Investigating Ryugu Samples: From Preparation to Molecular Characterization

Sample Preparation and Initial Characterization

Ryugu A0480 (11.9 mg) and C0370 (8.3 mg) aggregate samples were carefully allocated by JAXA for detailed analysis. Prior to their selection, comprehensive MicrOmega hyperspectral microscope images and near-infrared reflectance spectra (2.0–4.0 μm) were obtained.

These Ryugu samples contain organic matter and show functional groups such as –OH, –NH, and –CH.

For comparative analysis, the CI1 Orgueil meteorite (30.6 mg) served as a vital reference. Its selection was based on its established mineralogical and elemental similarities to Ryugu samples. To ensure data integrity, procedural blanks were also prepared using baked sea sand (14.4 mg) and baked serpentine (8.8 mg).

Organic Molecule Extraction

Rigorous control was maintained throughout the extraction and purification processes. All procedures occurred in an ISO Class 5 clean bench situated within an ISO Class 6 clean room, minimizing contamination risks.

Water Extraction

Each sample was initially suspended in 550 μl of ultrapure water and subjected to 15 minutes of ultrasonication at 25 °C. Following this, centrifugation for 10 minutes at 10,000 rpm allowed for the collection of the supernatant. The remaining residue was then rinsed with an additional 200 μl of ultrapure water, and this rinse was combined with the initial supernatant. This combined volume, totaling approximately ~640 μl, was designated as the 'H2O extract'. Eighty percent of the combined H2O extracts from samples A0480 and C0370 were subsequently freeze-dried for further processing.

Acid Extraction (HCl)

The residues obtained from the water extraction underwent a more robust treatment. They were suspended in 600 μl of 6 M HCl, transferred to glass ampoules, purged with N2 gas, and flame-sealed. The sealed ampoules were then heated at 110 °C for 12 hours.

This acid extraction process specifically aimed to isolate molecules that might decompose during acid treatment, thereby broadening the range of detectable compounds.

After acid extraction and centrifugation, the supernatant was collected. The residues were washed twice with 200 μl of ultrapure water, and these rinses were combined with the supernatant, forming the 'HCl extract'. It's important to note that secondary minerals also dissolved during this step. The combined HCl extracts from A0480 and C0370 were then freeze-dried, mirroring the treatment of the H2O extracts.

Desalting and Fractionation

The freeze-dried H2O and HCl extracts were redissolved in 0.5 ml of 0.1 M HCl, a crucial step for desalting using cation-exchange chromatography. A 0.5-ml aliquot of AG 50W-X8 cation-exchange resin was meticulously packed into a glass Pasteur pipette and preconditioned before the extract solution was loaded onto the column.

Initially, 2.5 ml of ultrapure water was used to elute acidic, neutral, and weakly basic compounds, creating the 'H2O fraction'. Subsequently, 2.5 ml of 10% NH4OH was applied to elute basic compounds, including most nucleobases, which formed the 'NH4OH fraction'.

This systematic approach resulted in the creation of four distinct fractions (H2O–H2O, H2O–NH4OH, HCl–H2O, and HCl–NH4OH) from the Ryugu samples, ensuring a comprehensive separation of molecular classes.

Trace amounts of N-containing molecules were detected in procedural blank NH4OH fractions, indicating the high sensitivity of the subsequent analyses. These fractions were freeze-dried and reconstituted in 50–100 μl of ultrapure water in preparation for instrumental analysis. Notably, while an Orgueil meteorite fragment was extracted using the same water and HCl protocol, its H2O extract did not undergo cation-exchange chromatography.

Elemental and Isotopic Analysis

Carbon (C, wt%) and nitrogen (N, wt%) contents, along with their isotopic compositions (δ13C and δ15N), were precisely measured in residues from A0480, C0370, and Orgueil using an ultrasensitive nano-EA/IRMS system.

Sample masses utilized for this analysis were 0.063 mg (A0480), 0.097 mg (C0370), and 0.125 mg (Orgueil). Isotopic compositions were expressed in δ notation, referenced to Vienna Peedee Belemnite for carbon and atmospheric air for nitrogen. Calibration was rigorously performed using international and inter-laboratory standards to ensure accuracy.

Analysis of Nucleobases and N-containing Molecules

The H2O and HCl extracts from Ryugu and Orgueil, alongside blanks and reference compounds, underwent detailed analysis using a high-resolution online HPLC/ESI-HRMS system. This sophisticated setup comprised an UltiMate 3000 HPLC coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer.

For quantification, purine nucleobases were separated using an isocratic elution program with a pentafluorophenyl column. Pyrimidine nucleobases, on the other hand, were analyzed using a HyperCarb column, employing a linear gradient of water and acetonitrile, both containing 0.1% formic acid. The eluent then entered a HESI-II ion source, where full-scan mass spectra were collected in positive ion mode.

To ensure confident identification, tandem mass spectrometry (MS/MS) was specifically performed for guanine and cytosine detected in the HCl–NH4OH fraction of Ryugu C0370.

Further cross-validation of the organic molecule analysis in Ryugu extracts was conducted using CE-HRMS.

This involved a capillary electrophoresis system coupled to a Q Exactive Plus mass spectrometer, with spectral data acquired in positive ion mode, providing an additional layer of verification for the molecular findings.