotection should be further optimised for arginine terminal residue. Therefore, we conclude that the silylation/desilylation procedure has a minimal impact on background molecules, which can still be effectively detected using various hyphenated analytical methods.

Hence, deprotection was quantitative for targeted molecules (amino acids, nucleobases, and peptides), with no interference from by-products in the LC-MS/MS analysis of pure standards. To be relevant for astrobiology, the strategy must, however, be evaluated on meteorite samples to assess potential matrix effects.

### 4.2. Application to Murchison meteorite

Our optimisation study shows that samples can be silylated, deprotected, and further analysed by LC-MS/MS. This protocol was applied to a real extraterrestrial sample. Hence, an aliquot of a Murchison meteorite aqueous extract was silylated for GC-MS/MS analysis and deprotected following the optimised protocol. The deprotected sample was then analysed by LC-MS/MS. An aliquot of the same extract was also directly analysed by LC-MS/MS for comparison. In all cases, no peptide used in the deprotection optimization was detected.

For comparison, we selected 30 amino acids and 5 nucleobases. Identified targets and their respective concentrations for each analysis are presented in Table 1/S1.

Interestingly, no compound was exclusively identified with direct LC-MS/MS, showing that GC and LC analyses provide complementary data. Twenty-four targeted molecules were identified by silylation GC-MS/MS, while 16 molecules were detected by direct chiral LC-MS/MS analysis. Silylation GC-MS/MS enabled the detection of glycine and nucleobases, which were not detected by the LC-MS/MS analysis. This confirmed the necessity of combining both analytical methods. Retention time shifts were observed in the LC-MS/MS analysis compared to standard solution experiments. However, unequivocal identification of each compound was achieved by spiking the sample with standard solutions (Fig. 4).

Deprotection resulted in no loss of targets (Table 1). The overall determined amounts were also in the same range for all the compounds but close to the LOQ levels (Table S1). D/L ratios for each target were different for the two LC-MS/MS experiments, but the same trend of D/L < 0.6 was observed. DL-serine and 5-aminovaleric acid were not detected in the direct LC-MS/MS analysis, at least above the detection limit. They were detected in GC-MS/MS and deprotected LC-MS/MS. Although contamination cannot be excluded, they were not detected in blank samples. Conversion of native molecules, to the best of our knowledge, has not been reported for serine and 5-aminovaleric acid, unlike arginine, which is detected as ornithine derivatives [29]. This difference in detection could more likely be a result of the silylation/deprotection procedure, which modifies the matrix effect.

To further validate the relevance of the present strategy, an additional experiment was conducted. Methyl chloroformate/methanol (MCF/MeOH)-GC-MS/MS analysis was selected because the effective enantiomeric separation of amino acids could not be achieved in our LC-MS/MS configuration (i.e., C₄ amino acids such as amino(iso)butyric acids) and because of its relevance for GC-combustion isotope ratio
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** LC-MS/MS chromatographic responses of L- and D- enantiomerically pure amino acids after deprotection (30 min, room temperature, H₂O + MeOH (1:1, v/v)) compared to that of the reference (direct analysis labelled*).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** LC-MS/MS extracted ion chromatogram (EIC) of deprotected peptides (5 µL of standard solution (10⁻⁶ M).</figcaption></figure>

mass spectrometry (GC-C-IRMS), a method widely used in astrobiology to determine the origin of amino acids [30,31]. C₄ amino acid enantiomers were identified and quantified in the concentration range used for LC-MS/MS. Acidic amino acids were not detected owing to a higher detection threshold. L-Methionine was the only target identified in the Murchison and procedural blank samples analysed by (MCF/MeOH)-GC-MS/MS. This amino acid was thus not included in Table 1 as it was not detected by any GC and LC analyses. This contamination was then attributed to the MCF/MeOH derivatization procedure.

Thus, combining GC and LC-MS/MS experiments on the same sample enables discrimination of some isomers and broadens the scope of detected molecules.

