Polysaccharides from Poria cocos are best extracted using alkali extraction with NaOH after ethanol reflux to remove impurities, followed by concentration, ethanol precipitation, dialysis, and purification, as hot water or enzyme extraction alone may yield low or no results.

Standard Operating Procedure for Preparation of Freeze-Dried Plant Samples Using BSTFA+TMCS (99:1) (SYLON BFT) KIT 144 0.1 ML/AMPUL 33154-U SUPELCO for GCMS Analysis

Objective:

To provide a detailed procedure for preparing freeze-dried plant samples using chemical derivatization with BSTFA+TMCS and Pyridine in a 50 ml Round Bottom Flask with septa and Argon balloon (without O2) at 50°C with Heidolph heating system with magnetic stirrer, followed by injection into GCMS with comparison to alkane standard (C8-C40) using Kovats Retention Index (KRI).

Requirements:

1. Freeze-dried plant samples (completely dry)

2. BSTFA+TMCS (99:1) (SYLON BFT) KIT 144 0.1 ML/AMPUL (Catalog No.33154-U )

3. Spectrophotometric grade Pyridine

4. 50 ml Round Bottom Flask with septa

5. Argon balloon (oxygen-free)

6. Heidolph heating system with temperature control

7. Magnetic stirrer with stir bar

8. GCMS system with SLBTM-5ms column (30 m × 0.25 mm, 0.25 μm film thickness)

9. Alkane standard (C8-C40)

10. Dry, moisture-free vials

11. Safety goggles, chemical gloves, and lab coat

Preparation Procedures:

1. Preparation of Freeze-Dried Samples:

• Ensure plant samples are completely dried using freeze dryer (Labconco FreeZone)

• Store dried samples in dry, sealed vials until analysis time

• Prohibited: Do not use moist samples (must be completely dry)

2. Derivatization Process using BSTFA+TMCS:

1. But 1 mg of freeze-dried sample in a 50 ml Round Bottom Flask

2. Add 100 μL of BSTFA+TMCS (99:1) (SYLON BFT) 0.1 ML/AMPUL 33154-U SUPELCO to the sample (1ampuls)

3. Add 50 μL of Pyridine (as catalyst)

4. Connect the oxygen-free Argon balloon to the flask via septa to maintain an oxygen-free environment

5. Place the flask on the Heidolph heating system with magnetic stirrer

6. Set temperature to 50°C (not 100°C as in some other protocols)

7. Turn on magnetic stirrer at appropriate speed (600-800 rpm)

8. Allow reaction to proceed for 60 minutes with continuous stirring

9. After completion, allow sample to cool to room temperature

10. Transfer 1 μL of the sample to an appropriate GCMS injectur

3. Kovats Retention Index (KRI) Calibration:

1. Dissolve 1 μL of each alkane (from C8 to C40) in 1 ml of hexane

2. Analyze the alkane standard mixture before sample analysis

3. Record retention times for each alkane

4. Calculate retention index for each compound in the sample using the formula: RI = 100 × n + 100 × (t_R - t_n)/(t_{n+1} - t_n) where:

1. n = number of carbon atoms in smaller alkane

2. t_R = retention time of compound

3. t_n = retention time of smaller alkane

4. t_{n+1} = retention time of larger alkane

4. GCMS Analysis Parameters:

1. Column: SLBTM-5ms (30 m × 0.25 mm, 0.25 μm film thickness)

2. Injector temperature: 220°C

3. Injection volume: 1 μL

4. Injection mode: Split mode

5. Carrier gas flow rate (helium): 1.0 mL/min

6. Temperature program:

1. Start at 60°C for 2 minutes

2. Ramp at 3°C/min to 170°C

3. Hold at 170°C for 3 minutes

4. Ramp at 3°C/min to 250°C

5. Hold at 250°C for 120 minutes

5. Data Analysis:

1. Analyze alkane standard mixture before sample analysis

2. Calculate Kovats Retention Index (KRI) for each compound in the sample

3. Compare calculated retention index with reference values from databases

4. Use internal standards to calculate absolute compound percentages

5. Record all data in logbook with sample details

Prohibited Actions:

1. Do not use BSTFA+TMCS from untrusted sources (must use Supelco33154-U )

2. Do not expose to moisture during preparation (BSTFA reacts strongly with water)

3. Do not exceed 15% of vial volume when adding solvents

4. Do not open the flask after BSTFA addition until reaction time is complete

5. Do not analyze samples without first analyzing alkane standard (for retention index calculation)

6. Do not rely solely on spectral match without retention index (may lead to incorrect identification)

7. Do not analyze sample more than 24 hours after derivatization (derivatives degrade over time)

Important Notes:

