Procedure for GC-MS Sample Preparation of Fungal Broth Culture

 

1. Sample Collection

• Fungal Broth Culture: Grow the fungal strain in a suitable liquid medium (e.g., Potato Dextrose Broth, Malt Extract Broth, or Czapek-Dox Broth) under optimized conditions (temperature, pH, and incubation time, typically 25–30°C for 5–14 days, depending on the fungal species).
• Sterility: Ensure all steps are performed under aseptic conditions to avoid contamination.
• Harvest: Collect the broth culture after the incubation period. Depending on the analysis, you may use the entire culture (mycelium + broth) or separate the mycelium from the broth via filtration or centrifugation.
  • Filtration: Use a vacuum filtration system with a 0.45 µm filter to separate the fungal biomass (mycelium) from the liquid broth.
  • Centrifugation: Centrifuge the culture at 8,000–10,000 rpm for 10–15 minutes at 4°C to pellet the mycelium. Collect the supernatant (broth) for analysis or use the pellet for intracellular metabolite extraction.

2. Extraction of Metabolites

The extraction method depends on whether you are targeting extracellular metabolites (in the broth) or intracellular metabolites (within the fungal biomass). Common methods include liquid-liquid extraction (LLE) or solid-phase microextraction (SPME).

a. Liquid-Liquid Extraction (LLE) for Extracellular Metabolites

1. Solvent Selection: Choose an organic solvent based on the target compounds’ polarity. Common solvents include ethyl acetate, dichloromethane, or chloroform, as they effectively extract a wide range of fungal secondary metabolites.

1. Procedure:
   • Transfer the filtered broth (e.g., 100 mL) to a separatory funnel.
   • Add an equal volume of the organic solvent (e.g., 100 mL ethyl acetate).
   • Shake vigorously for 5–10 minutes to allow partitioning of compounds into the organic phase.
   • Let the mixture stand until the organic and aqueous phases separate (typically 10–15 minutes).
   • Collect the organic phase in a clean glass container.
   • Repeat the extraction 2–3 times to maximize recovery, combining all organic phases.
2. Drying: Dry the combined organic phase over anhydrous sodium sulfate (Na₂SO₄) to remove residual water.
3. Concentration: Evaporate the solvent using a rotary evaporator under reduced pressure at 35–40°C to obtain a concentrated extract. Alternatively, use a nitrogen evaporator for small volumes.
4. Resuspension: Dissolve the dried extract in a small volume (1–2 mL) of a GC-MS-compatible solvent, such as methanol, hexane, or ethyl acetate, depending on the target compounds.

b. Solid-Phase Microextraction (SPME) for Volatile Organic Compounds (VOCs)

1. SPME Fiber Selection: Use a suitable fiber coating (e.g., polydimethylsiloxane (PDMS) or divinylbenzene/Carboxen/PDMS) based on the volatility and polarity of the target VOCs.
2. Procedure:
   • Transfer a small volume of the filtered broth (e.g., 5–10 mL) to a headspace vial.
   • Seal the vial with a PTFE/silicone septum cap.
   • Incubate the vial at a specific temperature (e.g., 40–60°C) to volatilize the compounds.
   • Insert the SPME fiber into the headspace of the vial and expose it for 20–60 minutes to adsorb VOCs.
   • Retract the fiber and immediately insert it into the GC-MS injection port for desorption.

c. Intracellular Metabolite Extraction (Optional)

1. Lysis of Fungal Biomass: If targeting intracellular metabolites, disrupt the fungal mycelium (pellet from centrifugation or filtration) using:
   • Mechanical Disruption: Homogenize the biomass in a solvent (e.g., methanol or ethyl acetate) using a bead beater or mortar and pestle.
   • Ultrasonication: Suspend the biomass in solvent and sonicate for 10–15 minutes in an ice bath to avoid overheating.
2. Solvent Extraction: Follow the same LLE steps as above to extract metabolites from the disrupted biomass.
3. Filtration and Concentration: Filter the extract through a 0.22 µm syringe filter and concentrate as described in the LLE section.

3. Sample Cleanup (Optional)

• Purpose: Remove impurities (e.g., proteins, lipids, or pigments) that may interfere with GC-MS analysis.
• Solid-Phase Extraction (SPE):
  • Pass the concentrated extract through an SPE cartridge (e.g., C18 or silica-based) preconditioned with methanol and water.
  • Elute the analytes with an appropriate solvent (e.g., methanol or hexane).
• Filtration: Filter the final extract through a 0.22 µm PTFE syringe filter to remove particulates.

4. Derivatization (If Required)

• Purpose: Some polar or non-volatile compounds (e.g., fatty acids, sugars, or amino acids) require derivatization to make them volatile for GC-MS analysis.
• Common Derivatization Methods:
  • Silylation: Add a silylating agent (e.g., N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)) to the sample. Incubate at 60–70°C for 30–60 minutes.
  • Methylation: Use reagents like diazomethane or methanolic HCl to convert carboxylic acids to methyl esters.
• Procedure:
  • Add 50–100 µL of the derivatizing agent to the dried extract.
  • Incubate under specified conditions (as per reagent instructions).
  • Dilute with a GC-MS-compatible solvent if necessary.

5. Preparation for GC-MS Analysis

• Dilution: Dilute the sample in a GC-MS-compatible solvent (e.g., hexane, methanol, or ethyl acetate) to a final concentration suitable for the instrument (typically 1–10 mg/mL, depending on sensitivity).
• Vial Preparation: Transfer 1–2 mL of the prepared sample to a GC-MS autosampler vial with a glass insert (for small volumes).
• Internal Standard (Optional): Add an internal standard (e.g., n-alkanes like hexadecane or deuterated compounds) to improve quantification accuracy.
• Blank and Control Samples: Prepare solvent blanks and control samples (e.g., uninoculated broth extracted similarly) to identify background contaminants.

6. GC-MS Instrument Setup

While this is not part of sample preparation, ensure the GC-MS parameters are optimized:

• Column: Use a non-polar capillary column (e.g., DB-5 or HP-5, 30 m × 0.25 mm, 0.25 µm film thickness) for most fungal metabolites.
• Carrier Gas: Helium at a flow rate of 1–1.5 mL/min.
• Injection Mode: Split or splitless, depending on sample concentration.
• Temperature Program: Typically, start at 50°C, hold for 2 minutes, ramp to 280–300°C at 5–10°C/min, and hold for 5–10 minutes.
• MS Settings: Electron ionization (EI) at 70 eV, scan range m/z 50–600.

7. Quality Control

• Sterility Check: Ensure no contamination during sample preparation by running blanks.
• Reproducibility: Prepare replicates (at least three) for each sample to ensure consistent results.
• Calibration: Use external or internal standards to quantify compounds and validate the method.

8. Storage

• Store prepared samples at -20°C in sealed vials to prevent degradation or volatilization of compounds. Analyze within 24–48 hours for best results.

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Notes

• Safety: Handle organic solvents and derivatizing agents in a fume hood, wearing appropriate PPE (gloves, goggles, lab coat).
• Optimization: The extraction solvent, incubation conditions, and derivatization method should be optimized based on the target metabolites and fungal species.
• Instrumentation Variability: Adjust sample preparation (e.g., concentration, solvent choice) based on the sensitivity and specifications of the GC-MS system.
• Data Analysis: After GC-MS analysis, use libraries (e.g., NIST, Wiley) to identify compounds by comparing mass spectra and retention times.