## 5. Conclusion

In this study, we demonstrated a practical and reproducible workflow for molecular deprotection and analysis, maintaining stereochemical integrity—an essential aspect for chiral biomarker detection in astrobiology. No major loss of molecules or racemisation was observed. This method is fast and simple, with no interference from subsequent analyses performed on the deprotected sample by LC-MS/MS or alternative derivatisation-based GC-MS/MS methods. It enabled at least three complementary analyses on the same sample. The work also has extended the analytical application of deprotection strategies to real-world extraterrestrial matrices, showing potential for sample handling in future space missions. The silylation/deprotection procedure was successfully applied to a Murchison extract, allowing enantiomeric and isomeric separation and detection of 24 amino acids and 3 nucleobases from a single sample. For future Martian or comet return samples, primary results from silylation could facilitate decision-making in selecting the most appropriate destructive analysis. For more complex molecules such as oligopeptides and a protein digest additional study should be conducted to ascertain a quantitative recovery.

## Authors contribution

C.G-R. directed and supervised the entire study; C.G-R. conceptualised the analysis strategy; R.T. and C.B performed the

<table><caption>**Table 1**
Comparison of identified amino acids and nucleobases in the Murchison extract by each type of analysis.</caption><thead><tr><th>Target compounds</th><th>TBDMS<br>GC-MS/<br>MS</th><th>Direct<br>LC-MS/MS</th><th>Deprotection<br>LC-MS/MS</th><th>MCF/<br>MeOH<br>GC-MS/<br>MS</th></tr></thead><tbody><tr><td>Glycine</td><td>✓</td><td>n.d</td><td>n.d</td><td>✓</td></tr><tr><td>Sarcosine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>D-Alanine</td><td>✓</td><td></td><td>✓</td><td>✓</td></tr><tr><td>L-Alanine</td><td></td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>β-Alanine</td><td>✓</td><td></td><td></td><td>n.d</td></tr><tr><td>N-Ethylglycine</td><td>n.d</td><td></td><td></td><td>✓</td></tr><tr><td>2-Aminoisobutyric<br>acid</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>D-2-Aminobutyric<br>acid</td><td></td><td></td><td></td><td>✓</td></tr><tr><td>L-2-Aminobutyric<br>acid</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>DL-3-Aminobutyric<br>acid</td><td>✓</td><td>n.d</td><td>n.d</td><td>n.d</td></tr><tr><td>D-3-Aminoisobutyric<br>acid</td><td></td><td></td><td></td><td>✓</td></tr><tr><td>L-3-Aminoisobutyric<br>acid</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>γ-Aminobutyric acid</td><td>✓</td><td></td><td></td><td>✓</td></tr><tr><td>D-Serine</td><td>✓</td><td>n.d</td><td>✓</td><td>x</td></tr><tr><td>L-Serine</td><td></td><td></td><td></td><td>x</td></tr><tr><td>D-Proline</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>L-Proline</td><td></td><td></td><td></td><td></td></tr><tr><td>D-Isovaline</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>L-Isovaline</td><td></td><td></td><td></td><td></td></tr><tr><td>D-Valine</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>L-Valine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>5-Aminovaleric acid</td><td>✓</td><td>n.d</td><td>✓</td><td>n.d</td></tr><tr><td>L-Threonine</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>D-Threonine</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>D-β-Leucine</td><td></td><td></td><td></td><td>n.d</td></tr><tr><td>L-β-Leucine</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>D-Isoleucine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>L-Isoleucine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>D-Leucine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>L-Leucine</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>D-Aspartic acid</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>L-Aspartic acid</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>D-Glutamic acid</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>L-Glutamic acid</td><td>✓</td><td>✓</td><td>✓</td><td>n.d</td></tr><tr><td>Uracil</td><td>✓</td><td>n.d</td><td>n.d</td><td>x</td></tr><tr><td>Adenine</td><td>✓</td><td>n.d</td><td>n.d</td><td>x</td></tr><tr><td>Guanine</td><td>✓</td><td>✓</td><td>✓</td><td>x</td></tr></tbody></table>

n.d: not detected.

x: cannot be detected (was not among targeted molecules for the analysis).

experiments and collected and processed the data; B.L. and L.R. provided the meteorite fragment and visualised sample treatment and analysis; M.D. supervised silylation/desilylation procedure; B.G., G.R., P.P. supervised the project and reviewed the paper; R.T. wrote the paper with contribution of all authors. All authors have given approval to the final version of the paper.

