In-time TMS derivatization and GC/MS determination of Sugars, Organic Acids and Amino Acids for High Throughput Metabolomics Studies

Význam tématu

Metabolomics aims to profile endogenous small molecules (<600 Da) in biological systems to understand responses to various stimuli. GC–MS, with its high chromatographic resolution, reproducibility and extensive spectral libraries, is widely used for analyzing sugars, amino acids, organic acids and other primary metabolites. However, GC–MS requires chemical derivatization to enhance volatility and thermal stability of polar analytes, and conventional batch derivatization workflows can introduce variability, prolong processing times and lead to degradation, especially in large sample sets.

Cíle a přehled studie / článku

This study demonstrates an automated in-time sample derivatization strategy using a GERSTEL MultiPurpose Sampler (MPS) robotic Pro coupled with an Agilent 7890B/5977B GC–MS system. The objectives are to:

• Minimize time differences between derivatization and injection to reduce degradation.
• Improve reproducibility (lower relative standard deviations, RSDs) across large batches (up to several hundred samples).
• Streamline reagent handling and reduce human intervention to enhance throughput and data quality.

Použitá metodika a instrumentace

Two-step derivatization comprises:

• Methoximation (MOX) to stabilize carbonyl groups of sugars and keto acids.
• Silylation with BSTFA + 1% TMCS to form trimethylsilyl derivatives.

Automation is achieved by programming a Maestro prep-sequence in PrepAhead mode that:

• Dispenses MOX reagent and mixes/incubates samples in an agitator.
• Adds BSTFA and an internal FAME standard, then mixes again.
• Imposes a controlled hold time (60–120 min) before injection.
• Switches syringes for reagent transfers (100 µL) and sample injection (10 µL) to minimize septum punctures.

Použitá instrumentace

• GERSTEL MPS robotic Pro with tool exchange, Peltier cooled stack, agitator and fast wash station.
• Agilent 7890B GC coupled to 5977B MSD with 2×15 m DB-5MS UI columns and backflush.
• Maestro software v1.5.3.67 for prep-sequence control and MassHunter GC/MS Acquisition and Qualitative Analysis for data processing.

Hlavní výsledky a diskuse

Automated in-time derivatization reduced RSDs for a mixed standard (14 metabolites) from up to 32.7% (offline) to 2.6–9.4% (in-time). Quantitation recovery at 250 ppb demonstrated tighter distribution and improved precision. The workflow supports 80 samples per run, expandable to 200–300 using multiple trays. Automation also cut manual preparation time significantly and optimized reagent usage.

Přínosy a praktické využití metody

• Enhanced data quality through consistent derivatization-to-injection timing.
• Higher throughput by scaling to hundreds of samples without multiple ThermoMixers.
• Reduced labor and reagent costs via precise automated dispensing and fewer manual steps.
• Lower variability in large-scale metabolomics studies, beneficial for clinical, nutrition and environmental research.

Budoucí trendy a možnosti využití

Future developments may include:

• Integration of Automated Liner Exchange (ALEX) for cleaner samples and extended maintenance intervals.
• Extension to larger polar analytes and integration with LC–MS workflows.
• Application of unknown identification using retention index libraries for discovery metabolomics.
• Adaptation to other derivatization chemistries or multi-omics platforms for comprehensive profiling.

Závěr

The GERSTEL MPS robotic Pro in-time derivatization workflow provides a robust solution to common GC–MS sample preparation bottlenecks in metabolomics. It achieves significant improvements in precision, throughput and labor efficiency, paving the way for reliable, high-capacity analysis of polar metabolites.

Reference

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• Sandra et al. 2014. Automated Sample Preparation for Metabolomics Studies Using the Gerstel MPS Dual Head Workstation. GERSTEL AppNote AN/2014/08.
• Sandra et al. 2015. Automated Sample Preparation for Metabolomics Studies Using the Gerstel MPS Dual Head Workstation. GERSTEL AppNote AN/2015/01.
• Sansone et al. 2007. The metabolomics standards initiative. Nature Biotechnology 25:846–848.
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