9.1 Extraction and Identification of Volatile Compounds from Agarwood

Target Compounds: Sesquiterpenes, chromones, essential oils, and other secondary metabolites.

1. Sample Preparation

  1. Collection:
    • Select resinous heartwood or induced agarwood chips.
    • Record tree ID, resin quality, and harvest date.
  2. Drying:
    • Air-dry at 40–50°C or in shade until constant weight.
    • Avoid high temperatures to prevent compound degradation.
  3. Grinding / Powdering:
    • Use a clean grinder to produce fine powder.
    • Store in airtight containers at low temperature until extraction.

2. Extraction Methods

A. Steam Distillation (for essential oils)

  • Procedure:
    1. Place powdered agarwood in distillation flask.
    2. Pass steam through the material.
    3. Condense vapors to collect essential oil.
  • Advantages: Produces volatile oil suitable for GC-MS analysis.

B. Solvent Extraction

  • Solvents: Ethanol, methanol, hexane, or dichloromethane.
  • Procedure:
    1. Mix powdered agarwood with solvent (1:10 w/v).
    2. Sonicate or macerate for 24–48 h.
    3. Filter and evaporate solvent under reduced pressure.
  • Advantages: Extracts both volatile and semi-volatile compounds.

C. Supercritical CO₂ Extraction (optional, advanced)

  • High selectivity for sesquiterpenes and chromones.
  • Solvent-free, high-purity extracts.

3. Identification Techniques

A. Gas Chromatography-Mass Spectrometry (GC-MS)

  • Purpose: Separate and identify volatile, low-molecular-weight compounds.
  • Procedure:
    1. Dissolve essential oil in suitable solvent (hexane or ethanol).
    2. Inject sample into GC-MS system.
    3. Separate compounds on a capillary column.
    4. Detect using mass spectrometer and compare spectra with reference libraries (NIST, Wiley).
  • Applications:
    • Sesquiterpene profiling
    • Quality assessment of agarwood oil
    • Chemotaxonomic studies

B. High-Performance Liquid Chromatography (HPLC)

  • Purpose: Separate and quantify semi-volatile or non-volatile compounds such as chromones.
  • Procedure:
    1. Dissolve extract in methanol or suitable solvent.
    2. Filter through 0.45 µm syringe filter.
    3. Inject into HPLC equipped with UV or PDA detector.
    4. Use C18 reverse-phase column; mobile phase: gradient of water/acetonitrile or water/methanol.
  • Applications:
    • Chromone quantification
    • Standardization of agarwood extracts
    • Correlation with resin quality

4. Data Analysis

  • GC-MS: Identify compounds by retention time and mass spectra; calculate relative abundances.
  • HPLC: Determine peak area, retention time, and quantify against standards (e.g., 2-(2-phenylethyl)chromone).
  • Reporting: Include chemical profile, total sesquiterpene and chromone content, and comparison with reference samples.

5. Quality Control and Considerations

  • Avoid contamination from solvents or equipment.
  • Use freshly prepared extracts for accurate analysis.
  • Ensure instrument calibration and standard references for reproducible results.
  • Store extracts in amber vials at 4°C to prevent degradation.

6. Applications

  • Resin quality assessment for commercial agarwood trade.
  • Selection of high-resin genotypes for plantations.
  • Correlation of induction methods with chemical profiles.
  • Research and product development in perfumes, cosmetics, and aromatherapy.