## CRediT authorship contribution statement

Ramzi Timoumi: Formal analysis, Data curation. Cedrick Bourseau: Formal analysis, Data curation. Boris Laurent: Writing – review & editing. Laurent Remusat: Writing – review & editing, Supervision, Funding acquisition. Marc David: Formal analysis. Guillaume Rioland: Supervision, Funding acquisition. Brian Grégoire: Writing – review & editing, Supervision. Pauline Poinot: Writing – review & editing, Supervision. Claude Geffroy-Rodier: Writing – original draft, Supervision, Methodology, Conceptualization.
<figure><img src="image_6.png" /><figcaption>Fig. 4. LC-MS/MS extracted ion chromatogram (EIC) of the deprotected Murchison sample and corresponding spiked sample (5 µL of standard solution (10⁻⁶ M)).</figcaption></figure>

## Funding sources

L.R. and B.L. are grateful to the European Research Council for funding (ERC Consolidator Grant No. 819587: HYDROMA-PI: L. Remusat). C.G. and P.P. are grateful to financial support from the French National Space Agency (CNES) [R&T RS11/SU-0006-013, RS15/SU-0006-20, 50 % PhD grant], the Nouvelle-Aquitaine Region PhD grant (50 %), and PEPR Origin (ANR-22-EXOR-0014-Plan France 2023).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors thank the Natural History Museum of London, especially Dr. Natasha Almeida and Dr. Helena Bates, for the Murchison meteorite fragment loan.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.microc.2025.115530.

## Data availability

Data will be made available on request.

## References

[1] M. Millan, S. Teinturier, C.A. Malespin, J.Y. Bonnet, A. Buch, J.P. Dworkin, J. L. Eigenbrode, C. Freissinet, D.P. Glavin, R. Navarro-González, A. Srivastava, J. C. Stern, B. Sutter, C. Szopa, A.J. Williams, R.H. Williams, G.M. Wong, S. S. Johnson, P.R. Mahaffy, Organic molecules revealed in Mars's Bagnold dunes by curiosity's derivatization experiment, Nat. Astron 6 (2022) 129–140, https://doi.org/10.1038/s41550-021-01507-9.

[2] R. Fkiri, R. Timoumi, G. Rioland, P. Poinot, F. Baron, B. Gregoire, C. Geffroy-Rodier, Gas chromatography fingerprint of Martian amino acids before analysis of return samples, Chemosensors 11 (2023) 76, https://doi.org/10.3390/chemosensors11020076.

[3] J.L. Eigenbrode, R.E. Summons, A. Steele, C. Freissinet, M. Millan, R. Navarro-González, B. Sutter, A.C. McAdam, H.B. Franz, D.P. Glavin, P.D. Archer, P. R. Mahaffy, P.G. Conrad, J.A. Hurowitz, J.P. Grotzinger, S. Gupta, D.W. Ming, D. Y. Sumner, C. Szopa, C. Malespin, A. Buch, P. Coll, Organic matter preserved in 3-billion-year-old mudstones at Gale crater, Mars, Science 360 (2018) 1096–1101, https://doi.org/10.1126/science.aas9185.

[4] K.T. Tait, F.M. McCubbin, C.L. Smith, C.B. Agee, H. Busemann, B. Cavalazzi, V. Debaille, A. Hutzler, T. Usui, G. Kminek, M.A. Meyer, D.W. Beaty, B.L. Carrier, T. Haltigin, L.E. Hays, C.S. Cockell, D.P. Glavin, M.M. Grady, E. Hauber, B. Marty, L.M. Pratt, A.B. Regberg, A.L. Smith, R.E. Summons, T.D. Swindle, N.J. Tosca, A. Udry, M.A. Velbel, M. Wadhwa, F. Westall, M.-P., Zorzano, preliminary planning for Mars sample return (MSR) curation activities in a sample receiving facility (SRF), Astrobiology 22 (2022) S–57, https://doi.org/10.1089/ast.2021.0105.

[5] C. Freissinet, D.P. Glavin, P.D. Archer, S. Teinturier, A. Buch, C. Szopa, J.M.T. Lewis, A.J. Williams, R. Navarro-Gonzalez, J.P. Dworkin, Heather.B. Franz, M. Millan, J.L. Eigenbrode, R.E. Summons, C.H. House, R.H. Williams, A. Steele, O. McIntosh, F. Gómez, B. Prats, C.A. Malespin, P.R. Mahaffy, Long-chain alkanes preserved in a Martian mudstone, Proceedings of the National Academy of Sciences 122 (2025) e2420580122. doi: https://doi.org/10.1073/pnas.2420580122.