1. Kovats Retention Index (KRI) is constant across instruments while retention time varies

2. Using oxygen-free Argon system prevents oxidation and improves derivative stability

3. Temperature of 50°C is suitable for sensitive samples that may degrade at higher temperatures

4. Ensure alkane standard is analyzed in every batch for accurate retention index calculation

5. For accurate analysis, compare sample with reference samples from the same geographical region

Quality Criteria:

1. Spectral match factor: must be >800 to confirm compound identification

2. Retention index: must be within ±10 units of reference value

3. Peak ratios: must be consistent with reference standards

4. Retention time reproducibility: must be within ±0.1 minute between replicates

Safety:

1. Always wear personal protective equipment (gloves, goggles, and lab coat)

2. Ensure adequate ventilation in the work area

3. Do not expose directly to BSTFA vapor for extended periods (causes eye and respiratory irritation)

4. Do not smoke or have spark sources in the work area

5. BSTFA is flammable - keep away from heat and ignition sources

STFA+TMCS Reaction with Polar Components in Freeze-Dried Samples

What BSTFA+TMCS Does

BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) with TMCS (trimethylchlorosilane) is a derivatization reagent used to convert polar compounds into less polar, more volatile derivatives suitable for Gas Chromatography-Mass Spectrometry (GCMS) analysis. This is particularly important for freeze-dried plant samples which contain many polar compounds that cannot be directly analyzed by GCMS due to:

1. Low volatility

2. Thermal instability

3. Strong adsorption to column surfaces

4. Poor peak shape in chromatograms

Chemical Reaction Mechanism

BSTFA+TMCS reacts with polar functional groups (primarily -OH and -COOH) to form trimethylsilyl (TMS) derivatives through the following reactions:

1. With Carboxylic Acids (-COOH)

R-COOH + BSTFA → R-COOSi(CH₃)₃ + CF₃CO₂H + TMSOH

2. With Alcohols/Phenols (-OH)

R-OH + BSTFA → R-OSi(CH₃)₃ + CF₃CO₂H + TMSOH

TMCS acts as a catalyst that enhances the reaction rate and completeness, especially for sterically hindered hydroxyl groups.

Examples with Polar Components in Freeze-Dried Samples

1. Phenolic Compounds

Benzoic acid (C₆H₅COOH)

1. Original: Polar, low volatility

2. After derivatization: Benzoic acid, trimethylsilyl ester (C₁₀H₁₄O₂Si)

3. Reaction: C₆H₅COOH + BSTFA → C₆H₅COOSi(CH₃)₃ + CF₃CO₂H + TMSOH

4. Result: Increased volatility, better peak shape, and thermal stability

2. Sugars (Critical for Freeze-Dried Samples)

Mannose (C₆H₁₂O₆)

1. Original: Highly polar, non-volatile, thermally unstable

2. After derivatization: Mannose, octakis(trimethylsilyl) ether (C₃₀H₇₄O₈Si₈)

3. Reaction: C₆H₁₂O₆ + 8 BSTFA → C₆H₁₂₋₈O₆(Si(CH₃)₃)₈ + byproducts

4. Result: Becomes sufficiently volatile for GCMS analysis (without derivatization, sugars cannot be analyzed by GCMS)

Glucose

1. Original: Polar sugar that decomposes before vaporizing

2. After derivatization: Glucose, octakis(trimethylsilyl) ether

3. Kovats Retention Index: ~2100

4. Key m/z values: 217, 204, 319

3. Organic Acids

Palmitic acid (Hexadecanoic acid)

1. Original: C₁₆H₃₂O₂ (fatty acid)

2. After derivatization: Hexadecanoic acid, trimethylsilyl ester (C₂₁H₄₆O₂Si)

3. Kovats Retention Index: ~1650

4. Key m/z values: 74, 255

4. Terpenoids

α-Terpineol

1. Original: Polar terpene alcohol

2. After derivatization: α-Terpineol, trimethylsilyl ether (C₁₃H₂₆OSi)

3. Kovats Retention Index: ~1350

4. Key m/z values: 93, 111

Why This is Essential for Freeze-Dried Samples

Freeze-dried plant samples contain high levels of polar compounds that would otherwise:

1. Decompose in the GC injector

2. Produce broad, tailing peaks

3. Adsorb to the column walls

4. Have poor detection sensitivity

The derivatization with BSTFA+TMCS:

1. Converts polar hydroxyl and carboxyl groups to non-polar TMS groups

2. Increases volatility by 10-100x

3. Improves thermal stability (allows analysis at 250°C)

4. Creates sharper, more symmetric peaks

5. Enables detection of compounds that would otherwise be invisible in GCMS

Important Considerations

1. Water-free environment is critical: BSTFA reacts violently with water (moisture must be