[6] Z. Cieslarova, M.F. Mora, A.C. Noell, C.W. Parker, P.A. Willis, Effect of pH on the release of amino acids from microorganisms via subcritical water extraction, ACS Earth Space Chem. 8 (2024) 274–280, https://doi.org/10.1021/acsearthspacechem.3c00277.

[7] M.F. Mora, M.G.M. Kok, A. Noell, P.A. Willis, Detection of biosignatures by capillary electrophoresis mass spectrometry in the presence of salts relevant to ocean worlds missions, Astrobiology 22 (2022) 914–925, https://doi.org/10.1089/ast.2021.0091.

[8] H. Naraoka, Y. Takano, J.P. Dworkin, Y. Oba, K. Hamase, A. Furusho, N.O. Ogawa, M. Hashiguchi, K. Fukushima, D. Aoki, P. Schmitt-Kopplin, J.C. Aponte, E.T. Parker, D.P. Glavin, H.L. McLain, J.E. Elsila, H.V. Graham, J.M. Eiler, F.-R. Orthous-Daunay, C. Wolters, J. Isa, V. Vuitton, R. Thissen, S. Sakai, T. Yoshimura, T. Koga, N. Ohkouchi, Y. Chikaraishi, H. Sugahara, H. Mita, Y. Furukawa, N. Hertkorn, A. Ruf, H. Yurimoto, T. Nakamura, T. Noguchi, R. Okazaki, H. Yabuta, K. Sakamoto, S. Tachibana, H.C. Connolly, D.S. Lauretta, M. Abe, T. Yada, M. Nishimura, K. Yogata, A. Nakato, M. Yoshitake, A. Suzuki, A. Miyazaki, S. Furuya, K. Hataakeda, H. Soejima, Y. Hitomi, K. Kumagai, T. Usui, T. Hayashi, D. Yamamoto, R. Fukai, K. Kitazato, S. Sugita, N. Namiki, M. Arakawa, H. Ikeda, M. Ishiguro, N. Hirata, K. Wada, Y. Ishihara, R. Noguchi, T. Morota, N. Sakatani, K. Matsumoto, H. Senshu, R. Honda, E. Tatsumi, Y. Yokota, C. Honda, T. Michikami, M. Matsuoka, A. Miura, H. Noda, T. Yamada, K. Yoshihara, K. Kawahara, M. Ozaki, Y. Iijima, H. Yano, M. Hayakawa, T. Iwata, R. Tsukizaki, H. Sawada, S. Hosoda, K. Ogawa, C. Okamoto, N. Hirata, K. Shirai, Y. Shimaki, M. Yamada, T. Okada, Y. Yamamoto, H. Takeuchi, A. Fujii, Y. Takei, K. Yoshikawa, Y. Mimasu, G. Ono, N. Ogawa, S. Kikuchi, S. Nakazawa, F. Terui, S. Tanaka, T. Saiki, M. Yoshikawa, S.
Watanabe, Y. Tsuda, Soluble organic molecules in samples of the carbonaceous asteroid (162173) Ryugu, Science 379 (2023) eabn9033. Doi: https://doi.org/10.1126/science.abn9033.

[9] T. Yada, M. Abe, T. Okada, A. Nakato, K. Yogata, A. Miyazaki, K. Hataakeda, K. Kumagai, M. Nishimura, Y. Hitomi, H. Soejima, M. Yoshitake, A. Iwamae, S. Furuya, M. Uesugi, Y. Karouji, T. Usui, T. Hayashi, D. Yamamoto, R. Fukai, S. Sugita, Y. Cho, K. Yumoto, Y. Yabe, J.-P. Bibring, C. Pilonget, V. Hamm, R. Brunetto, L. Riu, L. Lourit, D. Loizeau, G. Lequertier, A. Moussi-Soffys, S. Tachibana, H. Sawada, R. Okazaki, Y. Takano, K. Sakamoto, Y.N. Miura, H. Yano, T.R. Ireland, T. Yamada, M. Fujimoto, K. Kitazato, N. Namiki, M. Arakawa, N. Hirata, H. Yurimoto, T. Nakamura, T. Noguchi, H. Yabuta, H. Naraoka, M. Ito, E. Nakamura, K. Uesugi, K. Kobayashi, T. Michikami, H. Kikuchi, N. Hirata, Y. Ishihara, K. Matsumoto, H. Noda, R. Noguchi, Y. Shimaki, K. Shirai, K. Ogawa, K. Wada, H. Senshu, Y. Yamamoto, T. Morota, R. Honda, C. Honda, Y. Yokota, M. Matsuoka, N. Sakatani, E. Tatsumi, A. Miura, M. Yamada, A. Fujii, C. Hirose, S. Hosoda, H. Ikeda, T. Iwata, S. Kikuchi, Y. Mimasu, O. Mori, N. Ogawa, G. Ono, T. Shimada, S. Soldini, T. Takahashi, Y. Takei, H. Takeuchi, R. Tsukizaki, K. Yoshikawa, F. Terui, S. Nakazawa, S. Tanaka, T. Saiki, M. Yoshikawa, S. Watanabe, Y. Tsuda, Preliminary analysis of the Hayabusa2 samples returned from C-type asteroid Ryugu, Nat Astron 6 (2022) 214–220, https://doi.org/10.1038/s41550-021-01550-6.

[10] C. Potiszil, M. Yamanaka, C. Sakaguchi, T. Ota, H. Kitagawa, T. Kunihiro, R. Tanaka, K. Kobayashi, E. Nakamura, Organic matter in the asteroid Ryugu: what we know so far, Life 13 (2023) 1448, https://doi.org/10.3390/life13071448.

[11] D.N. Simkus, J.C. Aponte, J.E. Elsila, E.T. Parker, D.P. Glavin, J.P. Dworkin, Methodologies for Analyzing soluble organic compounds in Extraterrestrial samples: amino acids, amines, monocarboxylic acids, aldehydes, and ketones, Life 9 (2019) 47, https://doi.org/10.3390/life9020047.

[12] C. Meinert, P. Marcellus, L. Le Sergeant d'Hendcourt, L. Nahon, N.C. Jones, S.V. Hoffmann, J.H. Bredehöft, U.J. Meierhenrich, Photochirogenesis: photochemical models on the absolute asymmetric formation of amino acids in interstellar space, Phys. Life Rev. 8 (2011) 307–330. Doi: https://doi.org/10.1016/j.plrev.2011.08.005.

[13] M.P. Callahan, K.E. Smith, H.J. Cleaves, J. Ruzicka, J.C. Stern, D.P. Glavin, C.H. House, J.P. Dworkin, Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases, Proc. Natl. Acad. Sci. U.S.A 108 (2011) 13995–13998. Doi: https://doi.org/10.1073/pnas.1106493108.

[14] Y.V. Kissin, Hydrocarbon components in carbonaceous meteorites, Geochim. Cosmochim. Acta 67 (2003) 1723–1735, https://doi.org/10.1016/S0016-7037 (02)00982-1.

[15] L.D. Tunney, C.D.K. Herd, R.W. Hilts, Organic contamination on the surface of meteorites as a function of space and time: a case study of the buzzard coulee H4 chondrite, Meteorit. Planet. Sci. 55 (2020), https://doi.org/10.1111/maps.13551.

[16] C. Serra, J. Lange, Q.B. Remaury, R. Timoumi, G. Danger, B. Laurent, L. Remusat, C.G. Rodier, P. Poinot, Integrative analytical workflow to enhance comprehensive analysis of organic molecules in extraterrestrial objects, Talanta 243 (2022) 123324, https://doi.org/10.1016/j.talanta.2022.123324.

[17] J. Lange, F. Djago, B. Eddhif, Q.B. Remaury, A. Ruf, N.K.V. Leitner, L.L. S. Hendcourt, G. Danger, C.G. Rodier, S. Papot, P. Poinot, A novel proteomics-based strategy for the investigation of peptide sequences in Extraterrestrial samples, J. Proteome Res. 20 (2021) 1444–1450, https://doi.org/10.1021/acs.jproteome.0c00700.

[18] K. Thurow, Strategies for automating analytical and bioanalytical laboratories, Anal. Bioanal. Chem. 415 (2023) 5057–5066, https://doi.org/10.1007/s00216-023-04727-2.

[19] P. Schmitt-Kopplin, Z. Gabelica, R.D. Gougeon, A. Fekete, B. Kanawati, M. Harir, I. Gebefuegi, G. Eckel, N. Hertkorn, High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40 years after its fall, Proc. Natl. Acad. Sci. 107 (2010) 2763–2768, https://doi.org/10.1073/pnas.0912157107.

[20] Z. Martins, O. Botta, M.L. Fogel, M.A. Sephton, D.P. Glavin, J.S. Watson, J. P. Dworkin, A.W. Schwartz, P. Ehrenfreund, Extraterrestrial nucleobases in the Murchison meteorite, Earth Planet. Sci. Lett. 270 (2008) 130–136, https://doi.org/10.1016/j.epsl.2008.03.026.

[21] S. Pizzarello, G.W. Cooper, G.J. Flynn, The nature and distribution of the organic material in carbonaceous chondrites and interplanetary dust particles, Meteorites and the Early Solar System II (1) (2006) 625–651.

[22] Z. Martins, M.A. Sephton, Extraterrestrial Amino Acids, in: A.B. Hughes (Ed.), Amino Acids, Peptides and Proteins in Organic Chemistry, 1st ed, Wiley, 2009, pp. 1–42, https://doi.org/10.1002/9783527631766.ch1.

[23] F. Goesmann, W.B. Brinckerhoff, F. Raulin, W. Goetz, R.M. Danell, S.A. Getty, S. Siljeström, H. Mißbach, H. Steininger, R.D. Arevalo, A. Buch, C. Freissinet, A. Grubisic, U.J. Meierhenrich, V.T. Pinnick, F. Stalport, C. Szopa, J.L. Vago, R. Lindner, M.D. Schulte, J.R. Brucato, D.P. Glavin, N. Grand, X. Li, F.H.W. van Amerom, M.S.T. the, The Mars Organic Molecule Analyzer (MOMA) Instrument: Characterization of Organic Material in Martian Sediments, Astrobiology vol. 17 (2017) 655–685. Doi: https://doi.org/10.1089/ast.2016.1551.

[24] C. Rodier, R. Sternberg, F. Raulin, C. Vidal-Madjar, Chemical derivatization of amino acids for in situ analysis of Martian samples by gas chromatography, J. Chromatogr. A 915 (2001) 199–207, https://doi.org/10.1016/S0021-9673(01)00625-2.

[25] M. David, N.-Y. Musadji, J. Labanowski, R. Sternberg, C. Geffroy-Rodier, Pilot for Validation of Online Pretreatments for Analyses of Organics by Gas Chromatography–Mass Spectrometry: Application to Space Research, Anal. Chem. 88 (2016) 5137–5144. Doi: https://doi.org/10.1021/acs.analchem.6b00052.

[26] C. Meinert, U.J. Meierhenrich, Derivatization and multidimensional gas-chromatographic resolution of α-alkyl and α-Dialkyl amino acid enantiomers, ChemPlusChem 79 (2014) 781–785, https://doi.org/10.1002/cplu.201300328.

[27] R. Timoumi, P. François, A. Le Postollec, M. Dobrijevic, B. Grégoire, P. Poinot, C. Geffroy-Rodier, Focused ultrasound extraction versus microwave-assisted extraction for extraterrestrial objects analysis, Anal. Bioanal. Chem. 414 (2022) 3643–3651, https://doi.org/10.1007/s00216-022-04004-8.

[28] C. Serra, J. Lange, Q.B. Remaury, R. Timoumi, G. Danger, B. Laurent, L. Remusat, C.G. Rodier, P. Poinot, Integrative analytical workflow to enhance comprehensive analysis of organic molecules in extraterrestrial objects, Talanta (2022) 123324, https://doi.org/10.1016/j.talanta.2022.123324.

[29] J.M. Halket, D. Waterman, A.M. Przyborowska, R.K. Patel, P.D. Fraser, P. M. Bramley, Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS, J. Exp. Bot. 56 (2005) 219–243.

[30] S. Pizzarello, X. Feng, S. Epstein, J.R. Cronin, Isotopic analyses of nitrogenous compounds from the Murchison meteorite: ammonia, amines, amino acids, and polar hydrocarbons, Geochim. Cosmochim. Acta 58 (1994) 5579–5587, https://doi.org/10.1016/0016-7037(94)90251-8.

[31] R.G. Walsh, S. He, C.T. Yarnes, Compound-specific δ13C and δ15N analysis of amino acids: a rapid, chloroformate-based method for ecological studies, Rapid Commun. Mass Spectrom. 28 (2014) 96–108, https://doi.org/10.1002/rcm.6761.